Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2010 Apr 6;18(7):1310–1317. doi: 10.1038/mt.2010.48

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

Yoon-Sang Kim 1, Matthew M Wielgosz 1, Phillip Hargrove 1, Steven Kepes 2, John Gray 1, Derek A Persons 1, Arthur W Nienhuis 1
PMCID: PMC2911247  PMID: 20372106

Abstract

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Introduction

The ability to transfer genes into hematopoietic stem cells and achieve lineage-specific expression would create many therapeutic opportunities for blood disorders.1 Integrating retroviruses have been developed for this purpose because they result in permanent genetic modification of transduced cells and their progeny. Indeed, for immunological diseases such as X-linked and adenosine deaminase severe combined immunodeficiency (SCID), where gene-corrected lymphoid cells have a proliferative advantage over uncorrected cells, γ-retroviral vectors have proved corrective.2,3,4 However, improving the transduction efficiency of the most primitive stem cell pool will be necessary for successful treatment of various hemoglobinopathies in which gene-corrected repopulating cells do not have a proliferation advantage. Lentiviral vectors are particularly attractive in this regard, as they are able to package more genetic information, and are able to transduce nonmitotic cells.5 Indeed, amelioration of β-thalassemia and sickle cell anemia in mouse models using lentiviral vectors has been demonstrated.6,7,8,9

Lentiviral vectors can be pseudotyped with a broad array of viral envelope proteins.10 Particles generated with the membrane protein from vesicular stomatitis virus envelope glycoprotein (VSV-G) are stable, they can be concentrated to high titers and have a very broad tropism.11 Human cord blood CD34+ cells, the more primitive CD34+ CD38− hematopoietic cells from cord blood and bone marrow and mobilized peripheral blood CD34+ cells could be successfully transduced with VSV-G pseudotyped vector particles as determined by a variety of assays including transplantation into immunodeficient mice.12,13,14,15 The VSV-G protein is toxic to cells in which it is expressed but success has been obtained in deriving a stable packaging cell line by expressing this envelope protein from an inducible promoter by ourselves and others.16,17 If VSV-G proves to be the envelope of choice for transducing hematopoietic cells, producer clones derived from our packaging cell line could be used to derive clinical vector preparations.

In previous studies, we found that primitive cells capable of engrafting nonobese/severe combined immunodeficient (NOD/SCID) mice were transduced somewhat more efficiently with lentiviral vector particles pseudotyped with the amphotropic (AMPHO) envelope protein than with VSV-G pseudotyped particles.18 Based on these results, we developed a simian immunodeficiency virus-based lentiviral vector system19 and demonstrated that repopulating stem cells in the rhesus model could be transduced with reasonable efficiency and with sustained polyclonal hematopoietic marking and transgene expression 4 years after the original transplant procedure.20 However, our early experiments in the NOD/SCID mouse model involved an extended period of ex vivo culture. This extended culture period may result in reduction in the number of cells capable of engraftment in immunodeficient mice with preferential loss of those capable of longer term engraftment.21 Moreover, we found very low transduction of repopulating cells when using particles pseudotyped with the envelope protein of feline endogenous virus (RD114) which had worked well when used to pseudotype γ-retroviral vector particles.22 Subsequent work by others demonstrated that a chimeric RD114 envelope glycoprotein with the cytoplasmic domain from the AMPHO envelope demonstrated augmented transduction of primary lymphocytes and CD34+ cells from human and nonhuman primates.23 Work in the Bodine laboratory demonstrated improved transduction of human fetal sheep repopulating cells by γ-retroviral vectors pseudotyped with the RD114 and feline leukemia virus type C (FLVC).24 Based on all of these considerations, we undertook a re-evaluation of the comparison of VSV-G and AMPHO lentiviral pseudotypes using a significantly shortened transduction protocol and also have evaluated vector particles pseudotyped with the RD114 chimeric envelope and an analogous chimera which we assembled for the FLVC envelope protein. Engraftment and transduction efficiency were evaluated in an immunodeficient mouse strain which lacks the interleukin receptor common γ-chain (NOD/LtSz-scid IL2R γ−C).25 This strain is more receptive to human repopulating cells engraftment than the NOD/SCID strain we used previously.18

Results

Preparation of HIV-based vector particles with various envelope proteins

All vector preparations were made with the vector plasmid, pCL20cwMpGFP, and the helper plasmids we previously developed (Figure 1a).18,19 Our initial preparations of HIV-based lentiviral vector particles made with the FLVC envelope coding sequence (plasmid pCAG4 FLVC, Supplementary Figure S1a) were found to have a titer of only 1.3 ± 0.9 × 105 transducing units/ml (n = 3) when tested on HeLa cells. In an effort to improve the titer with the FLVC envelope protein, we constructed a chimeric envelope protein having a substantial portion of the transmembrane domain and all of the cytoplasmic domain of the AMPHO protein (Figure 1a) analogous to the RD114 TR protein assembled previously by others.23 This FLVC TR chimeric envelope protein (pCAG4 FLVC TR, Supplementary Figure S1e) yielded vector preparations with a sixfold higher transducing titer (8.2 ± 0.9 × 105, n = 3) (Figure 1b) which is statistically significant (P = 0.0014).

Figure 1.

Figure 1

Open in a new tab

Construction and testing of chimeric FLVC envelope protein having the transmembrane and cytoplasmic domains of the AMPHO envelope protein. (a) Amino acid sequence of the end terminal portion of the AMPHO envelope protein and the sequence differences between it and the chimeric RD114 TR and FLVC TR envelope proteins and the native FLVC envelope protein. Shown also in this panel is the diagram of the integrated proviral form of the HIV transfer vector used to estimate transduction with the various envelope pseudotypes. (b) Titers in transducing units/ml (TU/ml) of FLVC and FLVC TR pseudotypes on HeLa cells. The results were confirmed in three separate experiments and the difference in titers is statistically significant (P = 0.0014). AMPHO, amphotropic; FLVC, feline leukemia virus type C; GFP, green fluorescent protein; Mp, truncated version of the MSCV promoter15; WPRE, woodchuck post-transcriptional regulatory element.

