Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse D md gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3′-end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes, eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested in eukaryotic cells. Here, using a reconstituted mammalian translation system, we show that eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.

Many genetic diseases that are responsible for muscular disorders have been described to date. Gene replacement therapy is a state-of-the-art strategy used to treat such diseases. In this approach, the functional copy of a gene is delivered to the affected tissues using viral vectors. There is an urgent need for the design of short, regulatory sequences that would drive a high and robust expression of a therapeutic transgene in skeletal muscles, the diaphragm, and the heart, while exhibiting limited activity in non-target tissues. This review focuses on the development and improvement of muscle-specific promoters based on skeletal muscle -actin, muscle creatine kinase, and desmin genes, as well as other genes expressed in muscles. The current approaches used to engineer synthetic muscle-specific promoters are described. Other elements of the viral vectors that contribute to tissue-specific expression are also discussed. A special feature of this review is the presence of up-to-date information on the clinical and preclinical trials of gene therapy drug candidates that utilize muscle-specific promoters.