B6-hSMN2(SMA) Mice

Figure 1. Schematic illustration of gene editing targeting in B6-hSMN2(SMA) mice. The mouse Smn1 genome sequence will be replaced by the human SMN2 genome sequence (flanked with ~15 and 5 kb of upstream and downstream sequences).

B6-hSMN2(SMA) mice can be used for studies such as the pathogenic mechanism of spinal muscular atrophy (SMA) and the preclinical evaluation of therapeutic drugs

1. Detection of the gene expression

Figure 2. Detection of human SMN2 and mouse Smn1 gene expression in the brain, liver, heart, and skeletal muscle of 3-week-old female wild-type mice (WT) and homozygous B6-hSMN2(SMA) mice (hSMN2) (n=3). The expression of the human SMN2 gene and mouse Smn1 gene, as well as the proportion of SMN2 transcripts containing exon 7 (E7+), were examined by RT-qPCR. The results showed that compared with the wild-type, the expression of the human SMN2 gene was present in the brain, liver, heart, and skeletal muscle of B6-hSMN2(SMA) mice, while the expression of the mouse Smn1 gene was absent. In B6-hSMN2(SMA) mice, transcripts containing exon 7 (E7+) accounted for only a very small portion of total human SMN2 transcripts (E7+&E7-).

E7+&E7-: Total human SMN2 transcripts; E7-: Human SMN2 transcripts lacking exon 7; E7+: Human SMN2 transcripts containing exon 7; ND: Not detected.

 

2. Detection of SMN protein expression

Figure 3. Expression of SMN protein in the spinal cord, heart, skeletal muscle, brain, liver, and kidney of 3-week-old female wild-type mice (WT) and homozygous B6-hSMN2 (SMA) mice (hSMN2/hSMN2). The expression of SMN protein was detected by Western Blot. The results showed that the expression of SMN protein in the spinal cord, heart, skeletal muscle, brain, liver, and kidney of homozygous hSMN2 mice was severely down-regulated, indicating that only a restricted amount of SMN protein encoded by the SMN2 gene existed in homozygous B6-hSMN2 (SMA) mice.

 

3. Survival curves of B6-hSMN2(SMA) mice

Figure 4. B6-hSMN2(SMA) mice exhibit a rapid decline in survival compared to wild-type. The results indicate that homozygous B6-hSMN2 (SMA) mice begin to experience mortality around 13 days, reaching a 50% mortality rate at approximately 20 days, and nearly all mice have died before 40 days. In contrast, heterozygous hSMN2 mice exhibit similar survival patterns to wild-type mice, with no apparent survival abnormalities, consistent with the autosomal recessive inheritance characteristic of SMA.

 

4. Physical appearance status of B6-hSMN2 (SMA) mice

Figure 5. Physical appearance of 3-weeks-old homozygous and heterozygous B6-hSMN2(SMA) mice. Homozygous B6-hSMN2(SMA) mice (hMSN2/hSMN2) exhibit severe muscle atrophy, ataxia, dwarfism, shortened body length, taillessness, and edema of the limbs, while Heterozygous B6-hSMN2(SMA) mice (hSMN2/+) appear normal.

 

5. Histopathology of muscle in B6-hSMN2 (SMA) mice

Figure 6. H&E staining of muscle tissue from 3-week-old female homozygous B6-hSMN2(SMA) mice (hSMN2/hSMN2) and wild-type mice (WT). Homozygous B6-hSMN2(SMA) mice focal areas of muscle cell necrosis, characterized by cytoplasmic disruption and infiltration of a small number of lymphocytes (blue arrows). Surrounding muscle cells show atrophy, with decreased cell size and increased intermuscular space (black arrows). In contrast, muscle tissue from wild-type mice shows no evidence of muscle cell necrosis or atrophy.

