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Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

ABSTRACT: Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4⁺ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4⁺ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4⁺ T cell and up to 88% of CD34⁺ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4⁺ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

SUBMITTER: Knipping F

PROVIDER: S-EPMC8753564 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

Knipping Friederike F Newby Gregory A GA Eide Cindy R CR McElroy Amber N AN Nielsen Sarah C SC Smith Kyle K Fang Yongxing Y Cornu Tatjana I TI Costa Caroline C Gutierrez-Guerrero Alejandra A Bingea Samuel P SP Feser Colby J CJ Steinbeck Benjamin B Hippen Keli L KL Blazar Bruce R BR McCaffrey Anton A Mussolino Claudio C Verhoeyen Els E Tolar Jakub J Liu David R DR Osborn Mark J MJ

Molecular therapy : the journal of the American Society of Gene Therapy 20211102 1

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4<sup>+</sup> T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary ...[more]

PMID: 34737067

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Project description:BackgroundDuring the HIV-1 replication cycle, several molecules including chemokine receptors and cholesterol are crucial, and are therefore potential targets for therapeutic intervention. Indeed statins, compounds that inhibit cellular synthesis of cholesterol and have anti-inflammatory and immunomodulatory properties were shown to inhibit HIV-1 infection by R5 tropic strains but not by X4 strains in vitro, mainly by altering the chemokine receptor/ligands axes. Therefore, the objective of this study was to characterize in vivo, the capacity of statins to modulate in HIV seronegative and chronically HIV-1-infected adults the expression of CCR5 and CXCR4, of their ligands and the tropism of circulating HIV-1 strains.MethodsSamples from asymptomatic HIV-1-infected adults enrolled in a clinical trial aimed at evaluating the antiretroviral activity of lovastatin were used to evaluate in vivo the modulation by lovastatin of CCR5, CXCR4, their ligands, and the shift in plasma viral tropism over one year of intervention. In addition, ten HIV negative adults received a daily oral dose of 40 mg of lovastatin or 20 mg of atorvastatin; seven other HIV negative individuals who received no treatment were followed as controls. The frequency and phenotype of immune cells were determined by flow-cytometry; mRNA levels of chemokine receptors and their ligands were determined by real-time PCR. Viral tropism was determined by PCR and sequencing, applying the clonal and clinical model of analyses.ResultsOur study shows that long-term administration of lovastatin in HIV-infected individuals does not induce a shift in viral tropism, or induce a significant modulation of CCR5 and CXCR4 on immune cells in HIV-infected patients. Similar results were found in HIV seronegative control subjects, treated with lovastatin or atorvastatin, but a significant increase in CCL3 and CCL4 transcription was observed in these individuals.ConclusionsThese findings suggest that long-term administration of statins at therapeutic doses, does not significantly affect the expression of HIV-1 co-receptors or of their ligands. In addition it is important to point out that based on the results obtained, therapeutic administration of statins in HIV-infected patients with lipid disorders is safe in terms of selecting X4 strains.

Dataset Information

Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

Publications

Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

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