Regenerating an Immune System:
Gene Therapy and Stem Cell Transplantation for
Severe Combined Immune Deficiency
Brooke LaTour
Studentnr. 3729451
Supervisor: Dr Frank Staal
Leiden University Medical Center
Utrecht University Examiner: Dr Andries Bloem
Immunity & Infection, Graduate School of Life Sciences 2012-2013
Table of Contents
I.
Introduction
2
B-Cell Associated Immunodeficiences
2
Phagocytic Immunodeficiences
6
Combined Immunodeficiencies
6
II.
T-Cell Lymphopoiesis in Health and Disease
7
III.
Molecular Causes of SCID
10
Defective common  chain cytokine signaling associated SCID 10
Adenosine deaminase deficient SCID
12
Defective V (D) J recombination associated SCID
13
Defective pre-TCR/TCR signaling associated SCID
14
Genetically similar, symptomatically divergent
15
IV.
Current Therapy
15
V.
A History of Progression
18
Major setback: T-ALL due to insertional mutagenesis
VI.
VII.
Gene Therapy and Toxicity: The Evolution of Vectors
20
21
Retroviral Vector Insertional Mutagenesis in Gene Therapy
23
The Role of LMO2 in T-Cell Development
26
Patterns of Viral Integration
26
Predicting Genotoxicity: Animal Models
28
Future Treatments
Genomic editing
30
32
VIII.
Discussion
37
IX.
References
41
1
I.
Introduction
Primary immunodeficiencies (PIDs) are a subset of immunologically based,
inherited diseases that predispose affected individuals to a variety of infections,
allergies, and autoimmune disorders, and leave them at risk of developing cancer.
PIDs provide an invaluable model to dissect intrinsic mechanisms of the human
immune system. Examination of these mostly monogenic conditions has lead to the
identification of many of the associated genes and has produced a wealth of
information about the development and function of the immune system. These
pathologies give rise to immunological insights on the regulatory and genetic
complexities of the innate and adaptive immune system and allow for the
accreditation of scientific observations to underlying genotypes. Critical analysis
and understanding of these disorders is essential as it fosters the development of
new diagnostic and therapeutic tools for improved treatment of a wide variety of
pathologies.
B-cell associated primary immunodeficiencies
Multiple PIDs exist that arise from impairments in early B-cell development
(Figure 1). Mutations that prevent signaling via the pre-B-cell receptor (BCR) lead to
an absence of mature B-cells and consequently a lack of immunoglobulins. Studies of
these disorders have elucidated information on the process of normal B-cell
differentiation. Development of a mature and functional antibody repertoire occurs
via stochastic recombination, during which B-cells express rearranged
immunoglobulin heavy (IgH) () and light (L) genes 1, and response to antigen
engagement, which initiates Ig class switch recombination (CSR) and the generation
of somatic hyper mutations (SHM). These two major antibody maturation events
require close contact with T-cell 2. Following these processes, selection of B-cells
2
expressing functional non-autoreactive B-cell receptors (BCR) is initiated, resulting
in the generation of mature B-cells with a high affinity for antigen.
The majority of genetically characterized abnormalities that result in a lack
of mature B-cells involve defective signaling through the pre-BCR. Of these,
approximately 85% of patients that present with early onset recurrent bacterial
infections, hypogammaglobulinemia, and abnormally low levels of B-cells have
mutations in the X-chromosome encoded cytoplasmic Bruton’s tyrosine kinase
(BTK) 3. BTK, a member of the Tec family of tyrosine kinases, plays a central role in
B-cell signaling, development, and differentiation 4. Mutations in BTK result in Xlinked agammaglobulinemia (XLA). Other genetically defined cases of
agammaglobulinemia are caused by mutations in the  heavy chain, Ig, the 5
component of the pre-BCR surrogate light chain, or the B-cell adaptor protein BLNK
(also known as SLP65) (reviewed in 5) because these elements are necessary to
proceed past the pre-B stage during B-cell lymphopoiesis.
The phenotype of these disorders differs profoundly between species. For
example, substantial differences have been observed between the xid mouse model,
which carries a defective BTK allele 6 and XLA patients. XLA patients fail to produce
antibodies in response to vaccination, have substantially low levels of all
immunoglobulin isotypes, and have less than 1% of normal B-cell numbers. In
comparison, xid mice are able to generate an antibody response to most T-cell
dependent and some T-cell independent antigens. They exhibit very low serum
levels of IgM and IgG3 but have almost normal concentrations of IgG1, IgG2a, IgG2b
and they have approximately half the normal amount of B-cells. It is interesting to
note that defects in pre-BCR signaling generally elicit a much more severe
phenotype in humans than in mice. Mutations in 5 and BLNK results in a block of
early B-cell differentiation in both human and mice, however disruption of the gene
encoding Btk only causes a developmental block in humans. This strongly suggests
that there are differential needs for B-cell development between species. Further
support of this point is underscored by the fact that mutations in the interleukin-7
receptor (IL-7R) cause a complete abrogation of B-cell differentiation at the pre-B
3
pro-B stage in mice 7, 8 whereas a lack of IL-7 signaling in humans, as seen in specific
types of severe combined immunodeficiency (SCID), results in the absence of T-cells
and a normal or elevated number of immature B-cells. These distinctions illustrate
important species-specific criteria for B-cell development and highlight the
complexities of interspecies translational models.
Common variable immunodeficiencies (CVID), or acquired
hypogammaglobulinemia, is a collection of primary immune deficiencies
characterized by low serum levels of IgG, IgA, and/or IgM (reviewed in 9) with
varying underlying genetic causes. Unlike other PIDs, CVID generally presents
around the patient’s mid-20s but can manifest as late as the fourth decade of life 10.
While several genetic lesions have been identified, the majority of causational
biochemical and genetic abnormalities remain unknown. Mutations in which the
inducible T-cell costimulator (ICOS) protein and B-cell expressed CD19 are
disrupted account for approximately 1% of CVID cases. Most patients have altered
B-cell subsets, which indicate impaired B-cell differentiation. While specific
response to antigen is severely attenuated, detectable levels of autoantibodies and
autoreactive B-cells can be found 10.
Also characterized by low immunoglobulin levels, hyper IgM (HIGM)
syndrome is an amalgamation of several closely related but genetically divergent
disorders affecting B-cell development. It is characterized by defective
immunoglobulin CSR and consequently very low levels of IgG, IgA, and IgE. HIGM
syndrome led to the discovery that the collaboration of B and T-cells, via
CD40/CD40L interaction, in response to antigen recognition is required for CSR to
take place. T-cell surface protein CD40 ligand (CD40L also known as CD154) is
necessary for germinal center (GC) formation in lymphoid organs, which is the site
where B-cells interact with T-cells and undergo CSR. This finding was further
accredited by the discovery that mutations in the gene encoding CD40 results in the
inability of B-cells to undergo CSR 2. An intrinsic defect of B-cells is also observed in
HIGM syndrome. Utilizing genetic linkage analysis it was identified that a subset of
HIGM is caused by mutations of AICDA, the gene encoding the enzyme activation
induced deaminase (AID). In the absence of this enzyme, B-cells fail to undergo CSR
4
and are defective in their ability to generate somatic hyper mutations (SHM) in the
variable (V) segment of the immunoglobulin genes 11. Knowledge of this resulting
defect firmly linked the CSR and SHM events which both occur in the GC at
approximately the same time in B-cells development.
Figure 1: Primary Immunodeficiencies affecting B-cells development. Self-renewing HSCs exist
at the apex of the hematopoietic hierarchy. They have the capacity to differentiate into lymphoid or
myeloid progenitors. CLPs are the precursors of the lymphoid branch. They can divide to form Tcells, NK-cells and B-cells. Developing B-cells progress through a series of discrete stages to become
mature B-cell, in this diagram they are simplified into the Pro B, Pre B, and B-cell steps. Stages at
which the specific aforementioned PIDs arise are indicated. Affects on each isotype of
immunoglobulin are indicated in the table below the schematic diagram. Abbreviations are as
follows: HSC, hematopoietic stem cell; CLP, common lymphoid progenitor; BLNK, B-cell linker
protein; XLA, X-linked agammaglobulinemia; BTK, Bruton’s tyrosine kinase; CVID, common variable
immunodeficiency; ICOS, inducible T-cell co-stimulator; HIGM, hyper IgM syndrome; AICDA,
activation-induced cytidine deaminase; UNG, uracil-N-glycosylase; AICDA C, deletion of the Cterminal end of AICDA 16.
