Project Grant R01NS135175

MODULATING THE ADAPTIVE IMMUNE RESPONSE TO ENHANCE THE EFFICIENCY OF IN VIVO GENE EDITING IN MUSCLE - PROJECT SUMMARY DUCHENNE MUSCULAR DYSTROPHY (DMD) IS A DEBILITATING AND INCURABLE MUSCLE WASTING DISEASE CAUSED BY INHERITED MUTATIONS IN THE DMD GENE, WHICH ENCODES THE MUSCLE STRUCTURAL PROTEIN DYSTROPHIN. THERAPEUTIC APPROACHES THAT SEEK TO CURE DMD MUST INCLUDE A STRATEGY TO REPAIR OR REPLACE THE MUTANT DYSTROPHIN PROTEIN. A RECENT SERIES OF PAPERS FROM MY LAB AND OTHERS DEMONSTRATED THE POTENTIAL FEASIBILITY OF USING ADENO- ASSOCIATED VIRUS (AAV) MEDIATED DELIVERY OF CRISPR-CAS9 NUCLEASES TO MAKE SITE SPECIFIC DNA CLEAVAGES IN THE DMD GENE WITHIN SKELETAL MUSCLE FIBERS, CARDIOMYOCTYES AND MUSCLE STEM CELLS OF LIVING ANIMALS. WE HAVE SHOWN THAT THIS IN VIVO GENE EDITING STRATEGY IS SUFFICIENT TO RESTORE DMD READING FRAME AND ENABLE PRODUCTION OF FUNCTIONAL DYSTROPHIN PROTEIN THAT CAN INCREASE MUSCLE SPECIFIC FORCE AND PROTECT DMD MUSCLE FROM CONTRACTION-MEDIATED DAMAGE. THIS STRATEGY PRESENTS SOME ADVANTAGES WITH RESPECT TO OTHER "EXON SKIPPING" APPROACHES, IN THAT IT ALLOWS EFFICIENT RECOVERY OF DYSTROPHIN PROTEIN IN BOTH THE SKELETAL MUSCLE AND HEART, AND, BECAUSE IT EFFECTIVELY TARGETS MUSCLE STEM CELLS AND IRREVERSIBLY MODIFIES THE DMD LOCUS, IT OFFERS POTENTIALLY PERMANENT RESTORATION OF DYSTROPHIN EXPRESSION IN DYSTROPHIC TISSUES. YET, DESPITE ITS PROMISE, THERE REMAIN SIGNIFICANT POTENTIAL CHALLENGES FOR CLINICAL APPLICATION OF IN VIVO GENE EDITING. IN PARTICULAR, THE BACTERIALLY-DERIVED CAS9 NUCLEASE REPRESENTS A POTENTIAL ANTIGENIC TARGET FOR THE HOST IMMUNE RESPONSE. IN FACT, OUR PRELIMINARY DATA INDICATE THAT CAS9 IS RAPIDLY AND ROBUSTLY RECOGNIZED BY THE IMMUNE SYSTEM WHEN ECTOPICALLY EXPRESSED IN THE MUSCLE OF IMMUNE COMPETENT MICE. IN ADDITION, ANALYSES OF HUMAN SERA INDICATE THAT MORE THAN HALF OF POTENTIAL HUMAN RECIPIENTS OF CAS9 THERAPIES HARBOR PRE-EXISTING ANTIBODY AND T CELL RESPONSES TO CAS9 ORTHOLOGS DUE TO PRIOR INFECTION WITH THE BACTERIA FROM WHICH THESE PROTEINS DERIVE. THUS, INDUCED OR PRE-EXISTING ANTI-CAS9 IMMUNE RESPONSES MAY LOWER THE EFFICIENCY OF THERAPEUTIC GENE EDITING IN MUSCLE BY CAUSING THE IMMUNE-MEDIATED ELIMINATION OF CAS9 TRANSDUCED, GENE- EDITED CELLS. MOREOVER, EMERGING DATA INDICATING THAT THE IMMUNE SYSTEM AND ITS PRODUCTS CAN MODULATE THE EXPRESSION OF AAV-ENCODED TRANSGENES AND OF COMPONENTS OF CELLULAR DNA DAMAGE RESPONSE PATHWAYS RAISE THE POSSIBILITY THAT ANTI-CAS9 IMMUNITY COULD ALTER BOTH THE DEGREE OF ON-TARGET DMD EDITING AS WELL AS THE FREQUENCY AND TYPES OF OFF-TARGET MODIFICATIONS INDUCED. IN THIS PROJECT, WE WILL TEST THE HYPOTHESES THAT ROBUST ANTI-CAS9 RESPONSES REDUCE THE PRODUCTION AND DURABILITY OF GENE-EDITED CELLS AND ALTER THE FREQUENCY OF POTENTIALLY GENOTOXIC EDITS, WHEREAS OPPOSING CAS9 IMMUNITY THROUGH GENETIC OR PHARMACOLOGIC MANIPULATION WILL INCREASE THE EFFICIENCY, SAFETY AND DURABILITY OF DYSTROPHIN RECOVERY BY GENE EDITING. THIS WORK WILL ESTABLISH WHETHER THE ANTI-CAS9 IMMUNE RESPONSE HELPS OR HINDERS IN VIVO GENE EDITING AND WILL BE CRITICALLY IMPORTANT FOR THE DESIGN OF FUTURE CLINICAL EFFORTS USING THIS APPROACH.