scholarly journals Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair

Stem Cells ◽  
2018 ◽  
Vol 37 (2) ◽  
pp. 284-294 ◽  
Author(s):  
Anastasia Lomova ◽  
Danielle N. Clark ◽  
Beatriz Campo-Fernandez ◽  
Carmen Flores-Bjurström ◽  
Michael L. Kaufman ◽  
...  
Keyword(s):  
Dna Repair ◽  
Progenitor Cells ◽  
Gene Editing ◽  
Temporal Control ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

EFFICIENT AND NON-TOXIC MRNA DELIVERY AND GENE EDITING IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS USING NANOSTRAWS

2019 ◽  
Vol 76 ◽  
pp. S84
Author(s):  
Ludwig Schmiderer ◽  
Agatheeswaran Subramaniam ◽  
Kristijonas Žemaitis ◽  
Alexandra Bäckström ◽  
David Yudovich ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Gene Editing ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

scholarly journals S. Aureus Cas9 Leads to Higher Sickle Mutation Correction Compared to S. Pyogenes Cas9 in Patient Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1861-1861
Author(s):  
Byoungyong Yoo ◽  
So Hyun Julie Park Park ◽  
Yankai Zhang ◽  
Vivien A. Sheehan ◽  
Gang Bao
Keyword(s):  
Progenitor Cells ◽  
Research Funding ◽  
Gene Editing ◽  
Cell Phenotype ◽  
High Morbidity ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
Nucleotide Mutation ◽  
Stem And Progenitor Cells ◽  
Donor Template

Abstract Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied. Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS). Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs. Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

scholarly journals Thrombopoietin promotes NHEJ DNA repair in hematopoietic stem cells through specific activation of Erk and NF-κB pathways and their target, IEX-1

Blood ◽  
2014 ◽  
Vol 123 (4) ◽  
pp. 509-519 ◽  
Author(s):  
Bérengère de Laval ◽  
Patrycja Pawlikowska ◽  
Daniela Barbieri ◽  
Corinne Besnard-Guerin ◽  
Alba Cico ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Repair ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Stem And Progenitor Cells ◽  
Common Target

Key Points TPO specifically activates Erk and NF-κB pathways in hematopoietic stem and progenitor cells. Erk and NF-κB cooperate to trigger their common target, Iex-1, and DNA-PK-dependent NHEJ activation in HSPCs upon irradiation.

scholarly journals A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

2018 ◽  
Vol 24 (8) ◽  
pp. 1216-1224 ◽  
Author(s):  
Christopher A. Vakulskas ◽  
Daniel P. Dever ◽  
Garrett R. Rettig ◽  
Rolf Turk ◽  
Ashley M. Jacobi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Gene Editing ◽  
High Fidelity ◽  
Hematopoietic Stem ◽  
Ribonucleoprotein Complex ◽  
Efficient Gene ◽  
Stem And Progenitor Cells

scholarly journals Highly Efficient Editing of the Beta-Globin Gene in Patient Derived Hematopoietic Stem and Progenitor Cells to Treat Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2192-2192
Author(s):  
So Hyun Park ◽  
Ciaran M Lee ◽  
Daniel P. Dever ◽  
Timothy H Davis ◽  
Joab Camarena ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Progenitor Cells ◽  
Gene Editing ◽  
Globin Gene ◽  
Cell Disease ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Sickle cell disease (SCD) is an inherited blood disorder associated with a debilitating chronic illness. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution converts glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of red blood cells. Here we show that delivery of Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex (RNP) together with a short single-stranded DNA donor (ssODN) template into CD34+ hematopoietic stem and progenitor cells (HSPCs) from SCD patients' bone marrow (BM) was able to correct the sickling HBB mutation, with up to 33% homology directed repair (HDR) without selection. Further, CRISPR/Cas9 cutting of HBB in SCD HSPCs induced gene conversion between the HBB sequences in the vicinity of the target locus and the homologous region in δ-globin gene (HBD), with up to 4.4% additional gene correction mediated by the HBD conversion in cells with Cas9 cutting only. The erythrocytes derived from gene-edited cells showed a marked reduction of the HbS level, increased expression of normal adult hemoglobin (HbA), and a complete loss of cell sickling, demonstrating the potential in curing SCD. We performed extensive off-target analysis of gene-edited SCD HSPCs using the in-silico prediction tool COSMID and unbiased, genome-wide assay Guide-Seq, revealing a gross intrachromosomal rearrangement event between the on- and off-target Cas9 cutting sites. We used a droplet digital PCR assay to quantify deletion and inversion events from Day 2 to Day 12 after RNP delivery, and found that large chromosomal deletion decreased from 1.8% to 0.2%, while chromosomal inversion maintained at 3.3%. We demonstrated that the use of high-fidelity SpCas9 (HiFi Cas9 by IDT) significantly reduced off-target effects and completely eliminated the intrachromosome rearrangement events, while maintaining the same level of on-target gene editing, leading to high-efficiency gene correction with increased specificity. In order to determine if gene-corrected SCD HSPCs retain the ability to engraft, CD34+ cells from the BM of SCD patients were treated with Cas9/gRNA RNP and ssODN donor for HBB gene correction, cryopreserved at Day 2 post genome editing, then intravenously transplanted into NSG mice shortly after thawing. These mice were euthanized at Week 16 after transplantation, and the BM was harvested to determine the engraftment potential. An average of 7.5 ±5.4% of cells were double positive for HLA and hCD45 in mice injected with gene-edited CD34+ cells, compared to 16.8 ±9.3% with control CD34+ cells, indicating a good level of engraftment of gene-corrected SCD HSPCs. A higher fraction of human cells were positive for CD19 (66 ±28%), demonstrating lymphoid lineage bias. DNA was extracted from unsorted cells, CD19 or CD33 sorted cells for gene-editing analysis; the HBB editing rates were respectively 29.8% HDR, 2.4% HBD conversion, and 42.8% non-homologous end joining (NHEJ) pretransplantation, and editing rates at Week 16 posttransplantation were respectively 8.8 ±12% HDR, 1.8 ±1.7% HBD conversion, and 24.5 ±12% NHEJ. The highly variable editing rate and indel diversity in gene-edited cells at Week 16 in all four transplanted mice suggest clonal dominance of a limited number of HSPCs after transplantation. Taken together, our results demonstrate highly efficient gene and phenotype correction of the sickling mutation in BM HSPCs from SCD patients mediated by HDR and HBD conversion, and the ability of gene-edited SCD HSPCs to engraft in vivo. We also demonstrate the importance of genome-wide analysis for off-target analysis and the use of HiFi Cas9. Our results provide further evidence for the potential of moving genome editing-based SCD treatment into clinical practice. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G. B.), and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Eltrombopag promotes DNA repair in human hematopoietic stem and progenitor cells

2019 ◽  
Vol 73 ◽  
pp. 1-6.e6 ◽  
Author(s):  
Kacey L. Guenther ◽  
Patali S. Cheruku ◽  
Ayla Cash ◽  
Richard H. Smith ◽  
Luigi J. Alvarado ◽  
...  
Keyword(s):  
Dna Repair ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

scholarly journals Gene Editing of Human Hematopoietic Stem and Progenitor Cells: Promise and Potential Hurdles

2016 ◽  
Vol 27 (10) ◽  
pp. 729-740 ◽  
Author(s):  
Kyung-Rok Yu ◽  
Hannah Natanson ◽  
Cynthia E. Dunbar
Keyword(s):  
Progenitor Cells ◽  
Gene Editing ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells
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