Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

Ferrari, S., Jacob, A., Beretta, S., Unali, G., Albano, L., Vavassori, V., et al. (2020). Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking. NATURE BIOTECHNOLOGY, 38(11 (November 2020)), 1298-1308 [10.1038/s41587-020-0551-y].

Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking

Jacob A.;Beretta S.;Merelli I.;
2020

Abstract

Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.
Articolo in rivista - Articolo scientifico
Biotechnology; Gene therapy; Haematopoietic stem cells; Stem-cell biotechnology; Targeted gene repair;
English
29-giu-2020
2020
38
11 (November 2020)
1298
1308
none
Ferrari, S., Jacob, A., Beretta, S., Unali, G., Albano, L., Vavassori, V., et al. (2020). Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking. NATURE BIOTECHNOLOGY, 38(11 (November 2020)), 1298-1308 [10.1038/s41587-020-0551-y].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/311006
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