Revolutionizing Duchenne Muscular Dystrophy Research: Humanized Mouse Models Pave the Way

Imagine a world where children with Duchenne Muscular Dystrophy (DMD) can run, play, and live without the looming threat of muscle degeneration. This vision may soon become a reality, thanks to groundbreaking advancements in gene therapy and the development of humanized mouse models. In this article, we'll explore how these tiny yet powerful research tools are transforming the landscape of DMD treatment and bringing hope to thousands of families worldwide.

Key Takeaways:

• Discover the challenges of DMD gene therapy and why size matters
• Uncover the potential of exon skipping therapy in treating DMD
• Learn how humanized mouse models are accelerating drug development
• Explore Cyagen's innovative HUGO-GT^TM platform and its impact on DMD research

Join us on a journey through the cutting-edge world of DMD research, where small bodies carry big missions in the fight against this devastating genetic disorder.

Understanding Duchenne Muscular Dystrophy: A Rare but Devastating Genetic Disorder

Duchenne Muscular Dystrophy (DMD) is a severe genetic muscle disorder that primarily affects boys, with an incidence rate of 1 in 5,000 male births.^[1] The disease is caused by mutations in the DMD gene, which encodes dystrophin, an essential protein for muscle integrity. The absence of dystrophin leads to progressive muscle degeneration and weakness. Ultimately, this can result in lifelong wheelchair dependence and even respiratory failure.

Figure 1. Dystrophin: Stabilizing Muscle Structure and Ensuring Normal Contraction. ^[1]

Overcoming the Size Barrier Challenge of DMD Gene Therapy Development

The DMD gene is the largest known protein-coding gene in the human body, spanning approximately 2.4 Mb. Over 75% of patients have large deletions or nonsense mutations in the DMD gene, primarily concentrated in exons 3-9 and 45-55. These mutations cause dystrophin deficiency and trigger the breakdown of the dystrophin-glycoprotein complex (DGC), which disrupts the interaction between actin and the extracellular matrix, weakening muscle fiber connections and making them highly susceptible to damage. This muscle vulnerability ultimately results in progressive muscle deterioration and loss of mobility, as well as the development of cardiomyopathy.^[2] 

Traditional adeno-associated virus (AAV)-mediated gene therapy  has shown promise but is constrained by its limited vector capacity, which prevents the delivery of the full-length DMD gene. As a result, current AAV therapies are forced to rely on delivering a “mini-version” of dystrophin—known as the Micro-Dystrophin “mini-version” gene therapy approach.^[1-3]

Figure 2. The Diverse and Complex Types of DMD Mutations: Large Deletions Are the Most Common, Posing Challenges for Gene Therapy Design. ^[4]

Limitations of Micro-Dystrophin Gene Therapy: The Risk of Immunogenicity

Despite its advantages, micro-dystrophin therapy presents potential immunogenicity concerns. Some DMD patients have lost specific regions of dystrophin epitopes due to genetic deletions. If the Micro-Dystrophin used in gene therapy contains these "deleted regions," the patient’s immune system may recognize them as foreign "non-self" epitopes, triggering an immune rejection response and causing inflammatory damage.^{[5, 6]} Additionally,  these AAV-based therapies often require higher viral doses for effective treatment, which undoubtedly increases the risk of side effects and tissue toxicity.

Figure 3. Immunogenic Challenges in AAV Gene Therapy: Severe Immunotoxic Reactions in Some Patients. ^[6]

Exon Skipping Therapy: A New Approach to DMD Treatment

Exon skipping therapy has emerged as a promising alternative. This therapy utilizes antisense oligonucleotides (AONs) to modify pre-mRNA splicing, allowing cells to bypass mutated or deleted exons in the pre-mRNA of the DMD gene, effectively reshaping the mRNA reading frame. This results in the production of a truncated but still functional dystrophin protein.

