Background. The cellular introduction of short, interfering (si) RNA leads to sequence-specific degradation of homologous RNA, a process termed RNA interference (RNAi). Here, we report that adeno-associated virus-2 (AAV-2) can be modified to genetically transfer expression cassettes encoding siRNA targeting the HIV-1 transactivator protein tat. HIV-1 replication was reduced by 96% in H9 cells that expressed tat siRNA compared to control cells.

Purpose. To demonstrate that AAV-2 can be used as a genetic tool to permanently express HIV-1 specific siRNA.

Methods. The tat gene of HIV-1 encodes a transactivator protein that is essential for efficient viral replication. To determine the effects of RNAi on HIV-1 replication, we designed a 21-nucleotide (nt) siRNA that was homologous to the first exon of tat. The antiviral potency of this compound was confirmed by transiently co-transfecting 293T cells with tat siRNA and pNL4-3, an infectious molecular clone of HIV-1. HIV-1 replication was reduced by 91% in cells transfected with tat siRNA. To engineer stable cellular expression of tat siRNA using AAV-2, a vector was first constructed that contained a hairpin siRNA expression cassette. This cassette consisted of an upstream H1 pol III-type promoter followed by 21-nt tat sense and antisense sequences separated by a 6-nucleotide linker and a 3’ TTTTT termination signal. The gene for neomycin resistance was introduced downstream to the tat siRNA expression cassette. Recombinant AAV-2 viral particles were generated and used to infect H9 cells, a cell line derived from a human cutaneous T cell lymphoma. After two months of neomycin selection, the cells were infected with 65 TCID₅₀ of HIV_NL4.3. Culture supernatants were assayed for p24 antigen levels at four-day intervals over a one-month period.

Results. The level of HIV-1 replication was reduced by 96% in cultures of H9 cells expressing tat siRNA, compared to cultures of control cells. Expression of tat siRNA was confirmed by Northern blot analysis of total cellular RNA. Stable genomic integration of AAV-2 constructs was verified by Southern blotting. 

Conclusion. Our experiments demonstrate the feasibility of permanently expressing tat siRNA using AAV-2 as a delivery system. Control of HIV-1 replication by genetic transfer of HIV-1 specific RNAi to stem cells or lymphocytes ex vivo deserves further investigation as a therapeutic modality.