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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1882-1894
Transduction of Primitive Human Marrow and Cord Blood-Derived
Hematopoietic Progenitor Cells With Adeno-Associated Virus Vectors
By
Saswati Chatterjee,
Wei Li,
Christie Ann Wong,
Grace Fisher-Adams,
Di Lu,
Mausumee Guha,
James A. Macer,
Stephen J. Forman, and
K.K. Wong Jr
From the Division of Pediatrics, Department of Hematology and Bone
Marrow Transplantation, City of Hope National Medical Center, Duarte,
CA; and the Department of Obstetrics and Gynecology, School of
Medicine, University of Southern California and Huntington Memorial
Hospital, Pasadena, CA.
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ABSTRACT |
We evaluated the capacity of adeno-associated virus
(AAV) vectors to transduce primitive human myeloid
progenitor cells derived from marrow and cord blood in long-term
cultures and long-term culture-initiating cell (LTC-IC)
assays. Single-colony analyses showed that AAV vectors transduced
CD34+ and CD34+38 clonogenic
cells in long-term culture. Gene transfer was readily observed in
LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV
(rAAV) transduction was observed in every donor analyzed,
although a wide range of gene transfer frequencies (5% to 100%) was
noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5
LTC-ICs. Fluorescence in situ hybridization (FISH)
analyses performed to determine the fate of AAV vectors in transduced
cells showed that 9% to 28% of CD34+ and
CD34+38 cells showed stable vector
integration as evidenced by chromosome-associated signals in metaphase
spreads. Comparisons of interphase and metaphase FISH suggested that a
fraction of cells also contained episomal vector at early time points
after transduction. Despite the apparent loss of the episomal forms
with continued culture, the number of metaphases containing integrated
vector genomes remained stable long term. Transgene transcription and
placental alkaline phosphatase (PLAP) expression was
observed in CD34+, CD34+38
LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
EX VIVO GENE TRANSFER into pluripotent
hematopoietic stem cells represents an attractive treatment modality
for a variety of gene-based pathological processes, including inherited
diseases,1,2 oncogenic processes,3,4 and viral
infections.5 Despite the widespread use of retroviral
vectors in the majority of human gene therapy trials currently
underway, several limitations remain. Human trials and nonhuman primate
models of hematopoietic progenitor cell transplantation suggest that
the efficiency of retroviral gene transfer into hematopoietic cells is
low.6 Results of clinical gene therapy trials using
transplantation of retroviral vector-transduced CD34 cells derived from
either bone marrow,7,8 cord blood,2 or
mobilized peripheral blood8 have been informative in
determining the potentials of retroviral transduction of human hematopoietic stem cells (HSCs) in vivo. The majority of the results suggest a long-term marking frequency of approximately 1:10,000 to
1:100,000. Reports that marrow- and cord blood-derived human hematopoietic cells capable of multilineage repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice
exclusively present in the CD34+38
fraction were rarely transduced by retroviral vectors9 are in concordance with the results of the human trials.
Although gene transfer with retrovirus vectors10 in
lineage-committed colony-forming units (CFU) have been
successful, these studies have not been predictive of in vivo results.
Attempts to elucidate the reasons underlying poor transduction of HSCs by retroviral vectors suggested that the requirement for cell division11,12 to achieve retroviral
integration may impede their use for the genetic modification of
quiescent cell populations such as HSCs.13 Therefore, the
continued search for other vectors for the safe, stable, and efficient
introduction of transgenes into nondividing HSCs is critical. Vectors
based on adeno-associated virus (AAV) are emerging as efficient gene
transfer vehicles with biological properties quite distinct from retroviruses.
AAV is a single-stranded, replication-defective nonpathogenic human
parvovirus with a 4.7-kb DNA genome with palindromic inverted terminal
repeats (ITR).14,15 AAV requires coinfection with a helper
virus such as adenovirus or herpes simplex virus for productive
infection.16,17 In the absence of helper virus coinfection, wild-type AAV integrates site-specifically and in a stable fashion, via
the ITRs, into the AAVS1 site of human chromosome 19.18,19 However wild-type-free, rep-free AAV vectors integrate into
chromosomal sites other than AAVS1. AAV vectors20 have high
transduction frequencies in cells of diverse lineages and allow
efficient transgene expression from RNA polymerase II-and III-dependent
promoters.21 Recent studies from our laboratory and those
of others22 have shown that AAV vectors efficiently
transduce nondividing cells, including peripheral blood macrophages, a
variety of neurons, cardiac cells, skeletal cells, and smooth muscle
cells.23-27
We have previously reported the ability of AAV vectors to transduce
human marrow and cord blood-derived CD34+ hematopoietic
progenitor cells.28 Southern and fluorescence in situ
hybridization (FISH) analyses demonstrated the integration of AAV
vector sequences into CD34 chromosomal DNA. FISH analyses also showed
that integrated vector sequences replicated along with cellular DNA
during mitosis, suggesting stable integration. Transgene expression was
observed in transduced CD34 cells in suspension cultures and in myeloid
colonies differentiating from transduced CD34 cells regardless of
selective pressure. These results were in concordance with others who
have shown AAV transduction of colony-forming units granulocyte,
erythroid, monocyte, megakaryocyte (CFU-GEMMs).29-32 In addition, recombinant AAV
(rAAV) transduction of murine hematopoietic progenitor
cells has also been reported.33,34 Thus, the combined
ability of AAV vectors to transduce both nondividing cells and CD34
cells makes them attractive for further evaluation for gene
transduction into primitive human hematopoietic progenitor and stem
cells. Therefore, in this study, we evaluated the ability of AAV
vectors to transduce and integrate into primitive myeloid CD34 and
CD34+38 clonogenic progenitor cells from
marrow and cord blood. Investigation of AAV transduction of cells with
extended clonogenic capacity showed gene transfer into week-5, -8, and
-10 long-term culture-initiating cells (LTC-IC) by
individual colony analysis. Transduction levels varied from high to
low, depending on the donor. FISH analyses of CD34+ and
CD34+38 cells showed that, whereas
episomal and integrated forms of vector genomes could be detected at
early time points, only integrated forms persisted long term and
contributed to stable transduction. Sustained expression of
vector-encoded antisense RNA was detected in long-term cultures and
LTC-ICs in the absence of selective pressure.
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MATERIALS AND METHODS |
Cells and viruses.
