The human genome harbors two copies of the essential gene, Survival Motor Neuron. SMN1
mRNA expresses full-length transcript, whereas SMN2
produces only low levels of full-length transcript. The critical difference between SMN1
is a silent nucleotide transition in SMN
exon 7 (2
). Using comparative genomics, comparing the genes from mouse and human can provide a better understanding of the function of conserved genes and, additionally, how species have evolved and a gene changed. Indeed, by using this approach, we were able to show that both the mouse and human SMN
genes were regulated by the conserved exon 7 ESE, a suboptimal 5′ splice site and the intronic element ISS-N1. Analysis using and comparing the results from multiple minigene experiments allows an even deeper level of understanding of how a gene like the human SMN2
is regulated and which elements have been evolutionarily conserved.
It was our hypothesis that using the intermediate splicing level generated by the mouse pSmnC>T point mutation when compared with the pSMN2 minigene, it would be possible to generate a mouse that would live longer and be more amenable to testing new therapies for SMA. When the C>T alteration was engineered into the mouse Smn gene, the amount of full-length Smn protein is decreased and leads to a mouse with a very mild SMA phenotype. This mouse is a good model for adult-onset SMA (type III or IV). Though the mild phenotype of our mice could be due to differences in the regulation of the mouse and human SMN genes, it is also possible that mice may not be as sensitive to decreased SMN levels and that a difference in the threshold between mouse and human could also account for the differences in disease severity. Perhaps due to the difference in size, morphology or molecular make-up of the alpha-motor neurons, the mouse requires a more substantial loss of Smn protein before severe pediatric symptoms arise.
Analysis of the type II SMA Delta7 mouse models found that the testes had a higher level of full-length transcript being generated than any other tissue examined (41
). Likewise, splicing ratios generated in various tissues in the Smn
C>T/C>T mouse varied from tissue to tissue, suggesting that there may be tissue-specific splicing regulation of the Smn
gene. These differences seen in the Smn
C>T/C>T mice and the existence of an already identified factor binding in the testes of the SMA Delta7 mice suggest that the SMN
genes may be regulated in a tissue-specific manner. Such tissue-specific regulation could explain why the motor neurons are more sensitive to decreased SMN levels and are the cell type lost in SMA. Perhaps the motor neurons regulate exon 7 splicing of the SMN
genes in a unique way. Thus, the Smn
C>T/C>T mouse provides a tool that will allow us to better elucidate any tissue-specific function in the alternative splicing of the SMN
C>T/C>T mouse additionally offers us the opportunity to examine new modifiers of the Smn
gene. By crossing these mice to mice with other genetic modifications, the role other genes play in the severity of the SMA phenotype can be assessed. As the majority of the current mouse models are so severe that death occurs within the first 2 weeks of life, assaying genes that have a detrimental effect on RNA splicing or phenotype can become challenging, if not impossible. The mild SMA model that we have generated, which appears to be teetering between a normal and disease phenotype, is uniquely suited to assess these potential modifying genes such as NAIP
) or as-yet unidentified modifiers of the SMA disease and SMN2
) is an especially exciting gene to examine as a knock-out mouse has recently been generated and examined with respect to the splicing of the wild-type Smn
). Although complete knock-out of Tra2β
was embryonic lethal, in the heterozygous condition (Tra2β
+/−) mice were viable. When the endogenous Smn
gene was examined in the Tra2β
+/− mouse, there was a mild increase in exon 7 skipping. Since Tra2β
is a known splicing regulator of SMN
, a mouse carrying the Smn
C>T alleles and expressing Tra2β
in the heterozygous condition could yield an increase in exon 7 skipping and a more severe SMA like phenotype. This is just one example of a potential modifier of both SMN
splicing and the SMA phenotype that could be examined in our Smn
C>T/C>T mice. Similar experiments could be undertaken with other potential modifiers of SMA or SMN2
Additionally, new modifiers can be identified by crossing the Smn C>T mouse onto different genetic backgrounds and monitoring the disease phenotype. Backgrounds that increase the phenotypic severity can then be analyzed to identify the causative genetic changes. The work reported here was performed on mice from a mixed 129 Sv/Ev–C57BL/6 background. By breeding the mice onto a congenic background, variations in disease phenotype can be assessed to identify the possible modifier of the SMA phenotype. Understanding how other genes affect the SMA disease phenotype could provide unique points of therapeutic intervention and bring a greater understanding of SMA.
The completion of this research has provided a better understanding between the SMN genes of mice and humans and gives insight into conserved elements within the genes that could potentially be targeted to correct exon 7 splicing. Additionally, by generating the mouse knock-in model, we have a better understanding of how the Smn gene in mice is regulated, giving insight into the amount of Smn protein and transcript ratios that are required to produce the SMA phenotype.
To find a promising treatment for SMA, it is necessary to understand the dynamic interactions involved in the regulation of the SMN RNA and have models available that allow rigorous testing of new drug therapies. Unfortunately, the very mild phenotype of the Smn C>T/C>T mice makes measuring functional rescue of the SMA phenotype difficult. However, the extended lifespan of our mice allows the treatment of the disease at later developmental time points, beyond which other SMA mouse models do not survive. Additionally, crossing the Smn C>T/C>T with any of the currently available SMN2/SMN2 containing models could generate a mouse that could be used to gain more information on a compound's therapeutic effect. The Smn C>T/C>T; SMN2/SMN2 mouse would have the benefits of containing both the mouse Smn C>T and human SMN2 gene present, both containing their endogenous promoter. Additionally, the mouse Smn C>T gene would be in its correct genomic context. Compounds tested in the Smn C>T/C>T; SMN2/SMN2 mouse would allow the analysis of both SMN genes to get a better understanding of the effect of the drug on SMN splicing. A compound that affects splicing of only one gene may be acting through a non-conserved element, whereas changes in splicing of both genes would represent a compound acting on a conserved element. As animal testing represents an investment in both time and resources, being able to monitor an additional Smn gene and potentially better understand the mechanism of a new drug therapy is an excellent resource.
Development of the Smn C>T/C>T mouse provides a new and useful model organism of SMA and lends a deeper understanding of the Survival Motor Neuron gene and how the SMN gene is regulated in both mice and humans. The new Smn C>T/C>T SMA mouse model has the Smn gene regulated by natural epigenetic and transcriptional mechanisms and produces lower levels of Smn protein due to an increase in exon 7 skipping. The Smn C>T/C>T mouse can thus be of use in understanding the role of SMN protein in SMA and teasing apart the complex RNA regulation that is involved in the SMN genes. Furthermore, this new model for the Kugelberg–Welander disease can additionally be used for the identification of modifiers of the SMA phenotypes and to test new therapies for SMA aimed at correction of SMN2 splicing.