Project Grant R43AR083297

DEVELOPMENT OF OPTIMIZED ADENO-ASSOCIATED VIRAL CAPSIDS FOR MUSCLE GENE THERAPY - PROJECT SUMMARY. GENE THERAPEUTICS OFFER HOPE TO MANY PATIENTS WITH RARE MUSCLE AND NEUROMUSCULAR DISEASES. DESPITE SOME EARLY SUCCESSES, SEVERAL SERIOUS OFF-TARGET SAFETY CONCERNS HAVE COMPROMISED THEIR DEVELOPMENT DUE TO HEPATIC TOXICITIES AND RELATED IMMUNE RESPONSES TO THE ADENO-ASSOCIATED VIRAL (AAV) VECTORS. THE GENERATION OF NOVEL CAPSIDS WITH SUPERIOR MUSCLE SPECIFICITY COULD, THEREFORE, REVOLUTIONIZE THE MUSCLE GENE THERAPY SPACE BY AVOIDING THE OFF-TARGET EFFECTS THAT COMPROMISE DRUG EFFICACY AND SAFETY. OUR OBJECTIVE IS TO ENGINEER NOVEL AAV CAPSIDS WITH MUSCLE TROPISM THAT EXCEEDS THE CURRENT "MUSCLE TROPIC" SEROTYPES (E.G. AAV6, AAV8, AAV9, AAVRH74 & MYOAAVS) AS NONE IS ACTUALLY "MUSCLE-SPECIFIC". INDEED, ALL OF THESE SEROTYPES CAN TRANSDUCE OTHER TISSUES, ESPECIALLY THE LIVER, WHICH IS FUNCTIONALLY LINKED TO THE NOTED CLINICAL TOXICITIES. IN FACT, THE LIVER FUNCTIONS AS A SINK FOR THESE VECTORS, LIMITING MUSCLE TRANSDUCTION AND ELEVATING THE MINIMALLY EFFECTIVE DOSE. WE HYPOTHESIZE THAT DE-TARGETING THE LIVER WHILE SIMULTANEOUSLY ENHANCING MUSCLE TROPISM IS KEY TO IMPROVING MUSCLE GENE THERAPY SAFETY AND EFFICACY. OTHER GROUPS HAVE SOUGHT TO ENHANCE MUSCLE TROPISM USING DIRECTED EVOLUTION. THIS HIGH THROUGHPUT METHOD ARTIFICIALLY SELECTS CAPSIDS WITH IMPROVED MUSCLE TROPISM, BUT CANNOT ALSO DE-TARGET THE LIVER. BY CONTRAST, WE WILL USE A RATIONAL DESIGN APPROACH TO SIMULTANEOUSLY TARGET KNOWN EPITOPES FOR LIVER DE-TARGETING, ENHANCED AAV-RECEPTOR BINDING AND IMPROVED MUSCLE TARGETING. THESE INCLUDE THOSE FOR IMPROVED SIALIC ACID/AAV-RECEPTOR BINDING, IMPAIRED LIVER TARGETING/HEPARIN SULFATE BINDING, IMPROVED INTEGRIN BINDING AND CAPSIDS WITH COMBINED PROPERTIES. WE WILL ALSO USE AAV6 AS LIVER- AND MUSCLE-TARGETING EPITOPES ARE KNOWN FOR THIS SEROTYPE, BUT NOT FOR THE OTHER SEROTYPES. MILESTONE 1 WILL DEVELOP LIVER DE-TARGETED/MUSCLE TARGETED AAV6 CAPSIDS USING WELL-ESTABLISHED IN VIVO AND IN VITRO IMAGING APPROACHES. MILESTONE 2 WILL DEMONSTRATE FUNCTIONAL EFFICACY BY COMPARING A SMAD7 MUSCLE GENE THERAPEUTIC FEATURING A WILD-TYPE AAV6 (AVGN7) TO ONE WITH A NOVEL OPTIMIZED AAV6 CAPSID. THESE STUDIES ARE UNDERSTANDABLY HIGH RISK YET THEIR SIGNIFICANCE IS DISPROPORTIONATELY MUCH HIGHER AS THEY WILL CREATE A LIVER DE- TARGETED CAPSID WITH IMPROVED MUSCLE TROPISM AND AS A RESULT, VASTLY SUPERIOR SAFETY AND EFFICACY PROFILES. THIS WOULD SUBSTANTIALLY INNOVATE THE MUSCLE GENE THERAPY SPACE PRIMARILY BY REDUCING A MANUFACTURING BURDEN THAT LIMITS DRUG USE TO YOUNGER OR FEWER PATIENTS AND THUS, REDUCING THE OVERALL TREATMENT COSTS WHILE EXPANDING THE NUMBER OF POTENTIAL PATIENT THERAPIES.