AID acts on DNA to deaminate cytosine to uracil in immunoglobulin genes 12
and recently it was demonstrated that uracil residues are generated in
immunoglobulin genes within 24 hours of B-cell stimulation 11. Uracil-N- glycosylase
(UNG) is a DNA repair enzyme that acts cooperatively with AID to assist in shaping
the specificity of AID induced SHM 13. Loss of function recessive mutations of UNG
are associated with substantial impairment in CSR at a DNA precleavage step and
with partial distruption of SHM, these findings support the model that AID directly
deaminates cytosine into uracil in the switch (S) and variable (V) regions of
5
immunoglobin genes 14. An analysis of HIGM syndrome phenotypes lead
unexpectedly to the discovery that certain mutations of AID result in a nearly
normal frequency of B-cell SHM but impaired CSR 15. This phenotype was associated
with mutations in the 8-17 C-terminal residues of AID, suggesting that the Cterminus is required for CSR but not SHM. This is most likely due to cofactor binding
or recognition of specific conformations of the DNA immunoglobulin S region 16.
Phagocytic immunodeficiencies
Engulfment and destruction of invading pathogens by phagocytosis plays an
indispensible role in the innate immune response. Phagocytes also serve to clear
apoptotic bodies and other cellular debris during tissue homeostasis and
remodeling. There are several congenital disorders that hamper phagocytic function
(reviewed in Flannagan et al., 2012).
Phagocytic disorders can be divided into intrinsic and extrinsic based
disorders. Extrinsic factors that are impaired in phagocytic deficiencies include
opsonic abnormalities, impaired antibody function, and defective complement
factor activity. These issues can lead to severe neutropenia via suppression of
granulocyte production or autoantibodies directed again host neutrophils. Intrinsic
defects can result from impaired granulocyte development or exit into peripheral
circulation. They can also be the effect of impaired granulocyte killing ability or
chemotaxis. Examples of intrinsic disorders associated with impairment in
phagocytic killing include chronic granulomatous disease, glycogen storage disease
type Ib, Chediak-Higashi syndrome, and specific granule deficiency. PIDs associated
with deficiencies in chemotaxis include hyper-IgE syndrome, leukocyte adhesion
defects, Shwachman-Diamond syndrome, and syndromes with periodontitis.
Combined immunodeficiences
SCID is a heterogeneous disorder characterized by the aberrant development
and/or functional of T-lymphocyte. It often is fatal during the first few months of life
due to severe and recurrent infections unless transplantation of hematopoietic stem
cells (HSCs) restores the functional T cell compartment of the immune system.
6
Clinical manifestations of SCID, despite its wide range of underlying causes, share
several phenotypic characteristics including failure to thrive, extreme susceptibility
to infections, significant lymphopenia, a complete absence of T-cells, and reduced
thymic size and capacity17. The presence of B-cells is variable in different subtypes
of SCID, however even when B-cells appear in normal number, they have intrinsic
defects in addition to the impairments they suffer due to the absence of T-cells. As
previously noted, the lack of T and B-cell interaction results in immature B cells that
cannot undergo CSR and consequently cannot produce antibodies in response to
antigens 2.
II.
T-Cell Lymphopoiesis in Health and Disease
PIDs have propagated a myriad of information about immune development and
function. Critical species-specific differences in T-cell lymphopoiesis exist which
create challenges in deriving translational knowledge of human T-cell development
from mouse research. SCID, due to its characteristic, genetically determined blocks
in T-cell differentiation, has created the circumstance for modeling human T-cell
differentiation and function. These conditions have also revealed several nonredundant roles for cytokines and cytokine signaling molecules in human
lymphopoiesis. Moreover several interspecies dissimilarities in cytokine signaling
pathways have been elucidated via the comparison of human and mouse SCID
phenotypes.
Cytokines are paracrine factors that are classified as type I or II based on
three-dimensional structure. A shared feature of both types of cytokines is that, with
few exceptions, their associated receptors lack intrinsic tyrosine kinase capacity so
signaling is accomplished through the Janus-activated kinase (JAK)/ signal
transducer and activator of transcription (STAT) pathway (reviewed in 18).
Mutations that interfere with these signaling pathways result in deficiencies in
select hematopoietic cells types.
7
Figure 2: Differential phenotypes of T-cell development in the human versus mouse thymus.
During normal T-cell development, T-cell precursors migrate through different specialized regions of
the thymus. Different definitive phenotypic markers demarcate stages of T-cell development in
humans and mice. The main checkpoints associated with human T-cell development are indicated.
DN, double negative; ISP, immature single positive; DP, double positive; SP, single positive19
During physiologically normal human T-cell development (Figure 2), T-cells
arise from pluripotent progenitors cells in the bone marrow or fetal liver and
migrate to the thymus in order to differentiate. It is through a complex and highly
regulated process that a diverse and effective mature T-cell population is generated
and sustained. Naïve, single positive T-cells develop from thymocytes in the
specialized microenvironment of the thymus20. Maturation of T-cells requires
controlled rearrangement of the different T-cell receptor (TCR) genes to assemble
diverse TCR-complexes, this process is mediated by recombinase activating genes
(RAG) 1 and 2. It is important to note, that because RAG1 and 2 are also responsible
for the rearrangement of immunoglobulin gene segments in B-cells, mutations in
either gene results in an absence of mature T and B-cells. T-cells that recognize
foreign antigens in association with the self-major histocompatibility complex
(MHC) are positively selected for survival, while autoreactive T-cells are eliminated
via negative selection. Positive and negative selection events culminate in the
expression of mature TCR molecules.
8
T-cells progress through a series of discrete stages of development,
characterized by loss of potential for alternative lymphoid fates, that can be
discerned via the expression of membrane associated clusters of differentiation
(CD) such as CD4 and CD8 (Figure 2). The TCR lineage bifurcates from the TCR
lineage in murine models around the double negative (DN) 1 and DN2 subsets. In
human T-cell development TCR loci rearrangement begins around the DN1-DN2
stage when cells are at the immature CD34+CD38-CD1a-stage. TCRB rearrangement
occurs as soon as cells acquire expression of CD38. At the DN stage, pre-T-cells are
CD4 and CD8 negative. For murine thymocytes, there are 4 DN stages defined by the
gradual loss of CD34 and gain of committed T-cell associated genes; these stages are
defined by differential expression of CD44 and CD25. In humans there are 3
heterogenous subsets of the DN stage: the most immature being the CD34+CD38CD1a-stage, followed by the expression of CD38, and then finally CD1a expression
21
,TCR  selection occurs during this final DN stage. Only a few T-cell progenitors
enter the thymus per day, but once they have migrated to this new environment
they proliferate exponentially due to specific cytokine exposure, during the DN1 and
DN2 stages of development (reviewed in 22). By DN3 cells cease to proliferate and
undergo rearrangement of the TCRB locus 21. In mice TCRD rearrangement begins in
the DN1 stage followed by TCRG and TCRB in the DN2, TCRB rearrangement
continues in the DN3 stage. The discrete human stages resemble murine Tlymphopoiesis as follows: murine early DN1 (CD44+CD25-CD117-) corresponds to
the human CD34+CD38-CD1a- stage. The murine late DN1/DN2
(CD117+/CD44+CD25+) and DN3 (CD44-CD25+) correspond the human
CD34+CD38+CD1a- and CD34+CD34+CD1a+ respectively (Figure 2).
During the progression from DN3 to an immature single positive T-cell there
is surface expression of the TCR chain with the pre-TCR chain and together these
chains make up the pre-TCR complex. During this period the functional ability of the
mature TCR chain is tested, this process is known as -selection. At this time,
activation of the pre-TCR complex induces proliferation and initiates
rearrangements of the TCRA gene. It is at this point that the thymocyte expresses
9
both CD4 and CD8. Productive rearrangement results in the expression of the
mature TCR complex, which then allows these lymphocytes to undergo the
process of positive and negative selection. If the cell survives selection it matures
into a naïve CD4 or CD8 single positive T-cell.
TCR signaling is critical for T-cell development and lineage specification. Tcell homeostasis plays a central role in maintaining proper immune function.
Patients that present with the presence of a mature but non-functional T-cell
repertoire share several common features including impairment in positive and
negative T-cell selection, high levels of lymphocyte specific apoptosis resulting in
lymphopenia, susceptibility to viral and bacterial infections and high incidences of
autoimmune manifestations. The study of rare T-cell associated PIDs provides
insights not only into T-cell development but also in the molecular and biochemical
pathologies of other disorders. A novel form of SCID defined by a bi-allelic loss of
function mutation in the ZAP70 gene was first described in Mennoite Indians that
presented with a selective depletion of CD8+ lymphocytes. ZAP70 is a TCR
associated tyrosine kinase necessary for signaling TCR activation. These individuals
had detectable CD4+ T-cells in circulations but these cells failed to respond to CD3+
cross-linking. In mice, the phenotypic manifestation of a lack of functional ZAP70
results in a differentiation block at the DP stage of T-cell development, interestingly
development of TCR+ T-cells is unaffected (reviewed in 23).