Marrow samples were obtained from healthy donors for allogeneic
transplant recipients after obtaining informed consent and using a City
of Hope Institutional Review Board approved protocol. Umbilical cord
blood was procured from Huntington Memorial Hospital (Pasadena, CA)
using a protocol approved by both the COH and HMH Institutional Review
Boards. Light-density mononuclear cells were separated by
Ficoll-Hypaque (Pharmacia, Piscataway, NJ) centrifugation. After three
washes with phosphate-buffered saline (PBS), cell and viability counts
were performed and cells were resuspended in PBS with 0.5% bovine
serum albumin (BSA) and 2 mmol/L EDTA before use.
The CD34+ population of marrow cells was immunomagnetically
purified from mononuclear cells using CD34 Isolation kits (Miltenyi Biotech, Auburn, CA) as per the manufacturer's directions. CD34 cells
were passed through two columns sequentially to increase purity and
enrich for CD34+++ cells. CD34 purity was 96% to 98% as
assessed by flow cytometry after direct immunolabeling with fluorescein
isothiocyanate (FITC)-conjugated HPCA-2.
CD34+38 cells were cytofluorometrically
sorted using a Mo-Flo high speed flow cytometer (Cytomation, Fort
Collins, CO) after labeling with FITC-conjugated anti-CD34 and
phycoerythrin (PE)-conjugated anti-CD38 (Becton Dickinson, San Jose, CA).
Herpes simplex virus, type 1 (HSV-1), MP17 was used as the helper virus
for AAV vector encapsidation and was propagated and titered by plaque
assays as previously described.17
AAV vectors.
All AAV vectors used in this study were derived from the base vector,
CWRSV,5,28 which contains bases 1 through 189 and 4045 through 4680 of wild-type AAV2, including the 5' and 3'
ITRs and the endogenous AAV2 polyadenylation signal. CWRHIVASVN
(Fig 1A) contains two transcriptional
cassettes, one encoding the neomycin phosphotransferase
(NeoR) gene under the SV40 early promoter control and the
other encoding an antisense transcript complementary to human
immunodeficiency virus-type 1 (HIV-1) LTR sequences
under RSV LTR control.5 CWRHIVAPAP (Fig 1B) is identical to
CWRHIVASVN, except for the substitution of a gene cassette encoding the
thermostable human placental alkaline phosphatase (PLAP)
gene35,36 under the phosphoglycerate kinase (PGK) promoter
control for the SVNeo cassette.
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| Fig 1.
Maps of AAV vectors used. Bold arrows denote the
direction of transcription of each gene. The promoters, polyadenylation
signals (PA), and the AAV ITR are shown. Light arrows denote the
location of PCR primers. Primers RPSP and DLASP were used for
amplification from DNA templates. Primers 1a and 1b were used for
reverse transcription and amplification from RNA templates. Probes used
for FISH analyses and Southern hybridization of RT-PCRs and dot blot
analyses of vectors are designated. (A) vCWRHIVASVN encodes two
transgenes: (1) an antisense RNA (A) complementary to the HIV LTR,
including the TAR and the polyadenylation sequences under the
transcriptional control of the RSV LTR; and (2) the neomycin
phosphotransferase (NeoR) gene under the control of the
SV40 early promoter. (B) vCWRHIVAPAP contains the antisense RNA gene
cassette described in (B) in addition to the PLAP gene under the
control of the PGK promoter.
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The recombinant viral vectors vCWRHIVASVN, vCWRHIVAPAP, and vCWRAP were
encapsidated using HSV-1 MP17 as helper viruses, as described
previously.28,37 Briefly, semiconfluent 293 cells were
infected with HSV-1 MP17 (multiplicity of infection [MOI], 0.1) and
transfected 1 hour postinfection with 20 µg of the
vector plasmids by calcium phosphate coprecipitation (CellPhect;
Pharmacia Biotech, Uppsala, Sweden). AAV-encoded rep (DNA
replication) and cap (capsid proteins) gene functions were provided in
trans. Cells were harvested 72 hours posttransfection and were lysed by
three cycles of freeze-thawing and sonication. Vector stocks were
treated with 400 U DNase I (Boehringer Mannheim, Indianapolis,
IN) per 107 cells to digest residual plasmid
and cellular DNA. The lysate was digested with 0.25% trypsin and 1%
sodium deoxycholate for 30 minutes at 37°C before further
purification on two rounds of isopycnic cesium chloride (density, 1.41 g/mL) gradient centrifugation for 64 and 48 hours, respectively, at
40,000 rpm. Fractions of 0.5 to 1 mL were collected and
density-measured with a refractometer.
Particle titers were determined by dot blot analysis. DNA was isolated
from each fraction using standard procedures38 after proteinase K digestion, phenol and phenol:chloroform extractions, and ethanol precipitation. DNA was dot-blotted onto
nitrocellulose and hybridized with a vector-specific probe
(BamHI-SnaBI fragment of CWRHIVAPAP;
Fig 1). Duplicate blots were hybridized with a wild-type
AAV-specific probe (Sac II fragment of pTZAAV). Results were
analyzed on a phosphorimager and titers were determined from standard
curves. pBluescript DNA served as a negative control. A particle titer
of 109/mL was obtained for vCWRHIVAPAP and one of
108/mL was obtained for vCWRHIVASVN.
The functional titers of vCWRHIVAPAP and vCWRHIVASVN stocks were
determined by quantitation of specific alkaline phosphatase-expressing cells and neomycin resistant (NeoR) colonies after serial
dilutions on 293 cells. Functional titers of 106/mL to
107/mL were obtained for vCWRHIVAPAP and one of
105/mL was obtained for vCWRHIVASVN. The presence of
full-length vector genomes and the absence of contaminating wild-type
AAV was confirmed by either dot blot or electrophoretic analysis of DNA
extracted from vector stocks, followed by Southern hybridization with
vector-specific and wild-type-specific probes. All helper virus stocks
and cell lines were screened for and found to be free of wild-type AAV contamination.
For most experiments, transductions with vCWRHIVAPAP were performed at
functional MOI of 1 to 3 and with vCWRHIVASVN at MOI of 0.1 to 0.2. Specific multiplicities are stated for each set of experiments.
Transductions.
CD34 cells were transduced immediately upon isolation at the time of
culture initiation at 37°C in a humidified, CO2
incubator. Transductions were performed by the direct addition of
vector to cells at functional MOIs of 1 to 10 (corresponding to
particles MOIs of 200 to 2,000) and left undisturbed for 24 to 48 hours, after which cells were washed and replated. In separate
experiments, we have shown that there is no detectable free vector left
in the medium after washes. Two batches of vCWRHIVAPAP of equivalent titer and one batch of vCWRHIVASVN were used for all experiments reported here.
