Highly efficient in vivo hematopoietic stem cell transduction using an optimized self-complementary adeno-associated virus
<i>In vivo</i> gene therapy targeting hematopoietic stem cells (HSCs) holds significant therapeutic potential for treating hematological diseases. This study uses adeno-associated virus serotype 6 (AAV6) vectors and Cre recombination to systematically optimize the parameters for effective <i>in vivo</i> HSC transduction. We evaluated various genetic architectures and delivery methods of AAV6, establishing an optimized protocol that achieved functional recombination in more than two-thirds of immunophenotypic HSCs. Our findings highlight that second-strand synthesis is a critical limiting factor for transgene expression in HSCs, leading to significant under-detection of HSC transduction with single-stranded AAV6 vectors. We also demonstrate that HSCs in the bone marrow (BM) are readily accessible to transduction, with neither localized injection nor mobilization of HSCs into the bloodstream, enhancing transduction efficacy. Additionally, we observed a surprising preference for HSC transduction over other BM cells, regardless of the AAV6 delivery route. Together, these findings not only underscore the potential of AAV vectors for <i>in vivo</i> HSC gene therapy but also lay a foundation that can inform the development of both <i>in vivo</i> AAV-based HSC gene therapies and potentially <i>in vivo</i> HSC gene therapies that employ alternative delivery modalities.
Authors
- Carsten T. Charlesworth (ORCID: https://orcid.org/0000-0002-8588-6204)
- Tze-Kai Tan (ORCID: https://orcid.org/0000-0002-7359-5303)
- Sicong Wang
- Agnieszka Czechowicz (ORCID: https://orcid.org/0000-0003-2909-2669)
- M. Kyle Cromer (ORCID: https://orcid.org/0000-0002-8198-5010)
- Amanda M. Dudek (ORCID: https://orcid.org/0000-0003-0239-3576)
- Adam C. Wilkinson (ORCID: https://orcid.org/0000-0001-7406-0151)
- Hiromitsu Nakauchi (ORCID: https://orcid.org/0000-0002-9841-6973)
- Carla Dib (ORCID: https://orcid.org/0000-0002-7242-8327)
- Shota Homma (ORCID: https://orcid.org/0000-0001-8090-9916)
- Kyomi J. Igarashi (ORCID: https://orcid.org/0000-0002-4352-3835)
- Fabian P. Suchy (ORCID: https://orcid.org/0000-0002-7187-5360)
- Alvaro Amorin (ORCID: https://orcid.org/0000-0002-3500-5085)
- Sriram Vaidyanathan (ORCID: https://orcid.org/0000-0001-9167-3056)
- Anais K. Amaya (ORCID: https://orcid.org/0000-0002-8396-2226)
- Yusuke Nakauchi (ORCID: https://orcid.org/0000-0002-6943-3811)
- Masashi Miyauchi (ORCID: https://orcid.org/0000-0002-6301-3429)
Institutions
- Nationwide Children's Hospital (US)
- University of California, San Francisco (US)
- Wellcome/MRC Cambridge Stem Cell Institute (GB)
- The University of Tokyo (JP)
- Stanford University (US)
Publication Details
- Journal
- Molecular Therapy — Methods & Clinical Development
- Published
- 2025-02-21
- DOI
- https://doi.org/10.1016/j.omtm.2025.101438
- Citations
- 3
- Primary Topic
- Virus-based gene therapy research
- Type
- article
- Field-Weighted Citation Impact
- 5.45
Funders
- National Science Foundation
- Leukemia and Lymphoma Society
- Kay Kendall Leukaemia Fund
- National Institutes of Health
- Japan Society for the Promotion of Science
- Stanford Maternal and Child Health Research Institute