Step into the 'Ten Deadly Sins of Rare Diseases' series, where we unlock the mechanisms of occurrence and development of rare diseases, research advancements in the field of gene therapy, and innovative preclinical strategies related to model development and drug screening that drive translation of outcomes.

Previous issues:

1. Exploring RHO-Related Pathogenic Mechanisms and RHO Gene Therapy Research Progress [Ten Deadly Sins of Rare DiseasesⅠ]

2. Why are humanized mice more suitable for hemophilia research, which has a higher prevalence in men? [Ten Deadly Sins of Rare Diseases Ⅱ]

3. The TARDBP gene is closely associated with pathogenesis of amyotrophic lateral sclerosis (ALS), but is it an angel or a demon? [Ten Deadly Sins of Rare Diseases Ⅲ]

Muscular Dystrophy

The focus of this issue is muscular dystrophy (MD), which refers to a group of genetic disorders characterized by progressive muscle weakness and degeneration that affects motor control. Recently, there has been increased media attention from the news of the United States FDA’s accelerated approval of the first gene therapy drug (SRP-9001) to treat Duchenne muscular dystrophy (DMD), which is one type of muscular dystrophy.

In addition to DMD, other types of muscular dystrophy that receive significant attention are Facioscapulohumeral muscular dystrophy (FSHD), Myotonic dystrophy (DM), and Limb-girdle muscular dystrophy (LGMD). Now, let's take a look at these four major types of muscular dystrophy in terms of pathogenic mechanisms and gene therapy progress.

Pathogenic mechanisms of DMD and gene therapy developments

The pathogenic gene of DMD is well defined: the DMD gene encodes the production of the dystrophin protein, which is indispensable for normal muscle structure and function in humans. Among individuals with DMD, the DMD gene undergoes different mutations, primarily characterized by gene deficiency, which leads to the inability of DMD to be expressed and function properly.

Currently, the main types of gene therapy for DMD include CRISPR, ASO, and AAV. Regarding CRISPR and ASO based gene therapies [1], it is crucial to have animal models with higher similarity to the human genome, since these therapies involve editing human nucleotide sequences. However, due to the high cost of non-human primates, the development of full genomic DNA humanized mice as the next-generation of preclinical models is urgently needed for more effective evaluation and translation of gene therapy from bench to bedside.

Driven by these demands, Cyagen has self-developed several humanized DMD (hDMD) models that cover the hotspots of DMD gene mutations and drug target regions. In addition, we have developed humanized mouse models of disease with specific point mutations that are clinically-relevant for gene therapy research. These genomically humanized mice can provide more phenotypically and clinically relevant models of human disease, as well as the littermate controls, for highly effective preclinical evaluations.

Cyagen offers both wild-type (WT) and point mutations (MU) humanized models of DMD, which include the following features:

  • Wild-type humanized models encompassing hotspot mutation regions as baseline templates, and point mutation models built upon them.

  • Customized point mutations can be directly performed on existing wild-type humanized models, which enhances efficiency and success rates.

  • Humanized regions cover a significant portion of drug target areas, making them more suitable for drug screening and efficacy studies, particularly for gene therapy-related drugs such as ASOs, CRISPR, siRNAs, etc.

  • In situ insertion of the human DMD gene ensures a defined copy number and stable heredity.

Recommended Mouse Models Target Type
DMD(hE1-70) Humanized(WT)
DMD(hE8-30) Humanized(WT)
DMD(hE44-45) Humanized(WT、MU)
DMD(hE49-53) Humanized(WT、MU)

Pathogenic mechanisms of Facioscapulohumeral muscular dystrophy (FSHD) and gene therapy developments

FSHD is classified into two types: FSHD1 and FSHD2. Despite having different pathogenic mechanisms, both types ultimately result in an abnormally expressed or dysfunctional DUX4 (Double homeo-box 4) gene in skeletal muscle. FSHD1 is the most common type, accounting for 95% of cases. The pathogenic cause of FSHD1 is primarily associated with the deletion of multiple copies of D4Z4 macrosatellite repeat sequence within the subtelomeric region of the long arm of chromosome 4. This deficiency leads to reduced methylation of the DUX4 gene, allowing its re-expression in skeletal muscle and contributing to the disease progression.

Gene therapy primarily focuses on inhibiting expression of the toxic protein DUX4 through ASOs and siRNAs[2]. There are two types of mouse models commonly used for this study: FLExDUX4 mice (inducible)[3] and AAV-mediated DUX4 expression mice[4]. Cyagen has developed AAV-mediated DUX4 expression mouse models, which are highly suitable for preclinical research of gene therapy drugs treating FSHD.

Pathogenic mechanisms of Myotonic dystrophy (DM) and gene therapy developments

DM is classified into two types: DM1 and DM2, with DM1 being the most common type. The pathogenic mechanism involves abnormal expansion of CTG repeat segment in the 3' untranslated region of DMPK gene, leading to dysregulation of RNA-binding proteins and abnormal mRNA splicing. This, in turn, results in various tissue defects and multi-system involvement during DM. Gene therapy primarily focuses on suppressing DMPK expression through ASOs and siRNAs. The models commonly used for DM research include DMPK transgenic mice (carrying multiple CTG repeats)[5] and HSALR transgenic mice[6]. Cyagen has developed both the DMPK and HSALR  transgenic mouse models, which are suitable for preclinical research on DM1.

Pathogenic mechanisms of Limb-girdle muscular dystrophy (LGMD) and gene therapy developments

LGMD is a collective term for a group of neuromuscular diseases characterized by muscle wasting in the limb-girdle or shoulder and hip regions. LGMD is generally divided into autosomal dominant LGMD type 1 and autosomal recessive LGMD type 2. The pathogenic genes are mostly associated with mutations that affect the production of proteins required for muscle function, including: DYSF, SGCA, and SGCB, etc. Gene therapy for LGMD primarily involves the delivery of missing proteins using AAV vectors. In addition, ASO has also been studied by many research groups. Based on the pathogenic mechanisms, Cyagen has developed mouse models such as humanized DYSF (hDYSF; wild-type, mutant options), Dysf-KO, Scgs-KO, Sgcb-KO, etc.[7], which serve to empower preclinical drug development for LGMD for improved translational outcomes.

Next-Generation Humanized Mouse Models for Preclinical Gene Therapy Research: HUGO-GTTM

In addition to muscular dystrophy, the in-depth research of various diseases such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Parkinson's disease (PD)  requires long-segment or even whole-genomic DNA humanized mouse models. However, whole-genome replacement is technically challenging, since the introduction of exogenous sequences on a large scale might affect the expression and regulation of endogenous genes.

To address this, Cyagen has launched the Next-Generation Humanized Mouse Model Development Program: HUGO-GTTM (Humanized Genomic Ortholog for Gene Therapy) Program. Based on the proprietary TurboKnockout-Pro technology, mouse genes are replaced in situ, enabling development of whole genomic DNA humanized mice with a more diverse range of intervention targets. HUGO-GTTM mice carry more efficient large fragment integration, serving as a universal template for targeted mutation customization services. These models provide a better representation of real-world biological mechanisms compared to humanization models which merely implement the coding sequence (CDS), making them valuable tools for efficient preclinical drug research evaluations and improving translatability of results into clinical applications.


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[2] Lim K R Q , Maruyama R , Echigoya Y ,et al.Inhibition of DUX4 expression with antisense LNA gapmers as a therapy for facioscapulohumeral muscular dystrophy (vol 117, pg 16509, 2020)[J].Proceedings of the National Academy of Sciences of the United States of America.  2020(35):117.

[3] Jones T I , Chew G L , Barraza-Flores P ,et al.transgenic mice expressing tunable levels of dux4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity title: fshd-like transgenic mouse models of varying severity[J].  2019.DOI:10.1371/journal.pone.0150938.

[4] Ba L M W , Garwick S E , Mei W ,et al.DUX4, a candidate gene for facioscapulohumeral muscular dystrophy, causes p53-dependent myopathy in vivo.[J].Annals of Neurology, 2011, 69(3):540-552.DOI:10.1002/ana.22275.

[5] Mahadevan M S , Yadava R S , Yu Q ,et al.Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy.[J].Nature Genetics, 2006, 38(9):1066.DOI:10.1038/ng1857.

[6]Mankodi A, Logigian E, Callahan L, McClain C, White R, Henderson D, Krym M, Thornton CA. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science. 2000 Sep 8;289(5485):1769-73. doi: 10.1126/science.289.5485.1769. PMID: 10976074.

[7]Jakub,Malcher,Leonie,et al.Exon Skipping in a Dysf-Missense Mutant Mouse Model.[J].Molecular Therapy Nucleic Acids, 2018.DOI:10.1016/j.omtn.2018.08.013.