1. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy
- Author
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Giuliana Ferrari, Luigi Naldini, Maria Rosa Lidonnici, Francesco Boccalatte, Erika Zonari, Anna Kajaste-Rudnitski, Bernhard Gentner, Giacomo Desantis, Alessandro Aiuti, Carolina Petrillo, Zonari, Erika, Desantis, Giacomo, Petrillo, Carolina, Boccalatte, Francesco E., Lidonnici, Maria Rosa, Kajaste Rudnitski, Anna, Aiuti, Alessandro, Ferrari, Giuliana, Naldini, Luigi, and Gentner, Bernhard
- Subjects
0301 basic medicine ,purified HSCs ,HSC expansion ,Genetic enhancement ,Antigens, CD38 ,Cell Culture Techniques ,CD34 ,Antigens, CD34 ,CD38 ,HSC gene therapy ,Biochemistry ,0302 clinical medicine ,Mice, Inbred NOD ,Transduction, Genetic ,lcsh:QH301-705.5 ,Cell Engineering ,lcsh:R5-920 ,Hematopoietic Stem Cell Transplantation ,Cell biology ,Haematopoiesis ,030220 oncology & carcinogenesis ,Genetic Vector ,Stem cell ,lcsh:Medicine (General) ,Cell Culture Technique ,Human ,Genetic Vectors ,Biology ,Article ,Lentiviru ,03 medical and health sciences ,Genetic ,Genetics ,Animals ,Humans ,Progenitor cell ,Cell Proliferation ,prostaglandin E2 ,Animal ,Lentivirus ,Hematopoietic Stem Cell ,Genetic Therapy ,Cell Biology ,Hematopoietic Stem Cells ,ADP-ribosyl Cyclase 1 ,Molecular biology ,Transplantation ,030104 developmental biology ,lcsh:Biology (General) ,UM171 ,lentiviral vector transduction ,purified HSC ,Ex vivo ,Developmental Biology - Abstract
Summary Ex vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38− cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing., Highlights • CD34+CD38− cells as an HSC-enriched starting population for ex vivo gene therapy • Reduced culture time (, In this article, Gentner and colleagues undertake a comprehensive strategy to advance ex vivo genetic engineering of HSCs for gene therapy. They experimentally define an optimal strategy to purify HSCs, which allows uncoupling long-term from short-term hematopoietic reconstitution, and implement ex vivo conditions that best preserve their biological properties applying novel transduction-enhancing compounds and pyrimidoindole derivatives to support HSC expansion.
- Published
- 2017