Development of Novel Micro-dystrophins with Enhanced Functionality

• Published in: Molecular therapy Vol. 27; no. 3; pp. 623 - 635
• Main Authors: Ramos, Julian N., Hollinger, Katrin, Bengtsson, Niclas E., Allen, James M., Hauschka, Stephen D., Chamberlain, Jeffrey S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.03.2019; Elsevier Limited; American Society of Gene & Cell Therapy
• Subjects: AAV; Animals; Binding sites; Carrying capacity; Clinical trials; Cytomegalovirus - genetics; Dependovirus - genetics; Design; DMD; Duchenne muscular dystrophy; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Dystrophin - metabolism; Expression vectors; Extracellular matrix; Gene therapy; Genetic Therapy - methods; Humans; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal - metabolism; Muscle, Skeletal - pathology; Muscle, Striated - metabolism; Muscle, Striated - pathology; Muscular dystrophy; Muscular Dystrophy, Duchenne - metabolism; Muscular Dystrophy, Duchenne - pathology; Mutation; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase - metabolism; Nitric-oxide synthase; Original; Proteins; Sarcolemma; Skeletal muscle; Spectrin; gene therapy; DMD; clinical trials; dystrophin; Duchenne muscular dystrophy; AAV
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2019.01.002

Gene therapies using adeno-associated viral (AAV) vectors have advanced into clinical trials for several diseases, including Duchenne muscular dystrophy (DMD). A limitation of AAV is the carrying capacity (∼5 kb) available for genes and regulatory cassettes (RCs). These size constraints are problematic for the 2.2-Mb dystrophin gene. We previously designed a variety of miniaturized micro-dystrophins (μDys) that displayed significant, albeit incomplete, function in striated muscles. To develop μDys proteins with improved performance, we explored structural modifications of the dystrophin central rod domain. Eight μDys variants were studied that carried unique combinations of between four and six of the 24 spectrin-like repeats present in the full-length protein, as well as various hinge domains. Expression of μDys was regulated by a strong but compact muscle-restricted RC (CK8e) or by the ubiquitously active cytomegalovirus (CMV) RC. Vectors were evaluated by intramuscular injection and systemic delivery to dystrophic mdx4cv mice, followed by analysis of skeletal muscle pathophysiology. Two μDys designs were identified that led to increased force generation compared with previous μDys while also localizing neuronal nitric oxide synthase to the sarcolemma. An AAV vector expressing the smaller of these (μDys5) from the CK8e RC is currently being evaluated in a DMD clinical trial. Gene therapy for DMD is complicated by the enormous dystrophin gene. AAV vectors can only deliver partially functional micro-dystrophin genes. In this report, improved micro-dystrophin cassettes are described that increase muscle strength, avoid muscle-tendon injury, and persist more than 2 years. These improved vectors have great promise for DMD therapy.