Cell & Gene Therapy
Comparing Preclinical Models for Duchenne Gene Therapy
Cyagen Technical Content Team | July 18, 2025
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Contents
Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by mutations in the DMD gene, resulting in abnormal function of the dystrophin protein. This ultimately leads to progressive muscle degeneration. Duchenne muscular dystrophy (DMD) is a rare muscle disorder but it is one of the most frequent genetic conditions affecting approximately 1 in 3,500 male births worldwide. It is usually recognized between three and six years of age.
Types of Gene Therapy for DMD
The gene therapy for DMD can be classified into three main types: the first type involves AAV-mediated delivery of mini dystrophin or micro-dystrophin; the second type utilizes ASO-mediated exon skipping therapy; and the third type employs Targeted Gene Editing gene editing techniques.
AAV-Mediated Delivery of Mini Dystrophin or Micro-Dystrophin
The AAV-mediated delivery of mini dystrophin or micro-dystrophin has the potential to be applicable to a
wide range of patients, as this supplementation therapy may work for individuals with different types of
mutations. Companies like Sarepta, Roche, and Solid are actively developing AAV-based therapies
targeting mini dystrophin or micro-dystrophin. Among them, SRP-9001 has shown the most promising
progress. Recently, it has gained attention in the news as the FDA advisory committee experts
recommended accelerated approval for SRP-9001. Preclinical animal studies for this pipeline have
utilized the classic mdx model of DMD. The mdx mouse model carries a mutation in the CAA codon in exon
23, which leads to abnormal gene expression. While mdx mice exhibit disease phenotypes similar to DMD,
there are certain differences compared to real patients. First, the phenotype of mdx mice is milder,
particularly in terms of fibrosis and muscle atrophy. Second, the lifespan of mdx mice is approximately
80% of normal mice, whereas the average lifespan of DMD patients is only about one-third of healthy
individuals. Third, the mdx mouse mutation site in exon 23 is not a hotspot mutation, which limits its
disease homogeneity. Additionally, mdx mice show some variations in phenotypes depending on different
backgrounds, adding complexity to studies conducted on mdx mice.
ASO-Mediated Exon Skipping Therapy
ASO-mediated exon skipping therapy is one of the most prominent gene therapy approaches in the pipeline
for DMD. This is attributed to the strong targeting ability and ease of synthesis of ASOs. Companies
like Sarepta, Nippon, and DYNE have invested in this area. Exon skipping targets specific exons such as
44, 45, 50, 51, and 53, and multiple exon skipping strategies have been reported in the literature.
Nippon's pipeline NS-065/NCNP-01 utilizes ASO-mediated exon skipping of exon 53 and has been tested in
preclinical animal studies using mdx52 mice. These mice carry a deletion of exon 52 in the Dmd gene,
resulting in premature termination of translation at exon 53. While mdx52 mice are more suitable for
ASO-based gene therapy as the mutation site is closer to hotspot mutations in patients, there are risks
associated with selecting ASOs specific to human DMD gene from the mouse Dmd gene due to sequence
differences between humans and mice. On the other hand, mdx mice are not suitable for ASO-mediated exon
skipping, particularly when targeting exons located after exon 23, as mdx mice already have a
termination signal at exon 23, and skipping the exons following exon 23 does not provide behavioral
efficacy readouts. In the literature, the hDMDΔ52/mdx model has been used to evaluate the efficacy of
ASO-mediated exon skipping. This model involves the introduction of full-length human DMD gene with exon
52 deletion and subsequent hybridization with mdx mice to generate a DMD disease model. While the
hDMDΔ52/mdx model expresses full-length human DMD protein, the insertion site of the DMD gene is
unknown, it is not an X-linked gene, and further hybridization with mdx mice is required, making the
model construction complex.
Targeted Gene Editing Gene Editing Techniques
The progress of targeted gene editing therapy in DMD has been relatively slow, partly due
to the lack of DMD disease models that can facilitate specific drug target screening and drug
predictability. CRD-TMH-001 is the first Targeted Gene Editing-based pipeline for treating DMD, and it
has received Investigational New Drug (IND) approval. Several successful cases of Targeted Gene Editing
therapy for DMD have been reported in the literature. For example, the use of a cytosine base editor
(CBE) to remove premature termination codons and enable continued translation of the Dmd gene has been
demonstrated in the Δ50;h51KI model. This model involves humanization of the E51 region of the mouse Dmd
gene and deletion of E50, introducing a premature termination codon at E51. It is evident that compared
to mdx52, the Δ50;h51KI model used in Targeted Gene Editing gene editing therapy requires a higher
degree of humanization for the drug targeting sequence. This is an important factor to consider in
Targeted Gene Editing gene editing therapy, as off-target effects can have immeasurable negative impacts
on patients. However, it should be noted that the Δ50;h51KI model has limitations in terms of
humanization compared to the hDMDΔ52/mdx model.
