Aims: 1. To evaluate the therapeutic efficacy of scAAV9-shRNA mediated SOD1 silencing in the SOD1-G93A mouse model using a clinic ready vector. 2. To investigate the measurement of CSF SOD1 protein levels as a biomarker of effective dosing and efficacy of SOD1 knockdown. 3. To investigate and evaluate miRNA-like sequence specific off-target effects on gene expression resulting from the viral delivery of a therapeutic shRNA targeting hSOD1. Methods: Animals were treated at postnatal day 1 (P1, pre-onset) and P40 (onset). scAAV9-hSOD1si or scrambled control scAAV9-hSOD1ssi viral vectors were delivered via cisterna magna injection. Mice were tested using behavioural tests including weekly rotarod runs, neurological scoring and CatWalk gait analysis. Weekly body weight was also collected. Analysis of CSF by ELISA was used to determine SOD1 protein levels after treatment. Total RNA extracted from transduced isogenic Tetinducible FLP-In HEK293T cell lines expressing either wildtype or G93A hSOD1 transgenes was analysed by whole genome microarray to elucidate potential off target effects. Results: Cisterna magna injection of scAAV9-hSOD1si at P1and P40 improves motor performance and extends median survival in the mouse model by 42% and 13% respectively. Treatment at P1 also delays disease onset and significantly reduces SOD1 protein in the CSF as detected by ELISA, compared to scrambled controls. An in vitro assay using human cells demonstrated that the construct generated no off-target effects. Conclusions: These findings are evidently important translational aspects of the study, in as much as: 1) a lack of observable off-target effects in human cells in vitro suggest the therapeutic shRNA construct is specific to hSOD1 and unlikely to elicit detrimental gene regulation in patients and 2) most clinical approaches suffer from lack of reliable biomarkers that correlate with treatment benefit. As the collection and analysis of CSF from patients is a relatively simple procedure, the measurement of SOD1 protein in CSF from patients undergoing SOD1 knockdown therapy has the potential to be a very useful and informative biomarker of therapeutic efficacy. Our approach therefore reinforces an already promising gene therapy strategy for clinical application in SOD1- linked familial ALS with additional translational aspects absent from previous studies.