 

6. Histopathology of paws in B6-hSMN2 (SMA) mice

Figure 7. H&E staining of paw tissue from 3-week-old female homozygous B6-hSMN2(SMA) mice and wild-type mice (WT). In B6-hSMN2(SMA) mice (hSMN2), the joint structures of the toes are clear, with occasional infiltration of free granulocytes around them (black arrows). Local necrosis and dissolution of muscle fibers can be seen around the paw, with the structure disappearing and being largely replaced by proliferating connective tissue (green arrows), accompanied by a large amount of granulocyte infiltration (blue arrows), and occasional fractures of the metacarpal bones (red arrows). Subcutaneous edema is common, with loose connective tissue, widened gaps, and a small amount of granulocyte infiltration (orange arrows).

 

7. Histopathology of tails in B6-hSMN2 (SMA) mice

Figure 8. H&E staining of tail tissue from 3-week-old female homozygous B6-hSMN2(SMA) mice and wild-type mice (WT). The epidermis and dermis of the tail tissue of B6-hSMN2 (SMA) mice showed no obvious abnormalities, with local subcutaneous edema and loose connective tissue arrangement, occasional blood vessel dilation, and a small amount of lymphocyte infiltration (blue arrow). Compared with the control group, muscle cell atrophy, and reduced volume were more common in the muscle layer (yellow arrow); the center of the tissue was the tail vertebrae, with no obvious abnormalities.

 

8. SMN2-targeted antisense oligonucleotides (ASO) increase the expression of SMN proteins

Figure 9. Treatment of homozygous B6-hSMN2(SMA) mice (hSMN2/hSMN2) with ASO modulating SMN2 splicing pattern. Antisense oligonucleotides (ASO-10-27, synthesized by GenScript), structurally and functionally similar to Spinraza*, an FDA-approved SMA drug, were administered to B6-hSMN2(SMA) mice in different doses via intracerebroventricular (icv) and subcutaneous (s.c.) injections. Data show that icv-injected ASO can increase the expression of SMN protein in the brain (a) and the number of anterior horn motor neurons in the spinal cord (b) of B6-hSMN2(SMA) mice.

**Spinraza is the first approved drug for the treatment of SMA. It modifies the splicing pattern of SMN2 Pre-mRNA through antisense oligonucleotides (ASO), leading to the production of a large amount of normal SMN2 mRNA containing exon 7, which encodes functional SMN protein ^[4].

 

9. SMN2-targeted ASO alleviates the disease phenotype and improves the survival rate.

Figure 10. ASO-10-27 enhances the survival rate of homozygous B6-hSMN2(SMA) mice and delays tissue lesions. Following treatment with ASO-10-27, the survival rate of homozygous B6-hSMN2(SMA) mice significantly improved, with all ASO-treated mice beginning to die only at 140 days of age. In contrast, the median survival period for B6-hSMN2(SMA) mice not treated with ASO-10-27 was only 29 days, with toe necrosis and tail loss appearing at 35 days of age, and all mice dying around 40 days of age. However, B6-hSMN2(SMA) mice treated with ASO only showed slight toe swelling at 43 days of age, with intact tails and no toe necrosis. By 78 days of age, only a few mice showed tail loss, with no toe necrosis. At 85 days of age, although some mice lost their tails, their toes remained healthy. It was not until 120 days of age that some mice began to show signs of paw necrosis. These results indicate that ASO-10-27 treatment can significantly improve the survival and health status of B6-hSMN2(SMA) mice.

1. Basic information about the SMN2 gene

https://rddc.tsinghua-gd.org/en/gene/6607

 

2. SMN2 clinical variants

https://rddc.tsinghua-gd.org/en/ai/pathogenicity/result?id=6be9f925-234d-48e9-b3aa-c1c34fb77e45

 

3. Disease introduction

Spinal Muscular Atrophy (SMA) is an autosomal recessive genetic disease characterized by damage to the anterior horn motor neurons of the spinal cord, leading to progressive muscle weakness and atrophy. This can affect the muscles that control breathing, crawling, walking, head and neck control, and swallowing, thereby increasing the risk of pneumonia and respiratory infections in patients. SMA is the most common fatal neurogenetic disease in infancy, with an incidence rate of 1/6000 to 1/10000 in the population. The expression level of SMN protein is related to the severity of the disease. Based on the copy number of SMN2, SMA can be divided into five phenotypes, with type 0 being the most severe and type IV having the mildest symptoms. Currently, there are about 30,000 SMA patients in China, and the carrier rate of pathogenic variants is about 1/50.

