Functional Profiling of Single CRISPR/Cas9-Edited Human Long-Term Hematopoietic Stem Cells

Abstract

and Eric R. Lechman (elechman@uhnresearch.ca) regenerative therapies. Thus, LT-HSCs are essential for therapeutic genome editing to correct acquired and genetic hematopoietic disorders 3,4 . Furthermore, the pathogenesis of hematological malignancies like acute myeloid leukemia (AML) is associated with the presence of initiating mutations acquired in LT-HSCs, which lead to their competitive expansion 5,6 . Pre-leukemic LT-HSCs are a source of clonal evolution within blood malignancies and can act as a reservoir of relapse after chemotherapy treatment 7 . Thus, modeling and understanding the genetic complexity and cellular heterogeneity seen in human hematological malignancies requires novel methodologies that allow genome editing in LT-HSCs and their functional read-out 3,4 .Recently several studies have demonstrated efficient gene editing of bulk CD34 + populations that are enriched for human HSPCs [8][9][10][11][12][13][14][15][16] . Highly efficient non-homologous end joining (NHEJ) mediated gene disruption of up to 80-90% efficiency has been reported in CD34 + HSPCs 10,11,14,16 . In addition, homology-directed repair (HDR) mediated knock-ins, with or without selectable fluorescent reporter genes, have been established with an efficiency of up to 20% in CD34 + HSPCs 8,9,11,13,15 . Stable integration of a fluorescent reporter using rAAV6 combined with flow cytometrybased sorting enabled enrichment of CRISPR/Cas9 edited HSPCs 8,9,16 . Because LT-HSCs represent only 0.1 -1% of CD34 + populations, these studies did not address LT-HSC targeting in the most direct manner. Previous studies have reported long term engraftment of up to 16 weeks following xenotransplantation of CRISPR/Cas9 edited human CD34 + HSPCs 8,15,16 , suggesting that rare LT-HSCs within the CD34 +

WITHDRAWN: A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

“…However, there was a reduction in the percentage of chimeric cells at 12 weeks post-transplantation in the RNP+AAV group, which was consistent with the results of other studies and may reflect a reduced engraftment capacity of edited mixed cells [16,17]. The reason that the engraftment capacity of edited mixed HSPCs is decreased compared with that of unedited cells may be that the HDR donors are themselves toxic or that HSCs are intrinsically more di cult to edit than other cells [28]. We further demonstrated that GFP+HSPCs representing the gene-targeted HSPCs can be detected in the BM of NSI mice, which indicated that some of the targeted HSPCs were stable for up to 12 weeks post-transplantation, although the in vivo genetargeted HSPCs had a nearly 6-fold reduction compared with the in vitro gene-targeted HSPCs.…”

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures

“…As the seed cells for the blood system, their clinical potential for cellular therapies is vast and the need to understand their molecular program in different physiological states is critical for their therapeutic application. Recently, cell culture conditions have been reported to produce large numbers of functional mouse and human HSCs (hHSCs) ( Fares et al., 2017 ; Wilkinson et al., 2019 ) but, in all cases, the substantial majority of cells produced are non-HSCs ( Bak et al., 2018 ; Gundry et al., 2016 ; Shepherd and Kent, 2019 ; Wagenblast et al., 2019 ).…”

Implications of hematopoietic stem cells heterogeneity for gene therapies

“…As an additional (LT-)HSC marker CD133 was introduced, and for both CD34 and CD133 clinical largescale isolation technologies are available, but CD133+ selected cells featured superior ex vivo proliferation potential compared to CD34+ selected cells [38,42], and CD133 was successfully used as entry receptor for lentiviral gene transfer into LT-HSCs [38]. Their sustainable repopulation potential suggests LT-HSCs as a prime target cell type for gene therapies [43]. Further, CD45RA expression was found to distinguish multipotent (CD133+ CD34+ CD45RA−) from lympho-myeloid (CD133+ CD34+ CD45RA+) human HPC fractions [44].…”

“…To address these issues it might be useful to assess the genetic modifications for each cell type being present in the graft, and/or to develop manufacture concepts that target a specific cell type. In fact, high efficiency human LT-HSCs gene editing using CRISPR/Cas9 technology was recently reported [43].…”