Many rare inheritable diseases often result from duplication mutations. Unfortunately, there is difficulty finding animal models that accurately represent these

Duchenne muscular dystrophy (DMD) is a muscular disease caused by alterations in dystrophin expression, which has a functional role in muscle force transmission and sarcolemma stability. As such, a loss of dystrophin can lead to muscle weakening and wasting, cardiomyopathy, and several other health conditions. This study focuses on the efficacy of using NG-targeting base editors to restore dystrophin levels in mdx4cv mice. Five weeks following base editor AAv9-iNG injection, mdx4cv mouse hearts exhibited increased levels of dystrophin. Furthermore, approximately 42% of cardiomyocytes became dystrophin-positive following systemic AAV9-iNG treatment, whereas dystrophin expression remained almost completely absent in the control group. A long-term analysis conducted at the 10-month mark showed that dystrophin was almost completely rescued in mdx4cv mouse hearts. In addition to this, dystrophin rescue was also observed in skeletal muscles, including the gastrocnemius and diaphragm, of mdx4cv mice treated with AAV9-iNG. Long-term analysis following intravenous administration of AAV9-iNG also showed almost complete dystrophin restoration (95% of wild type level) in mdx4cv hearts. Through trichome staining analysis, the increased percentage of fibrotic areas seen in the muscle of mdx4cv mice was significantly reduced in those treated with the base editor. Lastly, to determine if AAV9-iNG could improve muscle function, the authors used Aurora’s 1300A 3-in-1 Whole Animal System to measure the maximum plantarflexion tetanic torque during supramaximal electric stimulation of the tibial nerve. The results showed that mdx4cv mice produced significantly less torque compared to wild type mice. Moreover, a significant increase in tetanic torque was observed in AAV9-iNG treated mdx4cv mice. These findings underscore the importance of optimized base editing techniques and their potential use in treating monogenic diseases such as DMD.

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by mutations in the DMD gene. These mutations disrupt the expression of dystrophin, a protein important for muscle fiber stability. DMD is characterized by a progressive loss of skeletal and cardiac muscle strength. This study focuses on characterizing the effects of rAAVrh74.MHCK7.micro-dystrophin, an adeno-associated virus vector containing a codon-optimized human micro-dystrophin transgene, on DMD mice. The authors administered systemic injections of the dystrophin-restoring vector to mdx mice at low, intermediate, and high doses. Three months post-treatment, they analyzed micro-dystrophin positive fibers via immunofluorescent staining. They observed mean expression percentages of 46.7%, 66.8%, and 78.3% for low, intermediate, and high doses, respectively, across all muscles assessed (TA, GAS, QD, PSO, TRI, GLUT and DIA). Fiber diameter and fibrosis were then assessed, where low and intermediate doses reduced fibrosis and normalized fiber size in the diaphragm, similar to that of wild type. Force production was analyzed using Aurora’s 1200A isolated muscle system. In vitro analysis of the dystrophic diaphragm revealed improvement in specific force output following high dose treatment similar to that of wild type. To determine if restoring dystrophin would have a functional effect on hindlimb muscle, the authors further assessed the tibialis anterior (TA) in situ using the 1300A Whole Animal System. rAAVrh74.MHCK7.micro-dystrophin not only improved force output in the TA but rescued the muscle from eccentric contraction-induced damage. Lastly, western blot analysis of tissues from injected mice showed micro-dystrophin protein expression across all skeletal muscles. No micro-dystrophin was detected in off-target organs at low and intermediate doses, with high dose exhibiting faint expression in only the liver. These findings demonstrate safety and efficacy of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin, supporting the use of this vector in Phase I/II safety study in boys with DMD.