THERAPEUTIC POTENTIAL FOR THE INHIBITION OF HIV FROM CD34+
HEMATOPOIETIC STEM CELL DERIVED INDUCED PLURIPOTENT STEM
CELLS EXPRESSING THREE ANTI-HIV GENES
Brian Patrick Fury
B.S., California State University, Sacramento, 2009
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
BIOLOGICAL SCIENCES
(Stem Cell)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
THERAPEUTIC POTENTIAL FOR THE INHIBITION OF HIV FROM CD34+
HEMATOPOIETIC STEM CELL DERIVED INDUCED PLURIPOTENT STEM
CELLS EXPRESSING THREE ANTI-HIV GENES
A Project
by
Brian Patrick Fury
Approved by:
__________________________________, Committee Chair
Thomas Peavy, Ph.D.
__________________________________, Second Reader
Jan Nolta, Ph.D.
__________________________________, Third Reader
Tom Landerholm, Ph.D.
________________________
Date
ii
Student: Brian Patrick Fury
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
_________________________, Graduate Coordinator
Susanne Lindgren, Ph.D.
Department of Biological Sciences
iii
_________________
Date
Abstract
of
THERAPEUTIC POTENTIAL FOR THE INHIBITION OF HIV FROM CD34+
HEMATOPOIETIC STEM CELL DERIVED INDUCED PLURIPOTENT STEM
CELLS EXPRESSING THREE ANTI-HIV GENES
by
Brian Patrick Fury
Human immunodeficiency virus continues to persist in millions of people worldwide.
While antiretroviral drug therapies have improved life for many, a cure remains elusive.
Long-term antiretroviral drug therapy can also lead to toxicity, the development of drug
resistant strains and the persistence and maintenance of latent reservoirs in resting cluster
of differentiation 4 positive T-lymphocytes. Gene therapy offers a promising alternative
strategy for eradicating human immunodeficiency virus infection through the
development of lentiviral vectors comprised of highly potent anti-HIV transgenes capable
of inhibiting human immunodeficiency virus infection during the preintegration phases.
Cellular reprogramming allows for the generation of induced pluripotent stem cells
(iPSC) that are similar to embryonic stem cell-like cells with the potential to differentiate
into any cell type in the body. In this study, hematopoietic stem cells were isolated from
umbilical cord blood, transduced with a triple combination lentiviral vector containing
three potent anti-HIV transgenes encoding a chemokine-chemokine receptor 5 short
hairpin RNA, a chimeric tripartite motif-containing protein 5, and a viral transactivation
iv
response element decoy.
These cells were then de-differentiated back into an
undifferentiated pluripotential state by transduction with specific transcription factors
octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2),
cytoplasmic c-MYC, and Krüppel-like factor 4 (KLF4). The resulting colonies were
analyzed and found to express the anti-HIV genes, by enhanced green fluorescent protein
detection. Additionally, the pluripotency markers were also determined to be expressed
indicating successful transduction and reprogramming. Reprogramming patient-specific
cells to derive personalized iPSCs is an excellent source for potentially transplantable
tissue that will not cause an immune response and subsequent rejection. This could be
used to treat a myriad of human blood and degenerative diseases without the ethical
concerns attached to the acquisition and use of ES cells. In addition, this iPSC approach
could provide a highly beneficial and personalized patient specific platform for future
treatments while diminishing the need to rely on potentially toxic drug therapies and
resistant strains.
_______________________, Committee Chair
Thomas Peavy, Ph.D.
______________________
Date
v
ACKNOWLEDGEMENTS
I would like to thank Thomas R. Peavy, Ph.D., Tom Landerholm, Ph.D., my
mentor Joseph Anderson, Jan Nolta, and Gerhard Bauer for opening their labs to our
program and for all of their support. This work was supported by the James B. Pendleton
Charitable Trust, the UC Davis Institute for Regenerative Cures, the California Institute
for Regenerative Medicine (CIRM), and the CSUS Biological Sciences Department. I
would also like to extend a special thank you to my wife Lori and my mother, Margery
Fury, whose endless support, guidance, and belief in my abilities made all of this
possible. Lastly, I would like to say thank you to all of those who supported me during
any aspect of this project.
vi
TABLE OF CONTENTS
Page
Acknowledgements ............................................................................................................ vi
List of Figures .................................................................................................................. viii
INTRODUCTION ...............................................................................................................1
METHODS ..........................................................................................................................7
Lentiviral constructs and production. .....................................................................7
Primary human CD34+ HSC isolation. ..................................................................8
Lentiviral vector transduction of CD34+ HSCs. ....................................................8
Generating and maintaining HSC derived iPSC. .....................................................9
Immunofluorescent assay for pluripotency. .............................................................9
RT-PCR, PCR, and Gel Electrophoresis assays for pluripotency. ........................10
RESULTS ..........................................................................................................................12
DISCUSSION ....................................................................................................................26
Literature Cited ..................................................................................................................33
vii
LIST OF FIGURES
Page
Figure 1. Isolation and purity of CD34+ HSC from umbilical cord blood. ......................13
Figure 2. Transduced CD34+ HSC derived anti-HIV induced pluripotent
stem cells iPSCs. ...............................................................................................15
Figure 3. HSC derived iPSC colony morphology and viability after
passaging. ..........................................................................................................19
Figure 4. Immunofluorescent expression of pluripotency markers in
CD34+ HSC derived wild type iPSCs. .............................................................21
Figure 5. Reverse Transcriptase-PCR shows expression of pluripotency
factors in wild type iPSCs. ...............................................................................25
viii
1
INTRODUCTION
Human immunodeficiency virus (HIV) infection continues to be a pervasive
disease and the World Health Organization estimates that over 33 million people
worldwide are currently living with HIV especially in developing countries. Acquired
immunodeficiency syndrome (AIDS) is the long-term result of an HIV-1 infection
leading to the depletion of CD4+ T-cells as well as the existence of latent viral reservoirs.
This allows opportunistic infections to occur, with a persistent viral rebound (1-9). While
there is no cure for HIV, current antiretroviral drug therapies have been effective in
inhibiting viral infection and replication while requiring an infected individual to
maintain a highly regimented schedule. Previous studies have shown that over time,
continued use of these treatments can lead to toxicity in the prescribed patient as well as
the development of drug resistant HIV strains (2, 3, 10, 11). Attempts at developing a
variety of vaccines against HIV has also proven challenging due to the high rate of
variation in HIV strains which further facilitates the evasion of an effective immune
response (3,12). Novel strategies need to be developed which supercede existing
technologies and their limitations.
HIV is a lentivirus that belongs to the family of retroviruses whose mechanism of
infection begins when HIV’s gp120 envelope protein interacts and binds cluster of
differentiation CD4, a glycoprotein expressed on the surface of T-cells, which regularly
functions in T-cell receptor antigen identification by interacting with MHC class II
molecules expressed on antigen-presenting cells (2, 3). The binding of gp120 to CD4
causes a conformational shift in gp120 such that HIV also binds a co-receptor, chemokine
2
receptor CCR5 or CXCR4 on the T-cell or macrophage respectively, which is required
for HIV to gain entry into the host cell. This leads to the fusion of the viral envelope to
the cell membrane where the contents of virus are released into the cytosol. The contents
of a single virion include two single stranded viral RNAs, reverse transcriptase, integrase,
and accessory proteins enclosed within a viral capsid, consisting primarily of p24
proteins (2, 3). Upon fusion, the viral capsid immediately destabilizes. The two single
stranded viral RNA genomes, together with reverse transcriptase, undergo rapid reverse
transcription resulting in double stranded viral cDNAs. Then, the cDNAs are translocated
into the nucleus where they are integrated into the host genome by viral integrase. At this
stage, HIV is able to use the machinery of the host cell to replicate itself. First, a portion
of the provirus is transcribed into mRNA and subsequently spliced into smaller
fragments. These fragments are exported to the cytoplasm where they are translated into
transcriptional regulatory protein, Tat. Trans-activator of transcription protein (Tat) is
able to interact with the proviral transactivation response element (TAR) located in the 5’
UTR of the viral genome. The binding of Tat to TAR results in the exponential
replication and production of new virus. Alternatively, the viral genome can go latent for
a period of years before becoming reactivated (2, 3, 18).
The emergence of gene therapy using lentiviral vector based delivery systems is
becoming an increasingly promising alternative (2, 3, 13-17). Lentiviral vectors are
derived from HIV. They are modified by deletion of specific genes needed for viral
replication rendering them replication incompetent and no longer capable of recombining
to generate infectious disease particles. They maintain the genes necessary for
3
attachment, reverse transcriptase enzyme production, a 5’ LTR Psi- localization
sequence, and host genome integrase enzymes (28, 30). HIV vectors can be constructed
using different plasmid expression systems including a transfer vector, for transferring a
therapeutic gene, a packaging plasmid (the virus backbone), and an envelope plasmid for
cell membrane targeting (30).
To be considered efficacious in a clinical setting, the lentiviral vector should
produce a minimal physiological effect on the cells targeted for infection and show
negligible toxicity. Once a vector’s DNA becomes integrated into a host genome, it can
be passed on to new cells as they divide (28). Since the site of integration is potentially
random, there is concern that the integrated provirus may interfere with nearby protooncogenes leading to tumorogenesis and cancer (28). However, previous studies have
found that integration of lentiviral vectors did not lead to increased tumorogenesis in a
mouse model with high incidence of tumorogenic activity and low oncogenic potential
when used in a gene therapy clinical setting for treating HIV (29).
In this study, a previously developed third generation HIV derived, replication
deficient lentiviral vector was used. The vector contained a combination of three highly
potent anti-HIV genes. These included a chimeric human/rhesus TRIM5α protein
designed to interfere with viral capsid destabilization, a CCR5 shRNA engineered to
knock down the expression of the co-receptor, and a TAR decoy that mimics HIV-1 TAR
by sequestering the Tat binding protein from interacting with viral TAR RNA preventing
the initiation of transcription (2, 3).
TRIM5α is a member of the tripartite motif (TRIM) protein family and consists of
4
a RING finger zinc binding domain, a B box zinc binding domain, and a coiled-coil
region. TRIM5α also contains a c-terminal b30.2 PRY-SPRY domain critical in its
function as a species-specific retroviral restriction factor for HIV-1 (31). Made up of 493
amino acids, TRIM5α’s antiviral activity has been isolated within 13 amino acid patch of
the SPRY domain, and is found in most primates (32). TRIM5α mediates HIV-1
restriction at the postentry phase and is thought to interfere with the process of capsid
destabilization by ubiquitination, stabilization of the capsid core, and subsequent
proteasomal degradation (33). In humans this patch of amino acids contains a deletion
and is inactivated and unable to target HIV-1. To solve this problem, a chimeric
human/rhesus TRIM5α was generated by replacing the human amino acid patch with the
rhesus monkey isoform conferring HIV restriction capacity while avoiding human
immune response activity (33).
Chemokine-chemokine receptor type 5 (CCR5) is a member of the beta
chemokine receptor family and is a G-coupled transmembrane protein receptor. CCR5 is
primarily involved in chemotaxis and binds ligands RANTES, MIP1α, and MIP1β
playing a role in the inflammatory response at the site of infection. Cells that express
CCR5 include T-cells, macrophages, and dendritic cells (34). Specifically, HIV-1 prefers
to infect T-cells and macrophages by taking advantage of the CCR5 co-receptor to gain
entry into a host cell. This type of interaction makes CCR5 a good target for exploring
antiviral strategies for preventing HIV infection (34). There is a subset of the human
population of Northern European descent that are homozygous for a 32 base pair deletion
in their CCR5 genes, a selective advantage in survivors of the Black Death, resulting in a
5
nonfunctional receptor capable of preventing HIV R5 entry (35). Because CCR5 is part
of a family of redundant receptors, humans lacking the expression of the CCR5 receptor
are unaffected and physiologically normal (34, 35). Using this in combination with the
power of RNA interference, a CCR5 shRNA was developed to recognize and
complementarily bind normal CCR5 transcripts, targeting them for degradation by the
cell’s natural processes thereby effectively silencing the expression of CCR5 and
preventing HIV entry in those cells (34, 35).
A third line of defense involves a post-integration block that was designed to
prevent transcription of an HIV provirus after it had already integrated. In order for all
classes of HIV to successfully undergo transcription, viral trans-activator of transcription
(Tat) protein must bind to transcriptional-response element (TAR), a stem loop structure
located in the 5’ UTR of the provirus genome (36). Therefore, a TAR decoy was
developed so that only the stem loop structure itself was being over-expressed in the
nucleus. Over-expressing the viral TAR mimics would mask native TAR and sequester
Tat binding proteins thus preventing the initiation of HIV proviral transcription (36).
Another important and emerging technology is the use of induced pluripotent
stem cells (iPSCs) which are embryonic-like stem cells (ESC) in that they are
undifferentiated and have the pluripotent capacity to generate any cell type in the body
(19-22). Using retroviral vector delivery systems, it was discovered that by over
expressing four specific transcription factors, OCT4, KLF4, SOX2 and c-MYC, tail tip
fibroblasts and mouse embryonic fibroblasts could be reprogrammed back into a
pluripotent state (19). Another study showed that OCT4 was essential to drive selection
6
for the reactivation of gene loci necessary for pluripotency and reprogramming of the
cells. Injection of the iPS cells into mouse blastocysts produced live mouse pups and
germ cells (3, 20, 22). Further studies have demonstrated that iPS cells, from neonatal
fibroblasts, could be isolated based on colony morphology, which displayed ES cell-like
traits including surface markers SSEA-3 and Tra-1-60. Additionally, these cells
expressed OCT4 and were capable of differentiating into all 3 germ layers. Microarray
analysis confirmed that these iPS cells had expression patterns that were more similar to
ES cells than to fibroblasts, including cell-specific ESC genes (24, 25). Alternatively, a
different group of factors was identified that was also able to induce fibroblasts back into
a pluripotent state including OCT4, SOX2, NANOG, and RNA binding protein Lin28.