Long-term marrow cultures (LTBMCs).
Heterologous stromal layers were established by plating 5 to 10 × 106 allogeneic marrow mononuclear cells in T75 flasks in
RPMI 1640 with 10% fetal bovine serum (FBS) and L-glutamine (cRPMI).
Stromal layers developed in 2 to 3 weeks and were used for LTC-IC
assays after at least three passages, after which they were harvested by trypsinization, washed, and plated in 12-well plates at 2 to 3 × 104/cm2 in cRPMI. The stroma was
irradiated with 15 Gy of 250 KVp x-ray when a confluency of
approximately 70% was reached, and media was removed and overlaid with
CD34 cells in long-term culture medium.
Purified CD34 cells were suspended in Myelocult (StemCell Technologies,
Vancouver, British Columbia, Canada) containing 1 µmol/L of
hydrocortisone (Sigma, St Louis, MO) and
penicillin-streptomycin and overlaid on irradiated heterologous stromal
layers in 12-well plates at a density of 2 to 6 × 104
cells/cm2 either with or without the addition of
vCWRHIVAPAP at a functional MOI of 2 to 3 (particle MOI, 400 to 600).
Suspension cells were washed after 24 to 48 hours and replated on
washed stromal layers. Cultures were maintained by demi-depopulation of
cells every 7 to 10 days.
LTC-IC assays.
Clonogenic assays to test for LTC-ICs were performed 5 to 10 weeks
after culture initiation by inoculation of suspension cells from LTBMCs
into methylcellulose media39,40 (StemCell Technologies). Cells were harvested from LTBMCs by trypsinization, washed, and resuspended in 500 µL Iscove's Modified Dulbecco's Medium (IMDM) with 30% heat-inactivated FBS (GIBCO, Grand Island, NY) and
L-glutamine. The cell suspensions were mixed with a suspension
containing 0.8% methylcellulose (MethoCult; StemCell Technologies),
30% fetal calf serum (FCS), 1% BSA, 104 mol/L
2-mercaptoethanol, 2 mmol/L L-glutamine, interleukin-3 (IL-3; 10 ng/mL;
R&D Systems, Minneapolis, MN), granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/mL; R&D Systems), and erythropoietin (50 U/mL; Amgen, Thousand Oaks, CA) and placed in
12-well plates at a concentration of 10,000 cells/well in duplicate. Colonies containing greater than 50 cells were scored microscopically approximately 14 days after plating. Individual colonies were plucked
and washed. DNA was extracted from colonies by standard methods after
RNase treatment, sodium dodecyl sulfate (SDS)-proteinase K digestion,
phenol:chloroform extraction, and ethanol precipitation.38 DNA extracted from each colony was resuspended in 25 µL TE.
Amplification of rAAV vector sequences.
LTC-IC colony DNA and controls were tested for the presence of vector
sequences by polymerase chain reaction (PCR) amplification using
specific primers in a Perkin-Elmer Model 9600 Thermal Cycler (Perkin-Elmer, Foster City, CA). Primers (RPSP [sense,
5'-GGTGGAAGTAAGGTGGTACG-3'] and DLASP [antisense,
5'-TGCCGCACTGAAGGTGGTCGAA-3']) were used to amplify a
418-bp product spanning a region from the Rous Sarcoma virus long
terminal repeat (RSV LTR) to the AAV polyadenylation site from RNA of
vCWRHIVASVN and vCWRHIVAPAP (Fig 1) transduced cells. Internal controls
for DNA concentration and template integrity were provided by
amplification of a 268-bp fragment from the ubiquitous human  -globin
gene (sense primer, 5'-CAACTTCATCCACGTTCACC-3'; antisense
primer, 5'-GAAGAGCCAAGGACAGGTAC-3'). One third of the RPSP
primer used was end-labeled with  [32P]-dATP using T4
polynucleotide kinase (New England Biolabs, Beverly, MA).
Amplified products were resolved by electrophoresis in a 5%
polyacrylamide gel in Tris borate-EDTA (TBE) buffer, dried, and exposed
to Kodak X-omat AR film (Eastman Kodak, Rochester, NY) at
 70°C with an intensifying screen. Radioautographic band intensities were quantitated by phosphorimage analysis.
FISH analyses.
CD34 cells were transduced with vCWRHIVAPAP at MOI of 1 to 3 (particle
MOI, 200 to 600) immediately upon isolation at the time of culture
initiation in IMDM containing 20% FCS, IL-3 (10 ng/mL; R&D Systems),
IL-6 (10 ng/mL; R&D Systems), and stem cell factor (1 ng/mL; R&D
Systems). FISH analysis was performed as described
previously.28 Briefly, at the time of analysis, suspension cells were harvested from cultures, washed, and resuspended in fresh
IMDM containing FCS, glutamine, and higher levels of cytokines (50 ng/mL IL-3, 50 ng/mL IL-6, and 5 ng/mL stem cell factor) for 72 hours
to boost the mitotic index. To block cells in metaphase, cells were
placed in fresh cytokine-containing media containing colcemid (0.025 mg/mL) for 16 to 24 hours. Harvested cells were treated with a
hypotonic solution (0.4% KCl) and fixed in 3:1 methanol:acetic acid
before dropping on slides to obtain nuclear spreads.41
A 3.6-kb Hpa I-SnaBI fragment from pCWRHIVASVN (Fig 1)
was labeled with digoxygenin-deoxyuridine triphosphate by nick
translation and used as the probe for FISH. The probe was derived from
CWRHIVASVN to avoid inclusion of the human PLAP gene while still
providing a large enough fragment for use as an efficient FISH probe.
The probe was specific for the RSV LTR, antisense to HIV-1, the
polyadenylation region, and the neomycin phosphotransferase gene under
the control of the SV40 promoter and was found to hybridize to
vCWRHIVAPAP and vCWRHIVASVN transduced cells with equivalent efficiency
(Fisher-Adams et al, unpublished data). A BamHI
fragment from AAVS118 (kindly provided by R. Kotin,
National Institutes of Health, Bethesda, MD) was used for
the chromosome 19 AAVS1 site. Hybridization and washes were performed
as described.41 Visualization and imaging was achieved
using a PSI imaging system (Perceptive Scientific Instruments Inc,
League City, TX) housed in the cytogenetics core facility at the City
of Hope National Medical Center. At least 100 nuclear spreads were
scored for each sample (except for HJ, for which there were
fewer suspension cells for analysis; see Table
2).
Reverse transcription-PCR (RT-PCR) analyses.
