scholarly journals Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

Cell Reports ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2606-2616 ◽  
Author(s):  
Caroline Y. Kuo ◽  
Joseph D. Long ◽  
Beatriz Campo-Fernandez ◽  
Satiro de Oliveira ◽  
Aaron R. Cooper ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Editing ◽  
Specific Gene ◽  
Hematopoietic Stem ◽  
Site Specific ◽  
Hyper Igm Syndrome ◽  
Hyper Igm

Identifying mechanisms that limit efficient site-specific gene modification by homology-directed repair in hematopoietic stem cells

2015 ◽  
Vol 43 (9) ◽  
pp. S54
Author(s):  
Carmen F. Bjurström ◽  
Michelle Mojadidi ◽  
Beatriz Campo ◽  
Andreas Reik ◽  
Michael C. Holmes ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Specific Gene ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Site Specific ◽  
Homology Directed Repair

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

Epigenetic Effects of IDH1/IDH2 Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-33-SCI-33 ◽  
Author(s):  
Ari M. Melnick ◽  
Ross L Levine ◽  
Maria E Figueroa ◽  
Craig B. Thompson ◽  
Omar Abdel-Wahab
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Covalent Modification ◽  
Specific Gene ◽  
Gene Promoters ◽  
Loss Of Function ◽  
Hematopoietic Stem

Abstract Abstract SCI-33 Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ∼20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss-of-function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCpG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using α-ketoglutarate (αKG) as a substrate. 2HG can disrupt the activity of enzymes that use αKG as a cofactor, including TET2 and the jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibits TET2 hydroxymethylation and jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards increased histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is in abundance in hematopoietic stem cells and displays specific distribution patterns, yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Disclosures: Thompson: Agios Pharmaceuticals: Consultancy.

Human hematopoiesis in murine embryos after injecting human cord blood–derived hematopoietic stem cells into murine blastocysts

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 719-721 ◽  
Author(s):  
Friedrich Harder ◽  
Reinhard Henschler ◽  
Ilse Junghahn ◽  
Marinus C. Lamers ◽  
Albrecht M. Müller
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Developmental Stages ◽  
Specific Gene ◽  
Human Cord Blood ◽  
Human Donor ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Murine Embryos

Abstract At different developmental stages, candidate human hematopoietic stem cells (HSCs) are present within the CD34+ CD38− population. By means of xenotransplantation, such CD34+CD38− cells were recently shown to engraft the hematopoietic system of fetal sheep and nonobese diabetic severe combined immunodeficient adult mice. Here it is demonstrated that, after their injection into murine blastocysts, human cord blood (CB)–derived CD34+and CD34+ CD38− cells repopulate the hematopoietic tissues of nonimmunocompromised murine embryos and that human donor contribution can persist to adulthood. It is further observed that human hematopoietic progenitor cells are present in murine hematopoietic tissues of midgestational chimeric embryos and that progeny of the injected human HSCs activate erythroid-specific gene expression. Thus, the early murine embryo provides a suitable environment for the survival and differentiation of human CB CD34+ CD38− cells.

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
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