560. Therapeutic Editing of the HBB Locus Using the Endogenous HBD Locus as a Donor Template

Abstract

The first approach is aimed at approximately 26% of patients that have point mutations in DMD gene. Here we used gene editing to correct DMD E2035X, a pathogenic mutation in DMD patient myoblasts by designing short RNA sequences complementary to the locus called guides, paired with the Streptococcus pyogenes Cas9. We were able to generate targeted double stranded DNA breaks that were corrected through homology directed repair using single stranded oligos carrying the corrected DNA sequence. Other pathogenic DMD mutations are duplications, which are found in approximately 13% of DMD patients. Utilizing a modified CRISPR strategy that involves a double nuclease approach we deleted a 145 kb (Exons 18-30) duplication and restored dystrophin expression in patient cells. Furthermore, as an universal, mutation-independent treatment approach, we have also utilized a third modified CRISPR system to upregulate utrophin expression to compensate for the loss of dystrophin. In DMD patient myoblasts, we utilized S.pyogenes Cas9, fused to transcriptional activator VP160 to target utrophin promoter. We demonstrated 2.5-5 fold utrophin upregulation resulting in increased expression of β-dystroglycan, providing evidence for functional significance of this strategy.Lastly, in order to determine if this tool is of potential clinical significance, we have began to interrogate CRISPR/Cas9 system in dystrophin-deficient mdx mouse. We have developed strategies for the delivery of CRISPR machinery via rAAV with the goal to achieve dystrophin editing and utrophin upregulation simultaneously in vivo. Taken together, our proof of concept studies provide insights into the potentially far-reaching impact of the therapeutic benefits of the CRISPR system that can be adapted to target a variety of mutations in DMD.