III.
Molecular Causes of SCID
Defective common  chain cytokine signaling associated SCID
There are at least 15 different underlying genetic causes of SCID 24. X-linked
SCID (SCID-X1) is the most common form of SCID accounting for approximately
50% of cases 25 (Figure 3). Its underlying genetic cause is attributed to a deficiency
in the cytokine receptor common  chain (c) also referred to as the Interleukin-2
receptor chain- (IL-2R). There have been hundreds of mutations described in the
c gene that are causative of SCID 26. The c chain is an essential subunit of several
10
cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 27. IL-7 receptor
signaling is indispensible for development, survival, and proliferation of mature
human T-cells. It is known that IL-7 is essential for T-cell development because
mutations in the gene encoding the -chain of the IL-7 receptor lead to an absence
of T-cells, but the retained presence of B and natural killer (NK)-cells 28. The
phenotypic lack of NK-cells associated with SCID-X1 is attributed to defective IL15/IL-15R interaction 29, 30 as murine models have demonstrated that the absence
of IL-15 or IL-15R leads to a phenotypic lack of NK-cell. A notable immunological
difference between humans and murine models is that c mutation in humans leads
to a form of SCID marked by the absence of T and NK cells, while in mice this same
mutation results in an animal completely devoid of lymphocytes demonstrating the
fact that c signaling is not required for B-cell development in humans but is
essential for proper B-cell development in mice. This could be attributed to the fact
that murine B-lymphopoiesis, as previously mentioned, is dependent upon IL-7/IL7R signaling (reviewed in 18).
A symptomatically indistinguishable form of SCID can also result from
mutations in the JAK3 gene 31 because the JAK3 tyrosine kinase is functionally
coupled to cytokine receptors that utilize the c chain. In order to signal interleukin
receptors, which lack intrinsic catalytic activity, require the c chain to associate
with the intra-cytoplasmic tyrosine kinases JAK1 and JAK3 32. At least 23 unique
mutations have been identified in different structural domains of the JAK3 gene that
contribute to the formation of SCID. Dysfunction or absence of c chain associated
signaling results in a block in T and NK lymphopoiesis leading to compromised
immune development. B-cells are present in normal or high numbers, but are not
functional.
11
Figure 3: Relative distribution of SCID subtypes. Data obtained from 33,34
Adenosine deaminase deficient SCID
Adenosine deaminase (ADA) deficient SCID is the second most common form
of SCID (Figure 2). It is an autosomal recessive metabolic disorder that affects
multiorgan systems. The ADA enzyme plays a central role in purine metabolism and
its absence results in the accumulation of the toxic metabolite, deoxyadenosine
triphosphate (dATP). Build up of adenosine and deoxyadenosine causes an
accretion of cellular dATP, which triggers cell death by apoptosis 33. The buildup of
toxic metabolites severely compromises hepatic, skeletal, and neurological function
12
35.
Because lymphocyte precursors are extremely sensitive to the toxic build up of
dATP, lack of ADA leads to an absence of T, B, and NK cells. Valerio and colleagues
characterized that this form of was due to the presence of an aberrant form of the
ADA protein, suggesting that the lack of ADA activity is not attributable to
transcriptional or translation defects but instead to changes in the configuration of
the protein that affect it enzymatic capacity 36. This disease is almost always fatal by
year 2 of life if not treated with immune reconstitution therapy (Giblett et al., 1972
republished 37). These patients can also be sustained on pegylated ADA enzyme
replacement therapy (ERT).
Defective V (D) J recombination associated SCID
The third most common type of SCID is caused by recombination defects in any
of three genes involved in this process. Variable, diverse, joining V (D) J
recombination ensures somatic diversification of immunoglobulin and TCR genes
and is an essential part of both B and T-cell differentiation. V (D) J recombination is
initiated by Rag1 and Rag2. The Rag1 and Rag2 complex creates nicks between the
V, D, J coding elements of the TCR and BCR genes. These nicks are converted to
double stranded DNA breaks, which form hairpin sealed coding ends and blunt
signal ends (reviewed in 38). Mutations in either RAG1 or RAG2 result in impaired V
(D) J recombination and incomplete B and T cell differentiation. In this form of SCID
NK development is spared. Another assortment of mutations that impair V (D) J
recombination can occur in proteins involved in the non-homologous end joining
repair (NHEJ) pathway. The NHEJ machinery is responsible for joining the two
coding ends that result from the double stranded DNA breaks. Mutations in the
endonuclease protein Artemis cause hypersensitivity to DNA double-strand breakinducing agents, like ionizing radiation, and an absence of T and B cells due to the
inability to repair DNA breaks 39. Mutations in DNA ligase IV, another NHEJ protein,
result in a partial T+ B-cell SCID-like immunodeficiency 40. Absence of functional
DNA ligase IV leads to a substantial reduction in the number of B-cells due to an
incomplete but substantial block in early B-cells differentiation, and a 10-fold
reduction in the number of  T-cells but patients had normal numbers of  T-cells
13
41.
SCID Phenotypes characterized by very low levels of B-cells and a complete
absence of T-cells have also been described for mutations in the gene encoding DNA
ligase IV 42.
Figure 4: Blockades in lymphopoiesis at different stages manifest as different subtypes of
SCID. Vertical arrows indicate the approximate stage where lymphopoiesis is block in each condition.
ADA deficiency results in an absence of mature B, T, and NK cells. Mutations in the genes encoding
the c chain or JAK3 result in a lack of T-cells and NK cells. Mutations in CD45 and the associated
subunits of CD3 prevent pre-TCR/TCR signaling and result in an absence of T-cells. Mutations that
interfere with V (D) J recombination, such as RAG1, RAG2, or Artemis lead to a deficiency of B and T
cells. Adapted from33
Defective pre-TCR/TCR signaling associated SCID
The TCR complex is made up of the  and  or the  and  variant chains. These
chains pair as heterodimers in association with the invariant CD3 associated chains,
CD3, CD3 , CD3, and CD3. Defective pre-TCR and TCR signaling due to
impairments in CD3 associated subunits result in the absence of T-cells. Mutations
in the CD45 phosphatase are rare but also result in the formation of the SCID
phenotype. Deficiencies in each of the pre-TCR/TCR signaling complex subunits CD3
 and CD3 and CD3 and CD3 have been described. For unknown reasons,
14
deficiency in CD3 43 yields a substantially less severe phenotype than those
corresponding with CD3 and CD3 mutations 44.
Genetically similar, symptomatically divergent
Omenn Syndrome (OS) is an autosomal recessive disease that is genetically
similar to SCID. Also known as “leaky SCID,” it is characterized by hypomorphic
mutations in RAG1 and 2, and often in other genes implicated in the SCID phenotype
(reviewed in 45), such as Artemis, DNA ligase 4, c chain, IL-7R, and ADA (Niehues
et al., 2010). Partial activation of RAG1 and RAG2 leads to the generation of a small
number of T-cell clones. The clinical manifestations of this disease are extremely
serious as these patients present with severe susceptibility to infection as seen with
classic SCID patients as well as extensive tissue inflammation due to autologous
oligoclonal hyper-autoreactive T-helper cell 2 (Th2) lymphocytes. These
lymphocytes proliferate extensively due to thymic and peripheral reactivity 46 and
create extreme levels of inflammation. Individuals with OS have markedly low to
absence levels of immunoglobulins with the exception of IgE, which is present in
elevated concentrations. The high levels of IgE are hypothesized to be due to the
expansion of Th2 cells (reviewed in 47). Other key symptoms include
hepatosplenomegaly, lymphadenopathy, often accompanied by recurrent infections,
and alopecia. Rapid diagnosis and intervention is essential as OS is fatal if not
treated 47.
IV.
