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PLENARY PAPER
From the Division of Experimental Hematology,
Department of Hematology/ Oncology, St Jude Children's Research
Hospital, Memphis, TN.
Limited expression of the amphotropic envelope receptor is a
recognized barrier to efficient oncoretroviral vector-mediated gene
transfer. Human hematopoietic cell lines and cord blood-derived CD34+ and CD34+,
CD38 The ability to transfer genes into hematopoietic
stem cells would provide the opportunity for somatic gene therapy for
malignant and nonmalignant disorders that affect bone marrow and
peripheral blood function.1,2 Extensive efforts have been
invested in adapting oncoretroviral vectors for gene transfer into stem
cells. Despite considerable success in murine models,
retrovirus-mediated gene transfer into human stem cells has
been difficult to achieve.3-8 Recognized barriers to human
stem cell gene transfer include low expression of viral receptors and
the relative quiescence of this target cell
population.9-11
A useful surrogate for human stem cells are primitive hematopoietic
cells that are able to establish human hematopoiesis in immunodeficient
mice, such as the nonobese diabetic/severe combined immunodeficiency
(NOD/SCID) strain. Human NOD/SCID repopulating cells (SRCs) can be
recovered from murine bone marrow months after transplantation, as
reflected by their ability to establish human hematopoiesis in
secondary recipients.12-15 This quality plus the multilineage engraftment observed in the NOD/SCID model suggest that
gene-transfer strategies that result in retroviral marking of a
significant proportion of the progeny of SRCs would be a better
predictor of stem cell gene transfer than surrogates evaluated by in
vitro assays.15 Accordingly, we used umbilical cord blood SRCs as targets in our experiments designed to improve the efficiency of oncoretrovirus-mediated gene transfer into primitive human hematopoietic cells.
Oncoretroviral vectors require cell division to achieve genome
integration and long-term gene expression.11 Many
combinations of cytokines have been tested in experiments designed to
initiate cell-cycle activation of primitive hematopoietic cells without loss of repopulating potential.16-18 Recent evidence
suggests that short-term cultures with high concentrations of
cytokines19,20 allow limited division and expansion of
SCID repopulating cells, raising the possibility that receptor
deficiency may be the major remaining barrier to stem cell-targeted
gene transfer.
Most vector preparations used for gene transfer into human cells
contain particles that have amphotropic specificity based on the
structure of their envelope protein. Amphotropic viral particles have a
broad host range that includes human cells,9 and they rely
on a phosphate transporter for cell entry21-23 that is now
known to be expressed at very low levels on primitive human hematopoietic cells.10 To overcome this barrier to
oncoretrovirus-mediated gene transfer, researchers have generated and
studied vector preparations pseudotyped with envelope proteins from
other viruses.
Oncoretroviral vectors pseudotyped with the envelope protein of the
gibbon ape leukemia virus (GALV) can transduce human clonogenic hematopoietic progenitor cells24 and SRCs from cord
blood.25,26 The GALV receptor, also a phosphate
transporter, is expressed at a somewhat higher level than the
amphotropic receptor on the CD34+ population of cells from
human and baboon bone marrow.21-23,27,28 The frequency of
retrovirus-marked human cells achieved with the GALV-pseudotyped
particles exceeds that achieved with amphotropic vector particles in
the NOD/SCID model under similar transduction conditions.25,26,29-33 GALV-pseudotyped murine
oncoretroviral vector particles are somewhat more efficient than
amphotropic particles at transducing canine and baboon repopulating
cells, but the gene-transfer efficiency, as reflected by the proportion of genetically modified peripheral blood or bone marrow cells in these
myeloablated animal models, remains less than or equal to
10%.8,28,34-36 This suggests that repeated exposure to
conditioned medium affects the long-term repopulating potential of
transduced cells, which may be reflected in the larger animal models
but not in the NOD/SCID assay.33
The G-envelope protein of vesicular stomatitis virus (VSV-G), a
rhabdovirus, has been used to pseudotype both lentiviral and murine
retroviral vector particles.37-41 VSV-G-pseudotyped
particles can be concentrated 100- to 1000-fold by centrifugation,
thereby removing the conditioned medium.42 Successful gene
transfer into SRCs with concentrated VSV-G-pseudotyped
oncoretroviral or lentiviral vector particles has been
reported.43,44 However, only with VSV-G-pseudotyped
lentiviral vector particles was the vector genome documented to be
present in human cells of multiple lineages in the NOD/SCID
recipients of transduced cells.44
Murine retroviral vector particles pseudotyped with the envelope
protein of the feline endogenous virus, RD114, have been shown to have
tropism for human hematopoietic cells.45,46 The RD114
retrovirus is a member of the large interference group 1 of
retroviruses, all of which use the same receptor on human
cells,47 recently identified as a neutral amino acid
transporter.46 We have evaluated the use of
RD114-pseudotyped oncoretroviral vector particles for transducing
primitive human hematopoietic cells, including those that repopulate
NOD/SCID mice.
