Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.
Animals
Base Sequence
Cell Lineage
Cell Tracking
Clone Cells
Dependovirus
/ metabolism
G2 Phase
Gene Editing
HEK293 Cells
Hematopoietic Stem Cells
/ cytology
Humans
Mice
Recombinational DNA Repair
Reproducibility of Results
S Phase
Transcription, Genetic
Transplantation, Heterologous
Tumor Suppressor Protein p53
/ metabolism
Up-Regulation
Viral Proteins
/ metabolism
Xenograft Model Antitumor Assays
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
06
12
2019
accepted:
07
05
2020
pubmed:
1
7
2020
medline:
16
12
2020
entrez:
1
7
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32601433
doi: 10.1038/s41587-020-0551-y
pii: 10.1038/s41587-020-0551-y
pmc: PMC7610558
mid: EMS118408
doi:
Substances chimiques
Tumor Suppressor Protein p53
0
Viral Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1298-1308Références
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