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

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA <i>trans</i>-splicing at the HIV-1 D4 donor site. AUG1-deficient <i>HSVtk</i> therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 <i>tat1</i>. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where <i>trans</i>-spliced HIV-1 <i>tat</i>/<i>HSVtk</i> mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in <i>HSVtk</i> _ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted <i>in silico</i> correlated with the propensity of opt 2 payloads to induce HIV-1 RNA <i>trans</i>-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.