Gene editing using a zinc-finger nuclease mimicking the CCR5Δ32 mutation induces resistance to CCR5-using HIV-1

Badía, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Esté, José A.; Ballana, Ester

doi:10.1093/jac/dku072

Cited by 27 publications

(17 citation statements)

References 15 publications

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“…Owing to the indispensable role of CCR5 for HIV-1 entry and the natural presence of the CCR5 D32 variant, a number of gene editing studies have targeted CCR5 by various approaches (Badia et al, 2014;Holt et al, 2010;Huang et al, 1996;Mussolino et al, 2014;Perez et al, 2008;Saito et al, 2014;Ye et al, 2014). In a recently finished Phase I clinical trial, autologous CD4 + T-cells with CCR5 modified by ZFN showed promise in alleviating the disease condition of some HIV-1 patients (Tebas et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Guan

et al. 2015

Journal of General Virology

184

139

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CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5 D32 variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4 + T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4 + Tlymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4 + T-cells utilizing adenovirus-delivered CRISPR/Cas9.

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Section: Discussionmentioning

confidence: 99%

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Guan

et al. 2015

Journal of General Virology

184

139

View full text Add to dashboard Cite

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“…Another study demonstrated safety and feasibility of ZFN-CCR5-modified autologous CD4 + T cell infusions in HIV-infected patients [31 ]. More recently, an optimized ZFN producing CCR5 modifications within the D32 region has been designed that confers high resistance against CCR5-tropic HIV infection with no significant offtarget activity [32]. Collectively, these studies have established the artificial CCR5 gene disruption by ZFNs systems followed by autologous cell transplantation as a reasonable and promising approach for the development of new strategies for HIV treatment.…”

Section: Host Genome Editingmentioning

confidence: 99%

CCR5Δ32 mutation and HIV infection: basis for curative HIV therapy

Allers

Schneider

2015

Current Opinion in Virology

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“…Since the unique and exceptional case of an HIV-1 sterilizing cure of a patient due to bone marrow transplantation with a matched donor homozygote for the CCR5⌬32 mutation (47,48), alternative strategies have aimed to reproduce the CCR5⌬32 phenotype using genome-editing tools. Indeed, ZFNs targeting the HIV coreceptor gene have been successfully developed to generate human CD4 ϩ T cells and human embryonic cell precursors and induce pluripotent stem cells that were refractory to HIV-1 infection in different mouse models (9)(10)(11)(12)(13).…”

Section: Discussionmentioning

confidence: 99%

“…The effect of ZFN on the PSIP1 alleles was assessed by performing PCR on the region surrounding the ZFN target site, followed by digestion with the Surveyor (CEL-1) nuclease assay (Transgenomic, Omaha, NE, USA), which cleaves DNA heteroduplex formations at the mismatch sites. The generated fragments were resolved by 10% polyacrylamide electrophoresis, as previously described (10,12).…”

Section: Vectorsmentioning

confidence: 99%

“…The development of genome-editing tools, such as zinc finger endonucleases (ZFNs) (2), transcription activator-like effector nucleases (TALEN) (3), and clustered regularly interspaced short palindromic repeats (CRISPR) (4)(5)(6), has introduced a promising alternative for the modification of essential host factors along the replication cycle of HIV (7,8). ZFNs have demonstrated their applicability to reproduce the CCR5⌬32 phenotype in vitro by successfully cleaving the CCR5 gene, generating human CD4 ϩ T cells refractory to HIV-1 infection (9)(10)(11)(12). Similarly, a ZFN approach successfully cleaved the alternative HIV-1 coreceptor CXCR4 in CD4 ϩ T cells in a humanized mouse model, resulting in impaired HIV-1 infection (13).…”

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confidence: 99%

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Zinc Finger Endonuclease Targeting PSIP1 Inhibits HIV-1 Integration

Badía

Pauls

Riveira-Muñoz

et al. 2014

Antimicrob Agents Chemother

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Genome editing using zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and infection. Gene therapy using ZFNs to modify the PSIP1 gene, which encodes the lens epithelium-derived growth factor (LEDGF) protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFN LEDGF ) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of the LEDGF/p75 protein. ZFN LEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of LEDGF expression at the mRNA and protein levels. Functional assays revealed that infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual infection was detected in the LEDGF knockout cells. Indeed, LEDGF knockout restriction was overcome at a high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through LEDGF/p75. However, the observed residual integration was sensitive to the integrase inhibitor raltegravir. These results demonstrate that the described ZFN LEDGF effectively targets the PSIP1 gene, which is involved in the early steps of the viral replication cycle; thus, ZFN LEDGF may become a potential antiviral agent for restricting HIV-1 integration. Moreover, LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.

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Gene editing using a zinc-finger nuclease mimicking the CCR5Δ32 mutation induces resistance to CCR5-using HIV-1

Abstract: ZFNCCR5Δ32 is a specific and efficient tool for the generation of CCR5 knockouts. Its ability to mimic the natural CCR5Δ32 phenotype in the absence of relevant off-site cutting events suggests that ZFNCCR5Δ32 might be safe in clinical research.

Cited by 27 publications

References 15 publications

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

CCR5Δ32 mutation and HIV infection: basis for curative HIV therapy

Zinc Finger Endonuclease Targeting PSIP1 Inhibits HIV-1 Integration

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