Preparation of cord blood cells
Retroviral vector preparation
Vector particles pseudotyped with the RD114 envelope protein were also generated in 293T cells that had been transfected with vector, helper, and envelope plasmids. For RD114-pseudotyped particles, the 293T cells were transfected with a plasmid encoding the vector genome (pMGirL22Y), a second encoding the GAG and POL proteins of murine leukemia virus (pEQPAM3-E), and a third plasmid encoding the gene for the RD114 envelope (pRDF48). Between 48 and 72 hours after transfection, conditioned medium was harvested and titered on HeLa cells. The titers ranged from 0.5 to 2 × 105 infectious vector particles per milliliter. In vitro analysis of gene transfer CD34+ or CD34+, CD38
purified cell populations were cultured in Iscove's modified
Dulbecco's medium plus 1% bovine serum albumin, human insulin (5 µg/mL), human transferrin (100 µg/mL), low-density lipoproteins (10 µg/mL), 0.1 mmol/L  -mercaptoethanol, stem cell factor (SCF, 300 ng/mL), Flt-3 ligand (300 ng/mL), interleukin-3 (IL-3, 10 ng/mL), and
IL-6 (10 ng/mL). The cytokines were obtained from R&D Systems
(Minneapolis, MN). The cells were incubated for 24 hours (or as
otherwise indicated) at 37°C in 5% CO2 before transduction.
Transduction of the CD34+ and CD34+,
CD38 After a total of 96 hours in culture, the CD34+ or
CD34+, CD38 To assay gene transfer into clonogenic progenitors, we replated transduced and control hematopoietic cells after 96 hours of culture into Methocult GF (H4434; Stem Cell Technologies, Vancouver, BC, Canada), which had been pretreated with 1.2 U/mL thymidine phosphorylase at 37°C for 2 hours. Cultures were established with or without 100 nmol/L trimetrexate. At this concentration of trimetrexate, no colonies formed in the cultures of control, untransduced cells. Hematopoietic cells were cultured in 35-mm plates (1 mL of medium per plate) at 37°C in a 5% CO2 humidified atmosphere for 10 to 15 days, after which the colonies were enumerated. Analysis of gene transfer into cells that establish human hematopoiesis in immunodeficient (NOD/SCID) mice In a series of 3 experiments, purified CD34+ cells were prestimulated in medium containing SCF, Flt-3 ligand, IL-3, and IL-6, as described earlier, for 24 hours or 48 hours at a concentration of 1 to 2 × 105 cells/mL. The cells in this medium were then transferred to retronectin-coated culture plates to which amphotropic or RD114-pseudotyped vector particles had been absorbed (preloaded) or to retronectin-coated plates without virus. All cultures were diluted 2-fold with serum-free medium containing cytokines at 48 hours and harvested for injection at 96 hours. Where indicated, amphotropic vector particles were also added in the form of conditioned medium at up to 50% of the culture volume daily for 2 days to maximize transduction of CD34+ cells with this pseudotyped virus.The NOD/SCID mice (original stocks kindly provided by M. Pallavicini, University of California, San Francisco, CA) were housed in sterile microisolator cages and supplied with sterile food, acidified water, and bedding. Each mouse was injected with 1 to 1.5 × 105 freshly isolated CD34+ cells (greater than 95% purity) or after expansion of this input cell number for 96 hours in culture. Six- to 8-week-old mice were used after sublethal irradiation (325 cGy-127Cs source). The mice were killed 8 to 10 weeks after injection, and bone marrow cells were harvested for flow cytometric analysis and in vitro culture. Bone marrow cells from animals injected with human cells were subjected to flow cytometric analysis with the use of conjugated antibodies against human surface antigens, as follows: (1) human hematopoietic cells, CD45-APC; (2) B lymphocytes, CD19-PE; and (3) myeloid cells, CD33-PE. These antibodies were obtained from Pharmingen (San Diego, CA). Bone marrow cells at 5 to 10 × 105 were mixed with either rat anti-mouse CD16/CD32 Fc block (clone 2.4G2; Pharmingen) or 10% heat-inactivated, pooled mouse serum to block nonspecific antibody binding and then incubated with saturating amounts of one of the conjugated antibodies. Cells from each animal were also stained with appropriate conjugated, isotype-matched, control antibodies obtained from Becton Dickinson or Pharmingen. After incubation, cells were resuspended in red cell lysis buffer (0.83% NH4Cl, 0.1% KHCO3, 0.004% EDTA) and washed twice in PBS containing 2% FCS. In all experiments, cells stained with the isotype control antibody were used to set the quadrant markers such that the negative quadrant contained at least 97% of the control cells. The percentage of engrafted human cells was determined by CD45 positivity, and lineage marker and EGFP expression were determined on the CD45+-gated population. Aliquots of bone marrow cells were assayed for total and
trimetrexate-resistant human clonogenic progenitors, as described earlier. After 14 to 21 days, individual colonies were plucked from the
methylcellulose and processed to recover DNA. The DNA samples were
assayed for EGFP coding sequences with the polymerase chain reaction
(PCR) methodology. After scoring the plates, we picked 20 colonies (or
fewer, if fewer were present) at random and incubated them in lysis
buffer (50 nmol/L Tris, pH 8.5, 1 mmol/L EDTA, 0.5% Tween 20, and 100 µg/mL proteinase K) at 56°C for 2 hours. To inactivate the
proteinase K, we heated the samples at 95°C for 10 minutes. PCR was
performed with the PCR Core Kit (Boehringer Mannheim) according to the