Figure 4. Mechanism of Exon Skipping Therapy for DMD Treatment. ^[7]

Currently, four AON drugs targeting exons 45, 51, or 53 have received FDA approval, offering new hope for some patients.^[8] However, there are still many patients who remain ineligible and have yet to benefit. Recent studies suggest that single or multiple exon skipping strategies could extend therapeutic benefits to a broader patient population. For example, the exon 51 skipping strategy benefits 17.2% of patients with large deletions, while multi-exon skipping strategies targeting exons 45-55 or 3-9 could respectively cover 70.6% and 19.2% of patients with large deletion mutations—significantly increasing the proportion of patients who can benefit.^[8]

Figure 5. Exon Skipping Strategy Shows Broad Application Potential, with the Promise of Covering a Wide Range of DMD Mutations.^[8]

Humanized Mouse Models: Accelerating Exon Skipping Therapy Development

To successfully translate exon skipping therapy from theory to clinical application, robust preclinical animal models are essential for safety and efficacy evaluations. Since exon skipping therapies specifically target the human DMD gene, humanized DMD mouse models—expressing the human DMD gene—provide the ideal platform for preclinical testing of AON candidates. Several research institutions and companies, such as VICO Therapeutics, Entrada Therapeutics, and BioMarin, have successfully used humanized DMD mouse models for in vivo screening and evaluation of next-generation AON molecules,^[9-13] playing a crucial role in the development of DMD therapies.

Figure 6. Humanized DMD Mouse Model for In Vivo Screening of AON Candidate Molecules. ^[9]

Cyagen's Humanized DMD Mouse Models: Advancing Therapeutic Development

To accelerate DMD therapeutic research, Cyagen has developed multiple humanized DMD mouse models through its HUGO-GT^TM (Humanized Genomic Ortholog for Gene Therapy) platform. These models incorporate high-frequency human DMD gene mutations, providing researchers with powerful tools to accelerate the development of exon skipping therapies.

The verification data for some of the humanized mice are as follows:

● B6-hDMD(E8-30) Mice

Figure 7. B6-hDMD(E8-30) Mice Successfully Express the Human DMD Gene and Full-Length Dystrophin Protein.

● B6-hDMD(E49-53) Mice

Figure 8. B6-hDMD(E49-53) Mice Successfully Express the Human DMD Gene and Full-Length Dystrophin Protein.

● B6-hDMD(E44-45) Mice

Figure 9. B6-hDMD(E44-45) Mice Successfully Express the Human DMD Gene and Full-Length Dystrophin Protein.

● B6-hDMD(E44-45)*Del E44 Mice: Simulate the Exon 44 Deletion Mutation

Figure 10. B6-hDMD(E44-45)*Del E44 Mice Exhibit Functional Impairment and Significant Muscle Damage Phenotype Due to Exon 44 Deletion.

With the continuous advancements in gene editing and nucleic acid drug technologies, new treatment approaches to DMD are emerging. Humanized mouse models play a pivotal role in facilitating these breakthroughs, bringing renewed hope to DMD patients and their families. Scientific innovation is paving the way for transformative treatments that could change the trajectory of this debilitating disease.

The HUGO-GT^TM Initiative: Partnering for Next-Generation Humanized Models

Cyagen has launched the HUGO-GT^TM (Humanized Genomic Ortholog for Gene Therapy) program, inviting global partners to collaborate in developing novel fully humanized models to accelerate new drug discovery.

Our scientific team has already established a range of genomically humanized mouse models to enable your preclinical studies of DMD, including: wild-type (wt), mutant (Mut), and knockout (KO) lines. Our models feature genomic integration for a variety of high-frequency exon mutation sites seen in patient populations.

Can’t find what you’re looking for? We are constantly innovating and can develop custom HUGO-GT^TM mice to suit your study—contact us for a free consultation and quote.

Key Features of HUGO-GT^TM mice:

• Full Genomic Coverage of Pathogenic Gene
• Efficient Drug Screening
• Applicable to Gene Therapy Methods including ASO/CRISPR/siRNA, etc

Cyagen HUGO-GT^TM mice are developed using the proprietary TurboKnockout-Pro technology, enabling in situ replacement of murine genes. These models encompass a broader range of intervention targets and incorporate an advanced large-fragment vector fusion technology. Serving as a versatile template, they allow for customized mutation services and provide a preclinical research model that more closely mimics real-world biological mechanisms.

References
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[7]Entrada Therapeutics. "Duchenne Muscular Dystrophy (DMD)." Entrada Therapeutics. Accessed October 31, 2023. https://www.entradatx.com/dmd.
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