Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease
Naoya Uchida, Claire Drysdale, Tina Nassehi, Jackson Gamer, Morgan Yapundich, Julia DiNicola, Yoshitaka SHIBATA, Malikiya Hinds, Bjorg Gudmundsdottir, Juan J. Haro‐Mora, Selami Demirci, John F. Tisdale
Abstract
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 βs-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.