832. Suppression of HIV-1 Replication by Delievery of multiple siRNA Genes Against p24, Tat/ Rev and CCR5

Abstract

Small interfering RNAs (siRNA) have been evaluated for their potential to treat infectious diseases such as HIV infection in humans. Several studies demonstrated that siRNAs targeted to different sites in the HIV-1 genome could lead to significant suppression of HIV-1 replication in cell culture. However, HIV-1 escape variants emerged quickly with the expression of a single siRNA and sequence analysis of these escape variants revealed that the siRNA target sites in the HIV-1 genome had nucleotide substitutions or deletion. The rapid replication kinetics of HIV-1 and the error-prone nature of viral reverse transcriptase most likely lead to the emergence and selection of viral quasispecies that are genotypically distinct in the siRNA target site, thereby eliminating the siRNA antiviral effect. These studies suggest that multiple siRNAs targeted to different regions of the HIV-1 genome needs to be developed to reduce the probability of generating these escape mutants. To down-regulate HIV-1 replication permanently, a generally taken approach is to stably express in the target cell a short hairpin RNA (shRNA) that can be processed into siRNA by endogenous RNAse III like enzyme Dicer. The shRNA gene is introduced into hematopoietic cells via either retroviral or HIV-based vectors. In the current study, we showed that using HIV-based vector for the delivery of a single shRNA gene against HIV-1 encounters the problem of reduced vector titers. This most likely is due to siRNA-mediated down-regulation of packaging plasmid-derived transcripts during the vector production process. Insertion of multiple shRNA genes against HIV-1 in a single vector led to further reduction in vector titers. Introduction of conservative changes that modified the siRNA target sequence without altering the amino acid sequence into the packaging plasmid restored the production of viral proteins from the packaging plasmid and increased the vector titers. Such modifications would be critical for large-scale production of HIV vectors containing multiple shRNA genes for clinical application to treat HIV-1 infection. In this study, we also compared three shRNA genes against the p24 region, a common region shared by both the tat and rev genes and HIV-1 co-receptor CCR5 for their ability to suppress HIV-1 replication in culture. Human H9 cells transduced with HIV vectors expressing either two or all three of these shRNAs were established and challenged with HIV-1NL4-3 at different doses. The efficacy of different combinations of these shRNAs compared with single shRNAs against HIV-1 will be discussed.