Gene Therapy - Progress and Prospects Review Series
Each Gene Therapy Progress and Prospects review provides a succinct summary of the last 2 years of progress in a specific aspect of
gene therapy research and will highlight prospects for the next 2 years. Written by the leaders in the field, the concise, targeted
content will cover the most significant as well as the 'hottest' topics. From identifying potential target diseases to the vectors,
technologies and systems being developed to detect efficiency, the whole range of the field will be covered. The Progress and
Prospects format has been specifically designed to be reader-friendly, including a bulleted section that allows a quick snapshot of the
Progress and Prospects as the expert authors see them.
Cancer gene therapy using tumour suppressor genes
I A McNeish, S J Bell and N R Lemoine
Noninvasive imaging of gene therapy in living subjects
J J Min and S S Gambhir
Gene therapy for severe combined immunodeficiency
H B Gaspar, S Howe and A J Thrasher
Parkinson's disease
E A Burton, J C Glorioso and D J Fink
Gene therapy of lysosomal storage disorders
S H Cheng and A E Smith
Adenoviral vectors
J A St George
Gene therapy for the hemophilias
C E Walsh
Gene therapy in organ transplantation
J Bagley and J Iacomini
Naked DNA gene transfer and therapy
H Herweijer and J A Wolff
Therapeutic angiogenesis for limb and myocardial ischemia
T A Khan, F W Sellke and R J Laham
Alpha-1 antitrypsin
A A Stecenko and K L Brigham
Nonviral vectors
T Niidome and L Huang
Post-intervention vessel remodeling
J Rutanen, H Puhakka and S Ylä-Herttuala
Cystic fibrosis
U Griesenbach, S Ferrari, D M Geddes and E W F W Alton
March 2004, Volume 11, Number 6, Pages 497-503
Table of contents
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Review
Gene Therapy Progress and Prospects: cancer gene therapy
using tumour suppressor genes
I A McNeish1, S J Bell1 and N R Lemoine1
Cancer Research UK Molecular Oncology Unit, Imperial College
1
School of Medicine, Hammersmith Hospital, London, UK
Correspondence to: Dr IA McNeish, Cancer Research UK Molecular
Oncology Unit, MRC Cyclotron Building, Hammersmith Hospital,
London W12 0HS, UK
Abstract
Targeting tumour suppressor gene pathways is an attractive therapeutic strategy in cancer.
Since the first clinical trial took place in 1996, at least 20 other trials have investigated the
possibility of restoring p53 function, either alone or in combination with chemotherapy, but
with limited success. Other recent clinical trials have sought to harness abnormalities in the
p53 pathway to permit tumour-selective replication of adenoviral vectors such as dl1520
(Onyx-015). Other tumour suppressor genes, such as retinoblastoma (Rb) and PTEN
(phosphatase, tensin homologue, deleted on chromosome 10), are the targets for imminent
clinical trials, while microarray technologies are revealing multiple new genes that are
potential targets for future gene therapy.
Gene Therapy (2004) 11, 497503. doi:10.1038/sj.gt.3302238
Published online 5 February 2004
Keywords
tumour suppressor gene; p53; Rb; PTEN; cinical trial
In brief
Progress







Clinical trials of p53 gene replacement have had limited success
Replicating adenoviral vectors targeting abnormal p53 function
have also had limited success in clinical trials
Targeting the Rb pathway: Rb mutants may be more potent
tumour suppressors than wild-type Rb
New oncolytic adenoviruses also target the Rb pathway
The INK4ARF locus provides two potential targets for gene
therapy
PTEN expression alters metastatic potential and reduces
neovascularization
Multiple new tumour suppressor genes offer new therapeutic
possibilities, especially mda-7 and OPCML
Prospects




The ability to induce growth arrest and apoptosis in vitro does
not guarantee clinical success.
Fuller understanding of downstream targets of p53 and Rb is
necessary.
Clinical trials of second-generation oncolytic viruses targeting
Rb pathway will be eagerly awaited
Combinations of tumour suppressor genes may offer new
greater therapeutic potential

