Pathways Toward a Functional HIV-1 Cure: Balancing Promise and Perils of CRISPR Therapy

Abstract

First identified as a viral defense mechanism, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) has been transformed into a gene-editing tool. It now affords promise in the treatment and potential eradication of a range of divergent genetic, cancer, infectious, and degenerative diseases. Adapting CRISPR-Cas into a programmable endonuclease directed guide RNA (gRNA) has attracted international attention. It was recently awarded the 2020 Nobel Prize in Chemistry. The limitations of this technology have also been identified and work has been made in providing potential remedies. For treatment of the human immunodeficiency virus type one (HIV-1), in particular, a CRISPR-Cas9 approach was adapted to target then eliminate latent proviral DNA. To this end, we reviewed the promise and perils of CRISPR-Cas gene-editing strategies for HIV-1 elimination. Obstacles include precise delivery to reservoir tissue and cell sites of latent HIV-1 as well as assay sensitivity and specificity. The detection and consequent excision of common viral strain sequences and the avoidance of off-target activity will serve to facilitate a final goal of HIV-1 DNA elimination and accelerate testing in infected animals ultimately for use in man.

Keywords: CRISPR-Cas; CRISPR-associated proteins; RNA.

Figure 1.. CRISPR Mechanisms and Potential Pitfalls.

(A) Pathways by which CRISPR can interdict HIV-1 replication in CD4+ T cells and MP are outlined. They include: (1) direct targeting of integrated proviral HIV-1 DNA; (2) excision of viral entry receptors and or co-receptors and modulation of host proteins linked to (3a) innate immunity, (3b) DNA transcription, (3c) endosomal processing (that is hijacked by HIV-1), and (3d) active viral transport. (B) CRISPR can mediate a functional HIV-1 cure. A cure must overcome a number of barriers. These include: (1) potential cleavage of host genes bearing homology to HIV-directed guide RNAs; (2) non-frameshift mutagenesis of proviral DNA that may lead to CRISPR-resistant HIV-1; (3) delivery of CRISPR to infected cells leading to (4) viral breakthrough and re-seeding of cell and tissue HIV-1 reservoirs.

Figure 2.. Combination ART and CRISPR-Cas9 Therapies for HIV-1 Elimination.

Viral entry and fusion require the CD4 receptor and CCR5 and CXCR4 coreceptors at the cell surface. Upon entering into virus susceptible CD4+ T cells and macrophages viral RNA is reverse transcribed into double-stranded circular DNA and integrated into host chromosomal DNA. The integrated HIV-1 provirus can remain quiescent. ART drug combinations serve to suppress virus production and target distinct stages of the HIV life cycle. Highly specific gRNA based CRISPR-Cas9 targets integrated proviral HIV-1 DNA. The CRISPR-Cas9 utilizes ~100 bp of gRNA to facilitate the Cas9 endonuclease activities at viral integration sites. This made complex then recognizes and cleaves a 20 bp double-stranded DNA target site (known as protospacer DNA) that is complementary to the 5′ end of the gRNA. The double-strand break excises the HIV-1 DNA from the host genome, which gets repaired by non-homologous end-joining to restore the break. This combination of ART and CRISPR-Cas9 has so far achieved viral elimination from a subset of HIV infected animals. Reprinted with permission from Ebiomedicine.