Human Immunodeficiency Virus Type 2 Lentivirus Vectors for Gene Transfer: Expression and Potential for Helper Virus-Free Packaging

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Abstract
In addition to the long-term expression of the transgene provided by all retroviral vectors, lentiviruses present the opportunity to transduce nondividing cells and potentially achieve regulated expression. The development of lentiviral vectors requires the design of transfer vectors to ferry the transgene with efficient encapsidation of the transgene RNA and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus production. 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 immunodeficiency 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 safer during design and production; its desirable nuclear import and undesirable cell-cycle arrest functions are segregated on two separate genes. In HIV-1 infection, it is less likely to recombine with the resident HIV-1, and it may itself downregulate HIV-1 expression. Evidently, elements located both upstream and downstream of the splice donor site in the leader sequence participated in RNA encapsidation and these sequences appeared necessary and sufficient. Deletion of both sequence elements resulted in a dramatic curtailment of RNA encapsidation and helper virus production. This was accompanied by some but acceptable loss of gene expression capability. The helper virus-free phenotype and expression capability of the double mutant was maintained upon replacement of its 3′ long terminal repeat with a minigene cassette containing a transcriptional termination signal and a drug resistance marker gene. Deletion of the splice donor site itself had a dramatic negative effect on gene expression, supporting the important role of this element in the life of RNA. The human immunodeficiency virus type 2 (HIV-2) lentivirus contains elements in its leader sequence located both upstream and downstream of the splice donor site that participate in RNA encapsidation. These elements appeared to be necessary and sufficient for packaging. Deletion of both upstream and downstream elements was clearly beneficial for curtailing helper virus protection, although this was accompanied by some but acceptable loss of expression capability. The helper virus-free phenotype of the packaging vector could be further ensured without additional loss of expression by replacing the 3′ long terminal repeat with a minigene containing a heterologous transcriptional termination signal and a selection marker gene. Relevant to the design of transfer vector, deletion of the splice donor site itself had a dramatic negative effect on viral gene expression.