Approximately 0.5 µg of total RNA or 1 µL of oligo-dT purified RNA
was used for each reaction. Reverse transcription was performed in 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, and 5 mmol/L MgCl2 with 1 µmol/L antisense primer 1a
(5'-ATGCTTCGAAATTACGAGTCAGGTATCTGGTGCCAAT-3'), 0.5U RNase
Inhibitor, and 1 mmol/L each of dGTP, dATP, dCTP, and dTTP. Reaction
tubes were prepared without RNA, and after RNA was added, each
preparation was divided into two separate reactions: (1) +RT, which
included 1.75 U Moloney murine leukemia virus (MuLV) reverse transcriptase (Perkin-Elmer), or (2) RT, which included dH2O. Reverse transcription was performed at 42°C for
45 minutes, followed by denaturing at 99°C for 3 minutes and
snap-cooling to 5°C for 5 minutes. The resulting cDNAs were
subjected to 30-cycle PCR, after annealing at 55°C, using the sense
primer 1b (5'-GATCCTCGAGCCATTTGACCATTCACCACATTGGTGT-3') and
the antisense primer 1a described above to amplify a 526-bp product
spanning a region from the RSV LTR transcriptional start site to the
AAV poly A region. A total of 2.5 µL of 10× Vent buffer was
added to each +/ RT reaction such that the final concentrations were 20 mmol/L KCl, 10 mmol/L
(NH4)2SO4, 2 mmol/L
MgSO4, 5 mmol/L MgCl2, 0.1% Triton X-100, and
1.8 mmol/L Tris HCl. Primers 1a and 1b were added to a final
concentration of 1 µmol/L. Deep Vent (0.25 µL; New England
Biolabs) was added immediately before thermal cycling.
Amplification conditions were as follows: 94°C for 40 seconds,
55°C for 40 seconds, and 72°C for 1 minute for 30 cycles, followed by a 72°C final extension for 5 minutes. Gel loading buffer was added to each tube and one third to one half of each reaction was electrophoresed on a 1.5% agarose gel and subsequently analyzed by Southern hybridization using a probe containing the BamHI-Xba I fragment isolated from pCWRHIVASVN.
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RESULTS |
AAV vector transduction of LTBMCs.
CD34 cells were purified from light-density bone marrow or cord blood
mononuclear cells and transduced with vCWRHIVAPAP28 encoding an antisense RNA complementary to the HIV-1 LTR under the
control of the RSV LTR and the gene encoding thermostable human PLAP
under the control of the phosphoglycerate kinase promoter (Fig
1) at a functional multiplicty of 3 (particle MOI, 600). CD34 cells were transduced immediately after isolation and plated in
long-term culture on irradiated heterologous stroma along with untransduced controls. No toxicity or major differences in cellularity were observed in transduced cultures as compared with untransduced controls.
To evaluate transduction of primitive hematopoietic progenitor cells
with AAV vectors, we first analyzed gene transfer into clonogenic CD34
cells in long-term cultures. Cells harvested from LTBMCs were assayed
for clonogenic potential in LTC-IC assays 5 weeks or later after
culture initiation and vector transduction. No reduction in the
clonogenic capacity of transduced cells was observed as compared with
untransduced cells, suggesting that AAV transduction was not toxic or
deleterious to myeloid differentiation from primitive progenitor cells
in vitro.
AAV vector sequences in individual colonies derived from LTC-ICs.
To determine transduction efficiencies of clonogenic cells in long-term
cultures, we analyzed individual colonies harvested from LTC-ICs from
LTBMCs described above. Entire LTBMC wells were harvested after
trypsinization of stromal layers from cultures initiated with
vCWRHIVAPAP-transduced and untransduced cells, washed, and placed in
LTC-IC assays at 5, 8, and 10 weeks after transduction. Colonies
developing from LTC-ICs were primarily CFU-GEMM and colony-forming
unit-granulocyte-macrophage (CFU-GM), few colony-forming
unit-granulocyte (CFU-G), colony-forming unit-macrophage (CFU-M), and
burst-forming unit-erythroid (BFU-E) were also observed. Individual colonies were plucked from methylcellulose in LTC-IC assays,
DNA extracted, and analyzed for the presence of vector sequences by
amplification of the HIV LTR antisense gene using primers RPSP and
DLASP (Fig 1). Figure 2 shows
representative PCR amplification analyses of DNA from LTC-ICs from
donors TS at weeks 5 and 8 (Fig 2A and B, respectively), donor HJ at
weeks 8 and 10 (Fig 2C), and donor FK at weeks 5 and 10 (Fig 2D).
Amplification of globin served as a control for template integrity.
The number of colonies showing vector specific bands from the total
number containing globin signals provided the transduction
efficiency. Figure 2E shows the amplification of 10 methylcellulose
samples harvested between colonies and subjected to DNA isolation
procedures. No vector-specific bands were observed from these samples,
indicating that free vector DNA was undetectable in these cultures and
that the vector signals observed in LTC-ICs originated from transduced cells.
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| Fig 2.
Amplification of vCWRHIVAPAP sequences from
individual LTC-IC colonies. CD34 cells were transduced with vCWRHIVAPAP
at a functional MOI of 3 (particle MOI, 600) on day 0 and plated in
long-term culture as described in Materials and Methods. Cells were
harvested from stromal layers at designated time points, washed, and
plated in methylcellulose with no G418 selection and colonies were
plucked after 2 weeks. DNA was extracted and amplified for either the
AAV vector using primers RPSP and DLASP or for globin. Globin
served as a control for template integrity. The 418 HIVA band denotes
the vector signal. The 268-bp Glo band denotes the globin signal.
Representative colonies from untransduced controls (Untd) are shown.
Copy number controls are included in each analysis. For HIVA, the copy
number corresponds to 12, 120, and 1,200 copies of the genome. For globin, copy number controls show amplification from 80, 160, and 1,000 cells. (A) Week-5 LTC-ICs from donor TS. All colonies except one had
intact DNA, albeit at varying quantities. The absence of a globin
signal from colony 9 indicated that the DNA template was inadequate.
(B) Amplification of vCWRHIVAPAP sequences from individual week-8
LTC-IC colonies from donor TS. All colonies analyzed had intact DNA
templates and 9 of 15 showed vector-specific signals. The standard
curve is reversed in this experiment. (C) Amplification of vCWRHIVAPAP
sequences of individual week-8 (transduced lanes 1 through 10) and
week-10 (transduced lanes 11 through 20) LTC-IC colonies from donor HJ.
