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

Int Conf AIDS. 2004 Jul 11-16;15:Abstract No. LbOrA03

L-J Chang, J He
University of Florida, Gainesville, United States

BACKGROUND: The high mutation rate of the human immunodeficiency virus (HIV) makes it difficult for any therapy employing a single anti-HIV mechanism to sustain prolonged effect. Lentiviral small interfering RNAs (siRNAs) targeting multiple highly conserved HIV-1 sequences represents a novel anti-HIV strategy.

METHODS: Lentiviral siRNA vectors targeting eleven highly conserved regions in the HIV-1 genome were constructed and tested against different subtypes of HIV-1. The anti-HIV effect was determined by reverse transcriptase (RT) assay, Northern analysis, progeny virus infectivity, and inhibition of M- and T-tropic syncytium-forming activity using reporter cell lines, primary human peripheral blood lymphocytes (PBLs), and HIV-1 long term producers. Lentiviral dual-siRNA vectors carrying both U6- and tRNA-driven siRNA cassettes were constructed and tested.

RESULTS: When tested against different HIV-1 strains including pNL4-3 (subtype B), p89.6 (subtype B) and p90CF402.1.8 (subtype A/E recombinant), the siRNAs targeting conserved gag, pol, int and vpu but not U3, nef or U5 regions, efficiently inhibited replication of all three viral strains. The lentiviral siRNAs protected host cells from syncytium-forming M- and T-tropic HIV-1 induced cytotoxicity. Transduction of a long term chronically infected human lymphoma cell line with the lentiviral siRNAs resulted in stable inhibition of HIV-1 replication. In primary human peripheral blood lymphocytes, the lentiviral siRNAs efficiently reduced HIV-1 infection and replication. Analysis of siRNA-protected HIV-1 producer cells indicates that both nuclear and cytoplasmic viral RNAs are significantly down-regulated. Analysis of detailed molecular mechanisms of U6- and tRNA-siRNA processing in both dividing and non-dividing cells by siRNA Northern blotting and RNase A/T1 mapping suggests that efficient nuclear export and siRNA processing contribute to the silencing effects.

CONCLUSIONS: The lentiviral siRNA targeting multiple highly-conserved HIV-1 sequences holds significant promise for the treatment of HIV/AIDS.

040711
LbOrA03

Copyright © 2004 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.