Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4⁺ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4⁺ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4⁺ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.