Evaluation of micro-dystrophin based and dystrophin-independent AAV gene therapies for Duchenne Muscular Dystrophy

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] Duchenne Muscular dystrophy (DMD) is the most common, progressive childhood muscular dystrophy with an X-linked inheritance. The major cause of the disease is the mutations in the dystrophin gene which results in the absence of a functional dystrophin protein. Currently there is no permanent cure for DMD. Many genetic and pharmacological approaches have resulted in tremendous improvements in animal models and advanced the mission of finding a permanent cure for DMD. Adeno associated virus (AAV) mediated micro-dystrophin gene therapy is the most promising approach to treat patients irrespective of their type of mutations. Dystrophin independent AAV gene therapies have also shown encouraging data in animal models in subsiding DMD pathology. In engineering micro-dystrophins, it is important to include the most essential regions or domains to achieve maximum benefits, that fits into the AAV. Our goal was to understand the impact of hinge 1 (H1) and hinge 4 (H4) regions in the function of a micro-dystrophin ([micro]Dys) construct. Two novel micro-dystrophins were engineered by complete deletion of either hinge 1 or hinge 4 and packaged in AAV9. Three separate groups of 3-month old male mdx4cv mice tibialis anterior muscles were injected with each novel AAV.[micro]Dys vector and parent vector separately. Three months post injection TA muscle contractile properties were evaluated. Hinge 1 deletion was tolerated by parent [micro]Dys although deletion of hinge 4 reduced the functional performance. Hinge domains played an important part in localization of [micro]Dys to the sarcolemma. Deletion of hinge 1 did not interfere with normal sarcolemmal localization whereas complete deletion of hinge 4 failed to localize [micro]Dys. Both novel [micro]Dys were able to restore dystrophin associated glycoprotein complex (DGC) proteins to the sarcolemma in dystrophin positive fibers. To further analyze which region of hinge 4 that could be devoid of [micro]Dys, we engineered additional four novel [micro]Dys with modifications in only the hinge 4 region, while hinge 1 is intact. Deletion of the region upstream of WW domain was shown to enhance the [micro]Dys function, and any other deletion reduced the performance of [micro]Dys. We also found that deletion of upstream region of WW domain did not interfere in [micro]Dys localization to sarcolemma and other deletions failed to fully restore [micro]Dys to sarcolemma. Next, we developed another micro-dystrophin that combined complete deletion of hinge 1 with deletion of the upstream region of WW domain. This latest [micro]Dys showed to preserve the muscle tetanic force similar to parent [micro]Dys. This is the first study of in-depth evaluation of the importance of the presence or absence of hinge 1 and hinge 4 in the functional performance of micro-dystrophin. These data provide valuable insights in engineering novel micro-dystrophins. One of the major cellular networks affected in DMD is the mitochondrial function and subsequent metabolic homeostasis. PGC-1a is a key transcriptional co-activator of mitochondrial biogenesis and oxidative metabolism in muscle. PGC-1a has previously studied in improving skeletal muscle pathology in mdx mouse model although its therapeutic effects on mdx cardiac pathology has not been evaluated. We delivered AAV9.PGC-1a vector systemically via the tail vein of 12-month old female mdx mice and 4-months post injected we evaluated the left ventricular hemodynamic parameters. AAV.PGC-1a treated mice showed normalization of several left ventricular hemodynamic parameters to the wild type level. Pathway protein analysis revealed overexpression of PGC-1a, resulted in the increased expression of several major transcription factors in oxidative phosphorylation, mitochondrial biogenesis, fatty acid metabolism, electron transport chain. This is the first study to report that cardiac hemodynamic improvements in 4-month treatment of AAV.PGC-1a in aged mdx mice. This study also shows that without replacing dystrophin, PGC-1a overexpression alone resulted in improving cardiac performance by improving cardiac metabolism in mdx mice. The data provided useful insights developing novel therapies in improving DMD cardiomyopathy. In the final study we used another novel isoform of PGC-1a family, PGC-1a4 which has shown to be expressed during resistance training and regulates muscle hypertrophy. As muscle hypertrophy induction has previously shown to be therapeutically effective in mdx mouse model, we delivered AAV.PGC-1a4 systemically and as intramuscular injections. In the mdx4cv mouse model, we could not overexpress the PGC-1a4 protein above the endogenous levels and no cardiac or skeletal muscle function was improved. Although intramuscular delivery of AAV.PGC-1a4 in wild type mice showed overexpression of PGC-1a4 protein above endogenous levels. Wild type mice showed improvements in eccentric force, although muscle cross sectional area or muscle weight did not reach statistical significance. Our study concluded that PGC-1a4 is not a suitable candidate for AAV gene therapy for DMD. In summary, this dissertation provides important discoveries related to development of next-generation micro-dystrophin vectors and dystrophin-independent AAV gene therapies.