Gene therapy against HIV infection should involve vector-mediated delivery of anti-HIV therapeutic genes into T-lymphocytes and macrophages or, alternatively, hematopoietic progenitors. Transduction of mature cells with defective vectors would have limited success because the vector would disappear with cell turnover. However, if a vector could be trafficked by wild-type HIV, initial transduction of a majority of the population would not be required, as the vector would be able to spread. We describe HIV-1-based lentiviral vectors that are efficiently packaged and trafficked by HIV-1, allowing a small number of cells initially transduced to spread the vector within a nontransduced cell population. We examined whether the presence or absence of the rev gene and the Rev-responsive element (RRE) would have a noticeable effect on the ability of lentiviral vectors to be trafficked and to inhibit HIV-1 replication. We found that replacement of rev/RRE with a constitutive transport element from Mason-Pfizer monkey virus had no apparent effect on trafficking and did not change the intrinsic inhibitory abilities of the vectors. We also constructed a rev/RRE-independent HIV-1-derived vector carrying a trans-dominant negative mutant of HIV-1 Rev, RevM10. This vector was less efficiently trafficked by HIV-1 and, despite the presence of an anti-HIV-1 gene, RevM10, was less efficient at inhibiting HIV-1 replication when introduced into a target T-cell population.
Lentiviral vectors, so far, have been optimized for the expression of a single open reading frame. Certain practical applications of gene therapy will, however, require expression of multiple genes. The goal of this study was to explore the feasibility of directing expression of two marker genes from a lentiviral vector. We designed two types of multigene lentiviral vectors. First, we used a strategy based on the natural splicing signals of HIV-1, by which multiple mRNAs are generated from a single transcriptional unit. A second strategy was construction of a polycistronic mRNA using a translational cis-acting element, the encephalomyocarditis virus internal ribosome entry site (IRES). Our studies show that the inclusion of multiple genes in lentiviral vectors does not result in reduction in virus titers or in the loss of ability to infect nondividing cells. We introduced mutations in tat and/or rev to test whether splicing modulates the relative levels of expression of reporter genes. We also developed a truncated version of tat, which is devoid of the apoptosis-associated domain. Inclusion of this tat mutant in a lentiviral vector resulted in the generation of virus with titers similar to those of lentivirus vectors expressing wild-type tat.
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