Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells
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University of Cincinnati / OhioLINK
2019
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ndltd-OhioLink-oai-etd.ohiolink.edu-ucin15439199402196772021-08-03T07:09:05Z Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells Jayavaradhan, Rajeswari Molecular Biology CRISPR Cas9 HSPC HSC Gene Editing DNA DSB Repair Outcomes Gene correction The CRISPR/Cas9 discovery has revolutionized precise gene correction of the genome. However, the site-specific double-strand breaks (DSB) caused by the Cas9 nuclease are still predominantly repaired by the `error-prone` non-homologous end-joining (NHEJ)/microhomology-mediated EJ (MMEJ) mechanisms, rather than by precise correction using homology-directed repair (HDR). Hematopoietic stem cells (HSC) remain among the most promising target cells for gene therapy because of their unique self-renewing, differentiation and expansion capabilities, which ensure permanent correction of the entire blood cell progeny for the lifetime of an individual. Currently, the only readout of the edited HSC is by analysis dependent on molecular assays for NHEJ- and MMEJ-induced insertions/deletions and HDR detection in total CD34+ population (of which 99% of cells are progenitors, not HSC; sorting the HSC population post editing requires huge numbers of HSPC for obtaining sufficient DNA for analysis). Herein, we report a unique platform that can precisely quantify NHEJ, MMEJ, HDR and random integration of the HDR donor in the human HSC populations using a flow cytometry at a single-allele level. Using this system, we were able to optimize different editing and cell parameters in both umbilical cord blood and mobilized peripheral blood-derived CD34+ HSPC and CD34+38-90+RA- HSC populations to achieve high viability and 80-87% NHEJ/MMEJ mediated editing efficiency and 40% HDR mediated editing efficiency in HSPC and HSC. The edited CD34+ HSPC, when injected into NSG mice resulted in robust human cell engraftment, with 45±10% total engraftment, and 39±16% edited cell engraftment (n=12 mice). We also found that myeloid cells had a higher rate of MMEJ than lymphoid cells. Using this platform, we manipulated the DNA repair pathways using small molecular inhibitors/agonist and found that NU7441, a DNA-PK inhibitor improves HDR frequency to two-fold in HSPC. However, the impact that NU7441 may have on genome integrity or Cas9 off-target activity remains to be investigated. We, therefore, developed a Cas9-DN1S fusion protein to specifically inhibit error-prone repair only at the DSB generated by Cas9, HDR efficiency and fidelity would be enhanced without compromising genome integrity, thereby promoting cellular homeostasis, cellular viability, and clinical safety. The DN1S protein fused with either SpCas9 or SaCas9 protein improves HDR frequency at multiple loci in different human cells, including leukocyte adhesion deficiency (LAD) patient-derived B lymphocytes. 2019-10-14 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543919940219677 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543919940219677 unrestricted This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Molecular Biology CRISPR Cas9 HSPC HSC Gene Editing DNA DSB Repair Outcomes Gene correction |
| spellingShingle |
Molecular Biology CRISPR Cas9 HSPC HSC Gene Editing DNA DSB Repair Outcomes Gene correction Jayavaradhan, Rajeswari Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| author |
Jayavaradhan, Rajeswari |
| author_facet |
Jayavaradhan, Rajeswari |
| author_sort |
Jayavaradhan, Rajeswari |
| title |
Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| title_short |
Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| title_full |
Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| title_fullStr |
Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| title_full_unstemmed |
Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells |
| title_sort |
optimization of gene editing approaches for human hematopoietic stem cells |
| publisher |
University of Cincinnati / OhioLINK |
| publishDate |
2019 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543919940219677 |
| work_keys_str_mv |
AT jayavaradhanrajeswari optimizationofgeneeditingapproachesforhumanhematopoieticstemcells |
| _version_ |
1719455035147943936 |