New tumour suppressor genes will be discovered
Introduction
It has long been recognized that the development of invasive malignancy requires multiple genetic
events, and modern technologies now suggest that tens, if not hundreds, of genes may be aberrantly
expressed in malignant cells.1,2 In the last decade, studies on p53 replacement have dominated the
literature and it remains the only tumour suppressor gene to be evaluated formally in clinical trials. Here,
we review the progress that has been made in the past 2 years in the field of tumour suppressor gene
therapy and the future prospects for utilizing pathways other than p53, including the well characterized,
such as retinoblastoma (Rb) and PTEN (phosphatase, tensin homologue, deleted on chromosome 10), as
well as those described more recently, such as melanoma differentiation associated gene-7 (mda-7) and
opioid binding proteincell adhesion molecule-like gene (OPCML).
Clinical trials of p53 gene replacement have had limited success
After the promise of the first clinical trial of p53 gene replacement in non-small-cell lung carcinoma in
1996, those published in the past 2 years3,4,5,6,7 have been somewhat disappointing. In a neoadjuvant
bladder carcinoma trial5, 12 patients received either intratumoral or intravesical injections of an
adenovirus-encoding wild-type p53 (Ad p53) 3 days prior to radical cystectomy. Interestingly,
transfection efficiency was much greater following intravesical administration and, overall, 711 (64%)
evaluable patients (including 79 of the intravesical cohort) had evidence of transgene expression by
vector-specific reverse-transcriptase PCR (RT-PCR), as well as some evidence of increased expression
(both mRNA and protein) of p21Waf1Cip1, a p53 target gene. By contrast, in patients with locally advanced
bladder cancer treated with intravesical Ad p53 at comparable doses,6 only 27 (29%) tumours
demonstrated p53 transgene expression, with no detectable changes in the expression of either
p21Waf1Cip1 or Bax. When comparing transgene expression in these two bladder cancer trials, it is possible
that the larger instillation volume (120 ml) and the use of a transfection-enhancing agent in the
neoadjuvant trial 5 contributed to the higher transfection rates.
In a phase I recurrent glioma trial, 12 patients received intratumoral Ad p53 at doses between 3  1010
to 3  1012 particles, followed by tumour resection, at which time more Ad p53 was injected into the
tumour bed.7 Before Ad p53 injection, only one of eight assessed tumours was p53 positive (by
immunohistochemistry), while 1012 showed nuclear p53 staining after injection and 78 showed positive
staining for p21Waf1Cip1. However, the zone of transfected cells extended no more than 8 mm from the
injection site and the median overall survival for the whole cohort was only 43 weeks.
In non-small-cell lung cancer, intratumoral injection of 7.5  1012 particles of Ad p53 every 21 or 28 days
produced transgene expression in 1725 (68%) tumours. Patients also received chemotherapy (either
carboplatin and paclitaxel or cisplatin and vinorelbine), but the frequency of overall tumour response was
the same in Ad p53-injected lesions and noninjected lesions (52 versus 48%, respectively). However,
there was a suggestion that the Ad p53-treated lesions reduced in size by a greater amount than the
noninjected controls.
Ovarian cancer is traditionally thought to be an appealing target for clinical gene therapy because the
disease tends to remain localized within the abdominal cavity, so that intraperitoneal vector delivery is a
rational strategy. The extensive experience of p53 gene therapy in this disease culminated in a
randomized phase III trial in which women with p53-null or p53 mutant tumours were randomized to
chemotherapy alone or chemotherapy plus intraperitoneal Ad p53 following optimum debulking primary
surgery. However, the first interim analysis indicated that not only did Ad p53 fail to improve
effectiveness but was also associated with increased toxicity. As a result, the study has been abandoned
(reported in Zeimet and Marth8).
Despite the limited clinical efficacy, some positive factors have emerged from these trials. Firstly, it is
noticeable that the trials have been designed with credible scientific as well as clinical end points.
Secondly, except for the experience in the ovarian phase III trial, of which few details are available,
treatment has largely been well tolerated with minimal toxicity. However, one must address why the
trials were relatively unsuccessful and two broad possibilities emerge. Firstly, there remains the
perennial problem of optimizing gene transfer. Improving gene transfer in the clinical setting with
delivery of vectors to tumours disseminated throughout the body is a huge problem and lies outside the
scope of this review. Secondly, there remains the possibility that p53 is the 'wrong' transgene. Although
p53 mutations are found in many malignancies and defective p53 function may be causally linked to
chemotherapy resistance,9 many aspects of p53 biology remain unanswered, especially what determines
whether cells undergo apoptosis or cell cycle arrest in response to p53 activation.10 There is some
evidence that low-level p53 expression, such as is likely to result from adenoviral gene transfer, causes
cell cycle arrest rather than cell death. Also, the proapoptotic function of p53 depends upon
transactivation of genes such as Bax, Apaf-1, Fas and PTEN, whose own expression or activity may be
abnormal in tumour cells.11 It is known that mutant p53 can act in a dominant-negative manner in p53
tetramers,12 which could negate the effect of ectopically expressed wild-type protein. Finally, there is
evidence that polymorphisms of the p53 gene (especially codon 72  arginine versus proline) can
determine the responsiveness of tumours to chemo- and radiotherapy by influencing inhibition of p73.13
Only once all these issues have been addressed is there likely to be any advance in the field of p53 gene
replacement.
Replicating adenoviral vectors targeting abnormal p53 function have also had limited success
in clinical trials
The adenovirus E1B 55 kDa protein suppresses p53 function in infected cells and E1B 55K-deleted
adenoviral vectors may be able to replicate within and cause cytolysis of tumours with defective p53
function. In the past 2 years, six separate phase III trials of such a virus (variously known as dl1520,
Onyx-015 and CI-1042) have been published, in a range of tumour types, including colorectal, 14,15
ovarian 16 and pancreatic carcinomas,17,18 and in patients with liver metastases from gastrointestinal
malignancies.19 A total of 93 patients received doses of up to 2  1012 viral particles per injection with no
objective clinical responses seen in any patient treated with dl1520 as a single agent. However, in
combination with chemotherapy, some responses were seen; with 5-FU, eight patients with colorectal
liver metastases demonstrated either partial or minor responses, at least five of whom had previously
been refractory to 5-FU.14,19 In primary pancreatic carcinoma, two patients had partial responses in
combination with gemcitabine.17
One complexity in analysing these results is that it is now apparent that cellular p53 status is not the
only determinant of the replication of this virus.20 There have been many reports of replication within
cells that are p53 wild type and there is contradictory evidence on the possible importance of the mdm2hdm-2 inhibitor p14Arf.21,22 Similarly, E1B 55K almost certainly has functions in addition to p53
suppression, including modulating viral and cellular mRNA nuclear transport and stimulating late viral
mRNA translation. Given this, the results of the trials and the uncertainties over p53 replacement, it
seems unlikely that any further significant progress will be made with dl1520.
Targeting the Rb pathway: Rb mutants may be more potent tumour suppressors than wildtype Rb
Rb is the paradigmatic tumour suppressor gene, originally postulated in 1971. It is the target for
transforming viral proteins such as HPV E7 and adenovirus E1A, inactivation of the Rb and p53 genes
alone can induce malignancy in mouse models23 and abnormalities in the Rb pathway and the G1S
checkpoint probably exist in all malignancies.24 The pathway has many components that are potential
targets for therapy (see Figure 1). Upon growth stimulation, cyclin D expression increases and it forms
complexes with cyclin-dependent kinase 4 (cdk4) or cdk6 and these complexes sequester the cdk
inhibitor p27Kip1 from cyclin Ecdk2. The cyclin Dcdk4 and cyclin Ecdk2 complexes are now able to
phosphorylate Rb and this phosphorylated form of Rb can no longer bind the E2F family of transcription
factors, freeing E2F to transactivate the genes necessary for S-phase entry. The activity of cyclin Dcdk is
also controlled by the INK4 family of inhibitors, of which p16 INK4A is perhaps the best known.
Rather surprisingly, there have been many fewer studies on replacement of Rb family members than
p53. Early reports suggested that the ability of Rb expression alone to inhibit tumour cell growth is
variable and Rb expression may, paradoxically, inhibit p53-induced apoptosis.25 Rb phosphorylation
mutants and truncated variants may have enhanced tumour suppressor function compared to the wildtype protein. One such derivative is Rb94, in which translation is initiated from a second AUG codon in the
Rb mRNA and which lacks the N-terminal 112 amino acids of the full-length protein. There is evidence
that Rb94 has a longer half-life than Rb itself and remains in the hypophosphorylated form for extended
periods. Two recent reports suggest that adenovirus-mediated Rb94 gene transfer can induce apoptosis in
models of head and neck26 and bladder27 cancers, with minimal effects on nonimmortalized normal cells.
Of note, Rb94 appears able to induce cell death regardless of the Rb status of tumours, unlike the full-
length protein, which is not effective in tumours bearing wild-type Rb. One potential explanation for this
is that Rb94, in addition to generating caspase-mediated apoptosis, appeared to induce cell cycle
blockade at G2M (rather than G1) and also rapid telomere erosion with ensuing chromosomal instability.
Although it had previously been reported that full-length Rb could inhibit telomerase, the cell cycle
findings are novel and as yet unexplained.
Another Rb variant, Rb56, is also a C-terminal derivative. It contains the regions necessary for E2F
binding and may be capable of inhibiting E2F-mediated transcription more efficiently than full-length Rb.
Recent work on Rb56 has demonstrated the ability of a fusion protein, consisting of Rb56 and the DP-1
binding domains of E2F to induce cell cycle arrest in vascular smooth muscle cells and inhibit smooth
muscle cell hyperplasia in response to intimal injury.28 Taken together, these reports suggest that Rb
mutants and splice variants may be more potent tumour suppressors than Rb. Data on the potential of
the other two members of the Rb family, p107 and p130, in gene therapy are very limited, but
retrovirus-mediated transfer of the p130 gene can suppress the growth of lung carcinoma cells in vitro
and in vivo.29
New oncolytic adenoviruses also target the Rb pathway
Following on from dl1520, a second generation of selectively replicating adenoviral vectors has now been
developed. The viruses nearest to clinical trial specifically target Rb function. The adenoviral E1A protein
contains two conserved regions, CR-1 (amino acids 3060) and CR-2 (amino acids 120127), the latter
critical for binding to and inactivating Rb and whose deletion prevents formation of E1ARb complexes.
Two similar mutants have been described recently; dl922947 is deleted in amino acids 122129,30 while
24 is deleted in amino acids 121128.31 Both have been assessed in in vitro and in vivo models of
cancer and dl922947 is capable of replicating with much greater efficiency within a panel of tumour cell
lines than dl1520, with minimal S-phase induction in quiescent nonimmortalized cells.30 Most recently,
24 has been modified further to include a RGD-4C peptide into the adenoviral fibre, which permits
infection of cells independent of the normal coxsackie adenovirus receptor that is frequently expressed at
very low levels on tumour cells.32 24-RGD is capable of lysing ovarian carcinoma and glioma cells in
vitro, as well as extending the survival of mice-bearing xenografts of both tumour types.32,33 Of note,
24-RGD appeared to have a significantly greater cytopathic effect than 24 and its replication on normal
human astrocytes was at least 3 log scales lower than a wild-type adenovirus.33 Clinical trials of both
24-RGD and dl922947 are imminent.
Further adenoviral mutants also explore targeting of the Rb pathway. Ar6pAE2fF34 and Onyx-41135 both
have an E2F promoter in place of the adenoviral E1A promoter. In addition, Onyx-411 has a second E2F
promoter to drive the expression of the E4 region and is also deleted in the E1A-CR-2 region, like
dl922947. The rationale behind these modifications is that the E2F promoter is selectively activated in
the presence of a defective Rb pathway and E4 gene products, especially E4 orf46, cooperate with E1A
and E1B proteins to create a cellular environment that permits efficient expression of viral genes and
thus productive viral infection.36 Both Ar6pAE2fF and Onyx-411 demonstrate tumour-specific replication
with minimal effect upon normal cells, including proliferating epithelial cells, and both were more potent
and tumour selective than dl1520.
INK4ARF locus provides two potential targets for gene therapy
The INK4ARF locus on chromosome 9 encodes two separate tumour suppressor genes from alternative
reading frames, p16INK4A and p14Arf (also known as p19Arf in mice), which serve to highlight the close link
between the p53 and Rb pathways.24 p16INK4A is a potent inhibitor of the cyclin Dcdk4 complex that
phosphorylates Rb, while p14Arf inhibits hdm-2 (mdm-2 in mice), whose functions are to prevent p53mediated transcription and to promote p53 ubiquitination. Homozygous deletions of the INK4ARF locus
are seen in many malignancies especially melanoma.37
Adenoviral delivery of the p16INK4A gene is able to induce cell cycle arrest in vitro and, in cooperation with
adenoviral p53 expression, induce apoptosis and inhibit tumour growth in vivo. It appears that p16INK4A
may be a more effective inducer of apoptosis than other members of the INK4 family (p15 INK4B, p18INK4C)
or the Waf1Cip1 family (p21, p27). Interestingly, it appears that p16 INK4A is able to induce apoptosis in
cells lacking Rb, suggesting that it may have alternative functions. This has been reiterated by more
recent work in which the effectiveness of p16INK4A and p53 gene delivery was compared in ovarian
carcinoma models with varied p16INK4A and p53 status (wild type, null and mutant).38 In all cell lines,
p16INK4A appears as a more efficient inducer of growth arrest, but not apoptosis, than p53. In vivo,
however, adenoviral p16INK4A (Ad p16) produces statistically greater survival in p16 INK4A- and p53-null or
wild-type models than Ad p53 alone or even Ad p16 and Ad p53 combined. Clearly, as has been
mentioned above, the limited efficacy of Ad p53 could result from abnormalities in downstream effectors
of p53-mediated apoptosis. However, the same may be true of pathways downstream of p16 INK4A.
Therefore, it remains possible that p16INK4A has additional functions, of which downregulation of vascular
endothelial growth factor (VEGF) is one possibility. Other groups have recently demonstrated the efficacy
of adenoviral p16INK4A delivery in lymphoma39 and glioma40 models, but one note of caution is necessary.
There is evidence that ectopic overexpression of p16INK4A can produce resistance to some chemotherapy
drugs, possibly by inducing G1 cell cycle arrest, as many chemotherapy drugs are at their most effect in
S phase.41
In the past 2 years, more interest has focused on p14Arf. Several reports have demonstrated that
adenoviral delivery of the p14Arf gene is capable of inducing cell cycle arrest and apoptosis in a wide
variety of tumour models42,43,44,45,46,47 and can sensitize cells to chemotherapy.48 Initial reports suggested
that intact p53 pathways were required for p14Arf-mediated cytotoxicity47,48,49 and that cotransfection
with wild-type p53 could enhance the p14Arf effect.46 It now appears that p14Arf is capable of affecting
proteins other than p53, such as E2F-1,50 HIF1
51
and topoisomerase I.52 Cluster analysis of gene
expression patterns in mouse embryo fibroblasts indicates that p19ARF induces expression of both p53dependent and -independent genes, the latter including members of the B-cell translocation family
(BtgTob) that can inhibit proliferation in cells regardless of p53 status.43 Recently, p14Arf has shown itself
capable of inducing apoptosis in p53- and Bax-null DU145 prostate carcinoma cells.42 In p53-null H358
lung carcinoma cells, p14Arf induces arrest in G2 phase, followed by apoptosis. This G2 arrest correlates
with inhibition of CDC2, inactivation of CDC25C and induction of p21Waf1. Of note, p14Arf is capable of
inducing tumour regression in H358 xenografts.53 One possible explanation for the discrepancy between
these results and earlier studies that suggested p53 was an absolute requirement for p14Arf-mediated cell
death is timing.49 In p53-null cells, it takes up to 6 days for G2 arrest to take place, in contrast to only
2448 h in p53-positive cells.53
PTEN expression alters metastatic potential and reduces neovascularization
PTEN, also known as MMAC1 and TEP-1, is a phosphatase whose importance as a tumour suppressor
gene is being increasingly recognized. Although PTEN can dephosphorylate proteins such as focal
adhesion kinase, its primary function is to degrade the products of phosphatidylinositol 3'-kinase (PI3kinase) by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4,bisphosphate at the 3' position. One of the main downstream targets of the PI-3kinase pathway is the
kinase Akt, also known as PKB (protein kinase B), which, in turn, can activate a wide range of signals
that lead to cell proliferation and decreased apoptosis (see Figure 2). Thus, the loss of PTEN activity,
which is seen in up to 40% of all malignancies,54,55 can have diverse effects on cell growth and
differentiation.
In the past 2 years, there have been a number of studies investigating PTEN gene replacement, many of
which have focused on prostate cancer. In PTEN-null prostate cancer, expression of PTEN causes a
decrease in Bcl-2 expression and sensitizes cells to doxorubicin and vincristine chemotherapy, 56 and also
sensitizes cells to death receptor-mediated apoptosis that could be overcome with Bcl-2
overexpression.57 In another prostate model, adenoviral PTEN (Ad PTEN) delivery to PC3 cells in vitro
leads to G1 arrest, but not apoptosis. 58 Interestingly, when the PC3 cells are transfected with Ad PTEN
and then implanted orthotopically into mice, there is no reduction in tumorigenicity, but a significant
reduction in the development of lymph node metastases, implying that PTEN may not be a critical
regulator of tumour formation and growth, but a controller of dissemination. When Ad PTEN is injected
directly into pre-existing prostate xenografts, there is no tumour regression, which further underlines
this point.
By contrast, injection of Ad PTEN into bladder xenografts produced demonstrable tumour regression and
induction of apoptosis, but only in PTEN-null UM-UC-3 tumours. In tumours that are PTEN wild type, Ad
PTEN injection produced only transient growth inhibition.59 Alongside reduction in phosphorylated Akt
expression, another observation from the UM-UC-3 tumours is a reduction in VEGF expression both in
vitro and in vivo, the latter accompanied by a reduction in tumour vessel formation. VEGF is known to be
an AktPTEN target,60 and neovascularization is a marker of transformation from low- to high-grade
gliomas in humans with PTEN mutations seen almost exclusively in high-grade tumours. Further
indication of the potential of PTEN to influence angiogenesis in glioma is shown with U87MG xenografts in
mice. In the presence of PTEN expression, in vivo growth is reduced, with marked reduction in
angiogenic activity.61 Even in the presence of proangiogenic signals such as constitutive EGFR activation
andor p53 inactivation, Ad PTEN delivery to glioma xenografts in mice produces a marked reduction in
tumour vascularity.62 Therefore, there may be a differential role for PTEN in different tumour types,
reducing invasion and metastatic potential in some models and inhibiting tumour vascularization in
others.
Multiple new tumour suppressor genes offer new therapeutic possibilities, especially mda-7
and OPCML
Mda-7 (also known as IL-24) is a member of the IL-10 family of cytokines and was first described as a
potential tumour suppressor gene, when shown to be expressed on differentiated melanocytes but not
melanoma cells. Subsequently, it was shown that adenoviral delivery of the mda-7 gene (Ad mda-7) is
able to induce apoptosis in malignant cells but not normal epithelial cells in both melanoma 63 and
NSCLC.64
Work in the past 2 years has extended knowledge on this gene. Expression is downregulated in a wide
variety of malignancies,65 while restoration of expression via Ad mda-7 can also induce growth arrest in
vivo.66 The mechanisms via which mda-7 induces growth arrest and apoptosis are complex. It appears to
upregulate the expression of TRAIL and its receptors DR45, which could sensitize tumour cells to death
receptor-mediated apoptosis.66 There is also evidence that mda-7 can increase the expression of the
RNA-dependent protein kinase PKR in some NSCLC cells.67 The normal role of PKR is to limit viral
infection by inhibiting protein synthesis and hence block viral protein production, but it may also function
as a regulator of tumorigenesis. Recently, microarray analysis suggests that Ad mda-7 transfection can
alter expression of members of both the
-catenin and PI3kinase signalling pathways in some breast
and NSCLC cell lines.68 Curiously, this analysis was performed on the same NSCLC line (H1299) as had
been studied previously,67 but PKR was not one of the genes whose expression was upregulated. Finally,
several reports suggest that mda-7 may have a role in angiogenesis. Ad mda-7 is able to inhibit
endothelial cell differentiation and reduce tumour vascularity in human lung cancer xenografts in mice, 66
and purified mda-7 protein is capable of inhibiting endothelial cell differentiation and migration more
effectively than endostatin.69
Finally, another potential tumour suppressor gene has been identified in ovarian cancer that may have
therapeutic potential. OPCML is a member of the family of Ig domain-containing
glycosylphosphatidylinositol-anchored cell adhesion molecules and its expression is completely absent in
over 80% of ovarian carcinomas, including both established cell lines and primary tumours. 70
Interestingly, the downregulation appears due mainly to CpG island methylation, and restoration of
OPCML expression was able to impair ovarian carcinoma cell growth both in vitro and in vivo.70 Clearly,
more work will be required to evaluate the pathways via which OPCML functions in ovarian carcinoma.
Conclusions and prospects
Although targeting tumour suppressor gene pathways is an attractive and logical strategy for cancer
gene therapy, results from clinical trials have not mirrored the preclinical studies. Clearly, the ability to
induce cell cycle arrest and apoptosis in vitro or growth arrest in mouse xenografts does not guarantee
responses in clinical trials. Several specific hurdles must be overcome if such therapies are to become
routine. Firstly, a greater understanding of the biology of the ubiquitous p53 and Rb tumour suppressor
genes pathways is vital, especially an understanding of their own downstream targets and how these
may be altered in malignancy. Secondly, other pathways need to be thoroughly evaluated, especially
those that appear to be tumour-type specific. Surprisingly, little gene therapy work has been published
on restoring well-known tumour suppressor genes such as BRCA1 in breast cancer and APC in colon
cancer. The novel genes, OPCML and mda-7, may offer new disease-specific pathways to target in
ovarian and melanomalung carcinoma, respectively. Thirdly, restoring tumour suppressor gene function
alone may be insufficient and combination treatments, either with multiple genes (eg one diseasespecific and one ubiquitous gene) or a tumour suppressor gene with an apoptosis inducer such as
chemotherapy or activated caspases, may be required. However, one thing is certain: extending our
knowledge of tumour suppressor genes and their normal roles must ultimately lead to improved
therapies for all malignancies.
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Figures
Figure 1 Rb pathway. In response to a mitogenic stimulus, cyclin
Dcdk4 complexes form and sequester p27 and other Waf1Cip1
family members. The cyclin Dcdk4 and cyclin Ecdk2 complexes are
then free to phosphorylate Rb. This frees members of the E2F family
to transactivate genes necessary for S-phase entry. p16INK4A inhibits
cyclin Dcdk4 and thus prevents Rb phosphorylation.
Figure 2 The AktPTEN pathway. Oncogenic and mitogenic stimuli
that activate PI3kinase can lead to Akt activation, either directly, via
the actions of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and
phosphatidylinositol 3,4,-bisphosphate (PI(3,4)P2) on the plectrin
homology (PH domain), or indirectly, via 3'-phosphoinositidedependent kinase 1 (PDK1) and PDK2-mediated phosphorylation at
positions T308 and S473. Activated Akt can then modulate multiple
cellular pathways, leading to the inhibition of apoptosis and
stimulation of cell growth. PTEN has intrinsic lipid phosphatase
activity that removes the phosphate moeity from the 3' position of
PIP3 and PI(3,4)P2, and thus counters the antiapoptotic and growth
stimulatory activities of PI3kinase and Akt.
Received 22 September 2003; accepted 19 December 2003; published online 5
February 2004
March 2004, Volume 11, Number 6, Pages 497-503
January (2) 2004, Volume 11, Number 2, Pages 115-125
Table of contents
Previous Article Next PDF
Review
Gene Therapy Progress and Prospects:
Noninvasive imaging of gene therapy in living subjects
J J Min1 and S S Gambhir1
Department of Radiology and Bio-X Program, Stanford University,
USA
1
Correspondence to: Dr SS Gambhir, Stanford University, James H
Clark Center, 318 Campus Drive, East Wing, 1st Floor, Stanford, CA
94305-5427, USA
Abstract
Recent progress in the development of noninvasive imaging technologies should allow
molecular imaging to play a major role in the field of gene therapy. These tools have recently
been validated in gene therapy models for continuous quantitative monitoring of the
location(s), magnitude, and time variation of gene delivery andor expression. This article
reviews the use of radionuclide, magnetic resonance, and optical imaging technologies, as
they have been used in imaging gene delivery and gene expression for gene therapy
applications. The studies published to date lend support that noninvasive imaging tools will
help to accelerate preclinical model validation, as well as allow for clinical monitoring of
human gene therapy.
Gene Therapy (2004) 11, 115125. doi:10.1038/sj.gt.3302191
Keywords
molecular imaging; radionuclide imaging; magnetic resonance imaging (MRI); optical imaging; positron
emission tomography (PET)
In brief
Progress
Diagnostic imaging technologies such as positron emission tomography (PET), single-photon emission
computed tomography (SPECT), optical imaging, and magnetic resonance imaging (MRI) have been used
to noninvasively monitor transgene expression for gene therapy.
These in vivo molecular imaging technologies have been applied for gene therapy of cancer,
cardiovascular, neurological, musculoskeletal, hepatic and inherited diseases.
Radionuclide imaging (PETSPECT) has been used for imaging the distribution of radiolabeled vector,
immune cell trafficking, and assessment of transgene expression using diverse reporter genes and
reporter probes.
MRI-based molecular imaging technology provides excellent three-dimensional spatial resolution, but
still requires a further improvement in sensitivity.
Bioluminescence imaging using cooled CCD camera shows a minimal background and a very high
sensitivity, but lacks detailed tomographic information.
Therapeutic transgene expression can be monitored indirectly using a fusion approach, bicistronic
approach, dual-promoter approach, bidirectional transcriptional approach, and two-vector administration
approach.
Tumor-restricted gene expression through use of a tissue-specific promoter can be amplified using
two-step transcriptional amplification (TSTA) strategies.
Prospects
The explosion in genetic engineering is expected to generate vectors with more robust gene transfer
efficiency, bicistronicbidirectional molecular vectors and tissue-specific amplification techniques.
Continued refinement in the chemistry of molecular probe development should give rise to a new
generation of molecular imaging probes with greater sensitivity and specificity.
Advances in detector technology and image reconstruction techniques for PET should help to produce
a newer generation of imaging instruments with better spatial resolution, sensitivity, and significantly
improved throughput time.
New optical technology development including 3-D tomography will expand gene therapy research
using small animals, and can play an important role.
Multimodality reporter gene approaches should help gene therapy investigators to more readily move
between the various imaging instruments, and should help to accelerate the testing of various preclinical
models.
Introduction
Diagnostic imaging technologies have been used to try to monitor transgene expression for gene therapy
using MRI, optical imaging, and radionuclide imaging techniques including PET and SPECT. 1,2,3,4,5,6,7,8,9,10
Reporter genes with optical signatures (eg, fluorescence and bioluminescence) are low-cost alternatives
for real-time analysis of gene expression in small animal models. Fluorescence imaging uses a
fluorescent protein such as green fluorescent protein (GFP) that is excited with external illumination, and
the emission is subsequently detected.11 GFP-encoding cDNA can easily be included in the myriad of
therapeutic vectors and serve as a monitoring tool for gene therapy. Nevertheless, excitation and
emission wavelengths in the range of 500 nm (eg, GFP) have limited penetration in mammalian tissues
(15 mm). Since mammalian tissues absorb light that is used to excite these fluors, the tissues also
fluoresce when excited at these wavelengths. The combination of absorption of specific signal and
autofluorescence of tissues can result in poor signal-to-noise.3 Recently, red-shifted mutants of GFP
(RFP) have been known to have an advantage over GFP that red light penetrates tissues more efficiently
than green.12 Bioluminescent photoproteins such as luciferase have been used as reporter proteins in
living animals.2,13,14 Firefly luciferase (FL) catalyzes D-luciferin to produce oxyluciferin in the presence of
oxygen, cofactors, Mg+2, and ATP to produce light with peak at 562 nm. Recently, validated renilla
luciferase (RL) catalyzes the oxidation of coelenterazine in the presence of oxygen, to generate a flash of
blue luminescence with a peak wavelength at 482 nm. The advantage of bioluminescence is the minimal
background noise, since luciferase is not a natural constituent of mammalian organisms.
Bioluminescence-based approaches currently lack detailed tomographic information, and are limited to
relatively small animals.10,15,16 A newer approach to fluorescence imaging of deeper structures uses
fluorescence-mediated tomography.17 The subject is exposed in an imaging chamber to continuous wave
or pulsed light from different sources, and detectors arranged in a spatially defined order capture the
emitted light. Mathematical processing of this information results in a reconstructed tomographic image.
Fluorescence-mediated tomography is still in its infancy, requiring extensive mathematical validation
prior to routine implementation. The advantage of MR for the imaging of gene expression is the excellent
three-dimensional spatial resolution (tens of
m range) at imaging. Owing to the indirect nature of
enhancement produced by MR contrast agents, much higher concentrations of injected material, on the
order of 10100
M concentrations and higher, are generally necessary to produce sufficient image
contrast.2,3,6,7,10 The low sensitivity often entails long imaging times, and consequently slow data
7
acquisition. While magnetic resonance spectroscopy (MRS) does not usually produce three-dimensional
images, the technique does provide accurate measurement of gene expression in short time frames, and
may eventually be harnessed to produce true spatial images, but at a much poorer spatial resolution
than MRI. Radionuclide imaging with PET and SPECT has been used to characterize enzyme activity,
receptortransporter status, and biodistribution of various radiolabeled substrates (tracers). 8 For these
reasons, it has made the most significant progress for imaging gene therapy by monitoring gene delivery
and identifying therapeutic andor reporter gene expression in living subjects. While sensitivity in PET
imaging is high (as little as 10-1110-12 M of tracer can be detected) and the speed of imaging is
relatively rapid (min), these techniques lack micrometer spatial resolution (12 mm with micro-PET).3,7,9
An alternative approach to PET is SPECT imaging. While the sensitivity of the single-photon system is
intrinsically about one to two orders of magnitude less than PET systems, the required
radiopharmaceuticals and imaging systems are more readily available. Further details of the
instrumentation available and relative advantages between the various types of imaging instrumentation
may be found elsewhere.1,2,18
In the following sections, we will review several imaging technologies for monitoring gene delivery or
transgene expression. Much of the current focus of molecular imaging in gene therapy is directed
towards oncological applications; however, preliminary studies for cardiovascular and neurological
applications have also been reported. All the applications are briefly described in the text and are also
summarized in Tables 1 and 2.
Oncology
Radionuclide imaging
Imaging the vector utilized for gene delivery: To assess the efficiency of vector delivery, radionuclide
imaging can be used to look at the distribution of the radiolabeled vector itself. The ideal gene therapy
paradigm for brain tumors may consist of a combination of intratumoral injection and intra-arterial
administration of vectors bearing therapeutic transgenes. In previous studies, herpes simplex virus
(HSV) was radiolabeled with lipophilic 111In-oxine complex to be administered to intracerebral gliomabearing rats. Intracarotid injection of radiolabeled HSV revealed the low efficiency of viral uptake in the
tumor (0.10% of the injected dose per gram of tissue) at 1 h. When animals received virus injections
stereotactically into the tumor, 71.335.0% of the total dose was found in the tumor at 24 h.19 An
alternative labeling approach based on 99mTc-labeled recombinant adenovirus serotype 5 knob (Ad5K)
has also been validated.3 Imaging data for the hepatic uptake studies were in agreement with the
biodistribution determined by removing and measuring tissues. Recently, we have investigated the
potential of labeling adenovirus with 99mTc or 124I to study the viral biodistribution and demonstrated
stable labeling with 99mTc using an Isolink carbonyl kit20 and 124I using the standard iodogen method21
without loss of viability or infectivity. This allows imaging viral biodistribution and reporter gene
expression.
Imaging of nonviral vector delivery has been studied for direct visualization of the distribution of doublestranded DNA (pCMV-GFP). The generic structure of the probe comprises three elements: (1) a peptidebased chelate that binds the 99mTc; (2) a positively charged linker for binding to DNA phosphodiester
backbone; (3) an intercalating psoralen group.22 The formation of a stable complex between the probe
and the DNA is achieved by ultraviolet crosslinking of psoralen and DNA. The feasibility of imaging the
delivery of plasmid DNA was shown in normal and tumor-bearing animals using gamma camera imaging.
The nonviral genetic vector pegylated liposome was also radiolabeled to monitor the gene delivery. A
preliminary study demonstrated gamma camera images after intravenous infusion of 111InDTPA-labeled
pegylated liposome revealed a high level of accumulation in the head and neck cancer lesions of
patients,23 and therefore strongly supported the use of pegylated liposome as a targeting vehicle of
therapeutic gene for solid tumors. The imaging of gene delivery in vivo could serve as a general predictor
of the ability of the viral or nonviral vectors to reach the tissue(s) of interest. However, mere
visualization of exogenous DNA accumulation at a certain site in the body might not correlate with the
expression levels of desired gene product.
Imaging cell trafficking: In vivo imaging of cell trafficking has been investigated in many immunological
and oncological studies to track the selective recruitment and time of arrival and departure of specific
cells. These studies may be useful for investigators using various cell types transfected with gene(s) of
interest. Tracking the migration of cells in living small animals has been performed with
radionuclide,24,25,26,27,28,29 MR,30,31 and both fluorescence32 and bioluminescence33 optical imaging.
Radionuclide imaging has been used to monitor the trafficking of therapeutic cells in living subjects. For
example, gene-modified ovarian cancer cells expressing HSV1-tk (PA1-STK) were radiolabeled with
99mTc and infused into the pleural space of patients with malignant pleural mesothelioma for suicide
gene therapy. Radiolabeled PA1-STK cells adhere preferentially to intrapleural mesothelioma deposits,
and are retained for at least 24 h in the chest cavity.24 Rat glioma (C6) cells and lymphocytes were
radiolabeled using 64Cupyruvaldehyde-bis(N4-methylthiosemicarbazone) (64CuPTSM) and imaged with
microPET in living nude mice.25 MicroPET images indicated trafficking of tail-vein-injected C6 cells to the
lungs and liver, and transient splenic accumulation of lymphocytes at 3.33 h postinfusion. Reporter gene
imaging is also being used to follow the specific localization and expansion of adoptively transferred
immune T lymphocytes to the antigen-positive tumor and other sites within the animal.28,29 This
approach can be used to assess the effects of immunomodulatory agents intended to potentiate the
immune response to cancer, and can also be useful for the study of other cell-mediated immune
responses, including autoimmunity.
Imaging therapeutic gene expression: Radionuclide imaging technologies, especially PET and SPECT, can
play a significant role in imaging gene expression using diverse reporter genes and reporter probes. A
reporter gene can be introduced into the target tissue(s) by various methods including viral and nonviral
delivery vectors. If the promoter leads to transcription of the reporter gene, then translation of the
imaging reporter gene mRNA leads to a protein product which can interact with the imaging reporter
probe (administered in trace amounts for PETSPECT and sometimes referred to as a tracer). This
interaction may be based on intracellular enzymatic conversion of the reporter probe with retention of
the metabolite(s), or a receptorligand-based interaction. Examples of intracellular reporters include
herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its mutant gene (HSV1-sr39tk).34 Note that
HSV1-tk or HSV1-sr39tk refers to the genes and HSV1-TK or HSV1-sr39TK refers to the respective
enzymes. Substrates that have been studied to date as PET reporter probes for HSV1-TK can be
classified into two main categories  pyrimidine nucleoside derivatives (eg, 5-iodo-2'-fluoro-2'-deoxy-1-D-arabinofuranosyluracil (FIAU)) and acycloguanosine derivatives (eg, 9-(4-fluoro-3hydroxymethylbutyl)guanine (FHBG)), and have been studied in terms of sensitivity and specificity. 35,36
Examples of reporters on or in the cell surface in the form of receptors include the dopamine 2 receptor
(D2R),37 receptors for human type 2 somatostatin receptor (hSSTr2),38 and the sodium iodide symporter
(NIS).39,40 Among these reporter genes, the HSV1-tk gene may alter the cellular behavior towards
apoptosis by changes in the dNTP pool,41 and receptors may result in second messenger activation such
as triggering of signal transduction pathways. For the D2R system, a mutant gene has been studied,
which shows uncoupling of signal transduction but preservation of the affinity of receptor for tracer
ligand.42
The reporter gene can itself be the therapeutic gene or can be coupled to the therapeutic gene. 9 In the
former approach, the reporter gene and therapeutic gene are one and the same. For example, anticancer
gene therapy using HSV1-tk and ganciclovir (GCV) can be coupled with imaging of the accumulation of
radiolabeled probes (18FFHBG or 131124IFIAU).35,36,43 Jacobs et al44 used 124IFIAU PET imaging of
humans in a prospective gene-therapy trial of intratumorally infused liposomegene complex (LIPOHSV1-tk), followed by GCV administration in five recurrent glioblastoma patients. These preliminary
findings showed that 124IFIAU PET is feasible and that vector-mediated gene expression may predict a
therapeutic effect. Recently, sodiumiodide symporter (NIS), which facilitates the uptake of iodide by
thyroid follicular cells, is also being applied in radioiodide gene therapy. 39,45 The conventional radioiodide
or 99mTcpertechnetate scintigraphy has been used to directly monitor NIS expression. 45,46,47,48 NIS has
many advantages as an imaging reporter gene that includes wide availability of its substrates, wellunderstood metabolism, and clearance of these substrates in the body, and no likely interaction with the
underlying cellular biochemistry. Since the iodine is not trapped, issue of efflux has to be optimized, but
initial studies show significant promise. Further studies are needed with regard to NIS as an imaging
reporter gene.
A second approach involves indirect imaging of therapeutic transgene expression using expression of a
reporter gene, which is coupled to a therapeutic transgene of choice. This strategy requires proportional
and constant co-expression of both the reporter gene and the therapeutic gene over a wide range of
transgene expression levels. An advantage of this approach is that it provides for a much wider
application of therapeutic transgene imaging, because various imaging reporter genes can be coupled to
various therapeutic transgenes, while utilizing the same imaging probe each time. Linking the expression
of a therapeutic gene to a reporter gene has been validated using PET through a variety of different
molecular constructs. Examples include fusion approaches,49,50,51 bicistronic approaches using internal
ribosomal entry site (IRES),52,53,54 dual-promoter approaches,55,56 a bidirectional transcriptional
approach,57 and a two-vector administration approach.58 An advantage of the fusion gene approach is
that the expression of the linked genes is absolutely coupled (unless the spacer between the two proteins
is cleaved). However, the fusion protein does not always yield functional activity for both of the
individual proteins andor may not localize in an appropriate subcellular compartment. Although the IRES
sequence leads to proper translation of the downstream cistron from a bicistronic vector, translation
from the IRES can be cell type specific and the magnitude of expression of the gene placed distal to the
IRES is often attenuated.53 This can lead to a lower imaging sensitivity, and methods to improve this
approach are currently under investigation.54 Two different genes expressed from distinct promoters
within a single vector (dual-promoter approach) may avoid some of the attenuation and tissue-variation
problems of an IRES-based approach.55 The potential problem of this approach is that the expression of
the two genes may become uncoupled if the two identical promoters have different transcriptional
activity based on where the vector integrates into the host genome or if a mutation occurs in one or both
promoters that changes transcriptional activity. A bidirectional transcriptional approach utilizes a vector
in which the therapeutic and the reporter genes are driven by each minimal CMV promoter induced by
tetracycline-responsive element (TRE), transcribing separated mRNA from each gene which would then
be translated into separate protein products.57 This system also avoids the attenuation and tissuevariation problems of the IRES-based approach, and may prove to be one of the most robust approaches
developed to date. It is limited by the fact that a fusion protein also needs to be co-expressed, but future
vectors should be able to encode for both the fusion protein and the bidirectional transcriptional system
on a single vector. Another way to image both the therapeutic and reporter genes can be through
administration of two separate vectors, by cloning of the therapeutic and reporter genes in two different
vectors, but driven by same promoter. This system may eliminate the need for making a new construct
for each therapeutic gene, and has been validated through the expression of two PET reporter genes and
showed good correlation.58 However, it is important to realize that the trans effects between two
promoters can potentially affect reporter gene expression, and that not all cells may be equally infected
with the PET reporter gene vector and therapeutic gene vector.
Tumor-restricted gene expression through tissue-specific transcriptional targeting is an attractive
approach for gene therapy. It has been demonstrated that gene expression of highly efficient gene
therapy vectors can be targeted to tumors using cell-type or tissue-type specific promoter elements.
Approaches have also been developed to image the transcriptional regulation of the PET reporter gene in
living animals.27,57,59 For example, transcriptional regulation at the level of induction has been reported in
living mice using two PET imaging reporter genes (HSV1-tk and D2R) under the control of a tetracyclineinducible promoter.57 Using PET, correlative expression of both reporters after doxycycline treatment was
measured in animals harboring stably transfected tumor. Low levels of imaging reporter gene expression
owing to relatively weak tissue-specific promoters were circumvented with VP16 transactivating domains
fused to yeast GAL4 DNA-binding domains. This two-step transcriptional amplification (TSTA) system
was valuable in demonstrating PSE- or CEA-driven reporter gene expression in vivo using HSV1-sr39tk
or HSV1-tk under the control of GAL4-responsive elements.60,61,62,63,64,65 Further studies are necessary to
link this system to amplify both therapeutic and reporter gene expression. These approaches hold
significant promise for the development of tissue-specific vectors with high levels of gene expression.
The lytic properties of herpes, adeno or Newcastle viruses are also being tailored for the destruction of
various tumors. Although these oncolytic viruses are promising agents for treatment of malignancy due
to their direct, selective toxicity for tumor cells, it is not easy to document viral replication in living
subjects, as serial tissue sampling was required to assess viral titers over time. 124IFIAU PET scanning
was capable of distinguishing a half-log difference between viral doses, and was able to document viral
proliferation in xenograft tumor infected by oncolytic HSV infection. 66,67 This PET data might provide a
new direction for evaluating viral infection and proliferation in future clinical trials involving oncolytic viral
therapy.
MRI and MRS
In spite of its high spatial resolution (10100
m), MR imaging has only a micromolar sensitivity to
paramagnetic contrast agents, so robust signal-amplification strategies are necessary. Relatively large
amounts of reporter probe (metals) have to accumulate in cells in order to lead to signal changes that
can be imaged in the MR scanner. Targeted MR contrast agents in conjunction with biochemical
amplification strategies have been preliminarily studied. Studies highlight the use of the transferrin
receptor (Tf-R) as a potential transporter for accumulation of contrast agents, which consist of human
holotransferrin covalently conjugated to low-molecular-weight dextrans coating monocrystalline iron
oxide nanoparticles (Tf-MION)68,69 or crosslinked iron oxide (Tf-CLIO).70 The transferrin is recognized by
the receptor and the entire particle is endocytosed by the cell, bringing in iron, a paramagnetic ion that
acts as a contrast agent by affecting the T2 rate. It has been demonstrated that overexpression of the TfR in rat gliosarcoma cells in conjunction with the Tf-MION successfully increases the iron content in the
cells, such that measurable MRI contrast can be achieved in living mice implanted with tumors. Further
studies are under investigation to verify whether the Tf-R can be engineered to coexpress with a
therapeutic gene,70 and are necessary to assess the effect of overexpressing Tf-R and increased level of
iron on normal cellular function.
Contrast agents that change the magnetic properties at enzymatic hydrolysis have been used recently to
image transgene expression. Contrast agents, (1-(2-( -galactopyranosyloxy)propyl)-4,7,10tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane)gadolinium(III) (EgadMe), are based on the
framework of a clinical constrast agent, Gd(HP-DO3A), that has been modified with a carbohydrate 'cap'
that blocks the access of water to the gadolinium. When access of water to gadolinium is blocked, signal
enhancement by the contrast agent is turned 'off'. The cap is attached to the contrast agent through a
-galactosidase-cleavable linker. Enzyme cleavage releases the cap and opens water access to the
gadolinium ion, turning the contrast agent 'on'.71 Experiments in Xenopus embryo system revealed
regions of higher intensity in the MR image correlates with regions expressing
-galactosidase, and
demonstrated the ability of MRI to detect gene expression in living animals. 71 In this report, the contrast
agent was introduced to animal systems by microinjection. Such application will require further
refinement of the contrast agents in order to be delivered to cells without direct injection.
The ability of MRS to distinguish signals from chemically distinct compounds also offers the potential to
measure gene expression. Conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to the
chemotherapeutic agent 5-fluorouracil (5-FU) by yeast cytosine deaminase (yCD) could be observed and
quantitated in colorectal tumor xenograft in living subjects, using 19F MRS.72 This study demonstrates the
feasibility of using MRS to noninvasively monitor therapeutic transgene expression in tumors.
Optical imaging
A variety of different optical imaging approaches have been used to image gene expression in living
subjects in optically transparent organisms. Using adenovirus encoding GFP as a reporter gene and
illumination in a light box by blue light fiber optics, noninvasive, whole-body, real-time fluorescence
optical imaging of transgene expression was demonstrated in the major organs of nude mice including
the brain and liver.11 GFP also has been used to image the transduction of lentivirus in nondividing
hepatocytes in living nude mice.73
Bioluminescence imaging exploits the emission of visible photons at specific wavelengths, on the basis of
energy-dependent reactions catalyzed by various luciferases.14,16 The emitted photons can be detected
and counted using low-light CCDs or photon-counting cameras. The kinetics of gene expression after
vector administration has been examined by injecting lentiviral vector encoding FL. 74
To improve the activity and specificity of prostate-targeted gene expression, enhanced promoters were
developed by multimerizing key regulatory elements in the prostate-specific antigen (PSA) enhancer and
promoter. The resulting PSE-BC construct was incorporated into an adenovirus vector with FL (AdPSEBC-luc), and applied in the prostate cancer model to identify metastases. Cooled CCD camera imaging
localized and illuminated metastases in the lung and spine, and demonstrated the potential use of
noninvasive imaging modality in therapeutic and diagnostic strategies for prostate cancer (Figure 1).75
This tissue-specific approach was also applied to the TSTA system, and revealed 20-fold higher levels of
expression than the cytomegalovirus enhancer.60,63,65 These approaches were partly validated in a
clinically relevant imaging modality such as micro-PET, as mentioned earlier;61,62 however, these
approaches are still in need of further studies.
Different kinds of nonviral vectors have been evaluated in the small animal model using optical imaging
techniques. The cationic lipid 1,2-dioleoyl-3-trimethyl ammonium-propane (DOTAP):cholesterol DNA
liposome complexes76 and transferrin targeted DNApolyethylenimine (PEI) complexes77 were evaluated
with FL reporter gene imaging.
Cardiovascular disease
Gene therapy holds much promise as a potential treatment for various cardiovascular diseases. These
treatments include the prevention of restenosis after angioplasty, promotion of angiogenesis, and
treatment of end-stage heart failure. 78
Initial studies using autoradiography detected the uptake of 125IFIAU in rat myocardium transduced with
adenoviral-mediated HSV1-tk reporter gene. The authors hypothesized that in vivo cardiac gene imaging
is feasible and may eventually be used for the noninvasive monitoring of gene therapy.79 The first
demonstration of cardiac reporter gene imaging in living subjects was reported with FL bioluminescence
imaging. This study demonstrated the feasibility of imaging the location, magnitude, and time course of
cardiac reporter gene expression in living rats.80 The optical study was validated in a clinically relevant
microPET (Figure 2). Rat myocardium was transduced with adenovirus carrying HSV1-sr39tk. The
presence of 18FFHBG uptake in microPET images was confirmed by gamma counting and the presence
of HSV1-sr39TK protein by thymidine kinase enzyme assay, while utilizing myocardial tissue samples. 81
More detailed quantitative microPET studies have also been performed using the same model. 82 Further
studies are under investigation to construct bicistronic vectors containing both therapeutic (eg, VEGF)
and PET reporter genes.83 Recently, we developed novel imaging approaches that allow noninvasive
assessment of myocardial response to cell therapy using embryonic cardiomyoblasts expressing HSV1sr39tk andor FL.84 The location, magnitude, and survival duration of the transplanted cells were
monitored noninvasively using PET and bioluminescence optical imaging.
Neurological applications
Both the HSV1-tkHSV1-sr39tk and the D2R PET reporter genes are not optimal for most central nervous
system application. All the reporter probes for HSV1-tkHSV1-sr39tk developed to date show very poor
penetration across the bloodbrain barrier (BBB). Therefore, imaging with any of these reporter probes
may be useful only if some BBB disruption is present (eg, brain tumors). The D 2R reporter gene with
18FFESP can work with brain-specific applications, but only if reporter gene expression is not in the
vicinity of normal D2R expression in the striatum.
Recently, in Parkinson's disease research, aromatic-amino-dopadecarboxylase (AADC) gene was
delivered to striatum as a therapeutic gene via an adeno-associate viral vector (AAV), and its expression
was imaged by 18Ffluoro-m-tyrosine (18FFMT) PET. The AADC tracer 18FFMT is both decarboxylated
and stored in the striatal neurons of monkeys providing a method for in vivo visualization of gene
expression in the brain.85 For monitoring of stem cells after grafting by a noninvasive imaging technique,
embryonic stem cells were labeled by a lipofection procedure with a MRI contrast agent SINEREM
consisting of ultrasmall super-paramagnetic iron-oxide particles (USPIO). MRI at 78-
m isotropic spatial
resolution permitted the observation of the implanted cells in the corpus callosum and their migration to
the ventricular walls.86
Miscellaneous diseases
Cellular radiolabeling techniques can be useful to assess the biodistribution of various cells for cellular
therapy applications. Hepatocyte transplantation has been shown to provide temporary liver function in
acute hepatic failure and various metabolic diseases. 111In-labeled hepatocytes were useful for the
short-term noninvasive analysis of the biodistribution of transplanted hepatocytes. 87 In another study,
myoblasts were radiolabeled with 99mTcbis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt) and injected to
assess the biodistribution of these cells in Duchenne-type human muscular dystrophy model.88
Creatine kinase (CK) and arginine kinase (AK) expression was monitored by using
31
P MRS in the liver
and skeletal muscle.89,90 Walter et al89 have introduced Drosophila melanogaster AK as a reporter gene
for MRS detection of gene therapy of muscle diseases. This enzyme phosphorylates arginine, leading to
the production of arginine phosphorylate, a unique metabolite that is not otherwise found in mammalian
tissues and is readily detected by
31
P MRS. CK is expressed primarily in the muscle (MM isoform) and
brain (BB isoform), but is absent in the liver, kidney, and pancreas. In a recent study, syngeneic enzyme
CK was used as a reporter gene for in vivo monitoring of gene expression after virally mediated gene
transfer to the liver, the key target for gene therapy applications.90
Future prospects
Molecular imaging strategies associated with gene therapy will likely expand significantly over the next
few years as gene therapy continues to evolve. The explosion in genetic engineering is expected to
generate more robust gene-transfer vectors, both viral and nonviral. Bicistronicbidirectional vectors that
can be easily modified, and tissue-specific amplification techniques, will likely expand. Continued
refinements in the chemistry of molecular probe development should give rise to a new generation of
probes with greater sensitivity and specificity. Advances in detector technology and image reconstruction
techniques for PET should help to produce a newer generation of imaging instruments with better spatial
resolution, sensitivity, and significantly improved throughput time. The optical technologies including
fluorescence tomography may allow optical strategies to be the method of choice for small animal gene
therapy research. Multimodality reporter gene approaches, so that gene therapy investigators may
readily move between the various technologies, should help to also test various preclinical models. It will
be very important for preclinical imaging strategies to be extended into patient studies where gene
therapy is directly or indirectly monitored throughout the use of state-of-art imaging. Ultimately, all of
the imaging technologies of gene delivery andor expression will be used as an early measure of
successful gene therapy in patients.
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Figures
Figure 1 Visualization of primary and metastatic lesions of
advanced human prostate cancer by a targeted gene transfer vector
and optical bioluminescence imaging. PSE-BC was incorporated into
an adenovirus vector encoding firefly luciferase (AdPSE-BC-luc) to
make the prostate-specific gene transfer vector. A total of 1.8  109
infectious units of AdPSE-BC-luc were injected into 7-mm diameter
tumors of severe combined immunodeficient (SCID) mice. (a) At 21
days postinjection, not only tumoral (arrowhead) but also lowmagnitude extratumoral signals (arrows) were visible (200
RLUmin), emanating from the lower back and chest. The signals in
the chest and lower back were found to originate from the lung and
spine, respectively, after re-imaging the isolated organs of the
mouse. (b) Histological analysis (H&E staining) of the spinal column
revealed an elongated lesion embedded in the spinal musculature,
characterized by large pleomorphic nuclei and a high mitotic rate
consistent with neoplasia (arrow); reproduced from Adams et al,65
with permission.
Figure 2 PET and optical bioluminescence imaging of cardiac
reporter gene expression in living rats. Replication-defective
adenovirus carrying cytomegalovirus promoter-driving herpes
simplex virus type 1 thymidine kinase (AdCMV-HSV1-sr39tk) and
replication-defective adenovirus carrying cytomegalovirus promoterdriving firefly luciferase (AdCMV-FL) viruses (1  109 pfu) were
injected into the anterolateral wall of the left ventricular
myocardium of a SpragueDawley rat. (a) The whole-body microPET
image of a rat shows focal cardiac 18FFHBG activity at the site of
intramyocardial AdCMV-HSV1-sr39tk injection. Liver 18FFHBG
activity is also seen because of systemic adenoviral leakage with
transduction of hepatocytes. Control rats injected with AdCMV-FL
show no 18FFHBG activity in either of the cardiac of hepatic
regions. Significant gut and bladder activities are seen for both the
study and control rats, because of the route of 18FFHBG clearance.
(b) Tomographic views of cardiac microPET images. The 13NNH3
(gray scale) images of perfusion are superimposed on 18FFHBG
images (color scale), demonstrating HSV1-sr39tk reporter gene
expression. (c) Bioluminescence image in rat myocardium
transfected with AdCMV-FL emits significant bioluminescence due to
cardiac firefly luciferase activity.
Tables
Table 1 Application of in vivo molecular imaging in gene therapy for
oncology
Table 2 Application of in vivo molecular imaging in gene therapy for
cardiovascular, neurological and miscellaneous diseases
Received 15 January 2003; accepted 16 October 2003
January (2) 2004, Volume 11, Number 2, Pages 115-125
November 2003, Volume 10, Number 24, Pages 1999-2004
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Review
Gene therapy progress and prospects: gene therapy for
severe combined immunodeficiency
H B Gaspar1, S Howe1 and A J Thrasher1
Molecular Immunology Unit, Institute of Child Health, London, UK
1
Correspondence to: Dr AJ Thrasher, Molecular Immunology Unit,
Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
Abstract
Severe combined immunodeficiencies have long been targeted as a group of disorders
amenable to gene therapy because of their defined molecular biology and pathophysiology,
and the prediction that corrected cells would have profound growth and survival advantage.
Recently, several clinical studies have shown that conventional gene transfer technology can
produce major beneficial therapeutic effects in these patients, but, as for all cellular and
pharmacological treatment approaches, with a finite potential for toxicity.
Gene Therapy (2003) 10, 19992004. doi:10.1038/sj.gt.3302150
Keywords
SCID-X1; ADA; PEG-ADA; LMO-2; insertional mutagenesis
In brief
Progress
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Sustained correction of X-severe combined immunodeficiency
(SCID) has been observed in clinical trials.
Cells in patients with adenosine deaminase (ADA) deficiency,
treated by lymphocyte or stem cell gene therapy, persist and
maintain transgene expression for many years.
Withdrawal of PEG-ADA from patients treated by lymphocyte
gene therapy for ADA-deficient SCID results in enhanced
immunological reconstitution.
Successful gene therapy for ADA-deficient SCID can be
achieved in the absence of PEG-ADA and in combination with
myelosuppression.
Animal models of RAG-2- and JAK-3-deficient SCID have
been corrected using similar strategies.
Insertional mutagenesis has been observed in human studies,
reinforcing the need to develop methods for optimization of
protocol safety.
Prospects
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As a group of well-defined disorders, SCIDs are amenable to
treatment by gene therapy, and an extended range will enter
clinical study over the next few years.
The durability of immunological reconstitution will determine
the effectiveness and the need for repeated administration.
The absolute risk of clinically manifesting mutagenesis using
retroviral vectors is at present unknown, and will only be
determined by the extended observation of more patients, and
by the development of clinically relevant models to test for
toxicity in a rigorous way.
The characteristics of retroviral and lentiviral integration in
human patients will be determined by mapping integration sites.
Risks of mutagenesis will be reduced by improved design of
vectors that restrict promoter activity to relevant cell types and
within the domain of the therapeutic transgene.
Development of targeted integration or of stable episomal
vector systems will also enhance safety.
Gene-repair strategies may have particular efficacy in SCID
because of the profound growth and survival advantage
conferred to corrected cells.
Sustained correction of X-severe combined immunodeficiency (SCID) has been observed in
clinical trials
The most severe forms of primary immunodeficiency are known as SCIDs. These are a group of diseases
in which T-lymphocyte development is invariably interrupted, and associated with diverse disorders of
development and functionality of B lymphocytes, and natural killer (NK) cells.1 X-linked SCID (SCID-X1)
accounts for approximately 5060% of all SCIDs, and is caused by mutations in the gene encoding the
common cytokine receptor gamma chain (
c). This is a subunit of the cytokine receptor complex for
interleukins (IL) 2, 4, 7, 9, 15 and 21.2 In the absence of
c signaling, many aspects of immune cell
development and function are compromised. The classical immunophenotype of SCID-X1 is the absence
of T and NK cells, and persistence of dysfunctional B cells (T-B+NK-SCID). If a genotypically matched
family donor is available, bone marrow transplantation is a highly successful procedure with a long-term
survival rate of over 90%. The high survival rates are partly due to the fact that the absence of T and NK
cells in SCID-X1 patients allows engraftment in the absence of myelosuppressive conditioning. For the
majority of individuals, this is not possible, and the survival from mismatched family (usually parental
donors) transplants is less good, and is associated with predictable toxicity.
Many incremental advances in gene transfer technology have recently been translated into successful
gene therapy for SCID-X1.3 These have included the activation of cells with high concentrations of
cytokines (thereby making them susceptible to gamma retrovirus vector-mediated gene transfer), and
transduction in containers coated with a recombinant fibronectin fragment (RetroNectin) that is
believed to facilitate the colocalization of the virus particle and the target cell.4,5 In the first landmark
study, a conventional amphotropic retroviral vector encoding a
c cDNA (regulated by Moloney murine
leukaemia virus long-terminal repeat sequences) was used to transduce ex vivo autologous CD34+ cells
(separated by conventional magnetic bead technology from a bone marrow harvest). The cells were
reinfused into the patients in the absence of preconditioning. The results obtained from the first five
patients have recently been reported in the scientific literature.6 To date, 10 infants in total have now
been treated, with good immunological reconstitution in all but one, in whom the graft appears to have
become sequestered in a pathologically enlarged spleen 6,7 In nearly all patients, NK cells appeared
between 2 and 4 weeks after infusion of cells, followed by new thymic T-lymphocyte emigrants at 1012
weeks. With some variation, the number and distribution of these T cells normalized rapidly (more
rapidly than observed following haploidentical transplantation). They also appeared to function normally
in terms of proliferative response to mitogens, T-cell receptor (TCR), and specific antigen stimulation,
and to have a complex phenotypic and molecular diversity of TCR. Functionality of the humoral system
was also restored, maybe not quite as effectively, but to a sufficient degree that discontinuation of
immunoglobulin therapy was possible. Persistent long-term marking in myeloid cells (between 0.1 and
1%) suggests that long-lived stem or progenitor cells have been successfully transduced. More recently,
we have initiated a similar study for the treatment of SCID-X1. The transduction protocols and vector are
very similar although we have used a gibbonapeleukaemia virus (GALV) pseudotype, and conditions
that obviate the requirement for foetal calf serum. Although the follow-up period for four children treated
in our study is short, all have cleared viral infections, and immunological reconstitution has followed a
similar pattern8 (Thrasher et al, manuscript in preparation).
The contribution to the initial burst of thymopoiesis from relatively late T-cell precursors in the original
transduced CD34+ cell population versus that from cells earlier in the haematopoietic differentiation
hierarchy, which have engrafted in the bone marrow, has not yet been determined. This may have
important implications for the durability of immunological reconstitution, and for sustained production of
new T cells. These issues may be resolved by longitudinal study of naive T-cell production, and by the
isolation of common integration sites between myeloid and lymphoid populations. Ultimately, the
longevity of functional reconstitution can only be determined by clinical monitoring, but it should also be
feasible to repeat gene therapy on multiple occasions. Unknown, however, are the time frames within
which this will be clinically effective, particularly bearing in mind that there may be age-related
restrictions to the reinitiation of thymopoiesis in these patients.
Cells in patients with ADA deficiency treated by lymphocyte or stem cell gene therapy persist
and maintain transgene expression for many years
Deficiency of the purine salvage enzyme adenosine deaminase (ADA) accounts for approximately
1020% of all SCIDs. ADA catalyses the deamination of deoxy-adenosine (dAdo) and adenosine to
deoxyinosine and inosine, respectively, and the lack of ADA leads to the build of the metabolites
deoxyATP (dATP) and dAdo, which have profound effects on lymphocyte development and function
through a number of cellular mechanisms. There is variation in the severity of the condition but most
ADA patients have very low numbers of T and B lymphocytes. Bone marrow transplantation is highly
successful in the genotypically matched setting, but human leucocyte antigen (HLA)-mismatched
transplants have poor survival outcomes. An alternative modality of treatment is exogenous enzyme
replacement with polyethylene glycol-conjugated bovine ADA, which as regular intramuscular injections
can result in the correction of metabolic and immunological abnormalities, albeit only partially in a
significant number of cases.9
The first human gene therapy studies were conducted on patients with ADA deficiency in the early
1990s. It is generally agreed that these initial studies were unsuccessful in correcting the immune defect
in ADA-SCID. This was in part due to the continued use of PEG-ADA enzyme replacement therapy, which
in itself improved the immune function but may also have blunted the survival advantage of genemodified cells (discussed further below). However, a decade on, more detailed analysis of the patients
originally treated does provide important information about the longevity and efficacy of gene transfer. 10
In the first human clinical gene therapy study, two patients were treated following repeated
gammaretroviral vector-mediated ADA gene transfer into stimulated peripheral blood lymphocytes.
Patient 1 still shows over 15% of gene-marked cells in peripheral blood mononuclear cells (PBMCs) and
ADA activity in PBMCs remains at 25% of the normal. The level of gene marking (0.1% of PBMCs) and
ADA activity (<5% of normal) is considerably less in patient 2, which may reflect the smaller number of
gene-transduced cells initially infused, but may also be due to the development of an immune response
against the retroviral envelope and lipoprotein components of the foetal calf serum used for culturing the
cells. However, these findings clearly demonstrate that human T cells have a lifespan greater than 10
years in the peripheral circulation, and also that transgenes regulated by gammaretroviral sequences
continue to express in peripheral T cells and resist in vivo silencing. Molecular analysis of TCR diversity,
combined with transgene integration analysis, reveals that within individual V
family clones, each cell
contains multiple unique integration sites. This suggests that, in the initial phase, cells were repeatedly
sampled and transduced.
In a later study, umbilical cord blood CD34+ cells were harvested from antenatally diagnosed ADA-SCID
patients, transduced and reinfused in the first week of life. In these patients, little clinical benefit was
seen, and the level of gene marking was low (110% of T lymphocytes) in all the three children. Recent
clonal integration analyses demonstrate that transgene-containing T lymphocytes are monoclonal or
oligoclonal (with 15 different integration sites) more than 8 years after gene therapy, and that single
prelymphoid clones contribute between 25 and 100% of genetically corrected lymphocytes. 11 Marking in
other lineages is consistently less than 1%. Again, the continuation of PEG-ADA throughout these early
studies almost certainly compromised the efficient engraftment of transduced cells.
Withdrawal of PEG-ADA from patients treated by lymphocyte gene therapy for ADA-deficient
SCID results in enhanced immunological reconstitution
Matched sibling donor transplants for ADA-SCID performed without conditioning result in rapid
engraftment and persistence of donor T lymphocytes, strongly suggesting that T lymphocytes expressing
ADA have a powerful proliferative and survival advantage. The failure of initial gene therapy studies to
demonstrate the proliferation and expansion of gene-modified cells seemed to question this premise,
although the continued administration of PEG-ADA in all these patients may have abrogated this
advantage. Recently, one patient participating in another study demonstrated convincingly that a
survival advantage for gene-transduced cells did exist in the absence of PEG-ADA. In this individual, who
had received multiple infusions of autologous transduced peripheral blood lymphocytes (PBLs), and who
had reached a plateau of 13% gene-transduced T cells, PEG-ADA-associated immune dysregulatory
problems led to a gradual discontinuation of enzyme replacement. As the dose was decreased and finally
stopped, the percentage of transduced T cells as assessed by PCR quantification increased, eventually
reaching nearly 100% of all T lymphocytes.12 Absolute CD3+ T-cell counts also increased and stabilized
at levels higher than prediscontinuation values. T-cell proliferative responses were also restored, and
analysis of ADA metabolites showed a rise in intracellular PBL ADA activity.
The blunting effect of PEG-ADA may also be responsible for the disappointing observations made in a
more recent study that employed enhanced vectors, and transduction conditions similar to those used for
SCID-X1 trials.13 As before, transduced CD34+ cells were returned to the patients without conditioning
and patients continued PEG-ADA treatment. ADA gene marking was only seen at low levels (in a range
from 0.0001 to 3.6%). On the basis of previous observations, the investigators plan to withdraw PEGADA 1 year after treatment.
Successful gene therapy for ADA-deficient SCID can be achieved in the absence of PEG-ADA
and in combination with myelosuppression
Results of a new study have clearly demonstrated that ADA-SCID can be successfully treated by gene
therapy.14 In this protocol, CD34+ cells were transduced with an amphotropic gammaretroviral vector
(originally used in PBL transduction studies) under current optimal conditions. Two important changes
were incorporated into the protocol. Firstly, for economic reasons, patients were not commenced on PEGADA and, secondly, patients received a mild dose of conditioning (4 mgkg of busulphan as 2 mgkg on
two successive days) prior to the return of gene-modified cells. A neutrophil and platelet nadir were seen
at approximately 3 weeks, but neither child required blood product support. One patient is now 20
months. Over 2 years, postgene transfer one patient has normal numbers of peripheral T, B and NK
cells. This patient has normal immunoglobulin production and is not receiving any prophylactic therapy.
There has also been impressive correction of the metabolic defects, with dATP levels falling to 10% of
that at diagnosis (comparable to that achieved following successful BMT). The second patient is now 12
months postgene therapy, but was an older child (approximately 2.5 years) at the time of treatment and
also received a lower cell dosage. In this patient, recovery has been slower and the T-cell reconstitution
at present is suboptimal, but significantly improved from pretransplant levels. There is evidence of some
immunoglobulin production, but the patient remains on replacement therapy. A third child has been
treated more recently using the same protocol,15 and is showing a recovery similar to that in patient 1.
Molecular analysis of the first two patients shows a diverse TCR repertoire and an increase in TCR
excision circle (TREC) levels, indicating the successful engraftment of prethymic progenitor populations.
Lineage-specific transgene analysis by quantitative PCR shows high level marking in T, NK and B cells,
and the persistence of gene-modified granulocytes, monocytes and megakaryocytes at levels between 5
and 20%, again suggesting that multipotent progenitors have engrafted. Somewhat surprising is the
level to which marking has persisted in myeloid cells, particularly at the dosage of conditioning
employed. This may suggest that a survival advantage is not restricted to lymphoid cells, and that it also
extends to myeloid and haematopoietic multipotent progenitor cells.
The results from this study are extremely encouraging. At present, it is difficult to determine whether the
success of the procedure is due to the lack of PEG-ADA or the use of nonmyeloablative conditioning, but
it is likely that the combination is important. The key to correction of the metabolic abnormalities in ADASCID seems to be the delivery of large amounts of ADA enzyme, whether exogenously in the form of
PEG-ADA or intracellularly as gene-modified cells. The use of conditioning may facilitate the initial
engraftment of a greater number of gene-modified cells. Certainly, if immune function in these patients
is sustained, and further patients show a similar safety profile and immune response, this strategy holds
great promise for ADA-SCID and potentially other haematopoietic conditions.
Animal models of JAK-3- and RAG-2-deficient SCID have been corrected using similar
strategies
The molecular basis of autosomal recessive T-B+NK-SCID is mutation of the receptor tyrosine kinase
gene JAK-3. The dependence of
c on signalling through JAK-3 is responsible for a clinical and
immunological phenotype identical to that of SCID-X1, and the rationale for gene therapy is therefore
similar. Correction of a murine model of JAK-3-deficient SCID has been achieved using both
myelosuppresssive, and more relevant to clinical studies, conditioning-free protocols.16 Patients with
mutations of the recombinase-activating genes RAG-1 and RAG-2 characteristically present with the
absence of both B and T cells. Moloney-based gammaretroviral vectors have recently been shown to
effectively reconstitute RAG-2-deficient mice, and in the absence of detectable toxicity, even though
gene expression was not tightly regulated.17 One way to obviate the toxicity arising from dysregulated
gene expression in any condition, and to achieve physiological activity, is to correct genetic mutations by
gene repair or homologous recombination. Recently, it has been shown that RAG2-- mutant murine
embryonic stem (ES) cells, repaired by standard homologous recombination technology, can be grown in
vitro to provide sufficient haematopoietic progenitors for engraftment and correction of RAG-2 mutant
mice.18 This is the first example of gene therapy combined with a therapeutic cloning strategy and,
clearly, has important implications for future treatment of many genetic disorders.
Insertional mutagenesis has been observed in human studies, reinforcing the need to develop
methods for optimization of protocol safety
For retroviruses, which depend on chromosomal integration for the stability of transduction, the most
prominent safety concern has been for insertional mutagenicity. 19 On the basis of numerous animal
studies and over 300 clinical trials in which patients have received retroviral vectors, and from
theoretical considerations, the risk of clinically manifesting insertional mutagenesis has been judged to
be low. However, in a recent murine HSC retroviral transduction study, insertion of the vector into the
oncogene Evi-1 led to development of myeloid leukaemia.20 This has been followed by the reported
development of uncontrolled clonal T-cell proliferation in two patients in the Paris SCID-XI clinical trial
(Table 1).7,21 Having initially achieved successful immunological reconstitution, both developed
lymphoproliferation approximately 3 years after the gene therapy procedure. In both patients, retroviral
vector insertion into or near the LMO-2 proto-oncogene resulted in high-level expression of LMO-2 in the
clones, almost certainly as a result of retroviral enhancer-mediated activation of transcription. Activation
of LMO-2 is known to participate in human leukaemogenesis by chromosomal translocation, and results
in the development of T-cell lymphoproliferation and leukaemia in mice, albeit with a long latency. It is
therefore likely that other contributing factors are required for these events to manifest. At present,
there is no evidence for the contribution of dysregulated
c expression in lymphoid cells, although this
remains a possibility and is being studied carefully. Cells with high proliferative potential, such as
thymocytes, are also likely to be more susceptible to transformation following an insertional event than
quiescent cells, if they acquire additional adverse mutations unrelated to the gene therapy itself. This
increased risk cannot yet be quantified. The integration of the vector into LMO-2 in both cases strongly
suggests that there is some preference for the survival of these clones or less likely, for integration at
this site (Hacein-Bey-Abina et al, submitted for publication). The detailed molecular analysis of insertion
events in patients undergoing gene therapy will greatly assist in the delineation of integration points
within the genome, but is unlikely to be able to predict potential for mutagenesis unless recurrent
hotspots associated with clinical disease become evident.22,23,24 In combination with conventional
monitoring of lymphocyte numbers and distributions, longitudinal monitoring of integration sites will
provide an important way of monitoring for pathological clonal expansions. The applicability of any novel
therapy, including gene therapy, ultimately depends on the balance of risks against those of alternative
treatments. The accurate characterization of adverse events, the utilization of protocols to test toxicity in
a rigorous way, and the development of methods to minimize risks are therefore essential.
Future prospects for SCID
Recent clinical trials have shown that at least some forms of SCID can be effectively treated by gene
therapy. Much, however, can be done to improve the efficiency and safety of current protocols. The
design of vectors used for gene delivery is clearly important and modifications may be possible, which
will limit the risks of mutagenesis, for example by the incorporation of DNA and RNA insulator sequences
in integrating vectors, by the use of self-inactivating vectors or by targeting safe regions in the
genome.25,26 Alternative vectors based on lentiviruses or foamy viruses that obviate prolonged ex vivo
culture may allow the preservation of larger numbers of multipotential progenitor cells, but at the same
time may produce higher numbers of insertion events in each cell. 27,28,29,30,31 Methods to minimize the
number of integration events per cell, and to limit the number of engrafting clones, for example by more
stringent purification of stem cell (or defined target cell) populations, may therefore also be beneficial.
Probably, the most straightforward way to improve safety is to dispense with the powerful viral enhancer
sequences that can dysregulate gene expression over large chromatin domains. Lentiviral vectors, in
particular, provide greater capacity for the incorporation of more complex and physiological regulatory
sequences. The relative risk for each type of vector modification needs to be determined in clinically
relevant animal-model systems, and the effectiveness of these models to predict side effects in humans
will have to be validated. The development of homologous recombination or gene repair to correct
mutations, or the construction of mitotically stable extrachromosomal vectors, would obviate many of
these problems, but current technologies are inefficient.32 Once again, SCID may be a perfect initial
target for this strategy, as even limited efficiency will be sufficient to provide clinical benefit. The future
for gene therapy of SCID is exciting, but has been clouded by the occurrence of toxicity. As for all novel
therapeutic modalities, increased understanding of mechanisms, and increased sophistication of
technology will translate into even more effective and safe application. There is no better paradigm for
this process than allogeneic bone marrow transplantation.
References
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model for molecular medicine. Lancet 2001; 357: 18631869.
Article PubMed
2 Leonard WJ. Cytokines and immunodeficiency diseases. Nat Rev
Immunol 2001; 3: 200208.
3 Cavazzana-Calvo M et al. Gene therapy of human severe
combined immunodeficiency (SCID)-X1 disease. Science 2000;
288: 669672. PubMed
4 Demaison C et al. A defined window for efficient gene marking
of severe combined immunodeficient-repopulating cells using a
gibbon ape leukaemia virus-pseudotyped retroviral vector. Hum
Gene Ther 2000; 11: 91100. PubMed
5 Hacein-Bey S et al. Optimization of retroviral gene transfer
protocol to maintain the lymphoid potential of progenitor cells. Hum
Gene Ther 2001; 12: 291301. PubMed
6 Hacein-Bey-Abina S et al. Sustained correction of X-linked
severe combined immunodeficiency by ex vivo gene therapy. N Engl
J Med 2002; 346: 11851193. Article PubMed
7 Hacein-Bey-Abina S et al.. LMO2-associated clonal T cell
proliferation in two patients after gene therapy for SCID-X1
((gamma) c deficiency). In press.
8 Thrasher AJ et al. Immune Recovery Following Retroviral
Mediated Common Gamma Chain Gene Therapy for X-linked Severe
Combined Immunodeficiency. American Society of Gene Therapy's
Sixth Annual Meeting Executive Summaries, Vol. 36; 2003.
9 Hershfield M. ESID 2002.
10 Muul LM et al. Persistence and expression of the adenosine
deaminase gene for twelve years and immune reaction to gene
transfer components: long-term results of the first clinical gene
therapy trial. Blood 2002; 101: 25632569.
11 Schmidt M et al. Clonality analysis after retroviral-mediated
gene transfer to CD34+ cells from the cord blood of ADA-deficient
SCID neonates. Nat Med 2003; 9: 463468. Article PubMed
12 Aiuti A et al. Immune reconstitution in ADA-SCID after PBL
gene therapy and discontinuation of enzyme replacement. Nat Med
2002; 8: 423425. Article PubMed
13 Candotti F et al. Corrective gene transfer into bone marrow
CD34+ cells for adenosine deaminase (ADA) deficiency: results in
four patients after one year of follow-up. Mol Ther 2003; 7: S448.
14 Aiuti A et al. Correction of ADA-SCID by stem cell gene therapy
combined with nonmyeloablative conditioning. Science 2002; 296:
24102413. Article PubMed
15 Aiuti A. Correction of the immune and metabolic defect of ADASCID by stem cell gene therapy combined with nonmyeloablative
conditioning. Mol Ther 2003; 7: S448.
16 Bunting KD, Lu T, Kelly PF, Sorrentino BP. Self-selection by
genetically modified committed lymphocyte precursors reverses the
phenotype of JAK3-deficient mice without myeloablation. Hum Gene
Ther 2000; 11: 23532364. Article PubMed
17 Yates F et al. Gene therapy of RAG-2-- mice: sustained
correction of the immunodeficiency. Blood 2002; 100: 39423949.
PubMed
18 Rideout III WM et al. Correction of a genetic defect by nuclear
transplantation and combined cell and gene therapy. Cell 2002;
109: 1727. PubMed
19 Baum C et al. Side effects of retroviral gene transfer into
hematopoietic stem cells. Blood 2003; 101: 20992114. Article
PubMed
20 Li Z et al. Murine leukemia induced by retroviral gene marking.
Science 2002; 296: 497. Article PubMed
21 Hacein-Bey-Abina S et al. A serious adverse event after
successful gene therapy for X-linked severe combined
immunodeficiency. N Engl J Med 2003; 348: 255256. Article
PubMed
22 Schroder AR et al. HIV-1 integration in the human genome
favors active genes and local hotspots. Cell 2002; 110: 521529.
PubMed
23 Laufs S et al. Retroviral vector integration occurs into preferred
genomic targets of human bone marrow repopulating cells. Blood
2002; 101: 21912198.
24 Wu X et al. Transcription start regions in the human genome
are favored targets for MLV integration. Science 2003; 300:
17491751. Article PubMed
25 Burgess-Beusse B et al. The insulation of genes from external
enhancers and silencing chromatin. Proc Natl Acad Sci USA 2002;
99 (Suppl 4): 1643316437. Article PubMed
26 Olivares EC et al. Site-specific genomic integration produces
therapeutic Factor IX levels in mice. Nat Biotechnol 2002; 20:
11241128. Article PubMed
27 Follenzi A et al. Gene transfer by lentiviral vectors is limited by
nuclear translocation and rescued by HIV-1 pol sequences. Nat
Genet 2000; 25: 217222. Article PubMed
28 Glimm H, Oh IH, Eaves CJ. Human hematopoietic stem cells
stimulated to proliferate in vitro lose engraftment potential during
their SG(2)M transit and do not reenter G(0). Blood 2000; 96:
41854193. PubMed
29 Demaison C et al. High-level transduction and gene expression
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virus promoter. Hum Gene Ther 2002; 13: 803813. Article PubMed
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Tables
Table 1 Current clinical trials of gene therapy for SCID
November 2003, Volume 10, Number 24, Pages 1999-2004
September 2003, Volume 10, Number 20, Pages 1721-1727
Table of contents
Previous Article Next PDF
Review
Gene therapy progress and prospects: Parkinson's disease
E A Burton1, J C Glorioso2 and D J Fink3,4
Department of Clinical Neurology, University of Oxford, Radcliffe Infirmary,
Oxford, UK
1
Department of Molecular Genetics and Biochemistry, University of Pittsburgh,
USA
2
Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA
3
Geriatric Research, Education and Clinical Center (GRECC), Pittsburgh VA
Healthcare System, Pittsburgh, PA, USA
4
Correspondence to: Dr DJ Fink, Department of Neurology, University of
Pittsburgh, Pittsburgh, PA, USA
Gene Therapy (2003) 10, 17211727. doi:10.1038/sj.gt.3302116
In brief
Progress
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Inhibition of apoptosis by gene delivery prevents development of the
disease phenotype in animal models
Transgene-mediated expression of glial cell line-derived neurotrophic
factor may prevent progression after an initial insult, and may even be
restorative in animal models
Combination of antiapoptotic and glial cell line-derived neurotrophic factor
(GDNF) gene therapy protects dopaminergic neurons against a toxic insult,
more effectively than either intervention alone
Transgene-mediated production of the inhibitory neurotransmitter -amino
butyric acid (GABA) in neurons of the subthalamic nucleus ameliorates the
behavioral phenotype and may be neuroprotective, in an animal model
Delivery of transgenes encoding enzymes involved in dopamine
biosynthesis enhances dopamine production in the striatum
Stem cells may be driven to differentiate into functioning dopaminergic
cells by genetic modification
Isolation of genes implicated in rare genetic forms of Parkinson's disease
(PD) has allowed generation of new animal models and identification of
new candidate targets for intervention
One human gene therapy trial is about to commence in PD
The optimal vector remains uncertain
Prospects
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Development of presymptomatic diagnostic tests will facilitate
neuroprotective studies
Better understanding of the pathogenesis may lead to the development of
improved animal models that more closely resemble the human disease
Studies may broaden their scope to include the important nonmotor
manifestations of PD
Further characterization of ES and adult stem cell populations will establish
whether ex vivo transduction can drive their differentiation into
dopaminergic neurons in a therapeutically useful way
Well-designed clinical trials for PD gene therapy may take their lead from
cell transplantation trials
PD is an attractive target for central nervous system (CNS) gene therapy for several reasons. First, the pathology in early
PD is, to a first approximation, limited to dopaminergic neurons projecting from the substantia nigra pars compacta (SNc)
to the caudate aputamenl, so that localized gene delivery is a viable therapeutic strategy. Second, the neurochemical
deficits and the functional consequences of dopaminergic cell loss on local basal ganglia circuitry are well characterized;
gene transfer can be designed either to improve cell survival, or to modify functional activity in the damaged basal
ganglia circuitry (summarized in Figures 1 and 2). Third, PD is common and disabling despite treatment; no current
intervention is uniformly accepted as altering the natural history of disease progression; hence, development of novel
therapeutics is desirable. A variety of therapeutic transgenes has been delivered in experimental models of PD, using a
number of different vectors. In this article, we survey the literature from 2000 to 2003, and briefly review recent progress
in the development of gene transfer strategies for treating PD.
Inhibition of apoptosis by gene delivery prevents development of the disease phenotype in animal models
The most frequently studied animal models of PD involve chemical induction of lesions to the SNc of rodents, using the
toxins 6-hydroxydopamine (6-OHDA) or 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In each case, the toxic
insult leads to a pathogenic cascade, resulting in apoptotic cell death of dopaminergic (DA) neurons. Local expression of
apoptotic inhibitors, in the SNc of 6-OHDA- and MPTP-lesioned animals, prevents both the loss of DA neurons and the
development of a PD-like phenotype, following the chemical insult. Our previous work showed that expression of bcl-2
from a nonreplicating herpes simplex virus (HSV)-based vector within the SNc protected dopaminergic cells from
apoptosis following administration of 6-OHDA, with the resulting preservation of motor function. These findings have now
been extended to other experimental paradigms. The human neuronal apoptosis inhibitor protein (NAIP), delivered to the
SN of rats by intrastriatal inoculation of a recombinant type 5 adenovirus (Ad) vector, 1 protected the animals against 6OHDA toxicity administered 1 week after the vector, as measured by immunohistochemistry and motor phenotype studies
up to 28 days later. Prevention of the apoptotic protease-activating factor-1 (apaf-1)-dependent activation of caspase 9,
using a recombinant adenoassociated virus (AAV) vector expressing a dominant-negative apaf-1 derivative,2 protected
mice against the effects of intra-peritoneal injections of MPTP administered 2 weeks after vector inoculation. At 1 week
following MPTP intoxication, neuronal survival was 75% on the transduced side of the brain compared with 25% on the
contralateral control.
These protein inhibitors of apoptosis must be expressed intracellularly in order to block the apoptotic cascade, so that
gene transfer is uniquely suited to this approach. However, the pathogenic trigger for PD in humans is unknown, and the
role of apoptosis in dopaminergic cell death in naturally occurring PD is controversial. To test the hypothesis that
inhibition of apoptosis within the SNc of humans could arrest or slow the progression of PD, it would be necessary to
identify and treat patients early in the course of their illness, or to generate better animal models that more directly
model the pathogenesis of human PD. Finally, the effects of prolonged expression of antiapoptotic factors in the brain
have not been fully explored; some of these proteins are proto-oncogene products, and there might be important issues
regarding their safety.
Transgene-mediated expression of glial cell line-derived neurotrophic factor may prevent progression after
an initial insult, and may even be restorative in animal models
GDNF was originally isolated by virtue of its trophic effects on dopaminergic cells in culture. It was subsequently
demonstrated that GDNF could promote the survival of dopaminergic neurons in the face of a toxic insult in both rat and
monkey models of PD, and a putative role for GDNF as a neuroprotective agent in PD was suggested. However, the
delivery of potent biologically active peptides with short half-lives to the brain is difficult, and attempts at intraventricular
infusion of recombinant GDNF were disappointing. The alternatives include continuous intraparenchymal infusion of
recombinant GDNF,3 transplantation of genetically modified cells as production sites for GDNF, 4,5,6,7,8 or vector-mediated
transfer of the gene encoding GDNF into the CNS parenchyma.
Several different vector systems have been successfully used to effect GDNF gene transfer in experimental models,
including lentivirus,9,10,11 adenovirus, adeno-associated virus12,13,14 and herpes simplex virus.15 Various points emerge from
these studies, which differ mainly in the details of the experimental paradigms used. First, robust GDNF expression can be
seen after gene transfer into the striatum or substantia nigra, and anterograde transport of GDNF to nerve terminals after
transduction of the neuronal soma seems to be a property of GDNF rather than the vector system used. Second, GDNF
appears to provide trophic support, preventing degeneration of dopaminergic cells and loss of dopaminergic nerve
terminals in both the 6-OHDA and MPTP models. This protection correlates both with some behavioral measures of
nigrostriatal integrity and neurochemical assays examining dopamine production. Finally, in many circumstances, the
application of GDNF is protective or restorative even after the toxic insult has taken place.
As is the case with antiapoptotic gene therapy for PD, the applicability of the experimental studies to human patients is
uncertain, because the etiology and pathogenesis of the human disease are likely to be different from the animal models.
However, GDNF appears to provide generic trophic support to dopaminergic neurons in the face of a range of challenges,
and a phase I study examining direct intraputaminal infusion of the recombinant protein in patients was recently
reported.16 Adverse events were limited to repositioning one infusion catheter and asymptomatic signal changes on MRI
that resolved when the concentration of infused GDNF was reduced. Secondary end points in this nonblinded
nonrandomized study implied possible clinical benefit and improvement in functional imaging surrogates of dopaminergic
terminal integrity. Should the recombinant factor prove efficacious in phase II trials, it is possible that gene delivery will
offer advantages for long-term focal treatment.
A recent study has sounded a note of caution for GDNF therapy.17 Careful study of 6-OHDA rats showed that, although
pharmacologically induced circling behavior, a marker of dopaminergic neural function, was ameliorated in animals
treated with lentivirus-expressing GDNF over periods of up to 9 months, the spontaneous motor behavior was abnormal.
This correlated with abnormal axonal sprouting within the pallidum and other brain areas where GDNF expression
occurred, and with loss of the tyrosine hydroxlase (TH)-positive phenotype in SNc neurons that were preserved by GDNF
treatment. Further characterization of these models, in conjunction with the outcome of clinical trials, will determine
whether these concerns relate appropriately to GDNF therapy for PD.
Combination antiapoptotic and GDNF gene therapy protects dopaminergic neurons against a toxic insult,
more effectively than either intervention alone
Two recent studies have exploited simultaneous delivery of genes encoding an antiapoptotic factor and GDNF, to enhance
the dopaminergic cell survival seen with the corresponding single interventions.
Adenoviral delivery of X-linked inhibitor of apoptosis (XIAP), aimed at preventing apoptosis of SNc neurons in MPTPtreated animals, led to preservation of DA cells but did not prevent loss of striatal DA nerve terminals, resulting in failure
of behavioral recovery.18 Combination of adenovirus-expressing XIAP, with another adenovirus-expressing GDNF,
however, produced a synergistic effect with functional recovery that was not seen in animals treated with the GDNFencoding vector alone.
In another study, GDNF was combined with bcl-2 gene delivery using two HSV vectors encoding expression cassettes for
each of the factors.15 The 6-OHDA rat model was used, and either intervention (GDNF or Bcl-2) increased cell survival
from 25% (control) to 55% (pretreated). However, coadministration of the two vectors increased cell survival to 75%,
indicating that the effects of the different modalities were additive.
Transgene-mediated production of the inhibitory neurotransmitter
-amino butyric acid (GABA) in neurons of the
subthalamic nucleus ameliorates the behavioral phenotype and may be neuroprotective, in an animal model
One functional disturbance found in the basal ganglia of PD patients is the overactivity of neurons within the subthalamic
nucleus (STN) that project to the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr).
These excitatory neurons serve to increase the firing rate of GPi and SNr neurons that, in turn, inhibit brainstem and
thalamic projections to downstream motor pathways, thereby inhibiting the initiation of voluntary movement. Inhibition of
over-active STN neurons by stereotactic ablation or deep brain stimulation has been shown to ameliorate motor signs in
late-stage PD.
A gene transfer strategy based on this approach has recently been reported. 19 Transduction of STN neurons with glutamic
acid decarboxylase (GAD), the rate-limiting enzyme for synthesis of the inhibitory neurotransmitter gamma-amino butyric
acid (GABA), using an adeno-associated virus vector, resulted in synthesis and activity-dependent release of GABA from
STN nerve terminals. Microelectrode studies in control animals showed that stimulation of the STN resulted in excitation
of the majority of SNr neurons from which recordings were obtained, consistent with the known glutamatergic
neurochemical phenotype of STN neurons. However, stimulation of GAD-transduced STN neurons produced a
preponderance of inhibitory responses in the SNr neuron pool, suggesting that expression of GAD and consequent
modification of the neurochemical phenotype had altered the physiological properties of the STN-SNr projection.
Intriguingly, GAD transduction of the STN appeared to protect SNc dopaminergic neurons from a neurotoxic insult
following administration of 6-OHDA. The protective effect seemed dependent on the induction of an inhibitory phenotype
in the STN neurons, as destruction of the STN using ibotenic acid did not protect the SNc DA neurons from 6-OHDA.
Combining neuroprotection with functional compensation is attractive; a phase I clinical trial has been approved to begin
soon (see below).
Delivery of transgenes encoding enzymes involved in dopamine biosynthesis enhances dopamine production
in the striatum
Pharmacologic therapy of PD involves correction of the neurochemical deficit by systemic delivery of the dopamine
precursor, L-DOPA, or by use of agents that act directly on striatal dopamine receptors. In the first gene therapy study of
PD, gene transfer was employed to deliver the rate-limiting enzyme for dopamine formation, tyrosine hydroxylase, to the
striatum. This resulted in enhanced dopamine production and observable behavioral benefit in a rodent model.
More recently, it has been demonstrated that simultaneous delivery of multiple genes encoding enzymes that drive DA
synthesis, more effectively corrects the DA-deficient phenotype than single-enzyme replacement. Synthesis of DA from
tyrosine depends on two reactions, catalyzed by the enzymes tyrosine hydroxylase (TH) and aromatic acid decarboxylase
(AADC). The former step is rate limiting, and requires a cofactor that is synthesized by GTP-cyclohydrolase I (GCH1).
Various vector systems have been used in recent preclinical studies to deliver different combinations of these enzymes.
These include multicistronic lentiviruses simultaneously encoding GCH1, TH and AADC; 20 combinations of AAV vectors
separately encoding GCH1 and TH21 or GCH1, TH and AADC;22,23 an HSV vector coexpressing AADC and TH.24 In all cases,
coexpression of the enzymes and functional recovery of the experimentally lesioned animals was observed.
Long-term dopamine therapy in PD is associated with declining therapeutic efficacy and increasing adverse effects as the
disease progresses. While transgene-mediated dopamine expression effectively corrects the motor phenotype in lesioned
rodent and primate models, it is unclear at present whether the nonphysiological sustained delivery of dopamine in the
striatum by these kinds of approaches will alleviate or exacerbate the problem of adverse effects. Since the therapeutic
and toxic doses of dopaminergic agents alter in individual patients over the course of the disease, control over the
production of dopamine following gene transfer will be essential before the use of this approach can be contemplated
clinically. This might be accomplished using either (i) vectors with inducible enzyme expression, (ii) enzymes with
controllable activity, or (iii) a prodrug approach using AADC to activate
L-DOPA.
Stem cells may be driven to differentiate into functioning dopaminergic cells by genetic modification
Restoration of the dopaminergic projection from the SNc to the striatum has been a major goal of cell transplantation
strategies. One major hurdle for this approach to therapy has been the difficulty in obtaining a suitable source of donor
tissue that is both accessible and acceptable. One potential means for achieving this might rely on the use of human stem
cell populations, driven to differentiate into dopaminergic neurons by appropriate manipulations. Much effort has been
invested in determining which extracellular cues to stem cells are important in directing their differentiation into the
desired cell population. It is possible to direct the differentiation of ES cells, for example, into dopaminergic neurons by a
series of tissue culture manipulations, with an efficiency of around 15%. 25 This can be enhanced to around 50% after
transfection of the cells with a transgene encoding Nurr-1,26 an orphan nuclear receptor of the retinoic acid receptor
superfamily, which has been implicated in the later stages of dopaminergic neuronal differentiation. It is possible that
directed differentiation of dopaminergic neurons from a variety of stem cell sources might depend on ex vivo transduction
of stem cell populations to effect genetic modifications that favor adoption of the desired cell fate. Dopaminergic cells
formed from ES cells appear to have similar functional and neurochemical properties as native dopaminergic neurons, 26
and can rescue an animal model of PD.26,27
Isolation of genes implicated in rare genetic forms of PD has allowed generation of new animal models and
identification of new candidate targets for intervention
Although the common form of PD is sporadic and of unknown etiology, rare genetic forms have allowed isolation of genes
involved in their pathogenesis, and thus highlighted cellular pathways that may be vulnerable in dopaminergic neurons
and form potential targets for molecular intervention in PD. Mutations have been described in the gene encoding Synuclein, resulting in autosomal dominant PD. -Synuclein is abundantly expressed in the brain and normally localized
at nerve terminals. Aggregates of -Synuclein comprise a major component of the Lewy body, which is the pathological
hallmark of the common sporadic form of PD. A second form of familial PD is autosomal recessive, and results from
mutations in the gene encoding Parkin, a ubiquitin ligase. Intriguingly, -synuclein is a substrate of Parkin,28 linking the
two dissimilar proteins into a common functional pathway. Pathogenic mutations resulting in a PD phenotype have
recently been described in two other genes: DJ-1, of unknown function,29 and NR4A2, encoding Nurr-1, a nuclear receptor
(see above).30
Elucidation of the pathways involved in genetic forms of PD has provided new animal models of specific SN degeneration,
which do not rely on toxicity caused by chemical insults. These may more closely resemble the pathogenesis of human
disease. Thus, transgenic mice overexpressing either wild-type31 or mutated forms of human -synuclein32,33,34 develop
neuronal inclusions and cell loss. In addition, rat35 and monkey36 models have been developed using virally mediated synuclein gene transfer. Finally, a transgenic Drosophila model of -synucleinopathy has been described.37 Work in
Drosophila may accelerate understanding of the human disease by identifying candidate pathways for disease
modification.38
The genetic forms of PD are uncommon, but gene therapy targeting the
turn out to be an appropriate intervention for idiopathic PD. Using
overexpression of
-synuclein prevented aggregation of
-synuclein, Parkin or other pathways may also
-synuclein transgenic mice, it was shown that
-synuclein and the resulting abnormal phenotype.39 In
addition, it appears that Parkin is capable of blocking the toxic effects of mutant
inhibition in catecholaminergic neurons in culture.40 If deposition of
-synuclein expression and proteasome
-synuclein, formation of Lewy bodies, and
proteasome dysfunction are pivotal events in the pathogenesis of PD, then
-synuclein or Parkin gene delivery might be
effective measures to disrupt the pathogenic cascade causing neurodegeneration.
One clinical gene therapy trial is about to commence in Parkinson's disease
Despite the wealth of experimental data on preclinical studies of gene therapy for PD, only one gene therapy trial is
poised to start recruiting patients.41 The trial is based on GAD gene transfer to the subthalamic nucleus using an adenoassociated virus vector, as detailed in the section above.
In all, 12 patients with asymmetric disease will be selected by standard criteria, to undergo unilateral STN stimulator
implantation.41 The trial is a dose-escalation safety study, and as approved by US Food and Drug Administration three
cohorts of patients will receive between 1011 and 1012 particles of rAAV-GAD at the time of STN stimulator implantation.
The assessors will monitor the patients' clinical state and PET scans. In the worst case, if GAD gene transfer has an
unanticipated deleterious effect, then the STN can be either electrically silenced or ablated, both standard treatments for
PD, using the stimulator leads without additional surgery.
Although the molecular strategy used in this trial is highly specific to PD, the wider field will view this pioneering study of
in vivo gene transfer to the brain to treat neurodegeneration with considerable interest.
The optimal vector remains uncertain
Is there an optimal gene transfer vector with special utility for the development of treatments for PD? Nonviral gene
transfer (liposomes or naked plasmids) is in general ineffective for gene transfer to the brain parenchyma, but each of the
major viral vectors have demonstrated utility in experimental models of PD. In both earlier and recent studies viral
vectors based on Ad1,42 lentivirus (LV),11,20 AAV13,19 and HSV15,24 have all been used to transfer relevant genes to the
substantia nigra or striatum of experimental animals. Owing to the differences between studies in the animal model of PD
employed, the site and volume of vector inoculation, the transgene and promoter constructs tested, the vector dose and
the outcome measures assessed, the published literature does not allow one to make a direct comparison between
vectors. The immunogenicity of Ad is likely to exclude that vector from human trials for PD, but each of the remaining
vector platforms are likely to come to trial in the next few years. Although AAV and LV result in high-level long-term
expression in brain, the results of the human trial using retroviral gene transfer to treat X-linked SCID in which two
treated children developed leukemia as a result of insertional mutagenesis, 43 coupled with the recent observation that
AAV integrates more frequently into active genes than noncoding regions, 44 may favor the use of a non-integrating vector
such as HSV for these applications.
Prospects for the next 2 years
The development of readily available presymptomatic diagnostic tests for PD will be necessary to enable the use of
neuroprotective strategies to retard the progression of SNc cell loss. Better understanding of the pathogenesis of the
common idiopathic form of PD may lead to the development of improved animal models that more closely resemble the
etiology and cellular pathophysiology of human PD. This advance might enable identification of further targets for
molecular intervention, in addition to providing a necessary resource for studies aimed at tackling the nonmotor features
of PD. Depression, cognitive and autonomic dysfunctions are important contributors to morbidity in PD; further research
aimed at addressing these components of the illness would be welcome. Further characterization of ES and adult stem cell
populations will establish whether ex vivo transduction can drive their differentiation into dopaminergic neurons in a
therapeutically useful way. Finally, further clinical trials for PD gene therapy are likely to commence using a variety of
strategies. The trial designs may take their lead from recent well-executed clinical trials of cell transplant therapy.45,46 It
will be important to measure disability, depression and quality of life in addition to motor outcome, in order to be certain
about which aspects of the illness are favorably altered by gene transfer, and whether there is likely to be an overall
beneficial effect from these interventions in patients.
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neurodegeneration and promotes functional recovery in a rat model of
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14 McGrath J et al. Adeno-associated viral delivery of GDNF promotes
recovery of dopaminergic phenotype following a unilateral 6-hydroxydopamine
lesion. Cell Transplant 2002; 11: 215227.
15 Natsume A et al. Bcl-2 and GDNF delivered by HSV-mediated gene
transfer act additively to protect dopaminergic neurons from 6-OHDA-induced
degeneration. Exp Neurol 2001; 169: 231238. Article PubMed
16 Gill SS et al. Direct brain infusion of glial cell line-derived neurotrophic
factor in Parkinson disease. Nat Med 2003; 9: 589595. Article
17 Georgievska B, Kirik D, Bjorklund A. Aberrant sprouting and
downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine
neurons induced by long-lasting overexpression of glial cell line derived
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19 Luo J et al. Subthalamic GAD gene therapy in a Parkinson's disease rat
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20 Azzouz M et al. Multicistronic lentiviral vector-mediated striatal gene
transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and
GTP cyclohydrolase I induces sustained transgene expression, dopamine
production, and functional improvement in a rat model of Parkinson's disease.
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21 Kirik D et al. Reversal of motor impairments in parkinsonian rats by
continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer.
Proc Natl Acad Sci USA 2002; 99: 47084713.
22 Muramatsu S et al. Behavioral recovery in a primate model of Parkinson's
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vectors expressing dopamine-synthesizing enzymes. Hum Gene Ther 2002;
13: 345354. Article PubMed
23 Shen Y et al. Triple transduction with adeno-associated virus vectors
expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and
GTP cyclohydrolase I for gene therapy of Parkinson's disease. Hum Gene Ther
2000; 11: 15091519. Article PubMed
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from a helper virus-free herpes simplex virus type 1 vector. Hum Gene Ther
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25 Lee SH et al. Efficient generation of midbrain and hindbrain neurons from
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26 Kim JH et al. Dopamine neurons derived from embryonic stem cells
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Article PubMed
27 Bjorklund LM et al. Embryonic stem cells develop into functional
dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl
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28 Shimura H et al. Ubiquitination of a new form of alpha-synuclein by parkin
from human brain: implications for Parkinson's disease. Science 2001; 293:
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29 Bonifati V et al. Mutations in the DJ-1 gene associated with autosomal
recessive early-onset parkinsonism. Science 2003; 299: 256259.
30 Le WD et al. Mutations in NR4A2 associated with familial Parkinson
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31 Masliah E et al. Dopaminergic loss and inclusion body formation in alphasynuclein mice: implications for neurodegenerative disorders. Science 2000;
287: 12651269. Article PubMed
32 Lee MK et al. Human alpha-synuclein-harboring familial Parkinson's
disease-linked Ala-53 > Thr mutation causes neurodegenerative disease with
alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci USA 2002;
99: 89688973. Article PubMed
33 Giasson BI et al. Neuronal alpha-synucleinopathy with severe movement
disorder in mice expressing A53T human alpha-synuclein. Neuron 2002; 34:
521533. PubMed
34 Richfield EK et al. Behavioral and neurochemical effects of wild-type and
mutated human alpha-synuclein in transgenic mice. Exp Neurol 2002; 175:
3548. Article PubMed
35 Lo Bianco C et al. alpha-Synucleinopathy and selective dopaminergic
neuron loss in a rat lentiviral-based model of Parkinson's disease. Proc Natl
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36 Kirik D et al. Nigrostriatal alpha-synucleinopathy induced by viral vectormediated overexpression of human alpha-synuclein: a new primate model of
Parkinson's disease. Proc Natl Acad Sci USA 2003; 100: 28842889.
37 Feany MB, Bender WW. A Drosophila model of Parkinson's disease.
Nature 2000; 404: 394398. Article PubMed
38 Auluck PK et al. Chaperone suppression of alpha-synuclein toxicity in a
Drosophila model for Parkinson's disease. Science 2002; 295: 865868. Article
PubMed
39 Windisch M et al. Development of a new treatment for Alzheimer's disease
and Parkinson's disease using anti-aggregatory beta-synuclein-derived
peptides. J Mol Neurosci 2002; 19: 6369.
40 Petrucelli L et al. Parkin protects against the toxicity associated with
mutant alpha-synuclein: proteasome dysfunction selectively affects
catecholaminergic neurons. Neuron 2002; 36: 10071019. PubMed
41 During MJ, Kaplitt MG, Stern MB, Eidelberg D. Subthalamic GAD gene
transfer in Parkinson disease patients who are candidates for deep brain
stimulation. Hum Gene Ther 2001; 12: 15891591. PubMed
42 Xia XG et al. Gene transfer of the JNK interacting protein-1 protects
dopaminergic neurons in the MPTP model of Parkinson's disease. Proc Natl
Acad Sci USA 2001; 98: 1043310438. Article PubMed
43 Check E. Regulators split on gene therapy as patient shows signs of cancer.
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genes in mice. Nat Genet 2003; 34: 297302. Article PubMed
45 Nakamura T et al. Blinded positron emission tomography study of
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Figures
Figure 1 The human basal ganglia. A coronal section of a human brain is
shown, illustrating the anatomical locations of the basal ganglia. (Photograph
of human autopsy specimen kindly provided by Dr Olaf Ansorge, Department
of Neuropathology, Radcliffe Infirmary, Oxford).
Figure 2 Gene therapy strategies for PD. The putative events and functional
consequences involved in loss of SNc neurons are depicted. The complex
pathogenic and pathophysiological cascade provides several candidate targets
for molecular intervention, which are labeled with black arrows and white text;
some of these are supported by experimental evidence, which is discussed in
the text. In addition, alternative strategies to gene delivery, involving
functional neurosurgery, cell transplantation and neuropharmacology are
shown for contextual comparison. Abbreviations: P, putamen; GPe, external
segment of globus pallidus; GPi, internal segment of globus pallidus; STN,
subthalamic nucleus; SNc, substantia nigra pars compacta; TH, tyrosine
hydroxylase; GTPCH1, GTP-cyclohydrolase-1.
September 2003, Volume 10, Number 20, Pages 1721-1727
August 2003, Volume 10, Number 16, Pages 1275-1281
Table of contents
Previous Article Next PDF
Review
Gene therapy progress and prospects: gene therapy of
lysosomal storage disorders
S H Cheng1 and A E Smith1
Genzyme Corporation, 31 New York Avenue, Framingham, MA, USA
1
Correspondence to: Dr SH Cheng, Genzyme Corporation, 31 New
York Avenue, Framingham, MA 01701-9322, USA
Abstract
Despite disappointments with early clinical studies, there is continued interest in the
development of gene therapy for the group of metabolic diseases referred to as lysosomal
storage disorders (LSDs). The LSDs are monogenic and several small and large,
representative animal models of the human diseases are available. Further, the successful
reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are
expected to be sufficient to correct the disease at least in the case of some of the LSDs. For
these reasons, they are perceived as good models for the evaluation of different gene delivery
vectors and of different strategies for treating chronic genetic diseases by gene transfer. In
this review, we will highlight the progress that has been made over the past 2 years in
preclinical research for this group of disorders and speculate on future prospects.
Gene Therapy (2003) 10, 12751281. doi:10.1038/sj.gt.3302092
Keywords
lysosomal storage disorders; lysosomal enzymes; metabolic diseases; central nervous system; depot
organs
In brief
Progress









