Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41

• Published in: mBio 2022-02, Vol.13 (1), p.e0358921-e0358921
• Main Authors: Maslennikova, Alexandra, Kruglova, Natalia, Kalinichenko, Svetlana, Komkov, Dmitriy, Shepelev, Mikhail, Golubev, Dmitriy, Siniavin, Andrei, Vzorov, Andrei, Filatov, Alexander, Mazurov, Dmitriy
• Format: Article
• Language: English
• Subjects: Antiviral Agents - pharmacology; CD4-Positive T-Lymphocytes; CD52; CRISPR/Cas9; fusion inhibitors; gp41; GPI proteins; HIV Envelope Protein gp41 - chemistry; HIV Infections; HIV Seropositivity; HIV-1 - genetics; Humans; knock-in; Peptide Fragments - chemistry; Peptides - pharmacology; Research Article; Virology
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.03589-21

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 embedded a series of HIV-1 fusion inhibitory peptides in CD52, the shortest glycosylphosphatidylinositol (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. The shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentration in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and peptide-specific antibodies, we sorted CEM/R5 cells with biallelic KI of MT-C34 and 2P23 peptides at the locus. In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and cloning donor DNA into a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into the locus and HIV-1 proviral DNA at levels of up to 35% in the T-cell line and up to 4 to 5% in primary CD4 lymphocytes. Compared to lentiviral delivery, KI resulted in the higher MT-C34 surface expression and stronger protection of lymphocytes from HIV-1. Thus, we demonstrate that KI is a viable strategy for peptide-based therapy of HIV infection. HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as AIDS. Antiretroviral therapy (ART) does not lead 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 modificatio