Vector particles pseudotyped with the AMPHO, VSV-G, RD114 TR and the FLVC TR proteins were generated by standard methodology.18,19 As shown in Figure 2a, each vector preparation had nearly equivalent relative concentrations of genome RNA, as estimated by quantitative real-time-PCR (qRT-PCR)of purified viral particle RNA. Despite this similarity, the various vector preparations exhibited a wide range of transducing titers on HeLa cells. VSV-G, AMPHO, RD114 TR and FLVC TR pseudotyped vectors had titers of 3.2 × 107, 7.8 × 106, 1.8 × 105, and 3.6 × 105 TU/ml, respectively (Figure 2b). The relative transducing titers of the various pseudotypes did not appear to correlate with receptor mRNA concentration in HeLa cells in that mRNA concentrations for the FLVC and RD114 receptors were ~6- or >30-fold higher, respectively, than the estimated concentration of the AMPHO receptor mRNA (Table 1).

Figure 2.

Figure 2

Open in a new tab

Genome concentrations and transducing titers of various pesudotypes on HeLa cells. (a) Comparison of the relative vector genome concentration, as determined by qRT-PCR of medium containing vector particles pseudotyped with various envelope proteins as indicated below. For this relative comparison, the VSV-G value was set at 1. The number of times each preparation was tested is indicated above each bar. There were no statistically significant differences in the titers. (b) Comparison of the transducing titers. The values are plotted on a log scale to facilitate direct comparison to the relative differences in transducing titers. Each preparation was tested three times in independent experiments. AMPHO, amphotropic; FLVC, feline leukemia virus type C; qRT-PCR, quantitative real-time-PCR; VSV-G, vesicular stomatitis virus envelope glycoprotein.

Table 1. Receptor mRNA levels in primitive human hematopoietic cells.

graphic file with name mt201048t1.jpg

Open in a new tab

Transduction of primitive human hematopoietic cells with vector particles pseudotyped with the various envelope proteins

CD34+ cells were isolated from peripheral blood mononuclear cells harvested from normal donors after cytokine mobilization.25 Cells from three donors were studied in three separate experiments. Following overnight prestimulation in cytokine containing media, CD34+ cells were transferred to retronectin-coated plates which had been preloaded with vector particles for 4 hours at 37 °C. A fixed volume of vector was used for the preloading and during culture, despite differences in transducing titers on HeLa cells because relative vector particle concentrations as determined by qRT-PCR of viral RNA were similar for the different vector preparations (Figure 2a). Following prestimulation, the CD34+ cells were transferred to plates containing preloaded retronectin in medium comprised of 50% by volume of conditioned medium containing vector particles. Cytokines were also present in the medium and the cells were cultured for 24 hours at 37 °C during which time there was an ~1.5–2-fold increase in cell numbers. At the end of this 24-hours culture, aliquots of cells were taken for continued culture in liquid medium and for culture in semisolid medium to assay clonogenic progenitors with the remainder of the cells injected into immunodeficient mice (see text below). After 5 days in liquid culture, the percentage of green fluorescent protein (GFP)+ cells were 62 ± 4, 74 ± 1, 26 ± 4, and 31 ± 6% (n = 3), for the VSV-G, AMPHO, RD114 TR and FLVC TR pseudotypes, respectively (Figure 3a). The proportion of transduced progenitor-derived colonies in methylcellulose (semisolid) medium which were GFP+, scored 12 days after culture initiation, were 61 ± 6, 83 ± 2, 32 ± 3, and 37 ± 7% with the four envelope pseudotypes, respectively (Figure 3b). The number of cells recovered after liquid culture and the plating efficiency in the progenitor culture did not vary significantly among the individual, transduced cell populations. Of interest is that we observed that a tenfold reduction in the concentration of AMPHO vector particles did not significantly reduce transduction efficiency in that 72 ± 5% of the cells in liquid culture were GFP+ and 78 ± 4% of the progenitor-derived colonies were positive (Figure 3a,b).

Figure 3.

Figure 3

Open in a new tab

Transduction of CD34+ cells by various pseudotypes. Transduction of populations of CD34+ cells purified from (a) cytokine mobilized peripheral blood mononuclear cells and (b) the relative transduction of clonogenic progenitors within these populations. Vector particles prepared with the various envelope proteins were used to transduce CD34+ cells as described in the Materials and Methods. The center bars labeled “1/10 AMPHO” are data from cells transduced with a tenfold lower concentration of vector particles pseudotyped with the amphotrophic envelope protein. The experiment was done independently three times with mobilized CD34+ cells from three donors. Shown are the means and SD. AMPHO, amphotropic; FLVC, feline leukemia virus type C; GFP, green fluorescent protein; VSV-G, vesicular stomatitis virus envelope glycoprotein.

The cells remaining after 24-hours exposure to vector particles were injected into NOD/LtSz-scid IL2R γ−C mice which had received a sublethal dose of busulfan (35 mg/kg) 24 hours earlier. The immunodeficient mouse recipients were sacrificed 12 weeks after transplantation and the proportion of human CD45+ cells in bone marrow was determined for mock-transduced cells that had not been exposed to vector particles and for repopulating cells exposed to vector particles prepared with the various envelope proteins. Between 15 and 20 mice were transplanted in each group and individual mice received CD34+ cells transduced from one of three donors. Although there was considerable variation from animal to animal, the average human engraftment was ~40% for all cell populations ranging from 38 to 43% (Figure 4a). Some of the variation among individual animals reflected a consistently higher level of engraftment of female versus male animals with the comparison being statistically significant for four of the five groups (Figure 4b). There was no significant difference in engraftment of mock-transduced cells compared to those transduced with the various vector pseudotypes indicating a lack of toxicity to engrafting cells.

