Introduction: Duchenne muscular dystrophy (DMD) or Xp21 dystrophy is a malady that causes severe proximal leg weakness. Mutation in the dystrophin gene located on the X chromosome is the cause of DMD disease because the protein encoded by it plays an essential role in maintaining skeletal muscle stiffness. Dystrophin protein is located under the basal lamina and extends to the sarcoplasm and acts as a linker between the intracellular skeleton and the extracellular matrix by binding to actin and the DAPC (Dystrophin-associated protein complex) complex. Lack of production of functional dystrophin also will lead to dysfunction of the heart muscle as well as the pulmonary diaphragm. Although some treatment options such as respiratory support have led to the reduction of clinical symptoms and improvement of the quality of life in these patients, there is still no definitive treatment for these patients. However, some features of the dystrophin gene, including its monogenicity and the long lifespan of muscle cells facilitate the use of gene therapy to treat this disease.
Methods: In a study by D.R. Asher and his colleagues, the AAVrh74 vector was used for the transfection of SRP-9001 microdystrophin transgene along with the MHCK7b muscle-specific promoter and
α-MHC (α-myosin heavy chain) and MCK (Muscle creatine kinase) enhancers to DMD patients lacking functional dystrophin protein. Since the cDNA of the dystrophin gene is larger than the capacity of the AAV viral vector, a shortened version of the dystrophin gene was used in this study, which includes only the essential parts of the protein, including N-ter (for binding to actin), C-ter (for binding to DAPC), R1-R3 region (to connect with sarcolemma), and hing domains (to maintain protein flexibility) and its middle parts were removed. The design of this transgene is based on shortened dystrophin protein in BMD patients who have milder symptoms than DMD patients.
Nicolas Wein et al. used the scAAV vector to deliver four molecules of U7 snRNA (U7 small nuclear RNA) to DMD patients with exon 2 duplication. Duplication of exon 2 leads to disruption of the protein reading frame and non-functional protein production in DMD patients. The U7 snRNA molecules prevent the splicing complex from joining to pre-mRNA by binding to the splicing sites of exon 2 repeats, and thus exon skipping occurs for one of exon 2 repeats.
Another common mutation in DMD patients is the exon 44 deletion. Deletion of exon 44 results in a premature termination codon in exon 45 following splicing of exons 43 and 45. In one study,
Yi-Li Min and colleagues used the CRISPR/Cas9 system to delete exon 43 or 45 and restore the reading frame of the dystrophin protein. In this study, four sgRNAs (small guide RNA) of 20 nucleotides (G1, G2, G3, G4) were used to connect to the splicing acceptor and donor regions in the vicinity of exon 43, and another four sgRNAs (G5, G6, G7, G8) were designed to bind to the 5' splicing site of exon 45. In this way, the deletion of each exon 43 or 45 led to the restoration of the protein reading frame and the production of functional dystrophin protein.
Results: In all the mentioned gene therapy methods, the production of functional dystrophin protein has been restored through healthy gene transfection or restoration of the protein reading frame during mRNA translation.
Conclusion: Today, various methods are used to treat DMD, including transfection of adenoviral vectors carrying microdystrophin (which includes the functional sequences of dystrophin protein), exon skipping during pre-mRNA splicing, and genome editing through CRISPR/Cas9 technology. All these methods lead to the production of functional dystrophin protein, although with a shortened length, which leads to a reduction in the clinical symptoms of DMD patients and an improvement in the quality of their life.