Current therapeutic options for lysosomal storage disorders have
been shown to be effective but limited
Lysosomal storage disorders are good candidates for therapy by
gene transfer
Early clinical studies using ex vivo gene therapy vectors were
ineffective
Proof of concept for use of in vivo gene therapy of LSDs has
been demonstrated with different vector platforms and in
several animal models
AAV serotypes with improved liver transduction activity are a
promising vector platform for gene therapy of LSDs
Feasibility of in vivo gene therapy of LSDs with retroviral
vectors has been demonstrated
Use of liver-specific promoters minimizes the induction of
antibodies to the transgene product
Relative ability of the liver, muscle, and lung to support the
secretion of lysosomal enzymes has been evaluated
Encouraging progress has been made in gene therapy of LSDs
with CNS disease
Prospects






Candidate in vivo gene transfer vectors such as AAV8 for
potential application in the treatment of LSDs will be selected
for further characterization
Issues pertaining to safety, readministration of vector and
manufacturing will be improved
Performance of the vector platforms in more relevant larger
animal models will be addressed
The feasibility and necessity of incorporating gene regulation
elements will be evaluated
Candidate lysosomal storage disorders with minimal CNS
disease will be targeted for clinical studies
Methods to deliver gene or cell therapies that result in the global
rescue of CNS disease will be developed
Current therapeutic options for lysosomal storage disorders have been shown to be effective
but limited
Lysosomal storage disorders are a group of more than 40 heritable diseases that are caused by the
pronounced deficiency of one or more lysosomal enzymes.1 This loss in enzymatic activity results in the
progressive accumulation of undegraded substrate within the lysosomes with resultant engorgement of
the organelle. This leads to cellular and tissue damage, subsequent organ dysfunction, and in some
diseases to early mortality. Although the incidence of individual LSDs can be quite low, as a group they
occur in approximately 1 in 7500 live births, and as such represent one of the more prevalent groups of
genetic diseases in humans.
Currently, treatments for these rare disorders are limited to bone marrow transplantation and enzyme
replacement therapy.2 The latter has been shown to be effective in the treatment of the non-neuropathic
form of Gaucher disease3 and of Fabry disease,4,5 and enzyme replacement therapy is currently being
developed for several other LSDs such as mucopolysaccharidosis I (MPS I), II and VI, Pompe disease,
and Niemann-Pick B disease. However, because these enzymes generally have short circulating and
intracellular half-lives, therapy requires regular, often biweekly, parenteral administrations of relatively
large amounts of the relevant enzyme. Repeated bolus administrations of enzyme increase the likelihood
of an immune response against the infused proteins, particularly in patients with null mutations, and this
could affect the efficacy of subsequent treatments. Furthermore, because systemically administered
enzyme is unable to traverse the bloodbrain barrier, enzyme therapy is only effective for those
manifestations that do not involve the central nervous system (CNS). Therefore, other approaches,
including the use of gene- and cell-based therapies that offer the opportunity for a more prolonged
therapeutic effect than can be realized with enzyme replacement therapy, as well as the possibility of
treating the CNS, are also being evaluated.
Lysosomal storage disorders are good candidates for therapy by gene transfer
There are several features of LSDs that make them particularly attractive candidates for intervention by
gene therapy. For one, they are generally well-characterized single gene disorders. Importantly, it has
also been shown that a proportion of many newly synthesized lysosomal enzymes are secreted into
systemic circulation. Enzymes secreted in this way are recaptured by adjacent and distant cells, primarily
through the cation-independent mannose-6-phosphate receptor, which is present, albeit in different
amounts, on the surface of virtually all cells.1 Localized gene transduction of a depot organ such as the
liver or muscle could allow for secretion of therapeutic levels of the affected enzymes into circulation.
The amount of enzyme required for correction will vary with each disease, but may be only 110% of
normal levels, based on the observed enzyme levels in individuals with milder, late-onset disease. In this
regard, the potency of the gene transfer vectors necessary for facilitating the production of therapeutic
levels of these enzymes may not need to be very high. Although it is unclear whether sustained
overexpression of the hydrolases will have untoward consequences, tight regulation of enzyme
production levels is unlikely to be necessary in part because the pH optimas for their enzymatic activities
are likely to render them inactive in circulation at neutral pH. Moreover, a number of genetically
engineered mouse models as well as naturally occurring mouse and large animal models of LSDs are
available, which should allow assessment of these predictions.6
Early clinical studies using ex vivo gene therapy vectors were ineffective
Early attempts at gene therapy for subjects with the lysosomal storage diseases Gaucher, Hunter and
Hurler were with transplanted retrovirally transduced bone marrow stem cells or mobilized peripheral
blood monocytes. However, it was determined that the efficiency of retroviral transduction of these cells
was low, and engraftment of the modified stem cells, particularly in the absence of myeloablation, was
transient and occurred at a low and ineffective level.7 Nevertheless, with ongoing improvements in
vectors and processes for transduction, this approach continues to be of interest for use in LSDs. 8,9,10,11
Recent improvements include the development and use of cytokine combinations that induce
hematopoietic cell cycling to enhance retroviral transduction, 12 pseudotyped virions with envelopes that
recognize different and perhaps more abundant receptors in the target cells,13 lentiviral vectors that
infect nondividing cells with greater efficiency,14 and methods to enrich for the transduced cells prior to
transplantation.10 The adaptation of some of these methods has led to clinical success in treating humans
with the genetic disorder severe combined immunodeficiency disease (SCID).15 However, this outcome
was due in part to the strong positive selective pressure provided to the corrected lymphoid progenitors
upon gene transfer and this is unlikely to occur in corrected cells from LSDs patients. Moreover, two
cases of a T-cell leukemia-like disease was recently noted in the treated SCID patients that may have
resulted from vector integration. This observation together with the finding that some myeloablation is
likely necessary for efficient engraftment of transduced stem cells makes this gene therapy approach less
compelling, particularly for those LSDs where enzyme replacement therapy is available.
Proof of concept for use of in vivo gene therapy of LSDs has been demonstrated with different
vector platforms and in several animal models
The concept of directly transducing a depot organ to effect the production and secretion of lysosomal
enzymes to treat the visceral disease has been demonstrated using a variety of gene delivery systems
(Table 1). Understandably, the initial focus of interest has been directed primarily at those LSDs with no
or minimal neurological involvement, and for which a viable animal model is available.
Since systemic delivery of recombinant adenoviral vectors results in efficient transduction of the liver and
high-level expression and secretion of lysosomal enzymes, a large number of studies have been
performed with this vector.16,17 Intravenous delivery of the corresponding recombinant adenoviral vector
provided high-level secretion from the liver, and importantly, re-uptake of the lysosomal enzymes by
other affected tissues. Depending on the dose of virus used, the levels of enzyme attained in the
different tissues varied from 10- to 1000-fold higher than normal levels. In all cases, reconstitution of
the enzymes to these levels was sufficient to reduce rapidly the abnormal storage in the lysosomes to
normal or near normal levels.
However, these studies also showed that expression of the desired protein was transient, declining to
basal levels within several weeks. Moreover, an inflammatory response that included a significant
cytotoxic T-lymphocyte response was observed that likely attenuated expression. Hence, while these
studies demonstrated the potential of in vivo gene therapy for LSDs, they also highlighted the need for
significant improvements in the performance of the adenoviral vectors before clinical studies can usefully
be contemplated. The so-called 'gutless', 'PAV', or helper-dependent vectors which are essentially devoid
of viral genes, are purportedly less inflammatory, effect less liver toxicity, and support greater longevity
of transgene expression. Although these vectors are presently difficult to produce in large quantities and
with high purity, their superior properties support their further evaluation in animal models of LSDs.
Synthetic vectors in the form of cationic lipids or polymers have also been considered for in vivo gene
therapy of LSDs.18 Since synthetic vectors are nonproteinaceous, a significant advantage is their ability
to be readministered following the attenuation of gene expression. However, the therapeutic window of
current formulations of synthetic vectors is quite narrow and improvements in their transduction activity
as well as their toxicity profile are necessary.19 Another nonviral approach is the use of encapsulated
gene-modified cells or organoids. Implantation of nonautologous cells expressing
-glucuronidase has
been shown to alleviate some of the storage burden in MPS VII mice. 20 However, expression was
transient and was associated with the induction of antibodies against the enzyme. Limitations associated
with the number of cells that can be encapsulated in the current devices, coupled with the potential for
dissolution of the membrane biomaterials with ensuing loss of cell viability or escape of the cells need to
be further addressed before this can be considered for human application.
AAV serotypes with improved liver transduction activity are a promising vector platform for
gene therapy of LSDs
A viral vector that is gaining increasing interest for use in lysosomal storage and other genetic diseases
is the adeno-associated viral vector.21 AAV reportedly exhibits low toxicity and supports long-term
transgene expression in mice as well as large animals.22,23,24 Although the vector has a limited capacity
for inserted sequences, this is not problematic for most of the cDNAs that encode lysosomal enzymes,
since they are relatively small. Several distinct AAV serotypes have been isolated, of which AAV2 has
been the most studied. Recombinant AAV2 vectors have so far been constructed for the LSDs, Fabry, 24,25
Pompe,26 MPS VII,27 MPS I, and MPS IIIB.28 Systemic delivery of recombinant AAV2 encoding galactosidase A into Fabry mice, or encoding
-glucuronidase into neonatal MPS VII mice, resulted in
the reconstitution of the respective enzymes in several tissues to 1080% of normal levels. These levels
are 100- to 1000-fold lower than those attained using recombinant adenoviral vectors, despite the use of
much higher doses of AAV2.
The kinetics of expression were consistent with those reported previously for AAV2 vectors containing
other transgenes, with peak expression levels generally attained between 2 and 4 weeks, and these
levels persisting for several months post-treatment. Although there was only a modest increase in
enzyme levels in some of the tissues, they were sufficient to reduce measurably the amount of the
substrates that had accumulated in the different animal models. This observation supports the notion
that continuous expression of low levels of enzyme activity is sufficient to reduce the extent of storage in
the lysosomes. However, the kinetics for the reduction of the storage materials were slower in the AAVthan in adenoviral-treated animals, with the AAV2-treated animals requiring several more weeks to
effect clearance.
When coupled with its positive safety profile, these results suggest that AAV2 vectors have great
potential for treating LSDs. However, since AAV2-mediated expression levels were relatively low and
close to the threshold for therapeutic efficacy in some of the affected tissues, an improvement in
transduction activity would be beneficial. In this regard, other AAV serotypes such as AAV1, and in
particular AAV8, have recently been shown to have substantially greater liver transduction activity than
AAV2.29 The 10- to 100-fold higher expression levels attained with AAV8 were correlated with a higher
number of transduced hepatocytes and greater persistence of vector DNA. Moreover, AAV8 was shown to
have a low reactivity to neutralizing antibodies directed to human AAVs. This relative lack of pre-existing
immunity to AAV8 coupled with its superior tropism for liver argues that it is a good candidate for further
evaluation as a vector for gene therapy of LSDs. However, the induction of a humoral response to the
viral proteins following the first administration will present challenges for subsequent readministrations.
Although several strategies to overcome this limitation have been reported, none are facile or involve the
use of clinically approved agents.
Feasibility of in vivo gene therapy of LSDs with retroviral vectors has been demonstrated
Yet another vector that is under consideration for in vivo gene therapy of LSDs is the oncoretroviral
vector. As these vectors have the capacity to integrate into the host genome, they offer the possibility
for long-term expression. Both Moloney murine leukemia virus (MLV)-based and lentiviral-based
retroviral vectors have been shown to be capable of transducing hepatocytes with sufficient efficiency to
facilitate the production and secretion of lysosomal enzymes. 30,31 Intravenous delivery to MPS VII mice of
either a MLV or lentiviral vector encoding
-glucuronidase resulted in expression of enzyme to
approximately 1% of normal levels in the liver. Expression was sustained for the duration of the studies
(3.55 months) and was associated with a reduction in lysosomal storage. As these vectors preferentially
transduce replicating hepatocytes, improved transfection was realized when the animals were pretreated
with hepatocyte growth factor. Greater transduction of hepatocytes (220%) could also be attained when
the retroviral vectors were administered to neonatal animals as was the case in the recent study in MPS
VII dogs.32,33 Clonal expansion of the transduced hepatocytes during liver development resulted in the
production of
-glucuronidase levels that were within the normal range. These expression levels were
sustained in the dogs for several months and were associated with dramatic improvements in the clinical
manifestations. Although this intervention is conceivable for humans, implementation would necessarily
require an assessment of the risk for insertional mutagenesis, as well as the detection of disease at a
very early stage.
Use of liver-specific promoters minimizes the induction of antibodies to the transgene product
Expression of lysosomal hydrolases following viral-mediated gene transfer to immunocompetent mouse
models of LSDs is invariably associated with the generation of a robust humoral response against the
enzymes. This has the effect of extinguishing transgene expression and thereby limiting the duration of
therapy. This problem is likely to be particularly pertinent in LSD subjects that harbor null mutations.
The proportion of patients among the different LSDs carrying null mutations is varied but can be as high
as 70% as in the case of Hurler syndrome. However, it has been shown that this immune response can
be circumvented in both adenovirus- and AAV-treated animals, provided a tissue-restricted promoter was
used to direct the transgene expression.34 Systemic injection of either a recombinant adenoviral or AAV
vector in which the transgenes were placed under the transcriptional control of liver-restricted promoters
reduced the extent of antibodies induced and increased the longevity of transgene expression in
immunocompetent mice. The reduced tendency to provoke an immune response is thought to be related
to the reduced expression of the transgene in antigen-presenting cells.
In Fabry mice, injection of an AAV vector encoding
-galactosidase A under the control of a chimeric
human liver-restricted promoter consisting of two copies of the prothrombin enhancer linked to a human
serum albumin promoter resulted in undiminished expression for up to 1 year. 35 In contrast to Fabry
mice treated with a CMV expression cassette, no antibodies to the transgene product were detected in
the animals treated with the liver-restricted promoter cassette. The ability of this chimeric promoter to
sustain prolonged expression in larger animals such as in a nonhuman primate has also been
demonstrated.
Relative ability of the liver, muscle, and lung to support the secretion of lysosomal enzymes
has been evaluated
Although early in vivo gene therapy efforts for LSDs have focused primarily on the use of the liver as a
depot for the production of lysosomal enzymes, consideration has also been given to skeletal muscle and
lung as alternate portals. The liver was selected in part because of the tropism of the adenoviral and AAV
vectors for this organ. The rationale for selecting hepatocytes as the target for genetic modification was
also supported by the knowledge that they are adept at secreting a variety of proteins. However,
parenteral administration of viral vectors, particularly adenoviral vectors, has been shown to be
associated with liver toxicity. This characteristic is obviously undesirable, especially for those LSDs where
liver function is already compromised. Systemic delivery also carries the potential risk of transfecting the
gonads and of subsequent germline alteration.
Using the skeletal muscle as the depot in lieu of the liver circumvents some of these concerns. Using
intramuscular injection to produce lysosomal enzymes has been reported for MPS VII,36 Pompe
disease,26,37,38 and Fabry disease.25 Although high levels of localized expression of the enzymes could be
realized in the muscle, only very low levels of the enzymes were secreted into the circulation. In the case
of MPS VII and Pompe disease, these levels were determined to be insufficient to provide therapeutic
benefit to affected tissues that were distant from the injected muscle. These findings were consistent
with the report by Raben et al showing that the muscle was significantly less efficient than the liver at
secreting the lysosomal enzyme -glucosidase for Pompe disease.39 However, these observations did not
extend to Fabry disease. Despite attaining only low-level secretion of -galactosidase A, correction of the
storage defect was observed not only in the injected muscle but also globally in AAV2-treated Fabry
mice. Therefore, it would appear that the selection of muscle as a depot organ might be applicable for
use in some but not all LSDs. Perhaps the use of other AAV serotypes, such as AAV1 or AAV7 that
reportedly exhibit higher transduction efficiencies in muscle than AAV2, could further improve the utility
of this organ for the production of lysosomal enzymes.29
Another organ that is gaining interest as a metabolic factory for the production and secretion of
therapeutic proteins into systemic circulation is the lung. Use of the lung as a portal for systemic delivery
of proteins offers several advantages. Foremost is the large lumenal surface area of the lung that can be
accessed noninvasively by liquid or dry powder aerosols. The lung also has an extensive capillary
network that could support the delivery of proteins into the systemic circulation. Presently, a number of
metabolic hormones such as insulin and growth hormone are under consideration for systemic delivery
by pulmonary inhalation.40 Restricting delivery to the lumen also limits the dissemination and therefore
any systemic toxicity that may be associated with the gene transfer vector. Finally, several vector
systems, including adenoviral, AAV5 and pseudotyped lentiviral vectors have been shown to transduce
airway epithelial cells after pulmonary delivery.41,42
The feasibility of genetically modifying the lung and using this as a portal to administer proteins into the
blood has been demonstrated for the lysosomal enzyme
-galactosidase A43 and also for erythropoietin
(epo) and factor IX.44 Instillation of a recombinant adenoviral vector encoding
-galactosidase A into
Fabry mice resulted in high level expression in the lung, secretion into the circulation and subsequent
uptake of the lysosomal enzyme by the visceral organs. The levels of enzyme detected in the different
organs were sufficient to reduce the burden of storage in the affected lysosomes. Although expression in
the lung was transient using a recombinant adenoviral vector, expression kinetics were dramatically
improved when an AAV5 vector was used for factor IX and epo. The availability of vectors that support
high and sustained expression of proteins in the lung and of methods to deliver these in a noninvasive
manner to the lumen suggest that the lung may be a viable alternative depot for the production of
lysosomal enzymes. As for the gene delivery vectors, the final selection of the depot organ of choice will
likely depend on the particulars of the LSDs. A better understanding of the relative abilities of the
different organs to confer the necessary post-translational modifications to facilitate effective targeting of
the enzymes to the lysosomal compartment may also aid with the selection process.
Encouraging progress has been made in gene therapy of LSDs with CNS disease
To treat LSDs with CNS manifestations requires the development of strategies that not only bypass the
physical and bloodbrain barriers but that also support the delivery of enzyme throughout the brain. A
further challenge for gene transfer within the CNS is the relatively quiescent state of the resident cells
that precludes the use of vectors requiring cell division for transduction. Nevertheless, there have been
ample demonstrations of the feasibility of gene therapy with several vector platforms in animal models of
LSDs with CNS involvement. Intraventricular or stereotactic injections of recombinant adenoviral, AAV,
lentiviral, and herpes simplex viral (HSV) vectors into various cerebral structures can result in
transduction of both neuronal and glial cells. Of these, AAV and lentiviral vectors appear to be gaining
increasing favor because of their safety profile, and their ability to transduce apparently quiescent cells
within the CNS and to confer prolonged expression.
Intracranial injections of recombinant AAV245 or feline immunodeficiency viral46 vectors encoding
-
glucuronidase into MPS VII mice resulted not only in the correction of the characteristic cellular
pathologies but also in improvements in cognitive function. A similar demonstration of protection from
disease-associated pathology was also reported for metachromatic leukodystrophy using a lentiviral
vector encoding the lysosomal enzyme arylsulfatase A.47 In all cases, although expression of the
enzymes was concentrated at the sites of injection, pathology resulting from storage was reduced in
most areas of the brain. This suggested that the enzyme was secreted from the transduced cells and
taken up by uninfected adjacent cells, resulting in a zone of correction that extended beyond the site of
injection. In addition to enzyme diffusion, evidence has also been presented suggesting that the
lysosomal enzymes could be further distributed by axonal transport and by cells of the rostral migratory
stream.48 Further improvement in the biodistribution of the enzyme in MPS VII-injected mice could also
be realized through the incorporation of protein transduction motifs such as that derived from HIV Tat. 49
While these results in LSDs mouse models are encouraging, confirmatory studies are required to
ascertain whether these observations are translatable to animals with larger brains. In addition, although
a few of the studies were performed in adult mice with developed CNS pathology rather than very young
animals, it will be important to determine the temporal window for effective interventional therapy.
Summary
Significant progress has been made in studies of gene therapy for lysosomal storage disorders. While the
ability to deliver therapy to the CNS is still at a formative stage, the ability to treat the visceral
component of the disease, particularly using the recently described gene transfer vectors, appears
feasible. Those LSDs that lack or have only moderate CNS disease could therefore be initially considered
for therapy using this approach. Examples of such LSDs include Type I Gaucher disease, Fabry disease,
Niemann-Pick B disease, Pompe disease, MPS IS, IHS, IV and VI.
Prospects
Although more validation is required, the reported higher liver transduction activity of AAV8, coupled
with its good safety profile and ability to confer long-term expression suggest that this is the vector
choice for evaluation in human clinical studies. Studies to confirm that AAV8 transduces human
hepatocytes with high efficiency, as seen in mouse models, and that pre-existing neutralizing antibodies
to AAV8 are present at a low frequency in the general population are undoubtedly ongoing. However,
depending on the dose of vector needed in humans, consistent manufacture of clinical grade AAV vectors
at scale may still present a technical challenge. Other issues include the possibility of neutralizing
antibodies generated against the newly synthesized enzymes and the ability to readminister the
recombinant virus. While the former could be addressed in part through the use of tissue-restricted
promoters, the possible incorporation of gene regulation or gene switch technologies may further
alleviate this safety concern.50 However, readministration of the recombinant virus without the unwieldy
use of additional manipulations remains a significant challenge. Most animal studies would predict that
AAV-mediated gene expression is likely to persist for a very significant period in time. It is quite possible
that the antibody titer to the virus will drop sufficiently during the intervening period to allow
readministration.
There is clearly much optimism for the potential use of gene therapy for LSDs, at least initially, for those
with minimal CNS involvement. Over the past few years, investigators have better defined what
constitutes an effective and safe gene transfer vector for use in this group of metabolic disorders. If the
remaining issues associated with the viral gene delivery vectors can be adequately addressed over the
next period, it is likely renewed clinical studies for LSDs will proceed.
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Tables
Table 1 Lysosomal storage disorders
August 2003, Volume 10, Number 16, Pages 1275-1281
July 2003, Volume 10, Number 14, Pages 1135-1141
Table of contents
Previous Article Next PDF
Review
Gene therapy progress and prospects: adenoviral vectors
J A St George1
Genzyme Corporation, Framingham, MA, USA
1
Correspondence to: Dr J St George, Genzyme Corporation, 31 New
York Avenue, Framingham, MA 01701-9322, USA
Abstract
In September 1999, the perceptions of the use of adenoviral (Ad) vectors for gene therapy
were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector
succumbed to the toxicity related to vector administration. Appropriately, concerns were
raised about continued use of the Ad vector system and, importantly, there were increased
efforts to more fully understand the toxicity. Today it is recognized that there is no ideal
vector system, and that while Ad vectors are not suitable for all applications, the significant
advantages over other vector systems including efficient transduction of a variety of cell
types, both quiescent and dividing, make it optimal for certain applications. These include
protocols where high levels of short-term expression are sufficient to provide a therapeutic
benefit. Potential target applications include therapeutic angiogenesis, administration into
immune-privileged sites such as the CNS, or treatments where the adjuvant effect of
adenovirus can be of benefit such as cancer vaccines. Broader applicability of Ad vectors will
require resolution of toxicity issues. This review will therefore focus on studies conducted
over the last 2 years that have advanced our understanding of the toxicity associated with Ad
vectors, studies that have employed methods to reduce toxicity and improvements in Ad
vectors themselves that will reduce toxicity by one of several mechanisms. These mechanisms
include retargeting vector to the tissue of interest, minimizing or eliminating viral gene
expression that is thought to result in loss of transduced cells, or by methods that seek to
reduce the vector dose required for therapeutic benefit. An area where there remains
significant room for improvement is when readministration of vector is required because
transgene expression has decreased to background levels.
Gene Therapy (2003) 10, 11351141. doi:10.1038/sj.gt.3302071
Keywords
adenovirus; targeting; toxicity
In brief
Progress




