Molecular and phenotypic reassessment of an infrequently used mouse model for spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) results from loss of the survival motor neuron 1 (SMN1) gene, with retention of its nearly identical homolog, SMN2. There is a direct correlation between disease severity and SMN2 copy number. Mice do not have a Smn2 gene, and thus cannot naturally replicate the disorder. However, two murine models of SMA have been generated using SMN2-BAC transgenic mice bred onto a mutant Smn background. In these instances mice die shortly after birth, have variable phenotypes within the same litter, or completely correct the SMA phenotype. Both models have been imported to the Jackson Laboratory for distribution to the research community. To ensure that similar results are obtained after importation to The Jackson Laboratory to what was originally reported in the literature, we have begun a molecular and phenotypic evaluation of these mouse models. Here we report our findings for the SMA mouse model that has been deposited by the Li group from Taiwan. These mice, JAX stock number TJL-005058, are homozygous for the SMN2 transgene, Tg(SMN2)2Hung, and a targeted Smn allele that lacks exon 7, Smn1(tm1Hung). Our findings are consistent with those reported originally for this line and clarify some of the original data. In addition, we have cloned and mapped the integration site for Tg(SMN2)2Hung to Chromosome 4, and provide a simple genotyping assay that is specific to the junction fragment. Finally, based upon the survival data from our genetic crosses, we suggest that this underused SMA model may be a useful compliment or alternative to the more commonly used “delta7” SMA mouse. We provide breeding schemes in which two genotypes of mice can be generated so that 50% of the litter will be SMA-like pups while 50% will be controls

Mouse survival motor neuron alleles that mimic SMN2 splicing and are inducible rescue embryonic lethality early in development but not late

Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, SmnC-T-Neo and Smn2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice

Mouse survival motor neuron alleles that mimic SMN2 splicing and are inducible rescue embryonic lethality early in development but not late

Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, SmnC-T-Neo and Smn2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice

Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality; it results from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Humans have a paralogue, SMN2, whose exon 7 is predominantly skipped, but the limited amount of functional, full-length SMN protein expressed from SMN2 cannot fully compensate for a lack of SMN1. SMN is important for the biogenesis of spliceosomal small nuclear ribonucleoprotein particles, but downstream splicing targets involved in pathogenesis remain elusive. There is no effective SMA treatment, but SMN restoration in spinal cord motor neurons is thought to be necessary and sufficient. Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients, reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model. We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection. Systemic administration of ASO-10-27 to neonates robustly rescued severe SMA mice, much more effectively than intracerebroventricular administration; subcutaneous injections extended the median lifespan by 25 fold. Furthermore, neonatal SMA mice had decreased hepatic Igfals expression, leading to a pronounced reduction in circulating insulin-like growth factor 1 (IGF1), and ASO-10-27 treatment restored IGF1 to normal levels. These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate