15th International AIDS Conference Bangkok, Thailand - July 11-16, 2004

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. WeOrA1309)

Morris KV, Innis L, Chung C, Witke W, Looney DJ
University of California San Diego, La Jolla, CA 92098-0678, United States

BACKGROUND: Small interfering RNA directed against HIV-1 gag, vif, tat, and rev, and cellular genes necessary for HIV-1 entry (CD4, CCR5) have been shown to inhibit HIV replication in vitro. Among several potential problems faced in the development of siRNA technology for use against HIV-1 is the sequence dependence of the inhibitory effect in the setting of extensive HIV variation, which may severely limit the number of potential targets. A second problem may be the concentration dependence of sequence specific and non-specific inhibitory effects, which may make delivery of appropriate amounts difficult.

METHODS: To address these issues seven siRNA candidate sequences in gag-pol of 21-23 bp length were selected based on: (1) suitability for use in the Ambion SilencerTM system, (2) GC content (<50%), (3) lack of homology with other sequences in the Genbank nt database, and (4) representation in 207 gag-pol sequences in the Los Alamos databank. The anti-HIV-1 siRNA's and an irrelevant control (directed against EGFP) were synthesized and co-transfected at various concentrations with HIV-1 into Hela-CD4 or 293FT cells.

RESULTS: Only 2 of the initial 7 siRNAs inhibited p24 production more than 50% at any one concentration, though siRNA targeting positions 2064 and 2949 displayed inhibitory activity over the broadest range of concentrations and cell types. In both cell types the 100 nM concentration was found to be optimal for 2949 and 2064 respectively, with both higher and lower concentrations resulting in lower degrees of inhibition.

CONCLUSIONS: Considerations of target sequence conservation, concentration dependence (and non-specific effects at higher concentrations), together with previous observations indicating the importance for RNA accessibility are likely to severely limit the number of siRNA candidates for further development as possible therapeutics.

040711
WeOrA1309

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