Application of Ad vectors to select applications such as
therapeutic angiogenesis has generated cautious optimism.
An understanding of Ad vector-induced toxicity is continuing to
grow and is leading to methods to minimize it's effects.
Refinements in targeting of Ad vectors are improving the
therapeutic index.
Advanced generation Ad vectors have reduced toxicity and
improved persistence of transgene expression.
Prospects




Methods to reduce toxicity will continue to be advanced and
will lead to safer use of Ad vector systems.
The use of targeting methods will be improved.
Production methods of fully deleted Ad vectors will continue to
improve.
Further efforts will be focused on readministration challenges.
Ad vectors continue to be tested in clinical trials and have demonstrated a therapeutic effect
Although many have questioned the use of Ad vectors, the number of clinical protocols employing Ad
vectors has not decreased. Ad vector use remains around 27% (636 protocols) and the percentage of
patients treated at 18% (3494) (Journal of Gene Medicine Website, www.wiley.co.uk/genmed/clinical/).
This proportion is second only to retroviral vectors. The success of an Ad vector in humans was reported
recently when the results of a randomized, placebo-controlled, double-blinded phase II trial
demonstrated that an Ad vector coding for VEGF gene increased vascularity after percutaneous
translumenal angioplasty.1 Currently, there are five phase IIIII or phase III clinical trials where Ad
vectors are being tested in either angiogenic or cancer applications.
Ad vector-induced toxicity is complex
As a reflection of the importance of Ad-induced toxicity, the entire January 2002 issue of Human Gene
Therapy was devoted to the topic. A call by the National Institutes of Health Recombinant DNA Advisory
Committee for a better understanding of the toxicity of Ad vectors included recommendations to develop
standards, so that data collected by various laboratories can be compared, to develop a database for the
collection and organization of safety and toxicity data and several recommendations to improve the
safety of research participants.2 A standard that has been developed includes adenoviral vector reference
material that is available to investigators for comparison to their vectors. It is apparent that only with a
thorough understanding of all aspects of the toxic interaction between a vector and the host that the full
utility of any vector system will be realized. However, the toxicity associated with the use of Ad vectors
is extremely complex involving both the innate and adaptive immune responses. It is dose dependent,
occurs in phases, is related to route of administration, is dependent on tissue and cell type targeted and
varies with species. In addition, the role of the response to a foreign or reporter transgene is often
ignored but can complicate interpretation of the findings. Finally, it is important to remember that
extrapolations from preclinical studies to clinical studies should take into account that the vast majority
of preclinical studies are performed in normal, naïve animals, whereas clinical studies are conducted in a
population likely not naïve to adenovirus and that has underlying disease.
The initial response to Ad vectors administered intravascularly occurs within minutes, occurs in the
absence of viral gene expression and is attributed to the innate response. Building on earlier studies has
shown that, as the vector distributes in the blood, it interacts with cells of the reticuloendothelial system
(RES) and induces the release andor production of several proinflammatory cytokines including IL-6,
TNF , IL-8, GM-CSF and MIP-2.3,4 Cytokine production appears to occur through the NF B pathway
possibly via RafMAP kinase phosphorylation of I B.4,5 Higginbotham et al4 reported that exposure of
normal human peripheral blood mononuclear cells to Ad vector resulted in the release of multiple
proinflammatory cytokines in vitro. The authors suggested that transcriptional stimulation of these
cytokines was the result of cellular binding of virionscapsids. In addition to the mechanism described
above, Ad vectors have been shown to activate complement in vitro,6 but the significance of this
response has yet to be demonstrated in vivo.
The immediate response peaks around 6 h and the primary cells thought to be responsible are
macrophages and dendritic cells that serve as a functional bridge between the innate and acquired
immune response. These cells are activated and induced to mature, which results in upregulation of MHC
antigens as well as co-stimulatory and adhesion molecules.7 The inadvertent targeting of these antigenpresenting cells leads to a systemic acquired immune response. 8 In addition, vector interaction with
epithelial cells results in release of C-X-C chemokines, especially IP-10.5 IP-10 is a potent
chemoattractant for activated T lymphocytes and pushes the reaction towards a Th-1 type or cytotoxic Tlymphocyte (CTL) response. Thus, the innate response bridges via multiple pathways with the acquired
response that follows.
Subsequent responses or possible sequelae to the initial response can be lethal, can occur within days
and can be correlated, at least in part, with Ad gene expression. For the most part, this response has
been well characterized and is correlated with the infiltration of lymphocytes, the formation of anti-Ad
antibodies and the loss of transduced cells. When a lethal response occurs following administration of
high doses, systemic changes have been described that include extensive endothelial damage and
disseminated intravascular coagulopathy.9 Several studies described a decrease in platelets and an
increase in the von Willebrand factor. 8,9,10 It is of note, however, that Ad vectors do not directly induce
platelet aggregation.11
The January 2002 issue of Human Gene Therapy mentioned above contains two studies describing the
responses in nonhuman primates.9,12 Studies in non-human primates may be the most instructive to our
understanding of Ad-mediated toxicity as the responses have been shown to mirror those described in
patients.10 The findings of studies conducted in primates, including humans, are summarized briefly in
Table 1. It is important to note that on balance, other manuscripts in that issue of Human Gene Therapy
concluded that in studies using several routes of local administration of low to intermediate doses of Ad
vectors were well-tolerated in humans.13,14
In summary, the toxicity associated with the administration of Ad vectors must be fully appreciated and
steps taken to reduce it. An additional complication underscoring the need for treatment to reduce toxic
effects, was the demonstration that the physical act of some methods of administration themselves,
even in the absence of vector, result in elevated levels of IL-6.15 A straightforward approach to reduce
toxic responses has been the pretreatment or coadministration of anti-inflammatory agents.
Pretreatment of animal models with steroids such as dexamethasone and budesonide was shown to
decrease transcription of chemokines and cytokines, thereby reducing the innate and downstreamacquired response including production of antibodies to Ad vectors. 16
Targeting of Ad vectors will improve the therapeutic index
There have been significant efforts in the last 2 years to retarget Ad vectors away from the primary
receptor (coxsackieadenovirus receptor  CAR) to a tissue or cell-specific receptor. The aims are (i) to
restrict transduction to the organ of interest, thereby gaining the greatest benefit with the lowest dose,
(ii) to minimize the innate response by limiting vector interaction with the RES and (iii) to potentially
avoid the effects of an anti-Ad neutralizing antibody response.17,18,19 Another rationale for targeting Ad
vectors is that CAR is present in low levels in some cell types that are potential targets for gene transfer.
The most straightforward method of detargeting of CAR binding is to pseudotype human serotypes with
capsids from other species20,21,22 or to simply employ Ad vectors from nonhuman serotypes.23 This
approach has the added benefit of avoiding the neutralization of input vector by preexisting antibodies to
human adenoviruses. Other targeting approaches are grouped into two categories; those that modify the
viral capsid through genetic alteration especially of fiber and fiber knob DNA and those that employ two
components where one binds to the Ad vector and is linked to another that will target the complex to a
specific receptor. Genetic retargeting of Ad vectors involves the ablation of the normal tropism by
mutating or deleting the CAR-binding sequences and incorporating foreign sequences into the loops
within the knob of the fiber shaft redirecting the vector to specific receptors on selected cell types. The
feasibility of this approach was demonstrated with the identification of a conserved receptor-binding
region on the side of three divergent CAR-binding knobs. However, the complexity of the fiber knob is
such that modifications may destabilize the fiber with failure to trimerize, rendering the vector
nonfunctional.24 A modified approach therefore incorporated the deletion of the entire knob domain of
the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization
function for the knobless fiber and specific binding to the target cell.25 These approaches have been
tested extensively and demonstrated efficient transduction (up to a three log increase) 26 of the desired
targets including endothelial27 and smooth muscle cells,22 brain microcapillary bed,28 synovial cells29 and
tumor cells.30 A Phase I clinical trial is underway in which an Ad vector genetically modified to target
integrins via the Arg-Gly-Asp (RGD) peptide motif is being tested in ovarian cancer and recurrent cancer
of the oral cavity.26
Ablation of native tropism requires the development of additional packaging cell lines and the size of the
modifications to the fiber knob is restricted to about 30 amino acids. Even with these limitations and
added complexities, the advantages of the single component system are that vector production and
qualification for clinical use are more straightforward than the challenges of characterizing the
heterogeneous mixture of vector, targeting ligand and vector-binding ligand of a two-component system.
Advantages of the two-component system are that many receptors can be targeted and there is no need
to genetically engineer the vector. Acommonly used example of the two-component method employs
bispecific antibodies,31 one to the fiber and the other to a cell receptor expressed for example on tumor
cells32 or cell surface antigen that is upregulated in angiogenic areas of tumors.33 This approach has been
used frequently to demonstrate proof of concept of a given targeting ligand.
In addition to the viral particle targeting methods described above, the use of tissue-'specific' promoters
can limit transcription to the tissue of interest.34 While this approach does not avoid vector interaction
with the RES, it has provided an unexpected benefit where in the case of muscle or liver-specific
expression, there was no detectable antibody response to the foreign transgene. 35 Transgene expression
in these instances was also prolonged.
These are but a fraction of the studies that have been published recently in the area of targeting of Ad
vectors and it is clear that this intense effort will advance the field. It will be extremely important to
evaluate the modified vectors comprehensively in vivo, as exemplified in the recent report from Smith et
al,36 who demonstrated that it is not sufficient to simply mutate the CAR-binding sites in fiber knob to
redirect transduction in vivo.36 These investigators mutated CAR binding in Ad5 fiber only to discover
that upon injection into mice there was increased transduction in the liver with the modified vector. This
observation may be related to CAR-independent transduction via heparin sulfate glycosoaminoglycans37
and may call into question the currently accepted cell entry pathway.
Advanced generations of Ad vectors provide increased persistence with reduced toxicity
Many applications of gene therapy will require lifelong transgene expression, but most current Ad vectors
result in an expression that is transient. Transgene expression is complex with at least three factors that
can impact persistence including the expression cassette itself, the immune response to the vector
andor the transgene product and turnover of the transduced cell. The importance of the expression
cassette was demonstrated by De Geest et al,38 who reported that gene transfer with an adenovirus
comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA and four apo E enhancers, all of
human origin, resulted in human apo A-I expression above 20 mgdl for up to 6 months in the absence of
significant hepatotoxicity.38 Another study concluded that 'strong promoters are the key to highly
efficient, noninflammatory and noncytotoxic adenoviral-mediated transgene delivery into the brain in
vivo'.39 These investigators demonstrated that by using a very strong promoter the required dose to
achieve sufficient transduction could be reduced 100-fold, thereby reducing the toxicity. However, an
often-ignored factor is what impact such a strong expression will have on normal cellular function, and
this will likely vary between cell types.
It is widely held that transgene expression will decrease to approximately background levels within 3
weeks following administration of first-generation vectors. However, there are examples of firstgeneration vectors that provide prolonged expression.40 In addition, partially deleted vectors have
resulted in persistent expression.41 It is therefore unclear how much of the viral genome must be deleted
to achieve the desired length of expression.42 There may, in fact, be a benefit in retaining some viral
genes as they function to modulate the cellular immune response. 43 However, studies characterizing the
function of the E4 gene products led the investigators to conclude that it is 'prudent' to remove all of the
E4 region.44 It is important to remember that with each deletion of a viral gene, a cell line must be
created to complement that gene45 and this can create production issues.
Helper-dependent, fully deleted Ad vectors offer great potential
With the recognition that viral gene expression even at low levels can lead to loss of transgene
expression, a significant effort is in progress to develop Ad vectors that do not contain any viral genes.
These are variously referred to as helper-dependent, high-capacity, gutted or gutless vectors, fully
deleted and pseudo-adenovirus (PAV). Although these vectors do not avoid the innate response, many
studies have demonstrated reduced toxicity with prolonged expression (for recent review see Kochanek
et al46). The helper-dependent (HD) Ad vectors are produced with a helper Ad that provides the
necessary viral functions, but cannot be packaged because the packaging signal is excised. This excision
is typically mediated by a bacterial phage P1 Cre recombinase that recognizes loxP sites that flank the
region essential for packaging. Another approach has been to employ a yeast recombinase (FLPe), where
the loxP sites are replaced with FLPe recombinant targets (FRT).47,48 Umana et al47 report greater
efficiency with the FLPe recombinase and claim that this should allow large-scale production of HD
vectors using column chromatography-based virus purification. A challenge of the HD vector system is
that the vector preparations are contaminated with low levels of helper Ad virus. Since the intent is to
move away from a vector with any viral genes, there are concerns of what impact the contaminating
virus will have on the toxicity and persistence of expression. One study attempted to address this by
adding back increasing levels of helper virus and did not detect a decrease in persistence with levels up
to 10%.49 A novel system for the production of HD vectors that does not require helper Ad, and is
therefore not contaminated with helper virus, has been described.50 The adenoviral genes are delivered
into producer cells by a baculovirusadenovirus hybrid, Bac-B4, carrying a Cre recombinase-excisable
copy of the packaging-deficient adenovirus genome. Although this resulted in preparations of vectors
that were free of contaminating Ad helper, scaling-up was prevented by the eventual emergence of
replication-competent adenovirus (RCA). Further optimization of HD vector production that provides
increased yields, minimal Ad helper, scalability and minimal levels of RCA has recently been described. 51
HD vectors have been used in a variety of applications with an almost uniform improvement in
persistence of transgene expression. Several of these were recently reviewed by Kochanek.46 For
example, lifetime correction of hyperlipidemia was demonstrated in a mouse model. 52 In this study,
expression was detected for 2.5 years. In a mouse model of hemophilia, the defect was corrected for
greater than 9 months with the expression of factor VIII.49 Along with the advantages of the increased
persistence of transgene expression, HD vectors have a greater capacity of up to 36 kb. Although HD
vectors have demonstrated advantages, they remain difficult to produce and purify in clinically relevant
quantities and it is clear that advances in this area are still required before products can be introduced
into the clinic.
Readministration with Ad vectors remains a challenge
Applications in which lifelong expression is necessary will require readministration of vector following the
eventual loss of therapeutic transgene expression. Without intervention or masking of the vector, the
neutralizing antibody response to a previous exposure to Ad either through natural infection or
administration of vector can preclude or significantly reduce effective readministration. However, several
studies have documented effective readministration in certain limited applications. For example, an initial
intramuscular administration of low-dose Ad vector producing low but detectable levels of transgene
expression did not preclude readministration into the muscle, where systemic readministration was not
effective.53 In a phase III trial for recurrent ovarian cancer where intraperitoneal readministration was
used, transgene expression was measurable in 17 of 20 samples obtained after two or three cycles. 54
Thus, it should be recognized that the ability to effectively repeat administer Ad vectors is dosedependent and site-specific.
Since effective intravascular readministration in the absence of some intervention is unlikely, various
approaches are being tested to circumvent the neutralizing response. A popular and straightforward
approach that has been tested involves a serotype switch such that the second administration would not
encounter a neutralizing response.55 There are, however, a finite number of serotypes and this approach
would represent a complex set of clinical products. An intriguing approach attempts to physically remove
the neutralizing antibodies. Plasmapheresis is a clinical procedure commonly used to reduce the antibody
levels in patients with some autoimmune conditions. A method similar to this clinical practice was used
to remove anti-Ad antibodies from serum by affinity chromatography and the resulting eluate was tested
both in vivo and in vitro for neutralization capacity.56 These authors reported that depletion of
antiadenoviral antibodies restores transduction in vivo during systemic Ad gene therapy in hosts
previously exposed to adenovirus. Although this approach shows promise, it remains to be determined to
what level the antibodies must be reduced and whether this is feasible in humans. Another approach has
involved the masking of vectors to both reduce the response to the initial Ad treatment and evade the
neutralizing response on subsequent exposures. Studies have employed a variety of molecules including
polyethylene glycol polymers,57 multivalent hydrophilic polymers that can incorporate targeting ligands in
addition to masking the vector58 and bilamellar cationic liposomes.59 These methods have potential, but
remain to be fully characterized in terms of toxicity.
Immune intervention or modulation has also been used to avoid or minimize the neutralizing immune
response. This approach seeks to minimize the response to the first Ad vector exposure, thereby
permitting subsequent administrations. A straightforward approach involves pretreatment or
coadministration of anti-inflammatory drugs such as steroids.16 A more complex method involves the
administration of monoclonal antibodies directed against costimulatory signalling molecules that are
required for the development of a fully mature immune response. One such approach employed
antibodies directed against CD40, CD80B7.1 and CD86B7.2.60 This treatment reportedly fully abrogated
the immune response against the Ad vector such that readministration was as effective as that in naïve
animals. In addition, these authors claimed that the animals were capable of mounting a normal
response to Ad. Another complex approach incorporates genes into the vector that upon expression will
attenuate the immune response. This method was tested using two genes that block costimulatory
signals that are required for an optimal immune response.61,62 These authors report that the
immunomodulatory genes CTLA4Ig and CD40Ig included in one HD vector and coadministered with an
Ad vector coding for a reporter gene resulted in higher levels of transgene expression, lower levels of
anti-Ad antibody and more prolonged transgene expression, when compared to controls. However,
prolonged expression of these immunomodulatory genes compromised the host immune response. Other
investigators have incorporated the immune modulatory gene CTLA4Ig into the vector containing the
transgene of interest and claimed that this vector was more effective in suppression of the immune
response than the two-vector system.63 They speculated that high levels of local expression may be
required to completely block both humoral and cellular responses.
Summary
Although the utility of Ad vectors for use in clinics has been seriously challenged in the last 2 years, they
remain viable, if not preferred, candidates for some gene therapy applications. With a better
understanding of the toxic response to Ad vectors, investigators have begun to utilize a variety of
methods to attenuate or avoid this potentially lethal response. Future Ad vectors incorporating features
outlined in this review would be fully deleted nonhuman Ads, modified to target away from the RES in
favor of the organ of interest and carry, in addition to the gene of interest under control of a tissuespecific promoter, controllable immunomodulatory gene(s) that would attenuate the immune response.
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adenoviruses blunt cell-mediated and humoral immune responses
against the virus and allow for significant gene expression upon
readministration in the lung. J Virol 2001; 75: 47924801. Article
PubMed
58 Fisher KD et al. Polymer-coated adenovirus permits efficient
retargeting and evades neutralising antibodies. Gene Therapy 2001;
8: 341348.
59 Yotnda P et al. Bilamellar cationic liposomes protect
adenovectors from preexisting humoral immune responses. Mol
Ther 2002; 5: 233241. Article PubMed
60 Ziller C, Stoeckel F, Boon L, Haegel-Kronenberger H.
Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition
to CD40CD40L interaction fully abrogates the immune response
following systemic injection of adenovirus vector. Gene Therapy
2002; 9: 537546.
61 Jiang Z, Feingold E, Kochanek S, Clemens PR. Systemic
delivery of a high-capacity adenoviral vector expressing mouse
CTLA4Ig improves skeletal muscle gene therapy. Mol Ther 2002; 6:
369376.
62 Jiang ZL et al. Local high-capacity adenovirus-mediated
mCTLA4Ig and mCD40Ig expression prolongs recombinant gene
expression in skeletal muscle. Mol Ther 2001; 3: 892900. Article
63 Thummala NR et al. A non-immunogenic adenoviral vector,
coexpressing CTLA4Ig and bilirubin-uridinediphosphoglucuronateglucuronosyltransferase permits long-term,
repeatable transgene expression in the Gunn rat model of
CriglerNajjar syndrome. Gene Therapy 2002; 9: 981990.
Tables
Table 1 Systemic Ad vector administration in primates
June 2003, Volume 10, Number 12, Pages 999-1003
Table of contents
Previous Article Next PDF
Review
Gene therapy Progress and Prospects: Gene therapy for the
hemophilias
Christopher E Walsh MD, Ph,D1
Mt Sinai School of Medicine, One Gustave Levy Pl, New York City,
NY, USA
1
Correspondence to: Dr CE Walsh, Mt Sinai School of Medicine, One
Gustave Levy Pl, Rm 24-42C Annenberg Bldg, New York City, NY
10029, USA
Abstract
Recent gene transfer trials for hemophilia A and B, bleeding disorders lacking either
functional factor VIII or IX, respectively, have produced tantalizing results, suggesting that
the potential to correct these bleeding disorders at a molecular level may be at hand. Genetic
correction of the hemophilias represents a model system to develop a basic understanding of
how gene therapy will be achieved. The goals for hemophilia gene transfer require the longterm therapeutic production of the coagulant protein without stimulating an immune response
to the transgene product or the vector. Based on a scientific understanding of the molecular
and cellular defects, leading to the bleeding phenotype, impressive strides have been made in
the last 2 years.
Gene Therapy (2003) 10, 9991003. doi:10.1038/sj.gt.3302024
Keywords
hemophilia, gene transfer, gene repair, stem cells, viral vectors
In brief
Progress




New AAV serotypes and lentiviral vectors have recently been
studied for the production of factor VIII or IX.
Organs other than the liver can be used as 'factories' for factor
VIII and IX production.
The immune response to endogenous factor synthesis and viral
vectors is a potential problem in hemophilia gene transfer.
Hemophilia clinical trials using either retrovirus, AAV or ex
vivo transfected fibroblasts have been carried out or are
ongoing.
Prospects




