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RNA interference (RNAi)-based gene therapy for the treatment of HIV-1 infection provides a novel antiviral approach. For delivery of RNAi inducers to CD4+ T cells or CD34+ blood stem cells, lentiviral vectors are attractive because of their ability to transduce nondividing cells. In addition, lentiviral vectors allow stable transgene expression by inserting their cargo into the host cell genome. However, use of the HIV-1-based lentiviral vector also creates specific problems. The RNAi inducers can target HIV-1 sequences in the genomic RNA of the lentiviral vector. As the RNAi-inducing cassette contains palindromic sequences, the lentiviral vector RNA genome will have a perfect target sequence for the expressed RNAi inducer. Vectors encoding microRNAs face the putative problem that the vector RNA genome can be inactivated by Drosha processing. Here, we describe the design of lentiviral vectors with single or multiple RNAi-inducing antiviral cassettes. The possibility of titer reduction and some effective countermeasures are also presented.

RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy.