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

Nicholas Liao

Nat Biotechnol. 2020 Jun 29. doi: 10.1038/s41587-020-0551-y. | PubMed

Samuele Ferrari1,2, Aurelien Jacob1,3, Stefano Beretta1, Giulia Unali1,2, Luisa Albano1, Valentina Vavassori1,2, Davide Cittaro4, Dejan Lazarevic4, Chiara Brombin5, Federica Cugnata5, Anna Kajaste-Rudnitski1, Ivan Merelli1,6, Pietro Genovese1,7, Luigi Naldini8,9

  1. San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.
  2. Vita-Salute San Raffaele University, Milan, Italy.
  3. Milano-Bicocca University, Monza, Italy.
  4. Center for Omics Sciences, IRCCS San Raffaele Scientific Institute, Milan, Italy.
  5. CUSSB-University Center for Statistics in the Biomedical Sciences, Vita-Salute San Raffaele University, Milan, Italy.
  6. National Research Council, Institute for Biomedical Technologies, Segrate, Italy.
  7. Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatric Oncology, Harvard Medical School, Boston, MA, USA.
  8. San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy. naldini.luigi@hsr.it.
  9. Vita-Salute San Raffaele University, Milan, Italy. naldini.luigi@hsr.it.

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.

Presented By Samuele Ferrari