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Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells.

Molecular therapy : the journal of the American Society of Gene Therapy

Authors: Erika Valeri, Giulia Unali, Francesco Piras, Monah Abou-Alezz, Giulia Pais, Fabrizio Benedicenti, Maria Rosa Lidonnici, Ivan Cuccovillo, Ilaria Castiglioni, Sergio Arévalo, Giulio Spinozzi, Ivan Merelli, Rayk Behrendt, Adrian Oo, Baek Kim, Nathaniel R Landau, Giuliana Ferrari, Eugenio Montini, Anna Kajaste-Rudnitski

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities, but they have not been extensively exploited because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of Cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared to standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Copyright © 2023. Published by Elsevier Inc.

PMID: 37990494

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