manufacturer's instructions. The amplification conditions were as
follows: 92°C for 2 minutes, then 35 cycles of 92°C for 1 minute,
60°C for 1 minute, and 72°C for 1 minute, followed by a final
elongation step of 7 minutes at 72°C. Primers that amplify an
829-base pair (bp) fragment of human Evaluation of RD114-pseudotyped vector preparations, the RD114/MGirL22Y producer clone, and genetically modified bone marrow cells for replication-competent retroviruses Murine retroviral particles pseudotyped with the RD114 envelope protein do not infect murine cell lines. Vector preparations were therefore screened for replication-competent virus by a marker rescue assay with HeLa or K562 cells (K562-ATCC CCL 243), which contained an integrated vector genome encoding neomycin resistance (G1Na).53 These cells were repeatedly exposed to RD114-pseudotyped, MGirL22Y vector particles in conditioned medium, resulting in 100% transduction on the basis of fluorescence-activated cell sorter (FACS) analysis for EGFP expression. The transduced cells were expanded for 2 weeks while conditioned medium was collected, filtered, and stored. Neomycin-sensitive HeLa and K562 cells were expanded and exposed twice daily to the collected medium over 7 to 10 days before being placed under G418 selection for 14 days in G418-containing medium, which was changed every 48 hours. Fresh medium without G418 was then added twice weekly for an additional 2 weeks. No viable colonies were detected in the target cells and negative control cells, whereas the positive control cells (G1Na-transduced HeLa cells) expanded exponentially. Additionally, EGFP-positive HeLa cells were mixed with unexposed (EGFP-negative) HeLa cells at a 1:10 dilution and expanded over 4 weeks. No increase in EGFP-expressing cells was detected.DNA was recovered from the RD114/MGirL22Y producer clone used in these experiments and from the bone marrow of 3 mice that engrafted with high levels of EGFP+ human CD45+ cells. The DNA samples were assayed by PCR for a potential recombination product that would indicate the presence of replication-competent retrovirus (RCR). The RCR primers were designed to specifically recognize a recombination event 3' to the packaging signal in the vector genome and 5' to a portion of the GAG gene that was not included in the vector genome but was present in the pEQPAM3-E plasmid within the RD18 packaging cell line. The primers used were 5'-GTGGAACTGACGAGTTCTGAACAC-3' and 5'-GAGGAGAACGGCCAGTATTGAAGC-3', which amplified a 995-bp sample in a positive control of rhesus DNA derived from an animal that had previously been shown to be infected with RCR.54
Enhanced transduction of human hematopoietic cells with RD114-pseudotyped vector Human cord blood CD34+ cells were cultured for 24 hours in cytokine-containing, serum-free medium (see "Materials and methods") and then transduced on retronectin-coated plates. Serum-containing (10% FCS), conditioned medium from RD114-, amphotropic-, GALV-, and VSV-G-pseudotyped producer cells was added in amounts necessary to achieve the specified MOIs. After 24 hours, fresh, serum-free, cytokine-containing medium in amounts equal to the culture volume (2× dilution) was added, and after an additional 48 hours, the cells were analyzed for EGFP expression. With a single exposure at an MOI of 5, the RD114-pseudotyped particles were far more efficient at transducing human CD34+ cells than were vector particles pseudotyped with the amphotropic, GALV, or VSV-G envelope proteins (Figure 1).
Aliquots of cells transduced with either RD114- or
amphotropic-pseudotyped particles were replated in methylcellulose
immediately after transduction (48 hours of culture) and incubated for
an additional 10 to 15 days, and resistance to trimetrexate was
determined (Figure 2). The
RD114-pseudotyped vector efficiently transduced CD34+ cells
and their progenitors at a very low MOI, indicating the presence of the
appropriate receptor and cycling of a significant proportion of the
cell population. Amphotropic-pseudotyped particles failed to
efficiently transduce the same population even at increased particle
concentrations, consistent with a block of transduction at the receptor
level. These preliminary results led us to focus our efforts on
evaluating RD114-pseudotyped particles for transducing primitive human
hematopoietic cells under conditions that preserved their repopulating
activity in the NOD/SCID model.
Medium conditioned by derivatives of the HT1080 cell line altered the immunophenotype of primitive human hematopoietic cells during in vitro culture We routinely monitored the immunophenotype of purified CD34+ and CD34+, CD38 cells after
in vitro culture and transduction. After 96 hours in serum-free culture
with high-dose cytokines, purified CD34+,
CD38 cells retained their phenotype during in vitro
culture (Figure 3B).19
Exposure of CD34+, CD38 cells to medium
conditioned by the RD114/MGirL22Y producer cells during transduction
caused the majority of CD34+, CD38 cells to
become CD38+ (Figure 3C). Although only a small decrease in
total clonogenic progenitors accompanied this phenotypic change
(95% ± 9%, P > .05), we found that CD34+
cells exposed to conditioned medium from the RD114/MGirL22Y producer cells failed to engraft in murine NOD/SCID recipients (n = 4). In the
same experiment, control CD34+ cells cultured identically
but without exposure to vector particles in conditioned medium during
in vitro culture exhibited multilineage engraftment (data not
shown).
