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

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.     

Efficiency of onco-retroviral and lentiviral gene transfer into primary mouse and human B-lymphocytes is pseudotype dependent

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.003) with ecotropic (11%) than with VSV-G pseudotyped onco-retroviral vectors (1%). Using high-titer cell-free ecotropic viral supernatant or by coculture with ecotropic onco-retroviral vector-producing cells, transduction efficiency increased significantly (p < 0.001) to approximately 50%, whereas transduction efficiency by coculture with VSV-G pseudotyped vector-producing cells remained low (< 2%). Similarly, transduction of mouse B lymphocytes was significantly more efficient (twofold, p < 0.01) with the ecotropic (7%) than with the VSV-G pseudotyped lentiviral vectors although gene transfer efficiency remained low because of dose-limiting toxicity of the concentrated vector preparations on the LPS-activated murine B cells. Consistent with murine B-cell transduction, human B cells activated with CD40L and IL-4 were also found to be relatively refractory to VSV-G pseudotyped onco-retroviral vectors (< 1%). However, higher transduction efficiencies could be achieved in activated primary human B lymphocytes using VSV-G pseudotyped lentiviral vectors instead (5%-6%). Contrary to the significant increase in mouse B-cell transduction efficiency with ecotropic vectors, the use of amphotropic onco-retroviral or lentiviral vectors did not increase transduction efficiency in primary human B cells. The present study shows that the transduction efficiency of onco-retroviral and lentiviral vectors in human and mouse B lymphocytes is pseudotype-dependent and challenges the widely held assumption that VSV-G pseudotyping facilitates gene transfer into all cell types.     

Comparison of Various Envelope Proteins for Their Ability to Pseudotype Lentiviral Vectors and Transduce Primitive Hematopoietic Cells from Human Blood

Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated ∼100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34⁺ cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34⁺ cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.     

Gene transfer to respiratory epithelia with lentivirus pseudotyped with Jaagsiekte sheep retrovirus envelope glycoprotein

A feline immunodeficiency virus (FIV)-based lentiviral vector was pseudotyped to identify envelope (env) glycoproteins that direct efficient gene transfer to pulmonary epithelia for the treatment or prevention of lung diseases. The envelope glycoprotein from the Jaagsiekte sheep retrovirus (JSRV) is a candidate under investigation. We utilized high titer FIV vector (>10(8) TU/ml) pseudotyped with the JSRV env glycoprotein (JSRVFIV) to study the transduction of polarized primary cultures of human airway epithelia and receptor/vector interactions. The reported receptor for JSRV, hyaluronidase 2 (HYAL2), is a GPI-linked protein. We expressed FLAG-tagged HYAL2 in polarized airway epithelia using an adenoviral vector and documented that the HYAL2 protein sorts predominantly to the apical surface. Of interest, the efficiency of gene transfer with apically applied JSRV-FIV was markedly less than FIV pseudotyped with VSV-G, even in Ad-HYAL2 complemented epithelia. The inefficient gene transfer with JSRV-FIV in HYAL2 complemented cells suggests that factors other than receptor abundance limit apical gene transfer efficiency with this envelope. JSRV-FIV transduced the distal lung epithelia of rabbits in vivo and transduced primary cultures of rabbit type II cells with 100-fold greater efficiency than primary cultures of rabbit tracheal cells. These data indicate that a lentivirus pseudotyped with the JSRV envelope glycoprotein transduces type II cells with greater efficiency than conducting airway epithelia and provides an example of glycoprotein-mediated cell-specific tropism within a tissue with a widely heterogeneous cell population.     

