Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer.

Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction. The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.     

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.

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.     

Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

Vesicular Stomatitis Virus G-Pseudotyped Lentivirus Vectors Mediate Efficient Apical Transduction of Polarized Quiescent Primary Alveolar Epithelial Cells

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.     

Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins

Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.     

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.     

Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296

Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.     

Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.     

PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.     

Efficient Transduction by an Amphotropic Retrovirus Vector Is Dependent on High-Level Expression of the Cell Surface Virus Receptor

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.     

Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio

Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.     

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background

The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34⁺/CD38^low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.

Methods

CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).

Results

Sorted CD34⁺/CD38^low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34⁺ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×10⁵ cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34⁺ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).

Conclusions

The best conditions for transduction and engraftment of CB SRCs were obtained with GALV‐pseudotyped vectors using serum‐free conditions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene delivery to airway epithelial cells in vivo: a direct comparison of apical and basolateral transduction strategies using pseudotyped lentivirus vectors

Lentivirus vectors are being investigated as gene delivery vehicles for cystic fibrosis airway gene therapy. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped vectors transduce airway epithelia via receptors that are located predominantly on the basolateral surface of the airway epithelium. Effective transduction with VSV-G-pseudotyped vectors requires the use of a pre-treatment that disrupts epithelial tight junctions, allowing access to these basolateral receptors. In contrast, it has been reported that apically targeted lentiviral vectors allow efficient gene transfer in the absence of any pre-treatment. In a direct comparison of transduction by a VSV-G-pseudotyped vector, in combination with a pre-treatment with lysophosphatidylcholine (LPC), and the same vector pseudotyped with the apically targeted baculovirus GP64 envelope (without any pre-treatment), the GP64 vector was found to be significantly less efficient. However, when a pre-treatment with LPC was used the level of transduction with the GP64-pseudotyped lentiviral vector was not significantly different to that resulting from the VSV-G-pseudotyped vector. The cell types transduced with each vector were essentially the same, with the majority of cells transduced being respiratory (ciliated cells). However, unlike the VSV-G-pseudotyped vector, which results in persisting gene expression, transduction with the GP64-pseudotyped vector resulted in gene expression that declined to undetectable levels over six months, whether or not an LPC pre-treatment was used.     

Retrovirus molecular conjugates. A novel, high transduction efficiency, potentially safety-improved, gene transfer system

Two significant barriers limit the use of amphotropic retrovirus for human gene transfer protocols: 1) low transduction efficiency in cells with low receptor expression and 2) safety concerns originating from the risk of formation and propagation of replication competent virus in vivo. In principle, if ecotropic retrovirus, which is incapable of infecting human cells, could be transiently modified to effectively transduce human cells, this safety risk could be alleviated. Here we demonstrate that formation of amphotropic retrovirus polylysine molecular conjugates (aMMLV-PL) enhanced gene transfer up to 10-fold in a variety of human cell lines over the equivalent of unconjugated vector (aMMLV). The polylysine modification and formation of ecotropic retrovirus molecular conjugates (eMMLV-PL) permitted effective and stable transduction of different human cell lines as well as primary human bone marrow stroma cells at frequencies of greater than 80%. It is conceivable that this novel ecotropic-based conjugate retrovirus vector could also potentially provide enhanced safety characteristics not only over amphotropic retrovirus vectors but also over genetically tropism-modified recombinant ecotropic vectors. In contrast to genetic modifications, physical or chemical modifications are not propagated. Thus, formation of replication competent eMMLV from conjugates would be self-limited and would not result in virus propagation in humans.     

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.     

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.     

A Packaging Cell Line for Lentivirus Vectors

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>10⁹ IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314–317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319–10323, 1997; L. Naldini et al., Science 272:263–267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10⁶ IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 10⁹ IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.