The observed phenotypic changes during culture and the loss of
primitive repopulating cells could have been due to a direct effect of
the RD114 envelope protein or to the action of some other component
within the medium conditioned by the RD114/MGirL22Y producer cells. The
latter seemed more likely when we found that medium from an amphotropic
producer cell population derived from FLYA13, also a derivative of the
HT1080 cell line used to generate FLYRD18,48 induced a
similar immunophenotypic change in CD34+,
CD38
Transduction of primitive human hematopoietic cells without immunophenotypic change by RD114-pseudotyped vector particles that had been preloaded onto retronectin-coated plates An alternative experiment to test whether the conditioned media from the HT1080 cells is causing the differentiation would be to absorb (preload) the particles on retronectin-coated plates. Retroviral vector particles can be preloaded onto retronectin-coated plates by a brief incubation of viral conditioned medium.55 The vector particles become concentrated on the retronectin, allowing the conditioned medium to be removed and replaced by medium containing the target cell population. Using conditioned medium from the RD114/MGirL22Y producer cells, we absorbed RD114-pseudotyped particles onto retronectin and then transduced CD34+, CD38 cells. By this approach, we maintained the
immunophenotype of the CD34+, CD38 cells
(Figure 4F) with preservation of transduction efficiency (data
not shown).
Our objective in these studies was to efficiently transduce SRCs. We
were concerned about potential toxicity to these cells because of the
manipulations required to repeatedly expose the CD34+ cells
to our vector particles, preloaded on retronectin-coated plates.
Therefore, we performed experiments with CD34+,
CD38 Transduction of human cells capable of establishing hematopoiesis in immunodeficient mice with RD114-pseudotyped vector particles Purified CD34+ cells that had been prestimulated for 24 or 48 hours in serum-free medium were transduced by a single exposure to RD114 vector particles that had been preloaded onto retronectin-coated plates. After a maximum of 96 hours of culture, expanded cells derived from an input volume of 1.0 to 1.5 × 105 cells were injected into NOD/SCID murine recipients. Control cells were either not exposed to retroviral vector particles or transduced with amphotropic vector particles preloaded alone or additionally exposed to fresh viral supernatant at 48 and 72 hours in vitro to maximize transduction efficiency (Figure 6). Each method of transduction was performed at least twice in a series of 3 experiments. Figure 5 shows the results of analysis of the bone marrow cells of 3 animals that had received CD34+ cells transduced with RD114 vector particles preloaded onto retronectin-coated plates after 24 or 48 hours of prestimulation. In each animal, there was a substantial population of bone marrow cells that reacted with a human CD45-specific monoclonal antibody. EGFP+ cells were found in both the lymphoid (CD19+) and myeloid (CD33+) cell populations (Figure 5).
Overall rates of human engraftment in the NOD/SCID recipients and the
level of EGFP expression are shown in Figure 6. EGFP+ cells
were found in significant numbers only in animals that received
RD114-transduced CD34+ cells. The overall level of EGFP
expression varied among experiments but was consistent within
individual cohorts of animals. Although we observed decreased human
engraftment in NOD/SCID mice that received CD34+ cells
transduced by the RD114-pseudotyped vector after only 24 hours of
prestimulation as compared with untransduced CD34+ cells
(P < .05 compared with controls), this effect could be ameliorated by slightly longer prestimulation (48 hours) before a
single transduction. Extending the period of prestimulation also
resulted in a higher proportion of gene-modified human cells in
transplant recipients, which correlated with our in vitro studies and
those of others,20,30 suggesting that the optimal timing for transduction of NOD/SCID repopulating cells is after 48 hours in
culture.
The efficiency of gene transfer was also evaluated by plating bone
marrow from the experimental animals engrafted with human cells into
methylcellulose in the presence of human cytokines. DNA recovered from
such individual, plucked human, secondary hematopoietic colonies was
analyzed by PCR analysis for the presence of EGFP (Figure
7). Progenitors from every animal that
received CD34+ cells transduced with RD114-pseudotyped
particles and that grew human progenitors were analyzed. We limited the
number analyzed per mouse to prevent skewing of the data (the best
engrafted mice had the best marking). In the 24-hour RD114 cohort, a
mean of 7 progenitors per engrafted animal were analyzed (maximum of
15). For the 48-hour RD114 cohort, a mean of 16 progenitors were
analyzed per animal (maximum of 20). The results for all animals in
each experimental cohort were combined. A high percentage of the
colonies from animals that had received cells transduced with
RD114-pseudotyped vector particles was positive by PCR analysis (Table
1).
