application): Lentiviral vectors, specifically those based on HIV-1, are generating interest in the gene therapy community due to their attractive property of stable integration into non-dividing cell types. Their use for human therapy ultimately depends on the development of a safe lentiviral-based vector. We have exploited HIV virion associated accessory proteins (Vpr and Vpx) as vehicles to deliver protein of both viral and non-viral origin into HIV particles. Recently, we demonstrated that trans RT and IN (derived from Vpr-RT-IN fusion protein) can mimic cis- RT and IN (derived from Gag-Pol). The trans- RT and IN proteins can effectively rescue the infectivity and replication of virions derived from RT-IN minus provirus through the complete life cycle. These findings demonstrate that the functions of these critical enzymes can be provided in trans, independent of Gag Pol. These findings offer us a unique approach toward designing a new generation of safe lentiviral-based gene delivery vectors. It is our hypothesis that Vpr-RT-IN can be expressed in trans to the Gag-Pro component of the packaging plasmid and support vector infectivity (transduction). An obvious corollary to this hypothesis is that this strategy will reduce the frequency of recombination events that might generate an infectious/replicating lentivirus (LTR gag-pol-LTR structure). To test this hypothesis we propose to: (1) construct a """"""""trans-lentivirus"""""""" vector and analyze its ability to transduce primary airway epithelial cells; and (2) analyze the expression of the CFTR gene in primary airway epithelial cells using the trans-lentivirus vector. Central to the """"""""trans-lentiviral"""""""" vector approach is that the RT and IN genes are separated from packaging components. This is distinct from conventional lentiviral packaging systems in which RT and IN are expressed in cis a part of Gag-Pol (Gag-PRRT-IN). By expressing the critical RT and IN functions in trans it will be possible to decrease the generation of pathogenic viral forms that could arise by genetic recombination. Importantly, our published data show that trans-RT-IN can support the infectivity and replication of RT-IN minus HIV-1 at a level of approximately 80% that of wild type virus. We believe that this new generation of lentiviral-based vectors (trans-lentiviral vectors) will facilitate their application into human clinical trials.
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