Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease
Open accessAbstract
Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.
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It states that AAV6 donor templates with homology arms efficiently deliver donor DNA into sensitive primary immune cells such as HSPCs and T cells.
“AAV6 homologous donor DNA templates are an efficient way to deliver donor templates for sensitive primary immune cells, including HSPCs and T-Cells15,35–40.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [37]
The Methods section (AAV6 Donor Construction and Virus Production; page unavailable) describes AAV donor constructs flanked by homology arms. The results report efficient targeted integration in primary human HSPCs using AAV6 donor delivery. Our copy does not report AAV6 donor delivery in T cells, so it supports the HSPC example but not the T-cell example. (LLM, conf=0.87)
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References 26 · Cited by 195
- Authors
- De Ravin, S. S., Reik, A., Liu, P.-Q., Li, L., Wu, X., Su, L., Raley, C., Theobald, N., Choi, U., Song, A. H., Chan, A., Pearl, J. R., Paschon, D. E., Lee, J., Newcombe, H., Koontz, S., Sweeney, C., Shivak, D. A., Zarember, K. A., Peshwa, M. V., Gregory, P. D., Urnov, F. D., & Malech, H. L.
- Affiliations
- Sangamo BioSciences (United States) (US)
- MaxCyte (United States) (US)
- Leidos Biomedical Research Inc. (United States) (US)
- National Institutes of Health (US)
- Corresponding author
- Malech, Harry L
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2016-03-07
- Volume
- 34
- Issue
- 4
- Pages
- 424-429
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4824656
- OpenAlex
- W2293120410
- Cited by
- 195
- References
- 26
- Citations by year
- 2026: 6 · 2025: 7 · 2024: 18 · 2023: 11 · 2022: 13 · 2021: 25 · 2020: 33 · 2019: 31 · 2018: 18 · 2017: 21 · 2016: 12
- FWCI
- 22.08
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health
- National Institute of Allergy and Infectious Diseases (Z01-AI-00988, Z01-AI-00644, Z01AI000644, Z01AI000988, ZIAAI000644)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- targeted gene integration, AAVS1 safe harbor, X-linked chronic granulomatous disease, CD34+ hematopoietic stem and progenitor cells, zinc finger nucleases, gp91phox, NADPH oxidase, gene therapy, electroporation, transgene delivery, monogenic disorders
- MeSH terms
- Animals, Cells, Cultured, Granulomatous Disease, Chronic, Hematopoietic Stem Cells, Humans, Mice, Transgenic, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Antigens, CD34, Mice
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