A humanized SMN gene containing the SMN2 nucleotide alteration in exon 7 mimics SMN2 splicing and the SMA disease phenotype

Department of Pediatrics, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA.
Human Molecular Genetics (Impact Factor: 6.39). 11/2010; 19(21):4239-52. DOI: 10.1093/hmg/ddq343
Source: PubMed


Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of the survival motor neuron (SMN)
protein. In humans, SMN1 and SMN2 encode the SMN protein. In SMA patients, the SMN1 gene is lost and the remaining SMN2 gene only partially compensates. Mediated by a C>T nucleotide transition in SMN2, the inefficient recognition of exon 7 by the splicing machinery results in low levels of SMN. Because the SMN2 gene is capable of expressing SMN protein, correction of SMN2 splicing is an attractive therapeutic option. Although current mouse models of SMA characterized by Smn knock-out alleles in combination with SMN2 transgenes adequately model the disease phenotype, their complex genetics and short lifespan have hindered the development
and testing of therapies aimed at SMN2 splicing correction. Here we show that the mouse and human minigenes are regulated similarly by conserved elements within
in exon 7 and its downstream intron. Importantly, the C>T mutation is sufficient to induce exon 7 skipping in the mouse minigene
as in the human SMN2. When the mouse Smn gene was humanized to carry the C>T mutation, keeping it under the control of the endogenous promoter, and in the natural
genomic context, the resulting mice exhibit exon 7 skipping and mild adult onset SMA characterized by muscle weakness, decreased
activity and an alteration of the muscle fibers size. This Smn C>T mouse represents a new model for an adult onset form of SMA (type III/IV) also know as the Kugelberg–Welander disease.

Download full-text


Available from: Jordan Gladman, May 09, 2014
  • Source
    • "Furthermore, the distal site does not appear to be strengthened indicating that the ISS is likely to be functionally weakened also in the mouse. These observations indicate that the human ISS would have to be inserted into the murine Smn1 gene if Smn1 exon 7 skipping was to be induced by an +6C>T mutation, but a mouse model containing a murine Smn1 to Smn2-like conversion has been published without insertion of the human ISS [43]. This model exhibited a milder SMA phenotype similar to human SMA type III, indicating that even though the Smn2-like exon 7 was skipped, the level of inclusion was still higher than human SMN2 exon 7, resulting in a mild phenotype. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.
    Full-text · Article · Jun 2014 · PLoS ONE
  • Source
    • "In the past, others have successfully used humanized knock-in mouse models to mimic the pathophysiological mechanisms underlying splice site mutations for several inherited diseases [27-30]. In this study, even though the humanization of mouse Cep290 was not compromising the gene expression in general, the splicing machinery of the mouse appeared not to be able to recognize the human splice acceptor site or the splice donor site that is generated by the intronic CEP290 mutation, suggesting differential recognition of splice sites in humans vs. mice. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
    Full-text · Article · Nov 2013 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ribonucleoprotein (RNP) complexes function in nearly every facet of cellular activity. The spliceosome is an essential RNP that accurately identifies introns and catalytically removes the intervening sequences, providing exquisite control of spatial, temporal, and developmental gene expressions. U-snRNPs are the building blocks for the spliceosome. A significant amount of insight into the molecular assembly of these essential particles has recently come from a seemingly unexpected area of research: neurodegeneration. Survival motor neuron (SMN) performs an essential role in the maturation of snRNPs, while the homozygous loss of SMN1 results in the development of spinal muscular atrophy (SMA), a devastating neurodegenerative disease. In this review, the function of SMN is examined within the context of snRNP biogenesis and evidence is examined which suggests that the SMN functional defects in snRNP biogenesis may account for the motor neuron pathology observed in SMA. WIREs RNA 2011 2 546–564 DOI: 10.1002/wrna.76 For further resources related to this article, please visit the WIREs website
    No preview · Article · Jul 2011 · WIREs RNA
Show more