Figure 4.

Figure 4

Open in a new tab

Engraftment and relative transduction of engrafting cells in the immunodeficient mouse model. (a) Relative engraftment of untransduced cells (mock) or cells transduced with the various envelope pseudotypes. Each point represents the results from an individual mouse. (b) Comparison of engraftment in female versus male mice. Overall, average engraftment was higher in female mice and in four of five groups the difference reached statistical significance. In contrast, the differences in engraftment in males and females among the various vector pseudotypes, compared to mock, were not statistically significant. (c) Proportion of engrafted human CD45+ cells that were GFP+. (d) Comparison of the proportion of CD45+, GFP+ cells in male versus female recipients; no significant differences were observed. AMPHO, amphotropic; FLVC, feline leukemia virus type C; GFP, green fluorescent protein; VSV-G, vesicular stomatitis virus envelope glycoprotein.

The important comparison in our experiments is the proportion of human CD45+ cells that were GFP+ in mice transplanted with CD34+ cells transduced with the various envelope pseudotype particles. As shown in Figure 4c, the VSV-G group exhibited the highest average proportion of GFP+ cells (48 ± 14%), followed by AMPHO (20 ± 7%), RD114 TR (8 ± 4%), and FLVC TR (3 ± 3%). Reducing the concentration of AMPHO particles by tenfold resulted in a modest reduction in GFP+ cells (15 ± 8%) which was not statistically significant (P = 0.0655) (Figure 4c). When the GFP-marking of human CD45+ cells with each vector pseudotype was analyzed as a function of mouse gender, we found that there was no statistically significant difference between the percentages of GFP+, CD45+ cells which engrafted in female versus male mice (Figure 4d). As shown in Figure 5a, the levels of engrafted genetically modified lymphoid (CD19+) or myeloid (CD33/15+) cells derived from CD34+ cells transduced with the various vector pseudotypes compared to the mock group were similar with the percentages of lymphoid and myeloid engraftment ranging between 80–86 and 12–17%, respectively (Figure 5c). The proportion of GFP+, CD45+ cells in the two lineages derived from cells transduced with the various envelope pseudotypes were also similar. The vector copy number per transduced cell was estimated by quantitative PCR in DNA recovered from GFP+ myeloid and lymphoid cells purified by flow cytometric cell sorting. The copy number in myeloid and lymphoid cells derived from CD34+ cells transduced with VSV-G pseudotyped particles were somewhat higher (2.8 ± 0.5 genome copies/transduced cell and 2.2 ± 0.5 genome copies/transduced cell n = 6, respectively) than in lymphoid or myeloid cells derived from CD34+ cells transduced with the AMPHO pseudotype (1.5 ± 0.3 and 1.0 ± 0.3, respectively, n = 6), or genetically modified cells derived from CD34+ cells transduced with the RD114 TR pseudotype (1.6 ± 0.3 and 1.6 ± 0.9, respectively, n = 5).

Figure 5.

Figure 5

Open in a new tab

Engraftment and relative transduction of cells contributing to the lymphoid or myeloid lineages. (a) Relative engraftment of lymphoid (filled bars: CD19+) and myeloid (open bars: CD33/15+) cells derived from repopulating cells transduced with the various envelope pseudotypes. (b) Proportion of CD45+ cells of the two lineages that were GFP+. (c) Vector copy number in sorted myeloid (CD15/33+) and lymphoid (CD19+) cells. AMPHO, amphotropic; FLVC, feline leukemia virus type C; GFP, green fluorescent protein; VSV-G, vesicular stomatitis virus envelope glycoprotein.

Receptor mRNA concentration in primitive human hematopoietic cells

RNA was prepared from CD34+ cells and CD38+, CD38− cells purified from cytokine mobilized peripheral blood mononuclear cells. After sorting, >98% of the population were CD34+ and either CD38− or low expressers of CD38 indicating that significant enrichment in the proportion of cells having the CD34+, CD38− phenotype had been achieved. RNA samples were prepared immediately upon thawing frozen aliquots of cells or after 24 hours in culture. Receptor mRNA levels were estimated by comparison to a standard curve generated with serial tenfold dilution of in vitro transcribed RNA. Because the in vitro transcribed RNA has a defined molecular weight and molar absorbtivity and integrity was established by gel electrophoresis (data not shown), the comparative cycle threshold values could be converted to allow estimation of the number of receptor mRNA molecules/nanogram of total cellular RNA. The receptor mRNA levels for RD114 (RD114R), FLVC (FLVCR), and the AMPHO envelope protein (AMPHOR) were all relatively low in both CD34+ and CD34+, CD38− cells with no significant difference in mRNA concentrations in the two purified cell populations (Table 1). The concentration of RD114R and FLVCR mRNAs increased during in vitro culture (Table 1) whereas that of the AMPHOR mRNA did not change significantly. Similar results were obtained with CD34+ cells isolated from bone marrow (Table 1).

Discussion

The overall goal of our work and that of our colleagues is to develop gene therapy for blood disorders including X-SCID,16 Wiskott–Aldrich syndrome26,27 and the severe hemoglobin disorders, sickle cell anemia, and thalassemia.8,9 The HIV-based lentiviral vector system developed in our laboratories18,19 consistently gives relatively high titer preparations with the potentially therapeutic vectors that we have developed. In planning for future clinical trials, the identification of an envelope protein for pseudotyping vector particles that gave the most efficient transduction of primitive human hematopoietic cells was critical. Although significant evidence suggested that the VSV-G pseudotype was a reasonable choice,11,12,13,14,15,28,29 other envelope proteins including AMPHO,18,19,20 RD114,10,22,23,24 and FLVC24 remained viable candidates for achieving a higher transduction efficiency. In results now reported in this study, the critical data regarding choice of envelope pseudotype was obtained in a highly immunodeficient mouse strain which permits the efficient engraftment of transduced human cells.25 Of interest is the fact that engraftment in female mice was significantly more efficient than engraftment in male mice leading to the observation that gender should be considered when evaluating engraftment of human hematopoietic cells in immunodeficient mice. Our work clearly established the VSV-G pseudotype as the envelope protein of choice among those which were compared.