These findings were suggestive of the possibility of multiple combinations for complete
reprogramming, which further established the potential for developing, and using iPSCs
in gene therapy related studies (24).
In this project, CD34+ hematopoietic stem cells (HSCs) were isolated from
umbilical cord blood transduced with self-inactivating doxycycline inducible lentivectors
containing SOX2, KLF4, c-MYC and OCT4 pluripotency genes, constitutively expressed
to
de-differentiate
HSCs
back
into
an
undifferentiated
pluripotential
state.
Simultaneously, these cells were also transduced with a triple combination vector
containing the aforementioned anti-HIV transgenes including a CCR5 shRNA, a chimeric
TRIM5α and a TAR decoy. Finally, these cells were characterized to show evidence of
cellular reprogramming by morphology and growth, in addition to the expression of
pluripotency markers and anti-HIV genes.
7
METHODS
Lentiviral constructs and production. The combination, anti-HIV lentiviral vector
was constructed using a third generation HIV-derived lentiviral vector backbone pCCLcMNDU3-x-PGK-EGFP. The anti-HIV genes incorporated included a MNDU3 promoter
controlled chimeric human/rhesus macaque TRIM5alpha gene, a human U6 promoter
driven CCR5 shRNA inserted directly downstream of the MNDU3-TRIM5alpha and a
human U6 promoter driven TAR decoy expression cassette cloned downstream of the
CCR5 shRNA gene. Finally, a PGK promoter driven EGFP reporter gene was cloned
downstream of the entire anti-HIV combination lentiviral vector construct. For generating
the iPSC pluripotency reprogramming lentiviral constructs, human OCT4, SOX2, KLF4,
and c-Myc cDNAs were individually cloned into their own pCCLc-TRE-Tight-PGKrtTA doxycycline-inducible lentiviral vectors. (TRE-Tight refers to the tetracyclineresponsive promoter element. rtTA refers to a reverse tetracycline transcriptional
activator). HEK-293T cells (ATCC# CRL-11268) were used to generate, assemble, and
package the different lentiviral vectors. The anti-HIV combination vector was
constructed using 25 ug of pCMV-∆8.9, containing gag and pol genes for packaging, 25
ug of the anti-HIV combination vector, and 5 ug of pMDG-VSVG envelope. Plasmids
were transfected into HEK-293T cells by lipofection (Mirus, TransIT®-293 Transfection
Reagent, Madison, WI). Vector supernatants were collected two days post-transfection
and concentrated by Centricon Plus-70 centrifugal filter ultrafiltration units (Milipore,
Billerica, MA). All lentiviral vector preparations were titered by quantitative RT-PCR
8
using TaqMan PCR Master Mix Kit (Applied Biosystems). A WPRE (woodchuck post
transcriptional response element) gene was used to evaluate vector expression levels.
330 ng of DNA, isolated using a Wizard® Genomic DNA Purification Kit (Promega,
Madison, WI), was used for each reaction with primers and a probe specific for the
WPRE gene: WPRE (f) 5’-TTACGCTATGRGGATACGCTG-3’ WPRE (r) 5’-TCATA
AAGAGACAGCAACCAGG-3’ WPRE (probe) 5-/56-FAM/AGGAGAAAATGAAAGC
CATACGGGAAGC/36-TAMSp/-3’.
Primary human CD34+ HSC isolation. CD34+ HSCs were isolated from
anonymously donated umbilical cord blood (UCDavis Med Center, Sacramento, CA or
NDRI, Philadelphia, PA) using Ficoll-Paque gradient centrifugation (GE Healthcare,
Piscataway, NJ) and subsequently selected for by magnetic LS bead column separation
(Miltenyi Biotec, Auburn, CA). CD34+ HSCs were cultured for 2 days in Iscove’s
modified Dulbecco’s medium, 10% fetal bovine serum, supplemented with cytokines
including stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3
(Flt3) at 50 ng/ml. To determine the routine purity of isolated CD34+ HSC, flow
cytometry was performed using a Beckman Coulter FC500 using Expo32 software
analysis suite.
Lentiviral vector transduction of CD34+ HSCs. 5x10E4, 1x10E5, and 1x10E6
CD34+ HSCs were transferred into sterile 2 ml cryovials in 60 µl of culture medium.
Each vial of cells were transduced with the four individual doxycycline-inducible
lentivectors expressing the individual pluripotency factors OCT4, SOX2, KLF4, and cMYC, with EGFP alone (control vector), or with anti-HIV combination vector. The vials
9
containing cells and vectors were incubated at 37ºC and 5% CO2 for 3 hours.
Transductions were stopped by the addition of 2 ml of culture media to dilute out the
vector. Transduced cells were immediately transferred to 6 well plates containing
2.5x10E5 irradiated mouse embryonic fibroblasts and incubated at 37ºC and 5% CO2.
Multiplicity of infections (MOIs) used were 20 and 40 based on titering of genomic DNA
extracted from previously transduced HEK-293T cells.
Generating and maintaining HSC derived iPSC. Transduced CD34+ HSC were
transferred onto 5x10^5 irradiated mouse embryonic fibroblasts (Globalstem, Rockville,
MD) per well of a 6-well plate one day after initial transduction. iPSC culture media used
consisted of Dulbecco’s modified Eagle’s medium-F12, 20% knockout serum
replacement, 1x L-glutamine, 1% nonessential amino acids, 20 ng/ml human basic
fibroblast growth factor, 2 µg/ml doxycycline, and 0.1 mmol/L β-mercaptoethanol and
was changed daily. Once morphologically similar hESC-like colonies were observed,
between 9-14 days, colonies were isolated by dot blot and manually passaged using a 1
ml pipet tip onto new 6-well plates containing 5x10^5 mouse embryonic fibroblasts per
well using the same culture media without doxycycline. Routine passaging at day 4 or 5
were performed to break up larger colonies into smaller fragmented pieces which
prevented differentiation from occurring and resulted in the formation and expansion of
new colonies.
Immunofluorescent assay for pluripotency. Immunofluorescence was performed
to establish that transduced CD34+ HSCs were successfully reprogrammed and
expressing the expected pluripotency and self-renewal markers characteristic of hESC.