Current Therapy
Hematopoietic stem cell transplantation (HSCT) is the primary therapy used
to effectively treat SCID. In most cases transplantation is from an unaffected, HLA
identical sibling. However, in the majority of situations HLA matched donors are not
available so transplantation from a closely HLA-matched, unrelated donor or a
haploidentical (parental) donor may be performed, in such cases of none matched
HLA, the grafts must be T-cell depleted. Survival rates post transplantation are
15
significantly lower with haploidentical donor transplantations and symptoms of
graft versus host disease, such as severe erythematous rash or chronic liver disease
often result from engraftment of maternal T-cells. Partially HLA incompatible
transplantation is responsible for a 30% mortality rate within the first year postHSCT 34. Allogeneic transplantation carries significant complications such as graft
versus host disease, lack of T cell reconstitution and consequently immune
deficiency, engraftment failure, and severe side effects from myelosuppressive
chemotherapeutic ablation.
The advent of bone marrow transplantation began in 1968, and since then it
has been shown to effectively alleviate the adverse symptoms associated with SCID
by substantial and sustainable immune reconstitution 48. Despite complications, the
cytokine dependent signaling defects seen in SCID foster an optimal situation for
allogeneic HSCT, even in the absence of a HLA matched donor. The absence of Tcells or a combined lack of NK and T cell substantially reduces possibilities of grafts
versus host disease. Because of this, individuals with SCID cannot reject grafts 48 so
prior conditioning is not required. It has, however in cases of ADA SCID been shown
to improve engraftment 49 due to its ability to dissolve bone marrow from the bone
cavity while preserving the thymic niche.
New approaches for reconstituting the adaptive immune compartment
following HSCT are in development. In most cases of SCID, patients maintain thymic
architecture similar to the fetal thymus seen before week 12 of gestation 50. The
thymus is made up of primarily undifferentiated epithelial elements, Hassall’s
corpuscles, and small blood vessels, and an absence of lymphoid cells. The
maintenance of this fetal thymic architecture suggests that migration and
differentiation of pre-thymic lymphoid cells are required for the induction of a
mature thymus. In a very small number of patients, autopsies or post
transplantation thymic biopsies have indicated the present of a normal thymic
microenvironment, suggesting that in the presence of lymphoid precursors, the
thymus is able to recover and develop normally. Immune reconstitution following
HSCT, with T-cell depletion, in SCID patients is 3-6 months, which may in part be
16
due to the need for additional thymic development and thymic niche availability
prior to T-lymphocyte generation.
In individuals suffering from ADA deficiency, polyethylene glycol-conjugated
bovine derived ADA (PEG-ADA) can be used ameliorate symptoms caused by the
defects in purine metabolism and minimize the risk of infection associated
complications. With PEG-ADA intervention, there is a continuous circulation of
enzymatically active ADA available to detoxify adenosine and deoxyadenosine
metabolites. PEG-ADA effectively increases the numbers of circulating lymphocytes
and level of protective immunity, however the long-term effectiveness of PEG-ADA
is not quite as promising. Assessments of patients, ages 5-15 years, treated with
PEG-ADA for 5 or more years a decreased lymphocyte count despite the initially
observed improvement as well as a gradual decline in mitogenic proliferative
response leading to impaired antigenic response. Despite this, in a studying looking
at the long term effects of PEG-ADA treat, Chan and colleagues noted that at the time
of assessment all patients, despite low B-cell counts, had normal levels of
immunoglobulins and were in overall good health 51. These patients will need to be
monitored closely to ensure they do not experience further decline in immune
function if they continue on this therapy. This research demonstrates ADA therapy
can result in a decrease in efficacy with continuous use, which is a major concern
with long-term enzyme replacement therapy.
Correcting genetic diseases at a DNA level is an attractive option because it
alleviates the issues associated with allogeneic transplantation and long term ERT
and it restores functional and persistent corrected cell types. It also provides an
alternative treatment for individuals without an HLA matched donor, thereby
overcoming the immunological barriers associated with HSCT. In standard protocols
of gene therapy, HSCs are obtained directly from the patient, corrected ex vivo, and
re-infused. SCID represents an excellent model for gene therapy because due to a
selective amplification effect of corrected cells, even a low numbers of transgenic
cells are capable of reconstituting the immune system. Also alteration of cells via the
introduction of a corrected copy of the relevant gene, will not elicit an immunogenic
response in the background of SCID.
17
V.
A History of Progress
Since the initial identification and cloning of genes responsible for human genetic
disease, new methods for the introduction of genetic material into mammalian cells
have evolved. Early gene therapy trials performed in the 1990s did not produce the
revolutionary success initially anticipated but they have laid the foundation for
important gene therapy research. ADA deficient SCID was one of the first monogenic
diseases for which gene therapy was a possibility because the human ADA gene had
been cloned since the early 1980s 52 and the disorder was well characterized both
genetically and biochemically 37.
In 1990 at the National Institute of Health (NIH) in the United States, two
girls with ADA deficient SCID were infused with genetically modified matured Tcells, transduced with a -retrovirus encoding the ADA cDNA 53. Although no
complications arose from this procedure, no discernable clinical benefits were
achieved. The procedure included several cycles of leukopheresis, transduction of
the ADA gene into isolated T-cells, and cell reinfusion. During the procedure the two
patients remained on ADA enzyme replacement therapy, which may have negated
the selective proliferation advantage to the corrected cells 54.
Subsequent trials focused on transference of the ADA gene into a
hematopoietic CD34+ progenitor population. While these trials were ostensibly
ineffective due to inefficient transduction of target T-cells, continued enzyme
replacement therapy, and no ablation prior to transplantation 55, 56, 57, they provided
a foundation for improvement in techniques for isolating, genetically modifying, and
ex vivo culturing of human HSCs. Vectors were produced at higher titers increasing
gene transfer efficiency and cell scaffolding material and cytokines were used in
culturing to improved cell survival. In 1999, a gene therapy trial for SCID-X1 was
initiated at Necker-Enfants Malades Hospital in Paris, France. SCID-X1 serves as an
excellent model for gene therapy, in part because of the potential proliferative
advantage conferred to genetically corrected cells. Once functional, cytokine
18
receptors are able to transmit survival and proliferation signals to lymphoid
progenitors. This has been corroborated by an unusual event in which a SCID-X1
patient exhibited a spontaneous reversion of a mutant c gene which led to partial
and sustained correction of their T-cell deficiency58.
In 2000 an ADA deficient SCID gene therapy trials began in Milan, Italy at the
San Raffaele Telethon Institute for Gene therapy. The -retrovirus vector was used
to stably express ADA in autologous bone marrow derived HSC from SCID patients.
A low dose of the chemotherapeutic agent busulfan was utilized prior to HSC
reinfusion to ablate the pre-existing marrow and improve engraftment, thereby
providing a selective growth advantage to the gene corrected HSCs.
The results from the 1999 Paris trial and the 2000 Milan trial were
remarkable. Analysis of the first two subjects from each trial showed immunological
competence in all four individuals. T-cell counts reached physiologically normal
levels and lymphocytes proliferated in response to mitogens and specific antigen
stimulation. Immunoglobulin levels were normal and patients no longer suffered
from opportunistic infections or immunodeficiency related complications. These
two trials were the first to demonstrate significant clinical benefit in treating genetic
diseases with gene therapy.
Beginning in 2004, in London, England, 10 SCID-X1 children were treated
with CD34+ HSCs that had been transduced with the conventional -retroviral
vector expressing the c transgene. As in previous SCID-X1 trials these patients
received no myelosuppressive conditioning. Upon extended follow up substantial
immune reconstitution was observed, notably in 9 out of 10 subjects a polyclonal T
cell repertoire was achieved. Humoral immunity was only partially restored as was
observable by suboptimal humoral response to antigen stimulation, low levels of
immunoglobulin production, and some infection associated complications, despite
this some patients were able to discontinue immunoglobulin replacement therapy.
There was an initial spike in NK cell numbers across the patients post engraftment
however this was not maintained, suggesting a diminished contribution level of
transgenic progenitor cells to this compartment 59.
19
In 2006, the same London based group began conducting an ADA deficient
SCID gene therapy trial. Six pediatric subjects were enrolled and administered HSCs
transduced with the ADA cDNA after low dosages of pretransplant chemotherapy.
Extended follow up of these patients demonstrated that four subjects achieved
immune reconstitution between 6 and 24 months post transplantation. Three of the
four patients in which gene therapy proved to be advantageous exhibited sufficient
B-cell function to taper off immunoglobulin replacement therapy. In two subjects,
gene therapy was ineffective: one subject received a very low dosage of HSCs while
the other had a low percentage of transduced HSCs infused60.
A major setback: T-ALL due to insertional mutagenesis
Leukemia-like T lymphoproliferation occurred in 5 of the 20 subjects (1 in
the London trial and 4 in the Paris trial) 61, 62 treated in the SCID-X1 trials between
Paris and London due to insertional mutagenesis as a result of the retroviral vector
integrating into proto-oncogene loci. Other genetic abnormalities were also found.
