Selection of RNAi-based inhibitors for anti-HIV gene therapy

In the last decade, RNA interference (RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAi-based inhibitors against the chronic infection with human immunodeficiency virus type 1 (HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules (shRNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 shRNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three shRNAs. These shRNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome, exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial shRNA vector will soon move forward to the first clinical studies.