Today, we'll be introducing a model of Spinal Muscular Atrophy (SMA) - B6-hSMN2(SMA) mice. But before delving into this mouse model, let's first introduce the basic pathology of SMA.

SMA is an autosomal recessive genetic disease caused by degeneration of the spinal cord's anterior horn cells (α-motor neurons), characterized by severe progressive muscle weakness and atrophy. It is a common lethal rare genetic disease in infancy and early childhood. The global incidence of SMA is estimated to be 1/6,000 to 1/10,000, which is relatively high among rare diseases; this has led to SMA often being referred to as a "common rare disease." [1]

Most SMA cases are caused by mutations in the SMN1 gene

SMA is associated with the survival motor neuron (SMN) protein, whose deficiency leads to widespread splicing defects and functional loss in spinal motor neurons. The human SMN gene is divided into SMN1 and SMN2, with SMN1 transcribing and translating into a fully functional protein, serving as the primary source of SMN protein in the body. SMN2 is highly homologous to SMN1, but it contains a nucleotide difference in exon 7 splicing enhancer, resulting in the majority of SMN2 mRNA lacking exon 7, encoding a truncated SMN protein with functional deficits that are rapidly degraded within cells. Only a portion of SMN2 pre-mRNA (10-15%) can be spliced into full-length mRNA, encoding a functional SMN protein (Figure 1).

Figure 1. A small amount of functional SMN protein encoded by the SMN2 gene cannot fully compensate for the loss of the SMN1 gene. [3]

Approximately 95% of SMA patients carry homozygous deletion mutations of exon 7 in SMN1 or mutations that convert SMN1 into SMN2. The expression of SMN2 cannot fully compensate for the loss of SMN protein in the body, thereby affecting movement, respiration, and swallowing, and involving organs such as the spleen, heart, and pancreas, even posing a threat to life. In the absence of SMN1, SMA can be classified into Type 0 to IV based on the copy number of SMN2 in the patient's body. Type I SMA patients have 2 copies of SMN2, Type II SMA patients have 3 copies of SMN2, while Type III and IV SMA patients have more copies of SMN2. Generally, the more copies of SMN2 present in the patient's body, the less severe the disease. Type I SMA is the most severe subtype, accounting for approximately 60% of the total SMA population. [4-5]

Construction Strategy of SMA Models

Based on the characteristics of the mouse Smn gene (only containing the Smn1 gene) and the genetic features of most SMA cases (inability of SMN2 to compensate for the loss of SMN1), Cyagen replaced the mouse Smn1 gene with the human SMN2 gene in situ, thus constructing a Type I SMA model with SMN1 deficiency and expression of human SMN2 (hSMN2) (Figure 2).


                                                      Figure 2. Gene editing strategy for B6-hSMN2(SMA) mice.


This strategy mimics the genetic characteristics of the majority of SMA cases, namely homozygous deletion of SMN1 and double copies of SMN2. Compared to traditional strategies involving knockout of the mouse Smn1 gene and random insertion of several copies of the SMN2 gene via transgenesis, this strategy does not involve random insertion of the SMN2 gene. Instead, B6-hSMN2(SMA) mice have a clear copy number of SMN2 genes and can stably inherit them, which is consistent with the majority of SMA cases. Additionally, this model can be crossed with Rosa26-hSMN2 mice, which carry SMN2 genes inserted into chromosome 6, to increase the copy number of SMN2 genes in mice and simulate different SMA subtypes.

B6-hSMN2(SMA) mice express the human SMN2 gene but do not express the mouse Smn1 gene

Compared to the wild-type control group (B6N), homozygous B6-hSMN2(SMA) mice do not express mouse Smn1 mRNA (Figure 3. d). Instead, they exhibit both transcripts of human SMN2, with and without exon 7 (E7- and E7+, respectively) (Figure 3. a-b). The SMN2 E7+ transcript comprises only a small fraction of the total SMN2 transcripts (E7+ & E7-), similar to human expression patterns (Figure 3. c).

Figure 3. Gene expression analysis of 3-week-old B6-hSMN2(SMA) mice and wild-type mice (B6N).

B6-hSMN2(SMA) mice lack SMN protein

The genetic alteration affects the level of SMN protein. Compared to the wild-type, homozygous B6-hSMN2(SMA) mice exhibit only a small amount of SMN protein encoded by the SMN2 gene in the spinal cord, heart, skeletal muscle, brain, liver, and kidneys (Figure 4). This pattern of protein expression is similar to that observed in human SMA patients.

Figure 4. Detection of SMN protein in 3-week-old wild-type mice (C57BL/6N) and B6-hSMN2(SMA) mice.

B6-hSMN2(SMA) mice exhibit multi-organ SMA pathology

Histological examination results show that homozygous B6-hSMN2(SMA) mice exhibit muscle tissue pathology including myocyte necrosis and atrophy, cytoplasmic disintegration, lymphocyte infiltration, and widened/loosened arrangement of muscle cell interstitium. Additionally, there are foot and toe pathologies such as localized muscle fiber necrosis/dissolution/disappearance, fibrous tissue proliferation, granulocyte infiltration, metatarsal fractures, and subcutaneous edema. Tail pathologies include localized subcutaneous edema, loosened arrangement of fibrous tissue, vascular dilation, muscle cell atrophy, and minimal lymphocyte infiltration (Figure 5).

Figure 5. Obvious pathological phenotypes observed in the muscle, footpads, and tail of 3-week-old homozygous B6-hSMN2(SMA) mice.