After being treated with chemotherapy 4 of the 5 patients recovered fully and 1 died
of refractory leukemia 61.
It is more than likely that disease-associated factors influenced the outcome
in these cases because similar gene transfer technology was used in the ADA
deficient SCID trials and in some cases, similar LMO2 integration patterns (reviewed
in 63) were observed, however none of these patients developed leukemia. A wide
array of reasons has been proposed to explain this apparent discrepancy. A possible
explanation is the difference in the specific transgenes, the c gene encodes a
cytokine receptor chain, essential for cellular growth and proliferation versus the
ADA gene, which is an essential housekeeping gene involved in purine salvage
metabolism and does not induce cellular activation or proliferation. The rapid
proliferation associated with progenitor rescue due to the introduction of the c
gene is much more rapid than that associate with ADA rescue due in part to the fact
that ADA expressing cells do not have the selective proliferation advantage seen
20
with cells expressing the c gene. This is because ADA has a compensatory effect,
which benefits untransduced cells as well.
In ADA deficient SCID, T-cell reconstitution takes approximately 18 months
whereas SCID-X1 patients have full reconstitution within a few months of treatment.
Rapid proliferation of T-cells may result in the accumulation of mutations in this
population. Also since the c gene is required for T-cell development and
proliferation, untransduced cells will not survive. ADA corrected cells have less of a
proliferation advantage due to the fact that ADA expression affects all lymphocytes
and provides cross correction to cell lacking the ADA transgene. Thymic damage
also occurs during ADA deficiency, which is likely to play a compliant role in a more
laconic immune recovery 64. HSCs from SCID-X1 patients may be intrinsically
different than HSCs from ADA deficient patients; evidence exists to support the
hypothesis that children with SCID-X1 are predisposed to cellular transformation 65.
NK cells play an important role in tumor surveillance, in cases of human acute
myeloid leukemia NK cells participate in clearance of leukemic cells 66 the absence
of NK cells post immune recover in SCID-X1 patients may also serve as a
contributing factor to the lack of tumor clearance observed in the setting of SCID. It
has also been theorized that IL-2R may act cooperatively with LMO2 to induce
oncogenesis 67, 68, however evidence to the contrary has also been presented 69, 70.
The set back of having 25% of SCID-X1 patients treated with gene therapy
develop leukemia between 3 and 6 years after treatment has inspired researchers
and clinicians to proceed with cautioned enthusiasm while designing future gene
therapy treatments.
VI.
Gene Therapy and Toxicity: The Evolution of Vectors
Gene therapy has evolved over the past 20 years to treat several kinds of monogenic
disorders. PIDs that can be treated with bone marrow transplantation pose as the
most credible models for gene therapy because it has been established that an intact
progenitor population can effectively treat these disorders. SCID is an especially
21
amiable model due to the fact that SCID patients cannot mount an immune response
to the protein encoded by the transgene or immunogenic vector encoded epitopes
as seen in other disorders treatable with gene therapy 71, 72.
Retroviral vectors are produced through recombinant DNA techniques to act
as gene delivery tools. Novel surface modifications and transgenes built using
synthetic components can increase targeting specificity and efficiency 73. Vectors can
integrate the transgene into chromosomal DNA of mammalian target cells with high
efficiency, where it is passed on to successive generations of progeny during mitosis.
Replication defective retroviral vectors have previously been based on murine
oncoretroviruses (the -retrovirus), simian and human lentiviral viruses, retroviruses, and spumaviruses (also known as foamy viruses) (reviewed in
, ).
16 73
The first-generation vectors that were used in these trials utilized a Maloney murine
leukemia -retrovirus (MLV) backbone with viral long terminal repeats (LTRs)
containing both an enhancer and promoter to drive transgene expression.
In response to concerns of vector-mediated leukemogenesis, a variety of
novel modifications have been made to new generation vectors to curb foreseeable
risks associated with genomic integration. Self-inactivating vectors (SIN), which are
able to delete the U3 enhancer and promoter in the LTR during the viral reverse
transcription process, have a greatly reduced capacity to transactivate endogeneous
genes after genomic integration. While the integration profile of SIN vectors is
generally comparable to wild type retroviral vectors of the same type, SIN vectors
demonstrate a significantly reduced level of genotoxicity 74. Additionally next
generation vectors make use of promoters derived from cellular gene to drive
transgene expression. Promoters from genes such as elongation factor-1 (EF1)
create more physiologically typical gene transcription and reduce the strong
enhancer activity associated with first generation vectors that utilize retroviral
enhancer/promoters to transcribe transgenes 75.
22
Figure 5: First and second generation -retroviral vectors. First generation vectors were
characterized by a region of long terminal repeats (LTR) situated at the 3’ and 5’ end of their genome.
These LTRs have the capacity to promote expression of cellular oncogenes if the retroviral vector
integrates in close proximity to them. Vector integration also has the capacity to silence tumor
suppressor genes if it occurs within an exon of one of these genes. Second generation retroviral
vectors or so called self inactivating (SIN) vectors are constructed such that following reverse
transcription the viral promoter and enhancer elements including the CAAT and TATA box are
deleted. Weaker promoters are also used such as human elongation factor-1 to reduce the potential
for endogeneous gene transactivation. E/P, enhancer/promoter; LTR, long terminal repeats; Prom,
promoter. 54
Retroviral Vector Insertional Mutagenesis in Gene Therapy
The principle potential risk factor of retroviral vector mediated gene transfer
is malignant transformation. Genotoxicity can result from several vector
integration-associated mechanisms including: endogenous promoter activation,
aberrant gene transcript truncation, and erroneous splicing. The most common
cause of insertional mutagenesis is when retroviral vector insertion results in either
the activation of proto-oncogenes or attenuation of tumor suppressor genes.
Initially the risk factor for this was considered to be very low because it had never
previously been observed in a clinical trial 76.
23
Review of the potential factors contributing to leukemogenesis suggests that
insertional mutagenesis is the initiating causational event. However acquisition of
secondary genetic abnormalities is essential for oncogenic transformation. Genetic
analysis indicated that in 4 of the 5 patients that development clonal T-cell acute
lymphoblastic leukemia (T-ALL), vector integration occurred in close proximity to
the proto-oncogene LIM domain only 2 (LMO2) gene promoter and expression
analysis indicated an increased level of LMO2 transcription 77, 61, 62.
Integration near LMO2 caused increased transcriptional output of nearby
genes and promoted clonal T-cell proliferation, however evidence of cooperative
elements, such as additional genetic aberrancies, contributing to transformation
were noted in the patients as well. Initially the two youngest patients (P4 and P5)
were identified with uncontrolled exponential clonal out growth of mature T-cell
that occurred approximately 3 years after gene therapy. The absence of replication
competent retroviral vectors was confirmed by analysis of the specific leukemic-like
T-cell clones. These clones did not have amphotropic envelope, reverse
transcriptase, or integrase genes present. LMO2 insertions as well as other genetic
aberrations were detected in both subjects. At the time of documented
lymphoproliferative disease, peripheral T-cell clones exhibited a single insertion in
both patients, where as prior to the onset of leukemia multiple integrations were
observed in patients’ circulating T-cells. LMO2 transcript was normal as confirmed
by exon specific reverse transcriptase PCR (RT-PCR) however its abundance of
expression was comparable in levels to those found in mouse erythroleukemia
(MEL) cell lines. Analysis of mRNA demonstrated colocalization of LMO2 and c
indicating that the LMO2 allele targeted by retroviral insertion was being actively
transcribed in both patients. These data confirmed retroviral cis-activation resulted
in monoallelic LMO2 expression in both P4 and P5 77. P5 went into remission and
recovered fully, however despite aggressive intervention P4 succumbed to
refractory leukemia 61.
A few years later an additional 3 patients who had developed leukemia were
described 61, 62. One patient (P10) had, in addition to integration near the LMO2
24
promoter, an insertion site near the proto-oncogene BMI1. The patient (P7) lacking
the LMO2 distal integration was found to have vector insertion near the promoter of
CCND2, a gene known to act as a proto-oncogene in lymphoid cells. Upon analysis of
patients’ blast cells, it became apparent that these cells were affected by
chromosomal translocations, gain of function mutations activating NOTCH1 (P5 and
P10), deletion of tumor suppressor gene CDKN2A (P4 and P7), 6q interstitial losses
(P4 and P7), and SIL-TAL1 rearrangements (P5) 61. Both patients P10 and P7
recovered fully after chemotherapy.