Our experimental design was unconventional in that we relied on physical particle concentration in deciding to use a fixed volume of each vector preparation for transduction. The alternative approach of relying on transducing titers on an indicator cell line such as HeLa cells may be confounded by the fact that the receptor mRNA concentrations for the individual pseudotypes may vary from cell to cell and that other cell-specific factors may determine transduction efficiency. Our experimental approach is supported by the fact that the transducing titer of VSV-G pseudotyped particles is ~100-fold greater than the transducing titer of either RD114 or FLVC pseudotyped particles on HeLa cells whereas the difference in the ability of the VSV-G versus the two feline envelope pseudotypes to transduce CD34+ cells and progenitors within the CD34+ population was only about two- to threefold. There was no evidence that any of the envelope pseudotype preparations were toxic to primitive hematopoietic cells in that expansion of cells during the liquid culture, plating efficiency with respect to progenitor numbers and engraftment of human cells in the immunodeficient mouse model were roughly equivalent with cells transduced with each of the pseudotypes and also equivalent to engraftment of control, mock-transduced cells. Thus, our experimental design provides a valid comparison of the ability of individual pseudotypes to transduce primitive hematopoietic cells regardless of their relative abilities to transduce other cell types such as HeLa cells. Our results contrast with those of Relander et al.30 in that they found a higher transduction efficiency with particles pseudotyped with a modified RD114 envelope protein than with AMPHO vector particles. Differences between their work and ours are that they transduced cord blood rather than mobilized peripheral blood CD34+ cells, they utilized a RD114 envelope modified to include the HIV protease site rather than a chimeric envelope protein and their vector particles were derived from stable producer clones rather than by transient transfection. Di Nunzio et al.31 also found that the RD114 TR pseudotype transduced cord blood CD34+ cells with equivalent efficiency to that of VSV-G pseudotyped particles but they used 48 hours of prestimulation and 24 hours of vector exposure. Engraftment of human cord blood CD34+ cells in immunodeficient mice was relatively low as reflected by the percentage of human CD45+ cells (average 8%) suggesting potential loss of repopulating cells during culture.31

Historically, attempts to transduce primitive hematopoietic cells with γ-retroviral vectors have relied on extended periods in culture for up to 4 days in the presence of multiple cytokines with a period of prestimulation and/or multiple rounds of exposure to vector particles in 24-hour intervals.32,33 We now appreciate that this approach may result in depletion of certain populations of primitive hematopoietic cells21 and also carries with it the risk of loss of engraftment potential, presumably due to changes in the expression of cell surface markers involved in cell homing.34 As a consequence, as the transition to lentiviral vectors has occurred, efforts have been made to shorten and simplify the period of ex vivo manipulation. Required for transduction of truly quiescent cells with lentiviral vectors is activation from G0 into the G1 phase without advancement into the later phases nor the actual mitosis of the target cell.35,36 The preintegration complex of lentiviral vectors can be recovered for up to 7 days from activated cells without mitosis36 and it can be expressed from a nonintegrated genome in CD34+ cells for up to 8 days.37 As a consequence, the brief periods of ex vivo prestimulation (18 hours) and of transduction (24 hours) we employed may be sufficient to allow vector uptake and the establishment of a stable preintegration complex with the potential for integration of the proviral genome after the cell is returned in vivo and engraftment initiated. Our results indicate that a relatively high level of transduction of cells capable of engraftment in immunodeficient mice can be achieved with these brief periods of prestimulation and transduction with VSV-G pseudotyped particles. Toxicity of VSV-G pseudotyped vector particles has been observed at high multiplicities of infection31 but was not observed in our experiments. While acknowledging that human cells engrafting in immunodeficient mice may be those capable of short-term hematopoietic reconstitution in the autologous recipient as has been shown in the baboon model,38 we infer that the relative transduction efficiencies of these cells with the various vector pseudotypes will be predictive of long-term reconstitution with genetically modified cells in vivo. Performing secondary transplants in mice is another strategy for evaluating transduction of cells capable of long-term reconstitution.

Early studies suggested that there might be a correlation between receptor mRNA levels and the ability to transduce various target cell populations.39 This inference was initially based on the fact that the mRNA levels for the AMPHO receptor, a phosphate channel protein, were relatively low in mouse bone marrow compared to the level of the ecotropic receptor mRNA and the levels appeared to correlate with the relative transducability with vector particles of each pseudotype.39 The human ortholog for the murine ecotropic virus receptor, which does not bind virus, is expressed at a significantly higher level in primitive human hematopoietic cells than is the receptor mRNA for AMPHO virus.39 Enforced overexpression of specific receptor mRNAs has been shown to increase transduction with vector particles having the cognate envelope protein.40,41,42,43 However, others have found, as we did, that receptor mRNA levels do not necessarily always correlate with infectability by specific retroviral pseudotypes.44 Such a lack of correlation is perhaps not at all surprising given the fact that mRNA levels may not necessarily predict the level of expression of the receptor proteins on the cell surface. In addition, recent studies have identified >250 host-dependency factors which influence HIV infectivity; some factors enhance transducability whereas others, e.g., APOBEC3 and the TRIM5α protein diminish transduction.45,46 Consistent with a broad tropism of the VSV-G pseudotyped particles, its receptor is thought to be a ubiquitous membrane lipid47,48 although recent studies suggested that it is not phosphatidylserine as was thought previously.49

Overall, our results provide a sound basis for proceeding to clinical trials with VSV-G pseudotyped vector particles. Not only did VSV-G particles provide the highest efficiency of transduction of cells which engraft in the immunodeficient mouse model, there was no evidence of toxicity despite very high multiplicities of infection. The results of the first such trial for an inherited disorder, adrenoleukodystrophy, have recently been reported.50 Polyclonal reconstitution with 9–13% genetically modified cells in multiple lineages was documented in two patients over 24–30 months with many integrations present in both myeloid and lymphoid cells reflecting stem cell transduction.50 Our producer clones derived from the packaging line which relies on the VSV-G envelope protein16 have been adapted for large-scale production in a WAVE bioreactor and current efforts are focused on developing methods for purification and concentration of vector particles that will be employed in the preparation of clinical lots in the future.

Materials and Methods

Cells. Adherent human embryonic kidney 293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium (CellGro, Herndon, VA), supplemented with 2 mmol/l glutamine (GIBCO; Invitrogen, Carlsbad, CA; cat. no. 25030), 10% heat-inactivated fetal calf serum (BioWhittaker, Walkersville, MD), 50 IU/ml penicillin G/50 µg/ml streptomycin (designated hereafter as 1× P/S) (GIBCO; Invitrogen; cat. no. 15070), henceforth designated D10 medium.

The protocol on which the CD34+ cells were obtained was approved by the St Jude Children's Research Hospital (Memphis, TN) institutional review board and the studies were conducted in accordance with the Declaration of Helsinki. Volunteers gave written informed consent and were compensated for their time and expenses. Human CD34+ cells derived from peripheral blood were mobilized with granulocyte colony stimulating factor 4 days before apheresis, and CD34+ cells were purified (≥94% purity) in the Human Applications Laboratory at St Jude Children's Research Hospital using the CliniMACS device from Miltenyi Biotech (Auburn, CA) according to the manufacturer's protocol. Human donor CD34+ cells were aliquoted and frozen, and later used in preliminary transduction experiments, or for the quantification of receptor mRNAs. For the transplantation experiments, CD34+ cells processed from the Human Applications Laboratory were immediately placed on ice and used without freezing. Human donor CD34+ cells derived from bone marrow were purchased from Lonza (Walkersville, MD; cat. no. 2M-101D). Frozen vials were stored in liquid nitrogen until needed. Upon thawing, BM CD34+ cells were cultured in CD34+ culture medium.

Plasmids and vector production. pCAG4 RTR2, pCAG-kGP1.1R, pCL20cwMpGFP, pCAG VSV-G, pCAG4 AMPHO Env have been previously described. pHCMVRD114 which encodes RD114 TR23 was a generous gift of Francois Loic–Cosset. The details of the construction of plasmid encoding FLVC TR are provided in the Supplementary Data and Supplementary Figure S2. as are the details of the construction of the plasmids containing receptor mRNA coding sequences.

Viral preparations were produced using the transient, HIV lentiviral system developed at St Jude Children's Research Hospital,18 collected in D10, and stored in aliquots of 5 ml at −80 ºC. When vector preparations were thawed and used for the transduction of CD34+ cells, the relative number of RNA genomes per ml of viral supernatant was quantified using a Lenti-X qRT-PCR Titration Kit (Clontech/Takara Bio USA, Madison, WI; cat. no. 632165) according to a protocol provided by the vendor. Relative vector RNA concentrations were determined in comparison to values obtained for the VSV-G preparation. Vector preparations were also titered on HeLa cells using standard methodology.18

Transduction and assay of CD34+ cells. Ten centimeter plates, coated with retronectin (Takara Bio USA; cat. no. T100B), were preloaded with 7 ml of conditioned medium for 4 hours at 37 °C. In each experiment involving transplantation into immunodeficient mice, three identical plates were processed for each vector pseudotype. The preliminary experiments to access transduction efficiency of CD34+ cells and progenitors were done under identical conditions with scaled back volumes and numbers. After aspiration of the medium containing vector particles, the plates were washed with phosphate-buffered saline and 5 × 106 CD34+ cells in 5 ml serum-free StemSpan (StemCell Technologies, Vancouver, British Columbia, Canada), supplemented with 200 ng/ml each of recombinant human Flt3 (CellGenix, Freiburg, Germany; cat. no. 1405), SCF (CellGenix; cat. no. 1401), and TPO (CellGenix; cat. no. 1407) 2× P/S, and 0.8 µg/ml protamine sulfate were added to each plate. Five ml of conditioned medium was then added to each plate resulting in a final cell density of 5 × 105 cells/ml. The cells were incubated overnight at 37 °C and then used in one of three assays. 2 × 105 CD34+ cells were seeded into wells of an untreated 6-well plate and cultured for 4 days at 37 °C in CD34+ culture medium after which the proportion of GFP+ cells was determined by flow cytometric cell sorting. Two hundred to five hundred CD34+ cells were mixed with 1 ml of methocult (StemCell Technologies; cat. no. H4434 GF Methocult), and plated onto 35-mm plates, incubated at 37 °C for 11–12 days and the proportion of GFP+ colonies was determined by fluorescent microscopy.

