Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in longterm 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 with 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.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells / Valeri, Erika; Unali, Giulia; Piras, Francesco; Abou-Alezz, Monah; Pais, Giulia; Benedicenti, Fabrizio; Lidonnici, Maria Rosa; Cuccovillo, Ivan; Castiglioni, Ilaria; Arévalo, Sergio; Spinozzi, Giulio; Merelli, Ivan; Behrendt, Rayk; Oo, Adrian; Kim, Baek; Landau, Nathaniel R; Ferrari, Giuliana; Montini, Eugenio; Kajaste-Rudnitski, Anna. - In: MOLECULAR THERAPY. - ISSN 1525-0024. - 32:1(2024), pp. 124-139. [10.1016/j.ymthe.2023.11.020]

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

Valeri, Erika
Primo
;
Unali, Giulia
Secondo
;
Piras, Francesco;Castiglioni, Ilaria;Ferrari, Giuliana;
2024-01-01

Abstract

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in longterm 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 with 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.
2024
antiviral restriction factors
gene therapy
hematopoietic stem cells
innate immunity
lentiviral vectors
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/161960
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