The last patient (P8) who entered into the SCID-X1 gene therapy trial at
Great Ormond Street Hospital in London, England demonstrated that along with
LMO2 transactivation, he also carried additional contributing genetic mutations.
Other genetic abnormalities identified included chromosomal translocations, gain of
function mutations activating NOTCH1 and reduced expression of tumor suppressor
gene locus cyclin dependent kinase 2A (CDKN2A). The genes surrounding LMO2
were also found to be upregulated, suggesting the potent ability of the viral LTR
enhancer to alter the expression of several nearby cellular genes. After
administration of chemotherapy P8 recovered fully. Post chemotherapy the clone
containing the LMO2 insertion was no longer detectable by linear amplification
mediated (LAM) PCR or specific PCR tracking 62.
Strong evidence exists that the disease environment created by SCID-X1 is
supportive to transformation. Whether or not the expression of the γc chain
transgene in thymocytes is a significant risk factor was previously a point of debate
however evidence now exists to strongly suggest that the expression of the γc chain
gene in and of itself is not permissive to oncogenesis. Instead, it is possible that
there is an accumulation of cells poised to acquire genetic mutations prior to the
rapid expansion of thymocytes. Once the development burden existing in SCID-X1
patients is lifted massive proliferation of these cells leads to oncogenic
transformation. Another possibility is that IL-7 may be an important extrinsic factor
in the support and proliferation of leukemic thymocytes, therefore overexpression
of IL-7 could lead to a overriding of the normal cellular mechanism to shut down
dysregulated growth 62.
25
The role of LMO2 in T-cell development.
The transcription factor LMO2 contains zinc finger-like binding motifs called
LIM domains and function as a bridging protein in multiprotein complexes that acts
to upregulate genetic transcription 78. The LIM domain is essential for proteinprotein interaction, LMO2 cannot bind DNA directly but it has the capacity to
arbitrate transcriptional activation and repression by binding to other transcription
factors. While LMO2 is a T-ALL associated oncogene, it is also essential for normal
hematopoiesis (reviewed in 79). LMO2 acts as a bridging molecule for GATA1, Ldb1,
E2A, and TAL1 and this DNA-binding transcription regulator complex is essential for
differentiation of all hematopoietic lineages 80. LMO2 is known to cause a subset of
human T-cell acute lymphoblastic leukemias (T-ALL). T-ALL arises after an
asymptomatic phase during which there is an accumulations of double negative
thymocytes 78.
Utilizing a transgenic mice model, McCormack and colleagues have
demonstrated that the origin of LMO2-induced leukemia is thymocytes with T-cell
differentiation capability that express several genes typical of multipotent
hematopoietic progenitors. Their data supports the hypothesis that LMO2 acts to
maintain or induce the self-renewal faculty of pre-leukemic progenitors and this
continued long term renewal of thymocytes results in the accumulation of
transforming mutations 81. During normal T-cell development in the thymus, LMO2
is down regulated during the early DN1-DN3 stages. If overexpression of LMO2 via
retrovirus mediation is sustained it attenuates the differentiation of T-cells while
leaving other lymphoid lineages unaffected 82.
Patterns of Viral Integration
Different retroviral backbones have differential patterns of integration and
these insertion patterns are characterized by the epigenetic factors of the cell,
nuclear translocation mechanisms, and the interaction of viral integrase with host
proteins. The chromosomal integration of any vector backbone requires cellular
DNA repair mechanisms. Insertion site selection is dependent upon both cellular
26
determinants and vector design, thereby giving each retroviral vector its own
unique integration spectrum. The activation status and character of the target cell
therefore plays a significant role in vector integration.
Epigenetic profiles that favor integration have recently been identified 83. The
LMO2 locus in hematopoietic progenitors has been shown to contain several
features that accommodate frequent local integration. In vitro analysis of vector
integration site selection has provided important information on the behavior of
integrating retroviral vectors, however a homogeneous cell population cultured in
isolation from biological complexities, such as selection pressure, is not predictive of
an in vivo setting. In vivo, in the framework of gene therapy, transduced cell
populations are subjected to post engraftment environments that influence survival,
homing, and proliferation. Genome wide patterns of gene expression remained
unchanged in transduced cells however analysis of the retroviral integration sites
between transduced progenitors and post thymic T-cells indicate that sites of
insertion influence cell survival, engraftment, and proliferation 84.
-viral vectors have been shown to preferentially integrate into promoters,
CpG islands, and DNase hypersensitivity sites 85. Data from the French SCID-X1,
British SCID-X1, and Italian ADA-SCID trial demonstrated that when looking at
transduced CD34+ progenitor cells derived from patients, over 65% of insertions
are very near highly expressed genes, suggesting that vector integrations are
correlated with the level of expression of genes in CD34+ target cells (reviewed in
86).
This becomes problematic when these specific genes need to be downregulated
during the later progressive development stages of T-cell differentiation. Genes that
maintain a high expression level due to vector integration poses a high risk for
insertional leukemogenesis.
Lentiviral vector integration is a risk factor for the disruption of
transcription units. This can occur via the introduction of aberrant cellular-vector
chimeric transcripts 87, 88 or the induction of haploinsufficiency
89
because they
favor integration into genes that are being actively transcribed 73. Haploinsufficency
occurs when a diploid organisms who is heterozygous for a specific mutation is
27
clinically affected because only a single functional copy of the gene is incapable of
providing sufficient gene product to assure normal function. SIN lentiviral vectors
can also induce aberrant transcript formation since proviral integration can
introduce a premature termination of transcription 87. It has also been described
that SIN lentiviral vectors are susceptible, albeit at a much lower frequency than
LTR containing lentiviral vectors, to transcriptional read through activity due to
their tendency to integrate into genes 90. -viral vectors have the most neutral
integration patterns and they can be designed such that they lack strong splice
signals which could interfere with the processing of cellular mRNA 91. Foamy viruses
have also been described as being less oncogenic than lenti- and retroviral vectors
because they do not preferentially integrate into genes or genetic regulator units 92.
Understanding preferred integration patterns of viral vectors and integration
site analysis are a powerful tool for predictive analysis of potential genotoxicity.
Vector integration may at some point in the future also be used as a research tool to
understand epigenetic characteristics and gene expression profiles of tissues.
Predicting Genotoxicity: Animal Models
Despite already described issues with interspecies translation models,
animal models pose as an invaluable tool for identifying risk factors and therapeutic
outcomes in human disease. At this point, it is not yet possible to predict all
potential severe adverse events that can occur as a result of vector integration,
however animal models provide insight that would not otherwise be accessible. For
example, it is known that pre-leukemic events arise in the thymus and sampling for
clonality studies are based on peripheral blood, so in a preclinical setting, animal
models are a salubrious solution for abstracting knowledge of leukemic
development and predicting genotoxicity.
Utilizing a tumor prone mouse model, Montini and colleagues were able to
infer low genotoxicity from an array of vector integration sites. Cdkn2a-/- mice are
highly susceptible to developing a broad range of tumor types in response to genetic
lesions, researchers compared tumorigenesis as a result of lenti versus classical
28
retroviral integration. Retroviruses integration resulted in a dose dependant
acceleration of tumor development based on LTR activity and insertion in
oncogenes and cell cycle associated genes were enriched in early onset tumors.
Transformation was unaffected by lentiviral vectors and no specific integration
patterns were enriched for in tumors 93.
High through put screening of insertional mutagenesis by replication
competent retroviruses was utilized as a tool to identify potential oncogenes in
mouse models of tumorigenesis and create a cancer gene database 94. These results
are also useful for identifying integration sites that are common across different
tumor types and for addressing, which genetic lesions contribute to transformation.
To assess unique risk factors that the SCID environment imparts upon the subject,
Shou et al development an SCID-X1 mouse model lacking both the Arf tumor
suppressor gene and the c gene. In comparing Arf-deficient immunocompetent
mice with Arf-deficient SCID-X1 mice, they found that the SCID background was
required for the high rate of T-cell transformation associated with insertional
mutagenesis.
In vitro immortalization assays (IVIM) serve to drastically decrease the
number of animals necessary for assessing genotoxicity. The IVIM assay relies on
the induction of a proliferative, survival advantage conferred by insertional
mutagenic activation of cellular oncogenes, which becomes clear during in vitro
expansion when transduced cells are plated with cytokines that induce
differentiation in a limiting dilution assay95. Mutants are also resistant to
differentiating in response to a myeloid growth factor cocktail, therefore
tremendous importance is placed upon growing conditions of the cells since slight
variation could impact the sensitivity of the assay and potentially the types of
mutants that can be isolated. Specifically the IVIM sensitively detects insertional
regulation of Evi1 and Prdm16 via enhancer-related mechanisms in myeloid
progenitors.
