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

• Published in: Molecular therapy. Nucleic acids 2016, Vol.5 (8), p.e352-e352, Article e352
• Main Authors: Romano Ibarra, Guillermo S, Paul, Biswajit, Sather, Blythe D, Younan, Patrick M, Sommer, Karen, Kowalski, John P, Hale, Malika, Stoddard, Barry, Jarjour, Jordan, Astrakhan, Alexander, Kiem, Hans-Peter, Rawlings, David J
• Format: Article
• Language: English
• Subjects: CCR5; gene editing; Gene therapy; Genetic engineering; HIV; Human immunodeficiency virus; Immune system; Lentivirus; Lymphocytes; megaTAL; Original; Retroviridae; Ribonucleic acid; RNA
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1038/mtna.2016.56

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.