Advantages and Limitations of Existing Models
Classic mdx Mouse Model:
- Involves a mutation in the CAA codon in exon 23, which leads to abnormal gene expression
- Milder phenotype compared to DMD patients
- Lifespan is approximately 80% of normal mice
- Not suitable for ASO-mediated exon skipping, particularly when targeting exons located after exon 23, as mdx mice already have a termination signal at exon 23,
- mdx mouse mutation site in exon 23 is not a hotspot mutation, which limits its disease homogeneity
- Exhibits variations depending on backgrounds
mdx52 Mouse Model:
- Involves a deletion of exon 52 in the Dmd gene, resulting in premature termination of translation at exon 53
- more suitable for ASO-based gene therapy as the mutation site is closer to hotspot mutations in patients, but there are risks associated with ASO selection
hDMDΔ52/mdx Model:
- Involves humanization of the DMD gene with exon 52 deletion
-
Expresses full-length human DMD protein but has limitations in terms of humanization, such as:
- Unknown insertion site of DMD gene, not X-linked, and;
- Requires further hybridization with mdx mice, making the model construction complex
Δ50;h51KI Model:
- Involves humanization of the E51 region of the mouse Dmd gene and deletion of E50, introducing a premature termination codon at E51
- Compared to mdx52, the Δ50;h51KI model used in Targeted Gene Editing gene editing therapy requires a higher degree of humanization in the drug targeting sequence
- Has limitations in terms of humanization compared to the hDMDΔ52/mdx model
Humanized DMD (hDMD) Models by Cyagen
Considering the mutation characteristics of the DMD gene and the shortcomings of existing models, such as the complexity of construction, uncertainty in Transgenic (Tg)-induced gene insertion sites, insufficient humanization regions, and non-hotspot mutations, Cyagen has independently developed humanized DMD (hDMD) research models. In addition to the classic model mdx mouse, we have also independently developed humanized mice with DMD hotspot mutations, including DMD (hE8-30), DMD (hE44-45), and DMD (hE49-53). Furthermore, we have modified wild-type humanized mice to construct hotspot mutation humanized disease models, which can simultaneously obtain better control group and disease models.
Advantages of Cyagen's hDMD Models Include:
- Construction of wild-type and point-mutation humanized disease models in the hotspot mutation region
- Customization of different point mutations on existing wild-type models, improving efficiency and success rate
- Humanization region includes most drug-targeting regions to improve suitability for drug screening and pharmacological research, especially for gene therapy-related drugs such as ASO, Targeted Gene Editing, and siRNA
- Human DMD gene is inserted in situ with a stable and confirmed copy number, ensuring stable inheritance
- Includes several DMD patient-relevant ‘hotspot’ mutations: DMD (hE8-30), DMD (hE44-45), and DMD (hE49-53).
| Cyagen DMD disease model | mdx(E23,C-T) |
| DMD(hE8-30) | |
| DMD(hE44-45), MT | |
| DMD(hE49-53), MT |
Reference:
- Ryder-Cook AS, Sicinski P, Thomas K, et al. Localization of the mdx mutation within the mouse dystrophin gene. [J]. EMBO,1988.
- Mizobe Y , Miyatake S , Takizawa H , et al. In Vivo Evaluation of Single-Exon and Multiexon Skipping in mdx52 Mice[J].Methods Mol Biol, 2018.
- Hoen P A C ' , Meijer E J D , Boer J M , et al. Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene[J]. Journal of Biological Chemistry, 2008, 283.
- Zhang Y, Li H, Nishiyama T, McAnally JR , et al. A humanized knockin mouse model of Duchenne muscular dystrophy and its correction by Targeted Gene Editing-Cas9 therapeutic gene editing[J]. Mol Ther Nucleic Acids. 2022.
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