Dataset Information

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.

ABSTRACT: A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4⁺ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4⁺ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4⁺ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.

SUBMITTER: Romano Ibarra GS

PROVIDER: S-EPMC5023401 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Publications

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.

Romano Ibarra Guillermo S GS Paul Biswajit B Sather Blythe D BD Younan Patrick M PM Sommer Karen K Kowalski John P JP Hale Malika M Stoddard Barry B Jarjour Jordan J Astrakhan Alexander A Kiem Hans-Peter HP Rawlings David J DJ

Molecular therapy. Nucleic acids 20160823 8

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4<sup>+</sup> T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in ...[more]

PMID: 27741222

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Project description:We present here an extensive epigenetic analysis of a 500 kb region, which encompasses the human desmin gene (DES) and its 5' locus control region (LCR), the only muscle-specific transcriptional regulatory element of this type described to date. These data complement and extend Encyclopaedia of DNA Elements (ENCODE) studies on region ENr133. We analysed histone modifications and underlying DNA methylation patterns in physiologically relevant DES expressing (myoblast/myotube) and non-expressing (peripheral blood mononuclear) primary human cells.We found that in expressing myoblast/myotube but not peripheral blood mononuclear cell (PBMC) cultures, histone H4 acetylation displays a broadly distributed enrichment across a gene rich 200 kb region whereas H3 acetylation localizes at the transcriptional start site (TSS) of genes. We show that the DES LCR and TSS of DES are enriched with hyperacetylated domains of acetylated histone H3, with H3 lysine 4 di- and tri-methylation (H3K4me2 and me3) exhibiting a different distribution pattern across this locus. The CpG island that extends into the first intron of DES is methylation-free regardless of the gene's expression status and in non-expressing PBMCs is marked with histone H3 lysine 27 tri-methylation (H3K27me3).Overall, our results constitute the first study correlating patterns of histone modifications and underlying DNA methylation of a muscle-specific LCR and its associated downstream gene region whilst additionally placing this within a much broader genomic context. Our results clearly show that there are distinct patterns of histone H3 and H4 acetylation and H3 methylation at the DES LCR, promoter and intragenic region. In addition, the presence of H3K27me3 at the DES methylation-free CpG only in non-expressing PBMCs may serve to silence this gene in non-muscle tissues. Generally, our work demonstrates the importance of using multiple, physiologically relevant tissue types that represent different expressing/non-expressing states when investigating epigenetic marks and that underlying DNA methylation status should be correlated with histone modification patterns when studying chromatin structure.

Dataset Information

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.

Publications

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.

Similar Datasets

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