1. Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking
- Author
-
Giulia Unali, Ivan Merelli, Stefano Beretta, Aurelien Jacob, Luisa Albano, Pietro Genovese, Samuele Ferrari, Anna Kajaste-Rudnitski, Davide Cittaro, Valentina Vavassori, Federica Cugnata, Luigi Naldini, Chiara Brombin, Dejan Lazarevic, Ferrari, S, Jacob, A, Beretta, S, Unali, G, Albano, L, Vavassori, V, Cittaro, D, Lazarevic, D, Brombin, C, Cugnata, F, Kajaste-Rudnitski, A, Merelli, I, Genovese, P, Naldini, L, Ferrari, S., Jacob, A., Beretta, S., Unali, G., Albano, L., Vavassori, V., Cittaro, D., Lazarevic, D., Brombin, C., Cugnata, F., Kajaste-Rudnitski, A., Merelli, I., Genovese, P., and Naldini, L.
- Subjects
G2 Phase ,Transcription, Genetic ,Genetic enhancement ,Transplantation, Heterologous ,Biomedical Engineering ,Bioengineering ,Biology ,Applied Microbiology and Biotechnology ,Stem-cell biotechnology ,Article ,S Phase ,03 medical and health sciences ,Mice ,Viral Proteins ,0302 clinical medicine ,Gene therapy ,Genome editing ,Animals ,Humans ,Cell Lineage ,Haematopoietic stem cell ,E2F ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Base Sequence ,Targeted Gene Repair ,HEK 293 cells ,Recombinational DNA Repair ,Reproducibility of Results ,Dependovirus ,Hematopoietic Stem Cells ,Xenograft Model Antitumor Assays ,Cell biology ,Clone Cells ,Up-Regulation ,Transplantation ,Haematopoiesis ,Targeted gene repair ,HEK293 Cells ,Cell Tracking ,Molecular Medicine ,Stem cell ,Tumor Suppressor Protein p53 ,030217 neurology & neurosurgery ,Biotechnology - 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. Transient p53 inhibition and induced cell-cycle progression increase clonal engraftment and homology-directed repair in hematopoietic stem cells.
- Published
- 2020
- Full Text
- View/download PDF