| Autor: |
Jing Zeng, My Anh Nguyen, Pengpeng Liu, Lucas Ferreira da Silva, Linda Y. Lin, David G. Justus, Karl Petri, Kendell Clement, Shaina N. Porter, Archana Verma, Nola R. Neri, Tolulope Rosanwo, Marioara-Felicia Ciuculescu, Daniela Abriss, Esther Mintzer, Stacy A. Maitland, Selami Demirci, John F. Tisdale, David A. Williams, Lihua Julie Zhu, Shondra M. Pruett-Miller, Luca Pinello, J. Keith Joung, Vikram Pattanayak, John P. Manis, Myriam Armant, Danilo Pellin, Christian Brendel, Scot A. Wolfe, Daniel E. Bauer |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
bioRxiv |
| DOI: |
10.1101/2023.05.27.542323 |
| Popis: |
SUMMARYGene editing theBCL11Aerythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of theBCL11A+58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of theBCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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