​What is Duchenne Muscular Dystrophy?​​

 

Duchenne Muscular Dystrophy (DMD) is a rare X-linked recessive genetic disorder primarily caused by mutations in the DMDgene, leading to the absence of dystrophin, a protein crucial for maintaining the stability of muscle cell membranes.

 

The incidence of DMD is approximately 1 in 3,500 to 5,000 male births, with an estimated 450,000 to 600,000 patients globally. In China, the prevalence is about 8.5 per 100,000 males, corresponding to approximately 60,000 cumulative cases.

 

DMD primarily affects skeletal and cardiac muscle. Patients often present with a waddling gait, toe walking, and lordosis. As the disease progresses, individuals typically lose ambulation around age 12. Cardiac involvement can lead to dilated cardiomyopathy, conduction abnormalities, and arrhythmias. Ultimately, the average life expectancy is reduced to around 26 years.

 

​Pathogenesis and Treatment Approaches​

 

The DMDgene is the largest gene in the human genome, spanning 2.2 million base pairs and containing 79 exons. DMD is mainly caused by mutations in the dystrophin gene on the X chromosome, resulting in the absence or dysfunction of the dystrophin protein. This leads to instability of the muscle cell membrane and progressive destruction of muscle cells. Dystrophin, encoded by this gene, is predominantly expressed in skeletal muscle, cardiac muscle, and the brain, where it functions to stabilize the cytoskeleton and protect muscle cells.

 

The substantial size of the dystrophin gene contributes to its high mutation rate (approximately 1/10,000), with diverse mutation types. Deletion mutations are the most common, accounting for 65% of cases, followed by duplication mutations (6%–10%). The remaining 25%–30% comprise point mutations, small deletions, and insertions. Deletions and duplications frequently occur in two hotspot regions: one near the 5' end (accounting for about 20% of large mutations) and a central hotspot region (accounting for about 80%). Point mutations and small indels are randomly distributed without prominent hotspots.

 

Currently, DMD remains incurable. However, with in-depth research into its pathogenesis and the development of emerging technologies like gene therapy and stem cell therapy, treatment options are expanding.

 

​​(I) Corticosteroid Therapy​

 

Corticosteroids are currently the only pharmacologic treatment proven to improve muscle strength and delay disease progression in DMD patients. Prednisone is commonly used, helping to enhance muscle strength and prolong ambulation. Its mechanism of action involves reducing inflammation-mediated muscle damage. However, corticosteroids do not address the underlying genetic cause of DMD, and their therapeutic benefits are limited.

 

​​(II) Stem Cell Therapy​

 

Stem cell therapy involves transplanting autologous or allogeneic stem cells into DMD patients with the aim of having them differentiate into muscle cells expressing dystrophin. Current research indicates that the capacity of stem cells to differentiate into functional muscle cells is limited, with only a small fraction of cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), demonstrating the ability to contribute to muscle fiber formation.

 

​​(III) Gene Therapy​

 

Current gene therapy approaches for DMD primarily include the following five strategies:Approximately 15% of DMD patients have nonsense mutations introducing a premature termination codon (PTC). Aminoglycosides can bind to specific sites on the ribosomal RNA and disrupt codon-anticodon recognition at the aminoacyl-tRNA acceptor site. This principle can induce missense mutations that bypass the PTC, allowing translation to continue and producing a full-length or near-full-length dystrophin protein.

 

Exon skipping aims to restore the reading frame of the DMDgene by skipping specific exons, resulting in a shorter but partially functional dystrophin protein. This is primarily achieved using antisense oligonucleotides (ASOs), which are short, chemically modified RNA fragments that bind to pre-mRNA and modulate splicing to exclude a target exon. Researchers are exploring various strategies, such as conjugation to muscle-targeting peptides or encapsulation in nanoparticles, to improve the efficiency and delivery of ASOs.

 

This approach uses engineered nucleases, like Zinc Finger Nucleases, to permanently remove essential splicing sequences within an exon (e.g., exon 51) of the dystrophin gene. This prevents the inclusion of that exon in the mature mRNA, thereby restoring the open reading frame. This method could potentially benefit approximately 13% of DMD patients and could be compatible with other gene or cell therapies.

 

Gene editing therapies aim to permanently correct the mutation in the DMDgene at its native locus using technologies like CRISPR-Cas9, restoring normal or near-normal dystrophin expression. Theoretically, a single treatment could provide a permanent correction. This approach holds promise for achieving better functional outcomes than micro-dystrophin gene therapy, as edited dystrophin expression would be under the control of the endogenous gene's regulatory elements.

 

The large size of the DMDgene poses a challenge for gene delivery. However, studies show that delivering a truncated, functional version of the gene (micro-dystrophin) via viral vectors (like AAV) can ameliorate the disease phenotype. Several clinical trials are currently evaluating the safety and efficacy of various micro-dystrophin constructs.

 

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Genetically Engineered Mouse Models​

 

The mdx mouse is the most widely used model for studying dystrophin expression and function. It carries a nonsense point mutation in exon 23, leading to a premature stop codon and the absence of full-length dystrophin. While sharing the same genetic defect as DMD patients, the mdx mouse exhibits a milder dystrophic phenotype with a lifespan only about 20% shorter than wild-type mice, compared to a 75% reduction in DMD patients. The skeletal muscle pathology in mdx mice is relatively mild and progresses slowly with fluctuations.

 

The most common is the dystrophin/utrophin double-knockout mouse (mdx/utrn-/-). This model displays a more severe dystrophic phenotype, characterized by significant muscle weakness, joint contractures, and kyphosis, with an average lifespan of only about 3 months. This supports the hypothesis that the related protein utrophin may partially compensate for the lack of dystrophin.

 

​MingCeler Biotech Supports Gene Therapy Development​

 

Gene therapy offers hope for rare diseases, but its development and validation rely heavily on animal models. Leveraging its proprietary TurboMice™ platform, MingCeler Biotech has developed numerous rare disease mouse models. The TurboMice™ technology overcomes challenges such as long modeling cycles and low success rates for complex models, enabling precise editing at virtually any target genomic locus. It allows for the generation of complete homozygous gene-edited mouse models directly from embryonic stem cells in as little as two months.

 

Mingxun Biotech offers custom services for developing various DMD mouse models, such as mdx and mdx/utrn-/- mice. We welcome inquiries regarding your specific research requirements.