Production and concentration of pseudotyped HIV-1-based gene transfer vectors

Strategies to generate highly concentrated HIV-1 vector pseudotypes involving different envelope (Env) proteins including the vesicular stomatitis virus (VSV) G glycoprotein, the Moloney murine leukemia virus (MLV) 4070A amphotropic Env and the rabies G glycoprotein were established. Virus stocks were prepared by transient transfection using standard cell culture media or serum-free media. Such stocks were concentrated 50- to 300-fold by ultracentrifugation or by ultrafiltration using Centricon Plus-80 units yielding titers of up to 109transducing units per milliliter. There was no loss in titer with any of the pseudotypes tested. Thus, like lentiviral vectors pseudotyped with VSV-G, HIV-1-based vectors pseudotyped with the MLV 4070A amphotropic Env and the rabies G glycoprotein resist inactivation during concentration. This opens up the possibility to generate highly concentrated HIV-1 vector stocks carrying alternative Env proteins on a large scale.     

Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively). Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry. By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.     

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.     

Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors

Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors.     

Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro

The envelope glycoprotein from vesicular stomatitis virus (VSV-G) has been used extensively to pseudotype lentiviral vectors, but has several drawbacks including cytotoxicity, potential for priming of immune responses against transgene products through efficient transduction of antigen-presenting cells (APCs) and sensitivity to inactivation by human complement. As an alternative to VSV-G, we extensively characterized lentiviral vectors pseudotyped with the gp64 envelope glycoprotein from baculovirus both in vitro and in vivo. We demonstrated for the first time that gp64-pseudotyped vectors could be delivered efficiently in vivo in mice via portal vein injection. Following delivery, the efficiency of mouse cell transduction and the transgene expression is comparable to VSV-G-pseudotyped vectors. In addition, we found that gp64-pseudotyped lentiviral vectors could efficiently transduce a variety of cell lines in vitro, although gp64 showed a more restricted tropism than VSV-G, with especially poor ability to transduce hematopoietic cell types including dendritic cells (DCs). Although we found that gp64-pseudotyped vectors are also sensitive to inactivation by human complement, gp64 nevertheless has advantages over VSV-G, because of its lack of cytotoxicity and narrower tropism. Consequently, gp64 is an attractive alternative to VSV-G because it can efficiently transduce cells in vivo and may reduce immune responses against the transgene product or viral vector by avoiding transduction of APCs such as DCs.     

Lack of superinfection interference in retroviral vector producer cells

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.     

Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins

Malignant gliomas are the most frequent primary brain tumors and have a dismal prognosis due to their infiltrative growth. Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In a previous study, we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoproteins (GPs) and demonstrated transduction of human malignant glioma cells in culture. In the current approach, we compared the transduction efficacy of LCMV-GP- and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo. LCMV-GP pseudotypes transduced almost exclusively astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes. LCMV-GP pseudotypes showed an efficient transduction of solid glioma parts and specific transduction of infiltrating tumor cells. In contrast, VSV-G-pseudotyped lentiviral vectors transduced only a few tumor cells in solid tumor parts and infected mostly normal brain cells in infiltrating tumor areas. In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.     

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.     

Efficient gene transfer of VSV-G pseudotyped retroviral vector to human brain tumor

A retroviral vector constructed from the murine leukemia virus (MLV) can only express transgenes in cells undergoing mitosis, indicating its suitability as a delivery vehicle for cancer gene therapy. However, the transduction efficiency (TE) of retroviruses embedding endogenous envelope proteins in human cancer cells was found to be unsatisfactory. Recently, several research groups have demonstrated the feasibility of a retroviral vector pseudotyped with a vesicular stomatitis virus G (VSV-G) protein. In this study, the potential of VSV-G pseudotyped MLV-based retrovirus was examined as a delivery vehicle in a variety of human cancer cells including brain tumor cells in vitro and in vivo. The transduction efficiency of the 293T/G/GP/LacZ retrovirus in cell culture was superior in most cancer cells, particularly in brain tumor cells, compared with that of other retroviruses, such as PA317- or PG13-derived. The relative growth rate and phosphatidylserine expression level on the plasma membrane of target cells mainly influenced the transduction efficiency of VSV-G pseudotyped retrovirus, which suggested that both the relative growth rate and phosphatidylserine expression level were major determinants of TE. Furthermore, 293T/G/GP/LacZ could efficiently transduce human cancer cells regardless of the presence of chemical additives, whereas in other retroviruses, cationic chemical additives such as polybrene or liposomes were essential during virus infection. Finally, an average of 10% gene expression was routinely obtained exclusively in the tumor mass when 293T/G/GP/LacZ concentrated by simple ultracentrifugation was directly administrated to pre-established brain tumors in animal models (U251-N nu/nu mice or C6 Wistar rats). All told, the present study suggests that the VSV-G pseudotyped retrovirus is a suitable vector for brain tumor gene therapy.     