Development of improved lentiviral, gutless adenovirus and
alternate AAV serotype vectors is in progress
Gene repair, RNA repair (Trans-splicing) offer opportunities for
the treatment of hemophila.
Gene-modified circulating endothelial progenitor may prove
useful in the future.
Gene-modified stem cell therapy may become available.
Introduction
Current treatment for hemophila-related bleeding episodes utilizes intravenous infusion of purified and
recombinant factor protein, which is effective, yet transient because of the short half-life of the proteins.
This treatment is expensive, restricts the prophylactic use of factors and can lead to crippling joint
disease and susceptibility to infectious agents. Effective hemophilia gene transfer requires that a
sustained, long-term (years) production of coagulation factor at therapeutic levels be generated. Thus,
the method of gene delivery must be safe, and the risk of immune response to potential neoantigens
must be minimal. Given recent scientifictechnical developments, genetic correction of hemophilic
patients is now viewed as an achievable goal.
The factor VIII and IX gene and protein products have been extensively studied. 1,2 Many tissues and cell
types (skeletal muscle, liver, spleen and skin) are capable of producing and expressing fully modified and
functional factor IX protein. A therapeutic factor level is considered to be
2% of normal levels. This is
sufficient to convert a severely affected patient with frequent spontaneous bleeding episodes (patients
with <1% factor level) to a moderate or mildly affected level. Coagulation assays are standardized and
animal models (knockout mice and hemophilic canines) that mimic the human phenotype are available
for testing. A variety of gene transfer approaches are currently being tested both in the laboratory and in
the clinic. Results of two clinical trials, using nonviral and viral-based gene transfer approaches, suggest
that despite low factor levels, patients required less factor infusion and reported fewer bleeding episodes.
Although neither trial included a placebo arm, these results enforce clinical observations that low levels
of factor dramatically reduce spontaneous bleeding. Despite the current excitement, results point to the
need for improved vectors. Here, we will review the recent advances over the past 2 years in this field
that mirrors the advances in the field of gene transfer in general.
New adeno-associated virus (AAV) serotypes and lentiviral vectors have recently been studied
for the production of factor VIII or IX
In general, viral vectors exhibit long-term gene expression (years), whereas nonviral methods produce
transient (weeks to months) factor expression. Hemostatic levels of factors VIII and IX were reached
with first- and second-generation adenovirus vectors. Unfortunately, the exuberant cell-mediated
immune response engendered by this vector leads to inflammatory response directed at transduced cells
with the attendant loss of protein expression. Newer gutless adenovirus with a minimum of endogenous
adenoviral genes may limit the immune response, but as a consequence gene expression is drastically
reduced. AAV has recently come to the fore based on its now relative ease in preparation, ability to infect
both dividing and nondividing cells,3 and although it engenders a humoral immune response, it does not
stimulate a cytotoxic lymphocyte response. Using this vector, therapeutic levels of factors IX and VIII
were demonstrated in knockout mouse and hemophilic canines. Eight serotypes of AAV, have been
isolated and cloned (AAV1-8). Of these AAV, type 2 was the first cloned and most extensively studied.
Surprisingly, other serotypes yield factor IX at levels two logs greater than AAV-2 following skeletal
muscle injection into mice4 and produce sustained supratherapeutic factor levels leading to complete loss
of the bleeding diathesis.5 Such levels are achieved as a result of more efficient effective skeletal muscle
gene transfer. Here, a linear relation exists between input vector and factor expression. A unique side
benefit is lack of an immune inhibitor response presumably because of continuous production of factor as
the major determinant for inducing tolerance. This result is reminiscent of immune tolerance strategies
currently used in the clinic, performed by repeated infusion of factor.
In particular, AAV1 produces robust transgene expression in muscle, but the exact mechanism is unclear.
Data using AAV1EGFP suggested that the number of transduced muscle fibers infected increases
significantly with AAV1 compared to AAV2 (see Figure 1). A linear relation between circulating levels of
canine factor IX protein and AAV1 dose suggests that this result is most likely because of the number of
myocytes productively infected. One working hypothesis explaining the serotype transduction differences
lies in the level of virus specificity for binding cell receptors. At present, the receptor for AAV type 1 has
not been identified but appears distinct from type 2, that is, not inhibited by heparin.
AAV serotypes 7 and 8 recently isolated from primates infected with high-dose adenovirus 6 are not
neutralized by heterologous antisera raised to the other serotypes. Recombinant serotypes 7 and 8
vector particles carrying the alpha-1-antitrypsin cDNA were compared for transducing effectiveness in
mice. AAV7 was equivalent to AAV1 in efficient expression in skeletal muscle, whereas AAV8 expressed
at a 10 to 100-fold greater rate in liver-directed expression than all other serotypes. These data confirm
that relatively small differences in the capsid structure produce striking differences in transgene
expression in a wide variety of tissues.
Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. One study
used human immunodeficiency virus (HIV)-based lentiviral vectors containing human factors VIII or IX
cDNA expression for portal vein injection into C57Bl6 mice. Increasing doses of hFIX-expressing
lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately
5060 ngml. Partial hepatectomy resulted in a 4- to 6-fold increase in serum hFIX of up to 350 ngml
compared with the nonhepatectomized animals. The expression of plasma hFVIII reached 30 ngml (15%
of normal), but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII
antibodies.7
Owing to the potential safety issues using an HIV-based vector, an alternate approach is to utilize
plasmid-based approaches that carry genetic elements that promote integration. Early attempts using
such a system have provided encouraging results.8
Organs other than the liver can be used as sources for factor VIII and IX production
The liver is the principal organ synthesizing the coagulation factors. However, other organs can
synthesize factors VIII and factor IX. Factor IX can be expressed from skeletal muscle, fibroblasts,
kerintinocytes, intestinal mucosa, cells lining the amniotic cavity and marrow stroma. 9 Factor VIII
transgene expressing circulating endothelial cells are capable of secreting high levels of factor VIII for a
sustained period in animal models.10 Circulating endothelial cells obtained from peripheral blood are
expanded ex vivo and then genetically modified to express a gene of interest. The biochemical elements
necessary for high-level factor expression including the endogenous coexpression of factor VIII and vWF
may explain the high factor levels observed in vivo. The regulation of infused endothelial precursor cell
growth kinetics and half-life of fully differentiated endothelium remains to be determined. Ex vivo gene
transfer of hematopoietic progenitor cells and marrow stroma are also capable of factor VIII secretion in
vivo.11
The immune response to endogenous factor synthesis and viral vectors is a potential problem
in hemophilia gene transfer
The antibody responses to exogenous factor replacement, termed inhibitors, effect nearly 20% of factor
VIII patients and 3% of factor IX patients. Inhibitory antibodies that bind to the particular regions of the
factor molecule inactivate by changing factor protein conformation. 2 In general, the immune response
may in part be related to the type of mutation. For example, a large deletion in the factor VIII gene and
complete loss of protein typically leads to a greater incidence of inhibitor formation. Bleeding episodes of
patients with inhibitors are difficult to manage, relying on activated bypass factors and recombinant
factor VIIa.12 Will a constant source of factor engender high titer inhibitory antibodies negating any
positive benefit and with consequences of worsening bleeding? The clinical trials described below
carefully screen for noninhibitor patients or patients with frequent infusions where the chances of
inhibitor are reduced. CD4+ subset activation in humans and Th-1 and Th-2 lymphocytes in mice
suggests that both MHC class I and II mechanisms are involved. Involvement of both central (marrow,
thymus) and peripheral (lymph nodes, Peyers patch) tolerance mechanisms to factor VIII is described
but poorly understood.13,14,15 In addition to the immune response to the transgene factor proteins,
immune response to viral vectors is well described for adenovirus and AAV and thus prevent
readministration of vector.
Hemophilia clinical trials using either retrovirus, AAV or ex vivo transfected fibroblasts have
been carried out or are ongoing
Within the past 2 years, five gene transfer trials were approved (three for hemophilia A, two for
hemophilia B) in the US. Biotech firms that developed vectors specifically for factors VIII and IX sponsor
all five trials. In a phase I dose escalation study, 13 subjects with hemophilia A received by peripheral
intravenous infusion an amphotropic retroviral vector carrying a B-domain deleted human factor VIII
gene. Infusions were administered to patients with HIV and HCV infections and were well-tolerated.
Factor VIII was measured and no subject had sustained repeated FVIII levels >1% of normal levels.
Patients were treated with vector ranging from 3  107 to 9  108 vpkg. Overall, there was no significant
change in bleeding frequency. And there was no correlation between vector dose and time to FVIII
activity response. This clinical outcome is consistent with the limited capability of retroviral integration
into nondividing liver cells and the lack of a liver-specific promoter in the retroviral vector used.
A trial using an AAV2 vector carrying the human factor IX cDNA injected intramuscularly was carried out
in eight patients in a dose escalation study.16,17 One patient receiving the lowest dose (2  1011 vpkg)
was reported to maintain factor levels at 12% and reported a 50% reduction in factor usage and
bleeding episodes for a period up to 40 months postinjection. No evidence of inhibitor was reported
despite preclinical data in dogs of a transient inhibitory response. Virus dissemination was transiently
detectable in all body fluids, excluding semen. At higher doses, no significant plasma factor levels were
reported. However, reduced frequency of factor usage was one end point signifying treatment
effectiveness. Molecular analysis of virus dissemination was detectable in all body fluids (saliva, blood
and urine) but not in germ cells. All patients had low (1:1001000) preinjection anti-AAV2 neutralizing
titers that increased after vector administration. An increase in the number of injection sites from 10 to
90 produced no significant increase in factor level. High titer neutralizing antibodies to AAV developed in
all patients at levels sufficient to preclude readministration of vector. Muscle biopsy confirmed previous
observations in animals that slow twitch muscle fibers expressed factor IX.
A dose-escalation study based on AAV2 vectors carrying human factor IX cassettes delivered via the
hepatic artery has begun. Two patients received the lowest dose of virus (2  1011kg) via intra-arterial
delivery without adverse effects. However, the trial was temporarily halted because of detection of the
transgene in seminal fluid. Trial resumption was based on data that germ cells were not infected with the
virus. No detectable levels of factor FIX were observed above background in the two patients receiving
the lowest dose of vector. Two patients received moderate doses of vector (1  1012kg). One patient
developed FIX levels up to 1012% within 23 weeks after injection; however, factor levels subsequently
dropped coincident with an elevation of the liver transaminases. No data were presented on the second
patient. Whether this represents toxicity of the vector at this dose remains to be determined. It is
interesting to note that based on doseresponse experiments in hemophilic dogs, this moderate dose of
virus produced 414% of canine FIX for 12 years without significant toxicity.
A nonviral approach used a factor VIII plasmid electroporated into autologous skin fibroblasts: cells were
expanded in vitro and 100 or 400 million cells injected into the greater omentum. 18 Levels of factor VIII
above pretreatment levels were measured in four of six patients with either a concommitant reduction in
the use of recombinant factor VIII or decreased number of spontaneous bleeding episodes. However,
factor VIII decreased to pretreatment levels in all the patients after 12 months. The explanation for the
decline in factor expression may have been because of gene silencing, immunological clearance or
senescence of the fibroblasts after reimplantation.
What do these clinical results tell us? No significant toxicity in any of the patients was reported.
Furthermore, the factor levels predicted from animal models were not observed in patients. Thus,
although testing of new factor proteins in hemophilic animals is traditionally used because of similar
pharmacokinetic profiles seen in man, such extrapolation using gene transfer vectors may not be clearcut. A review of the preclinical data also suggests that animal studies may not be predictive of the
clinical outcome. For example, vector dosing based on a vector particle-to-weight ratio produced
discrepant results when comparing equivalent AAV vector dosing in mice and hemophilic dogs. Whereas
experiments in hemophilic mice are dose-dependent and can produce supraphysiologic levels of factor IX
(300% of normal), equivalent doses in hemophilic dogs produce factor IX at levels around 5% of normal
and do not appear to be dose-dependent.19 Recent data testing AAV2human factor IX vectors in nonhuman primates produced 410% factor IX,20 similar to data generated in hemophilic dogs,21 for a period
of 1 year. These outcomes reflect species differences in terms of the rate of cell infectivity, gene
expression, protein modification and processing. However, testing in different animal models serves to
confirm the validity of each new approach.
Future prospects
RNA repair (Trans-splicing) offers opportunities for the treatment of hemophila
A novel approach for genetic correction involves the use of premessenger RNA (pre-m-RNA) repair. RNA
trans-splicing utilizes endogenous splicing mechanisms to correct a portion of the defective RNA. A premRNA is designed to base pair with a pre-mRNA transcribed from the defective gene. The pre-mRNA also
contains all the requisite splicing signals that allows two independent mRNAs to splice together, resulting
in a correct copy of mRNA that is translated into a normal protein. 22 The advantage of this system is that
large genes, unable to be packaged into viral vectors, or genes that contain large regulatory elements
could be corrected by using the smaller spliced sequences. We developed such a system for factor VIII
correction . Using the factor VIII exon 16 knockout mice as a model to test trans-splicing, we
demonstrated that by injecting plasmid or AAV carrying pre-mRNA encoding for exons 1626 around
26% of factor VIII, that prevented bleeding challenge, was generated for 35 days and 34 months,
respectively (Chao et al, submitted). As trans-splicing efficiency improves, this may be useful for the
treatment of autosomal dominant disorders involving other coagulation and thrombotic defects.
Gene-modified circulating endothelial progenitor may prove useful in the future
The use of blood outgrowth endothelial cells (BOEC) as a source of cells synthesizing factor VIII has been
described.10 These circulating endothelial progenitor cells are isolated from peripheral blood, expanded in
culture, and modified genetically to carry the normal FVIII gene. A significant advantage includes the
expansion of BOEC clones that synthesize vWF, the carrier protein necessary for FVIII stability in plasma.
Major questions of BOEC use include the half-life of these cells in vivo, and potential for uncontrolled
growth following transplantation.
Gene-modified stem cell therapy may become available
Recent reports on the plasticity of stem cells derived from adult tissue forming liver, brain, muscle, skin,
fat have generated enormous interest on their use for the genetic correction of hemophilia. A
multipotential subset of mesenchymal stem (MAPC) cells derived from marrow stroma can be induced to
differentiate into cell types with neuroectoderm, endoderm and mesoderm characteristics. 23 When MAPCs
are injected into irradiated animals, they differentiate into hematopoietic lineages as well as epithelium
of the liver, gut and lung. Potentially MAPCs could be genetically modified to synthesize coagulation
factors before re-transplantation. Advantages include ex vivo expansion and gene modification with
selected clones producing high levels of factor. Autologous stem cells derived from each patient would
avoid transplantation rejection and immunosuppression. Current disadvantages include the long lead
time (months) required to generate the number of cells for transplantation and the ability to control the
differentiated fates of the transplanted multipotential cells.
Summary
A body of data suggests that genetic correction of the hemophilias is feasible. The subjective reporting
by patients of decreased bleeding episodes and an apparent self-declared reduction in bleeding episodes
at nominal levels of factor strongly hint that reasonable factor levels if reached will achieve a major
breakthrough in the treatment of hemophilia. Hemophilia gene transfer represents the combination of
vector delivery systems, animal models and clinical studies designed to answer specific questions. Not
only will these studies benefit hemophilic patients, but should also instruct others in the field as well.
Hopefully, this work will represent a milestone in the use of genetics for treatment of human ailments.
References
1 Kaufman R, Antonarakis S. Structure, biology, and genetics of
factor VIII, In: Hoffman R, Benz EJ, Shattil S, Furie B, Cohen H,
Silberstein L, McGlave P (eds). Hematology: Basic Principles and
Practice, Vol. VIII-108, 3rd edn. Church Livingstone: New York,
2000, pp 18501868.
2 Lillicrap D. Hemophilia treatment. Gene therapy, factor VIII
antibodies and immune tolerance: hopes and concerns.
Haematologica 85 (Suppl 10): 2000.
3 Monahan P, Samulski R. AAV vectors: is clinical success on the
horizon? Gene Therapy 2000;7:2430. Article PubMed
4 Chao H et al. Several log increase in therapeutic transgene
delivery by distinct adeno-associated viral serotype vectors. Mol
Ther 2000;2:619623. Article PubMed
5 Chao H et al. Sustained and complete phenotype correction of
hemophilia b mice following intramuscular injection of aav1
serotype vectors. Mol Ther 2001;4:217222. Article
6 Gao G et al. Novel adeno-associated viruses from rhesus
monkeys as vectors for human gene therapy. Proc Natl Acad Sci
USA 2002;99:1185411859. Article PubMed
7 Park F, Ohashi K, Kay M. Therapeutic levels of human factor
VIII and IX using HIV-1-based lentiviral vectors in mouse liver.
Blood 2000;96:11731176. PubMed
8 Yant S et al. Somatic integration and long-term transgene
expression in normal and haemophilic mice using a DNA transposon
system. Nat Genet 2000;25:3541. Article PubMed
9 Krebsbach P, Zhang K, Malik A, Kurachi K. Bone marrow
stromal cells as a genetic platformfor systemic delivery of
therapeutic proteins in vivo: human factor IX model. J Gene Med
2003;5:1117. Article
10 Lin Y et al. Use of blood outgrowth endothelial cells for gene
therapy for hemophilia A. Blood 2002;99:457462. Article
11 Chuah M et al. Long-term persistence of human bone marrow
stromal cells transduced with factor VIII-retroviral vectors and
transient production of therapeutic levels of human factor VIII in
nonmyeloablated immunodeficient mice. Hum Gene Ther
2000;11:729738. Article PubMed
12 Poon M. Use of recombinant factor VIIa in hereditary bleeding
disorders. Curr Opin Hematol 2001;8:312318. Article
13 Chao H, Walsh C. Induction of tolerance to human factor VIII in
mice. Blood 2001;97:33113312. Article
14 Qian J, Collins M, Sharpe A, Hoyer L. Prevention and
treatment of factor VIII inhibitors in murine hemophilia A. Blood
2000;95:13241329.
15 Brown B, Lillicrap D. Dangerous liaisons: the role of 'danger'
signals in the immune response to gene therapy. Blood
2002;100:11331140. Article
16 Kay M et al. Evidence for gene transfer and expression of factor
IX in haemophilia B patients treated with an AAV vector. Nat Genet
2000;24:257261. Article PubMed
17 Manno C et al. AAV-mediated factor IX gene transfer to skeletal
muscle in patients with severe hemophilia B. Blood, Prepublished
online Dec. 19, 2002.
18 Roth D et al. Nonviral transfer of the gene encoding coagulation
factor VIII in patients with severe hemophilia A. N Engl J Med
2001;344:17351742. Article PubMed
19 Wang L et al. Sustained expression of therapeutic level of factor
IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol
Ther 2000;1:154158. Article PubMed
20 Nathwani A et al. Sustained high-level expression of human
factor IX (hFIX) after liver-targeted delivery of recombinant adenoassociated virus encoding the hFIX gene in rhesus macaques. Blood
2002;100:16621669. Article
21 Mount J et al. Sustained phenotypic correction of hemophilia B
dogs with a factor IX null mutation by liver-directed gene therapy.
Blood 2002;99:26702676. Article
22 Puttaraju M et al. Messenger RNA repair and restoration of
protein function by spliceosome-mediated RNA trans-splicing. Mol
Ther 2001;4:105114. Article PubMed
23 Jiang Y et al. Pluripotency of mesenchymal stem cells derived
from adult marrow. Nature 2002;418:4149. Article PubMed
Figures
Figure 1 AAV serotype transduction of skeletal muscle. Fluorecence
of skeletal muscle samples following injection of equivalent doses of
serotype AAV1 and two vectors carrying an EGFP expression
cassette. A uniform fluorescence pattern is observed with AAV1
compared to the patchy appearance of AAV2.
April 2003, Volume 10, Number 8, Pages 605-611
Table of contents
Previous Article Next PDF
Review
Gene Therapy Progress and Prospects: Gene therapy in organ
transplantation
J Bagley1 and J Iacomini1
Transplantation Biology Research Center, Massachusetts General Hospital
and Harvard Medical School, MGH-East, Boston, MA 02129, USA
1
Correspondence to: Dr J Iacomini, Transplantation Biology Research Center,
Massachusetts General Hospital and Harvard Medical School, MGH-East,
Building 149, 13th Street, Boston, MA 02129, USA
Abstract
One major complication facing organ transplant recipients is the requirement for life-long systemic
immunosuppression to prevent rejection, which is associated with an increased incidence of malignancy
and susceptibility to opportunistic infections. Gene therapy has the potential to eliminate problems
associated with immunosuppression by allowing the production of immunomodulatory proteins in the
donor grafts resulting in local rather than systemic immunosuppression. Alternatively, gene therapy
approaches could eliminate the requirement for general immunosuppression by allowing the induction of
donor-specific tolerance. Gene therapy interventions may also be able to prevent graft damage owing to
nonimmune-mediated graft loss or injury and prevent chronic rejection. This review will focus on recent
progress in preventing transplant rejection by gene therapy.
Gene Therapy (2003) 10, 605611. doi:10.1038/sj.gt.3302020
Keywords
transplantation; gene transfer; tolerance
In brief
Progress









Gene therapy-mediated CD28B7 costimulatory blockade can prolong graft survival, but results in nonspecific
immunosuppression.
Gene therapy-mediated CD40CD154 costimulatory blockade can prevent acute allograft rejection, but does
not prevent chronic alloreactivity.
Hyporesponsiveness to virally encoded MHC genes has been induced in both rodent and large animal
models.
Induction of stable long-term T-cell tolerance to MHC class I antigens through central deletion of alloreactive
T cells following induction of molecular chimerism.
Evidence for induction of regulatory cells that can prevent allograft rejection in gene therapy models.
Induction of B-cell tolerance by molecular chimerism.
Induction of tolerance in sensitized recipients.
Gene therapy-mediated cytokine production, or immune deviation, prolongs graft survival, but does not
prevent rejection.
Transduction of dendritic cells with genes encoding immunomodulatory proteins can moderately prolong
graft survival.

Improved graft survival through antiapoptotic and antiproliferative gene therapy.
Prospects