The remainder of the CD34+ cells from the three plates (2.0–3.0 × 107 cells) were collected in 2.4 ml of phosphate-buffered saline and 300 µl was injected into the tail veins of eight NOD/LtSz-ILR γ−C mice that were 8–12-weeks old. The mice were injected intraperitoneally with 35 mg/kg busulfan (Busulfex; PDL BioPharma, Redwood City, CA) 24 hours before transplantation. Housing and care of the animals were in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Twelve to thirteen weeks post-transplantation, mice were sacrificed and bone marrow cells were collected from tibias and femurs into phosphate-buffered saline supplemented with 2% heat-inactivated fetal calf serum and antibiotics as indicated earlier. Samples from individual mice were prepared as single-cell suspensions and passed through a nylon cell strainer (BD Biosciences, San Jose, CA)) to remove debris and then stained with the following monoclonal antibodies per the manufacturer's instructions: allophycocyanin-conjugated antihuman CD45 (Clone HI30 BD cat. no. 555485), phycoerythrin-Cy7-conjugated antihuman CD19 (Clone SJ25C1 BD cat. no. 557835), phycoerythrin-conjugated antihuman CD15 (Clone HI98 BD cat. no. 555402), and phycoerythrin-conjugated antihuman CD33 (Clone WM-54 DAKO Code No. R0745). The efficiency of gene transduction was defined by the percentage of human cells expressing GFP. The GFP-expressing hCD45+ population was further analyzed for either myeloid (CD33/15) or B lymphoid (CD19) subpopulations. Dead cells stained with 4′-6-diamidino-2-phenylindole were excluded from the analysis. This analysis (four-color flow cytometric analysis) was conducted using a FACS Aria (BD Biosciences). Human CD45+ cells stained for CD19+ and CD15/33+ were sorted into lymphoid or myeloid populations, respectively (n = 1–6 mice per vector pseudotype). Total DNA was isolated from each sorted cell population, and the average vector copy number was determined by qRT-PCR, using a probe set specific for the lentivirus ψ-region, and normalized to values obtained using a probe set specific for human N-RAS. Details are provided in the Supplementary Data.

Receptor mRNA quantitation. The pTST3 FLVCR, pTST3 AMPHOR, and pTOPO TA RD114R plasmids were digested with DraI, and the DNA bands containing the receptor complementary DNA amplicons were resolved on, and purified from, 1% agarose gels. Up to 1 mg of each DNA template was used for the generation of in vitro transcribed RNA, using the T3 or T7 MEGAscript kits according to the manufacturer's protocol (Applied Biosystems/Ambion, Austin, TX; cat. no. AM1338 and cat. no. AM1333, respectively). In vitro transcribed RNA was purified using the MEGAclear kit (Applied Biosystems/Ambion; cat. no. AM1908), quantified by UV-spectrophotometry, and the sizes and integrities of each in vitro transcribed receptor RNA population was analyzed on a 1.2% agarose gel, in 10 mmol/l MOPS buffer (Millipore/Chemicon, Temecula, CA; cat. no. S4601), using 2× RNA loading buffer (Bioline, Randolph, MA; cat. no. BIO-38025). The sizes of each receptor RNA transcripts was compared against RNA Markers (Promega, Madison, WI; cat. no. G3191). Aliquots of each in vitro transcribed RNA were stored at −80 °C until needed, at which time, they were serially diluted tenfold, and used to generate standard copy number curves, ranging from 1,000,000 to 1,000 molecules, or 100,000 to 100 molecules.

When total cell numbers were ≥1 × 106, as was the case for HeLa cells and mobilized peripheral blood CD34+ cells from one donor, total cellular RNA was isolated using RNABee (Tel-Test, Friendswood, TX; cat. no. 100 ml CS-104B), according to the manufacturer's protocol. When the total cell number from a given cell source ranged from 15,000 to 500,000 cells (e.g., sorted mobilized peripheral blood CD34+CD38− cells), total cellular RNA was isolated using the RNAqueous Micro kit, according to the manufacturer's protocol (Applied Biosystems; cat. no. AM1931). CD34+CD38-cells were sorted by flow cytometry, following antibody staining using antihuman CD34+ FITC antibody (Miltenyi Biotec; cat. no. 130-081-001), and antihuman CD38+ allophycocyanin antibody (Miltenyi Biotec; cat. no. 130-092-261).

Sixty to three hundred nanogram of total cellular RNA was used as template in qRT-PCR, using probe assays specific for receptor exon junctions contained in the pT3TS FLVCR, pT3TS AMPHOR, and pTOPO TA RD114R plasmids, in a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA; serial no. 272000161). The cycle threshold value corresponding to a given amount of total cellular RNA for a particular cell source was directly compared to cycle threshold values obtained from known copy numbers of in vitro transcribed RNA used to generate the receptor standard curves. Thus, the number of receptor mRNA copies number from a given cell source were calculated using a direct linear equation of the form y = mx + b.

Statistical analysis. We assigned significance using the F-test function (Microsoft Excel 2007) to compare sets of data so as to ascertain the appropriate “type” to use in a two-tailed, Student's t-test function (Microsoft Excel 2007). Significance, using the Student's t-test, was assigned at P < 0.05.

A recent report confirms that engraftment of human stem cells is more efficient in female than male mice.51

SUPPLEMENTARY MATERIAL Figure S1. Assembly of the plasmids encoding the FLVC envelope and chimeric FLVC envelope protein. Figure S2. Constructs used to derive in vitro transcribed RNA for use as a standard in quantitating the various receptor mRNAs by real-time qRT-PCR as described in the methods. Supplementary Data.

Acknowledgments

We thank Pat Streich (St Jude) for her assistance in the preparation of the manuscript. None of the authors have financial interests that could be considered to pose a conflict of interest related to the submitted manuscript. This work was supported by a Program Project Grant P01 HL53749 from the NHLBI, by the Cancer Center Support Grant 5P30-021765-31 from the NCI, the Assisi Foundation of Memphis, and the American Syrian Lebanese Associated Charities.

Supplementary Material

Figure S1.

Assembly of the plasmids encoding the FLVC envelope and chimeric FLVC envelope protein.

Click here for additional data file. (22.8MB, tiff)
Figure S2.

Constructs used to derive in vitro transcribed RNA for use as a standard in quantitating the various receptor mRNAs by real-time qRT-PCR as described in the methods.

Click here for additional data file. (4.2MB, tiff)
Supplementary Data.
Click here for additional data file. (43.5KB, doc)

REFERENCES

  1. Nienhuis AW. Development of gene therapy for blood disorders. Blood. 2008;111:4431–4444. doi: 10.1182/blood-2007-11-078121. [DOI] [PubMed] [Google Scholar]
  2. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000;288:669–672. doi: 10.1126/science.288.5466.669. [DOI] [PubMed] [Google Scholar]
  3. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science. 2002;296:2410–2413. doi: 10.1126/science.1070104. [DOI] [PubMed] [Google Scholar]
  4. Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, Sinclair J, et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet. 2004;364:2181–2187. doi: 10.1016/S0140-6736(04)17590-9. [DOI] [PubMed] [Google Scholar]
  5. Sadelain M, Boulad F, Lisowki L, Moi P., and, Riviere I. Stem cell engineering for the treatment of severe hemoglobinopathies. Curr Mol Med. 2008;8:690–697. doi: 10.2174/156652408786241357. [DOI] [PubMed] [Google Scholar]
  6. May C, Rivella S, Callegari J, Heller G, Gaensler KM, Luzzatto L, et al. Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin. Nature. 2000;406:82–86. doi: 10.1038/35017565. [DOI] [PubMed] [Google Scholar]
  7. Levasseur DN, Ryan TM, Pawlik KM., and, Townes TM. Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells. Blood. 2003;102:4312–4319. doi: 10.1182/blood-2003-04-1251. [DOI] [PubMed] [Google Scholar]
  8. Zhao H, Pestina TI, Nasimuzzaman M, Mehta P, Hargrove PW., and, Persons DA. Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene. Blood. 2009;113:5747–5756. doi: 10.1182/blood-2008-10-186684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Pestina TI, Hargrove PW, Jay D, Gray JT, Boyd KM., and, Persons DA. Correction of murine sickle cell disease using γ-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin. Mol Ther. 2009;17:245–252. doi: 10.1038/mt.2008.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frecha C, Szécsi J, Cosset FL., and, Verhoeyen E. Strategies for targeting lentiviral vectors. Curr Gene Ther. 2008;8:449–460. doi: 10.2174/156652308786848003. [DOI] [PubMed] [Google Scholar]
  11. Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272:263–267. doi: 10.1126/science.272.5259.263. [DOI] [PubMed] [Google Scholar]
  12. Guenechea G, Gan OI, Inamitsu T, Dorrell C, Pereira DS, Kelly M, et al. Transduction of human CD34+ CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol Ther. 2000;1:566–573. doi: 10.1006/mthe.2000.0077. [DOI] [PubMed] [Google Scholar]
  13. Liu Y, Hangoc G, Campbell TB, Goodman M, Tao W, Pollok K, et al. Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells. Exp Hematol. 2008;36:947–956. doi: 10.1016/j.exphem.2008.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tesio M, Gammaitoni L, Gunetti M, Leuci V, Pignochino Y, Jordaney N, et al. Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors. Stem Cells. 2008;26:1620–1627. doi: 10.1634/stemcells.2008-0161. [DOI] [PubMed] [Google Scholar]
  15. Benhamida S, Pflumio F, Dubart-Kupperschmitt A, Zhao-Emonet JC, Cavazzana-Calvo M, Rocchiccioli F, et al. Transduced CD34+ cells from adrenoleukodystrophy patients with HIV-derived vector mediate long-term engraftment of NOD/SCID mice. Mol Ther. 2003;7:317–324. doi: 10.1016/s1525-0016(03)00002-9. [DOI] [PubMed] [Google Scholar]
  16. Throm RE, Ouma AA, Zhou S, Chandrasekaran A, Lockey T, Greene M, et al. Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection. Blood. 2009;113:5104–5110. doi: 10.1182/blood-2008-11-191049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Broussau S, Jabbour N, Lachapelle G, Durocher Y, Tom R, Transfiguracion J, et al. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Mol Ther. 2008;16:500–507. doi: 10.1038/sj.mt.6300383. [DOI] [PubMed] [Google Scholar]
  18. Hanawa H, Kelly PF, Nathwani AC, Persons DA, Vandergriff JA, Hargrove P, et al. Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther. 2002;5:242–251. doi: 10.1006/mthe.2002.0549. [DOI] [PubMed] [Google Scholar]
  19. Hanawa H, Hematti P, Keyvanfar K, Metzger ME, Krouse A, Donahue RE, et al. Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system. Blood. 2004;103:4062–4069. doi: 10.1182/blood-2004-01-0045. [DOI] [PubMed] [Google Scholar]
  20. Kim YJ, Kim YS, Larochelle A, Renaud G, Wolfsberg TG, Adler R, et al. Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector-transduced CD34+ cells. Blood. 2009;113:5434–5443. doi: 10.1182/blood-2008-10-185199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mazurier F, Gan OI, McKenzie JL, Doedens M., and, Dick JE. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood. 2004;103:545–552. doi: 10.1182/blood-2003-05-1558. [DOI] [PubMed] [Google Scholar]
  22. Kelly PF, Vandergriff J, Nathwani A, Nienhuis AW., and, Vanin EF. Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. Blood. 2000;96:1206–1214. [PubMed] [Google Scholar]
  23. Sandrin V, Boson B, Salmon P, Gay W, Nègre D, Le Grand R, et al. 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. 2002;100:823–832. doi: 10.1182/blood-2001-11-0042. [DOI] [PubMed] [Google Scholar]
  24. Lucas ML, Seidel NE, Porada CD, Quigley JG, Anderson SM, Malech HL, et al. Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes. Blood. 2005;106:51–58. doi: 10.1182/blood-2004-11-4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R γ null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174:6477–6489. doi: 10.4049/jimmunol.174.10.6477. [DOI] [PubMed] [Google Scholar]
  26. Strom TS, Gabbard W, Kelly PF, Cunningham JM., and, Nienhuis AW. Functional correction of T cells derived from patients with the Wiskott-Aldrich syndrome (WAS) by transduction with an oncoretroviral vector encoding the WAS protein. Gene Ther. 2003;10:803–809. doi: 10.1038/sj.gt.3301950. [DOI] [PubMed] [Google Scholar]