Nine of 10 week-8 LTC-ICs had intact templates. Six of these showed
vector-specific signals. All 10 week-10 colonies analyzed had intact
DNA, and 6 of these were transduced. (D) Amplification of vCWRHIVAPAP
sequences of individual week-5 and -10 LTC-IC colonies from donor FK.
Colony 4 had intact albeit very low amounts of DNA as evidenced by a
faint globin signal on a prolonged radioautographic exposure. (E)
Amplification of methylcellulose samples from between colonies in
transduced LTC-IC wells. Ten separate samples of methylcellulose were
amplified with primers RPSP and DLASP using PCR conditions identical to
above. NT, no template; C, CWRHIVAPAP control (10 copies). This film
was exposed for 36 hours longer than exposures in (A) through (D)
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Table 1 summarizes the transduction
efficiencies of individual colonies derived from week-5, -8, and -10 LTC-ICs from seven donors. Week-5 LTC-ICs were consistently observed in
each donor analyzed. The presence of week-8 and -10 LTC-ICs
representing more primitive progenitors varied from donor to donor.
Transduction frequencies varied from 5% to 100% of colonies sampled,
depending on the donor. Donors SM and BE showed the lowest level of
transduction, ranging from 5% to 7% at 5 weeks to 25% (SM) at 10 weeks. No week-8 or -10 LTC-ICs were present in cells from donor BE. No
overall significant decline in transduction frequencies was observed
with increasing time in culture except for donor TS, suggesting that the level of AAV vector transduction observed at 5 weeks was stable. In
some donors (HJ and SM), the frequency of transduced LTC-ICs was higher
in week-8 and -10 LTC-ICs as compared with week-5 LTC-ICs, perhaps due
to transduction of more primitive cells. These results suggest that
both early (week-5) LTC-ICs and late (week-8 and -10) LTC-ICs can be
transduced with AAV vectors in the absence of selective pressure.
Integration of AAV vector sequences in marrow CD34 cells in long-term
cultures.
Because AAV vectors can exist intracellularly in either double-stranded
episomal or chromosomally integrated forms, we analyzed the frequency
of vector integration into chromosomal DNA in CD34 cultures in LTBMCs
by FISH analyses. CD34 cells were transduced with
vCWRHIVAPAP16 at a functional MOI of 3 (particle
MOI, ~600) immediately after isolation and culture isolation. After
boosting the mitotic index with higher concentrations of cytokines,
cells were blocked in metaphase and harvested for FISH analyses.
Metaphase spreads of transduced and untransduced cells were hybridized
with a vector-specific probe representing the RSV LTR, antisense
sequences to the HIV LTR, and the polyadenylation region but not
including the alkaline phosphatase sequences.
Figure 3 shows FISH analyses of integrated
vCWRHIVAPAP sequences in transduced cells. Vector-specific signals were
never detected in untransduced controls (Fig 3A) from any sample,
indicating that there was no nonspecific hybridization of the probe to
untransduced cells. The clonal cell line HIIC21, transduced with
vCWRHIVASVN and containing one copy of the vector genome per cell,
served as the positive control (Fig 3B). Figure 3C, D, and E show
representative metaphase spreads from CD34 cells 5 weeks after
transduction. FISH signals were not digitally amplified and metaphases
were scored as positive only if vector-specific signals were clearly
visualized on both sister chromatids. The presence of vector-specific
signals on each sister chromatid indicated that the integrated vector
sequences had replicated with cellular chromosomal DNA, suggesting
stable transmission of the transgene to progeny cells. CD34 cells from
10 donors were analyzed 2 to 9 weeks after transduction and culture
initiation. Six percent to 29% of metaphases from different donors
were found to have integrated vector signals on both chromatids. By the
later time points in culture, the nonadherent progeny of transduced
CD34 cells analyzed for FISH were found to differentiate primarily into
myeloid cells under these culture conditions as determined by
immunophenotyping and flow cytometric analysis. However, because at the
time of transductions the population was highly enriched for CD34
cells, vector-positive cells most likely represent the progeny of
transduced CD34 cells.
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| Fig 3.
FISH analysis of vCWRHIVAPAP transduced and untransduced
cells. CD34 were transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI, 600) and placed in culture as described above.
Suspension cells were harvested at designated time points for analysis.
(A) A metaphase spread from an untransduced CD34 culture showing no
hybridization of the vector-specific probe to cellular sequences. (B)
Hybridization analysis of HIIC21, a G418-resistant clonal 293-based
cell line derived after transduction with vCWRHIVASVN. This clone
carries 1 copy of the vector genome per cell (28) and served as a
positive control. (C, D, and E) Representative metaphases from
vCWRHIVAPAP transduced CD34 cultures at 5 weeks posttransduction. Note
that vector-specific signals are observed on both sister chromatids.
Signals were not digitally enhanced in this analysis. (F)
Representative interphase nuclei from a vCWRHIVAPAP transduced CD34
culture 3 weeks posttransduction. Three nuclei with three or more
signals, two nuclei with two signals each, and three nuclei with no
signals are seen.
|
|
To determine the stability of vector integration, cells from three
donors (FF, BE, and MM) were evaluated at early and late time points
after transduction (Table 2). Twenty-seven
percent and 19% of metaphases from FF were vector-positive at 3 and 5 weeks posttransduction, respectively. Twenty percent and 22% of metaphases from donor BE hybridized with the vector-specific probe at 2 and 4 weeks, respectively. Cells from donor MM showed 27% and 20%
vector-positive metaphases at 2 and 8.5 weeks, respectively. These
results suggest that the frequency of vector integration was largely
stable in CD34 cells and that transduced cells containing integrated
AAV vector genomes did not have any growth disadvantage in tissue
culture.
To evaluate whether cells with episomal copies of vector genomes could
be detected in long-term culture, interphase nuclei were scored for
vector-specific signals per nucleus and compared with the frequency of
metaphase signals from the same time points. Because both episomal and
integrated genomes should hybridize with the vector-specific probe in
interphase nuclei, we reasoned that the presence of episomal copies
would result in the detection of greater than two signals in
interphases as compared with metaphases. Figure 3F shows interphase
nuclei with several nuclei containing three or more signals and two
nuclei with two signals. To date, we have never observed more than a
single integration event per metaphase spread in CD34 cells, perhaps
due to the relatively low MOI used in these studies. Therefore, we made
the assumption that interphase nuclei with three or more signals likely
contain episomal genomes either exclusively or in combination with
integrated signals. Most CD34 samples analyzed did not show a higher
frequency of vector-positive interphase nuclei compared with metaphase
spreads (Table 2), suggesting that most signals observed approximately 4 weeks after transduction likely represented integrated signals. However, due to the possibility of differential probe entry into permeabilized interphase nuclei as compared with metaphase spreads, a
precise estimation of the frequency of episomal forms is difficult. A
comparison of early and late time points after transduction showed some
decrease in positive nuclei at the later time points. Whether this was
due to the loss of episomal copies or transduced cells is not clear.
We performed two-color hybridization to determine if the rep-negative,
wild-type-free vector stocks used in our experiments integrated at
sites distinct from AAVS1, the integration site for wild-type AAV.
Colocalization of the AAVS1 probe and the vector probe was never
observed in any transduced cell (0/100 nuclei) indicating that, in the
absence of either rep78 or wild-type AAV, vCWRHIVAPAP did not integrate
into AAVS1.
Integration of AAV vector sequences in marrow-derived
CD34+38 cells.
To determine if AAV vector genomes could integrate into the more
primitive hematopoietic fraction of CD34 cells, we analyzed cultures
initiated with marrow CD34+38 cells by
FISH. Flow-sorted CD34+38 cells were
transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI,
~600) immediately after isolation and placed in culture on irradiated
heterologous stroma. Cells from all three donors were harvested at 4 weeks after transduction and culture initiation. Integration levels
ranging from 11% to 28% were observed, indicating that this
population was also transducible with AAV vectors (Table 2B). Cells
from two donors (KL and NT) were also analyzed at 8 weeks
posttransduction. The frequency of positive metaphases was comparable
at both time points for a given donor, again indicating that the
fraction of cells with integrated vector was stable over time. However,
for both donors, higher levels of positive interphases were observed at
4 weeks than at 8 weeks, suggesting the presence of episomal copies of
the vector at the earlier time point. Interestingly, by 8 weeks, the
frequency of positive interphases was comparable with that of
metaphases, possibly due to the loss of the episomal forms by this time
point and persistence of the integrated forms. These results are
consistent with the longer survival of episomal copies of rAAV in the
more primitive CD34+38 cells.
Expression of AAV vector-encoded transgenes in CD34 cells.
Because AAV vectors appeared capable of transducing and integrating
into primitive CD34+ and
CD34+38 hematopoietic progenitor cells,
we evaluated expression of vector-encoded transgenes in long-term CD34
cultures transduced with vCWRHIVAPAP at a functional MOI of 3 (particle
MOI, 600) under conditions similar to that for FISH
analyses. Table 2 shows PLAP expression in four CD34 cultures and three
CD34+38 cultures. In most cases, the
level of PLAP expression approximated the level of integration seen by
FISH, suggesting that most cells containing vector genomes also
expressed the transgene. In some cases, notably MM, a decrease in PLAP
expression was observed at the 8-week time point as compared with the
earlier (week-2 or -4) samples from corresponding donors. This could
either be due to expression from double-stranded episomal copies at the early time points that were lost with cell division and/or
silencing of transgene expression later in culture.
In addition to PLAP, we also analyzed transcription of antisense RNA
complementary to the HIV-1 LTR in cells harvested from LTBMCs and
LTC-ICs. Transcription of the antisense RNA is under the control of the
RSV LTR and represents the clinically relevant transgene present in
both vCWRHIVAPAP and vCWRHIVASVN. Cells were transduced at functional
MOI of 1 to 2 (particle MOI, ~500 to 1,000) with vCWRHIVASVN.
Figure 4A shows cDNA amplification using primers 1a and 1b (Fig 1) after reverse transcription of RNA extracted from transduced cultures 5 weeks after culture initiation and transduction. A 530-bp product representing the HIV-1 LTR specific antisense RNA transcribed from the vector was detected in transduced cells from each donor tested but not from untransduced cells. The
presence of the amplified band only in samples subjected to reverse
transcription before amplification confirmed that the product was of
RNA origin. All RT-PCR reactions were performed under conditions
determined to be in the linear range of the reaction. The levels of HIV
LTR antisense transcript detected in cultures from donors 13, 17, and
20 (lanes 5 through 14) were comparable to that observed in the clonal
cell line, HIIC21, which contains one copy of vCWRHIVASVN per
cell.28 However, donor 18 (lane 3) showed lower levels of
transcription, suggesting variability in either transduction efficiency
or transgene expression in this donor. To control for artefactual
sources of signal, cells from patient no. 20 were exposed to a control
sham stock prepared in an identical fashion to vector except for the
exclusion of AAV rep and cap genes, thus preventing the generation of
encapsidated transducing virus particles. As seen in Fig 4A, lanes 17 and 18, no vector-specific signals were detected in RNA from cells
exposed to this control stock, demonstrating that antisense RNA
transcription in long-term cultures directly resulted from AAV vector
transduction. Overall, these results demonstrated that AAV vector
transduction of long-term cultures resulted in readily detectable
expression of antisense RNA in the absence of selective pressure.
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| Fig 4.
Southern analysis of HIV antisense transcription from
vCWRHIVASVN in transduced marrow and cord blood-derived hematopoietic
progenitor cells after RT-PCR amplification. (A) RNA was extracted from
week-5 to -7 LTBMCs and antisense sequences were reverse transcribed
and amplified using primers 1a and 1b. The amplified products were
resolved on a 1.2% agarose gel, transferred to nitrocellulose, and
hybridized with an RSV LTR and antisense-specific probe. HIIC21,
containing 1 copy of integrated vector per cell, served as the positive
control. The 530-bp antisense transcript-specific product is shown.
+RT and RT refer to the presence or absence, respectively, of
reverse transcription before amplification. The absence of signals in
these lanes indicates that the antisense signals were RNA-specific.
 , RT-PCR analysis of cells (donor 20) exposed to a sham stock of
vector prepared in the absence of AAV rep and cap genes. Untransduced
(Untd) cells from each donor tested served as negative controls. Td,
transduced cultures. (B) HIV LTR antisense transcription in LTC-ICs
initiated with CD34+CD38 cord blood cells
8 weeks after transduction. A representative sample, CB6, was
transduced with vCWRHIVAPAP at MOI of 3 (particle MOI, 600) and
vCWRHIVASVN at MOI of 0.1 (particle MOI, 50). Cells were harvested from
6 week LTBMCs, washed, and placed in colony-forming assays. RNA was
extracted from colonies after 3 weeks, reverse transcribed, and
amplified with primers 1a and 1b. The 530-bp antisense product was
evident only after reverse transcription.
|
|
LTC-IC colonies derived from CD34+38
cells from umbilical cord blood were also tested for AAV vector
transduction. Cells were transduced at the time of culture initiation
with either vCWRHIVASVN at MOI of 0.1 (particle MOI, 50) or vCWRHIVAPAP
at MOI of 3 (particle MOI, 600). As with marrow cells, cord blood
cultures were passaged approximately every 10 days. Six weeks after
culture initiation, cells were harvested from stroma, washed, and
assayed for LTC-ICs. Week 6 LTC-IC colonies were harvested,
polyadenylated RNA was extracted from transduced and untransduced
cells, and RT-PCR of the HIV LTR antisense RNA was performed as
described above. Figure 4B shows the amplified antisense transcript
from a representative cord blood sample, CB6. Cultures
transduced at a functional MOI of 3 (particle MOI, ~600) with
vCWRHIVAPAP showed levels of antisense transcription
comparable to that of the single copy clonal cell line,
HIIC21,16 whereas cultures transduced at MOI of 0.1 (particle MOI, ~50) with vCWRHIVASVN showed lower levels of
transcript, consistent with the lower multiplicity of transduction.