10
iPSC were plated in 6-well plates and cultured for 3 days. Cells were washed with
phosphate-buffered saline (PBS), fixed using 4% paraformaldahyde in PBS for 15
minutes at room temperature, then washed three times with PBS. iPS cells were blocked
with 1% goat serum in PBS, permeabilized with 2% Triton x-100 and 1% goat serum.
Cells were incubated at 4˚C overnight with primary antibodies specific for OCT3/4
(rabbit antihuman; Spring Bioscience, Pleasanton, CA), SOX2 (rabbit antihuman; Spring
Bioscience, Pleasanton, CA), SSEA-4 (mouse antihuman; R&D Systems), and NANOG
(mouse antihuman; BD Biosciences, San Jose, CA). Colonies were stained either using
NANOG with SOX2 in the same wells or SSEA-4 and OCT3/4 together in the same
wells. The next day, cells were washed with PBS three times and incubated in secondary
antibodies: OCT3/4 (Alexa 594 goat antirabbit, Invitrogen), SOX2 (Alexa 488 (FITC)
goat antirabbit, Invitrogen), SSEA-4 (Alexa 488 (FITC) goat antimouse, Invitrogen), and
NANOG (Alexa 594 (red) goat antimouse, Invitrogen) at a 1:1000 dilution. Cells were
incubated for 1 hour at room temperature. After one hour, cells were washed with PBS
three times, 4 drops of DAPI mounting solution were added to each well and imaged
using a Nikon inverted fluorescent microscope.
RT-PCR, PCR, and Gel Electrophoresis assays for pluripotency. To determine
whether CD34+ HSC derived iPSC expressed hESC-like pluripotency and self-renewal
markers, total RNA was extracted from undifferentiated iPSCs using RNA-Stat-60 (TelTest, Friendswood, TX) according to the manufacturer’s protocol. Total RNA
concentrations were ascertained using a NanoDrop Spectrophotometer (Thermo
Scientific). A Taqman Reverse Transcription Reagents kit (Applied Biosystems,
11
Carlsbad, CA) was used to reverse transcribe iPSC total RNA and generate cDNA. For
each reaction, 200 ng of cDNA was used and RT-PCR was performed using specific
primer sets for each of the pluripotency and self-renewal genes:
NANOG (f) 5′-
CAGCCCCGATTCTTCCACCAGTCCC-3′ NANOG (r) 5′-CGGAAGATTCCCAGTC
GGGTTCACC-3′ OCT3/4 (f) 5′-AAACCCTGGCACAAACTCC-3′
OCT3/4 (r) 5′-
GACCAGTGTCCTTTCCTCTG-3′ SOX-2 (f) 5′-CACATGTCCCAGCACTACC-3′
SOX-2 (r) 5′-CCATGCTGTTTCTTACTCTCCTC-3′ c-MYC (f) 5′-GCGTCCTGGGA
AGGGAGATCCGGAGC-3′ c-MYC (r) 5-TTGAGGGGCATCGTCGCGGGAGGCTG3′ TDGF1 (f) 5′-CTGCTGCCTGAATGGGGGAACCTGC-3′ TDGF1(r) 5′-GCCACGA
GGTGCTCATCCATCACAAGG-3′ and (Control) GAPDH (f) 5′-ACAGTCAGCCGCA
TCTTC-3′ GAPDH (r) 5′-CTCCGACCTTCACCTTCC-3′. Gel-electrophoresis was run
at 70 volts for 2 hours and products were visualized on an ethidium bromide, 0.8%
agarose gel under UV light.
12
RESULTS
CD34+ HSC isolation and culture. Hematopoietic stem cells (HSCs) are stem
cells found in the bone marrow, are multipotent and undergo asymmetrical division to
form all of the blood cell types including monocytes, macrophages, and lymphocytes.
Because of this, a small number of “starter” HSCs can be expanded into large numbers of
daughter cells making them an excellent resource for bone marrow transplantation and
repopulation. In this study, CD34+ HSCs were isolated from umbilical cord blood using
Ficoll-Paque gradient centrifugation, stained with CD34+ antibodies conjugated to
magnetic beads, and finally isolated by LS magnetic bead columns. These cells were
further stained with CD34-PE antibodies and then analyzed by flow cytometry for purity.
Representative purity levels were shown to be around 98.0% (Figure 1). Many cords
were processed and analyzed similarly and typically showed a yield > 92.0% on average.
13
Figure 1. Isolation and purity of CD34+ HSC from umbilical cord blood. Anonymously
collected umbilical cord blood was separated by ficoll gradient centrifugation. CD34+
HSCs were isolated from total white blood cells by CD34+ antibody conjugated to a
magnetic bead and then run over 2 LS magnetic bead columns. CD34+ HSC purity was
evaluated by CD34-PE antibody staining on flow cytometry. The representative plots
show about 98% purity of CD34+ HSC isolated although multiple assessments produced
>92% on average.
14
CD34+ HSC derived anti-HIV iPSCs. CD34+ HSCs were transduced by a third
generation self-inactivating lentivector CCLc-MNDU3-x-PGK-EGFP anti-HIV triple
combination vector containing a chimeric human/rhesus macaque TRIM5α driven by a
retroviral MNDU3 promoter, a CCR5 shRNA with a U6 pol-III promoter, and a TAR
decoy directly downstream of the shRNA driven by a human U6 promoter. Additionally,
another set of transducing vectors were used to construct the four individual pluripotency
iPSC vectors. Individually, OCT4, SOX2, KLF4, and c-MYC genes were cloned into
separate self-inactivating, doxycycline-inducible lentivector, CCLc-TRE-Tight-PGKrtTA. Then, CD34+ HSCs were transduced with either the four iPSC reprogramming
vectors alone, the four vectors together with enhanced green fluorescent protein (EGFP)
alone, or with the triple combination anti-HIV vector in equal ratios. Transduced CD34+
HSCs were transferred onto irradiated mouse embryonic fibroblast in doxycycline
positive ESC media to induce the expression of the individual factors. Culture media was
changed daily and cultures were visually inspected for the formation of hESC-like
colonies. Between 9-14 days post transduction, tightly packed colonies of fused cells
were observed for wild type (Figure 2a), EGFP alone (Figure 2b), and anti-HIV (Figure
2c, d). Cultures were analyzed under fluorescent microscopy for EGFP expression. When
compared, EGFP- iPSC, showed no EGFP expression, while both the EGFP-alone and
anti-HIV colonies showed continuous expression of EGFP up through passage 2 (P2).
This successfully demonstrated the transduction capability of the corresponding lentiviral
vectors. Morphologies were also comparable across all cultures further demonstrating
lack of adverse effects on the generation and formation of iPSCs using these vectors.