Utilizing the IVIM assay, Modlich and colleagues have shown that relocation
of the -retroviral enhancer promoter sequence from the LTR to an internal
29
position, thus creating the SIN vector, reduces the fitness of mutants. Mutations in
transcription factor binding sites that reduce enhancer activity and the use of
insulators also decreases transformation capacity. Certain cellular promoters when
used in SIN vectors reduce the genotoxic risk of transformation below detection
level 75. The IVIM assay has been used to demonstrate that the post-translational
regulatory element of the woodchuck hepatitis virus (WPRE) does not affect
insertional transformation, however it has been shown previously to improve
vector generated transcription termination, thereby decreasing read through
transcripts, and increase vector titration when placed up stream of the
polyadenylation signal of the transgene 90. Lentiviral vector integration patterns
demonstrate a substantially reduced risk of insertional mutagenesis when
compared to -retroviral vectors in the IVIM assay.
Zychlinksi and colleagues also demonstrated that specific cellular promoters
reduce the risk of insertional mutagenesis when compared to viral promoters 75 and
different insulator elements have significantly different functional capacity 96.
Chromatin insulators are additional regulatory elements that can be utilized to
decrease both vector silencing as a result of chromosomal position and genotoxicty.
Upon utilizing the IVIM to test a battery of different insulators Baum and colleagues
found that only a subset were potent enough to reduce the transforming potential of
strong enhancer crosstalk capacity to any significant degree.
VII.
Future treatment
Stem cell-based therapy is a quickly evolving field of medicine that makes use of our
growing understanding of genetics to cure previously untreatable disease. Progress
in viral vector designs provides an appreciable advancement in the therapeutic
treatment of monogenic diseases. Integrating gene delivery systems allow for stable
and sustained gene expression, however continued efforts are being made to
increase precision and decrease risks associated with genomic insertion. SIN vectors
offer distinct safety advantages compared with their wild type counterparts (Figure
30
7). Research has shown that they substantially less likely to interact with
neighboring cellular genes 97, read through transcripts are significantly reduced 90,
and multi-lineage lymphoid reconstitution, as seen in murine models of SCID-X1 and
phase I clinical trials of Wiskott-Aldrich Syndrome (WAS) and
adrenoleukodystrophy (ALD) (reviewed in 98), is similar to those achieved with LTR
driven transgene expression 99. They nevertheless require the addition of promoters
and enhancers within the body of the vector to attain high levels of vector
expression and these elements potentially possess the capacity to alter the
expression of adjacent cellular genes. Tissue specific regulatory elements have been
shown to reduce this risk 75. Chromatin insulators are naturally occurring DNA
regulatory units block the interaction between vectors and the surrounding host
genome by forming boundaries between adjacent chromatin domains and can be
used to mitigate vector silences and insertional mutagenesis due to vector mediated
gene activation (Figure 7).
The addition of suicide genes to vectors could be utilized to subvert vector
activation in the event of a serious adverse reaction such as abnormal clonal
outgrowth or malignant transformation. This technique has been under
investigation in fields of cancer therapy and regenerative medicine. It has been
proven effective in cases of graft versus host disease post adoptive cell therapy.
Transgenic herpes simplex thymidine kinase (HSV-TK) expression in donor cells has
been effectively utilized as a safety switch in patients receiving cellular therapy.
Significant drawbacks of this system exist as the mechanism of interference requires
alterations in DNA synthesis so cell suicide can take several days and is not 100%
effective in eliciting cell death 100. HSV-TK is also potentially immunogenic as it is
virally derived. The antiviral drug ganciclovir is necessary for suicide gene
activation, which makes this class of antiviral drugs no longer available for
therapeutic use in these patients.
In an investigation of the effectiveness of a different suicide gene to abrogate
adverse events associated with cell transfer therapies; Di Stasi and colleagues
utilized an inducible T-cell safety switch based on the fusion of human caspase-9 to
a modified human forkhead binding protein. A synthetic dimerization drug could be
31
administered in the event of graft versus host symptoms, which could activate the
caspase-9 fusion protein leading to rapid apoptosis of cells expressing the construct
101.
Previous studies indicated that the use of T-cells depleted of alloreactive
progenitor cells provide immune protection against viral disease during recovery
from HSCT 102, this technique also spares leukemia reactive cells 103. The addition of
inducible caspase-9 to donor T-cells allows for rapid destruction of these cells in the
event of graft versus host disease. The additional of a suicide gene, such as inducible
caspase-9, to the vectors used in gene therapy would allow for the destruction of
transduced cells in the event of monoclonal outgrowth or transformation. HSV-TK
would not be an ideal choice as a suicide gene due to the factor that the class of
antiviral drug used to activate the suicide gene is an important therapeutic agent
used in immunocompromised individuals due to their extreme susceptibility to viral
infection and the prevalence of herpes simplex and herpes varicella.
Antibodies could also be used to eradicate transduced cells. In a mouse
model of SCID-X1, Scheumann and colleagues transduced murine HSCs with a c
myc-tag chimeric transgene. The transcription of this gene resulted in a myc tagged
c fusion protein that could be targeted with myc antibodies and depleted in the
presence of complement activity 104. This strategy could be potentially used in SCID
however it could not be effectively used as a safety strategy in other disorders
treated with gene therapy due to the immunogenicity of the myc tagged protein.
Addition of the myc tag could also alter the intended functional activity of the c
chain subunit.
Genomic editing
The ability to apply site-specific genomic manipulation provides a platform
for genetic research and therapeutic intervention. Homologous recombination is the
process by which cells are able to resolve stalled DNA replication forks, repair
double stranded DNA breaks, and foster genetic recombination 105. Gene targeting is
accomplished by utilizing an exogenous template DNA molecule that is introduced
into a cell to replace a corresponding chromosomal segment via homologous
32
recombination, this process represents a directed and specific way of altering the
genome. Two recently developed designer nucleases are Transcription ActivatorLike Effector Nucleases (TALENS) and Zinc finger nucleases (ZFN). TALENS are
synthetic restriction enzymes made up of a transcription activator-like effector DNA
binding domain fused to a DNA cleavage domain that can be engineered to targeted
to any specific site within the genome for genetic editing.
Figure 6: Gene editing utilizing designer endonucleases. ZFN made up of a zinc finger DNA
recognition domain and a FokI endonuclease domain joined by a short linker bind the genomic DNA
target site, which initiates the dimerization of the nuclease domain. Dimerization induces the enzyme
to become catalytically active and it cleaves the genomic DNA, generating a double stranded DNA
break. The double stranded DNA break can be repair via NHEJ pathway resulting in gene disruption,
or homology directed repair. Homology directed repair can either lead to gene correction of
transgene cassette insertion. Homology directed repair is homology driven because the two sister
chromatids are in close proximity to each other. If the donor template contains homology to the DNA
sequences on either side of the break, the exogeneous sequence can be incorporated at the site
allowing for transgene insertion or mutation correction 98.
Both TALENS and ZFN mediate site-specific gene addition by introducing
double stranded DNA breaks, which results in gene integration due to homologydirected repair (HDR) utilizing the supplied DNA fragment as a template. In the
33
absence of a supplied DNA template ZFN induced double stranded DNA breaks
stimulate the endogenous error prone NHEJ repair pathway, which results in gene
disruption.
DNA specific ZFN and template DNA for gene correction can be incorporated
into separate integrase-deficient lentiviral vectors and targeted to affected cells.
Transiently expressed ZFN made up of a zinc finger DNA recognition domain and a
FokI endonuclease domain joined by a short linker bind the genomic DNA target
site, which initiates the dimerization of the nuclease domain. Dimerization induces
the enzyme to become catalytically active and it cleaves the genomic DNA,
generating a double stranded DNA break (Figure 6). Cellular mechanisms then
induce the activation of exonucleases, which cuts the 5’ end from the broken ends,
leaving single stranded 3’ sticky ends. The donor DNA template complex can then
anneal to their homologous sequences within the genomic DNA and act as a
template for DNA synthesis upon recruitment of DNA polymerase. The site of the
double stranded DNA break is thereby repaired via DNA synthesis utilizing the
donor DNA as a template resulting in the correction of the disease causing mutation.
ZFN provide genetic correction in function as well as controlled expression.
