HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-2 LENTIVIRUS VECTORS FOR GENE-TRANSFER - EXPRESSION AND POTENTIAL FOR HELPER VIRUS-FREE PACKAGING

In addition to the long-term expression of the transgene provided by a ll retroviral vectors, lentiviruses present the opportunity to transdu ce nondividing cells and potentially achieve regulated expression, The development of lentiviral vectors requires the design of transfer vec tors to ferry the transgene with efficient encapsidation of the transg ene RNA and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus prod uction. For both vectors, a knowledge of packaging signal is required- the signal to be included in the transfer vector but excluded from the packaging vector. Among the human lentiviruses, human immunodeficienc y virus type 1 and type 2 (HIV-1 and HIV-2), we think HIV-2 is better suited for gene transfer than HIV-1, It is less pathogenic and thus sa fer during design and production; its desirable nuclear import and und esirable cell-cycle arrest functions are segregated on two separate ge nes. In HIV-1 infection, it is less likely to recombine with the resid ent HIV-1, and it may itself downregulate HIV-1 expression. Evidently, elements located both upstream and downstream of the splice donor sit e in the leader sequence participated in RNA encapsidation and these s equences appeared necessary and sufficient. Deletion of both sequence elements resulted in a dramatic curtailment of RNA encapsidation and h elper virus production. This was accompanied by some but acceptable lo ss of gene expression capability. The helper virus-free phenotype and expression capability of the double mutant was maintained upon replace ment of its 3' long terminal repeat with a minigene cassette containin g a transcriptional termination signal and a drug resistance marker ge ne. Deletion of the splice donor site itself had a dramatic negative e ffect on gene expression, supporting the important role of this elemen t in the life of RNA.