In support of August being annually recognized as International SMA Awareness Month, Part V of our "Ten Deadly Sins of Rare Diseases" article series will focus on Spinal Muscular Atrophy (SMA). Today's captivating column will reveal the underlying pathological mechanisms of SMA, explore the cutting-edge advancements of related gene therapy research, journey through the intricacies of preclinical model development, and unravel ingenious strategies for drug screening. These endeavors collectively steer the course of translational research towards more impactful outcomes.

Catch up on our past articles:

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]

4. Reviewing Muscular Dystrophy: Types, Pathology, and Gene Therapy Research [Ten Deadly Sins of Rare Diseases Ⅳ]

Spinal Muscular Atrophy (SMA) is an autosomal recessive genetic disorder characterized by progressive muscle weakness and atrophy, affecting the spinal anterior horn motor neurons. It is a relatively common and fatal neurogenetic disease in infants and young children, with an estimated incidence rate of approximately 1 in 6000 to 1 in 10000 individuals.

There are currently limited therapeutic drugs available to treat and/or mitigate SMA, but gene therapy has brought good news for patients with this condition. Recent research has suggested that the SMN2 gene plays a crucial role in treatment from a gene therapy approach, quickly transforming it into the most promising ‘key’ to a cure for SMA.

Pathogenesis of Spinal Muscular Atrophy (SMA)

In the human genome, there are highly homologous SMN1 and SMN2 genes, differing by only a few nucleotides. A crucial nucleotide difference, c.840C>T, exists in exon 7 of the SMN2 gene. This disparity in the pre-mRNA splicing of both genes results in the predominant production of an unstable SMNΔ7 protein with exon 7 deletion from the SMN2 gene [1].

The vast majority of SMA patients have mutations in SMN1 gene, while the highly homologous SMN2 gene cannot produce sufficient levels of full-length SMN protein to compensate for the functional loss of SMN1, leading to the onset of the disease. Consequently, in the process of SMN protein production, SMN1 can be considered the protagonist, while SMN2 plays a supporting role.

Gene therapy for SMA: SMN2 Gene Models

Currently, the main treatment approaches for SMA primarily involve supplementation therapy and targeting SMN2 to modify its splicing pattern and increase the expression of full-length SMN protein. In this process, the supporting role (SMN2 gene) undergoes a transformation and becomes a key player in the treatment by expressing full-length SMN protein. For instance, Ionis Pharmaceuticals' ASO drug, Nusinersen sodium, binds to the mRNA transcribed from the SMN2 gene, altering the RNA splicing process, thereby increasing the expression of normal SMN protein [2].

In preclinical research related to SMA, a commonly used disease model is the Δ7 mouse, which is a triple-homozygous transgenic mouse: Smn1-/-; SMN2tg/tg; SMNΔ7tg/tg. To advance drug development for this disease, Cyagen has self-developed the humanized SMN2 (hSMN2), which is a full-length genomically humanized mouse model, which provides an unparalleled degree of genetic humanization. In this mouse model, the endogenous Smn1 gene is replaced with the full-length human SMN2 gene, providing researchers with a humanized disease model that more closely resembles the biological mechanisms that are observed in human pathologies.

Fully Humanized SMN2 (hSMN2) Model: Validation Data

1. Detection of hSMN2 and mSmn1 Gene Expression in hSMN2 Mice

Figure 1. mRNA Expression of Human SMN2 Gene and Mouse Smn1 Gene in hSMN2 and Wild-Type Mice (B6N).

The qPCR results of brain and liver indicate that, compared to the control group B6N, hSMN2 mice successfully express human hSMN2 within their bodies and do not express mouse Smn1. (Note: E7+ & E7- represent all hSMN2 transcripts; E7- represents hSMN2 transcripts lacking exon 7.)

Figure 2. SMN Protein Expression in the brain and liver hSMN2 and Wild-Type (B6N) Mice.

The expression of SMN protein in the brain and liver of hSMN2 mice is significantly lower than that in wild-type (WT) B6N mice, indicating a reduced ability of SMN protein synthesis in hSMN2 mice.

2. Abnormal Body Size in hSMN2 Mice

Figure 3. Comparison of Body Length between homozygous (KI/KI) and heterozygous (KI/+) hSMN2 mice.

hSMN2 homozygous mice (KI/KI) exhibit muscular atrophy, instability while standing, smaller body size, shorter body length, and tail truncation compared to age-matched heterozygous mice (KI/+), which carry both fully humanized and murine SMN2 gene alleles.

3. Abnormal Muscle Tissue in hSMN2 Mice

Figure 4. Muscle Histological Staining (Hematoxylin and Eosin Staining)

Compared to the wild-type mice (B6N strain background), muscle tissue from hSMN2 mice shows a degree of muscle cell necrosis and cytoplasmic disintegration, which is accompanied with a small amount of lymphocyte infiltration (blue arrows). Surrounding areas exhibit muscle cell atrophy, reduced volume, widened intercellular spaces, and more loosely arranged muscle cells and tissues (black arrows).

In conclusion, hSMN2 mice successfully express the human SMN2 gene while exhibiting significantly reduced SMN protein expression. Additionally, these mice demonstrate abnormal muscle tissue and body size development phenotypes, effectively mimicking certain symptoms observed in SMA patients.

Next-Generation Humanized Mouse Models: Unlocking New Avenues for Disease Research

In addition to Spinal Muscular Atrophy (SMA), a multitude of diseases such as Retinitis Pigmentosa (RP), Age-Related Macular Degeneration (AMD), Parkinson's Disease (PD), and more, require an in-depth exploration of their underlying disease mechanisms. Unveiling these intricacies often necessitates the utilization of long-segment or whole genomic DNA humanized mice. However, the intricate challenge of replacing the entire genomic DNA segment poses hurdles, including potential disruptions to native gene expression.

Next-Generation Humanized Mouse Model Development Program: HUGO-GTTM

To surmount these hurdles, Cyagen introduces the groundbreaking initiative - the Humanized Genomic Ortholog for Gene Therapy (HUGO-GTTM) Program, a remarkable leap in Next-Generation Humanized Mouse Model Development. Powered by our pioneering TurboKnockout-Pro technology, this program enables the seamless in situ replacement of mouse genes. This innovation paves the way for the creation of fully humanized mice carrying entire genomic DNA segments, opening a realm of possibilities for diverse intervention targets. 

HUGO-GTTM mice redefine large fragment integration efficiency, standing as a universal template for targeted mutation customization services. These models transcend the limitations of traditional humanization approaches that solely focus on coding sequence implementation (CDS). The result? A truer reflection of real-world biological mechanisms and pathogenesis that enables effective and translatable preclinical research. The advanced HUGO-GTTM mice model platform provides indispensable gene modeling tools for efficient preclinical drug research evaluations, serving to enhance the translation of research outcomes into impactful clinical applications.


[1]Gladman JT, Chandler DS. Intron 7 conserved sequence elements regulate the splicing of the SMN genes. Hum Genet. 2009 Dec;126(6):833-41. doi: 10.1007/s00439-009-0733-7. PMID: 19701774; PMCID: PMC2891348.

[2]Hill SF, Meisler MH. Antisense Oligonucleotide Therapy for Neurodevelopmental Disorders[J]. Dev Neurosci. 2021;43(3-4):247-252. doi: 10.1159/000517686.