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

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 upon 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 significantly shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserved 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.

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering

“… 30 , 33 , 34 The two compounds were used in combination, since we have recently demonstrated that GSE56 alone does not increase editing efficiency but leads to an improved engraftment and higher clonality of edited cells upon transplantation in vivo . 35 To this aim, after 2 days of prestimulation, HSPCs were transduced in the presence or absence of CsH, with a donor template-carrying IDLV, which harbors a GFP cassette. After 24 h post-transduction, HSPCs were electroporated with a Cas9 ribonucleoprotein (RNP) targeting the AAVS1 locus in the presence or absence of GSE56.…”

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper‐IgM syndrome

“…Editing CD40LG in long-term repopulating human HSPC Since the reconstituted repertoire and chimerism of edited T cells might be limiting in the long term and their transfer alone cannot correct all affected hematopoietic lineages, we investigated application of our editing strategy to HSPC. We thus performed CD40LG gene editing in human CD34 + male HSPC ( Fig 4A), using the same reagents as above in the context of recently optimized protocols from our laboratory (Schiroli et al, 2019;Ferrari et al, 2020). When using a donor template comprising only the corrective cDNA, we achieved up to 40% targeted integration in the most primitive subpopulation (CD34 + CD133 + CD90 + , Fig 4B), while preserving the culture composition after the manipulation ( Fig 4C).…”

Longitudinal tracking reveals sustained polyclonal repopulation of human-HSPC in humanized mice despite vector integration bias

“…Overall, the clonal repopulation of human HSPC in hu-BLT mice resembles that of mouse HSPC after autologous transplant. Cord blood HSPC transplanted in NGS mice also showed similar clonal behavior, with clonal stabilization starting near week 18 to 20 post-transplant 33,34 . Although the timescales compare well with other studies, caution is due considering high incidence of graft versus host-disease-related illnesses in xenograft mouse models.…”

“…A recent study demonstrated use of CRISPR/Cas to introduce barcodes in the long-term HSPC and longitudinally tracked a very limited number of HSPC clones 34 . Comparatively, using LoVIS-Seq we have tracked ~10 times more HSPC clones per animal with high accuracy and reproducibility.…”