15
A
B
C
D
Figure 2. Transduced CD34+ HSC derived anti-HIV induced pluripotent stem cells
iPSCs. (a) CD34+ cord blood HSCs were transduced with the four lentiviral vectors
expressing OCT4, SOX2, KLF4, and c-MYC alone to be used as a negative control
16
showing no background when compared to EGFP alone at an MOI of 30. (b) CD34+
HSCs from cord blood were transduced with four lentiviral vectors expressing the four
individual pluripotency factors OCT4, SOX2, KLF4 and c-MYC in addition to a
lentivector expressing EGFP alone at an MOI of 30 as a positive control. (c) HSC derived
iPSC were generated using the four iPSC factors in addition to a lentivector expressing an
anti-HIV triple combination expression cassette including a chimeric TRIM5a, a CCR5
shRNA, a TAR decoy, and EGFP at an MOI of 30. (d) iPSC generated from the four
iPSC factors in combination with an anti-HIV triple combination vector at MOI of 40.
All images were captured at 100 x magnification.
17
Morphology and viability of passaged iPSC. To determine if iPSCs generated
were fully reprogrammed and able to persist, colonies were manually passaged every
three to four days and then transferred to new plates containing mouse embryonic
fibroblast (MEFs) feeders. Passaging was accomplished by using a dot blotter microscope
attachment used to mark and visualize the location of mature colonies by eye in a six well
plate. Once the colonies were marked, a 1 ml pipet was used to scrape the colonies loose
from the plate. The newly free floating iPSC colonies were broken into smaller chunks by
gentle trituration with the same 1 ml pipet. Wild type iPSC, provided by Joseph Anderson
Ph.D., were used as a positive control and are characteristic of human embryonic stem
cells containing tightly packed, fused cells with highly defined edges (Figure 3a). It was
expected that continued passaging should result in the expansion of viable,
undifferentiated iPSC colonies with similar characteristics.
As shown in Figure 3b, this colony displayed a generally opaque appearance with
centrally flattened cells. Further, the colony was surrounded by dark, flattened cells
growing out from the edges of the central formation. It is also worth noting that while
there is a distinct outline of the central formation, there are no observable well defined
edges. This is characteristic of a colony undergoing differentiation and moving away
from pluripotency. In Figure 3c, small chunks of previously passaged iPSC were
observed and contained dark centers. Again, flattened cells were seen growing out from
the central cluster as well as a small cluster of differentiating cells in the top left corner of
the panel. Based on prior experiments within the lab, it was believed that when a colony
is broken into fragments too small (< 1000 cells), there is an insufficient number of cells
18
necessary for the colony to persist and grow. Figure 3d shows a passaged colony
fragment with a large dark center. This fragment came from a larger colony that was
allowed to grow for too long before passaging (6 days) displaying these characteristics
the next day after its first passage. Ultimately, this colony did not persist and lifted off of
the plate by day 3 after passage 1. In Figure 3e irregularly shaped colonies formed with
well-defined edges containing tightly packed cells that appeared flattened and opaque.
Again, flattened groups of cells were obsserved growing from around the outer edge of
the colony’s top border. Figure 3f shows a blebbing formation with multiple lobes that
are swollen in appearance. None of the cells shown in this panel survived to a second
passage and therefore could not be characterized to show whether these were pluripotent
fully reprogrammed CD34+ HSCs.
19
Figure 3. HSC derived iPSC colony morphology and viability after passaging. (a) Wild
type iPSC (positive control) showing tightly packed fusion of cells in a granular-looking
colony with highly defined edges. No flattened cells observed in or surrounding the
formation indicative of “healthy” well formed iPSC colony. (b-f) Observed were a
variety of misshapen malformed colonies after the manual passaging. (b) The colony is
opaque, centrally fused cells were flattened and with dark, flattened cells bordering the
colony indicative of differentiation. (c) Initial iPSC, that were manually passaged with a
small number of cells per “chunk”, showed dark clumps of cells with irregular edges and
differentiation. (d) A dark region appears in the center of the formation after first manual
passage typically observed in “unhealthy” colonies. (e) iPSCs are shown with crisp,
highly defined edges and an irregular shape that may suggest partial reprogramming. (f)
A blebbing cellular formation was observed, with a cystic-like morphology, with no
clustering or fusion of cells. All images were taken at 100 x magnification.
20
iPSCs express pluripotency markers by immunhistochemistry. To determine
whether wild type iPSC were fully reprogrammed, expression of pluripotency markers
was evaluated by immunofluorescence. CD34+ HSC derived iPSCs were stained with
four different antibodies specific for pluripotency markers including OCT4, SSEA4,
NANOG and SOX2. As displayed in Figure 4a, OCT4 expression was seen in the
nucleus, as expected, while stage-specific embryonic antigen-4 (SSEA4), a glycolipid
carbohydrate epitope, was shown to be expressed on the surface of the wild type CD34+
HSC derived iPSCs. This is what would be expected to be seen in control embryonic
stem cell (hESC) lines, which are known to express all of the aforementioned
pluripotency markers and is not shown here. Figure 4b further demonstrates the
expression of the same pluripotency markers in another randomly expanded iPSC culture.
In Figure 4c, nuclear staining NANOG was readily expressed along with SOX2, also
found in the nucleus. Figure 4d shows a higher magnification of the same iPSC colony
shown in 4c. The expression and subsequent immunofluorescent detection of these
factors demonstrates their embryonic-like pluripotent state.
21
Figure 4. Immunofluorescent expression of pluripotency markers in CD34+ HSC derived
wild type iPSCs. (a) CD34+ HSC derived, wild type (WT) induced pluripotent stem cells
(iPSCs) were stained with antibodies specific for human stage-specific embryonic antigen
22
4 (SSEA-4) and octamer-binding transcription factor 4 (OCT4) at 100x magnification.
DAPI was used to localize nuclei and is not shown in overlay. (b) Wild type iPSC stained
similarly as in (a). DAPI was included to localize nuclei with respect to SSEA-4 and
OCT4 at 100x magnification. (c) WT iPSC stained with antibodies specific for
pluripotency markers NANOG and sex determining region Y-box 2 (SOX2) and
observed under 40x magnification. (d) The same WT iPSC colony shown in (c) observed
under 100x magnification looking at the same pluripotency markers.
23
Reverse Transcriptase-PCR analysis for the expression of pluripotency markers
in iPSCs. To further validate the findings of the immunofluorescent antibody staining,
reverse transcriptase PCR (RT-PCR) was performed to demonstrate the expression of
OCT4, SOX2, and NANOG pluripotency genes as well as teratocarcinoma-derived
growth factor 1 (TDGF1), found in embryonic stem cells functioning to promote
tumorogenesis, stimulate proliferation, and maintain self-renewal. In Figure 5, total RNA
from wild type iPSCs were reverse transcribed by PCR and run on a 0.8% agarose gel
with ethidium bromide and visualized under UV light. All of the pluripotency genes
tested for were shown to express in the wild type iPSC, which is what we would expect to
see in an embryonic stem cell positive control line like H9 ESCs. GAPDH was also run
as a control and is not shown here. Cytoplasmic c-MYC was run on a separate gel and did
not express. A single blurred band was observed in the primer dimer size range (data not
shown). A no template control was not performed. Overall, the results of the RT-PCR
support the immunocytochemistry results showing that the pluripotency genes were
actively expressing factors associated with pluripotency.
Ideally, this assay would have been run against multiple cell types to serve as
positive and negative controls. CD34+ HSCs would have been a good negative control as
they were the starting cells iPSC were generated from and shouldn’t show any gene
expression of pluripotent factors. Human embryonic stem cell line H9 would have been a
good positive control because it should express all of the pluripotent factors and would
have provided positive expression patterns for comparison. Additionally, multiple iPSC
lines should be used to compare against each other including, wild type, EGFP alone, and
24
anti-HIV iPSC lines. The expected result would have been to show that all iPSC lines
contained the genes actively expressing the factors associated with pluripotency in hESC
lines thus demonstrating conclusively that these were truly reprogrammed pluripotent
iPSC colonies.
25
Figure 5. Reverse Transcriptase-PCR shows expression of pluripotency factors in wild
type iPSCs. Total RNA was isolated from undifferentiated wild type induced pluripotent
stem cells (iPSCs) and reverse transcribed to determine the expression of pluripotency
genes octamer- binding transcription factor 4 (OCT4), sex determining region Y-box 2
(SOX2), NANOG, and teratocarcinoma-derived growth factor 1 (TDGF1). Cytoplasmic
c-MYC was also assessed and was observed as a diffused “fuzzy” primer dimer band on
a separate gel (data not shown). H9 hESCs would have made a good positive control
while cord blood would have been used as a negative control to show the lack of
expression in the initial CD34+ HSC cells.
26
DISCUSSION
After nearly two decades of research, there is still no cure for HIV. While current
drug regimens manage the disease and prolong life, they can become toxic over time with
drug resistant strains resulting. New and innovative strategies are required. In this study,
CD34+ HSCs were evaluated for their ability to be genetically modified with anti-HIV
genes conferring resistance to infection from HIV. These cells were assayed for
programmed dedifferentiation back into a pluripotential state while maintaining
constitutive expression of the inserted anti-HIV genes. The newly generated iPSC should
be capable of continuous self-renewal and proliferation making them an ideal model for
generating and studying cellular and gene modifications and potential therapeutic
benefits. One significant advantage of this strategy is the potentially continuous supply of
cells that are tailored to patient specific requirements in addition to their anti-HIV
proclivity.
In this study, CD34+ hematopoietic stem cells (HSCs) were routinely isolated
from umbilical cord blood, transduced with a lentiviral vector containing a triple
combination anti-HIV expression cassette engineered to express a chimeric TRIM5α, a
CCR5 shRNA, a TAR decoy. These cells were simultaneously transduced with four
pluripotency reprogramming factors to generate anti-HIV induced pluripotent stem cells
(iPSCs). Here, we demonstrated that none of the vectors utilized caused any deleterious
effects on iPSC generation, morphology, nor did the lentivectors interfere with each
other. Further, it was shown by immunofluorescent detection and RT-PCR that wild type
27
CD34+ HSC derived iPSC did express embryonic stem cell-like pluripotency markers as
a result of transcriptionally active genes necessary for cellular reprogramming.
While working on a project of this scope, there were many hurdles to overcome
and plenty of obstacles to navigate. Some of the many details that came into play dealt
with the successful transduction of CD34+ HSC, sterility issues in cell culture, lentiviral
vector preparation, vector tittering, quality of starting plasmids, transduction design and
optimization, and maintenance of newly generated iPSCs. Since each experiment or sets
of experiments were built off of the one prior, many things had to go right before the next
experiment could be executed. CD34+ hematopoietic stem cells are found in the bone
marrow. They undergo asymmetric division where one daughter cell maintains its
“stemness” while the other daughter is free to go down a lineage and become any of the
other blood cell types. They are suspension cells in that they do not natively sit down on a
substratum to attach and proliferate. Instead, HSCs are free-floating cells that are
relatively difficult to transduce. Add to that the idea that we are trying to transduce as
many single cells in suspension with a total of five different vectors simultaneously for a
complete transduction and it’s easy to see the difficulty in this challenge with the
expected outcome of true reprogramming as well as HIV resistance. The process actually
starts with the initial collection and culturing of isolated CD34+ HSCs. If the cells we are
initially starting with are not healthy, they will likely not even be expressing as many
surface receptors necessary for our vectors to achieve attachment and fusion.
Additionally, if the cells are so stressed in culture, they may not be actively dividing as
28
frequently. In culture, healthy cells appeared as bright, round, grape-like clusters when
actively dividing. This was believed to be the best time for successfully transducing the
cells with lentivectors.
Since this project relies on so many cell types, cell culture and aseptic technique
were critical. First, HSCs were always in limited supply. These came to our study by
anonymous donation from various donations and at irregular intervals. Umbilical cord
blood was also purchased from a tissue bank and was relatively expensive, as a limiting
factor. If any of these cells became contaminated after isolation and subsequent culturing,
it could be days or even a week before a new source of blood could be secured. HEK293t cells were used to package our lentiviral vectors. At one point in our experiments, a
large portion of our frozen stock of 293t’s was discovered to be contaminated resulting
not only in the loss of cells but also of vector stocks and most importantly time. There
weren’t any other stocks of 293t’s in our lab and we were down for 3-4 weeks trying to
find cells that weren’t already contaminated. Bad cells made bad vector and everything
packaged by those cells was unusable. This also resulted in a loss of otherwise healthy
cord blood cells that would not transduce with “bad” vector. The problem was recognized
and finally corrected. Once we were able to isolate an uncontaminated source of cells,
they had to be grown up and expanded to large numbers and then frozen down to
replenish the largely contaminated stocks.
Another issue concerned bacterial transformations of our packaging, anti-HIV
vector, and iPSC reprogramming plasmids. There were several instances where the
bacteria used for transformations would either add or delete base pair segments to or from
29
our plasmids resulting in an unusable plasmid. It was discovered when transduced cells
were not expressing EGFP as was expected with the EGFP alone plasmid or the anti-HIV
plasmid. This was remedied by purifying our plasmids, performing a restriction enzyme
digest, and then running the digested plasmids on an agarose gel with a known control to
check for correct size of vector and insert.
The inherent project design was somewhat complex from the outset. It was made
known that only two individuals in the lab were successfully generating iPSCs using our
pCCLc- lentivector backbone and that it had taken one of them nine months to put
together a working protocol for a single cell type, that being fibroblasts. This project was
focused on using cord blood CD34+ HSC since one of the hypotheses was that they
already had the characteristic of partial stemness, therefore they should be more easily
reprogrammable, and then more likely to go back down the same lineage to ultimately be
used for differentiation into immune cells. Questions that needed to be considered
included the idea that since these cells were suspension cells, how many should be
transduced and at what volume? How much of each vector should be used? Can we even
get a reliable titer on the vectors to be used? If too much c-MYC is used, it could drive
cells towards apoptosis. Too much vector in general may overwhelm and kill our cells.