We regard the scoring of individual colonies for the proviral genome by PCR as being the most valid measure of gene transfer into SRCs because silencing or expression variegation may affect the percentage of cells scored as EGFP+ or trimetrexate resistant. This is best illustrated by the analysis of human progenitors derived from the bone marrow of NOD/SCID mice that received human CD34+ cells transduced by RD114/MGirL22Y at 48 hours. In 2 separate experiments, 3 mice received CD34+ cells transduced by RD114/MGirL22Y at 48 hours (Figure 6). One cohort of animals was 71% positive by EGFP expression and 66% positive (39 of 59) by progenitor analysis. Analysis of the other cohort of animals showed that only 10% of the engrafted human cells expressed EGFP, but 49% (19 of 39) of the progenitors were positive for the proviral genome. Thus, the marking in the human progenitors (58 of 98 combined) derived from the NOD/SCID mouse bone marrow was equivalent to the gene-transfer efficiency into the progenitors from the transduced CD34+ population used for transplantation (45% to 66%; Table 1). The significant marking of the SRCs achieved by a single exposure to RD114-pseudotyped retroviruses raised the possibility of RCR contamination. We therefore performed PCR analysis on DNA from the producer cell clone and the bone marrow of 3 NOD/SCID recipients that had received human CD34+ cells transduced with RD114-pseudotyped retroviral particles and had the highest levels of EGFP expression. Primers specific for a recombination product that would reflect RCR were used.54 The RCR recombination product was not present in either the producer clone DNA or bone marrow DNA. Sensitivity of this assay, based on the positive control rhesus DNA, containing 36 copies of RCR per cell was 0.1% (data not shown).
Our results demonstrate that murine oncoretroviral vector
particles pseudotyped with the envelope protein of feline endogenous virus (RD114) transduced human hematopoietic cell lines and the CD34+ and CD34+, CD38 The nature and identity of the substance(s) produced by derivatives of
the FLYRD18 cell line that induced phenotypic changes and
depletion of NOD/SCID repopulating cells are unknown. The evidence
indicates that the RD114-pseudotyped particles themselves are not
responsible because the effect was observed with amphotropic particles
produced by a derivative of this HT1080-derived cell line and was not
observed with RD114 particles produced by human 293T cells.
CD34+ cells stained with the cell membrane dye PKH26 on day
0 and analyzed after 96 hours in culture showed that virtually all
cells divided 1, 2, or 3 times in culture. There was no difference in
the mean fluorescence pattern of CD34+ cells exposed to
either preloaded RD114-pseudotyped virus or HT1080-derived conditioned
medium compared with untransduced controls (P. Kelly, unpublished
observations). Thus, the substance in the conditioned medium seems to
alter the immunophenotype without altering proliferation. The CD38
antigen is a bifunctional ectoenzyme that participates in signal
transduction pathways involved in the regulation of cell growth and
differentiation.56 The retinoic acid
receptor- The proportions of genetically modified cells within the human myeloid
and lymphoid lineages achieved by transduction with RD114-pseudotyped
particles have not been achieved with oncoretroviral vector particles
pseudotyped with other envelope proteins. In addition, we achieved
high-frequency transduction of human NOD/SCID repopulating cells with a
single exposure to vector particles at a low MOI, whereas the lower
levels of gene transfer into these cells achieved with
amphotropic-pseudotyped particles25,31 or GALV-pseudotyped
particles25,26,29-33 required multiple exposures to vector
particles at generally higher MOIs. VSV-G-pseudotyped oncoretroviral
vector particles have been reported to transduce NOD/SCID repopulating
cells with a single exposure at an MOI of greater than 100, resulting
in marking of 25% of the progenitors in animals undergoing
transplantation under conditions that were toxic to the repopulating
cells.43 Moreover, we have found that CD34+,
CD38 The frequency of genetically modified human progenitors present in bone marrow 8 to 10 weeks after transplantation was approximately equivalent to the frequency of genetically modified primary progenitors in the transduced population used for transplantation of the immunodeficient mice. These results imply that the frequency of transduction of NOD/SCID repopulating cells was equal to the transduction frequency of progenitors detected in vitro. In this respect, the results obtained in transducing human cord blood cells with RD114-pseudotyped vector particles are comparable to those generally obtained in transducing murine hematopoietic cells with ecotropic vector particles.3,5 These results contrast with the general experience with amphotropic- and GALV-pseudotyped oncoretroviral vector particles, in which the transduction frequency of more mature human CD34+ cells and progenitors exceeds that of repopulating cells.25,26,29-31 It is interesting that RD114- and ecotropic-pseudotyped particles use amino acid transporters as receptors.46,60 We infer that the neutral amino acid transporter that serves as the receptor for RD114-pseudotyped particles is expressed at functionally higher levels on primitive human hematopoietic cells than are the phosphate transporters that serve as receptors for amphotropic- and GALV-pseudotyped vector particles. In a recent study, a significant proportion of engrafted human hematopoietic cells were genetically modified (mean, 18%) after multiple exposures of CD34+ cells to a GALV-pseudotyped vector over 96 hours of culture, but with a decline in SRC frequency believed to be secondary to the presence of serum in the conditioned