Development of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells

Only two patients have ever been cured of HIV-1. The latent HIV-1 reservoir is the major roadblock to this and necessitates lifelong therapy. In the ‘shock and kill’ approach to eliminate cells harbouring dormant virus the latency reversing agent (LRA) vorinostat increased HIV-1 RNA levels, but this failed to enable a reduction in reservoir size by immune- or virus-mediated cytotoxicity. To leverage LRAs’ ability to heighten HIV-1 transcription, we are developing a ‘kill’ strategy that hijacks the HIV-1 splicing process with a therapeutic trans-splicing RNA, encoding an incomplete Herpes simplex virus thymidine kinase (HSV-tk) that gains functionality by trans-splicing onto HIV-1 tat pre-mRNA. HSV-tk activates the exogenous prodrug ganciclovir for selective killing of HIV-1-infected cells. Following proof-of-principle transfection studies, therapeutic constructs were engineeredfor lentivirus-mediated delivery to HIV-infected cells in vitro. We optimized lentiviral production for high infectious titer (>106 TU/mL) and low vector plasmid carryover (<1 copy/cell). In a tissue culture model of HIV-1 infection we confirmed lentiviral delivery of therapeutic (and control) vectors. Successful trans-splicing of the HSV-tk RNA onto HIV-1 RNA in cells co-transduced with HIV-1 and therapeutic vector at an MOI as low as 4 was confirmed by sequencing. In MTT assays the most potent therapeutic vector killed approximately 80% of HIV-1-expressing cells. Next-generation vectors are being evaluated for enhanced therapeutic potential and improved HIV-1-targeting RNA expression. The lead candidate culminating from this work will be assessed for selective killing of HIV-1-infected cells both productively infected and with virus reactivated from latency.