These results demonstrated that primitive clonogenic
CD34+38 cord blood cells transduced with
AAV vectors demonstrated transgene expression at 8 weeks
posttransduction in the absence of G418 selection. These findings are
consistent with both the analyses of transduction of LTC-ICs and FISH
analyses of CD34 cells.
| |
DISCUSSION |
In this study we evaluated AAV vectors for their capacity to transduce
genes into primitive human hematopoietic progenitor cells with
replating and differentiative capacity in long-term culture.
CD34+ and CD34+38 cells from
marrow and cord blood were evaluated for AAV vector transduction by
single-colony analysis, FISH, and transgene expression from 22 CD34
samples. rAAV transduction levels varied from donor to donor. Despite
variability in transduction efficiencies, every sample analyzed showed
evidence of transduction. AAV vector transduction had no effect on
colony formation, cobblestone area formation, or cell numbers in
long-term cultures.
Amplification of vector sequences from single colonies derived from
early and extended LTC-ICs indicated that primitive myelo-erythroid progenitors could be transduced with AAV vectors. The transduction frequency of extended (week-8 and -10) LTC-ICs from most donors was
observed to be equal to or higher than week-5 LTC-ICs, suggesting that
primitive progenitors also serve as good targets for AAV transduction.
Comparable transduction levels in LTC-IC assays initiated at early and
late time points after culture initiation suggested that (1) cells with
extended clonogenic capacity were transducible with AAV vectors and (2)
transduction levels attained by week 5 were largely stable. These
results suggest that primitive myelo-erythroid progenitor cells capable
of long-term survival in vitro and possessing delayed replating and
differentiative capacity are amenable to genetic modification by AAV vectors.
The genomes of both wild-type AAV and AAV vectors are known to exist in
either chromosomally integrated or double-stranded episomal forms.
Either of these double-stranded forms of the vector genome can direct
transgene transcription and serve as templates for DNA amplification.
Because DNA amplification from LTC-IC colonies analysis does not
distinguish between episomal and integrated states of the vector, we
analyzed the fate of vector genomes by FISH after long-term
transduction. Metaphase FISH analyses provided an estimate of
integration frequencies, because vector signals were exclusively
associated with chromosomes. A vector integration frequency of 9% to
28% was observed in marrow CD34+ and
CD34+38 cells over the 2- to 9-week
period of study in the absence of selective pressure. Similar results
were obtained with CD34+ and
CD34+38 cells from umbilical cord blood
(data not shown). Importantly, the fraction of cells containing
integrated vector remained comparable throughout the culture period for
a given donor, suggesting that AAV vector integration was stable.
Interphase nuclei contained both episomal and integrated vector
genomes. Therefore, comparisons of the frequency of vector-positive
interphase and metaphase nuclei by FISH allowed a relative
approximation of the proportion of nuclei containing episomal vector.
At early time points after transduction, a fraction of
CD34+38 cells appeared to contain
episomal vector, perhaps in addition to cells containing integrated
vector. However, the episomal forms appeared to be lost over time in
culture, whereas the proportion of cells containing integrated vector
remained stable and contributed to long-term transduction. By 8 weeks
posttransduction, only the stably integrated population was evident.
Interestingly, episomal forms appeared to persist longer in the more
primitive and possibly more slowly cycling
CD34+38 cells than in CD34+
cells. This could be due to the longer persistence of episomal forms of
AAV vectors in slowly dividing cells. Thus, there appears to be two
populations of cells transduced with rAAV vectors. Stable chromosomal
integration is observed in one population, whereas the episomal form of
the vector persists in the other and is eventually lost over time.
The factors that determine AAV vector genome integration are not fully
defined. The virus-encoded rep68/78 protein has been implicated in
site-specific integration of wild-type AAV into the AAVS1 locus on the
human chromosome 19. The AAV rep protein recognizes and binds to a
consensus sequence present on both the viral ITRs as well as in
chromosome 1942-44 and has ATPase, endonuclease, and DNA
helicase activities45 that are likely instrumental in site-specific integration of wild-type AAV and rAAV vectors. The complete absence of rAAV integration into AAVS1 observed in our studies
is in accordance with previous reports46,47 and provided independent evidence that the vectors used here were free of wild-type AAV. Integration of AAV vectors into chromosomal DNA has been previously demonstrated by us and others. However, wild-type-free rAAV
has consistently been found to integrate at sites other than AAVS1,28,46-48 and viral proteins have not been implicated
in rAAV integration. It is likely that cellular enzymes, including those involved in DNA repair and replication, play a role in this event. The differences between cell populations exhibiting stable integration of AAV vectors and those containing transient episomal genomes may be useful in further defining factors important for AAV
vector integration.
Evaluation of transduction of CD34 cells by both single-colony PCR and
FISH analyses showed comparable gene transfer frequencies at similar
time points for some donors; however, somewhat higher frequencies were
observed by PCR in others. This was attributed to (1) the higher levels
of sensitivity of PCR amplification as compared with FISH, especially
without digital amplification of hybridization signals; (2) the
possible persistence of episomal forms of the vector in some colonies;
and (3) differences in the target cell populations being assayed in the
two systems. In LTC-IC assays, clonogenic cells capable of giving rise
to myelo-erythroid colonies were evaluated, whereas in FISH, all cells
capable of undergoing mitosis from 2 to 9 weeks after culture
initiation were analyzed.
Each donor evaluated in this study showed evidence of some level of AAV
transduction. However, cells from some donors showed significantly
lower levels of transduction than others. These results are in
concordance with those of Ponnazhagan et al,49 who reported donor to donor variability of CD34 transduction by AAV
vectors. In keeping with these results, we also observed that LTBMCs
processed in parallel and transduced with the same batch of vector
showed variability in transduction frequencies. Whether this reflects
genetic variability in putative cell surface receptors for AAV,
receptor density, second-strand synthesis, or differences in the
physiologic status and therefore the intracellular milieu of cells from
different donors at the time of transduction is currently unknown.
Antisense RNA to the HIV LTR encoded by AAV vectors used in this study
has previously been shown to potently inhibit HIV-1 replication.5 Transcriptional analysis of transduced
CD34+, CD34+38, and LTC-IC
colonies showed that cells from each donor tested transcribed antisense
RNA to the HIV LTR, with the transcript levels from several donors
being comparable to that of a single-copy clone. Importantly, no
selective pressure was required to observe high levels of transgene
transcription in either marrow or cord blood progenitor cells. The
level of antisense RNA transcription observed was proportional to the
functional multiplicity of transduction. These results also suggest
that the RSV LTR remain transcriptionally active in a significant
proportion of hematopoietic progenitor cells over the 5- to 9-week
culture period analyzed in this study. Whether these levels of
antisense RNA transcription confers resistance to challenge with HIV in
the monocyte-macrophages differentiating from CD34 cells in culture is
currently being determined.
These results extend our previous studies showing AAV vectors
transduction and integration into CD34+ marrow and cord
blood-derived lineage-committed progenitor cells to primitive
progenitors in long-term culture. The cells evaluated here were
functionally more primitive than CD34 cells previously studied in CFU-C
assays.28 However, it must be noted that the culture
conditions used in this study and the cells assayed here represent
myelo-erythroid progenitors and do not address gene transfer into
lymphoid progenitor cells. LTC-ICs also most likely do not represent
true HSCs capable of long-term engraftment in vivo.9
Efficient transduction of LTC-ICs by retroviral vectors, despite
inefficient long-term in vivo engraftment with transduced cells as
evidenced by several human gene therapy trials, suggests that other
studies with in vivo models of hematopoiesis are necessary to determine
stem cell transduction. Nevertheless, this is the first systematic
report of AAV-mediated gene transfer into primitive clonogenic cells in
long-term culture with analysis of gene transfer at the single-colony
level and integration at the chromosomal level.
Despite several recent reports of AAV-mediated gene transfer into
CD34+ human and murine hematopoietic progenitor cells in
vitro and in vivo,28-34,50 some groups have reported
difficulty in transducing CD34 cells with AAV vectors. There may be
several possible explanations for these differences. (1) The source of
CD34 cells may be critical in determining transduction frequencies.
Preliminary observations from our group suggest that marrow CD34 cells
may have different rAAV transduction frequencies than
cytokine-mobilized peripheral blood stem cells (Wong et al, unpublished
data). We have previously observed that rapidly dividing
cells show poor transduction with AAV vectors possibly due to a quicker
loss of episomal vector genomes before integration. (2) Cytokine
concentrations used in culture media may also contribute to altered
cell cycle time and therefore AAV transduction frequencies. We have
found that the use of lower cytokine concentrations correlates with
higher levels of AAV transduction of CD34 cells (Fisher-Adams et al,
data not shown). (3) Attachment and entry of AAV virions to cells is
likely mediated by one or more cell surface receptors.51
Heparan sulfate proteoglycan has recently been identified as one AAV
binding molecule that may facilitate binding and internalization of
virions.52 Thus, the choice of collection media and culture
conditions may influence AAV transduction of cells by either
facilitating binding or blocking attachment sites. (4) Genetic factors
may influence receptor polymorphism and cell surface density affecting
virus entry. Genetic polymorphism or the intracellular milieu may also influence postentry processes53 such as second-strand
synthesis and genome integration that would ultimately
affect the outcome of AAV transduction. (5) Lastly,
differences in vector backbones and/or production and
purification methods used by different groups may lead to different
outcomes. Clarification of these issues is essential for better insight
into the potentials of AAV vectors and must await further delineation
of AAV vector biology and elucidation of cellular processes associated
with AAV transduction.
Recently, numerous studies have been published showing AAV transduction
of primary nondividing cells. AAV vector transduction of postmitotic
neurons in vivo,54 cochlear, retinal and glial cells of the
human central nervous system, nonproliferating respiratory epithelial
cells, alveolar stem cells, adult skeletal cells,26,27 and
cardiac muscle25 have been documented. Thus, it has become abundantly clear that AAV vectors are capable of transducing
nondividing cells. AAV transduction of skeletal muscle and brain
accompanied with prolonged gene expression has been demonstrated in
vivo28,29 with little or no evidence of AAV-specific T-cell
responses. Early reports from a human gene therapy trial testing AAV
vectors for cystic fibrosis indicate the lack of any vector-associated
toxicity or immune rejection.55 These results, together
with our findings of long-term transduction, vector integration, and
transgene expression in primitive myeloid progenitor cells, suggest
that AAV vectors merit further evaluation as gene transfer vehicles for
quiescent HSCs. Further studies with lymphomyeloid progenitors and in
vivo models of human hematopoiesis should provide more information about AAV transduction of HSCs. However, ultimately, in vivo human trials testing long-term, multilineage engraftment of AAV-transduced cells will be necessary to evaluate true stem cell transduction by AAV vectors.
| |
ACKNOWLEDGMENT |
The authors are indebted to the members of the COH Hematology/BMT unit
for providing bone marrow and to the physicians and nurses of the Labor
and Delivery Unit and the Department of Obstetrics and Gynecology at
Huntington Memorial Hospital for collecting umbilical cord blood
samples. We thank Drs Navtej Juty, Robert Krouse, Elizabeth
Shaughnessy, Li Jing Li, Edna Rosborough, Deepinder Brar, James Bolen,
Marilyn Slovak, and Christine Wright for their assistance and comments.
The following COH Cancer Center Core facilities were used in this
study: Flow Cytometry, Cytogenetics, Phosphorimaging, and DNA synthesis
and sequencing.
| |
FOOTNOTES |
Submitted March 31, 1998; accepted November 10, 1998.
Supported in part by Grants No. AI-R0140001 and AI-U1938592 from the
National Institute of Allergy and Infectious Diseases, National
Institutes of Health (NIH), and by Grants No. CA-R0171947, CA-P0159308,
CA-P0130206, and CA33572 from the National Cancer Institute, NIH.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Saswati Chatterjee, PhD, Division of
Pediatrics, City of Hope National Medical Center, 1500 E Duarte Rd,
Duarte, CA 91010; e-mail: schatterjee{at}coh.org.
| |
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