medium containing vector particles.32 Of greater concern are the findings by Demaison et al33 of preferential marking of myeloid lineage-committed progenitors after 72 hours in culture and marking of lymphoid-committed progenitors after an additional 24 hours of exposure to vector in vitro. These findings suggest significant heterogeneity in the population scored as SRCs after repeated exposure to vector containing conditioned medium over 96 to 120 hours in culture. The ability to achieve a high frequency of marking after a single exposure to RD114-pseudotyped vector particles preloaded on retronectin-coated plates in the NOD/SCID model may be advantageous with respect to genetic modification of true multilineage repopulating cells. In this report, experiments were performed using umbilical cord blood-derived CD34+ cells, which are not the most clinically relevant stem cell source. Indeed, studies are now ongoing using a similar transduction strategy on peripheral blood CD34+ cells. In initial studies, we have achieved greater than 80% transduction efficiency of peripheral blood CD34+ cells (n = 3) on the basis of EGFP expression with only a single exposure to retronectin-coated plates preloaded with RD114-pseudotyped particles after 48 hours of prestimulation (unpublished observations, P. Kelly and K. Pollok). We anticipate that given the more quiescent nature of peripheral blood CD34+ cells, additional exposures to vector may be necessary to achieve therapeutic levels of gene transfer but that this may be determined only by larger animal models, such as the rhesus monkey autologous transplant model. In conclusion, we report that RD114-pseudotyped murine retroviral vectors can efficiently transduce umbilical cord blood SRCs after only 24 to 48 hours of prestimulation before a single exposure to the viral particles. Preloading the viral particles onto retronectin-coated plates with removal of the conditioned medium allowed significant gene transfer to occur. Such an approach may bypass deleterious effects to the true long-term repopulating cells not appreciated when using other pseudotyped retroviruses to mark SRCs. We predict that RD114-pseudotyped retroviral vectors may result in gene-transfer efficiencies at levels that may be curative for hematopoietic genetic diseases.
We acknowledge the expert assistance of Jean Johnson in preparation of the manuscript.
Submitted December 23, 1999; accepted April 14, 2000.
Supported by NHLBI Program Project Grant P01 HL 53749, The ASSISI Foundation of Memphis 94-00, Cancer Center Support CORE Grant P30 CA 21765, and the American Lebanese Syrian Associated Charities (ALSAC).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Patrick F. Kelly, Division of Experimental Hematology, St Jude Children's Research Hospital, 332 N Lauderdale, Room D-4026, Memphis, TN 38105.
1.
Anderson WF.
Prospects for human gene therapy.
Science.
1984;226:401-409 2. Sorrentino BP, Nienhuis AW. The hematopoietic system as a target for gene therapy. In: Friedmann T, ed. The Development of Gene Therapy. New York, NY: Cold Spring Harbor Laboratory Press; 1999:351-426. 3. Allay JA, Persons DA, Galipeau J, et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med. 1998;4:1136-1143[Medline] [Order article via Infotrieve]. 4. Bunting KD, Sangster MY, Ihle JN, et al. Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer. Nat Med. 1998;4:58-64[Medline] [Order article via Infotrieve]. 5. Persons DA, Allay JA, Riberdy JM, et al. Use of the green fluorescent protein gene as a marker to identify and track genetically modified hematopoietic cells. Nat Med. 1998;4:1201-1205[Medline] [Order article via Infotrieve].
6.
Donahue RE, Wersto RP, Allay JA, et al.
High levels of lymphoid expression of enhanced green fluorescent protein in non-human primates transplanted with cytokine mobilized peripheral blood CD34+ cells.
Blood.
2000;95:445-452
7.
Dunbar CE, Cottler-Fox M, O'Shaughnessy JA, et al.
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood.
1995;85:3048-3057
8.
Kiem HP, Andrews RG, Morris J, et al.
Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor.
Blood.
1998;92:1878-1886
9.
Kurre P, Kiem HP, Morris J, et al.
Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor.
J Virol.
1999;73:495-500
10.
Orlic D, Girard LJ, Jordan CT, et al.
The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.
Proc Natl Acad Sci U S A.
1996;93:11097-11102
11.
Miller DG, Adam MA, Miller AD.
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection [published erratum appears in Mol Cell Biol. 1992;12:433].
Mol Cell Biol.
1990;10:4239-4242
12.
Kamel-Reid S, Dick JE.
Engraftment of immune-deficient mice with human hematopoietic stem cells.
Science.
1988;242:1706-1709
13.
Vormoor J, Lapidot T, Pflumio F, et al.
Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.
Blood.
1994;83:2489-2497
14.
Cashman JD, Lapidot T, Wang JC, et al.
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood.
1997;89:4307-4316 15. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med. 1996;2:1329-1337[Medline] [Order article via Infotrieve].
16.
Laneuville P, Chang W, Kamel-Reid S, et al.
High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors.
Blood.
1988;71:811-814
17.
Hughes PF, Eaves CJ, Hogge DE, et al.
High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture.
Blood.
1989;74:1915-1922
18.
Dick JE, Kamel-Reid S, Murdoch B, et al.
Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays.
Blood.
1991;78:624-634
19.
Bhatia M, Bonnet D, Kapp U, et al.
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med.
1997;186:619-624
20.
Glimm H, Eaves CJ.
Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture.
Blood.
1999;94:2161-2168
21.
van Zeijl M, Johann SV, Closs E, et al.
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family.
Proc Natl Acad Sci U S A.
