B6-3*hSMN2 Mice

Catalog Number: C001681

Strain Name: C57BL/6NCya-Smn1tm1(hSMN2/hSMN2)Gt(ROSA)26Sortm1(hSMN2/+)/Cya

Genetic Background: C57BL/6NCya

One of Cyagen's HUGO-GT™ (Humanized Genomic Ortholog for Gene Therapy) Mouse Strains

 

Strain Description

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterized by the degeneration of motor neurons in the anterior horn of the spinal cord, leading to severe progressive muscle weakness and atrophy. SMA is one of the most common fatal neurogenetic diseases in infancy, with an incidence of 1/6,000 to 1/10,000 [1]. SMA is primarily caused by biallelic loss-of-function mutations in the SMN1 gene. The SMN1 gene is ubiquitously expressed throughout the body, with the highest expression in the spinal cord, and its encoded survival motor neuron (SMN) protein is crucial for the survival and function of motor neurons [2]. In addition, humans possess a highly homologous gene to SMN1, called SMN2, with only a few nucleotide differences. The SMN2 gene contains a c.840C>T point mutation at an exon 7 splicing enhancer, which disrupts the splicing enhancer and/or creates a splicing silencer. This mutation results in a different pre-mRNA splicing pattern for SMN2 compared to SMN1, with most mRNA lacking exon 7, leading to the production of a non-functional, truncated SMN protein that is rapidly degraded. Only about 10%-15% of SMN2 pre-mRNA is spliced into full-length mRNA, encoding functional SMN protein [2-3]. Approximately 95% of SMA patients have homozygous deletions of exon 7 in SMN1, resulting in the inability of SMN2 to adequately compensate for the deficiency of SMN protein, leading to disease onset [2-4]. Therefore, the copy number of SMN2 is currently considered an important modifier influencing SMA disease phenotype. In most patients, the SMN2 copy number ranges from 1 to 6, and a higher copy number correlates with increased production of full-length SMN protein, typically resulting in a milder clinical phenotype [5-6]. Consequently, modulating SMN2 splicing to generate normal functional SMN protein has become a major research focus for SMA treatment. Unlike humans, mice have only one Smn1 gene encoding SMN protein, and its homozygous knockout is lethal, which presents limitations in constructing SMA models and mimicking the compensatory mechanism of the human SMN2 gene [7]. Therefore, the development of humanized mouse models that can both simulate the pathogenic mechanism of SMA (especially SMN2 copy number) and recapitulate the human disease course is of great significance for the development and validation of SMN2-targeted therapies.

Cyagen Biosciences has developed two foundational humanized SMN2 strains based on different strategies and used them to breed SMA disease models containing varying SMN2 copy numbers. One foundational strain (Smn1hSMN2/hSMN2) was constructed by in situ replacement of both copies of the endogenous mouse Smn1 gene with the human SMN2 gene, i.e., knocking out the mouse Smn1 gene while simultaneously introducing two copies of the human SMN2 gene, aiming to mimic SMA patients carrying two copies of SMN2. The other foundational strain was constructed by inserting two copies of the human SMN2 gene into the mouse ROSA26 safe harbor locus (ROSA26hSMN2/hSMN2). Through multiple breeding crosses of Smn1hSMN2/hSMN2 mice and ROSA26hSMN2/hSMN2 mice using different strategies, SMA models carrying 2, 3, and 4 copies of the SMN2 gene on an Smn1-deficient background can be obtained. B6-3*hSMN2 mice (genotype: Smn1hSMN2/hSMN2ROSA26hSMN2/+) are a humanized disease model carrying three copies of the human SMN2 gene, which can be used to mimic SMA patients with three SMN2 gene copies. Since the SMN2 gene primarily produces SMNΔ7 protein lacking exon 7, rather than full-length SMN protein, the humanized SMN2 gene cannot fully compensate for the abnormalities caused by Smn1 deficiency, resulting in the manifestation of SMA-like phenotypes in this model.

Strain Strategy

  • Schematic diagram of gene editing strategy of ROSA26hSMN2/hSMN2 mice

 


Figure 1. Schematic diagram of gene editing strategy of ROSA26hSMN2/hSMN2 mice. Using embryonic stem cell (ES) gene editing technology, the human SMN2 genomic sequence (containing approximately 15kb upstream and 5kb downstream non-coding regions) was reverse cloned into the first intron of the ROSA26 locus.

 

  • Schematic diagram of gene editing strategy of Smn1hSMN2/hSMN2 mice


Figure 2. Schematic diagram of gene editing strategy of Smn1hSMN2/hSMN2 mice. Using embryonic stem cell (ES) gene editing technology, the region spanning approximately 10kb upstream to 0.5kb downstream of the mouse Smn1 gene was replaced by the human SMN2 gene sequence spanning approximately 15kb upstream to 5kb downstream.

 

Application

B6-3*hSMN2 mice can be used for studying the pathogenic mechanisms of spinal muscular atrophy (SMA) and for preclinical screening and evaluation of targeted drugs.

 

Validation Data

1. Detection of Human SMN2 and Mouse Smn1 Gene Expression


Figure 3. Detection of human SMN2 and mouse Smn1 gene expression in the heart, skeletal muscle, brain, and liver of wild-type (WT) mice, B6-2*hSMN2 mice, B6-3*hSMN2 mice, and B6-4*hSMN2 mice (3 weeks old, female). The expression of full-length human SMN2 gene transcripts containing exon 7 (E7+) and endogenous mouse Smn1 gene transcripts were detected by RT-qPCR. The results showed that compared to wild-type mice, full-length human SMN2 gene expression was present in the brain, liver, heart, and skeletal muscle of B6-2*hSMN2, B6-3*hSMN2, and B6-4*hSMN2 mice, while the expression of the endogenous mouse Smn1 gene was completely absent. Furthermore, as the SMN2 copy number increased, the expression of full-length SMN2 transcripts in B6-2*hSMN2, B6-3*hSMN2, and B6-4*hSMN2 mice increased accordingly, consistent with the genetic constitution of SMN2 genes in each model (n=3, Bars represent mean ± SEM).

B6-2*hSMN2 mice (Catalog Number: C001504) Genotype: Smn1hSMN2/hSMN2ROSA26+/+;

B6-4*hSMN2 mice (Catalog Number: C001682) Genotype: Smn1hSMN2/hSMN2ROSA26hSMN2/hSMN2;

 

2. Detection of SMN Protein Expression


Figure 4. Detection of human SMN protein expression in the brain and skeletal muscle of wild-type (WT) mice, B6-2*hSMN2 mice, B6-3*hSMN2 mice, and B6-4*hSMN2 mice (3 weeks old, female)*. The expression levels of human SMN protein in each group of mice were detected by Western Blot. The results showed that consistent with the changes in full-length SMN2 transcript expression observed in RT-qPCR, the expression of SMN protein in B6-2*hSMN2, B6-3*hSMN2, and B6-4*hSMN2 mice gradually increased with increasing SMN2 copy number (n=3, Bars represent mean ± SD; *P<0.05, **P<0.01, ***P<0.001).

*Note: The SMN protein antibody used for detection is human-specific, and the band in wild-type mice may be due to high homology between human and mouse proteins. Considering the potential differences in affinity of this antibody for human and mouse SMN proteins, direct quantitative comparison of SMN protein bands between wild-type mice and SMN2 humanized mice is not recommended.

 

3. Growth Curve


Figure 5. Comparison of growth curves of wild-type control mice, B6-3*hSMN2 mice, and B6-4*hSMN2 mice (0-14 weeks old). The results showed no significant difference in growth between B6-3*hSMN2 mice and B6-4*hSMN2 mice compared to the wild-type group during the observation period, with only a slight decrease (n≥7, Bars represent mean ± SD).

 

4. Tail Length


Figure 6. Comparison of tail lengths of wild-type control mice, B6-3*hSMN2 mice, and B6-4*hSMN2 mice (0-14 weeks old). The results showed that compared to the wild-type group, most B6-3*hSMN2 mice experienced complete tail loss around 10 weeks of age, with a few mice retaining a small portion of the tail. B6-4*hSMN2 mice also showed tail truncation, but the tail length essentially remained unchanged from around 8 weeks of age (n≥7, Bars represent mean ± SD).

 

5. Incidence of Common Disease Symptoms


Figure 7. Comparison of the incidence of external disease symptoms in wild-type control mice, B6-3*hSMN2 mice, and B6-4*hSMN2 mice (0-100 days)**. The data showed that the survival of B6-3*hSMN2 mice and B6-4*hSMN2 mice was similar to that of wild-type mice, with all groups surviving to 100 days (therefore, survival curves are not shown). Regarding disease symptoms, B6-3*hSMN2 mice exhibited significantly more severe symptoms than B6-4*hSMN2 mice. Most B6-3*hSMN2 mice developed symptoms such as tail necrosis, tail disappearance, toe/paw edema, ear necrosis, and toe necrosis during the observation period, while the detection results for other disease symptoms in B6-4*hSMN2 mice were similar to the wild-type control group, except for a higher proportion of tail necrosis (n≥7).

**Note: The y-axis of the above graphs represents the proportion of mice that have not yet developed the indicated symptom. When the y-axis value is 100, it indicates that none of the mice in that group have developed the symptom; when the y-axis value is 0, it indicates that all mice in that group have developed the symptom.

 

6. Timeline of model symptom development


Figure 8. Schematic diagram showing the approximate timeline of the development of various external disease symptoms in B6-2*hSMN2, B6-3*hSMN2, and B6-4*hSMN2 mice. This figure depicts only the overall incidence of each lesion type within the mouse cohort at specific time points. It does not indicate that these lesions originate exclusively at these time points, as some mice may have already manifested lesions earlier.

 

References
[1]Wirth B, Karakaya M, Kye MJ, Mendoza-Ferreira N. Twenty-Five Years of Spinal Muscular Atrophy Research: From Phenotype to Genotype to Therapy, and What Comes Next. Annu Rev Genomics Hum Genet. 2020 Aug 31;21:231-261.
[2]Nicolau S, Waldrop MA, Connolly AM, Mendell JR. Spinal Muscular Atrophy. Semin Pediatr Neurol. 2021 Apr;37:100878.
[3]Kolb SJ, Kissel JT. Spinal Muscular Atrophy. Neurol Clin. 2015 Nov;33(4):831-46.
[4]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.
[5]Cuscó I, Bernal S, Blasco-Pérez L, Calucho M, Alias L, Fuentes-Prior P, Tizzano EF. Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy. Neurol Genet. 2020 Nov 18;6(6):e530.
[6]Calucho M, Bernal S, Alías L, March F, Venceslá A, Rodríguez-Álvarez FJ, Aller E, Fernández RM, Borrego S, Millán JM, Hernández-Chico C, Cuscó I, Fuentes-Prior P, Tizzano EF. Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases. Neuromuscul Disord. 2018 Mar;28(3):208-215.
[7]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.