Due to direct genomic DNA editing the corrected gene is under the control of
endogenous regulatory elements. This type of correction has the added benefited of
leaving the rest of the genome unperturbed. However, co-delivery of the template
DNA and the ZFN represents a major hurdle to exploiting the use of ZFN mediated
genetic manipulation. Also genomic instability as a potential result of ZFN gene
targeting needs to be more fully understood. There is a low efficiency of correction
associated with ZFN gene targeting however, in instances of correcting genes like
the c chain, the resolution of the mutation will give these cells a strong selective
growth advantage, such that a low percentage of corrected cells will be permissive
to immune recovery, at least in theory.
Control of vector site integration can also be achieved with the integrase
binding cellular protein lens epithelium derived growth factor (LEDGF). The Cterminal portion of LEDGF interacts with lentiviral integrase. Tethering of viral
34
integrase results in directed integration and in the presence of LEDGF viral
integration into active genes is reduced. Introduction of chimeras composed of
LEDGF integrase-binding domains fused to alternate chromatin binding domains
that program integration outside of transcription units can be used to obviate
random gene insertion 106
35
Figure 7: Future implements for improved safety and efficacy of HSC-based gene therapy. The
implementation of SIN vectors in gene therapy has been a substantial step towards improving the
safety of vectors used in for transgene incorporation. It is also necessary to ensure that the corrective
gene is expressed at physiologically relevant levels and can persist in the progeny of transduced cells.
Physiological regulation of transgene expression via cellular promoters as well as the addition of
chromatin insulators has lead to a decreased likelihood of genomic perturbation by promoter
transactivation or by positional silencing. Selection of gene integration sites could potentially lead to
better transgene transfer and reduced risk by obviating random genomic insertion. Targeting of such
sites with designer endonucleases can lead to site directed genetic correction via induced
homologous recombination (also refered to as homology directed repair). Non-chemotherapy based
clearing using agents such as monoclonal antibodies can ablate bone marrow and lead to improved
engraftment. 107
36
VIII.
Discussion
Exceptional advances have been made in the therapeutic treatment of mongenic
disorders (Figure 7). Improvements in vector safety and efficiency have provided
momentum for increasing the accessibility of gene therapy for a variety of disease
types including immunological and lysosomal storage related disorders, as well as
neurodegenerative diseases and cancer.
Currently HSCT is a curative therapy for SCID and other immunological
primary immunodeficiencies. Despite this, its inaccessibility to many patients and
its several life threatening complications render it a suboptimal intervention,
therefore alterative treatment options are necessary. Symptom-alleviating enzyme
replacement therapy is available for certain monogenic diseases such as metabolic
disorders and various lysosomal storage disorders, however the cost of sustaining
patients over the course of their lifetime is astronomically high and in some
situation impossible. Gene therapy serves as a wide platform for the curative
venture of an extensive array of diseases that were previously considered
insurmountable.
SCID is an ideal disorder for modeling gene therapy due to the fact that HSCT
is curative for individuals afflicted with SCID, and these patients do not have the
immune capacity to reject transduced HSCs. However a number of obstacles and
safety concerns exist with gene therapy, such as insertional mutagenesis and vector
immunogenicity in disorders other than SCID. Insertional mutagensis is of particular
concern with SCID because the genetic landscape of this disorder is particularly
permissive to oncogenic transformation. SCID patients are predisposed to various
cancers due to a variety of factors. The absence of NK cells as seen in approximately
70% of SCID cases 108 results in insufficient tumor surveillance. SCID is also often
observed in the backdrop of other genetic abnormalities, so when transformation
occurs there is not a cellular system or tumor suppressor-signaling pathway in place
to subvert oncogenesis. Murine models of SCID demonstrate an increased
population of bone marrow lymphoid progenitors, which may be posed for
transformation once the differentiation block is lifted.
37
Understanding viral vector integration patterns in a variety of cell types is
essential for optimal vector backbone selection. As HIV infection has not culminated
in oncogenic transformation it is reasonable to hypothesize that lentiviral vectors
might be safer alternatives to oncogenic retroviral vectors. Lentiviral vectors have
not been associated with insertional mutagenesis in pre-clinical research or clinical
trials, however an instance of monoclonal outgrowth has been observed in one
patient from a -thalassemia trial (reviewed in 109). SIN -viruses and foamy viruses
have also recently been shown to have a non-oncogenic pattern of integration, as
they do not preferentially target transcriptional regulatory sites.
While targeted vector integration is a progressive and promising mode of
overcoming insertional mutagenesis, additional research must be done to benefit
from the full potential of technologies such as designer endonucleases. Targeted
integration requires that vectors accurately home to a specific genomic site that is
permissive to robust transgene expression, not susceptible to insertional
mutagenesis, epigenetically not conducive to vector silencing, and resistant to
transcriptional disruption of the surrounding genome. Issues associated with off
target sites of genomic instability may also arise due to the fact that nucleases create
double stranded DNA breaks. These double stranded DNA breaks result in the
activation of intrinsic DNA repair mechanisms, however nonspecific DNA cleavage
could result in the unintended disruption of genes.
Integration tethering protein lens epithelium–derived growth factor (LEDGF)
could act as a potential alternative for integration targeting, as it has previously
been associated with reduced integration into genes. The cellular transcriptional
coactivator LEDGF/p75 is a cellular factor harnessed by HIV for chromosomal
integration. The HIV viral integrase tethers to the chromatin via the LEDGF/p75,
which thereby increases the efficiency of targeted integration into transcriptional
units 110. The engineering of LEDGF integrase binding domains fused to specific
chromatin binding domains could act to direct viral integration into specific
predetermined sites.
38
An additional potential resource of precise genomic editing is the RNAguided nuclease technology known as clustered regularly interspaced short
palindromic repeats (CRISPR)/Cas system. The CRISPR/Cas is a part of the type II
prokaryotic adaptive immune system, which aids in RNA-guided site-specific DNA
cleavage. Cong and colleagues recently demonstrated that in mammalian cells, the
Cas9 nucleases can induce highly precise cuts at endogenous genomic loci via the
guidance of short RNAs. Several different guide sequences can be loaded into a
single CRISPR array, which allows for simultaneous editing of several genomic sites
111
. Utilizing RNA to direct specific genomic editing poses as a powerful tool for
biotechnology and genomic manipulation.
Induced pluripotent stem (IPS) cells represent an interesting potential future
prospect for gene therapy. While several safety concerns could potentially arise due
to the insertional mutagenesis and the proto-oncogenes used for cellular
reprogramming, IPS cells pose as a great source for disease modeling. IPS cells are
created with viral vectors and have induced expression of factors associated with
pluripotency, this represent a potential hazard due to the fact that these genes are
well established oncogenes. These cells can be derived from patient tissue,
reprogrammed into pluripotent progenitors, and then differentiation into
hematopoietic precursors to be used for the modeling of individual patients. Disease
modeling in this context could be used to test viable therapies in the future. IPS cells
have already been generated from individuals with Parkinson's disease,
amyotrophic lateral sclerosis (ALS), and β-thalassaemia to further investigate
mechanisms of these diseases.
Genetic engineering is in the early stages of investigating potential
mechanisms of immune resistance to pathologies such as cancer and HIV. T-cell
engineering could lead to novel immune functions as oppose to simply correcting
specific defects. HIV leads to the progressive destruction of CD4+ T-cells, as these
levels decline, patients progress towards AIDS. If it were possible to create a
population of T-cells resistance to HIV mediated CD4+ T-cell destruction, patients
would be protected against immune decline. Genomic editing is one method that
could be used to modify the T-cell genome. The use of retroviruses to express anti39
HIV genes that inhibit steps in the viral infection or replication cycle or the
introduction of small interfering RNA molecules could be used to prevent infection
of cells 112. ZFN could also be used to modify the viral genome directly and knock out
essential genes required for viral replication.
Genetic modification of T-cells could provide treatment against specific
tumor types. Genetically engineered T-cells have shown impressive efficacy and
clinical benefit in human anti-cancer therapy trials 113. Modified antigen receptors
could be highly effective in directing T-cells to mount a strong immune response
again a number of different tumor types. Recent research has suggested that the
tumor microenvironment plays a central role in oncogenic immune evasion 114. Tcells programmed to be unsusceptible to this inactivation could ensure effective
tumor eradication. An important consideration however would be to ensure that
these T-cells do not create an autoimmune event due to their inability to be shut
down by normal immunosuppressive mechanisms. Genetically engineered, epitope
specific programmed T-cells could be a potent tool for overcoming specific immune
evasion events associated not only with cancer but with other pathologies as well. In
conclusion, with its continuous evolution, and the furthered understanding of
potential adverse effects associated with gene therapy, this field offers great hope
for the eradication of a wide variety of previously debilitating diseases.
40
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1.
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5.
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