Engineering T cell resistance to HIV-1 infection via knock-in of peptides from the heptad repeat 2 domain of gp41

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we cloned a series of HIV-1 fusion inhibitory peptides and embedded them in CD52, the shortest GPI-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. Unlike membrane-bound peptides, the shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentrations in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and cell sorting with antibodies raised against gp41 peptides, we generated CEM/R5 cells with biallelic KI of MT-C34 (embedded in CD52 for expression in lipid rafts) and 2P23 (N-terminally fused to CXCR4). In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and substituting PCR donor DNA with a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into CXCR4 loci and HIV-1 proviral DNA at levels of up to 35% in CEM/R5 cells and increased KI occurrence from undetectable to 4-5% in CD4 lymphocytes. Comparative analysis of lentiviral and HDR-based delivery strategies showed that KI led to the higher MT-C34 expression and stronger protection of primary CD4 lymphocytes from HIV-1 than lentiviral transduction, albeit the efficiency of KI needs further improvements in order to meet clinical requirements. Thus, the developed CRISPR/Cas9 platform offers a new opportunity for antiviral peptide delivery with a concomitant precise genetic modification of targeted locus that can be employed to strengthen cell protection against HIV. AUTHOR SUMMARY HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as acquired immunodeficiency syndrome. Antiretroviral therapy (ART) is not leading to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2-peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1. ### Competing Interest Statement The authors have declared no competing interest.