  27. Strom TS, Turner SJ, Andreansky S, Liu H, Doherty PC, Srivastava DK, et al. Defects in T-cell-mediated immunity to influenza virus in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene transfer into repopulating hematopoietic cells. Blood. 2003;102:3108–3116. doi: 10.1182/blood-2002-11-3489. [DOI] [PubMed] [Google Scholar]
  28. Trobridge GD, Beard BC, Gooch C, Wohlfahrt M, Olsen P, Fletcher J, et al. Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors. Blood. 2008;111:5537–5543. doi: 10.1182/blood-2007-09-115022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M, et al. Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther. 2003;14:509–519. doi: 10.1089/104303403764539305. [DOI] [PubMed] [Google Scholar]
  30. Relander T, Johansson M, Olsson K, Ikeda Y, Takeuchi Y, Collins M, et al. Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV- or RD114-pseudotyped HIV-1-based vectors from stable producer cells. Mol Ther. 2005;11:452–459. doi: 10.1016/j.ymthe.2004.10.014. [DOI] [PubMed] [Google Scholar]
  31. Di Nunzio F, Piovani B, Cosset FL, Mavilio F., and, Stornaiuolo A. Transduction of human hematopoietic stem cells by lentiviral vectors pseudotyped with the RD114-TR chimeric envelope glycoprotein. Hum Gene Ther. 2007;18:811–820. doi: 10.1089/hum.2006.138. [DOI] [PubMed] [Google Scholar]
  32. Kiem HP, Heyward S, Winkler A, Potter J, Allen JM, Miller AD, 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. [PubMed] [Google Scholar]
  33. Hu J, Kelly P, Bonifacino A, Agricola B, Donahue R, Vanin E, et al. Direct comparison of RD114-pseudotyped versus amphotropic-pseudotyped retroviral vectors for transduction of rhesus macaque long-term repopulating cells. Mol Ther. 2003;8:611–617. doi: 10.1016/s1525-0016(03)00239-9. [DOI] [PubMed] [Google Scholar]
  34. Takatoku M, Sellers S, Agricola BA, Metzger ME, Kato I, Donahue RE, et al. Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates. J Clin Invest. 2001;108:447–455. doi: 10.1172/JCI12593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sutton RE, Reitsma MJ, Uchida N., and, Brown PO. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol. 1999;73:3649–3660. doi: 10.1128/jvi.73.5.3649-3660.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lewis PF., and, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol. 1994;68:510–516. doi: 10.1128/jvi.68.1.510-516.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Haas DL, Case SS, Crooks GM., and, Kohn DB. Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. Mol Ther. 2000;2:71–80. doi: 10.1006/mthe.2000.0094. [DOI] [PubMed] [Google Scholar]
  38. Mezquita P, Beard BC., and, Kiem HP. NOD/SCID repopulating cells contribute only to short-term repopulation in the baboon. Gene Ther. 2008;15:1460–1462. doi: 10.1038/gt.2008.108. [DOI] [PubMed] [Google Scholar]
  39. Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP., and, Bodine DM. 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 USA. 1996;93:11097–11102. doi: 10.1073/pnas.93.20.11097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sabatino DE, Do BQ, Pyle LC, Seidel NE, Girard LJ, Spratt SK, et al. Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines. Blood Cells Mol Dis. 1997;23:422–433. doi: 10.1006/bcmd.1997.0161. [DOI] [PubMed] [Google Scholar]
  41. Bertran J, Miller JL, Yang Y, Fenimore-Justman A, Rueda F, Vanin EF, et al. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors. J Virol. 1996;70:6759–6766. doi: 10.1128/jvi.70.10.6759-6766.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kurre P, Kiem HP, Morris J, Heyward S, Battini JL., and, Miller AD. 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. doi: 10.1128/jvi.73.1.495-500.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Relander T, Brun AC, Olsson K, Pedersen L., and, Richter J. Overexpression of gibbon ape leukemia virus (GALV) receptor (GLVR1) on human CD34(+) cells increases gene transfer mediated by GALV pseudotyped vectors. Mol Ther. 2002;6:400–406. doi: 10.1006/mthe.2002.0678. [DOI] [PubMed] [Google Scholar]
  44. Uckert W, Willimsky G, Pedersen FS, Blankenstein T., and, Pedersen L. RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes. Hum Gene Ther. 1998;9:2619–2627. doi: 10.1089/hum.1998.9.17-2619. [DOI] [PubMed] [Google Scholar]
  45. Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science. 2008;319:921–926. doi: 10.1126/science.1152725. [DOI] [PubMed] [Google Scholar]
  46. Wolf D., and, Goff SP. Host restriction factors blocking retroviral replication. Annu Rev Genet. 2008;42:143–163. doi: 10.1146/annurev.genet.42.110807.091704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schlegel R, Tralka TS, Willingham MC., and, Pastan I. Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site. Cell. 1983;32:639–646. doi: 10.1016/0092-8674(83)90483-x. [DOI] [PubMed] [Google Scholar]
  48. Schlegel R., and, Wade M. Neutralized vesicular stomatitis virus binds to host cells by a different “receptor”. Biochem Biophys Res Commun. 1983;114:774–778. doi: 10.1016/0006-291x(83)90848-3. [DOI] [PubMed] [Google Scholar]
  49. Coil DA., and, Miller AD. Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus. J Virol. 2004;78:10920–10926. doi: 10.1128/JVI.78.20.10920-10926.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009;326:818–823. doi: 10.1126/science.1171242. [DOI] [PubMed] [Google Scholar]
  51. Notta F, Doulatov S., and, Dick JE.2010Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgcnull recipients Blood(epub ahead of print). [DOI] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Assembly of the plasmids encoding the FLVC envelope and chimeric FLVC envelope protein.

Click here for additional data file. (22.8MB, tiff)
Figure S2.

Constructs used to derive in vitro transcribed RNA for use as a standard in quantitating the various receptor mRNAs by real-time qRT-PCR as described in the methods.

Click here for additional data file. (4.2MB, tiff)
Supplementary Data.
Click here for additional data file. (43.5KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES