CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination

Jonathan Herskovitz, Mahmudul Hasan, Milankumar Patel, Wilson R. Blomberg, Jacob D. Cohen, Jatin Machhi, Farah Shahjin, R. Lee Mosley, Jo Ellyn McMillan, Bhavesh D. Kevadiya, Howard E. Gendelman

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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 (tat1-2/rev1-2/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.

Original languageEnglish (US)
Article number103678
JournalEBioMedicine
Volume73
DOIs
StatePublished - Nov 2021
Externally publishedYes

Keywords

  • CRISPR delivery
  • Clustered regularly interspaced short palindromic repeat
  • Human immunodeficiency virus type one
  • Latent infection
  • RNA loaded Lipid nanoparticles (rLNP)
  • Viral eradication

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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