Abstract
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.
Highlights
Introduction published maps and institutional affilGenetic editing of T cells continues to show great promise in many clinical efforts
In our efforts in finding pragmatic solutions for gene therapy towards a HIV cure, we present an efficient gene delivery and editing platform for resting CD4+ T cells
As we have shown, lentiviral vectors often have difficulties transducing certain cell types, which limits the range of diseases that can be successfully treated using gene/cell therapy
Summary
Introduction published maps and institutional affilGenetic editing of T cells continues to show great promise in many clinical efforts. The delivery of modified lentiviral vectors is proceeded at very high multiplicities of infection (MOI) to overcome many lentiviral limitations Whilst this enables genetic manipulation of T cells, this does come at a cost, as cell “molding” lowers the future proliferation and engraftment in vivo [4,5] and the use of very high viral MOIs can often render many approaches cost-prohibitive at the clinic. Both can impact treatment at the clinic. The potency and efficacy of CAR T cells has been shown to rapidly iations
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