In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods
Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurre...
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Elsevier,
2019-09-01T00:00:00Z.
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| 001 | doaj_5c5d0cc4529c44adb9273c8f4a9f26d9 | ||
| 042 | |a dc | ||
| 100 | 1 | 0 | |a Sowmya Pattabhi |e author |
| 700 | 1 | 0 | |a Samantha N. Lotti |e author |
| 700 | 1 | 0 | |a Mason P. Berger |e author |
| 700 | 1 | 0 | |a Swati Singh |e author |
| 700 | 1 | 0 | |a Christopher T. Lux |e author |
| 700 | 1 | 0 | |a Kyle Jacoby |e author |
| 700 | 1 | 0 | |a Calvin Lee |e author |
| 700 | 1 | 0 | |a Olivier Negre |e author |
| 700 | 1 | 0 | |a Andrew M. Scharenberg |e author |
| 700 | 1 | 0 | |a David J. Rawlings |e author |
| 245 | 0 | 0 | |a In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods |
| 260 | |b Elsevier, |c 2019-09-01T00:00:00Z. | ||
| 500 | |a 2162-2531 | ||
| 500 | |a 10.1016/j.omtn.2019.05.025 | ||
| 520 | |a Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34+ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ−/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation. Keywords: sickle cell disease, gene editing, rAAV6, ssODN, homology-directed repair, Crispr/Cas9, hemoglobin disorders, NHEJ versus HDR, in vivo engraftment, NBSGW41 mice, CD34, hematopoietic stem cells, stem cell cures | ||
| 546 | |a EN | ||
| 690 | |a Therapeutics. Pharmacology | ||
| 690 | |a RM1-950 | ||
| 655 | 7 | |a article |2 local | |
| 786 | 0 | |n Molecular Therapy: Nucleic Acids, Vol 17, Iss , Pp 277-288 (2019) | |
| 787 | 0 | |n http://www.sciencedirect.com/science/article/pii/S2162253119301568 | |
| 787 | 0 | |n https://doaj.org/toc/2162-2531 | |
| 856 | 4 | 1 | |u https://doaj.org/article/5c5d0cc4529c44adb9273c8f4a9f26d9 |z Connect to this object online. |