Too little vector may not be able to drive the cells backwards toward an undifferentiated
pluripotential state. The fact that our system relies on trying to get five different vectors
to transduce all of the cells at equal ratios was a challenge. It was likely that not all of the
cells would even receive all five of the vectors. Even seemingly, simple details needed to
be addressed. For example how many MEFs should be plated per well of a six well plate?
30
How many are too many and what’s not enough? After discussions with the two
individuals who had successfully generated iPSCs, it was decided to go with 50 000 –
100 000 thousand cells per transduction in 100 µl reactions. Multiplicity of infections
(MOI = ratio of the number of infectious particles to the number of target cells) varied
between 5, 10, 20, 30, and 40 to determine a range of reprogramming efficiency.
Another area that presented challenges dealt with the continued maintenance of
iPSCs once they had been generated. If cells were left in culture too long, they would
differentiate and no longer retain their pluripotency, overgrow the plate, and in many
cases die and lift off the plate. If colonies were passaged in small fragments (< 1000
cells), they tended to turn a brownish color by day two, differentiate and die. Finding the
right manual dissection and passaging tools was also a technical challenge. ESC training
at the Buck Institute demonstrated the use of modified glass Pasteur pipets as dissection
tools. These frequently broke apart either during their creation or into the cultures while
attempting to dissect. There was no time or resources to sit for hours trying to create
usable tools only to have them break off in culture. Not to mention, if you were able to
perform this passage successfully, if any glass did break off, the fragmented colonies and
fibroblasts removed tended to cling to those shards and interfere with subsequent plating
and growth characteristics of the colonies. During early attempts at learning to passage,
needles were suggested as a working alternative and already being used by others to
manually passage and actively maintain currently growing cell lines. This approach was
fraught with problems! First, in order to use this strategy, dissections had to be done
under a microscope, outside of a hood, exposing your cultures to the outside atmosphere.
31
This was certain to introduce all sorts of contamination, which it did, and is still currently
an ongoing challenge in the lab. Second, using a needle to cut colonies off of a plastic
substrate presented the same kinds of problems as the glass pipets. Scraping plastic with a
metal needle created plastic shards, which frequently became entangled with the colonies
resulting in the same kinds of growth characteristics previously mentioned. Both of these
approaches required a significant time investment, the sacrifice of limited resources, and
were neither suitable for consistency nor forward moving. Finally, I learned that a simple
1 ml plastic pipet tip was perfect for this application and avoided all previous
catastrophes. All that was required was to visually isolate and mark colonies using the
blotter, and then the 1 ml pipet tip could be used to simply scrape the entire colony up off
of the plates in one large chunk. There was no scratching of the substrate, no broken
fragments of glass pipet, no need to expose the cultures to outside air, and dissections
yielded large chunks that were easily broken down into smaller pieces by the natural
action of trituration. All that remained was to transfer the colonies to new MEF plates
with fresh media. This technique resulted in the exponential expansion of iPSC cultures
over a period of several weeks.
Overall, time and resources were the single biggest factor for not moving the
project forward to completion. If allowed to continue, assays involving the differentiation
of iPSC into actual immune cells would be achieved, with phenotype and functional
assays performed. Additionally, HIV challenges would be performed in vitro, and finally
tested in an appropriate animal model, an immune deficient NOD-SCID RAG1-null IL2
common gamma chain knockout mouse, for example. These could be engrafted with
32
human iPSC-derived, anti-HIV immune cells and challenged with various strains of HIV.
Alternative approaches for future study would focus more closely on the pre-integration
phase of HIV. One strategy would be to incorporate other potent anti-HIV genes into the
already successful triple combination vector. Theta-defensins, or Retrocyclins, are small
18-residue peptides that can bind SIV gp41 and inhibit fusion in old world monkeys. This
same gene exists for humans, but is inactivated by an in-frame stop codon in its signal
sequence. The mutation could be corrected using site directed mutagenesis and then
cloned into the triple combination anti-HIV lentivector. Other strategies for improving
reprogramming efficiency would look at the development and use of a 4-in-1 iPSC vector
system where a single vector would contain all four of the iPSC reprogramming factors.
In conclusion, the data presented in this project is an early step that provides evidence
showing that the ultimate goal of inhibiting HIV infection using a cellular and gene
therapy strategy is achievable.
33
LITERATURE CITED
1. Lopez, CA.,Vazquez M., Hill, M, Colon, M., Porrata-Doria, T., Johnston, I., and
Lorenzo, E. (2010). Characterization of HIV-1 RNA forms in the plasma of
patients undergoing successful HAART. Arch. Virol. 10.1007/s00705-010-0659-3.
2. Anderson, JS., Javien, J., Nolta, JA., and Bauer, G. (2009). Preintegration HIV-1
inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha
protein, a CCR5 shRNA, and a TAR decoy. Mol Ther. 17 (12):2103-14.
3. Anderson, JS., Walker, J., Nolta, JA., and Bauer, G. (2009). Specific transduction of
HIV- susceptible cells for CCR5 knockdown and resistance to HIV infection: a
novel method for targeted gene therapy and intracellular immunization. J Acquir
Immune Defic Syndr. 52 (2):152-61.
4. Finzi, D., Blankson, J., Siliciano, JD., Margolick, JB., Chadwick, K., Pierson, T.,
Smith, K., Lisziewicz, J., Lori, F., Flexner, C., Quinn, TC., Chaisson, RE.,
Rosenberg, E., Walker, B., Gange, S., Gallant, J., and Siliciano, RF. (1999). Latent
infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1,
even in patients on effective combination therapy. Nat Med. 5:512–517.
5. Gunthard, HF., Frost, SD., Leigh-Brown, AJ., Ignacio, CC., Kee, K., Perelson, AS.,
34
Spina, CA., Havlir, DV., Hezareh, M., Looney, DJ., Richman, DD., and Wong, JK.
(1999). Evolution of envelope sequences of human immunodeficiency virus type 1
in cellular reservoirs in the setting of potent antiviral therapy. J Virol. 73:94049412.
6. Pomerantz, RJ. (2003). Reservoirs, sanctuaries, and residual disease: the hiding spots
of HIV-1. HIV Clin Trials. 4:137–143.
7. Pomerantz, RJ. (2004). Ludwik Hirszfeld Memorial Lecture: HIV-1reservoirs: major
molecular obstacles to viral eradication. Arch Immunol Ther Exp (Warsz.). 52:297306.
