Investigating the feasibility of incorporating Vpx into lentiviral gene therapy vectors: at a global and targeted scale

Lentiviral gene therapy has the potential to target HIV-1 reservoirs. However, a major obstacle in effectively targeting the HIV-1 latent reservoir is the lack of efficient gene delivery that occurs within cells. The viral protein Vpx from the SIVSM/HIV-2 lineage is capable of increasing lentiviral reverse transcription (RTn); by antagonizing the restriction factor SAMHD1. This study aimed to investigate the feasibility of incorporating Vpx into lentiviral vectors. Primary macrophages and dendritic cells were utilised as resting cell models. Firstly, we determined the short and long-term effects post-Vpx exposure. Results revealed Vpx exposure led to increased permissiveness of cells to HIV-1 infection over a period that exceeded two weeks and was associated with lower potency of HIV antiretrovirals that target HIV RT or integration. Secondly, we incorporated Vpx with lentiviral gene therapy approaches that target HIV pre- and post-RTn and determined whether the inclusion of Vpx increased their effectiveness. When Vpx was incorporated into anti-HIV gene therapy constructs, transduction levels were significantly increased. However, increase in gene delivery only translated into an increase in HIV protection with constructs that inhibit pre-RTn phases of the virus life cycle. Thirdly, we utilised proteomics to determine any hidden phenotypes, in macrophages and dendritic cells, post-Vpx exposure, which could affect the usage of Vpx in gene therapy protocols. Significant protein changes were further investigated by generating a panel of THP1 knockdown/overexpression cell lines and examining whether individual changes, similar to SAMHD1 depletion, could increase HIV-1 infection. No detrimental hidden phenotypes were revealed in both macrophages and dendritic cells. Importantly, two knockdown cell lines, CALR and FKBP5, were shown to enhance HIV-1, in the absence of Vpx. In conclusion, this study supports the use of Vpx with gene therapy vectors as a way to overcome the major genetic delivery obstacle presented in resting cells. This effect was the greatest when incorporating anti-HIV constructs that target HIV-1 prior to reverse transcription. Importantly this approach can target cells of the existing HIV-1 latent reservoir or in resting cells that require HIV-1 protection