Spinal Muscular Atrophy (SMA), a common autosomal recessive motor neuron disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy gene, is present in all SMA patients but differs by a critical nucleotide that alters exon 7 splicing efficiency. This results in low SMN levels which are not enough to sustain motor neurons. Thus, SMA is not a true loss-of-function disease, but rather one of dosage in which clinical presentation results in severe (Type I), intermediate (Type II) and mild (Type III) forms. The goals of this project are to define SMN dosage requirements for normal health, determine the timing requirements of SMN function and the therapeutic window of treatment for severe and mild forms of SMA. These have yet to be determined and are vitally important for developing a rational treatment strategy. We are using mice, which only have one Smn gene to model SMA. Previously, we have used gene targeting strategies to engineer two different mutations within Smn exon 7 splice enhancer elements (ESE). This has allowed us to model human SMN2 exon 7 splicing in mice, at the endogenous Smn locus. The first allele, SmnC-T, is a C-T nucleotide transition that mimics SMN2. The second allele, Smn2B, alters the central ESE within Smn exon 7. Both alleles produce a mixture of transcripts, some contain while others lack exon 7 and the amount of splicing depends upon the mutation. In addition, the progenitor lines of these mice, SmnC-T/Neo and Smn2B-Neo contain a LoxP flanked Neomycin resistance cassette in intron 7. Its presence causes exon 7 to be excluded from transcripts; however, these progenitor alleles are """"""""repairable"""""""" as excision of the floxed Neo cassette by Cre recombinase allows exon 7 to be included into transcripts. Hence these lines of mice are inducible Smn alleles that can be used to return Smn expression temporally by combining it with a tamoxifen-inducible Cre line.
In Aim 1, we will determine the minimum amount of SMN that is required for health. This will be achieved by breeding our novel Smn mice to generate an allelic series that titrates Smn dosage from 0- 100%.
In Aim 2, we will use our Cre-inducible Smn alleles, SmnC-T/Neo and Smn2B-Neo, to generate and characterize a novel SMA mouse model that presents with severe SMA in which Smn expression can be induced at different times pre- and post-natally.
In Aim 3, we will use our severe Smn-inducible mouse model of SMA to test whether pathological changes in motor neurons due to reduced SMN levels is reversible, and if so, we will define the therapeutic window. This will directly address the timing requirements of SMN function in motor neurons. Finally, in Aim 4, we will use a mild mouse model of SMA and induce Smn expression at various post-natal times to determine the therapeutic window of treatment for mild (Type III) SMA, which we predict will be completely different from severe (Type I) SMA. Overall the research presented in this proposal will provide important information that is critical towards the development of a treatment strategy for all forms of SMA. ? ? ?
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