Gene transfer in human skin with different pseudotyped HIV-based vectors

Pseudotyping lentiviral vector with other viral surface proteins could be applied for treating genetic anomalies in human skin. In this study, the modification of HIV vector tropism by pseudotyping with the envelope glycoprotein from vesicular stomatitis virus (VSV), the Zaire Ebola (EboZ) virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), Rabies or the rabies-related Mokola virus encoding LacZ as a reporter gene was evaluated qualitatively and quantitatively in human skin xenografts. High transgene expression was detected in dermal fibroblasts transduced with VSV-G-, EboZ- or MuLV-pseudotyped HIV vector with tissue irregularities in the dermal compartments following repeated injections of EboZ- or LCMV-pseudotyped vectors. Four weeks after transduction, double-labeling immunofluorescence of beta-galactosidase and involucrin or integrin beta1 demonstrated that VSV-G-, EboZ- or MuLV-pseudotyped HIV vector effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies. Among the six different pseudotyped HIV-based vectors evaluated, VSV-G-pseudotyped vector was found to be the most efficient viral glycoprotein for cutaneous transduction as demonstrated by the highest level of beta-galactosidase expression and genome copy number evaluated by TaqMan PCR.     

Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor

A major obstacle that limits the potential of human gene therapy is the inefficiency of gene delivery to appropriate sites in vivo. Previous studies demonstrated that the physiological surveillance function performed by von Willebrand factor (vWF) could be incorporated into retroviral vectors by molecular engineering of the MuLV ecotropic envelope (Env) protein. To advance the application of vWF targeting technology beyond laboratory animals, we prepared an extensive series of Env proteins bearing modified vWF-derived matrix-binding sequences and assembled these chimeric proteins into targeted vectors that are capable of transducing human cells. Initially, a dual envelope configuration was utilized, which required coexpression of a wild-type amphotropic Env. Subsequently, streamlined "escort" Env proteins were constructed wherein the inoperative receptor-binding domain of the targeting partner was replaced by the vWF-derived collagen-binding motif. Ultimately, an optimal construct was developed that exhibited properties of both extracellular matrix (ECM)-targeting and near wild-type amphotropic infectivity, and could be arrayed as a single envelope on a retroviral particle. On intraarterial instillation, enhanced focal transduction of neointimal cells (approximately 20%) was demonstrated in a rat model of balloon angioplasty. Moreover, transduction of tumor foci (approximately 1-3%) was detected after portal vein infusion of a matrix-targeted vector in a nude mouse model of liver metastasis. We conclude that the unique properties of these targeted injectable retroviral vectors would be suitable for improving therapeutic gene delivery in numerous clinical applications, including vascular restenosis, laser and other surgical procedures, orthopedic injuries, wound healing, ischemia, arthritis, inflammatory disease, and metastatic cancer.     

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.     

Activation of Transgene-specific T Cells Following Lentivirus-mediated Gene Delivery to Mouse Lung

Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)–deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vector–mediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells.     

Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV-, or RD114-pseudotyped HIV-1-based vectors from stable producer cells.

A novel, stable human immunodeficiency virus type 1 vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation into NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used: the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+), and a modified feline endogenous virus RD114 envelope (RDpro). Gene transfer to freshly thawed CD34+ cells in the absence of cytokines was very low. Addition of cytokines increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 h. Concentration of the vectors (15-fold) by low-speed centrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 65-70% of the cells under the same conditions. Transplantation of CD34+ cells prestimulated for 24 h and then transduced with a single exposure to concentrated vector revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells, which was significantly higher than the MLV-A (12.6%)- or GALV+ (25.1%)-pseudotyped vectors.