Preclinical R&D Interest Soars as the First AAV Gene Therapy for DMD Hits $200 Million in Sales

Jointly developed by Sarepta Therapeutics and Roche, ELEVIDYS is an adeno-associated virus vector-based gene therapy indicated for the treatment of ambulatory pediatric patients aged 4 - 5 years with Duchenne muscular dystrophy (DMD) with a confirmed mutation in the DMD gene. The United States Food and Drug Administration authorized accelerated approval for this indication based on expression of ELEVIDYS micro-dystrophin in skeletal muscle observed in patients treated with ELEVIDYS in June 2023. It is reported that ELEVIDYS had sales of approximately $130 million in the fourth quarter of 2023. Together with the net product revenue of $69.1 million in the third quarter, its total annual sales reached about $200 million. This achievement far exceeded Wall Street's sales forecasts for ELEVIDYS. The news of Elevidys (delandistrogene moxeparvovec, SRP-9001) receiving FDA accelerated approval has sparked heated discussions while also initiating a new wave of preclinical research for DMD.

DMD Gene Mutations Lead to Duchenne Muscular Dystrophy

DMD is an X-linked recessive genetic disorder primarily affecting males, characterized by progressive muscle atrophy and weakness. It is caused by a deficiency or dysfunction of dystrophin, a protein that helps keep muscle cells intact. The DMD gene, which codes for dystrophin, is the largest human gene and is responsible for protein-coding, providing the genetic instructions for producing dystrophin proteins. In patients with DMD, over 75% have deletions of single or multiple exons in the DMD gene, or point mutations leading to nonsense mutations. These mutations result in a lack of dystrophin, further causing the breakdown of the dystrophin-associated glycoprotein complex (DGC), disrupting the interaction between actin in the muscle cells and the extracellular matrix. This increased susceptibility to damage ultimately leads to the progressive loss of muscle tissue and function, as well as the development of cardiomyopathy.^[1]

Figure 1. Different types of DMD mutations lead to Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD), respectively.

Classic DMD Research Model: The mdx Mouse

The X-linked muscular dystrophy (mdx, DMD^mdx) mutation originates from a spontaneously occurring premature termination codon (PTC) in the mouse Dmd gene. Under the influence of the nonsense-mediated mRNA decay (NMD) mechanism, Dmd mRNA carrying the PTC is partially degraded. The remaining undegraded mRNA encodes a truncated dystrophin protein lacking functionality, which cannot be properly localized at the sarcolemma of muscle fibers.

Cyagen has developed the C57BL/6J background DMD-Q995* mouse (product code: C001518), which carries the same mutation as the mdx mouse. In this mouse, part of the mutated Dmd mRNA is degraded, and the remaining undegraded mRNA cannot encode the full-length dystrophin protein, leading to the absence of this protein.

Figure 2. DMD-Q995* mice lack the expression of dystrophin (Dystrophin).

DMD-Q995* Mice Exhibit Severe Muscle Damage

Creatine kinase (CK) levels are positively correlated with the extent of muscle and cardiac damage, and gender differences significantly affect serum CK levels and muscle damage phenotypes in mdx mice. Studies have found that serum CK levels in male mdx mice are generally more than twice those of female mice ^[2-3], suggesting more severe muscle damage in male mdx mice. Similarly, the serum CK levels of 9-week-old DMD-Q995* mice are consistent with existing literature reports and also show significant gender differences.

Figure 3. Serum creatine kinase (CK) levels significantly increase in DMD-Q995* mice and exhibit certain gender differences.

Histopathology of Muscle Tissues in DMD-Q995* Mice

The genetic background has a certain impact on the muscle atrophy phenotype of mdx mice. The original genetic background of mdx mice was C57BL/10, but through multiple breedings, several genetic backgrounds have been derived, among which DBA/2J, C57BL/10, and C57BL/6 are the most common.^[4] DBA/2J-mdx mice have weaker muscle regeneration capabilities, severe muscle damage, worsened intramuscular fibrosis, leading to severe muscle atrophy and weakness, similar to the phenotype of severe DMD patients. However, the DBA/2J strain carries various gene mutations, which may lead to hearing loss and eye abnormalities in mice. In contrast, C57BL-mdx mice have a slightly longer lifespan, higher serum CK values, and higher levels of inflammation, and their skeletal muscles undergo cycles of degeneration and regeneration, which aligns more closely with the disease mechanism, biochemical, and cellular level changes in DMD patients.^[5-6] Cyagen Biosciences' self-developed DMD-Q995* mice show disease phenotypes such as varying muscle fiber sizes, nuclear aggregation, and inflammatory cell infiltration, similar to model phenotypes reported in the literature.