1994;91:1168-1172
22.
Miller DG, Edwards RH, Miller AD.
Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus.
Proc Natl Acad Sci U S A.
1994;91:78-82
23.
Kavanaugh MP, Miller DG, Zhang W, et al.
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.
Proc Natl Acad Sci U S A.
1994;91:7071-7075
24.
Bauer TRJ, Miller AD, Hickstein DD.
Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope.
Blood.
1995;86:2379-2387
25.
van Hennik PB, Verstegen MMA, Bierhuizen MFA, et al.
Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.
Blood.
1998;92:4013-4022 26. Marandin A, Dubart A, Pflumio F, et al. Retrovirus-mediated gene transfer into human CD34+38 low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo. Hum Gene Ther. 1998;9:1497-1511[Medline] [Order article via Infotrieve].
27.
Orlic D, Girar LJ, Anderson SM, et al.
Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors.
Blood.
1999;91:3247-3254
28.
Kiem HP, Heyward S, Winkler A, et al.
Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.
Blood.
1997;90:4638-4645
29.
Schilz AJ, Brouns G, Knob H, et al.
High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions.
Blood.
1998;92:3163-3171 30. Hennemann B, Conneally E, Pawliuk R, et al. Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables. Exp Hematol. 1999;27:817-825[Medline] [Order article via Infotrieve].
31.
Conneally E, Eaves CJ, Humphries RK.
Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential.
Blood.
1998;91:3487-3493
32.
Dorrell C, Gan OI, Pereira DS, et al.
Expansion of human cord blood CD34+CD38 33. Demaison C, Brouns G, Blundell MP, et al. A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector. Hum Gene Ther. 2000;11:91-100[Medline] [Order article via Infotrieve].
34.
Tisdale JF, Hanazono Y, Sellers SE, et al.
Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability.
Blood.
1998;92:1131-1141 35. Sellers SE, Tisdale JF, Bodine DM, et al. No discrepancy between in vivo gene marking efficiency assessed in peripheral blood populations compared with bone marrow progenitors or CD34+ cells. Hum Gene Ther. 1999;10:633-640[Medline] [Order article via Infotrieve].
36.
Whitwam T, Haskins ME, Henthorn PS, et al.
Retroviral marking of canine bone marrow: long-term, high-level expression of human interleukin-2 receptor common gamma chain in canine lymphocytes.
Blood.
1998;92:1565-1575 37. Yang Y, Vanin EF, Whitt MA, et al. Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum Gene Ther. 1995;6:1203-1213[Medline] [Order article via Infotrieve].
38.
Emi N, Friedmann T, Yee JK.
Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.
J Virol.
1991;65:1202-1207 39. Friedmann T, Yee JK. Pseudotyped retroviral vectors for studies of human gene therapy. Nat Med. 1995;1:275-277[Medline] [Order article via Infotrieve]. 40. Akkina RK, Walton RM, Chen ML, et al. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J Virol. 1996;70:2581-2585[Abstract]. 41. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272:263-267[Abstract].
42.
Burns JC, Friedmann T, Driever W, et al.
Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.
Proc Natl Acad Sci U S A.
1993;90:8033-8037
43.
Rebel VI, Tanaka M, Lee JS, et al.
One-day ex vivo culture allows effective gene transfer into human nonobese diabetic/severe combined immune-deficient repopulating cells using high-titer vesicular stomatitis virus G protein pseudotyped retrovirus.
Blood.
1999;93:2217-2224
44.
Miyoshi H, Smith KA, Mosier DE, et al.
Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.
Science.
1999;283:682-686 45. Porter CD, Collins MKL, Tailor CS, et al. Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors. Hum Gene Ther. 1996;7:913-919[Medline] [Order article via Infotrieve].
46.
Rasko JE, Battini JL, Gottschalk RJ, et al.
The RD114/simian type D retrovirus receptor is a neutral amino acid transporter.
Proc Natl Acad Sci U S A.
1999;96:2129-2134 47. Sommerfelt MA, Weiss RA. Receptor interference groups of 20 retroviruses plating on human cells. Virology. 1990;176:58-69[Medline] [Order article via Infotrieve]. 48. Cosset FL, Takeuchi Y, Battini JL, et al. High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol. 1995;69:7430-7436[Abstract]. 49. Cheng L, Du D, Murray D, et al. A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells. Gene Ther. 1997;4:1013-1022[Medline] [Order article via Infotrieve]. 50. Persons DA, Mehaffey MG, Kaleko M, et al. An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol Dis. 1998;24:167-182[Medline] [Order article via Infotrieve].
51.
Miller AD, Garcia JV, von Suhr N, et al.
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J Virol.
1991;65:2220-2224
52.
Ory DS, Neugeboren BA, Mulligan RC.
A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.
Proc Natl Acad Sci U S A.
1996;93:11400-11406 53. McLachlin JR, Mittereder N, Daucher M, et al. Factors affecting retroviral vector function and structural integrity. Virology. 1993;195:1-5[Medline] [Order article via Infotrieve].
54.
Vanin EF, Kaloss M, Broscius C, et al.
Characterization of replication competent retroviruses from nonhuman primates with virus induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.