 

4. SMN1 gene and mutations

The SMN1 gene is an important pathogenic gene for SMA. The SMN protein it encodes is a housekeeping protein essential for the survival of eukaryotic cells and maintains the survival of motor neurons. Pathogenic mutations in both alleles of SMN1 can cause SMA. The human genome contains highly homologous SMN1 and SMN2 genes. The SMN1 and SMN2 genes differ by only a few bases. However, the critical base pair difference, c.840C>T, in exon 7 of the SMN2 gene causes the pre-mRNA splicing of the SMN2 gene to differ from that of the SMN1 gene. As a result, the SMN2 gene mainly produces an unstable SMNΔ7 protein that lacks exon 7^[1]. The vast majority of SMA patients have mutations in the SMN1 gene, and the highly homologous SMN2 gene cannot produce enough full-length SMN protein to compensate for the loss of SMN1 gene function, leading to disease onset.

Among SMA patients, about 95% of cases are caused by the homozygous deletion of exon 7 of the SMN1 gene (type 0+0), usually accompanied by the deletion of exon 8. About 5% of cases are caused by compound heterozygous mutations of the SMN1 gene (type 0+1d), that is, one allele is deleted and the other allele has a small pathogenic variation. A very small number of patients are caused by small pathogenic variations in both alleles of the SMN1 gene (type 1d+1d)^[2].

 

5. SMN2-targeted gene therapy

Currently, the treatment of SMA is focused on the following three aspects:
First, target the transcription and splicing process of the SMN2 gene to change its splicing pattern and increase the expression of full-length SMN protein. However, mice are the most common preclinical experimental subjects, and they only have the Smn1 gene and Smn1 homozygous knockout is lethal. Therefore, it is crucial to construct a mouse model that can simulate human SMA pathogenesis and conforms to human disease progression for the development and validation of targeted drugs and other therapies. The application of fully humanized animal models can help promote the further transformation of potential SMA-related therapies into clinical trials. For example, Nusinersen sodium injection (Spinraza) from IONIS is an Antisense Oligonucleotide (ASO) drug, and its preclinical animal model is the classic SMA model Δ7 mice.

This model carries a full-length human SMN2 gene and SMN cDNA with exon 7 deletion (SMN2 delta7) based on knocking out the mouse Smn1 gene^[3]. This triple homozygous mouse is significantly smaller than normal littermates at birth and exhibits progressive muscle weakness, with an average lifespan of 17.7 days. Spinraza targets the splicing site of the intron 7 of the SMN2 gene, modifies the splicing pattern of SMN2 Pre-mRNA, generates a large amount of normal SMN2 mRNA containing exon 7 to encode functional SMN protein, and extends the lifespan of Δ7 mice^[4].

The second type is a replacement therapy that supplements the SMN1 gene. The SMN1 gene is delivered to the patient’s body by an adeno-associated virus (AAV) vector, which can compensate for the protein expression deficiency caused by SMN1 gene mutations. Zolgensma, developed by Novartis, is such a drug and has been approved for marketing.

In addition, small molecule drugs that act on muscles such as CK-2127108 and SRK-015, as well as stem cell transplantation therapy, are under development.

 

6. Summary

The SMN1 gene is an important pathogenic gene for SMA. Developing SMA disease models with copy number determination and stable inheritance is crucial for SMA-related research. The SMN2 full gene humanized model from Cyagen not only has similar disease homology and behavioral manifestations to SMA patients but also has a determined copy number and stable inheritance, making it a better preclinical research model for SMA-related gene therapy.