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

Abstract

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34 ؉ hematopoietic progenitor cells. The relatively low level 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؇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.

Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives

“…Lentiviral vectors make up a subset of retroviral vectors, which will transduce regardless of cellcycle stage. These vectors are often constructed using HIV-1 provirus pseudotyped with a VSV-G protein coat, which is highly effective at transducing a vast majority of mammalian cells due to its binding mechanism (Schlegel et al 1983;Akkina et al 1996;Naldini et al 1996;Reiser et al 1996). The typical time frame of a pooled lentiviral infection takes approximately two weeks from generation of virus to expansion of the genetically modified cell line.…”

Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance

“…is RNA packaging is coupled with viral assembly and released out of the cell by virion budding. is budding takes place at the cellular membrane, where the ENV glycoprotein is also incorporated to the budding virion [28][29][30][31][32]. Once the virion is released, the viral protease will exert its activities on the GAG/GAG-POL polyproteins, releasing the structural proteins and conferring infectivity to the newly formed virion [6,33].…”

Efficient, Long‐term Transgene Expression in Xenopus laevis Dermal Melanophores

“…Lentivirus vectors based on HIV-1 have been shown to efficiently transduce non-dividing mammalian cells in vitro and in vivo (18)(19)(20)(23)(24)(25). This is an important consideration regarding the transduction of melanophores, since these amphibian cells divide approximately once every 3-4 days.…”