Project Summary/Abstract Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes loss of motor neurons, results in paralysis, and in the most severe forms leads to death. The incidence of SMA is 1 in 10,000 live births, making this disease one of the leading causes of infant mortality. SMA is caused by low levels of the survival motor neuron (SMN) protein. Recent experiments have shown a remarkable rescue of phenotype in SMA mice upon delivery of SMN using scAAV9. Likewise, correction of SMN2 splicing using antisense oligonucleotides (ASO) can expand survival and rescue electrophysiology defects. Such promising pre-clinical studies have led to several clinical trials for the treatment of SMA. It is crucial that biomarkers are established for SMA that can help predict treatment response and to measure therapeutic effect. A panel of protein markers known as SMA- MAP shows correlation with function in SMA patients. Yet, it is unknown if these markers can quantify therapeutic response. A subset of these plasma markers are both abnormal in SMA mice and normalize in SMA mice treated presymptomatically with anti-sense oligonucleotides (ASO) to increase SMN. The ability of these markers to quantify treatment response following treatment with ASO to increase SMN will be tested in SMA mice treated at different disease stages. These findings will be further investigated by testing the SMA- MAP panel in blood samples from treated SMA type I infants enrolled in the phase 1 gene therapy clinical trial at our center. These samples will be compared with samples from untreated SMA type 1 infants, matched for age and SMN2 copy number, from the NeuroNext clinical trial to allow determination of the markers that respond to treatment. While SMN therapies are very effective when given early, they are less effective late in the course of disease. In aim 2, combinatorial therapies to improve muscle function in combination with SMN therapies, will be tested in SMA mice. Mutations in the troponin C gene that result in increased calcium sensitivity will be delivered using adeno associated virus (AAV) vectors and tested for an effect on muscle contraction force even with reduced motor neuron input. Self-complementary AAV follistatin will be used to increase muscle mass to determine if there is an additive effect of increased muscle size when combined with SMN. In these combinatorial therapy experiments, mice will be treated with ASOs to increase SMN at different disease stages to mimic the clinical trial situation. We will monitor muscle force and electrophysiological measures of motor unit function in these mice. Finally, aim 3 will investigate whether higher levels of SMN can enhance motor neuron repair and improve therapeutic response in post symptomatically treated mice. Lastly intron 6 and 7 will be investigated using the CRISPR/Cas9 system to find new regulatory sites that control splicing of exon 7. Identification of new sites will expand therapeutic...