B6-hSMN2(SMA) mice exhibit severe developmental defects and a progressive disease course

The absence of SMN protein leads to developmental defects in B6-hSMN2(SMA) mice, manifested as severe phenotypes including muscle atrophy, unstable gait, smaller body size, shorter body length, tail kinking, and limb edema (Figure 6).

Figure 6. Appearance of 3-week-old heterozygous B6-hSMN2(SMA) mice (hSMN2/+) and homozygous B6-hSMN2(SMA) mice (hSMN2/hSMN2).


In addition, homozygous B6-hSMN2(SMA) mice begin to die around day 20, with a mortality rate of about 50% by day 30. Surviving homozygous B6-hSMN2(SMA) mice exhibit toe necrosis and complete tail loss around 30 days of age, ear necrosis around 50 days of age, and whitening of hair around the ears by day 66 (Figure 7). Heterozygous B6-hSMN2(SMA) mice show no abnormalities similar to wild-type mice, consistent with the autosomal recessive inheritance pattern observed in SMA patients.


Figure 7. Survival curve and disease progression of B6-hSMN2(SMA) mice.

Small nucleic acid drugs alleviate disease phenotypes and increase survival rate in B6-hSMN2(SMA) mice

Currently, three SMA drugs, Zolgensma, Spinraza, and Evrysdi, have been approved for marketing. Spinraza, the first approved drug, modulates the splicing pattern of SMN2 mRNA using antisense oligonucleotides (ASOs), thereby increasing the production of normal SMN2 mRNA containing exon 7 to encode functional SMN protein. This approach represents one of the main directions in SMA therapy.[3] Based on publicly available information about Spinraza, an ASO (ASO10-27, synthesized by GenScript) with a structure and function similar to Spinraza was synthesized. Different doses of ASO10-27 were administered to B6-hSMN2(SMA) mice via intracerebroventricular (icv) and subcutaneous (s.c.) injections. Data show that intracerebroventricular (icv) injection of ASO increases the expression of SMN protein in the brain (Figure 8. a) and the number of motor neurons in the spinal cord anterior horn (Figure 8. b) in B6-hSMN2(SMA) mice.

Figure 8. The effects of ASO targeting SMN2 on SMN protein and motor neurons in B6-hSMN2(SMA) mice.

B6-hSMN2(SMA) mice treated with intracerebroventricular (icv) injections showed a significant improvement in survival rate, with mice surviving up to 78 days of age. Untreated B6-hSMN2(SMA) mice exhibited toe necrosis and tail loss by day 35, while mice in the ASO-treated group only showed slight toe swelling by day 43, with no toe necrosis and the tail still intact. By day 78, some mice in the ASO-treated group exhibited tail loss, but toe necrosis was still not observed (Figure 9). The ASO treatment of B6-hSMN2(SMA) mice improves survival rates and delays SMA-related tissue pathology.


Figure 9. ASO treatment improves survival rate and delays tissue pathology in B6-hSMN2(SMA) mice.

Conclusion

In summary, B6-hSMN2(SMA) mice express the human SMN2 gene and exhibit selective splicing of SMN2 mRNA, resulting in the loss of SMN protein in mice and phenotypes similar to classical SMA models. Small nucleic acid drugs such as ASOs, siRNAs, and miRNAs have rapidly emerged in recent years as promising therapeutics due to their ability to target mRNA rather than genes, thereby avoiding genetic risks. They have garnered significant attention, particularly in the field of genetic rare diseases. B6-hSMN2(SMA) mice mimic the genetic mechanism and disease progression of human SMA and show a good response to ASO drugs targeting SMN2. This suggests that this model can be used for research on the development, screening, and evaluation of small nucleic acid drugs targeting SMN2 and other types of drugs.

In addition, Cyagen also possesses a variety of spontaneously occurring, induced, or transplanted disease models and humanized research models in fields such as ophthalmology, immunology, neuroscience, metabolism, oncology, rare diseases, and COVID-19 research.

Cyagen HUGO Plan

Cyagen has initiated the HUGO (Humanized Genomic Ortholog) program, inviting global partners to collaborate in developing novel fully humanized models to aid in drug discovery efforts.

Powered by our innovative TurboKnockout-Pro technology, HUGO-GT enables the seamless in situ replacement of mouse genes with fully-human genes, resulting in a broader range of intervention targets and complete coverage of pathogenic gene mutation sites. The fully humanized target genes significantly enhance drug screening efficiency and can be widely used in preclinical experiments for various types of drugs.




References:
[1] Nicolau S, Waldrop MA, Connolly AM, Mendell JR. Spinal Muscular Atrophy. Semin Pediatr Neurol. 2021 Apr;37:100878.
[2] Kolb SJ, Kissel JT. Spinal Muscular Atrophy. Neurol Clin. 2015 Nov;33(4):831-46.
[3] Day JW, Howell K, Place A, Long K, Rossello J, Kertesz N, Nomikos G. Advances and limitations for the treatment of spinal muscular atrophy. BMC Pediatr. 2022 Nov 3;22(1):632.
[4] Edens BM, Ajroud-Driss S, Ma L, Ma YC. Molecular mechanisms and animal models of spinal muscular atrophy. Biochim Biophys Acta. 2015 Apr;1852(4):685-92.
[5] Keinath MC, Prior DE, Prior TW. Spinal Muscular Atrophy: Mutations, Testing, and Clinical Relevance. Appl Clin Genet. 2021 Jan 25;14:11-25.

 

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