Translational development of novel CRISPR approach to treat genomic duplications

PROJECT ABSTRACT The goal of this Phase I STTR project is to accomplish key milestones in commercializing a novel SaCas9 nickase-based system (CRD-002) to treat dystrophinopathies in order to restore the production of healthy, endogenous full-length dystrophin protein otherwise compromised by duplication mutations. Duchenne muscular dystrophy (DMD) is characterized by progressive muscle loss and cardiomyopathy ultimately leading to death in the third decade of life. There is thus a strong rationale for mutation-specific therapies that can restore endogenous full-length dystrophin in order to restore the entire function of the dystrophin protein that cannot be achieved by current microdystrophin gene replacement therapy. Duplication mutations are ideal candidates for mutation-specific correction as they are amenable to full-length gene restoration by removal of duplicated exons to restore the normal reading frame. Pathogenic duplications underlie 10% to 15% of DMD mutations, but there is no specific therapy for duplication mutations downstream of exon 5. The Lek lab developed a novel single nickase approach that efficiently removes DMD duplications at in vitro efficiencies comparable to nuclease approaches. Importantly, nickase produces a scarless edit at the target site, whereas nucleases produce a characteristic insertion/deletion mutation. The novel exon 18-30 tandem duplication mouse model for DMD (Dup18-30), provides a valuable tool to test DMD human therapies. Sequences in DMD exons 18-30 that are shared between the human and mouse genes will be targeted with this novel nickase strategy to induce a scarless edit that restores the gene sequence without introducing frameshifts. The objective of this Phase I STTR project is to characterize the efficacy and pharmacology of the novel single- guide nickase strategy to remove duplications using human-mouse compatible targets for direct clinical translation. Myos, Inc., develops and commercializes novel biomedical therapeutics to improve outcomes for patients. This STTR project is a collaboration with Monkol Lek, Ph.D. of Yale University to optimize the CRD- 002 CRISPR nickase approach. Aim 1 is to characterize human-mouse compatible editing targets to identify lead candidate for in vivo validation. This work will assess the in vitro editing efficiency of three candidate CRISPR guides in human and mouse cell lines, and will validate the predicted target sites and characterize off- target effects. Aim 2 is to perform a functional assessment of CRD-002 in the Dup18-30 mouse model. CRD- 002 packaged in rAAV9 will be injected systemically into Dup18-30 mice followed by molecular analysis of the splice site, analysis of DMD protein sequence, and functional analysis of muscle structure. Successful completion of this project will demonstrate the feasibility of using CRD-002 to restore full-length dystrophin protein. A future Phase II project will support the manufacturing development and the pivotal GLP toxicology study to support the submission of an IND for future clinical trials.