scholarly journals 754. Site-Specific Genome Editing in Human Long-Term Repopulating Hematopoietic Stem Cells for Correction of SCID-X1

2014 ◽  
Vol 22 ◽  
pp. S291
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Hematopoietic Stem ◽  
Site Specific

scholarly journals Gene Correction for SCID-X1 in Long-Term Hematopoietic Stem Cells

2018 ◽  
Author(s):  
Mara Pavel-Dinu ◽  
Volker Wiebking ◽  
Beruh T. Dejene ◽  
Waracharee Srifa ◽  
Sruthi Mantri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Limit Of Detection ◽  
Severe Combined Immunodeficiency ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
High Frequencies ◽  
Monogenic Diseases

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. High frequencies of reproducible targeted integration of a wild-type cDNA into the endogenous start codon of a gene in LT-HSCs could provide a robust genome editing approach to cure genetic diseases in which patients have different mutations throughout the gene. We describe a clinically relevant method for correcting X-linked severe combined immunodeficiency (SCID-X1). By using a highly specific and active CRISPR/Cas9-AAV6 based strategy and selection-free approach, we achieve up to 20% genome integration frequencies in LT-HSCs of a full-length IL2RG cDNA at the endogenous start site as demonstrated by serial transplantation and analysis of genome edited human cells eight months following initial transplantation. In addition to high frequencies of functional gene correction in LT-HSCs we observed no evidence of abnormal hematopoiesis following transplantation, a functional measure of the lack of genotoxicity. Deep analysis of potential off-target activity detected two sites with low frequency (<0.3%) of off-target mutations. The level of off-target mutations was reduced to below the limit of detection using a high fidelity Cas9. Moreover, karyotype evaluation identified no genomic instability events. We achieved high levels of genome targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect of patient derived cells both in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

scholarly journals Genome Editing Via NHEJ-Mediated Targeted Integration Retains Engraftment Potential of Edited Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3480-3480
Author(s):  
Gene I. Uenishi ◽  
Keith Abe ◽  
Bee-Chun Sun ◽  
Cornell Mallari ◽  
Andrew M. Scharenberg ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Post Injection ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Feasible Method ◽  
Targeted Integration ◽  
Equity Ownership

Abstract The ability to edit the genome of long-term-engrafting hematopoietic stem cells (LT-HSCs) would provide a curative therapy for a wide range of hematological disorders. While mixed LT/ST-HSC (ST, short-term) pools edited via non-homologous end joining (NHEJ) yield engraftable transgenic cells, pools made transgenic via homology-dependent repair (HDR) do not. This observation is likely explained by the absence of DNA replication required for HDR in LT-HSCs. Quiescence renders LT-HSCs refractory to transgene insertion via HDR, with the transgene-positive population consisting overwhelmingly of actively dividing, short-term-engrafting HSCs. In contrast to HDR, NHEJ is a DNA repair mechanism active in non-dividing cells. We hypothesized that CRISPR/Cas9-mediated transgenesis of HSC pools via NHEJ-mediated targeted integration (NHEJ-TI) would allow modification of LT-HSCs and produce edited, long-term engrafting progeny. First, we investigated whether NHEJ-TI is a feasible method of genome editing in HSCs. Utilizing CRISPR/Cas9 to induce a DSB in the AAVS1 (PPP1R12C) locus and a self-complementary rAAV6 (scAAV6) to deliver a GFP expression cassette dependent on the endogenous promoter, we optimized RNP nucleofection, scAAV6 infection, and HSC culture conditions for efficient NHEJ-TI in HSCs. Flow cytometry for eGFP and in-out PCR was used to confirm transgene integration at frequencies ranging from 4-6%. Next, we investigated whether the NHEJ-TI edited pool of mixed LT/ST-HSCs contained engraftable, edited cells by injection into irradiated NOD/SCID/IL2Rg-/- (NSG) mice. We compared the engraftment potential of transgenic HSCs cultured for either 2 days (2D) or 2 hours (2H) prior to editing. While the overall editing efficiency was ~20% higher in the 2D condition, the near absence of cell prestimulation in the 2H condition resulted in increased levels of overall engraftment, doubling the total engraftment level of edited hematopoietic cells at 10 weeks post-injection. Importantly, the percentage of edited cells among the engrafted human hematopoietic cell population remained essentially unchanged relative to the starting efficiency (2D: 4.0%, 2H: 3.6%; 10 weeks post-injection). Analysis of bone marrow-resident CD34-positive cells at 16 weeks post-engraftment will reveal the fraction of edited LT-HSCs. Our results show that NHEJ-TI is a feasible method of inserting transgenes into HSCs whilst retaining their engraftment potential. To our knowledge, this is the first study to achieve successful NHEJ-mediated targeted integration in HSCs while retaining engraftment potential and (perhaps) transgenesis as well. Disclosures Uenishi: Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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