Figure 4. Muscle pathology in DMD-Q995* mice.

DMD-Q995* Mice Exhibit Behavioral Deficit Phenotypes

Through rotarod test, grip strength test, treadmill test, and gait test, the muscular ability, limb strength, and motor capacity of mice were evaluated. The results indicate that DMD-Q995* mice began to exhibit significant deficits in limb strength, gait, and motor abilities as early as 6 weeks of age.

(1) Rotarod Test

Compared to wild-type mice, DMD-Q995* mice show a significant reduction in fall latency in the rotarod test starting from 6 weeks of age, indicating a deficit in their motor coordination abilities.

Figure 5. Rotarod test for DMD-Q995* mice and wild-type (WT) mice.

(2) Grip Strength Test

Compared to wild-type mice, DMD-Q995* mice exhibit a significant reduction in grip strength, indicating limb strength impairment due to muscle tissue damage.

Figure 6. Grip strength test for DMD-Q995* mice and wild-type (WT) mice.

(3) Treadmill Test

Compared to wild-type mice, DMD-Q995* mice show a significant reduction in the total distance traveled during treadmill tests, suggesting a decline in their exercise capacity and endurance. Additionally, the number of shocks times required to encourage mice to run increases significantly, indicating deficits in motor coordination and learning abilities. Furthermore, DMD-Q995* mice have shorter falling latency times when using the accelerating rotarod after treadmill exercise, which may indicate a higher level of fatigue.

Figure 7. Treadmill test for DMD-Q995* mice and wild-type (WT) mice.

(4) Gait Test

Compared to wild-type mice, DMD-Q995* mice exhibit an increased number of steps and prolonged walking time, with a gradual decline in the proportion of normal steps. Additionally, gait symmetry analysis reveals a trend towards asymmetrical gait. Data reveals a reduction in muscle strength, decreased coordination, lower movement efficiency, deterioration in gait health, as well as a lack of gait balance and stability in DMD-Q995* mice that resemble classic DMD phenotypes.

Figure 8. Gait analysis for DMD-Q995* mice and wild-type (WT) mice.

Recommended Models For Neurodegenerative Diseases

Cyagen has leveraged advanced animal model construction technologies to offer over 16,000 ready-to-use knockout (KO) and conditional knockout (cKO) mouse models, as well as over 20 types of gene-edited and drug-induced rodent models for neurodegenerative diseases. These models cover a variety of genetic targeting methods including gene knockout (KO), conditional knockout (CKO), point mutation (PM), transgenic, and humanization. In addition to ready-to-use mice, custom or collaborative development is also available to meet researchers' specific project needs.

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References:

[1]Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy. Nat Rev Dis Primers. 2021 Feb 18;7(1):13.

[2]Hermes TA, Kido LA, Macedo AB, Mizobuti DS, Moraes LHR, Somazz MC, Cagnon VHA, Minatel E. Sex influences diaphragm muscle response in exercised mdx mice. Cell Biol Int. 2018 Dec;42(12):1611-1621.

[3]Salimena MC, Lagrota-Candido J, Quírico-Santos T. Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice. Histochem Cell Biol. 2004 Nov;122(5):435-44.

[4]Sztretye M, Szabó L, Dobrosi N, Fodor J, Szentesi P, Almássy J, Magyar ZÉ, Dienes B, Csernoch L. From Mice to Humans: An Overview of the Potentials and Limitations of Current Transgenic Mouse Models of Major Muscular Dystrophies and Congenital Myopathies. Int J Mol Sci. 2020 Nov 25;21(23):8935.

[5]Fukada S, Morikawa D, Yamamoto Y, Yoshida T, Sumie N, Yamaguchi M, Ito T, Miyagoe-Suzuki Y, Takeda S, Tsujikawa K, Yamamoto H. Genetic background affects properties of satellite cells and mdx phenotypes. Am J Pathol. 2010 May;176(5):2414-24.

[6]Coley WD, Bogdanik L, Vila MC, Yu Q, Van Der Meulen JH, Rayavarapu S, Novak JS, Nearing M, Quinn JL, Saunders A, Dolan C, Andrews W, Lammert C, Austin A, Partridge TA, Cox GA, Lutz C, Nagaraju K. Effect of genetic background on the dystrophic phenotype in mdx mice. Hum Mol Genet. 2016 Jan 1;25(1):130-45.

[7]Rodrigues M, Echigoya Y, Maruyama R, Lim KR, Fukada SI, Yokota T. Impaired regenerative capacity and lower revertant fibre expansion in dystrophin-deficient mdx muscles on DBA/2 background. Sci Rep. 2016 Dec 7;6:38371.