scholarly journals Targeted gene editing of human hematopoietic stem cells (HSCs)

2014 ◽  
Vol 7 (25) ◽  
pp. 752-752
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Hematopoietic Stem ◽  
Targeted Gene Editing

scholarly journals Clinically Relevant Gene Editing in Hematopoietic Stem Cells for the Treatment of Pyruvate Kinase Deficiency

2021 ◽  
Author(s):  
Sara Fañanas-Baquero ◽  
Oscar Quintana-Bustamante ◽  
Daniel P. Dever ◽  
Omaira Alberquilla ◽  
Rebeca Sanchez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Pyruvate Kinase ◽  
Gene Editing ◽  
Hematopoietic Stem ◽  
Pyruvate Kinase Deficiency ◽  
Relevant Gene

scholarly journals CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich’s Ataxia

2020 ◽  
Vol 17 ◽  
pp. 1026-1036
Author(s):  
Celine J. Rocca ◽  
Joseph N. Rainaldi ◽  
Jay Sharma ◽  
Yanmeng Shi ◽  
Joseph H. Haquang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Friedreich’S Ataxia ◽  
Friedreich's Ataxia ◽  
Hematopoietic Stem

scholarly journals A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1108-1108
Author(s):  
Masoud Nasri ◽  
Perihan Mir ◽  
Benjamin Dannenmann ◽  
Diana Amend ◽  
Yun Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Gene Editing ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Editing Efficiency ◽  
Human Ipscs

Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction

2019 ◽  
Vol 12 ◽  
pp. 175-183 ◽  
Author(s):  
Christopher T. Lux ◽  
Sowmya Pattabhi ◽  
Mason Berger ◽  
Cynthia Nourigat ◽  
David A. Flowers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Fetal Hemoglobin ◽  
Hematopoietic Stem

scholarly journals 123. Helper-Dependent Ad5/35 Vectors for ZFN Mediated Gene Editing in Hematopoietic Stem Cells

2015 ◽  
Vol 23 ◽  
pp. S51
Author(s):  
Kamola Saydaminova ◽  
Hongjie Wang ◽  
Xun Ye ◽  
HongZhuan Chen ◽  
Ning Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Hematopoietic Stem

scholarly journals TALEN®-Based Gene Editing in Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5796-5796
Author(s):  
Brian W Busser ◽  
Sonal Temburni ◽  
Alex Boyne ◽  
Alexandre Juillerat ◽  
Laurent Poirot ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Target Site ◽  
Regulatory Sequences ◽  
Transcription Activator ◽  
Hematopoietic Stem ◽  
Exogenous Dna ◽  
Repair Template ◽  
Template Dna

Abstract Autologous gene therapy in human hematopoietic stem cells (HSCs) has shown the potential to transform the treatment of genetic diseases of the blood and immune system. However, viral vector delivery has shown issues with insertional mutagenesis, unregulated transgene expression and durability. Genome editing via homology directed repair (HDR) in human HSCs can mitigate these issues by correcting and repairing the endogenous gene permitting precise spatio-temporal gene expression. Transcription activator-like effector nuclease (TALEN®) are a designer nuclease that enables the site-specific introduction of double-stranded breaks (DSBs) in the genome. Repair of these DSBs occurs largely through one of two pathways, non-homologous end joining (NHEJ) and HDR. NHEJ is an error-prone pathway that often results in insertions or deletions (indels) whereas HDR is a precise mechanism that uses a homologous piece of DNA (usually the homologous chromosome or sister chromatid) to accurately repair the lesion by recombination. However, HDR can be manipulated by supplying an exogenous DNA template with homology arms that start at the DSB as single-stranded oligonucleotides or as double-stranded DNA to introduce any genetic modifications encoded in the template DNA. Recent studies have shown efficient HDR in numerous cell types using adeno-associated virus (AAV) to deliver template DNA. Here we combined TALEN® with AAV containing a repair template to identify the conditions required to efficiently modify disease-relevant alleles in HSCs. We first describe a strategy to remove the nuclease target site in the template DNA even when the target site was located in regulatory sequences. Optimization of the transfection, transduction and culturing conditions of HSCs shows highly efficient allele modification using low doses of AAV and nuclease. Importantly, alleles more often show evidence of HDR as opposed to NHEJ. Further, colony forming unit assays and a xenograft model confirm that modified HSCs are multipotent. Lastly, we show that differentiated progeny of modified HSCs produce similar amounts of mRNA from the modified allele as wild-type unmodified alleles. In total, our results show that combining TALEN® with repair template delivery via AAV is a robust gene editing system for HSCs. Disclosures Busser: Cellectis: Employment, Patents & Royalties: Cellectis. Temburni:Cellectis: Employment, Patents & Royalties: Cellectis. Boyne:Cellectis, Inc: Employment, Patents & Royalties: Cellectis. Juillerat:Cellectis: Employment, Patents & Royalties: Cellectis. Poirot:Cellectis: Employment, Patents & Royalties: Cellectis. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis.

scholarly journals SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5803-5803
Author(s):  
Abisha Crystal C ◽  
Saravanabhavan Thangavel ◽  
Shaji Ramachandran Velayudhan ◽  
Alok Srivastava ◽  
Aneesha Nath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Migration ◽  
Hematopoietic Stem Cells ◽  
Small Molecules ◽  
Gene Editing ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Hematopoietic Stem ◽  
Colony Forming Cell ◽  
Ex Vivo Culture

Abstract Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process. The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+). Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. Disclosures No relevant conflicts of interest to declare.

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

2020 ◽  
Vol 38 (11) ◽  
pp. 1298-1308 ◽  
Author(s):  
Samuele Ferrari ◽  
Aurelien Jacob ◽  
Stefano Beretta ◽  
Giulia Unali ◽  
Luisa Albano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Hematopoietic Stem ◽  
Efficient Gene
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