Duchenne muscular dystrophy is caused by genetic mutations in the DMD gene that result in loss of dystrophin expression in muscle cells. Nonsense mutations arising from the conversion of a sense codon to a stop codon within the DMD gene account for 10-15% of cases, with patient mutations occuring at one of over 800 unique locations spanning the length of the DMD gene. Transfer RNAs (tRNAs) engineered to read through the premature termination codon (PTC) created by a nonsense mutation have the potential to restore expression of full-length dystrophin in these patients. This therapeutic approach is not in principle affected by the immense size of the DMD gene, nor the large number of distinct nonsense mutations observed in Duchenne muscular dystrophy patients. tRNA therapeutics could overcome limitations of gene replacement, mRNA therapeutics, and gene editing approaches, and more generally could potentially unify the treatment of thousands of rare and ultrarare stop codon diseases.
To advance the application of tRNA therapeutics to muscle stop codon disease, Alltrna applied its tRNA engineering platform to optimize tRNA sequences for their ability to read through a PTC and then delivered these engineered tRNAs to muscle using Adeno-associated virus (AAV). Systemic delivery of an engineered tRNA using AAV resulted in robust tRNA expression in mouse skeletal and cardiac muscle. Evaluation of these AAV-delivered tRNAs in mouse models engineered with a PTC reporter gene or a DMD PTC mutation demonstrated the ability of AAV-delivered suppressor tRNAs to restore full-length protein production from a nonsense mutation in muscle. These promising preclinical proof-of-concept results provide a path towards developing a novel class of medicines to restore disrupted protein production from nonsense mutations in DMD as well as from nonsense mutations in other rare and ultrarare muscular dystrophies and congenital myopathies.