Proximal spinal muscular atrophy (SMA), the leading monogenic cause of infant mortality, is caused by the loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical gene is present in all SMA patients; however, it cannot provide protection from disease due to the aberrant splicing of exon 7. Hence only a small amount of SMN protein is made from SMN2 and this is not enough to maintain motor neurons. As all SMA patients have at least one copy of SMN2 it provides a natural target for therapeutic intervention. However, an important point that has yet to be addressed is """"""""How much SMN is enough?"""""""" Thus, the overall goal of this project is to determine the minimal amount of SMN that is required for normal development and postnatal health of motor neurons. Once identified this will be the target point for al future therapies aimed at upregulating SMN2. This has important implications for SMA therapy and has yet to be addressed or answered in humans or mouse models of SMA, most likely because the current models are not useful to answer this question, so a new generation of mice must be made. In this application we will use two novel Smn alleles that we have recently generated that mimic the alternative splice seen in human SMN2 exon 7 to titrate Smn dosage. These alleles were generated through subtle mutation of exonic splice enhancer elements that are present within Smn exon 7. We hypothesize that when combined with either the wild type or Smn null al ele a wide titration range can be achieved to provide a """"""""clean"""""""", consistent way of titrating Smn levels in a relevant way to what occurs in SMA and provide us with an opportunity to systematically define the minimal amount of SMN that is required to prevent an SMA phenotype. To test this, we will analyze two different subtle mutations of exonic splice elements within Smn exon 7 that we introduced into the murine germline. The first allele contains a C-T mutation that mimics the nucleotide transition found in SMN2, while the second al ele corresponds to a mutation within the central splice enhancer region of Smn exon 7. In the first aim of this grant, we will determine the molecular consequences of these """"""""knockin"""""""" mutations by analyzing Smn exon 7 splicing and determining Smn protein levels. In our second aim, we will determine the titration range that can be achieved using these new Smn alleles in combination with the Smn wild type and null allele. Based upon Smn dosage, we will choose three genotypes to characterize at the molecular, pathologic and phenotypic level to 1) validate these animals as new models of SMA. 2) gain insight into Smn dosage and its effects on physiological outcomes 3) determine the minimal level of SMN that is required for normal health and maintenance of motor neurons. Spinal muscular atrophy is the most common genetic cause of infant mortality. In this application we propose to use two novel Smn alleles that we have recently generated that mimic the alternative splice seen in human SMN2 exon 7 to titrate Smn dosage. This was achieved through subtle mutation of exonic splice enhancer elements that are present within Smn exon 7. We predict that these animals will provide a """"""""clean"""""""", more consistent way of titrating Smn levels in a relevant way to what occurs in SMA and provide us with an opportunity to systematically define the minimal amount of SMN that is required to prevent an SMA phenotype, which has yet to be determined in human or mouse. ? ? ?