8. Ibanez, A., Puig, T., Elias, J., Clotet, B., Ruiz, L., and Martínez, MA. (1999).
Quantification of integrated and total HIV-1 DNA after long-term highly active
antiretroviral therapy in HIV-1-infected patients. AIDS. 13:1045–1049.
9. Pomerantz, RJ. (2001). Residual HIV-1 infection during antiretroviral therapy: the
challenge or viral persistence. AIDS. 15:1201–1211.
10. Winters, MA., Baxter, JD., Mayers, DL., Wentworth, DN., Hoover, ML., Neaton, JD.
and Merigan, TC. (2000). Freuency of antivertroviral drug resistance mutations in
HIV-1 strains from patients failing triple drug regimens. The Terry Beim
35
Community Programs for Clinical Research on AIDS. Antivir Ther. 5(1): 57-63.
11. Gupta, R., Hill, A., Sawyer, AW., and Pillay, D. (2008). Emergence of drug
resistance in HIV type 1-infected patients after receipt of first-line highly active
antiretroviral therapy: a systematic review of clinical trials. Clin Infect Dis.
47(5):723-735.
12. Barouch, DH. (2008). Challenges in the development of an HIV-1 vaccine. Nature.
455: 613-619.
13. Nazari, R., and Joshi, S. (2008). CCR5 as target for HIV-1 gene therapy. Curr Gene
Ther. 8(4):264-72.
14. Asparuhova, MB., Barde, I., Trono, D., Schranz, K. and Schümperli, D. (2008).
Development and characterization of a triple combination gene therapy vector
inhibiting HIV-1 multiplication. J Gene Med. 10(10):1059-70.
15. Li, MJ., Kim, J., Li, S., Zaia, J., Yee, JK., Anderson, J., Akkina, R., and Rossi, JJ.
(2005). Long-term inhibition of HIV-1 infection in primary hematopoietic cells by
lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5
ribozyme, and a nucleolar-localizing TAR decoy. Mol Ther. 2(5):900-9.
36
16. Chang, LJ., Liu, X., and He, J. (2005). Lentiviral siRNAs targeting multiple highly
conserved RNA sequences of human immunodeficiency virus type 1. Gene
Ther. 12(14):1133-44.
17. Mautino, MR., and Morgan, RA. (2002). Enhanced inhibition of human immune
deficiency virus type 1 replication by novel lentiviral vectors expressing HIV type
1 envelope antisense RNA. Hum Gene Ther. 13(9):1027-37.
18. Monini, P., Sgadari, C., Toschi, E., Barillari, G., and Ensoli, B. (2004). Antitumour
effects of antiretroviral therapy. Nature Reviews Cancer. 4: 861-875.
19. Takahashi, K. and S. Yamanaka. (2006). Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126: 663676.
20. Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K.,
Bernstein, BE., and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into
a pluripotent ES cell-like state. Nature . 448: 318–324.
21. Takahashi, K., K. Okita, M. Nakagawa, and S. Yamanaka. (2007). Induction of
pluripotent stem cells fromfibroblast cultures. Nat. Protoc. 2: 3081–3089.
37
22. Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M.,
Yachechko, R., Tchieu, J., Jaenisch, R., Plath, K., and Hochedlinger, K. (2007).
Directly reprogrammed fibroblasts show global epigenetic remodeling and
widespread tissue contribution. Cell Stem Cell. 1: 55–70.
23. Meissner, A., M. Wernig, and R, Jaenisch. (2007). Direct reprogramming of
genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25:
1177–1181.
24. Park, I.H., Zhao, R., West, JA., Yabuuchi, A., Huo, H., Ince, TA., Lerou, PH.,
Lensch, MW., and Daley, GQ. (2008). Reprogramming of human somatic cells to
pluripotency with defined factors. Nature. 451: 141–146.
25. Yu, J., Vodyanik MA, Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, JL., Tian, S.,
Nie, J., Jonsdottir, GA., Ruotti, V., Stewart, R., Slukvin, II., and Thomson, JA.
(2007). Induced pluripotent stem cell lines derived from human somatic
cells. Science. 318: 1917–1920.
26. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and
Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell. 131: 861–872.
38
27. Rossi, JJ. (1999). The application of ribozymes to HIV infection. Curr Opin Mol
Ther. 1:316–322.
28. Amado, RG., and YI. S. Chen. (1999). Lentiviral vectors - the promise of gene
therapy within reach? Science. 285: 674-76.
29. Montini, E., Cesana, D., Schmidt, M., Sanvito, F., Ponzoni, M., Bartholomae, C.,
Sergi, Sergi L., Benedicenti, F., Ambrosi, A., Di Serio, C., Doglionim, C., von
Kalle, C., and Naldini, L. (2006). Hematopoietic stem cell gene transfer in a
tumor-prone mouse model uncovers low genotoxicity of lentiviral vector
integration. Nat. Biotechnol. 24: 687-96.
30. Naldini, Blömer, U., Gallay, P., Ory, D., Mulligan, R., Gage, FH., Verma, IM., and
Trono, D. (1996). In vivo gene delivery and stable tranduction of nondividing
cells by a lentiviral vector. Science. 272: 263-267.
31. Reymond, A, Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Riganelli, D.,
Zanaria, E., Messali, S., Cainarca, S., Guffanti, A., Minucci, S., Pelicci, PG., and
Ballabio, A. (2001). The tripartite motif family identifies cell compartments.
EMBO J. 20: 2140-2151.
39
32. Cullen, B. (2006). Role and mechanism of action of the APOBEC3 family of
antiretroviral resistance factors. J. Virol. 80 (3): 1067–76.
33. Anderson, J. and Akkina, R. (2005). TRIM5arh expression restricts HIV-1 infection
in lentiviral vector-transduced CD34+-cell-derived macrophages. Mol Ther. 12:
687-696.
34. Samson, M., Labbe, O., Mollereau, C., Vassart, G., and Parmentier, M. (1996).
Molecular cloning and functional expression of a new human CC-chemokine
receptor gene. Biochemistry. 35: 3362–67.
35. Samson, M., Libert, F., Doranz, BJ., Rucker, J., Liesnard, C., Farber, CM., Saragosti,
S., Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G., Verhofstede,
C., Burtonboy, G., Georges, M., Imai, T., Rana, S., Yi, Y., Smyth, RJ., Collman,
RG., Doms, RW., Vassart, G., and Parmentier, M. (1996). Resistance to HIV-1
infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine
receptor gene. Nature. 382: 722–725.
36. Li, Ming-Jie., Kim, J., Li, S., Zaia, J., Yee, JK., Anderson, J., Akkina, R., and Rossi,
JJ. (2005). Long-term inhibition of HIV-1 infection in primary hematopoietic cells
by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti
CCR5 ribozyme, and a nucleolar-localizing TAR decoy. Mol Ther. 12: 900-909.