The therapeutic application of CRISPR/Cas9 technologies for HIV

Abstract

Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system.

Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus.

Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

Keywords: CRISPR/Cas9; HIV-1; RNA therapy; gene activation; gene editing; gene excision; latency.

Figure 1. The life cycle of HIV with targeted intervention points

HIV binds to the cell surface via the CD4+ receptor and the CCR5/CXCR4 co-receptors. Following fusion with the cell membrane the viral particle enters the cell and its genome is transcribed from RNA to DNA. It is the DNA that is integrated into the host cell genome, and that provides the template to drive transcription of HIV RNA, producing progeny virions that bud off from the cell, completing the infectious cycle. Nuclease-directed disruption of the HIV life cycle could occur at any of the following stages: A. Targeting and preventing the integration of proviral DNA into the genome. B. Proviral DNA, once integrated into the genome, is a target for excision, or deactivation by mutagenic disruption. C. Cellular factors necessary for the HIV life cycle present further targets, and include the co-receptor CCR5 or other host dependency factors.

Figure 2. Mechanisms of CRISPR/Cas9-directed cleavage

The Cas9 protein forms a complex with a sgRNA, which guides the nuclease to a specific genomic address for cleavage. A. Cas9 catalyzed DNA cleavage is guided by a 17-20 nucleotide sequence within the sgRNA. B. The Cas9 protein “scans” genomic DNA for regions of homology with the guide sequence , where it unwinds the DNA and its nuclease domain directs site specific cleavage . This results in deletions generated by non-homologous end joining (NHEJ) at the binding site, or in homologous-dependent repair (HDR) (reviewed in ).

Figure 3. Targeting the HIV genome

The HIV provirus is flanked by identical viral long terminal repeat (LTR) sequences. Therefore CRISPR/Cas9 targeted to the LTR could cleave at both ends of the virus. DNA repair of the excised region between the cleavage sites would result in a single LTR “footprint” within the genome providing a reference to identify the position of the HIV proviral DNA ^{, ,} . Guide RNAs targeting two or more sites within the 5' LTR can result in loss of promoter activity, leading to deactivation of the provirus . Guide RNAs can also be targeted to specific viral reading frames, causing indels that affect viral protein function, and concomitant virion production .

Figure 4. Strategies to activate HIV provirus using CRISPR/Cas9

Gene specific transcriptional activation using engineered forms of CRISPR/Cas9 targeted to the activation “hotspot” within the 200bp upstream of the HIV proviral transcriptional start site (TSS). A. Nuclease deficient mutant of Cas9 (dCas9) fused to a C-terminal VP64 activation domain, which is boosted using multiple sgRNAs are targeted to this region . B. A modified dCas9 that uses a polypeptide scaffold termed SunTag, can recruit multiple antibody-fusion proteins resulting in enhanced activation . C. Aptamers that selectively bind to the dimerized MS2 bacteriophage coat protein can create a synergistic activation mediator (SAM) system that can recruit multiple activation domains.

Figure 5. The different CRISPR/Cas9 therapeutic approaches against HIV

New HIV infection can be blocked by directly targeting the (A) pre-integrated proviral dsDNA (and possibly 2-LTR circular DNA) ^, and (B) by disrupting early-stage host dependency factors such as the co-receptor CCR5 ^-, . By targeting the integrated provirus for cleavage, downstream viral production can be blocked by (C) excision of the viral genome by targeting the LTRs (or using multiple sgRNAs) ^- and (D) by disrupting viral genes thereby preventing viral genome assembly and budding . Lastly, by targeting upstream regulatory sequences using CRISPR/Cas9-based activators in latent cells, subsequent viral output can be restricted by ART and immune clearance in a “shock and kill” approach.