Journal
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
Volume 21, Issue -, Pages 121-132Publisher
CELL PRESS
DOI: 10.1016/j.omtm.2021.02.022
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Funding
- National Heart, Lung, and Blood Institute (NHLBI)
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at NIH
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The development of a non-integrating lentiviral system encoding Cas9 protein, guide RNA, and donor DNA allows for efficient one-time correction of hereditary diseases, improving prospects for genome editing.
Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous beta s-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.
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