Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection

Abstract

Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.

Keywords: B cells; C-peptides; CD4 T cells; CRISPR/Cas; HIV; co-receptors; knock-in; restriction factors.

Figure 1

A flowchart showing CRISPR/Cas anti-HIV approaches used to protect cells from HIV infection. Depending on the gene-editing techniques (shown on top of each block) and cellular or viral genes selected for targeting (shown below) they were divided into five groups. Some strategies may overlap. For example, gene knockout or provirus deletion can be accompanied by targeted integration of a therapeutic gene; all of them are inheritable and effects are stable in comparison to CRISPR-based transcription regulation. This image was created with BioRender.

Figure 2

Perspective HDR-based strategies that provide strong and broad protection against HIV. (A) Mature B lymphocytes engineered via knock-in of bNAb into IgH locus are capable to proliferate after adoptive transfer to syngeneic mice and immunization with cognate antigen and produce a high titer of anti-HIV Ig of different classes. Hopefully, preclinical trials on primates and clinical studies will be successful as well. The concept of full-length light chain and variable domain heavy chain knock-in-out at IgH locus was adapted from the papers (Hartweger et al., 2019; Nahmad et al., 2020). The single-chain antibody was introduced between VDJ and C segments that preserves mechanism of Ig class switch, while additional IgK gene KO prevents mispairing of light chain with transgenic heavy chain. (B). Knock-in-out of MT-C34 peptide from gp41 protein into the CXCR4 locus with a parallel ablation of CCR5 gene ensures full tropism-independent protection of engineered CD4⁺ T cells from HIV. (C). A combination of secreted and GPI-anchored forms of MT-C34 via peptide concatemeric construct knocked-in into CXCR4 gene will provide the selection of CD4⁺ T cells that are resistant to HIV and secrete the MT-C34 peptide which will shield non-edited cells from HIV infection. The GPI-anchored C-peptide KI concept was described previously in our paper (Maslennikova et al., 2022) and here it was complemented with CCR5 KO and peptide secretion strategy which was reported earlier and called SAVE (Egerer et al., 2011). The symbol and pictogram labels (grouped for panels B, C) are shown at the bottom of pictures. All images were created with BioRender.