CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination

Abstract

Background A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. Methods A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons ( tat 1-2 / rev 1-2 / gp41 ) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. Findings Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. Interpretation These results provide “proof of concept’ for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. Funding The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Inhibition of HIV-1 replication using the CRISPR/cas9-no NLS system as a prophylactic strategy

“…Studies have demonstrated that using lentiviral-based CRISPR-Cas9 and several gRNA targets for different regions of HIV-1 provirus DNA has reduced HIV-1 replication by more than 96% [ 59 ]. Thus, the use of viral vectors to express Cas9 and multiple gRNA targets can be more successful in targeting HIV-1 provirus DNA [ 63 , 64 , 65 ]. Some studies have suggested the concomitant use of CRISPR/Cas9 and antiretroviral drugs or RNA interference (RNAi) [ 66 ].…”

“…Thus, the use of viral vectors to express Cas9 and multiple gRNA targets can be more successful in targeting HIV-1 provirus DNA [63][64][65]. Some studies have suggested the concomitant use of CRISPR/Cas9 and antiretroviral drugs or RNA interference (RNAi) [66].…”

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

“…Each method has advantages and disadvantages, and depending on the intended application and target cell type, the right delivery method should be selected. These methods can be based on viral delivery [ 51 , 52 , 53 , 54 ] or lipid vector delivery of the CRISPR/Cas9 system [ 55 ], but genome editing can also be facilitated through fusion of the Cas9 protein to cell-penetrating peptides [ 56 ], or by directly electroporating the CRISPR/Cas9 system into cells of interest [ 27 , 55 , 57 , 58 ]. Interestingly, recently, the mechanism HIV-1 employs to target CD4 + T cells has been repurposed as a tool to direct viral particles containing the Cas9 system specifically to cells expressing CD4, i.e., T cells [ 59 ], opening up new potential avenues with this addition to the CRISPR/Cas9 toolbox [ 60 ], e.g., in vivo gene editing of CD4 + T cells.…”

“…CRISPR could also be used to directly target the HIV-1 genome and remove it from infected cells, for instance by targeting the HIV viral promoter long terminal repeat [ 55 ]. Importantly, in vitro experiments showed up to 100% viral excision [ 55 ]. While currently not employed as a therapeutic strategy yet, the approach is interesting.…”

Promising Stem Cell therapy in the Management of HIV and AIDS: A Narrative Review

“…In the future, CRISPR/SaCas9 and Ribonucleoprotein (RNP) administration should be explored in the further investigations. 108 As well as some different title studies were explained the effectiveness of the CRISPR gene editing technology on the management of HIV/AIDS including: CRISPR view of hematopoietic stem cells: Moving innovative bioengineering into the clinic, 104 CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukaemia, 109 Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice, 110 Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA, 111 HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells, 112 CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination, 113 RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection, 114 CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Pro viral DNA Circles with Reformed Long Terminal Repeats, 115 CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice, 116 Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9, 117 Transient CRISPR-Cas Treatment Can Prevent Reactivation of HIV-1 Replication in a Latently Infected T-Cell Line, 118 CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection, 119 CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo. 109 …”