Better understanding of activation of alloreactive cells will allow for the development of more targeted gene
therapy aimed at preventing this activation.
Increased understanding of costimulatory pathways will reveal additional targets for gene therapy
interventions.
Elucidation of the nature of regulatory cells may lead to gene therapy approaches designed to efficiently
generate these populations.
Induction of tolerance through molecular chimerism will be tested in large animal models.
Development of improved vectors for transduction of terminally differentiated tissue will improve efficacy of
gene therapy approaches that depend on intracellular protein expression.
Gene therapy-mediated CD28B7 costimulatory blockade can prolong graft survival, but results in
nonspecific immunosuppression
The major effectors of transplant rejection are host CD4 and CD8 T cells. In order for T cells to become activated and
participate in transplant rejection, they must first receive a signal through the T-cell receptor (TCR), which occurs after
either direct or indirect recognition of alloantigens on the surface of antigen-presenting cells (APCs). However, in order
for T cells to become fully activated and acquire effector function, such as the ability to produce cytokines, they must
also receive signals from the interaction of costimulatory molecules expressed on their surface with ligands expressed
on APCs. Costimulatory molecule interactions can result in T-cell activation or inhibition. For example, following TCR
ligation, the interaction of the costimulatory molecule CD28 expressed on T cells with CD80 and CD86 (B7.1 and B7.2,
respectively) expressed on APCs results in T-cell activation (reviewed in Sharpe and Freeman1). In contrast, signaling
through cytolytic T-lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells following ligation by CD80 and
CD86 downregulates immune responses.
T cell costimulatory pathways are central to T-cell activation, and therefore several groups have tried to block or
manipulate these pathways using gene therapy approaches in order to prevent T-cell responses. Blocking the
interaction of CD28 with CD80 and CD86 using an immunoglobulin fusion protein containing the extracellular portion of
CTLA-4 (CTLA-4Ig) can result in immunosuppression in vivo, and prevent transplant rejection. To overcome systemic
immunosuppression as a consequence of CTLA-4Ig administration, groups have tried to prevent transplant rejection by
expressing CTLA-4Ig locally within transplanted tissues or organs. Expression of the gene encoding CTLA-4Ig in donor
organs has generally resulted in the prolongation of transplant survival, but has not permitted permanent acceptance
(reviewed in Guillot et al2). Recently, indefinite graft survival was achieved in a cardiac transplantation model. 3
However, in this model, it is likely that the transplanted organs themselves may have participated in the maintenance
of hyporesponsiveness that was initially induced by transient expression of CTLA-4Ig. Indeed, cardiac allografts
themselves can be tolerogenic. In experimental systems involving transplantation of tissues expressing CTLA-4Ig which
are not thought to be tolerogenic, such as skin,4 hepatocytes,5 corneal grafts,6 and fetal cardiomyocytes,7 only modest
graft prolongation was achieved. In these studies, graft rejection was associated with a loss or decrease in gene
expression.
Analysis of rat cardiac allografts expressing adenovirus encoded CTLA-4Ig revealed that while permanent acceptance of
the genetically modified transplants could be achieved in allogeneic hosts, acceptance was associated with nonspecific
inhibition of T-cell responses to unrelated third-party antigens.3 Responses to third-party antigens were diminished
immediately after transplantation, and hyporesponsiveness was apparent even after a five-fold reduction in serum
levels of CTLA-4Ig was observed 120 days after transplantation. These data suggest that intragraft expression of CTLA4Ig can result in systemic immunosuppression, and therefore long-term expression of this gene product may be
detrimental to host immunity. In an attempt to overcome the long-term immunosuppressive effects of CTLA-4Ig
expression, adenovirus vectors carrying the gene encoding CTLA-4Ig flanked by two loxP sequences were developed.8
Following intravenous injection of adenoviruses carrying this novel CTLA-4Ig gene construct, subsequent administration
of adenoviruses carrying the gene encoding Cre recombinase permitted excision of the CTLA-4Ig gene, terminating
expression in vivo and vitro. Pancreatic islets transplanted into the liver of mice receiving loxP-flanked adenovirus
encoded CTLA-4Ig remained functional 40 days after serum CTLA-4Ig was no longer detectable following Cre-mediated
excision of the CTLA-4Ig gene.8 However, long-term islet function after cessation of CTLA-4Ig expression was not
examined, so it is not possible to determine if long-term survival occurred. Skin allograft survival was also prolonged,
however, all skin grafts were eventually rejected. In addition, Cre-mediated deletion of CTLA-4Ig was associated with
the restoration of responses to adenovirus, suggesting that long-term hyporesponsiveness was averted using this
approach, effectively allowing one to control the duration of immunosuppression.
Gene therapy-mediated CD40CD154 (CD40 ligand) costimulatory blockade can prevent acute allograft
rejection, but does not prevent chronic alloreactivity
In addition to the CD28-CD8086 costimulatory pathway discussed above, the interaction of CD40 ligand (CD154)
expressed on T cells with CD40 on APCs has also been shown to be an important component in the initiation and
maintenance of T-cell responses. Gene therapy approaches have been developed in which an adenovirus encoded
CD40-Ig fusion protein is used to block intragraft CD40-CD154 interactions in order to prevent graft rejection.
Adenoviral-mediated transfer of the gene encoding CD40-Ig into rat livers resulted in long-term survival following
transplantation into allogeneic recipients.9 Immunocompetence was tested by analyzing skin allograft survival 120 days
after liver transplanta-tion, long after CD40-Ig production had ceased. Therefore, it is unclear whether recipients were
able to respond to third-party antigens at earlier time points while CD40-Ig was expressed in the transplanted livers.
Expression of CD40-Ig in rat cardiac transplants has also been shown to delay acute rejection, although, at early time
points after transplantation, recipients of gene-modified hearts exhibited nonspecific immunosuppression. 10 At later
time points, nonspecific immuno-suppression abated and responses to third-party alloantigens were restored. However,
when immune responses to third-party antigens were restored, antidonor T-cell activity returned in these animals, and
was associated with chronic rejection.10 The development of chronic rejection suggests that intragraft expression of
CD40-Ig needs to be complemented by other therapeutic strategies to obtain long-term transplant survival.
Several other molecules involved in T-cell costimulation have recently been identified, but the function of many newly
discovered costimulatory pathways has not been fully elucidated.1,11 However, it is becoming apparent that other
costimulatory pathways, such as the inducible costimulator (ICOS)-B7 related protein-1 (B7RP-1) pathway, are
important for the activation of effector T cells.12 Indeed, it has been shown that an ICOS-Ig recombinant protein used in
conjunction with the CD40CD154 blockade may prevent chronic rejection of cardiac allografts.13 While no gene transfer
approach targeting ICOS-B7RP-1 interactions has been described to date, inhibiting this costimulatory pathway may
represent an interesting approach for preventing chronic graft rejection.
Hyporesponsiveness to virally encoded MHC genes has been induced in both rodent and large animal
models
It has been known for many years that a state of mixed hostdonor hematopoietic cellular chimerism, induced by
allogeneic bone marrow transplantation, leads to long-term stable donor-specific tolerance (reviewed in Sykes14).
Building on the concept of mixed chimerism, it has been suggested that a state of molecular chimerism involving the
transfer of genes encoding allogeneic donor-type MHC proteins, or other antigens, into autologous hematopoietic stem
cells may also result in tolerance.15 The induction of molecular chimerism through genetic modification of autologous
hematopoietic stem cells has the potential to induce donor-specific tolerance without the complications associated with
allogeneic bone marrow transplants, such as graft-versus-host disease. Expression of retrovirally transduced allogeneic
donor-type MHC genes in bone marrow-derived cells has been shown to be sufficient to induce donor-specific
hyporesponsiveness to the introduced gene product, allowing for prolonged survival of cardiac and skin allografts
without affecting rejection of third-party control grafts.15 Hyporesponsiveness to allogeneic renal transplants has also
been induced in pigs following induction of molecular chimerism, although it is not clear to what extent the transplanted
organ itself contributed to establishing allograft acceptance.16 The induction of hyporesponsiveness to marker genes
expressed in bone marrow-derived cells has also been achieved in rhesus macaques.17 Thus, the induction of molecular
chimerism has been shown to be capable of inducing hyporesponsiveness in multiple animal models.
Induction of stable long-term T-cell tolerance to MHC class I antigens through central deletion of
alloreactive T cells following induction of molecular chimerism
The ability to induce hyporesponsiveness through molecular chimerism has been well established; however, it has also
been shown that this hyporesponsiveness can be abrogated by providing T-cell help (reviewed in Bagley et al15). In
order for molecular chimerism to become clinically relevant, it is important to demonstrate that stable long-term T-cell
tolerance can be achieved using this approach. Recently, it has been shown that efficient expression of an allogeneic
MHC class I gene in bone marrow-derived cells is sufficient to allow for permanent survival of MHC class I disparate skin
grafts without affecting rejection of third-party grafts.18 Cytotoxic T cells capable of lysing donor-type targets remained
undetectable in vitro even after rigorous antigen challenge. These results are in contrast to previous studies in which Tcell hyporesponsiveness induced by genetic engineering of bone marrow could be broken by provision of T-cell help.
These results suggest that donor-specific tolerance can be established by inducing molecular chimerism.
The mechanism by which induction of molecular chimerism induces CD8 T-cell tolerance has been recently elucidated
using a T-cell receptor transgenic mouse model. In this system, H-2Kb-specific transgenic CD8 T cells were observed to
undergo negative selection in the thymus upon encountering bone marrow-derived cells expressing the transduced MHC
class I gene.19 Expression of the transduced antigen on T cells appears to participate in trafficking alloantigen to the
thymus to facilitate negative selection, as suggested by a study that demonstrated that in the absence of transduced
MHC class I expression on T and B cells, mice failed to become tolerant. 20 Since central deletion of alloreactive T cells is
the most stable form of tolerance, the proof that deletion occurs in molecular chimeras is encouraging. Collectively,
these data strongly suggest that gene therapy can be used to permanently reshape the T-cell repertoire.
Evidence for induction of regulatory cells that can prevent allograft rejection in gene therapy models
In recent years, a significant amount of evidence suggests that subpopulations of CD4 T cells can suppress allograft
rejection. These regulatory cells can be induced using a variety of approaches including administration of nondepleting
anti-CD4 antibodies and exposure to a tolerizing antigen in the form of donor-specific transfusions.21,22,23 Often
regulatory cells are able to induce tolerance to both the tolerizing antigen and third-party antigens, as long as they are
both expressed on the same graft, suggesting that inhibition occurs locally. 21,22 Recently, it has been shown that
treatment of immunocompetent mice with nondepleting anti-T-cell antibodies together with syngeneic bone marrow
infected with adenoviruses carrying an allogeneic MHC class I gene can lead to the acceptance of fully-allogeneic
cardiac transplants which share the same MHC class I antigen carried by the adenovirus construct. 24 This approach
essentially mimics results obtained using donor-specific transfusion, coupled with the use of nondepleting anti-T-cell
antibodies to induce tolerance. Although the mechanism by which acceptance was achieved is unknown, transplant
acceptance was observed even though gene expression was very short lived in vivo. These experiments suggest that
the use of gene therapy-modified bone marrow may be capable of inducing regulatory T cells that can inhibit the
responses to the transduced gene product as well as additional transplantation antigens when combined with other
therapies. This is a potentially important extension of the use of molecular chimerism. However, it is not clear whether
similar approaches would work in models in which the organ itself does not participate in the maintenance of tolerance,
or prevent chronic rejection.
Induction of B-cell tolerance by molecular chimerism
In addition to inducing T-cell tolerance, molecular chimerism has been shown to be capable of inducing B-cell tolerance.
Natural antibodies specific for the carbohydrate epitope Gal 1-3Gal
1-4GlcNac-R ( Gal) are the main mediators of
hyperacute xenograft rejection in pig to primate xenotransplantation. Mutant mice, which lack
(1,3)galactosyltransferase ( GT), the enzyme that synthesizes the
antibodies, as do humans. Expression of porcine
shown to prevent the production
Gal epitope, produce
GT in bone marrow-derived cells of
Gal-specific natural
GT knockout mice has been
Gal-reactive natural antibodies, resulting in stable long-term tolerance to
Gal even
after rigorous antigen challenge.15,25,26 Importantly, tolerance remained intact even after mice received cardiac
transplants from wild-type mice that expressed
Gal, and
Gal antibody-mediated rejection was prevented in
molecular chimeras.27 Analysis of B cells from mice reconstituted with
GT-transduced bone marrow revealed that B
cells which produce
Gal-specific antibodies were eliminated from the immunological repertoire following gene therapy.
Collectively, these data suggest that gene therapy may be used to induce both B- and T-cell tolerance through the
establishment of molecular chimerism.
Induction of tolerance in sensitized recipients
The extent to which pre-existing host immune responses can affect the induction of molecular chimerism and
establishment of donor-specific tolerance is an important issue. Many patients have been presensitized to organ
allografts by blood transfusions or previous transplants, and all humans have natural antibodies to
Gal epitopes on
xenogeneic organs. To determine the extent to which a preformed immune response was a barrier to the induction of
molecular chimerism,
GT knockout mice were immunized with
Gal-expressing pig cells. Since
Gal is a T-dependent
antigen28 this immunization resulted in T-cell priming and increased titers of anti-Gal antibodies. Despite high titers of
serum
Gal-specific antibodies in immunized hosts, molecular chimerism could be established by increasing the dose of
transduced bone marrow used for reconstitution of lethally irradiated recipient. 29 Once molecular chimerism was
established in sensitized hosts, tolerance to
Gal resulted and production of
Gal-specific antibodies ceased. These
data demonstrated that the induction of molecular chimerism could be used to reshape the pre-existing B-cell
repertoire in appropriately conditioned sensitized hosts.
Gene therapy-mediated cytokine production, or immune deviation, prolongs graft survival, but does not
prevent rejection
Acute rejection events in immunosuppressed patients correlate with intragraft production of T helper type 1 (Th1)
cytokines, such as IFN-
, while the lack of acute rejection is associated with production of T helper type 2 cytokines
(Th2), such as IL-10. It has therefore been hypothesized that production of Th2 cytokines might act to downregulate
the immune response to organ allografts. Thus, the concept of immune deviation was proposed as a way to prevent
organ allograft rejection by fostering a Th2-type response, rather than Th1. Since systemic administration of cytokines
often results in unacceptable side effects, gene therapy approaches were developed to test the hypothesis that
modifying the local graft environment to promote Th2 rather than Th1 responses would result in prolonged transplant
survival. The ability of several Th2-type cytokines to prolong allograft graft survival when expressed locally has been
assessed both with and without additional immunosuppression. In general, prolongation of graft survival has been
observed using this approach. However, expression of cytokine genes within donor tissues did not lead to permanent
graft acceptance. More recently, it has been shown that localized liposome-mediated IL-10 gene transfer into rabbit
cardiac transplants could induce alloreactive T-cell apoptosis and prolong cardiac allograft survival.30 In addition,
transfer of the gene encoding viral IL-10 delayed graft rejection in a rat model.31 However, in both these studies,
prolongation was extremely modest, and it is therefore unclear what kind of clinical significance can be attached to such
results in model systems that are relatively sensitive to tolerance induction.
Transduction of dendritic cells with genes encoding immunomodulatory proteins can moderately prolong
graft survival
An increased understanding of the role of dendritic cells in T-cell activation has led to an interest in modification of
dendritic cells to induce tolerance. While mature dendritic cells express high levels of costimulatory molecules and
induce strong T-cell responses, there is some evidence that dendritic cells expressing low levels of costimulatory
molecules can induce anergy in T-cells.32 Attempts have been made to modify graft rejection by genetically modifying
dendritic cells to express cytokines associated with delayed graft rejection. Retroviral delivery of TGFinto myeloid
dendritic cells has been shown to decrease their ability to stimulate alloreactive cells, resulting in moderately prolonged
graft survival of cardiac allografts in mice.33 Human myeloid dendritic cells transduced with the gene encoding IL-10
prolonged human skin allograft survival in a humanized NOD-scid chimeric model.34 However, other studies have
indicated that adenovirus transduction of the IL-10 gene into murine myeloid dendritic cells can enhance alloreactive
responses to cardiac grafts.35 Overall, the results obtained with cytokine-gene-transduced dendritic cells were similar to
those obtained with transduced organs. Both resulted in only a modest increase in transplant survival. Similar results
have been observed in models where dendritic cells were transduced with genes encoding CTLA-4Ig.36
Dendritic cells have also been genetically engineered to express Fas ligand (CD95L). 37,38,39 Engagement of Fas (CD95)
on the surface of T cells by CD95L leads to the induction of apoptosis in activated T cells. Apoptosis induced by
CD95CD95L interactions is thought to play a role in the establishment of immunoprivileged sites such as the eye and
testis, and may be involved in the killing of CD4 T cells. Dendritic cells genetically engineered to express CD95L on their
surface are able to inhibit alloreactive T-cell proliferation in vitro, and cause a slight prolongation of cardiac graft
survival when administered in vivo.38 However, as discussed above, since mouse cardiac transplants are relatively
sensitive to tolerance induction, it is unclear how significant these results will be in more rigorous transplantation
models.
Improved graft survival through antiapoptotic and antiproliferative gene therapy
In addition to host immunity, other nonimmunological factors play a role in transplant survival. Therefore, approaches
have been developed to protect donor organ or tissue grafts at the time of transplantation from damage owing to
nonimmune-mediated inflammation and ischemiareperfusion injury. The use of gene transfer to prevent damage that
occurs at the time of transplant has the advantage that it does not require long-term gene expression, making these
therapies potentially clinically relevant.
A significant proportion of cellular transplants such as islets or hepatocytes are lost due to anoxiaischemia reperfusion
injuries at the time of transplantation that trigger the generation of reactive oxygen species leading to cellular death
and localized inflammation. Endogenous scavenger systems can eliminate toxic radicals. However, the components of
these systems are usually degraded rapidly when given exogenously. The expression of scavenging factors locally
through genetic engineering of transplants would therefore be one way to protect organs and tissues. In a rat liver
transplantation model, introduction of the gene encoding copperzinc superoxide dismutase through adenoviral
transduction allowed for the survival of 100% of recipients, whereas only 25% of mock-treated controls survived.
Expression of copperzinc superoxide dismutase in transplanted livers also significantly reduced necrosis within the
transplant.40,41 These data suggest that expression of copperzinc superoxide dismutase decreases injury resulting from
the generation of reactive oxygen species during transplantation. Similarly, gene therapy approaches using other
cytoprotective genes such as hemeoxygenase-142,43 and catalase44 have also been shown to protect organs against the
effects of ischemiareperfusion. Expression of hemeoxygenase-1 in rat liver transplants following adenovirus-mediated
gene transfer resulted in an increased survival of recipients from 50% in controls to 80% in those receiving
hemeoxygenase-1-transduced livers. Gene transfer in these studies was performed from 4 to 24 h in advance of
transplant, which may be compatible with clinical application of these techniques.
The overexpression of antiapoptotic genes in transplanted tissue may also protect the graft from nonimmune as well as
immune-mediated injury. Bcl-2, an antiapoptotic cell survival factor, blocks the release of cytochrome 3 from the
mitochondria and subsequent activation of caspases, proteases involved in cell death. The overexpression of bcl-2 in
macaque pancreatic islets was able to enhance insulin production after transplant into diabetic SCID mice 45 and protect
porcine islets after exposure to rhesus monkey serum.46 In addition, the expression of bcl-2 protected human
endothelial cells from CTL activity.47 However, since the function of bcl-2 is intracellular, the effectiveness of this
approach is dependent on a high transduction efficiency that will be more difficult to achieve in the terminally
differentiated cells of vascularized organs.
Prospects
In the coming years, in order for gene therapy to be used to prevent transplant rejection clinically, it will be crucial to
gain a better understanding of how alloreactive T cells are activated and regulated, so that rational gene therapy
interventions can be designed to prevent transplant rejection (Figure 1). Blockade of yet to be defined costimulatory
pathways needs to be evaluated in order to determine if targeting these pathways using gene therapy approaches will
exhibit utility for clinical transplants. In addition, a greater understanding of the mechanisms by which regulatory T
cells are induced needs to be achieved in order to design strategies to exploit these cells to achieve transplant
acceptance. Tolerance induction remains a major goal in the field, and if it can be achieved clinically may represent the
most attractive approach, completely overcoming the need for immunosuppression. Results obtained in molecular
chimeras have demonstrated that gene therapy approaches can be used to induce tolerance. Coupling the induction of
molecular chimerism to, for instance, induction of regulatory cells may allow for survival of organs mismatched at
major and minor histocom-patibility loci, extending the clinical utility of this approach. However, the ability of molecular
chimerism to induce tolerance must also be assessed in preclinical non-human primate models to determine if similar
approaches could eventually be used in humans. In addition, methods must be developed to allow the induction of
molecular chimerism using clinically acceptable host conditioning. Lastly, as has been true for many years, the
development of more efficient vectors for gene transfer continues to be of great importance, allowing for more efficient
transduction and improved gene expression.
Acknowledgements
We thank Drs David KC Cooper and Yong Guang Yang for critical review of
the manuscript, and members of the Iacomini Laboratory for helpful
suggestions and comments. This work was upported by NIH grants RO1
AI43619 and RO1 AI44268 to JI. JB is supported by NIH Training grant T32
AI07529, and in part by a grant from the Children's A-T Project.
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18 Bagley J et al. Induction of T-cell tolerance to an MHC class I alloantigen
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19 Kang ES, Iacomini J. Induction of central deletional T cell tolerance by
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20 Tian C, Bagley J, Iacomini J. Expression of antigen on mature
lymphocytes is required to induce T cell tolerance by gene therapy. J
Immunol 2002; 169: 3776.
21 Zelenika D et al. The role of CD4+ T-cellsubsets in determining
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22 Waldmann H, Cobbold S. Regulating the immune response to
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23 Graca L et al. Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells
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tumour rejection. Immunology 2000; 101: 233241.
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"killer" hybrids created by fusing donor- and recipient-derived dendritic cells.
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39 Min W et al. Fas ligand-transfected dendritic cells induce apoptosis of
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transplantation in the rat. Transplantation 2000; 69: 10511057. PubMed
41 Lehmann TG et al. Gene delivery of CuZn-superoxide dismutase
improves graft function after transplantation of fatty livers in the rat.
Hepatology 2000; 32: 12551264.
42 Kato H et al. Heme oxygenase-1 overexpression protects rat livers from
ischemiareperfusion injury with extended cold preservation. Am J Transplant
2001; 1: 121128.
43 Coito AJ et al. Heme oxygenase-1 gene transfer inhibits inducible nitric
oxide synthase expression and protects genetically fat Zucker rat livers from
ischemiareperfusion injury. Transplantation 2002; 74: 96102.
44 Zhu HL et al. Blocking free radical production via adenoviral gene
transfer decreases cardiac ischemiareperfusion injury. Mol Ther 2000; 2:
470475.
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after transplantation using gene therapy. Kidney Int 2002; 61(Suppl 1):
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46 Contreras JL et al. Gene transfer of the Bcl-2 gene confers
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Figures
Figure 1 Preventing transplant rejection by gene therapy. Gene therapybased strategies can be used to prevent activation of alloreactive host T cells
in peripheral lymphoid tissue, such as the lymph node (shown), by blocking
costimulatory molecule interactions between T cells and APCs (a). Such
interventions leave T cells in a non-responsive state, and can also result in
anergy. Reconstitution of conditioned hosts with autologous hematopoietic
stem cells engineered to express donor type transplantation antigens using
retroviruses allows for expression of donor type antigen on bone marrowderived cells (b). Expression of retrovirally transduced antigen on bone
marrow-derived cells within the recipient thymus mediates negative selection
of newly formed alloreactive T cells (c). It is unclear whether expression in
the thymus can also lead to the generation of regulatory T cells capable of
controlling allograft rejection. Direct modification of the donor graft itself (d)
using a wide variety of gene delivery systems prior to transplantation offers
the possibility of reducing graft damage owing to ischemiareperfusion injury.
This approach has also been proposed as a method to allow the destruction of
alloreactive T cells that reach the graft.
Received 24 September 2002; accepted 30 January 2003
April 2003, Volume 10, Number 8, Pages 605-611
March 2003, Volume 10, Number 6, Pages 453-458
Table of contents
Previous Article Next PDF
Review
Progress and prospects: naked DNA gene transfer and
therapy
H Herweijer1 and J A Wolff1
Mirus Corporation and University of Wisconsin-Madison Madison,
WI, USA
1
Correspondence to: Dr J Wolff, University of Wisconsin-Madison,
Department of Pediatrics, Waisman Center, 1500 Highland Avenue,
Madison, WI 53705, USA
Abstract
Increases in efficiency have made naked DNA gene transfer a viable method for gene therapy.
Intravascular delivery results in effective gene delivery to liver and muscle, and provides in
vivo transfection methods for basic and applied gene therapy and antisense strategies with
oligonucleotides and small interfering RNA (siRNA). Delivery via the tail vein in rodents
provides an especially simple and effective means for in vivo gene transfer. Electroporation
methods significantly enhance direct injection of naked DNA for genetic immunization. The
availability of plasmid DNA expression vectors that enable sustained high level expression,
allows for the development of gene therapies based on the delivery of naked plasmid DNA.
Gene Therapy (2003) 10, 453458. doi:10.1038/sj.gt.3301983
Keywords
gene transfer; naked DNA; nonviral; intravascular; hydrodynamic
In brief
Progress
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Injection of naked DNA in muscle results in transfection of cells in vivo.
Electroporation enhances uptake of injected plasmid DNA into muscle and skin.
Intravascular delivery of plasmid DNA results in a very effective gene transfer of hepatocytes.
Tail vein pDNA delivery is a simple and effective method for transfecting liver cells in mice and
rats.
Effective transfection of skeletal muscle cells in mice, rats, dogs, and monkeys via
intravascular delivery of plasmid DNA.
Novel pDNA expression vectors allow for high-level sustained expression.
The mechanism of intravascular naked DNA delivery is thought to involve active cellular pDNA
uptake.
Naked DNA delivery has entered clinical studies for peripheral arterial occlusion disease
(PAOD).
Small interfering RNA (siRNA) can be delivered very efficiently using intravascular gene
transfer methods and results in potent knock out of the target gene expression.
Prospects
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Nonviral gene transfer will become more important as better delivery methods become
available.
Clinical trials for genetic diseases (eg, Duchenne muscular dystrophy, ischemia, hemophilia)
will be initiated in the next few years.
Tail vein injections in rodents will become a widely used technique for rapidly testing
expression vectors and gene therapy approaches.
RNA interference will become a major element in the gene therapy field.
Injection of naked DNA in muscle results in transfection of cells in vivo
Gene therapy would be a lot simpler if we could forget about recombinant viruses or complexing DNA
with polymers or lipids. It has been known for years that naked DNA can be delivered to cells in vivo and
result in gene expression. However, the efficiency of gene transfer into skeletal or cardiac muscle is
relatively low and variable. Attempts to increase pDNA uptake, for instance by inducing muscle
regeneration, have not increased efficiency to a level that would allow for clinical use in gene therapy
protocols. As a result, naked DNA gene transfer has been mainly used for genetic immunization studies.
In recent years, work in several laboratories has shown that naked plasmid DNA (pDNA) can be delivered
efficiently to cells in vivo either via electroporation, or by intravascular delivery, and has great prospects
for basic research and gene therapy.
Electroporation enhances uptake of injected plasmid DNA into muscle and skin
The last few years has seen a marked increase in the number of studies employing intramuscular or
intradermal injection of naked DNA followed by electroporation. Technical improvements in
electroporation equipment made in recent years, as well as better methodology following years of
experimentation, have enabled increases both in gene transfer efficiency and safety. 1 A very large
number of publications in the last few years have demonstrated gene transfer to a variety of different
cell types in vivo. Expression levels in muscle are at least 10-fold higher compared to injection of pDNA
without electroporation, but are accompanied by elevation of serum creatine kinase levels. 2 It is not clear
whether these increases in transgene expression (especially of secreted proteins) are due to enhanced
gene transfer into myofibers, or to simultaneous transfer into different cell types (eg, endothelial cells).
Expression levels are considered sufficient to warrant further investigation of this method for gene
therapy, for instance for chronic anemia3 or muscular dystrophies.4 Yet, expression levels are not as high
as those achieved following intravascular delivery,5 (see below). Injection of naked DNA in conjunction
with electroporation into skeletal muscle or skin should enhance the efficacy of genetic immunization
procedures.6
Intravascular delivery of plasmid DNA results in very effective gene transfer of hepatocytes
Intravascular delivery of genes is attractive because it avoids the necessity for multiple intraparenchymal
injections into the target tissue. The gene is disseminated throughout the tissue since the vascular
system accesses every cell. Vascular delivery could be systemic or regional in which injections are into
specific vessels that supply a target tissue.7 The intravascular delivery of adenoviruses or cationic
lipidDNA complexes in adult animals mostly results in expression in vascular-accessible cells such as
endothelial cells or hepatocytes reached via the sinusoid fenestrae. Efficient transgene expression in
hepatocytes throughout the liver can be obtained following delivery of naked pDNA via the portal vein,
the hepatic vein, or the bile duct in mice and rats. The use of hyperosmotic injection solutions and
occlusion of the blood outflow from the liver substantially increased the expression levels, although it
was later shown that a hyperosmotic solution is not absolutely necessary.
Tail vein pDNA delivery is a simple and effective method for transfecting liver cells in mice and
rats
A major advance in the intravascular delivery of pDNA was the recent development of the tail vein
delivery procedure in the Wolff and Liu laboratories. As a logical extension to rapid delivery of a relatively
large volume into a vessel leading into or from the liver, the tail vein itself can be employed. The tail vein
drains into the vena cava. Delivery of a large bolus presumably results in a liquid volume in the vena
cava that is too large for the heart to handle rapidly. The fluids back up and end up (predominantly) in
the liver, resulting in gene transfer. This explains the rather delicate sensitivity of this method of gene
delivery upon delivery volume and speed. Several groups have found that the optimal volume is around
10% of the body weight of a mouse or rat.7,8 The delivery time should be between 5 and 7 s in mouse;
1520 s in rat, these times being the fastest rates that can be practically achieved by skilled operators.
Tail vein or hydrodynamic delivery results in very high levels of gene transfer. Typically, 1015% of the
hepatocytes are transfected in mouse liver following injection of 10 μg pDNA, but levels up to 40% have
been reported. We have measured high levels of reporter genes (about 2 μg luciferase, 50 μg secreted
alkaline phosphatase) and supranormal levels of therapeutic genes (55 μgml human factor IX, 1.6 μgml
human factor VIII, 915 ngml rhesus erythropoietin) one day after gene delivery. Transgene expression
is also found in heart, spleen, and kidneys, at levels about 100-fold lower than in liver. While the
procedure seem harsh, nearly all animals survive (99%) and show no ill effects. Liver enzymes are
transiently elevated. For instance, we observed serum alanine aminotransaminase (ALT) levels around
1000 Uml 24 h after injection in mice. The ALT levels diminished to normal over the next few days. Liver
histology shows minimal damage that resolves within a week, which is similar to the data we observed
for intravascular delivery into liver vessels.9
The tail vein method has been adopted remarkably quickly in the gene therapy field for basic research
and gene therapy evaluation. At the recent meeting of the American Society of Gene Therapy (June 59,
2002), 22 abstracts were presented employing this novel technique. Owing to its simplicity and
reproducibility, it allows for the rapid testing of novel expression vectors (whether or not the expression
cassette will eventually be used in a viral or a nonviral vector). We, and others, have used tail vein
injections to evaluate the expression cassettes capable of driving sustained high level expression in the
liver.9,10,11,12,13,14 As it is easy to regulate the level of gene expression by adjusting the amount of plasmid
DNA, it is now possible to test accurately the level of transgene expression required for achieving a
physiological effect in a disease model (eg, the level of phenylalanine hydroxylase (PAH) expression in
PAH-deficient phenylketonurea (PKU) mice; Cary Harding, personal communication).
As the liver is the organ that is predominantly transfected, testing gene therapy approaches may be
limited to those diseases where liver transgene expression is appropriate. However, the liver may be
used as an ectopic expression site for secreted proteins (eg, erythropoietin), or in a situation where
hepatocytes can take over the function of other cells (eg, as in clearing of metabolites). An often asked
question is how this technique can be translated to the human situation. Since the human tail is on the
short side, other entry points need to be found. We have extensively investigated intravascular delivery
to the liver in large animals (unpublished data). It appears that similar efficiencies can be obtained in
monkeys as in mice by injecting pDNA into the afferent or efferent vessel of the liver. Such injections can
be done via catheters in humans, making this a relatively simple procedure. Eastman et al15 recently
presented data in rabbits, reporting high expression levels following delivery of pDNA via balloon
occlusion catheters introduced into the portal vein. Expression levels were somewhat lower than in mice
following tail vein injections; toxicity measurements showed a similar transient rise in liver enzymes.
These studies demonstrate the feasibility of intravascular delivery to the liver using catheters, and are a
step in the direction of human clinical trials. Intravascular delivery to other organs is also being explored
(eg, Zhang et al,7 and Maruyama et al16).
Effective transfection of skeletal muscle cells in mice, rats, dogs, and monkeys via
intravascular delivery of plasmid DNA
The intravascular delivery of naked pDNA to muscle cells is also attractive particularly since many muscle
groups would have to be targeted for intrinsic muscle disorders such as Duchenne muscular dystrophy.
An intravascular approach would avoid the limited distribution of pDNA through the interstitial space
following intramuscular injection. Muscle has a high density of capillaries that are in close contact with
the myofibers. Delivery of pDNA to muscle via capillaries puts the pDNA into direct contact with every
myofiber and substantially decreases the interstitial space the pDNA has to traverse in order to access a
myofiber. However, the endothelium in muscle capillaries is of the continuous, nonfenestrated type and
has low solute permeability, especially to large macromolecules. Nonetheless, rapid delivery of relatively
large volumes of pDNA solutions (10 ml injected into rat iliac artery) resulted in very efficient gene
transfer into myofibers. With the best injection condition, up to 50% of myofibers expressed βgalactosidase in many areas of the muscles. Experiments have successfully been performed in mice,
although these are technically difficult to do. Expression levels and percentage of transfected cells do
vary significantly from mouse to mouse, yet up to 20% of transfected myofibers have been observed
regularly.
Studies in larger animals have now demonstrated the clinical relevance of this method of gene delivery.
Intravascular delivery to limb skeletal muscle has successfully been performed in rabbits, dogs, and
rhesus monkeys (Zhang et al17; unpublished results). Several alternative methods for delivering the
pDNA solution and blocking limb blood flow have been evaluated. Delivery via a catheter to regional
target muscle groups, in combination with blocking blood flow with a tourniquet or blood pressure cuff, is
very effective in larger animals. In rhesus monkeys, transfection efficiencies of 40% have been
observed,17 a number that is considered sufficient for treatment of muscle defects such as Duchenne
muscular dystrophy. A transient increase in serum creatine kinase levels was measured, which resolved
within a few days. The short time required for occluding blood flow to skeletal muscle should be well
tolerated in a human clinical setting since ischemia can be tolerated by muscles for 23 h. In fact, a
common anesthetic procedure for distal limb surgery (eg, carpal tunnel repair) involves the placement of
a tourniquet to block both venous and arterial blood flow and the intravenous administration of a local
anesthetic (eg, lidocaine) distal to the tourniquet. Surgery in humans can be performed for a couple of
hours using these anesthetic procedure. Similarly, histologic analyses of the rat, dog, and rhesus
muscles in our experiments indicated that the ischemia did not cause myofiber damage. Besides gene
therapy for muscle defects, it appears worthwhile to evaluate this method for delivery of angiogenic
genes for the treatment of ischemia. Delivery is not limited to skeletal muscle, but can also target
cardiac muscle (retrograde delivery via standard angioplasty catheters) and can thus potentially be used
for treating heart ischemia.18
Novel pDNA expression vectors allow for high level sustained expression
One of the problem areas in gene therapy is the sustained expression of transgenes at high levels. We
have recently analyzed five explanations for the rapid loss of expression observed after intravascular
pDNA gene delivery to the liver.9 While these analyses were performed following portal vein delivery, the
conclusions appear valid for other intravascular delivery routes to the liver. First, the injection procedure
or the presence of intracellular pDNA may induce cell death causing loss of the vector. As described
above, it is clear that the injection procedure (with pDNA or with vehicle alone) does induce damage.
But, all pathological reactions observed were transient and liver morphology was restored at day 4 after
manipulation.
Second, intravascular delivery of pDNA may result in increased cell cycling causing the loss of the
nonintegrated pDNA. While an increase in the number of cycling cells was found (BrdU labeling studies),
peaking at 2 days at 11.5%, cycling appears to be insufficient to fully account for the 5000-fold drop in
expression observed for CMV promoter-driven luciferase expression (day 1 to day 7). Third, pDNA may
be lost independent of cell replication. Southern blotting experiments allowed us to determine the time
course of pDNA loss. pDNA is rapidly lost in the first 24 h, likely reflecting the loss of extracellular pDNA
or pDNA associated with Kupffer cells.19 The decline in pDNA levels from day 1 to day 7 was 1040 fold,
much less than the loss of reporter gene expression in the same period (5000 fold).
Fourth, the promoter driving reporter gene expression may be inactivated, thus resulting in the loss of
reporter gene expression. Comparison of viral- and tissue-specific promoters revealed that (1) viral
promoters generally express at very high levels 1 day after injection; (2) viral promoter-driven reporter
gene expression falls precipitously after day 1; (3) the liver-specific albumin promoter expresses at much
lower levels on day 1, but expresses at a similar level on day 7. We therefore hypothesized that the
enormous drop in expression measured for the viral promoters is the result of promoter inactivation.
Fifth, the expressed proteins may induce an antigen-specific immune response, resulting in a further loss
of expression after 23 weeks.
Our observations indicate that loss of transgene expression following pDNA gene transfer to the liver
occurs in two phases. There is a rapid loss of expression in the first few days, followed by a slower
decrease after about 1 week. The loss of expression in this latter phase appears to be the result of an
antigen-specific immune response in normal, immunocompetent mice, as expression is much prolonged
in immunocompromised mice. The early phase of expression loss appears multifactorial. The delivery
procedure does result in liver damage, as evidenced by histological observation and an increase in
hepatocyte cell cycling. It is therefore likely that part of the transfected cells are destroyed, or
nonintegrated plasmid DNA is lost during cell cycling. Overall, the loss of pDNA during this phase is not
nearly as great as the loss of expression. Relatively sustained expression driven by the liver-specific
albumin promoter (albeit at low levels) supports this hypothesis. This has recently been confirmed more
conclusively by Miao et al, who described long-term expression of human factor IX in mice following tail
vein injection of a plasmid DNA vector employing an α1-antitrypsin promoter in conjunction with a
hepatic control region.10,11 Similar expression vectors with liver-specific promoters and transcriptional
regulatory elements developed in our lab have corroborated these results.
It is now well established that certain sequences in bacterial DNA stimulate the immune system.20 This
appears to be based on the absence of CpG methylation in bacterial DNA, whereas in mammalian DNA
most CpG sequences are methylated. By inclusion of these sequences in genetic vaccines, an enhanced
immune response can be induced that is skewed to Th-1.21 If the objective is long-term expression,
minimizing the CpG content of the pDNA vector is beneficial.13 Combination of liver-optimized expression
cassettes in CpG-minimized vectors has made sustained, high-level transgene expression in the liver
following naked pDNA delivery a reality.
The mechanism of intravascular naked DNA delivery is thought to involve active cellular pDNA
uptake
The combined intraparenchymal and emerging intravascular data indicates that the uptake and
expression of naked DNA is a general property of animal cells within a tissue architecture. It is common
to cells of all three lineages: endoderm (eg, hepatocytes), mesoderm (eg, muscle), and ectoderm (eg,
skin). This property is typically lost when the cells are removed and maintained in culture. Tissue
disruption and cell isolation may modify the cell so that it can no longer take up naked DNA.
The intravascular injection conditions presumably enhance DNA transfer to hepatocytes by opening
transiently the hepatic endothelial barrier. Under normal conditions, the 0.1 μm size of the fenestrae
would prevent the exit from the sinusoids of plasmid DNA, has a gyration radius of 0.1 μm. Raising the
intraportal pressure may transiently enlarge their size and thereby increase the extravasation of the
pDNA complexes. In fact, results using fluorescent-labeled DNA showed that the increased pressure was
required for movement of the DNA out of the sinusoids and to the hepatocytes. Several observations
suggest that the mechanism of pDNA uptake may involve native cellular uptake processes. pDNA uptake
was time-dependent, suggesting that it is due in part to a cellular process. 19 A receptor-mediated process
is suggested by the inhibition of expression (from intramuscularly injected pDNA) by excess salmon
sperm DNA, dextran sulfate,19 or heparin.22 These cellular processes may be aided or initiated by the
rapid injection of the large volumes.
After traversing the plasma membrane, the pDNA must enter the nucleus since it is highly unlikely that
plasmid DNA containing RNA polymerase II promoters could be expressed anywhere else. Although it is
often assumed that DNA enters the nucleus from the cytoplasm little is known about the actual nuclear
uptake process despite many advances toward an understanding of protein and RNA nuclear transport.
Our preliminary model of DNA nuclear uptake is as follows. After cytoplasmic delivery, the small amount
of DNA that avoids binding to or sequestration by cytoplasmic elements enters the intact nucleus through
the nuclear pore. The relatively rare entry of DNA into the nucleus (in comparison with karyophilic
proteins) could be explained by its rapid and substantial cytoplasmic sequestration and its low rate of
transport through the nuclear pore. This understanding of DNA nuclear transport provides a basis for
future efforts to increase the efficiency of this process and is consistent with efforts to increase the
amount of DNA delivered to the cytoplasm. For example, we have observed increased nuclear entry in
digitonin-permeabilized cells of pDNA containing covalently attached SV40 T antigen nuclear localizing
signal.
Naked DNA delivery has entered clinical studies for peripheral arterial occlusion disease
(PAOD)
Several studies have been initiated for the treatment of limb ischemia or PAOD, using direct injection of
pDNA into skeletal muscle as the gene transfer method. While notoriously difficult to evaluate, a benefit
was noted in several early-phase clinical studies. Simovic et al23 performed a dose-escalation study in 29
patients with critical leg ischemia. After intramuscular injection of naked pDNA expressing the human
vascular endothelial growth factor (VEGF) driven by a CMV promoter, patients had significant clinical
improvements in examination scores and vascular ankle-brachial index in the treated limb.23 This was
accompanied with improvements in electrophysiologic measures 6 months after gene transfer. Similar
results were reported by Comerota et al 24 in a trial involving FGF-1 gene transfer into 51 patients with
lower leg ischemia.24 A significant decrease in reported pain and ulcer size was observed, as well as an
improved transcutaneous oxygen pressure and ankle-brachial index. No increase in FGF-1 serum levels
was measured, suggesting that localized expression may be effective. While clearly encouraging, the
gene therapy clinical trials to date have been predominantly small and noncontrolled (open label). Initial
results show encouraging improvements, although absolute increases in study parameters remain
relatively small.
Several questions are in need of answering prior to entering large-scale clinical trials: (1) Which
angiogenic gene, or cocktail of genes, should be used? VEGF and FGF-1 appeared to work in these early
trials, but are likely not optimal factors. The basic biology of angiogenesis needs to be developed more
fully to provide a better understanding of this complicated process, allowing for more rational choices of
therapeutic genes. (2) What naked DNA gene transfer method is optimal, direct injection (with or without
electroporation) resulting in localized expression, or intravascular delivery resulting in widespread gene
delivery and expression throughout the limb? This can likely be studies in an animal model, although no
current model closely mimics human disease. (3) What duration of expression is desired? A benefit of
naked DNA gene transfer into skeletal muscle is that generally long-term expression is obtained:
however, that may not be optimal for the expression of angiogenic factors for the treatment of PAOD.
While one could use regulated expression vectors, these introduce recombinant transcription factors that
may lead to immune rejection and other problems. Different expression vectors therefore may need to
be developed. Also, do we need to use optimized expression vectors (eg, CG-less) to avoid activating the
host immune system. (4) What criteria for successful gene therapy are used? Subjective measures (eg,
leg pain) are notoriously inaccurate. Measurement of increased blood flow appears a much better choice,
but may underestimate benefit of therapy. (5) When can new gene therapy approaches be tested in
clinical trials? Beyond basic safety testing, it may be difficult to assess effectiveness in animal models.
Patients with severe limb ischemia (near amputation) may benefit from gene therapy, even while not all
details (eg, optimal gene, optimal delivery method) are known. Based on recent history, it appears
prudent to approach clinical trials with great care.
Small interfering RNA (siRNA) can be delivered very efficiently using intravascular gene
transfer methods and results in potent knockout of target gene expression
RNA interference (RNAi) has been demonstrated to be highly effective for gene knockdown in a number
of experimental organisms, as well as in mouse oocytes and preimplantation embryos. RNAi is mediated
by double-stranded RNA (dsRNA), where mRNAs with sequence identity to the double-stranded RNA are
degraded rapidly. It has been demonstrated using cultured mammalian cells that RNAi can be
accomplished by delivering short interfering RNAs (siRNAs, dsRNA of 2125 bp length), which
circumvents the induction of an interferon response normally associated with the delivery of longer
dsRNA. We and others have determined that siRNAs can be delivered to tissues in vivo by intravascular
delivery.25,26 Expression of reporter genes (luciferase from codelivered pDNA or GFP in transgenic mice)
and endogenous genes can be downregulated by 90% in a majority of liver cells following one injection
with an appropriate siRNA. We have also observed efficient reporter gene downregulation in heart, lung,
spleen, and other tissues following tail vein delivery in mice. It is likely that several hurdles, associated
with gene delivery, are easier to overcome for siRNA delivery. siRNAs are much smaller than pDNA, and
therefore vascular extravasation and cellular uptake may be easier. siRNAs exert their effect in the
cytoplasm; therefore, the slow step of nuclear entry is avoided. It is not clear yet how long the RNAi
effect lasts following delivery of a single dose of siRNA. Nonetheless, it appears that RNAi is becoming a
very important extension of what we should call now the naked nucleic acid transfer field.
Summary
The direct intramuscular injection of naked DNA is in common use for genetic immunization and an HIV
vaccine clinical trial is in progress. The efficiency of gene expression following the intravascular delivery
of naked DNA is high in muscle and liver, even approaching what can be achieved with viral vectors.
High expression is also possible in larger animals including primates and there are potential applications
of the technique for treating human disorders. Given that tail vein injections of naked DNA under certain
conditions lead to high levels of foreign gene expression in hepatocytes in rodents and is convenient, the
technique is gaining increasing favor for studying gene expression in vivo. The technique also enables
delivery of siRNA to the liver and other organs in mice and should be useful for attenuating gene
expression.
References
1 Somiari S et al. Theory and in vivo application of electroporative
gene delivery. Mol Ther 2000; 2: 178187. Article PubMed
2 Hartikka J et al. Electroporation-facilitated delivery of plasmid
DNA in skeletal muscle: plasmid dependence of muscle damage and
effect of poloxamer 188. Mol Ther 2001; 4: 407415.
3 Terada Y et al. Efficient and ligand-dependent regulated
erythropoietin production by naked DNA injection and in vivo
electroporation. Am J Kidney Dis 2001; 38: S50S53.
4 Vilquin JT et al. Electrotransfer of naked DNA in the skeletal
muscles of animal models of muscular dystrophies. Gene Ther
2001; 8: 10971107. Article
5 Jiang J, Yamato E, Miyazaki J. Intravenous delivery of naked
plasmid DNA for in vivo cytokine expression. Biochem Biophys Res
Commun 2001; 289: 10881092.
6 Drabick JJ, Glasspool-Malone J, King A, Malone RW.
Cutaneous transfection and immune responses to intradermal
nucleic acid vaccination are significantly enhanced by in vivo
electropermeabilization. Mol Ther 2001; 3: 249255. Article
7 Zhang G et al. Surgical procedures for intravascular delivery of
plasmid DNA to organs. Methods Enzymol 2002; 346: 125133.
8 Maruyama H et al. High-level expression of naked DNA delivered
to rat liver via tail vein injection. J Gene Med 2002; 4: 333341.
9 Herweijer H et al. Time course of gene expression after plasmid
DNA gene transfer to the liver. J Gene Med 2001; 3: 280291.
10 Miao CH et al. Inclusion of the hepatic locus control region, an
intron, and untranslated region increases and stabilizes hepatic
factor IX gene expression in vivo but not in vitro. Mol Ther 2000; 1:
522532. Article PubMed
11 Miao CH, Thompson AR, Loeb K, Ye X. Long-term and
therapeutic-level hepatic gene expression of human factor IX after
naked plasmid transfer in vivo. Mol Ther 2001; 3: 947957.
12 Yew NS et al. High and sustained transgene expression in vivo
from plasmid vectors containing a hybrid ubiquitin promoter. Mol
Ther 2001; 4: 7582. PubMed
13 Yew NS et al. CpG-depleted plasmid DNA vectors with
enhanced safety and long-term gene expression in vivo. Mol Ther
2002; 5: 731738.
14 Chen ZY et al. Linear DNAs concatemerize in vivo and result in
sustained transgene expression in mouse liver. Mol Ther 2001; 3:
403410. Article PubMed
15 Eastman SJ et al. Development of catheter-based procedures
for transducing the isolated rabbit liver with plasmid DNA. Hum
Gene Ther 2002; 13: 20652077.
16 Maruyama H et al. Kidney-targeted naked DNA transfer by
retrograde renal vein injection in rats. Hum Gene Ther 2002; 13:
455468.
17 Zhang G et al. Efficient expression of naked DNA delivered
intraarterially to limb muscles of nonhuman primates. Hum Gene
Ther 2001; 12: 427438. Article PubMed
18 Herweijer H et al. Retrograde coronary venous delivery of
naked plasmid DNA. Mol Ther 2000; 1: S202.
19 Budker V et al. Hypothesis: naked plasmid DNA is taken up by
cells in vivo by a receptor-mediated process Review. J Gene Med
2000; 2: 7688. Article PubMed
20 Krieg AM. Now I know my CpGs. Trends Microbiol 2001; 9:
249252. Article PubMed
21 Krieg AM, Davis HL. Enhancing vaccines with immune
stimulatory CpG DNA. Curr Opin Mol Ther 2001; 3: 1524.
22 Satkauskas S, Bureau MF, Mahfoudi A, Mir LM. Slow
accumulation of plasmid in muscle cells: supporting evidence for a
mechanism of DNA uptake by receptor-mediated endocytosis. Mol
Ther 2001; 4: 317323.
23 Simovic D et al. Improvement in chronic ischemic neuropathy
after intramuscular phVEGF165 gene transfer in patients with
critical limb ischemia. Arch Neurol 2001; 58: 761768. PubMed
24 Comerota AJ et al. Naked plasmid DNA encoding fibroblast
growth factor type 1 for the treatment of end-stage
unreconstructible lower extremity ischemia: preliminary results of a
phase I trial. J Vasc Surg 2002; 35: 930936.
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Received 22 July 2002; accepted 13 December 2002
March 2003, Volume 10, Number 6, Pages 453-458
February 2003, Volume 10, Number 4, Pages 285-291
Table of contents
Review
Previous Article Next PDF
Gene therapy progress and prospects: therapeutic
angiogenesis for limb and myocardial ischemia
T A Khan1, F W Sellke1 and R J Laham2
Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, MA, USA
1
Division of Cardiology and Angiogenesis Research Center, Beth
Israel Deaconess Medical Center and Harvard Medical School,
Boston, MA, USA
2
Correspondence to: Dr RJ Laham, Angiogenesis Research Center,
Beth Israel Deaconess Medical Center, 330 Brookline Ave., SL-423,
Boston, MA 02215, USA
Abstract
After extensive investigation in preclinical studies and recent clinical trials, gene therapy has
been established as a potential method to induce therapeutic angiogenesis in ischemic
myocardial and limb disease. Advancements in viral and nonviral vector technology including
cell-based gene transfer will continue to improve transgene transmission and expression
efficiency. An alternative strategy to the use of transgenes encoding angiogenic growth
factors is therapy based on transcription factors such as hypoxia-inducible factor-1 (HIF-1
) that regulate the expression of multiple angiogenic genes. Further understanding of the
underlying biology of neovascularization is needed to determine the ability of growth factors
to induce functionally significant angiogenesis in patients with atherosclerotic disease and
associated comorbid conditions including endothelial dysfunction, which may inhibit blood
vessel growth. The safety and tolerability of therapeutic angiogenesis by gene transfer has
been demonstrated in phase I clinical trials. However, limited evidence of efficacy resulted
from early phase II studies of angiogenic gene therapy for ischemic myocardial and limb
disease. The utility of therapeutic angiogenesis by gene transfer as a treatment option for
ischemic cardiovascular disease will be determined by adequately powered, randomized,
placebo-controlled phase II and III clinical trials.
Gene Therapy (2003) 10, 285291. doi:10.1038/sj.gt.3301969
Keywords
angiogenesis; gene transfer; ischemic heart disease; coronary artery disease; peripheral vascular
disease; vascular endothelial growth factor; fibroblast growth factor
In brief
Progress
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Improvements in adenoviral vector and plasmid DNA technology have improved transmission
and expression efficiency in myocardial and skeletal muscle tissue.
Newer techniques of gene transfer including AAV vectors and liposome complexes have been
shown to be effective in preclinical studies of angiogenesis for myocardial and vascular
disease.
Studies in animal models of therapy based on HIF-1
using an approach of transcriptional regulation.
have provided evidence of angiogenesis
Preclinical studies of angiogenesis have identified disease processes that may contribute to
attenuated angiogenesis such as endothelial dysfunction.
Phase I and II clinical trials have demonstrated the safety and suggested the efficacy of gene
transfer in therapeutic angiogenesis for CAD and PVD.
Minimally invasive catheter-based and surgical techniques of delivery have been effective in
clinical trials.
Prospects
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Further improvements in viral technology will increase transmission efficiency and reduce
toxicity because of inflammatory and immune responses.
The advancement of nonviral vector technology will allow for efficient gene transfer while
avoiding the safety concerns associated with viral vectors.
Refinements in cell-based gene transfer to allow multigene and sequential gene expression in
addition to regulatable expression with organ and tissue specificity will be needed.
Gene transfer using transcription factors that regulate the expression of multiple angiogenic
genes may be preferable for the induction of angiogenesis.
Current investigations of the molecular mechanisms of disease states that have been shown
to inhibit angiogenesis will provide vital information to develop an effective therapeutic
strategy of neovascularization.
The method of delivery of angiogenic agents and timing of treatment will become essential
factors in the strategy of therapeutic angiogenesis.
Phase II and III clinical trials will be critical in determining the utility of angiogenic gene
therapy in the treatment of ischemic limb and heart disease.
Gene therapy is a potential method for therapeutic angiogenesis
Therapeutic angiogenesis has emerged as a promising investigational strategy for the treatment of
patients with ischemic limb and heart disease. After over a decade of preclinical studies and recent
clinical trials, gene therapy has been established as a potential method to induce therapeutic
angiogenesis in patients with ischemic cardiovascular disease. Therapeutic angiogenesis using gene
therapy vectors will be the subject of this review with emphasis on recent advancements and future
directions in the treatment of ischemic limb and myocardial disease.
Neovascularization involves a complex series of events that likely include the coordinated action of
several cytokines to produce new conduits of blood flow. Vasculogenesis, angiogenesis, and
arteriogenesis are the three processes that may contribute to the growth of blood vessels. 1
Vasculogenesis is the formation of new vessels from pluripotent stem cells as seen in embryonic
development. Increasing evidence suggests that vasculogenesis also occurs in the adult as seen in the
mobilization of endothelial progenitor cells from bone marrow and the incorporation of these cells into
foci of neovascularization. Angiogenesis describes capillary growth from enlarged venules that sprout
capillary buds, become divided by periendothelial cells (intussusception), or are separated by
transendothelial cell bridges (bridging) to form capillaries. The process involves vasodilation and
increased permeability to allow extravasation of proteins to form an extracellular matrix, endothelial cell
proliferation and migration, and vessel formation. Endothelial cell differentiation follows in response to
the local tissue environment.2 Angiogenesis is the manner by which capillaries proliferate in healing
wounds and along the border of myocardial infarctions. Arteriogenesis is the process that produces
arteries possessing a fully developed tunica media resulting in true collateral arteries. Smooth muscle
cells may differentiate from various cell types including endothelial cells and bone marrow precursors.
Arteriogenesis involves smooth muscle cell growth and proliferation, migration, and differentiation to a
contractile phenotype.2 An example of arteriogenesis is the development of angiographically visible
collaterals in patients with advanced obstructive atherosclerotic disease (Figure 1).
In patients with coronary artery disease (CAD) and peripheral vascular disease (PVD), progressive
occlusion of arteries often leads to the development of collateral vessels that supply the ischemic tissue.
However, this natural compensatory process of neovascularization is often not sufficient as evidenced by
the large number of revascularization procedures performed annually. The lack of an adequate
angiogenic response in part may be related to reduced production of angiogenic factors. Therapeutic
angiogenesis describes the method of improving blood flow to ischemic tissue by the induction of
neovascularization by angiogenic agents administered as recombinant protein or by gene transfer. The
administration of recombinant protein or the genes that encode these proteins both have been used as
techniques of angiogenic therapy in preclinical and clinical trials. Advantages of gene transfer include
persistent expression of the angiogenic factor providing prolonged, local exposure, potential for singledose regimens, and cell-specific angiogenic therapy. However, low efficiency of gene expression and
immune deactivation of the foreign material are limiting factors. Furthermore, induction of an
inflammatory response, nonspecific gene transfer to other cell types, and lack of regulation of gene
expression and the resulting uncontrolled level of growth factor are additional risks to the patient. 3,4
Protein formulations provide predictable pharmacokinetics and tissue therapeutic levels that allow for
controlled dosing at the time of growth factor administration. In using protein-based angiogenic therapy,
the administration of viral vectors and foreign genetic material is avoided. The short half-life of
angiogenic proteins limits the duration of exposure and presents a possible need for additional doses.
However, slow-release delivery systems may circumvent this issue and repeat dosing may be more
effective because of the relative lack of significant inflammatory and immune responses to protein
therapy.
As important as the type of angiogenic therapy is the delivery of the angiogenic agent. Intravascular
delivery may lead to nonspecific and systemic exposure, while intramyocardial delivery techniques can
allow for a local and sustained angiogenic effect.5 In addition, therapy based on single growth factor
agents may not be adequate to induce functionally significant angiogenesis in humans because of the
complexity of the angiogenic process, particularly in the context of advanced atherosclerotic disease and
associated comorbid conditions. Multi-agent therapy may be necessary to achieve angiogenesis and
provide significant improvements in myocardial perfusion and function as well as clinical outcome in
patients.
Viral and nonviral gene transfer agents have been successfully studied
Among the current clinical trials of therapeutic angiogenesis for CAD and PVD, adenoviruses and
plasmids are the vectors most often studied likely because of the ease of production, reasonable
transfection efficiency, and expression in nonproliferating cells. First-generation E1-deleted adenoviral
vectors were limited in vascular gene transfer because of endothelial injury and the inflammatory
response. The development of attenuated adenoviral vectors with further deleted elements of the viral
genome has produced vectors that result in increased transgene expression and reduced inflammation in
cardiovascular gene transfer. Deletions of the E1 and E4 regions to produce a second-generation
adenoviral vector have been shown to result in improved transgene expression with reduced
inflammatory response and preserved endothelium-dependent relaxation. However, lack of prolonged
transgene expression at 28 days may have been representative of residual, although substantially less,
inflammation induced by the second-generation adenoviral vector because of low-level, late gene
expression.6 Fully deleted adenoviral vectors may potentially eliminate this late expression and any
associated inflammatory response. This technology may improve transgene expression despite the
presence of antiadenoviral neutralizing antibodies.7 Production of fully deleted vectors is made possible
by a helper virus that provides viral proteins required for replication and packaging. A study of fully
deleted adenoviral vectors, also known as gutless or helper-dependent, carrying a marker transgene,
erythropoietin, demonstrated that intramuscular delivery resulted in efficient and prolonged expression
in both immunocompetent mice and those immunized against the adenovirus serotype. 7 The results of
the study are clinically important considering the significant proportion of the population with preexisting immunity to adenovirus. In addition, recombinant adeno-associated viruses (AAV) are potential
vectors for therapeutic angiogenesis. Advantages of the AAV vectors for gene transfer include the
transduction of nonproliferating cells, lasting transgene expression, and reduced inflammatory response,
while limitations involve difficulty with production and a small packaging capacity.
Nonviral methods of gene transfer studied in clinical trials include plasmid DNA and liposomal complexes.
The use of nonviral techniques avoids the concerns over the toxicity associated with viral vectors. While
the inflammatory response to adenoviral vectors is well described, both plasmid DNA and liposomal
complexes also potentially induce inflammation.8,9 Despite the ease of production and scale-up of
plasmid and liposomal complexes, low transmission efficiency and transgene expression are limiting.
Transmission efficiency of plasmid DNA may be improved by the use of ultrasound. Ultrasound exposure
with microbubble echocontrast agents increase transgene expression significantly after naked DNA
transfection by cell membrane permeabilization. This technique of membrane permeabilization, or
acoustic cavitation, with microbubble echocontrast was reported to increase transgene expression by
approximately 300-fold through the creation of transient small holes in the cell surface membrane
through which naked DNA is rapidly translocated.10 In a study of plasmid DNA transmission efficiency,
luciferase plasmid transfection using ultrasound with microbubble echocontrast was increased
approximately 10-fold compared to plasmid alone in cultured human skeletal muscle. In the same report,
gene transfer of a hepatocyte growth factor (HGF) plasmid in a rabbit model of hindlimb ischemia
produced increased angiographic score and capillary density in animals transfected using ultrasound with
microbubble echocontrast versus transfection with plasmid alone.11 The use of ultrasound with
microbubble contrast also has been demonstrated to increase the transfection efficiency of plasmid DNA
in human aortic endothelial and vascular smooth muscle cells without apparent toxicity.12 This technique
has the potential to improve the efficiency of plasmid DNA transfection in human myocardial tissue as
well. The transmission efficiency of liposomes may be enhanced by improvements in cationic polymers. 13
Liposomes have been shown to be effective in the transfer of growth factors in animal models of
angiogenesis. In a rabbit ischemic hind limb model, vascular endothelial growth factor (VEGF) gene
transfer by cationic liposome resulted in neovascularization and improved blood flow in the ischemic
limb.14 Liposome carriers also have been demonstrated to be effective in angiogenesis based on HGF.
Transfer of HGF with the hemagglutinating virus of Japan (HVJ)liposome method has been shown to
induce angiogenesis in normal and infarcted myocardium.15
Cell-based gene transfer is a novel strategy that utilizes autologous cells as vectors after in vitro
transfection with a transgene of interest.16 Such a system is able to circumvent the inflammatory
response by using autologous cells and achieves prolonged expression by stable transfection using
various measures including electroporation and in vitro retroviral or lentiviral transfection. In addition,
complex constructs can be synthesized that would allow stable, regulatable expression and multiple
transgene expression. Recent investigations have focused on gene transfer by cellular transplantation to
induce neovascularization in ischemic tissue using skeletal myoblasts and angioblasts.17,18,19
VEGF and FGF are the most widely studied growth factors
Numerous growth factors and transcription factors have been associated with physiologic and pathologic
angiogenesis. Among the transgenes under investigation in clinical trials for CAD and PVD, genes that
encode growth factors predominate. VEGF and fibroblast growth factor (FGF) are the agents most widely
studied in clinical trials, specifically the 121 and 165 amino-acid isoforms of VEGF1, VEGF2, FGF1, and
FGF4. Gene transfer of VEGF and FGF has been shown to induce functionally significant angiogenesis in
numerous preclinical studies of angiogenic therapy for ischemic heart disease20 and peripheral arterial
disease.21 Another strategy under investigation in clinical trials is a therapy based on the transcription
factor hypoxia-inducible factor-1
(HIF-1 ). The expression of many angiogenesis-related genes,
including VEGF and the VEGF receptor FLT-1, is regulated by HIF-1 . A hybrid, constitutively active form
of HIF-1 , has been synthesized from the DNA-binding and dimerization domains of the HIF-1
subunit
and the transactivation domain of the VP16 protein of the herpes simplex virus. Angiogenic gene transfer
of this hybrid form has been reported in preclinical studies as described below. Another potential factor
that regulates angiogenesis is the peptide PR-39. This peptide increases the cellular levels of HIF-1
inhibiting its degradation in the ubiquitinproteasome complex. PR39 has been shown to increase the
expression of VEGF, the VEGF receptors KDR and FLT-1, and the FGF receptor 1.22 Concerns over a
nonspecific action of PR39 related to charge are being investigated.
by
Preclinical and clinical trials in PVD have demonstrated safety and suggested efficacy
Angiogenic responses to growth factor gene transfer using plasmid and adenoviral vectors have been
well documented in animal models of chronic limb ischemia. VEGF gene transfer techniques using
nonviral and adenoviral vectors have been the more common methods in preclinical studies. Recently,
angiogenesis has been induced in animal models of limb ischemic using AAV vector-mediated therapy
with VEGF. In rat hind limb models of ischemia, VEGF-based therapy via AAV vectors produced increased
blood flow and capillary growth in treated, ischemic limbs compared to controls. 23,24 HGF also has
emerged as a potential agent in therapeutic angiogenesis. In a rabbit ischemic hind limb model,
intramuscular injection of human HGF plasmid resulted in enhanced collateral development by
angiography and increased blood flow and blood pressure in the ischemic limb. 25 Furthermore, evidence
has recently been published that suggests FGF-2 contributes to the regulation of HGF expression.26
Recent results of clinical trials of therapeutic angiogenesis for peripheral arterial disease have provided
further information on the clinical response to angiogenic gene therapy including measures of
neovascularization as well as known side effects such as edema formation. Lower extremity edema was
evaluated in 90 patients that were treated with VEGF165 by intra-arterial or intramuscular gene transfer.
Edema was observed in 34% of patients, being more common in those patients with rest pain and
ischemic ulcers as compared to those with claudication only. The increased vascular permeability was an
effect attributed to the VEGF therapy.27 Recently, the results of a phase II clinical trial of VEGF-1 gene
therapy for chronic limb ischemia were published. In the randomized, placebo-controlled, double-blinded
study of catheter-based VEGF-1 gene therapy after percutaneous transluminal angioplasty (PTA),
patients in the treatment groups received intra-arterial VEGF-1 by adenoviral vector or liposomeplasmid
carrier while those in the control group received crystalloid solution. Digital subtraction angiography
(DSA) revealed increased vascularity in both treated groups. Both the VEGF-adenoviral and VEGFliposomeplasmid groups showed increased vascularity distal to the site of gene transfer. In addition, the
VEGF-adenoviral group demonstrated significantly increased vascularity in the clinically most severe
region of ischemia. However, the ratio of lower extremity compared to upper extremity blood pressure,
or ankle-brachial index (ABI), was not significantly different between treated and control groups. In
addition, antiadenoviral antibodies increased in 11 of 18 patients administered VEGF-1 by adenoviral
vector.28
Phase III clinical trials in patients with myocardial ischemia have been completed
Several preclinical studies have demonstrated efficacy of angiogenic gene therapy for myocardial
ischemia. FGF- and VEGF-based protocols have been the most widely studied. A recent report of VEGF 121
gene transfer by an adenoviral vector in a porcine model of myocardial ischemia demonstrated that
intramyocardial delivery resulted in transient, focal VEGF expression in the target, ischemic area of the
myocardium. The localized VEGF expression was 10-fold greater than in intracoronary delivery and
produced regional improvement in myocardial blood flow.29 Other studies of late have reported
alternative strategies producing favorable results in animal models of myocardial angiogenesis. In a
mouse model of myocardial ischemia, an AAV-VEGF vector injected around ischemic myocardium
resulted in neovascularization without evidence of inflammation. 30 Gene transfer of the HIF-1 VP16
hybrid in a rabbit model of hind limb ischemia was associated with increased regional blood flow and
capillary density.31 In a rat model of acute myocardial ischemia, intramyocardial delivery of the HIF-1
VP16 hybrid plasmid was able to reduce the size of myocardial infarction and increase capillary density
in the border zone of the infarct area. The induction of angiogenesis was similar to the neovascularization
that resulted from VEGF therapy by plasmid gene transfer in the same model.32 In a study of transgenic
mice with porcine PR39 cDNA, angiogenesis was induced in the PR39 mice compared to age-matched
controls as seen by increased CD-31-stained vascular structures.22 These results suggest that agents
that produce a multifactorial angiogenic response such as HIF-1 and PR39 are effective in
neovascularization and may represent a more effective strategy in therapeutic angiogenesis.
Results of several clinical trials of gene transfer for therapeutic angiogenesis have been reported. These
trials include uncontrolled, open label designs primarily investigating safety and feasibility. The results of
these studies should be interpreted with caution considering the significant placebo effect observed in
patients with CAD. In a study of 129 patients enrolled in control groups of phase I and II clinical trials of
therapeutic angiogenesis and laser myocardial revascularization, the mean CCS angina class was 3.00.5
at baseline and 2.10.6 at 6 months (P<0.001), with 24.8% of patients improved by two or more angina
classes. Mean follow-up was 306 months and at last follow-up, mean CCS angina class was 2.30.8
(P<0.001). The results of this study underscore the significance of the placebo effect in this patient
population of severe CAD.33
Two studies of surgical angiogenic therapy using gene transfer have been published recently. A phase I
study evaluated the surgical delivery of VEGF165 plasmid DNA through a mini-thoracotomy in seven
patients. As previously reported, both nitroglycerin intake and CCS angina class were significantly
reduced, while improved myocardial perfusion was suggested by single-photon emission computed
tomography (SPECT) and coronary angiography.34 A study of intramyocardial gene transfer through a
limited thoracotomy was initiated using VEGF-2 plasmid DNA in patients with chronic stable angina. An
initial report on 11 patients revealed reductions in angina episodes and nitroglycerin use as well as
improvement in exercise tolerance testing.35
Two trials of catheter-based myocardial gene transfer of plasmid DNA encoding for VEGF-2 have been
reported of late. An initial randomized, single-blind, placebo-controlled study investigated the safety and
feasibility of VEGF-2-based therapy in six patients. Patients that received VEGF-2 plasmid DNA
experienced reduced angina compared to control patients at 90 days of follow-up. Reduction in ischemia
and improvement in myocardial perfusion were suggested by electromechanical mapping and SPECT
imaging, respectively.36 A multicenter, randomized, double-blind, placebo-controlled phase III clinical
trial followed. VEGF-2 plasmid DNA was administered as part of a dose-escalating protocol to patients
with Canadian Cardiovascular Society (CCS) class III or IV angina. The catheter-based delivery to the
endocardial surface of the left ventricle resulted in no hemodynamic alterations, sustained ventricular
arrhythmias, or electrocardiographic evidence of infarction. A recent interim report after enrollment of 19
patients described a significant reduction in CCS angina class in the treated group compared to
controls.37 Although these results as a whole suggest the therapeutic efficacy of angiogenesis by gene
transfer, a study of intramyocardial gene transfer of plasmid DNA encoding VEGF-A and VEGF-B as an
adjunct to CABG in 24 patients was published recently that provided only modest evidence of improved
perfusion.38
The angiogenic gene therapy (AGENT) study was the first randomized, double-blind, placebo-controlled
trial of therapeutic angiogenesis by gene transfer for myocardial ischemia. FGF-4 carried by an
adenoviral vector was given intracoronary to 79 patients with chronic stable angina randomized in a 1:3
ratio to produce 19 in the control group and 60 in the treatment group. Overall, FGF-4 therapy using an
adenoviral vector was well tolerated without significant safety concerns. No significant difference was
observed in stress-induced wall motion by echocardiography between groups. The results of the primary
end point, exercise treadmill testing (ETT), after 4 and 12 weeks of follow-up demonstrated a
nonsignificant trend towards improvement in the FGF-4-treated group. Subgroup analysis revealed a
significant improvement in those patients with baseline ETT of less than 10 min. 39
Patient selection and end-point evaluation are important factors in clinical trial design
Therapeutic angiogenesis has provided a new treatment strategy for patients with end-stage CAD and
PVD. Currently, results of adequately powered, randomized, double-blind, placebo-controlled trials are
lacking. For enrollment in clinical trials, randomization should include baseline angiogenic response
manifest as collateralization. Patients selected for trials of therapeutic angiogenesis often previously have
had multiple percutaneous and surgical revascularization attempts. These individuals may possess
resistance to stimulation of neovascularization, considering they likely suffer from failure of natural
angiogenic responses. Thus, patients enrolled in current trials may represent the group least likely to
respond. Ideal candidates are those with ischemic but viable myocardium and diffuse multivessel
disease. Exclusion criteria generally include a history of malignancy or proliferative retinopathy because
of concerns of pathogical angiogenesis. Patients with abnormal baseline renal function and proteinuria
are excluded from trials of FGF therapy because of the risk of renal toxicity.
End points of cardiac morbidity and mortality including myocardial infarction and death may provide
objective measures of outcome. However, the low frequency of these events in clinical trials of treatment
of myocardial disease indicate that a prohibitively large study population may be required to show
significant reductions in these outcomes. Limb salvage from amputation may be used in a patient
population with advanced peripheral vascular disease. Other end points in cardiac trials include exercise
tolerance testing and measures of myocardial perfusion using single-photon emission computed
tomography (SPECT) and magnetic resonance imaging (MRI), while end points in peripheral vascular
trials may include vascularity by DSA or magnetic resonance angiography, lower extremity blood
pressure by ABI, and healing of ischemic ulcers. The response to angiogenic gene therapy for limb
ischemia may be assessed through improvements in claudication and rest pain. Relief of myocardial
ischemic symptoms and improvements in quality of life may be determined by methods such as the
Seattle Angina Questionnaire or the Canadian Cardiovascular Society angina classification. However, the
placebo effect has been shown to be very powerful in studies of patients with severe CAD and PVD.
Prospects
Recent advances in gene transfer have allowed therapeutic angiogenesis to become a potential
treatment for CAD and PVD.
Continued development of viral and nonviral vector technology will improve transgene transmission and
expression efficiency. Refinements in cell-based therapy may provide for regulatable and multiagent
therapy. The attenuated angiogenic response in patients enrolled in clinical trials may be related to
comorbid pathophysiology that is associated with CAD and PVD. Endothelial dysfunction is associated
with atherosclerotic disease, and may play a role in a reduced angiogenic response. We investigated the
effect of endothelial dysfunction secondary to hypercholesterolemia on therapeutic angiogenesis by
perivascular delivery of FGF-2 through a mini-thoracotomy.40 In our pig ameroid constrictor model of
chronic myocardial ischemia, hypercholesterolemic animals showed significant endothelial dysfunction
and impaired angiogenesis manifest as decreased perfusion compared to the control, normal diet group.
Thus, endothelial dysfunction may represent one of many factors that prevent a significant angiogenic
response in patients with CAD and PVD. The future development of treatment options for endothelial
dysfunction may allow for an improved angiogenic response to growth factor therapy in humans with
atherosclerotic occlusive disease.
Improvements in delivery modalities with increased local distribution and retention and reduced systemic
circulation are needed. Intramyocardial and intramuscular delivery techniques currently under
investigation in phase I and II clinical trials may provide evidence of efficacy that was lacking in studies
using intravascular routes of administration. Delivery optimization studies also should be conducted for
specific agents prior to preclinical and clinical investigation. Multiagent therapy may be needed to
achieve the complex process of angiogenesis in humans. The natural angiogenic response is likely a
result of the actions of multiple growth factors at various points in time. The sequential or concomitant
administration of the agents may be an important factor as well. In addition, a synergistic mechanism of
action between growth factors in angiogenesis has been suggested. Furthermore, it has become clear
that the timing of growth factor therapy may be critical in inducing an angiogenic response. A recent
study of VEGF-based angiogenesis has demonstrated that a critical duration of growth factor exposure is
required to prevent regression of the newly formed vasculature.41 Thus, therapeutic angiogenesis using
methods with prolonged presence of growth factors over a few weeks may be necessary, as suggested
by the promising results of the clinical trial using surgically implanted heparin-alginate microcapsules
that release FGF-2 over a course of 34 weeks.42
Conclusions
Therapeutic angiogenesis is a promising therapy for patients with CAD and PVD not amenable to current
revascularization techniques. Clinical trials of gene transfer for therapeutic angiogenesis in the treatment
of ischemic limb and heart disease have been limited to predominantly uncontrolled phase I studies that
have demonstrated safety and preliminary reports of phase II trials that have provided modest evidence
of clinical efficacy. As further investigation of gene transfer proceeds, monitoring of potential toxicities
must be continued to maximize the benefit and minimize the risk of angiogenic therapy. Overall, the role
of therapeutic angiogenesis by gene transfer as a potential treatment option for ischemic limb and heart
disease will be determined by adequately powered, randomized, placebo-controlled phase II and III
clinical trials.
Acknowledgements
Supported in part by NIH Grants MO1-RR01032 and HL63609 (RJL),
and HL46716 and HL69024 (FWS). Dr Khan is supported by an NIH
Individual National Research Service Award, HL69651-01.
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Figures
Figure 1 Arteriogenesis in an animal model of myocardial ischemia.
Batson casts were performed on explanted hearts following left
circumflex coronary artery ameroid constrictor placement. After
myocardial digestion, vessels were visualized (blue for occluded left
circumflex, red for left anterior descending (LAD), and white for
right coronary arteries). Arrows point to epicardial collateral vessels
going from the LAD and right coronary arteries to the ischemic left
circumflex territory.
Received 10 September 2002; accepted 3 December 2002
January 2003, Volume 10, Number 2, Pages 95-99
Table of contents
Previous Article Next [PDF]
Review
Gene Therapy Progress and Prospects: Alpha-1 antitrypsin
A A Stecenko and K L Brigham
Center for Translational Research in the Lung, McKelvey Center for Lung
Transplantation and Division of Pulmonary and Critical Care Medicine,
Department of Medicine, Emory University School of Medicine, Atlanta,
GA, USA
Correspondence to: KL Brigham, Department of Medicine, Emory
University School of Medicine, Whitehead Biomedical Research Building,
615 Michael Street, Suite 205, Atlanta, GA 30322, USA
Abstract
Over the 2 years covered here, there has been one clinical study in which a normal alpha-1 antitrypsin
(AAT) gene was delivered to the nasal epithelium of AAT-deficient subjects using plasmidliposome
complexes; a second study using an adeno-associated vector should begin soon. Although progress in
clinical studies has been slow, advances in both viral and nonviral vector designs show considerable
promise. Strategies that combine liposome technology with imaginative vector design may permit
long-term expression of a normal transgene that is sufficient to achieve therapeutic serum AAT
concentrations. While reproducing the normal physiology by targeting normal AAT gene expression to
the liver is logical, local expression in lung cells may be less demanding of the technology and offers
therapeutic benefits that are produced neither by AAT protein therapy nor by AAT gene therapy
targeted to the liver. Developing technologies may permit direct correction of the mutant AAT gene
using innovative approaches to in vivo gene repair.
Gene Therapy (2003) 10, 9599. doi:10.1038/sj.gt.3301947
Keywords
alpha-1 antitrypsin; organ-targeted gene therapy; gene repair; emphysema
In brief
Progress




