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Cited by 223CRISPR and Genetic EngineeringVirus-based gene therapy researchCAR-T cell therapy research

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

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Sather, Blythe D., Romano Ibarra, Guillermo S., Sommer, Karen, Curinga, Gabrielle, Hale, Malika, Khan, Iram F., Singh, Swati, Song, Yumei +7 more·2015·Science Translational Medicine
DOI 10.1126/scitranslmed.aac5530PMC PMC4790757
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Abstract

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

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Authors
Sather, B. D., Romano Ibarra, G. S., Sommer, K., Curinga, G., Hale, M., Khan, I. F., Singh, S., Song, Y., Gwiazda, K., Sahni, J., Jarjour, J., Astrakhan, A., Wagner, T. A., Scharenberg, A. M., & Rawlings, D. J.
Affiliations
  • Seattle Children's Research Institute (US)
  • Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children’s Research Institute, Seattle, WA 98101, USA.
  • Bluebird Bio (United States) (US)
  • University of Washington (US)
Corresponding author
  • Sather, Blythe D.
  • Scharenberg, Andrew M.
  • Rawlings, David J.
ORCID
Venue
Science Translational Medicine
Publisher
American Association for the Advancement of Science (AAAS)
Published
2015-09-30
Volume
7
Issue
307
Pages
307ra156-307ra156
ISSN
  • 1946-6234 (ISSN-L)
  • 1946-6242
DOI
10.1126/scitranslmed.aac5530
PMC
PMC4790757
OpenAlex
W2229738114
Cited by
223
References
9
Citations by year
2026: 9 · 2025: 9 · 2024: 13 · 2023: 17 · 2022: 19 · 2021: 15 · 2020: 17 · 2019: 28 · 2018: 27 · 2017: 28 · 2016: 35 · 2015: 6
FWCI
13.90
Type
journal article
Language
en
Open access
Licence
www.sciencemag.org/about/science-licenses-journal-article-reuse (vor, 366 d)
Locations
  • Science Translational Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Heart, Lung, and Blood Institute
  • National Institute of General Medical Sciences (PL1 HL092557, R42 GM085876)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Health Sciences › Medicine › Oncology › CAR-T cell therapy research
Keywords
CCR5, HIV drug resistance, meganucleases, TALEN, adeno-associated virus vectors, homology-directed recombination, CD4+ T cells, chimeric antigen receptor T cells, CD34+ hematopoietic stem cells, genome editing, HIV treatment
MeSH terms
Dependovirus, Adult, Cells, Cultured, Deoxyribonucleases, DNA Repair, Hematopoietic Stem Cells, Humans, Receptors, Antigen, T-Cell, T-Lymphocytes, Genetic Therapy, CD3 Complex, RNA Editing, Antigens, CD34, Receptors, CCR5, Green Fluorescent Proteins, Genetic Loci

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