J Virol.
1994;68:4241-4250 55. Williams DA. Retroviral-fibronectin interaction in transduction of mammalian cells. Ann N Y Acad Sci. 1999;872:109-113[Medline] [Order article via Infotrieve]. 56. Mehta K, Umar S, Malavasi F. Human CD38: a cell surface protein with multiple functions. FASEB J. 1996;10:1408-1417[Abstract].
57.
Drach J, McQueen T, Engel H, et al.
Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha.
Cancer Res.
1994;54:1746-1752
58.
Mehta K, McQueen T, Manshouri T, et al.
Involvement of retinoic acid receptor-
59.
Evans JT, Kelly PF, O'Neill E, et al.
Human cord blood CD34+CD38
60.
Albritton LM, Kim JW, Tseng L, et al.
Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.
J Virol.
1993;67:2091-2096
© 2000 by The American Society of Hematology.
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|
  E. Verhoeyen and F.-L. Cosset Engineering the Surface Glycoproteins of Lentiviral Vectors for Targeted Gene Transfer CSH Protocols, August 1, 2009; 2009(8): pdb.top59 - pdb.top59. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Vera, B. Savoldo, S. Vigouroux, E. Biagi, M. Pule, C. Rossig, J. Wu, H. E. Heslop, C. M. Rooney, M. K. Brenner, et al. T lymphocytes redirected against the {kappa} light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells Blood, December 1, 2006; 108(12): 3890 - 3897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
B. P. Sorrentino Improved human stem-cell transduction: the cat's meow Blood, July 1, 2005; 106(1): 8 - 8. [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 M. C. Walters Stem Cell Therapy for Sickle Cell Disease: Transplantation and Gene Therapy Hematology, January 1, 2005; 2005(1): 66 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 K. N. Thimmaiah, J. Easton, S. Huang, K. A. Veverka, G. S. Germain, F. C. Harwood, and P. J. Houghton Insulin-like Growth Factor I-mediated Protection from Rapamycin-induced Apoptosis Is Independent of Ras-Erk1-Erk2 and Phosphatidylinositol 3'-Kinase-Akt Signaling Pathways Cancer Res., January 15, 2003; 63(2): 364 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Horn, M. S. Topp, J. C. Morris, S. R. Riddell, and H.-P. Kiem Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells Blood, December 1, 2002; 100(12): 3960 - 3967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. E. Nicolini, S. Imren, I.-H. Oh, R. K. Humphries, P. Leboulch, M. E. Fabry, R. L. Nagel, and C. J. Eaves Expression of a human beta -globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells Blood, July 30, 2002; 100(4): 1257 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Sandrin, B. Boson, P. Salmon, W. Gay, D. Negre, R. Le Grand, D. Trono, and F.-L. Cosset Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates Blood, July 18, 2002; 100(3): 823 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Tsai, H. L. Malech, M. R. Kirby, A. P. Hsu, N. E. Seidel, C. D. Porada, E. D. Zanjani, D. M. Bodine, and J. M. Puck Retroviral transduction of IL2RG into CD34+ cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras Blood, June 17, 2002; 100(1): 72 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Walters, A. W. Nienhuis, and E. Vichinsky Novel Therapeutic Approaches in Sickle Cell Disease Hematology, January 1, 2002; 2002(1): 10 - 34. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Gatlin, M. W. Melkus, A. Padgett, P. F. Kelly, and J. V. Garcia Engraftment of NOD/SCID Mice with Human CD34+ Cells Transduced by Concentrated Oncoretroviral Vector Particles Pseudotyped with the Feline Endogenous Retrovirus (RD114) Envelope Protein J. Virol., October 15, 2001; 75(20): 9995 - 9999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
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|
 A. M. Davidoff, C. Y. C. Ng, P. Brown, M. A. Leary, W. W. Spurbeck, J. Zhou, E. Horwitz, E. F. Vanin, and A. W. Nienhuis Bone Marrow-derived Cells Contribute to Tumor Neovasculature and, When Modified to Express an Angiogenesis Inhibitor, Can Restrict Tumor Growth in Mice Clin. Cancer Res., September 1, 2001; 7(9): 2870 - 2879. [Abstract] [Full Text] [PDF]
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T. Dervieux, J. G. Blanco, E. Y. Krynetski, E. F. Vanin, M. F. Roussel, and M. V. Relling Differing Contribution of Thiopurine Methyltransferase to Mercaptopurine versus Thioguanine Effects in Human Leukemic Cells Cancer Res., August 1, 2001; 61(15): 5810 - 5816. [Abstract] [Full Text] [PDF]
|
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith Gene Therapy 2000 Hematology, January 1, 2000; 2000(1): 376 - 393. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-satellite DNA,
5'-AATTTCAGCTGACTAAACA-3' and 5'-TTTAGTTAGGTGCAGTTAT-3', were used to
confirm the presence of human DNA in each sample. The EGFP gene was
amplified using the primers 5'-ACCCCGACCACATGAAGCAGC-3' and
5'-CGTTGGGGTCTTTGCTCAGGG-3', giving a 417-bp product. Primers specific
to the 