Human gene therapy trials: cautious optimism
Strategies for reducing the cellular immune response and eliminating need for receptors may improve
utility of adenoviral vectors
Adeno-associated viral vectors have low toxicity and effectively deliver an AAT transgene in animals
Nonviral vectors can deliver a functioning AAT gene in vivo and can be designed to prolong transgene
expression
Prospects




Lung-directed AAT gene transfer may be therapeutic without 'therapeutic' protein levels in body fluids:
applications beyond AAT deficiency
RNADNA oligonucleotide (RDA)-directed repair of the defective AAT gene
Extension of AAT gene therapy to treat acquired emphysema
Expanded use of AAT gene therapy as 'antiprotease therapy' (antiviral, anti-inflammatory)
Human gene therapy trials: cautious optimism
Alpha-1 antitrypsin (AAT) is the most prominent endogenous serine proteinase inhibitor (SERPIN) in humans. An
inherited AAT deficiency is second in prevalence only to cystic fibrosis among inherited diseases of the lungs. The
genetic abnormality is thoroughly described, current therapy is suboptimal and multiple vectors capable of
delivering the normal AAT gene in vivo have been developed. In addition, unlike the cystic fibrosis
transmembrane conductance regulator, AAT is a secreted protein so that gene therapy should be less demanding
of the technology. However, in contrast to the situation with cystic fibrosis, there have been few clinical trials of
gene therapy with AAT. There are currently no active AAT gene therapy trials in progress.
A general surrogate criterion for efficacy of AAT gene therapy has been achieving 'therapeutic' concentrations
(that is approaching normal) in circulating blood or (receiving more emphasis) in bronchoalveolar lavage fluid. If
the goal is to reproduce normal physiology, production and secretion of sufficient amounts of normal AAT protein
by the liver to mimic the normal condition, those criteria are appropriate. The criteria are based primarily on the
rationale used to approve AAT protein for clinical use as an orphan drug without proven effect on the course of
the disease. Although some of the more recent technologies are approaching levels of gene delivery and
expression sufficient to meet these criteria, recent evidence suggests that if the AAT gene is expressed in the
lungs (ie at the site of the disease for which the therapy is intended), the level of AAT transgene expression
required may be less than previously thought.
There are two clinical trials in humans either imminent or nearly so that propose delivery of a normal AAT
transgene to the respiratory tract of humans. One proposed study is based on animal studies indicating that the
normal AAT gene delivered in an adeno-associated viral vector (AAV-AAT) can produce prolonged 'therapeutic'
serum concentrations of the normal protein (see discussion of the animal studies below). 1 This proposal would
inject AAV-AAT intramuscularly with the goal of achieving normal blood AAT concentrations. 2 Studies would be
done in 812 relatively healthy subjects with the common PiZZ phenotype AAT deficiency. According to the
information on the Alpha-1 web site,2 these studies are nearing approval by the appropriate regulatory agencies
and should begin soon.
The one reported study in human subjects used an unmodified cationic liposome (DOTMA:DOPE) and a plasmid
vector containing the normal human AAT gene driven by a CMV promoter.3 In this study, the lipoplex was instilled
into one nostril of five subjects with ZZ phenotype AAT deficiency, with the contralateral nostril serving as a
control. AAT concentrations were measured in serial samples of nasal lavage fluid. As shown in Figure 1, AAT
concentrations increased significantly in the transfected nostril, peaking on day 5 after transfection and returning
to baseline by day 14. The mean peak AAT concentration in the transfected nostril in these five subjects was
about one third the normal level (Figure 2). Those studies demonstrate that lipoplex technology can deliver a
normal AAT gene to human respiratory epithelium in vivo, but delivery to the entire lung presents other
challenges.
A new proposal is to conduct a similar investigation in subjects with cystic fibrosis where lung destruction may be
partly attributable to the presence of large amounts of protease, but to extend the study to determine the effects
of repeated dosing of the lipoplex. Two subjects have completed this protocol, but the study is now on clinical
hold by the FDA awaiting production of new plasmid and liposome reagents.
Lung-directed AAT gene transfer may be therapeutic without 'therapeutic' protein levels in body
fluids: applications beyond AAT deficiency
Proteolytic events are ubiquitous in both physiologic and pathologic processes. AAT is normally produced primarily
in the liver, reaching the lung via the circulation. Since AAT is a relatively large hydrophilic protein, it is excluded
from intracellular and other cryptic spaces where pathologic proteolytic events occur. If lung cells are made to
produce AAT, the antiproteolytic activity might locate inside cells where it is produced as well as in interstitial and
intercellular spaces not usually available to circulating AAT. Thus, locally expressed AAT might have potentially
therapeutic activities similar to those of small molecule antiproteases.
Figure 2 summarizes data from nasal lavage fluid in studies on AAT-deficient subjects who had a single nostril
transfected with a normal human AAT gene delivered as a lipoplex. 3 Shown are measurements of AAT and of
interleukin-8, a proinflammatory cytokine measured in these studies as an indicator of inflammation. When on no
therapy, AAT concentrations in nasal lavage fluid were very low as expected, and IL-8 concentrations were
significantly elevated compared to normal, indicating some level of continuing inflammation. While these subjects
were receiving intravenous AAT protein therapy, nasal lavage fluid concentrations of AAT were in the normal
range, but IL-8 levels remained elevated. In contrast, when nasal epithelium was expressing the normal AAT
transgene, nasal lavage fluid AAT concentrations were only about one-third the normal mean, but IL-8
concentrations were normal. These findings suggest that if the normal AAT gene is expressed locally, it may not
be necessary to achieve 'therapeutic' AAT concentrations in plasma or bronchoalveolar lavage fluid in order to
have a therapeutic effect. In addition, the anti-inflammatory effect of local expression may expand the disease
targets for this therapy to include a broad range of chronic lung diseases in which inflammation is a major part of
the pathophysiology.
Strategies for reducing the cellular immune response and eliminating need for receptors may improve
utility of adenoviral vectors
Replication-deficient adenoviral vectors, originally promising as vectors for delivering therapeutic transgenes to
the lungs, have been disappointing for several reasons. Human respiratory epithelium is not rich in the requisite
specific cell receptors, so that adenoviral vectors are inefficient at delivering transgenes to the lungs of humans. 4
In addition, even replication-deficient adenoviruses cause an acute inflammatory response and immunity is
common. Several strategies have been used in attempts to improve efficiency and decrease toxicity of these
vectors.
One strategy to overcome pre-existing immunity is to employ species-specific adenoviral vectors in a
heterologous species. This approach should diminish the issue of pre-existing immunity. An ovine adenoviral
vector was used to deliver an AAT transgene to mice by intramuscular injection.5 Moderate doses of the vector
resulted in high serum AAT levels, and expression of the gene was limited to the site of injection, but expression
was transient and viral DNA was rapidly cleared and was accompanied by a cellular immune response. Viral doses
low enough to minimize the immune response still produced potentially therapeutic serum AAT levels (>100
ngml) and a second intramuscular injection produced serum AAT levels similar to the first. These studies address
some of the problems limiting the clinical potential of adenoviral vectors, but requirements for long-term repeated
dosing with unknown efficacy and safety are limitations to clinical application.
Although the bulk of work with liposomes as a technology for delivering genes in vivo has been with lipoplex
formulations (plasmid-cationic liposome complexes), a recent report uses liposomes as an escort for an
adenoviral vector. Yotdna et al6 encapsulated adenoviral vectors into bilamellar DOTAP:chol liposomes with the
hypothesis that the lipid-coated virus could enter cells independent of specific receptors and would also be
unaffected by virus-specific antibodies. Adenoviral vectors expressing either the reporter gene,
-galactosidase,
or the human alpha-1 antitrypsin gene were coated with liposomes and their transfection efficiency in cultured
cells with and without adenoviral receptors was compared with uncoated vector. The liposomeviral complexes
infected cells independent of receptors. The complexes also protected the virus from neutralization by human
antisera. In mice, intravenous delivery of viralliposome complexes expressing the AAT gene produced detectible
serum AAT levels for up to 30 days (uncoated vector expressed for only about a week). The coated vector also
expressed more exuberantly after a second dose delivered a month after the first. The acute inflammatory
response (as measured by serum levels of IL-6) was significantly less in animals given the liposomeviral complex
than those given the uncoated viral vector. More advanced formulations employing combinations of liposome and
viral technologies for gene delivery may eventually be clinically useful.
Adeno-associated viral vectors have low toxicity and effectively deliver an AAT transgene in animals
As it becomes obvious that adenoviral vectors are less than optimal, other viral vectors are being explored.
Adeno-associated virus (AAV) vectors have the advantage of apparent high in vivo transgene expression and low
toxicity. A single injection of an AAV vector expressing the AAT gene into the liver can achieve potentially
therapeutic serum AAT levels that persist for at least several weeks.7
However, in order to achieve potentially therapeutic serum AAT levels with intramuscular injection, it is necessary
to give fairly high doses of vector. A more efficient system is desirable both to minimize toxic potential and
because production of AAV vector is complicated and expensive. In addition, AAT deficiency causes liver as well
as lung disease. The liver disease results from aberrant intracellular trafficking of the misfolded mutant AAT
protein. So, although the lung disease might be prevented by achieving sufficient extracellular AAT
concentrations, correcting the liver disease will likely require expression of a normal AAT transgene in the
majority of hepatic cells since suppression of synthesis of the abnormal protein by expressing the normal gene
would only occur in cells expressing the normal transgene.
The group that undertook the original studies with intramuscular injection of an AAT-containing AAV vector now
report studies in which such vectors were injected into the portal vein of adult mice. 7 They compared results to
those with intravenous injection. They injected mice intravenously with an AAV construct containing the AAT gene
driven by a CMVactin hybrid promoter at varying doses and followed serum AAT protein concentrations. There
was a dose-related elevation in serum AAT with potentially therapeutic levels sustained for up to 50 weeks at the
higher viral doses. Southern blot analysis of liver tissue indicated that the vector was predominantly episomal at
an average density of 25 copies per cell. However, immunohistochemical analysis of liver tissue using an antibody
specific for the human protein showed a patchy distribution of the protein and a relatively small fraction of cells
(6.4%) containing the protein. There was no evidence of toxicity.
AAV vectors appear to have some distinct advantages over adenoviral vectors. The lack of an acute inflammatory
response, even on direct injection into the liver, and the prolonged expression of the normal AAT gene at levels
sufficient to produce potentially therapeutic serum AAT concentrations are encouraging for possible treatment of
the lung disease in AAT-deficient subjects. However, the efficacy of intraportal delivery of the normal AAT gene in
an AAV vector for treating the liver disease of AAT deficiency will likely require strategies to increase the fraction
of hepatocytes expressing the transgene.
Nonviral vectors can deliver a functioning AAT gene in vivo and can be designed to prolong transgene
expression
Nonviral vectors for gene therapy are limited by low efficiency of transgene expression and short duration of
expression. They continue to be attractive, however, because plasmids are not human pathogens in any setting
and therefore carry less potential for harm than most viral vectors. Recent studies attempt to address these
issues.
There are several reasons for the short duration of expression of transgenes delivered to mammals in plasmid
vectors. One reason is that plasmids do not replicate in mammalian cells so that host cell replication obligatorily
dilutes the vector. However, plasmids can be constructed to include eukaryotic replication initiation sites that
render them capable of replicating in cycle with replication of the host cell. Stoll et al8 constructed a plasmid
expression vector that contained the AAT gene and incorporated eucaryotic replication initiation sequences from
EpsteinBarr virus, EBNA1 and its family of binding sites. After intravenous injection using the interesting
technique of 'hydrodynamic injection' of the naked plasmid into mice, they found greater than 300
gml of AAT
in serum, and increased serum AAT concentrations were maintained for greater than 9 months after a single
administration of the vector. Although it is well established that these vectors containing EBV eukaryotic
replication initiation sequences undergo extrachromosomal replication in dividing cells in culture, the fact that
essentially nondividing liver cells in vivo retain the expressing vector for long period of time is somewhat
unexpected. The favorable safety profile of plasmid vectors over viral vectors makes these observations
interesting as the technology for clinical application develops.
Intravenous injection of linear DNA encoding the AAT gene driven by an RSV promoter into mice is reported to
achieve 10100 fold higher serum AAT concentrations than similar administration of circular DNA with expression
persisting for at least 9 months.9 The administered DNA localized to the liver and rapidly formed large
unintegrated concatemers. This physical form of the DNA may contribute to more exuberant and prolonged in
vivo expression than seen with circular vectors. However, those results were obtained after rapid intravenous
injection of 40
g of DNA in 2 ml saline. Assuming a mouse of approximately 25 g, similar administration to a 50
kg human would require rapid intravenous injection of 80 mg of DNA in 4 l of saline, so that the clinical
applicability of this approach is questionable at present.
The clinical potential of cationic liposomeplasmid complexes (lipoplexes) remains interesting because of their
apparent safety, but their limitations are similar to those of other nonviral technologies. Dasi et al10 covalently
coupled an asialoglycoprotein (asialofetuin) ligand for a unique hepatocyte receptor to either cationic or anionic
liposomes and administered lipoplexes containing these modified liposomes and a plasmid vector containing the
AAT gene intravenously to mice. They produced serum AAT levels of 50300 ngml that persisted for up to 12
months with DNA doses of 200 ngmouse. The asialoglycoprotein conjugate conferred considerably greater
efficacy than unmodified liposomes, either cationic or anionic.
RNADNA oligonucleotide (RDA)-directed repair of the defective AAT gene
Although not uniformly accepted as a valid approach to therapy, a newly reported technology under development
may permit site-specific correction of a single base carried in a chromosome and therefore, in theory, provide a
basis for correcting the most common genetic mutations leading to AAT deficiency. 11 This technology (designated
Genoplasty (Valigen, Lawrenceville, NJ, USA)) uses RNADNA chimeric molecules with short targeting sequences
that are completely homologous with the region of interest except for the single base to be corrected. These
regions are thought to be recognized and corrected by the cell's endogenous DNA repair systems. In vivo
feasibility of this technology has apparently been reported for some genes in mice, but application to AAT
deficiency will require confirmation of the concept and developing efficient systems for delivering the chimeric
molecules to the liver that is efficient enough to correct the requisite number of hepatocytes.
Conclusions
AAT deficiency should be a good target for gene therapy since it is a single gene defect, the molecular biology is
well-understood and the gene encodes a secreted protein. The possibility that expressing the normal gene locally
in lung cells may preclude the necessity for normal circulating levels of the protein may make gene therapy more
practical. Developing vectors and technologies encourage cautious optimism that toxicity can be reduced and
efficacy increased.
References
1 Flotte TR. Recombinant adeno-associated virus gene therapy for cystic
fibrosis and alpha1-antitrypsin deficiency. Chest 2002; 121: 98S102S.
2
http:www.alphaone.orgresearchclinical_trialsGene_Therapy_Trials.htm.
2002.
3 Brigham KL et al. Transfection of nasal mucosa with a normal alpha-1
antitrypsin (AAT) gene in AAT deficient subjects: comparison with protein
therapy. Human Gene Therapy 2000; 11: 10231032.
4 Albelda SM, Wiewrodt R, Zuckerman JB. Gene therapy for lung
disease: hype or hope? Ann Int Med 2000; 132: 649660.
5 Loser P, Hillgenberg M, Arnold W, Both GW, Hofmann C. Ovine
adenovirus vectors mediate efficient gene transfer to skeletal muscle.
Gene Ther 2000; 7: 14911498.
6 Yotdna P et al. Bilamellar cationic liposomes protect adenovectors
from preexisting humoral immune responses. Mol Ther 2002; 5:
233241. Article PubMed
7 Song JE et al. Stable therapeutic serum levels of human alpha-1
antitrypsin (AAT) after portal vein injection of recombinant adenoassociated virus (rAAV) vectors. Gene Ther 2001; 8: 12991306. Article
8 Stoll SM et al. Epstein-Barr virushuman vector provides high-level
long-term expression of 1-antitrypsin in mice. Mol Ther 2001; 4:
122129. Article PubMed
9 Chen Z-Y et al. Linesa Dnas concatemerize in vivo and result in
sustained transgene expression in mouse liver. Mol Ther 2001; 3:
403410. Article PubMed
10 Dasi F, Benet M, Crespo A, Alino SF. Asialofetuin liposomemediated human 1- antitrypsin gene transfer in vivo results in stationary
long-term gene expression. J Mol Med 2001; 79: 205212.
11 Metz R et al. Mode of action of RNADNA oligonucleotides: progress in
the development of gene repair as a therapy for alpha1-antitrypsin
deficiency. Chest 2002; 121: 91S97S.
Figures
Figure 1 Concentrations of transgene-derived normal alpha-1 antitrypsin
protein in nasal lavage fluid from subjects with PiZZ AAT deficiency. Time
course of responses to intranasal lipoplex delivery of a normal AAT gene
with the untreated contralateral nostril serving as control. Data are
normalized to total protein concentration. (Reprinted with permission
from reference 3.)
Figure 2 AAT and IL-8 concentrations in nasal lavage fluid from PiZZ
AAT-deficient subjects when on no therapy, while receiving treatment
with intravenous AAT protein at weekly intervals and at day 5 (time of
peak transgene expression) after lipoplex intranasal transfection with the
normal AAT gene. (Reprinted with permission from reference 3.)
January 2003, Volume 10, Number 2, Pages 95-99
December 2002, Volume 9, Number 24, Pages 1647-1652
Table of contents
Previous Article Next [PDF]
Review
Gene Therapy Progress and Prospects: Nonviral vectors
T Niidome and L Huang
Center for Pharmacogenetics, School of Pharmacy, University of
Pittsburgh, Pittsburgh, PA, USA
Correspondence to: L Huang, Center for Pharmacogenetics, School of
Pharmacy, 633 Salk Hall, University of Pittsburgh, Pittsburgh, PA
15213, USA
Abstract
The success of gene therapy is largely dependent on the development of the gene delivery
vector. Recently, gene transfection into target cells using naked DNA, which is a simple and safe
approach, has been improved by combining several physical techniques, for example,
electroporation, gene gun, ultrasound and hydrodynamic pressure. Chemical approaches have
been utilized to improve the efficiency and cell specificity of gene transfer. Novel gene carrier
molecules, which facilitate DNA escape from the endosome into the cytosol, have been
developed. Several functional polymers, which enable controlled release of DNA in response to
an environmental change, have also been reported. Plasmids with reduced number of CpG
motifs, the use of PCR fragments and the sequential injection method have been established for
the reduction of immune response triggered by plasmid DNA. Construction of a long-lasting gene
expression system is also an important theme for nonviral gene therapy. To date, tissue-specific
expression, self-replicating and integrating plasmid systems have been reported. Improvement
of delivery methods together with intelligent design of the DNA itself has brought about large
degrees of enhancement in the efficiency, specificity and temporal control of nonviral vectors.
Gene Therapy (2002) 9, 16471652. doi:10.1038/sj.gt.3301923
Keywords
non-viral vector; gene therapy; cationic lipid; cationic polymer; naked DNA
In brief
Progress





Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene
expression in vivo
Gene delivery using a chemical carrier: to establish functional gene delivery in vivo
Nonviral vector modifications with peptides to increase intracellular gene delivery
Reduction of immune responses by modifying the administration protocol or the composition of
the DNA
Design of tissue-specific, self-replicating and integrating plasmid expression systems to facilitate
long-lasting gene expression
Prospects






Physical techniques for gene delivery into cells such as electroporation, with and without
adjuvants, will be significantly optimized
Knowledge of the interaction of naked DNA with serum components and cell surface receptors
will continue to accumulate. Immune responses originating from CpG motifs and nonviral gene
carriers will diminish
The structure of gene carriers will be further optimized and tailored for specific uses such as
systemic administration, local injection or organ-specific delivery
Novel ligands for targeted delivery of DNA will be found
Translocation mechanisms for plasmid DNA within the cell will be identified  these may provide
novel strategies for efficient delivery
More tissue-specific, site-specific integrating or self-replicating plasmid vectors are likely to
appear
Introduction
The development of gene carriers for effectively delivering genes into cells has attracted a great deal of
attention in recent years. Nonviral vectors should circumvent some of the problems occurring with viral
vectors such as endogeneous virus recombination, oncogenic effects and unexpected immune response.
Further, nonviral vectors have advantages in terms of simplicity of use, ease of large-scale production and
lack of specific immune response. These techniques are categorized into two general groups: (1) naked DNA
delivery by a physical method, such as electroporation and gene gun and (2) delivery mediated by a
chemical carrier such as cationic polymer and lipid. In this review, we focus on the progress made over the
last two years and discuss techniques in these two categories.
Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene
expression in vivo
Many mechanical techniques are included in this section. The simplest way for administration of DNA is
direct injection of naked plasmid DNA into the tissue or systemic injection from a vessel. Use of naked DNA
without any carrier molecule is also the safest method. Little attention needs to be paid on issues of
complex formation and its safety assessment. So far, site of the direct injection includes skeletal muscle,
1
liver, thyroid, heart muscle, urological organs, skin and tumor. Systemic injection is also a convenient
route for gene administration. However, owing to rapid degradation by nucleases in the serum and
clearance by the mononuclear phagocyte system, the expression level and the area after injection of naked
DNA are generally limited. Various physical manipulations have been used to improve the efficiency.
Electroporation, bio-ballistic (gene gun), ultrasound, hydrodynamics (high pressure) injection and others
2
have been established (Figure 1).
Electroporation, the application of controlled electric fields to facilitate cell permeabilization, is used for
3
enhancement of gene uptake into cells after injection of naked DNA. In addition, electroporation can
achieve long-lasting expression and can be used in various tissues. Skin is one of the ideal targets because
4
of the ease of administration. Drabick et al established cutaneous transfection method for the purpose of
DNA vaccination. To optimize the condition of electroporation, factors such as dose of DNA, electrode shape
and number, electrical field strength and duration have been optimized for expression of hepatitis B surface
4
5
6
antigen, erythropoietin and IL-12. High ionic strength in the injection medium is also favorable for gene
7
expression in the skin. Muscle is also a good candidate for electroporation. Most of reports published
recently relate to immunological applications. For DNA vaccination, potent immune responses against
hepatitis B surface antigen and HIV gag protein were obtained by electroporation of muscle after
8
9
intramuscular injection of naked plasmid DNA. Therapeutic effect of cytokines, such as IL-12 and IFN10
,
for inhibition of tumor growth located at a distant site has been demonstrated. IL-12 was also employed
11
for electroporation after intratumor injection.
Our laboratory recently reported the use of a syringe
electrode, with which same transfection efficiency could be achieved by using much lower electric field
12
strength than that of conventional electrode. Tissue damage by the electric field is thus minimized.
13
Electrically mediated DNA delivery to hepatocellular carcinoma in the liver was reported by Heller et al.
All
of the electroporation protocols employ local injection of the plasmid DNA. However, our group recently
demonstrated efficient gene transfer to the liver by electroporation following tail-vein injection of naked
14
DNA.
Comparing with local injection of DNA to the liver, systemic injection has the advantage of
delivering genes more evenly to the liver.
Gene gun can achieve direct gene delivery into tissues or cells. Shooting gold particles coated with DNA
allows direct penetration through the cell membrane into the cytoplasm and even the nucleus, bypassing
the endosomal compartment. Majority of the efforts reported in the last 2 years are to introduce genes for
15,16
17,18,19
antigen or cytokines such as IL-12 into skin
or liver
for vaccination and immunotherapy,
respectively. However, a disadvantage of this method is the shallow penetration of DNA into the tissue.
Ultrasound can increase the permeability of cell membrane to macromolecules such as plasmid DNA.
Indeed, enhancement of gene expression was observed by irradiating ultrasonic wave to the tissue after
20,21
injection of DNA.
Since ultrasound application is flexible and safe, its use in gene delivery has a great
advantage in clinical use. Recently, it was reported that combination of microbubble with ultrasound could
further increase the gene expression level. Microbubbles, or ultrasound contrast agents, lower the threshold
for cavitation by ultrasound energy. In most cases, perfluoropropane-filled albumin microbubbles or Optison
(Mallinckrodt, San Diego, USA) were used as microbubbles. It was modified with plasmid DNA before
injection, followed by irradiation of ultrasound. At present, this technique is used for gene delivery to
22,23,24,25,26
26,27
28
vascular cells,
muscle
and fetal mouse.
Hydrodynamic injection, a rapid injection of a large volume of naked DNA solution (eg 5
g plasmid DNA
injected in 58 s in 1.6 ml saline solution for a 20 g mouse) via the tail vein, can induce potent gene
transfer in internal organs, especially the liver. Budker et al hypothesized that naked plasmid DNA is taken
29
up by receptor-mediated pathway by hepatocytes.
Certain DNA receptors have been found in various
30
tissues;
however, their function has not been elucidated. It has been proposed that the injected DNA
solution accumulates mainly in the liver because of its flexible structure, which can accommodate large
volume of solution, and the hydrostatic pressure forces DNA into the liver cells before it is mixed with blood.
Furthermore, breaking of the endothelial barrier by the pressure has been proposed as the major
mechanism responsible for the highly efficient expression in the liver. Recently, our group reported that
external massage of the abdomen after small-volume injection of DNA via the tail vein can enhance gene
31
expression in the liver.
The observation suggests that mechanical stretching of the endothelial barrier
may affect uptake of DNA into the hepatocytes. This pressure-mediated transfection method can be
applicable to other tissues. Wolff's group showed that large-volume injection with high speed via the portal
vein of liver or the artery of limb muscles achieved high gene expression in the respective organ.
29,32
Our group has demonstrated that significant gene expression can be achieved in the liver by transiently
restricting blood flow through the liver immediately following peripheral intravenous injection of naked
33
DNA.
Occlusion of blood flow either at vena cava or at hepatic artery and portal vein increased the
expression level in the liver. Presumably, the injected DNA is internalized into the hepatic cells by receptor29
mediated mechanism as proposed by Budker et al
or via a nonreceptor-mediated pathway. However, the
binding of DNA to the surface of hepatic cells might be so weak that DNA could be easily dissociated and
washed away by the blood flow in the normal physiological condition. Only when the blood flow is
transiently stopped, the DNA can stably bind with the receptor and be internalized into cells. A similar
uptake of DNA by the diaphragm muscle cells was achieved by a brief occlusion of the blood flow through
34
the diaphragm immediately after peripheral intravenous injection of DNA.
Gene delivery using a chemical carrier: to establish functional gene delivery in vivo
Novel carriers to achieve high-level gene expression and functional delivery have been designed. Gene
carriers can be categorized into several groups: (1) those forming condensed complexes with the DNA to
protect the DNA from nucleases and other blood components; (2) those designed to target delivery to
specific cell types; (3) those designed to increase delivery of DNA to the cytosol or nucleus; (4) those
designed to dissociate from DNA in the cytosol and (5) those designed to release DNA in the tissue to
achieve a continuous or controlled expression. Lipids and polymers are mainly used for gene delivery.
Lipid-mediated gene delivery
Liposome-based gene delivery, first reported by Felgner in 1987, is still one of the major techniques for
gene delivery into cells. In 1990s, a large number of cationic lipids, such as quaternary ammonium
detergents, cationic derivatives of cholesterol and diacylglycerol, and lipid derivatives of polyamines, were
reported. However, the development of novel types of lipid molecules appears to be saturated, and most of
the efforts have shifted to improving efficacy by the modification listed above, as well as to specific in vivo
applications. We will highlight a number of new concepts that have appeared in the last 2 years.
The reductionoxidation (redox) sensitive character of thiol groups has been exploited to control DNAlipid
35
complex formation. Dauty et al
reported a dimerizable cationic detergent, which contains free thiol, amine
and alkyl groups. This alkylated ornithinyl cysteine derivative forms a complex with plasmid DNA.
Subsequent oxidation of the thiol groups to disulfides converts the complex into stable nanometric particles.
The particle is made of a single molecule of condensed plasmid DNA with a uniform diameter of less than 40
nm and showed reasonable transfection activity in vitro. Practical advantages include the small size for in
vivo gene delivery (improved particle diffusion) and that the disulfide bonds should be reduced to thiols in
the cytosol because of the reductive environment provided by intracellular glutathione, thus resulting in
DNA release.
Peptide-mediated gene delivery
36
Redox-sensitive thiols have also been incorporated into peptide gene carriers. McKenzie et al
developed
peptides containing a cysteine residue and a continuous sequence of lysine residues, for example, Cys-Trp36
Lys18.
This peptide can also condense plasmid DNA, and the thiol group is spontaneously oxidized,
resulting in a highly stable complex with potent transfection activity in vitro. Cross-linking the peptide
caused elevated gene expression, without increasing DNA uptake by the cells, suggesting that intracellular
37
release of the DNA triggered by disulfide bond reduction played a key role. Furthermore, Park et al
have
also synthesized sulfhydryl cross-linking poly(ethylene glycol)-peptides (for stealth activity) and
glycopeptides for targeted delivery of genes in vivo.
Polymer-mediated gene delivery
38
Wightman et al
systematically compared the ability of branched and linear PEIDNA complexes to
transfect cells in vitro and in vivo at various aminephosphate ratios and salt concentrations. They showed
that salt-free DNA complexes of linear PEI (22 kDa), which showed high transfection efficiency in the lung,
were small, but subsequently aggregated when salt was added. In contrast, DNA complex of branched PEI
(25 kDa), which showed low transfection efficiency in most of the organs, remained small even after salt
was added. The greater efficiency of linear PEI in vivo might be because of a dynamic structure change of
the complex under high salt concentrations as found in blood. Understanding of the interaction between
linear PEI and DNA could help in designing future vectors.
Biodegradable polymers are known for their low toxicity and high biocompatibility. Recently, a
biodegradable polymer, poly -(4-aminobutyl)-L-glycolic acid (PAGA), a derivative of poly-L-lysine, in
39
which the ester link is substituted with amide, was designed by Kim's group.
This biodegradable and
water-soluble polymer condenses DNA and subsequently releases DNA upon hydrolysis of the polymer. The
complex showed higher in vitro gene transfection efficiency with lower cytotoxicity than poly-L-lysine.
Significant expression of murine IL-10 was observed in the serum after tail-vein injection of PAGADNA
complexes, and the systemic administration of murine IL-10 gene with PAGA into NOD mice markedly
40
reduced insulitis.
The murine IL-12 gene was also injected with PAGA into subcutaneous tumors in BALBc
41
mice. Significant level of the protein expression and reduction of tumor growth was observed.
Recently,
other types of biodegradable polymers were reported by Kim's and Leong's groups, who have synthesized
42
43
cationic copolymers derived from PEI and polyethylene glycol (PEG)
and cationic polyphosphoester,
respectively.
Thermosensitive polymers can control the release of encapsulated DNA in response to temperature changes
44
that lead to swelling or de-swelling of the hydrated polymer. Kurisawa et al
synthesized a
thermosensitive copolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate
(DMAEMA)-co-butylmethacrylate (BMA), and investigated its thermosensitive character and transfection
44
efficiency at different incubation temperatures.
A polymer containing 8 mol% DMAEMA and 11 mol% BMA
had a low critical solution temperature of 21°C and complex formationdissociation was modulated by
temperature alteration. Transfection efficiency in vitro also depended on the incubation temperature. Kim's
group have developed the biodegradable and thermosensitive polymer, PEGpoly(D,L-lactic acid-coglycolic acid) (PLGA)PEG triblock co-polymer. This nonionic, hydrophilic polymer shows temperature45,46
dependent solution-to-gel transitions
and can be loaded with plasmid DNA in aqueous phase at
420°C. At above 303°C (eg, at the body temperature), the solution-to-gel transition occurs. It is
conceivable that DNA could be formulated and injected in the polymer solution at room temperature, and
slowly released from the hydrogel for prolonged transfection at the injection site.
PEG-PLL block contains a hydrophilic part consisting of PEG and a DNA-binding moiety consisting of PLL and
forms self-assembling particles with DNA in a core-shell structure with electrostatic interaction as the main
driving force. These polyion complex micelles are water-soluble and nuclease-resistant nanoparticles,
suitable for in vivo gene delivery. Thus, DNA in the complex remained intact in the blood stream for 30 min,
47
although gene expression after injection via the tail vein of mice was only seen in the liver.
Nonviral vector modifications with peptides to increase intracellular gene delivery
48
49
Many anionic pH-sensitive peptides
and cationic fusogenic peptides
show an enhancing effect on gene
expression mediated by cationic liposome and PEI, respectively. These peptides show membrane disrupting
activities in weakly acidic condition, which is similar to that in the endosome compartment, and could
50
enhance the translocation of the DNA to cytosol. Rittner et al
reported that a bifunctional peptide with
both DNA-binding and membrane-disrupting activities showed significant gene expression in the lung after
50
tail-vein injection.
Inefficient entry of DNA into the nucleus is a major limiting step in the development of nonviral gene
delivery system. The problem is particularly serious in nondividing cells, where entry into the nucleus is
thought to occur only through the nuclear pore complex. To achieve active transport to the nucleus, nucleus
localizing signal (NLS) peptides have been widely used. Recent effort has been summarized in excellent
51,52,53
reviews.
In most cases, NLS is conjugated with a gene carrier such as PEI, or with DNA directly.
Reduction of immune responses by modifying the administration protocol or the composition of
the DNA
Although it is well known that nonviral gene delivery produces a less severe immune responses than virusmediated delivery, problems still remain. The DNAgene complex is recognized by macrophages, dendritic
and other immune cells. For cationic liposomes, toxicity relates to the rapid induction of proinflammatory
54
cytokines such as TNF- , IL-6, IL-12 and IFN- .
This response stems from the stimulation of the
immune cells by the unmethylated CpG motifs in the plasmid DNA. Various approaches have been taken to
55
reduce this inflammatory toxicity, including elimination of CpG motifs in the plasmid DNA,
use of PCR
56
fragments with reduced numbers of CpG motifs
and active targeting of the DNA to the endothelium,
57
which minimizes interaction with immune cells.
Furthermore, sequential injection of cationic liposomes
14
58
followed by naked plasmid DNA, first reported by Liu's group,
reduces the inflammatory response.
Thus, when plasmid DNA was injected into the tail vein of mice 25 min after the injection of cationic
liposome, 5080% lower levels of proinflammatory cytokines (compared to lipoplexes) were observed,
without affecting gene expression level in the lung.
Design of tissue-specific, self-replicating and integrating plasmid expression systems to facilitate
long-lasting gene expression
Producing sustained gene expression is also an important goal for nonviral gene therapy. Tissue-specific
expression systems can produce stable expression by reducing the probability of inducing an immune response to the transgene. Thus, Kay's group constructed a plasmid DNA containing the apolipoprotein E locus
control region, 1-antitrypsin promoter, human factor IX minigene sequence including a portion of the first
59
intron, 3'-untranslated region, and the bovine growth hormone polyadenylation signal.
When the plasmid
DNA was delivered to mouse liver by hydrodynamic injection, it produced not only increased gene
expression of factor IX (in the therapeutic range), but also maintained these levels for at least 10 months.
Furthermore, a linear DNA expression cassette originating from this plasmid showed 10- to 100-fold higher
60
expression than the closed circular DNA for a period of 9 months.
The EpsteinBarr virus (EBV)-based plasmid vector is known to self-replicate in cells. It carries two genetic
elements from EBV, the EBV nuclear antigen 1 (EBNA1) gene and the oriP element. The EBNA1 protein
binds to oriP, and facilitates the replication of the plasmid in synchrony with chromosomal DNA.
Furthermore, the EBNA1 also facilitates nuclear localization of the plasmid DNA. This approach has been
61
used for tumour suicide therapy
(coupled to a polyamidoamine dendrimer), long-term expression of the
62
2-adrenergic receptor in cardiomyocytes,
and efficient and long-lasting luciferase expression in murine
63
64
liver after hydrodynamic injection.
Stoll et al
have also reported high-level and long-lasting expression
of the 1-antitrypsin gene in mouse liver using the hydrodynamic injection protocol.
Controllable integration of plasmid DNA into the genome of mammalian cells would also provide long-lasting
gene expression. Reconstitution of an ancient transposon, Sleeping Beauty, from sequence alignment of
nonfunctional remnants of members in the Tc1mariner superfamily of transposons within the genomes of
65
salmonids, provided the first functional transposon for use in vertebrate species.
Sleeping Beauty has
been used to accomplish stable chromosomal integration of functioning genes in somatic cells of adult
66
mice.
In addition, several phage integrases and their corresponding recognition elements, which can
67,68,69,70
mediate integration into mammalian chromosomes, were reported by Calos's group.
Although
the integration efficiency of integration system is still low, this technology may one day enable site-specific
and high-efficiency integration into the host chromosome without the potential for mutagenesis.
Summary
To establish efficient and safe gene delivery in vivo, a number of new techniques and concepts have been
introduced in the last 2 years, with improvements in targeted or controlled delivery of genes. However, we
are still far from the perfect gene carrier suitable for clinical use. We have come a long way in
understanding the cellular barriers which prevent proper delivery of DNA, but still relatively ignorant about
factors controlling the stability, pharmacokinetics and biodistribution of nonviral vectors. Much of the above
effort has been carried out in rodents and whether the new improvements are applicable to larger animals
remains to be seen. We are still far from the perfect gene carrier suitable for clinical use, and much more
work is still ahead of us.
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Figures
Figure 1 Overview of nonviral gene delivery technologies. Different
injection routes of naked DNA and enhancement strategies are
outlined.
November 2002, Volume 9, Number 22, Pages 1487-1491
Table of contents
Previous Article Next [PDF]
Progress And Prospects
Post-intervention vessel remodeling
J Rutanen1, H Puhakka1 and S Ylä-Herttuala1,2,3
AI Virtanen Institute, University of Kuopio, Kuopio, Finland
1
Department of Medicine, University of Kuopio, Kuopio, Finland
2
Gene Therapy Unit, University of Kuopio, Kuopio, Finland
3
Correspondence to: S Ylä-Herttuala, Department of Molecular
Medicine, AI Virtanen Institute, University of Kuopio, PO Box 1627,
FIN-70211 Kuopio, Finland
Abstract
By-pass surgery and percutaneous transluminal (coronary) angioplasty, PT(C)A, are standard
techniques for the treatment of vascular occlusions. Their usefulness is limited by by-pass
graft failure and restenosis occuring after the procedures. Twenty percent of patients treated
with PTCA/PTA need a new revascularization procedure within 6 months, despite a successful
procedure. Stents are used to prevent restenosis in selected lesions, but in-stent restenosis
also remains an important clinical problem. In this review we discuss progress of gene
therapy for the treatment of post-PT(C)A restenosis, in-stent restenosis and by-pass graft
stenosis over the last 2 years (20002002).
Gene Therapy 2002 9, 14871491. doi:10.1038/sj.gt.3301866
Keywords
artery; stenosis; intimal hyperplasia; vein graft; neointima
Progress





Low gene transfer efficiency in blood vessels is still a significant problem
Identification of new treatment genes has provided more opportunities for therapy
Combination therapy may be more efficient than single gene treatment and first evidence of
prolonged therapeutic effect has been achieved
Inhibition of vein graft stenosis using gene therapy has been successful in animal models
Clinical trials have indicated cautious optimism only in vein graft stenosis
Prospects










Low gene transfer efficiency in blood vessels is still a significant problem
Identification of new treatment genes has provided more opportunities for therapy
Combination therapy may be more efficient than single gene treatment and first evidence of
prolonged therapeutic effect has been achieved
Inhibition of vein graft stenosis using gene therapy has been successful in animal models
Clinical trials have indicated cautious optimism only in vein graft stenosis
Identification of new treatment genes should continue
Improved gene delivery methods to blood vessels need to be developed
Regulated, targeted vectors, gene cocktails and combination therapies need to be studied
Randomized, blinded, controlled phase II/III studies are needed to establish safety and
efficacy of gene therapy
Other novel methods, such as arterial grafting and drug eluting stents may overcome some
problems related to post-intervention vessel occlusion
Low gene transfer efficiency in blood vessels is still a significant problem
1,2
Various types of catheters are available for gene transfer into the vessel wall.
Unfortunately, even
with powerful viral vectors gene transfer efficiency through human atherosclerotic lesions and lipid-rich
3
atheroma is 5%, and results in a potentially harmful biodistribution of the vector. Therefore, there is
great interest in developing better strategies for efficient, targeted gene delivery into the vessel wall.
4,5,6,7,8,9,10
Accordingly several biologic targeting systems have recently been introduced.
In spite of
relatively low transfection efficiency adenoviruses are still the most widely used viral vectors for vascular
2,11
applications since they can transfect both proliferating and non-proliferating cells.
However,
3
adenoviruses also transfect many unwanted organs and peripheral blood monocytes. Therefore, novel
6,9,10,12,13
methods to target adenoviruses to the vessel wall have been developed.
These include
matrix metalloproteinase-2 and -9 (MMP-2 and -9) targeted tissue inhibitor of matrix metalloproteinase10
14
1 (TIMP-1) encoding adenoviruses,
integrin targeted human interleukin-2 encoding adenoviruses
9
and endothelial cell targeted adenoviruses. However, longer expression times are probably needed for
better clinical efficacy. Thus, adeno-associated viruses (AAV) have been successfully used to transduce
15
rabbit jugular veins with expression lasting beyond 30 days,
and a method to target AAV gene therapy
8
to vascular endothelial cells has been described. Furthermore, modified retrovirus, Semliki Forest virus,
Baculovirus and herpes simplex virus vectors have been used for vascular gene transfer in vivo, but
7,16,17,18
these vectors have not yet solved all problems related to vascular gene transfer.
The most
widely used non-viral vector for vascular gene therapy is plasmid DNA with or without carrier molecules.
Due to the safety aspects and relatively easy processibility, there is a great interest in the development
of targeted non-viral gene delivery methods. However, relatively low tranfection efficiency has limited
their use in vascular applications. It should be pointed out that different treatments strategies require
different gene transfer efficiencies, eg with secreted compounds, such as growth factors or NO, even a
relatively inefficient vector may be useful, whereas antisense or decoy constructs for cell cycle mediators
need to be delivered in the majority of vascular cells before a therapeutic effect can be expected.
Therapeutic ultrasound has also proven efficient in augmenting intravascular gene delivery.
19
For the
treatment of in-stent restenosis a good method to deliver vectors locally to the target area is the stent
itself. Klugherz et al succeeded in transducing porcine coronary arteries with a plasmid DNA eluting
20
polymer-coated stent.
Also, they reported approximately 6% transfection efficiency using a stent21
based antibody-tethered adenoviral gene transfer system.
An ex vivo approach using genetically
engineered cells attached to the stent surface could be another possibility to achieve long-term
transgene expression. Panetta et al have demonstrated GFP expression 1 month after implantation of a
22
stent carrying engineered autologous smooth muscle cells (SMCs) into porcine coronary artery.
However, these approaches still suffer from fast wash-out of the vectors from the stent coating and
detachment of the seeded cells from the stent struts.
Identification of new treatment genes has provided more opportunities for therapy
Smooth muscle cell migration and proliferation are key factors in the development of restenosis, in-stent
23
restenosis and vein graft stenosis
and most gene therapy strategies are directed towards these targets
(Table 1). Early markers of SMC activation, such as oncogenes are detectable shortly after arterial injury.
Antisense oligonucleotides directed against oncogenes and cell cycle regulators have been used to
decrease neointimal thickening in animal models. Promising results have been obtained with antisense
24
25
26
oligonucleotides against NF B,
E2F
and c-myc.
Also, adenoviral Gax (growth arrest homeobox)
27
gene transfer has been shown to reduce neointimal hyperplasia in stented rabbit iliac arteries.
The
platelet-derived growth factor (PDGF) gene family is one of the most potent chemoattractants of vascular
SMC. PDGFs mediate neointimal growth after vascular injury and this can be prevented by inhibition of
28
PDGF expression with antibodies against PDGF or its receptors.
However, clinical use of antibodies
carries the risk of immunoreactivity and thus gene-based approaches against PDGFs have been
29
introduced.
MMPs are also important effectors in the migration of SMCs and can be inhibited by
overexpressing tissue inhibitors of metalloproteinases (TIMPs). Gene transfer of TIMP-1 has been shown
10
to reduce neointimal growth in rat and rabbit denudation models.
Since intravascular manipulation causes damage to endothelium, it is hypothesized that rapid reendothelialization of arterial wall after balloon dilatation should reduce restenosis. Members of the
vascular endothelial growth factor (VEGF) family induce endothelial cell proliferation and migration.
Hiltunen et al demonstrated that both VEGF-A and VEGF-C gene therapy reduced restenosis after arterial
30
31
injury.
Also, gene therapy with VEGF-D attenuates neointimal growth in rabbits.
In addition to reendothelization, the effects of VEGFs may be at least partially due to increased nitric oxide (NO)
production which inhibits SMC migration and proliferation. Hepatocyte growth factor (HGF) is another
gene that is reported to reduce neointimal hyperplasia through re-endothelization and increased NO
32
production.
Aiming at increased NO production Janssens et al have previously shown that eNOS gene
transfer reduced neointima formation in a rat model and Varenne et al noticed the same effect with
eNOS gene transfer in a pig model. Also, van der Leyen et al have achieved prevention of restenosis with
iNOS gene transfer. Further, extracellular superoxide dismutase (EC-SOD) gene transfer has been shown
33
to reduce neointima formation in a rabbit model.
Platelet-activating factor (PAF)-like phospholipids are
characteristic of proinflammatory conditions. These phospholipids are inactivated by PAF-acetylhydrolase
(PAF-AH). Quarck et al showed that PAF-AH prevents neointima formation and reduces spontaneous
34
atherosclerosis in apolipoprotein E-deficient mice.
It has also recently been demonstrated that a
therapeutic effect on intimal hyperplasia can be achieved by inhibiting tissue factor (TF) with TF pathway
35,36,37
inhibitor (TFPI).
Genes that have an effect on various pathological processes in the vessel wall,
such as VEGFs, HGF or E2F decoy may offer the most promising tools for gene therapy against post-
intervention vessel occlusion. On the other hand, similar effects may be achieved with combination
therapy.
Combination therapy may be more efficient than single gene treatment and first evidence of
prolonged therapeutic effect has been achieved
The majority of gene therapy studies for restenosis, in-stent restenosis and vein graft stenosis have
evaluated only single genes. Considering the complex pathophysiology of these events it is possible that
combination therapy or 'gene cocktails' may provide a better treatment effect. Thus, utilization of
different mechanisms which are known to reduce restenosis may lead to an additive treatment effect. So
far, there have been only a few reports on combination therapy and its effects on these remodeling
processes. Puhakka et al studied peptide re-targeted TIMP-1 plus VEGF-C combination gene therapy in a
38
rabbit model and found a prolonged treatment effect.
Leppänen et al have shown that combination of
VEGF-C gene transfer and treatment with PDGF receptor kinase inhibitor STI571 leads to a long-term
39
reduction in neointima formation in a balloon-denuded rabbit aorta.
These studies have shown
28,30
prolonged therapeutic effect compared with single treatment with VEGF-C or STI571.
As another
example of combination therapy Atsuchi et al reduced neointima formation in a rabbit model by brief
irrigation with tissue factor pathway inhibitor (TFPI) combined with adenovirus-mediated local TFPI gene
37
transfer.
Inhibition of vein graft stenosis using gene therapy has been successful in animal models
Surgical approach provides an opportunity to use ex vivo approach, which allows longer contact time
with vector and may result in a better gene transfer efficiency. After surgical revascularization vein grafts
are exposed to high blood pressure, which causes endothelial cell damage, platelet and leukocyte
adhesion, thrombosis, matrix destruction and SMC proliferation. Although neointimal hyperplasia caused
by SMC proliferation and migration may not be the most prominent factors producing significant
stenosis, it exposes veins to the later development of graft atheroma. Vein graft stenosis has similar
features to neointimal hyperplasia in arteries, and thus the same therapeutic approaches may apply.
40
Suppressing SMC migration and proliferation by inhibiting MMPs with TIMP-1, TIMP-2 or TIMP-3
has
been shown to inhibit neointima formation. TIMP-2 reduces neointima formation in human saphenous
40
veins and TIMP-3 decreased stenosis in human and porcine veins.
Also, long-term stabilization of the
vein grafts with ex vivo pressure-mediated E2F decoy oligonucleotide gene transfer has been
41
achieved.
Endothelial damage predisposes vein grafts to thrombus formation and stimulates SMC
migration to intima. Thus, re-endothelization of the graft should be useful. Ohno et al demonstrated
accelerated re-endothelization with suppressed thrombogenesis and neointimal hyperplasia using gene
42
transfer of C-type natriuretic peptide (CNP).
An emerging new area of gene therapy is prevention of
stenosis in dialysis access grafts and arterio-venous loops.
Clinical trials have indicated cautious optimism only in vein graft stenosis
In spite of promising results achieved in preclinical animal models, only a few clinical trials have been
started (Table 2). To date, there are only two successful reports on the prevention of neointimal
hyperplasia in clinical trials. Previously, Mann et al reduced bypass vein graft failure rate with E2F
antisense decoy by using an ex vivo gene transfer method and Grube et al have now reported significant
43
efficacy of the same construct in coronary vein grafts.
Laitinen et al demonstrated the safety and
feasibility of intravascular catheter-mediated VEGF-A plasmid/liposome gene transfer to human
coronaries in conjunction with PTCA in a randomized, controlled phase I study, but no efficacy was
1
detected in control angiography. In a randomized, controlled phase II study of catheter-mediated
VEGF-A gene transfer with plasmid/liposome or adenovirus to infrainguinal arteries after PTA, increased
vessel formation was seen in the VEGF treatment groups in the follow-up angiography 3 months after the
44
gene transfer, but no effect was found on restenosis.
The first anti-sense-based clinical study for in45
stent restenosis was recently published by Kutryk et al.
They used antisense oligonucleotides against
c-myc to inhibit cell proliferation in stented lesions. Although the pre-clinical results in preventing intimal
hyperplasia with this cell cycle regulator were very promising, the study did not show any therapeutic
45
effect measured by intravascular ultrasound and angiography.
This may be explained by compromised
efficacy of intravascular delivery compared with ex vivo approach used for vein graft stenosis. Thus,
vascular gene therapy still faces the same problem as any pharmacological therapy: how to transfer
positive pre-clinical results to clinically successful therapy.
Summary
Gene therapy offers an alternative approach for the treatment of vessel remodelling. First clinical trials
have established the safety of gene therapy for the treatment of these remodeling processes and some
positive clinical results have been reported for vein graft stenosis. Currently several genes and proteins
have shown promising results in pre-clinical studies. However, randomized, double-blinded, placebocontrolled phase II/III studies are needed to determine usefulness of gene therapy for these diseases.
Acknowledgements
This study was supported by grants from Finnish Academy and
Sigrid Juselius Foundation. We thank Ms Marja Poikolainen for
preparing the manuscript.
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Tables
Table1 Gene therapy approaches to reduce restenosis or vein graft
stenosis
Table2 Clinical gene therapy trials to prevent restenosis and vein
graft stenosis
Received 2 July 2002; accepted 24 July 2002
October 2002, Volume 9, Number 20, Pages 1344-1350
Table of contents
Previous Article Next [PDF]
Review
Gene Therapy Progress and Prospects: Cystic fibrosis
U Griesenbach, S Ferrari, D M Geddes and E W F W Alton
Department of Gene Therapy, National Heart and Lung Institute,
Imperial College, Faculty of Medicine, London, UK
Correspondence to: U Griesenbach, Department of Gene Therapy,
Faculty of Medicine, National Heart and Lung Institute, Imperial
College, London, SW3 6LR, UK
Abstract
Since the cloning of the cystic fibrosis gene (CFTR) in 1989, 18 clinical trials have been carried
out, including five in the 2 years reviewed here. Most trials demonstrated proof-of-principle
for gene transfer to the airway. However, gene transfer efficiency with each of the three gene
transfer agents (adenovirus (Ad), adeno-associated virus 2 (AAV2) and cationic liposomes)
was low, and most likely insufficient to achieve clinical benefit. Here, we will review the
clinical and pre-clinical progress for the last 2 years (20002001) and briefly speculate on
future prospects for the next 2 in CF gene therapy.
Gene Therapy 2002 9, 13441350. doi:10.1038/sj.gt.3301791
Keywords
cystic fibrosis; gene therapy; airway gene transfer
In brief
Progress

Five clinical trials for CF have been carried out between 2000 and 2001

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Encouraging progress has been made to overcome some of the extracellular barriers to airway
gene transfer, such as mucus and the glycocalyx
Important intracellular barriers, such as cytoplasmic nucleases and the nuclear membrane
have been identified and first attempts been made to overcome these barriers
Viruses that recognise receptors on the apical surface of airway epithelial cells have been
identified
Targeted receptor-mediated endocytosis of synthetic vectors has increased transfection
efficiency of airway epithelial cells
Repeated administration of viral vectors, but not non-viral vectors, remains a significant
problem
Expression cassettes have been improved to enable prolonged transgene expression
Intravenous and in utero gene delivery have been evaluated
Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced
for CF
Prospects

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Five clinical trials for CF have been carried out between 2000 and 2001
Encouraging progress has been made to overcome some of the extracellular barriers to airway
gene transfer, such as mucus and the glycocalyx
Important intracellular barriers, such as cytoplasmic nucleases and the nuclear membrane
have been identified and first attempts been made to overcome these barriers
Viruses that recognise receptors on the apical surface of airway epithelial cells have been
identified
Targeted receptor-mediated endocytosis of synthetic vectors has increased transfection
efficiency of airway epithelial cells
Repeated administration of viral vectors, but not non-viral vectors, remains a significant
problem
Expression cassettes have been improved to enable prolonged transgene expression
Intravenous and in utero gene delivery have been evaluated
Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced
for CF
Integrating viral and non-viral GTAs will prolong gene expression in the airways and
expression will be regulated through on/off cassettes
Repeated administration of viral vectors will be improved
Physical methods of gene delivery will be developed and will overcome some of the barriers to
topical gene transfer
Better regulatory elements will be developed
Stem cell gene therapy will advance
Better animal models will begin to be developed
New imaging techniques to monitor the success and effect of gene transfer and more clinically
relevant endpoint assay will be developed
Five clinical trials for CF have been carried out between 2000 and 2002
Three out of the five trials used liposome-mediated gene transfer strategies, one trial used Ad2 and one
AAV2. Noone et al transfected the nasal epithelium of CF patients and was able to detect vector-specific
1
DNA up to 10 days after gene transfer, but not mRNA or functional correction of the CFTR defect. The
discrepancy between this trial and previously published nose trials that were able to detect CFTR mRNA
and partial correction of the CFTR defect, is likely due to the lower transfection efficiency of the cationic
lipid used. In a dose-escalating safety trial, liposome DNA complexes were aerosolised into the lungs of
CF subjects, half of which, had a transient fever, muscle and joint pain shortly after liposome/DNA
2
administration. This was attributed to an immunological response against the liposome/DNA complexes;
similar results were previously reported in a lung trial, using the same liposome/DNA formulation.
Interestingly, Hyde et al demonstrated for the first time, that liposome/DNA complexes could be
successfully re-administered to the nose of CF patients. Each subject received three doses, administered
4 weeks apart and samples were analysed 4 days after each treatment. Six out of 10 treated subjects
were positive for CFTR gene transfer after each dose.
3
Perricone et al reported the results of a phase I clinical trial, in which recombinant adenovirus
(Ad2/CFTR) was administered through bronchoscopic instillation or aerosolisation to the lungs of CF
patients. In contrast to previous studies, the authors carefully determined that inefficient gene
expression was due to the very low transduction efficiency of Ad in human airways. In addition, the vast
4
majority of transfected cells (97%), did not appear to be epithelial cells. The authors therefore
concluded that further improvements in Ad vector design are urgently required. An AAV2 vector was
nebulised into the lung of CF subjects to assess delivery and safety. AAV2 administration to the lung
appeared to be safe and vector genome was detected, at the highest dose, up to 30 days after
administration. However, vector-specific mRNA could not be detected and therefore evidence for gene
5
transfer was not provided.
In summary, these new studies consolidate the view that proof-of-principle of gene transfer can be
demonstrated in some, but not all studies, but that gene transfer efficacy is currently insufficient to
warrant phase II/III trials. Thus, significant improvements in all aspects of gene transfer need to be
made.
Encouraging progress has been made to overcome some of the extracellular barriers to airway
gene transfer, such as mucus and the glycocalyx
A major function of the airway epithelium is to prevent uptake of foreign materials, including gene
transfer agents (GTAs). For this purpose several very effective extracellular barriers, such as mucus, the
gycocalyx, tight junctions and mucociliary clearance have evolved (Figure 1). Mucus, for example,
reduces the transfection efficiency of most viral and non-viral gene transfer agents. However,
transfection efficiency could be increased through pre-treatment with mucolytics or the antichiolinergic
6
drug glycopyrolate in vitro and in vivo. In addition to mucus, sputum and bronchoalveolar lavage fluid
7
8
recovered from CF patients have been shown to inhibit liposome, adenovirus and AAV-mediated gene
9
transfer efficiency. Although recombinant DNase reduced sputum viscoelasticity and improved
10
nanosphere migration in vitro,
the effect of DNase on gene transfer in vivo is unclear. To avoid the
confounding effect of sputum in vivo, gene transfer should ideally be studied in CF children, before their
lungs become filled with secretions. Indirect evidence has indicated, that the glycocalyx is also a barrier
11
to gene transfer and that neuroaminidase enhances Ad transfection of polarised cells in vitro.
Receptors for Ad and AAV2 are located on the basolateral membrane of human airway epithelial cells and
uptake of non-viral gene transfer agents also appears to be enhanced on the basolateral site, due to the
presence of heparan receptors and a higher endocytosis rate. The effect of several tight junction
12,13
14
15
'openers', including EGTA,
anti-E-cadherin antibody,
sodium caprate,
a blend of sucrose,
16
17,18
mannitol and Pluronic F68
or perfluorochemical
on gene transfer has been studied in vitro and in
vivo. Although a 10- to 50-fold increase in virus- and non-virus-mediated gene transfer was generally
seen in vitro, it is unlikely that the opening of tight junctions, even if transient, will be clinically
applicable, given that the airways of most CF patients are heavily colonised with bacteria.
In summary, although encouraging progress has been made partially to overcome some extracellular
barriers, it is unlikely that these strategies on their own will significantly improve gene transfer in the
human airways. However, they may prove beneficial in combination with other strategies (see below).
Targeted receptor-mediated endocytosis of synthetic vectors has increased the transfection
efficiency of airway epithelial cells
Liposomes and other synthetic vectors are also unable to transfect airway epithelial cells efficiently via
the apical membrane. Targeted receptor-mediated endocytosis may increase gene transfer and proof-of-
principle for this approach has recently been provided by Ziady et al, who have shown that targeting of
19
the serpin enzyme complex receptor (sec-R) increases gene transfer to the nasal epithelium of mice.
The sec-R ligand was linked to the CFTR plasmid via a poly-L-lysine bridge, and this formulation partially
corrected the CFTR defect (chloride efflux) in the nose of CFTR knockout mice. Most remarkably, it was
demonstrated for the first time in vivo that CF-related secondary defects (nitric oxide synthase-2 and
19
sodium hyper-absorption) were partially correctable through gene therapy as well.
Targeting of sec-R
has been successful in increasing airway gene transfer and several groups are currently trying to identify
new peptides that bind to airway epithelial cells and promote targeted receptor-mediated endocytosis,
20
using phage display libraries.
Viruses that recognise receptors on the apical surface of airway epithelial cells have been
identified
As mentioned above, the density of receptors for Ad and AAV2 on the apical membrane of human airway
epithelial cells is low, which explains in part the inefficient transfection efficiency. Multiple approaches
have been studied to overcome this problem. Firstly, several new recombinant viruses have been
identified, that appear to be able to enter epithelial cells via the apical membrane efficiently. In addition
21
22
to respiratory syncytial virus (RSV),
Sendai virus (SeV)
has recently been shown to transfect
bronchial epithelial cells efficiently in vivo. Two days after infection with SeV 80% of airway epithelial
cells expressed the
-galactosidase transgene (Figure 2a). SeV uses binding to cholesterol and sialic
acid as receptors, which are both present at the apical membrane of airway epithelial cells. In addition,
SeV is not greatly inhibited by mucus. AAV entry into airway epithelial cells appears to be serotypespecific and AAV5 has been shown to transfect airway epithelial cells five times more efficiently than
23,24
AAV2.
AAV in general is a very promising gene transfer agent. Not only does the virus maintain
prolonged transgene expression due to integration or concatemerisation of the vector genome, but it is
also thought that the virus might be less immunogenic, due to the fact that it does not appear to
transduce antigen-presenting dendritic cells. Some reports have suggested that AAV could be repeatedly
administered to the lung. However, this possibility has recently been ruled out unless different serotypes
25
or some form of immunosuppression are used.
An important limitation of AAV as gene transfer vector
for CF has been its limited packaging capacity. However, it has recently been demonstrated that this
26
problem can be overcome by using trans-splicing or overlapping vectors
or by shortening the CFTR
27
cDNA.
Secondly, proof-of-principle has been established that adeno and AAV vectors can be retargeted to apical
28
29
surface receptors, such as the bradykinin,
the urokinase plasminigen
and the P2Y230
purinoreceptor
in vitro. However, it remains to be established if these approaches will increase
transfection efficiency in vivo.
A third strategy involves systematic pseudotyping of enveloped virus with different envelope
glycoproteins. Glycoproteins from viruses that naturally infect the airway epithelial cells, such as human
31
32
coronavirus 229E,
influenza A strains
and members of the Filovirus family, such as Marburg or Ebola
virus, are good candidates. Pseudotyping of lentiviral vectors such as the human, feline or equine
immunodeficiency virus is a particularly attractive approach. Lentiviral vectors have the ability to
integrate into the genome of dividing and non-dividing, differentiated cells. Expression is therefore
prolonged when compared with episomally maintained vectors and may last forever, if integration into a
stem cell occurred. Kobinger et al have recently demonstrated that an Ebola-pseudotyped HIV vector
33
efficiently transfects airway epithelial cells in vivo.
Most interestingly, expression was low 7 days after
administration, but strong expression was visible in airway epithelium and submucosal gland cells 28
days after transfection (30% tracheal epithelium expressed
-galactosidase). This persisted at least
until day 63 (24% of the tracheal epithelium expressed a
-galactosidase reporter gene) Figure 2b.
Currently large-scale, high titre production of pseudotyped lentiviral vectors is technically difficult and a
critical limiting factor.
The identification of viruses that are capable of efficiently transfecting the airway epithelium via the
apical membrane has been one of the most exciting findings in the last 2 years. Although big problems
such as repeated administration still exist, proof-of-principle studies, demonstrating that the primary
CFTR defect can be consistently corrected in humans, should now become possible.
Repeated administration of viral vectors, but not non-viral vectors, remains a significant
problem
Transgene expression will remain transient, unless lung repopulating stem cells can be targeted with a
stably maintained vector. The treatment of CF with gene therapy will therefore require repeated
administrations of gene transfer agents, which is a particular problem for viruses. Strategies such as
administration of immunosuppressants and corticosteroids, and treatments aimed at transiently blocking
+
34
CD4 T cells
have been evaluated for repeated administration of adenovirus. However, the success of
these strategies has been limited. In general, repeated administration was possible a few times, but
ultimately lead to reduced and finally absent transgene expression. The only exception so far is a report
by Kolb et al, who demonstrated that administration of the steroid budesonide enabled adenovirus to be
35
re-administered at least five times without loss of transfection efficiency.
Importantly, treatment with
an anti-CD40 ligand monoclonal antibody did not prevent a virus specific antibody response in non34
human primates.
Another and perhaps more promising approach is based on generating a 'stealth virus', which is invisible
to the immune system, by coating the virus capsid with polyethylene glycol (PEG). PEGylation of the
virus capsid reduced cytotoxic T cells and antibody production and significantly prolonged transgene
expression from 4 to 42 days. Repeated administration of the adenovirus modified with the same PEG
was not successful, however, when different PEG formulations were used, significant transgene
36
expression was detected after repeat administration.
Although, it has been demonstrated that repeated administration of liposome/DNA complexes to the
3
nose of CF patients is possible, there have been concerns raised regarding the inflammatory
2
components of bacterial DNA. The abundance of unmethylated CpG motifs in the bacterial plasmid DNA
may at least in part be responsible for the inflammatory response. Several strategies are currently being
explored to decrease these unwanted properties, such as (1) methylation of CpG sequences; (2)
reduction of the CpG frequency by eliminating non-essential regions or by site-directed mutagenesis;
37
and (3) the use of specific inhibitors of the CpG signalling pathway, such as chloroquine or quinacrine.
In summary, progress in enabling repeated administration of viruses has been rather slow and concerns
regarding viral and non-viral GTA-induced inflammation have been raised. A more detailed
understanding of the immune responses against viral and non-viral GTAs is therefore crucial for further
improvements in gene transfer.
Important intracellular barriers including cytoplasmic nucleases and the nuclear membrane
have been identified and first attempts been made to overcome these barriers
Intracellular barriers limiting transgene expression have also been identified. Endosomal and cytoplasmic
2+
degradation are a particular problem for most non-viral gene transfer agents. In the cytoplasm, Ca 38
sensitive cytosolic nucleases restrict the half-life of plasmid DNA to 5090 min.
Interestingly, even
recombinant viruses are inhibited by intracellular degradation. In polarised cells, the AAV capsid is
39,40
ubiquinated and subsequently enters the ubiquitin proteasome-dependent degradation pathway.
Thus, proteasome inhibitors augmented AAV2-mediated gene transfer in the mouse lung from
39
undetectable levels to 10% of airway epithelial cells transfected.
41
In airway epithelial cells the nuclear membrane is an important barrier for non-viral gene transfer.
It is
42
currently uncertain if strategies, such as the addition of nuclear localisation signals
or lactosylated
43
poly-L-lysine,
which increased nuclear transfection in other cell types, will be beneficial in these.
Although, it has been demonstrated that AAV is degraded by proteasomes, non-viral GTAs are probably
more affected by intracellular degradation and nuclear entry than viruses. Thus, new strategies are
urgently required to overcome the intracellular barriers. It is also important to note, that these barriers
are likely to be cell-type specific and therefore studies need to be carried out in the 'relevant' cell type.
Expression cassettes have been improved to enable prolonged trangene expression
Current non-integrating viral and non-viral GTAs are unable to maintain sustained expression. Immuno44
suppressants have been reported to prolong transgene expression,
due to their ability to reduce the T
cell-mediated response. In addition, Scaria et al showed that incorporation of the adenoviral gene ICP47
+
into an adenoviral vector, which reduced MHC class I antigen presentation in CD8 T cells, prolonged
45
expression in primate lung up to 21 days.
The strong CMV promoter has traditionally been used in most GTAs. However, more recently evidence
46
has been provided that weaker eukaryotic or hybrid promoters, such as the polyubiquitin C promoter,
46
47
the elongation factor 1 promoter
and the CMV-Ubiquitin B hybrid promoter
enable prolonged
transgene expression. In addition, there is growing evidence that genomic sequences, either within or
48
flanking the gene, might be essential to provide in vivo long-term expression.
Alternative strategies to
prolong transgene expression include the development of artificial chromosomes and other selfreplicating systems. Huertas et al have developed a circular yeast artificial chromosome (YAC) carrying
49
the human CFTR sequence and the oriP and EBNA-1 genes from EpsteinBarr (EBV) virus.
However, it
remains to be established if these large constructs are able to transfect airway epithelial cells in vivo.
Intravenous and in utero gene delivery routes have been evaluated
As noted above topical gene transfer to the lung is severely affected by many extra-cellular barriers. In
an attempt to overcome these barriers GTAs or oligonucleotides (ODN) have been administered
intravenously (i.v.). It is likely that i.v. administered GTA have to reach the bronchial circulation and
avoid lodging in the pulmonary circulation to reach relevant target cells for CF therapy. Fox et al have
recently demonstrated that 'naked' ODN are able to leave the bronchial circulation and transfect the
50
cytoplasm, but not the nuclei of airway epithelial cells.
In addition, Kohler et al have shown that i.v.
injection of lipid-complexed
-galactosidase plasmids lead to expression in bronchial epithelium and
tracheal submucosal glands of mice. However, this was only achieved with some, but not all of the
51
cationic lipids tested.
Importantly, in the past it has sometimes been difficult to discriminate
recombinant from endogenous
-galactosidase expression and it is therefore important for these
potentially exciting findings to be expanded upon.
The possibility for in utero gene therapy for CF has also been investigated. Injection of GTAs into the
amniotic fluid, would provide contact with most relevant target sites for CF (pulmonary, gastrointestinal
52
and sinus epithelium). Injection of adenovirus, retrovirus and more recently AAV
resulted in reporter
gene expression in both pulmonary and gastrointestinal epithelium with persistence of transgene
expression in the lung ranging from 14 to 30 days after infection. The possibility that in utero gene
53
therapy would tolerise recipients to viral GTAs, has recently been ruled out.
Larson et al have
postulated a role for CFTR during development and provided preliminary evidence that transient
expression of CFTR cDNA in utero alleviates the intestinal defect in the CF knockout mouse long
54,55
term.
These controversial findings, if replicated, may have far reaching implications for traditional
gene therapy approaches, as well as other areas of CF research.
Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced
for CF
Gene repair of the endogenous CFTR gene has two major advantages over traditional gene therapy. If
successful, gene repair will ensure gene expression for the lifetime of the cells and appropriate control of
gene expression will be guaranteed because the endogenous CFTR promoter is utilised. Preliminary
results indicated that the genomic CFTR locus could be modified in primary rat hepatocytes using
56
chimeraplasts (DNA/RNA hybrid oligonucleotides).
Hepatocytes have previously been shown to be
easily amenable for gene repair strategies, most likely due to efficient uptake of repair molecules into the
nucleus. In addition, a similar approach using small fragment homologous recombination (SFHR) was
able to reintroduce the wild-type CFTR sequence into the lungs of CF knockout mice, albeit at very low
57
frequency.
However, the specificity of targeted gene repair is currently unknown.
Down-regulation of gene expression through antisense molecules may be of therapeutic benefit in CF.
Lambert et al showed that antisense inhibition of the B cell antigen receptor-associated protein (BAP) 31
increased expression of both wild-type CFTR and mutant CFTR and partially restored CFTR chloride
58
channel function.
The exact function of BAP31 is unclear, although the authors speculated that the
protein may be involved in retaining mutant CFTR in the ER. Several other chaperone proteins, mucins or
the epithelial sodium channel (ENaC), which is up-regulated in CF may be suitable candidates for
antisense strategies.
Spliceosome-mediated trans-splicing (SMaRT) has recently been introduced as a means to generate wildtype CFTR mRNA in CF xenograft models. Cells were transfected with very high titres of adenovirus that
produced so-called pre-therapeutic wild-type CFTR mRNA molecules (PTMs), which are designed to
promote trans-splicing with the endogenous CFTR mRNA and 22% of wild-type CFTR function could be
59
restored.
Similar to gene repair, SMaRT ensures cell-type specific expression of wild-type CFTR mRNA,
however efficiency and specificity of the reaction require further improvements, before clinical trials can
be considered.
Prospects
Together with traditional GTAs, new approaches are being developed in order to increase gene transfer
60
efficiency to the airway epithelium. Gersting et al
recently reported that application of a magnetic field
to human primary airway epithelial cells transfected with plasmid DNA mixed with superparamagnetic
nanoparticles (magnetofection) resulted in more than 100-fold increase in gene transfer. Stem cell-based
gene therapy approaches are also being investigated. Two strategies can be envisaged: either targeting
lung-resident stem cells with integrating vectors or engineering bone marrow/embryonic cells ex vivo
and inducing engraftment into the airway epithelium. Thus, bone marrow-derived stem cells have
61
recently been observed to diferentiate into airway epithelial cells in the lung.
Prolonged and sustained
CFTR expression may not be beneficial, especially if integrating vectors are used. To this end,
62
epithelium-specific regulated cassettes are being developed.
Each of the vectors and technologies will
have to be tested in relevant in vivo models. Ideally, in contrast to CF mice, these would reproduce the
63
lung pathology seen in CF patients. For this reason efforts are being made to develop a CF ferret
and a
64
CF sheep,
their lung biology being more similar to humans than the mouse. Finally new imaging
techniques to monitor the efficiency of gene transfer in living animals/patients will also have to be
developed. In addition to positron emission tomography (PET) and magnetic resonance imaging (MRI)65
based strategies, laser-induced fluorescence bronchoscopy
has recently been developed as a way to
detect gene expression in a non-invasive way in human airways.
Conclusion
Although topical gene transfer to the airways for CF gene therapy is more challenging than originally
thought, significant progress has been made in pre-clinical research over the last 2 years to overcome
some of the hurdles. Most importantly, the identification of more effective viral and non-viral gene
transfer agents for airway gene delivery, development of new delivery routes and most recently
alternative strategies are keeping the development of CF gene therapy well on track.
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Figures
Figure 1 Scanning electron micrograph of human airway epithelium
showing fields of cilia covered at their tips by flakes of mucus. In
hypersecretory disease the mucus usually forms a continuous sheet
or 'blanket' overlying the cilia (supplied by courtesy of Professor
Peter Jeffery, Imperial College, London, UK).
Figure 2 Viral gene transfer to the lung. (a) Recombinant Sendai
virus (SeV) mediated -galactosidase expression 48 h after gene
transfer.48 (b) Filovirus pseudotyped lentivirus-mediated galactosidase expression 63 days after gene transfer (with
permission from Ref. 33)..
October 2002, Volume 9, Number 20, Pages 1344-1350