PROJECT SUMMARY / ABSTRACT Background: Huntington's disease (HD) is a fatal, dominant neurogenetic disorder caused by polyglutamine repeat expansion in exon 1 of the HD gene encoding huntingtin (HTT). It is estimated that 1−3 in every 10,000 persons (nearly 30,000 in the USA) have HD. Patients suffer from progressive neurodegeneration in the basal ganglia and the cortex, leading to progressive movement disorders and severe psychiatric disturbances. Although the disorder was described over 100 years ago, effective treatments do not exist. Genome editing using the precise and potent CRISPR-Cas9 system represents an exciting therapeutic approach to treat HD. The proposed study will employ the CRISPR-Cas9 system delivered via novel engineered capsid of adeno-associated-virus (AAV), AAV-LC.V1. Objective: We propose to determine efficacy and safety of the dual AAV-LC.V1 genetic therapy in the brain using animal models. The results of these studies are essential components for our Investigational New Drug (IND) application to the Food and Drug Administration (FDA) for clinical testing of our HD program. Specific Aims: Aim 1 will focus on in vitro screening a pair of gRNAs for the most frequent mutant human HTT gene haplotypes. These studies will test the efficacy of knocking down the mutant HTT allele as well as off-target editing. Aim 2 will determine the efficacy of knocking down the mutant HTT allele in two murine models of HD via AAV-CRISPR. The vectors will be injected by convection-enhanced delivery directly into the mouse brain (striatum and thalamus). To assess overall therapeutic benefit, efficiency of editing at the molecular level will be tested in collected tissues, while assessment of rescue from an HD phenotype will be evaluated by a battery of behavioral tests. Aim 3 will examine toxicity and biodistribution of different doses of our AAV- CRISPR system in rats in a GLP-compliant setting as required for IND-enabling studies. Tissues will be collected at 3- and 9-months for histopathological evaluation to prepare the required GLP toxicology report. As required by the FDA, acute and long-term efficacy, safety, and biodistribution of the two-AAV-CRISPR vector system will be also assessed in the brains of non-human primates (Aim 4). Many proposed experiments will be performed by specialized preclinical CROs in a GLP- compliant setting. Impact: By demonstrating the safety and efficacy of AAV-LC.V1-CRISPR gene therapy, the proposed suite of studies may lead to clinical testing of this system to treat the progressive neurodegeneration associated with HD.