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CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice.

ABSTRACT: In this preclinical study, we evaluated the efficacy and feasibility of creating broad human immunodeficiency virus (HIV) resistance by simultaneously disrupting the human CCR5 and CXCR4 genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient ex vivo delivery of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of CCR5 and CXCR4 in T lymphocyte cells significantly reduced surface expression of the co-receptors, thereby establishing resistance to HIV-1 infection by CCR5 (R5)-tropic, CXCR4 (X4)-tropic, and dual (R5/X4)-tropic strains. Similarly, disruption of CCR5 alleles in human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) successfully led to the differentiation of HIV-resistant macrophages. In a humanized mouse model under HIV-1 challenge, CXCR4-disrupted CD4⁺ T cells were enriched in the peripheral blood and spleen, indicating survival advantage because of resistance to viral infection. However, in human CD4⁺ T cells with both CCR5 and CXCR4 disruption, we observed poor engraftment in bone marrow, although significant changes were not observed in the lung, spleen, or peripheral blood. This study establishes a clinically scalable strategy for the dual knockout of HIV-1 co-receptors as a therapeutic strategy, while also raising caution of disrupting CXCR4, which may abate engraftment of CD4⁺ T cells in bone marrow.

SUBMITTER: Li S

PROVIDER: S-EPMC8847835 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice.

Li Shasha S Holguin Leo L Burnett John C JC

Molecular therapy. Methods & clinical development 20220122

In this preclinical study, we evaluated the efficacy and feasibility of creating broad human immunodeficiency virus (HIV) resistance by simultaneously disrupting the human CCR5 and CXCR4 genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient ex vivo delivery of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of CCR5 and CXCR4 in T lymphocyt ...[more]

PMID: 35229006

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Project description:BackgroundA 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.MethodsA 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.FindingsVirus 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.InterpretationThese 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.FundingThe 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.

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CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice.

Publications

CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice.

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