Liver-directed somatic gene correction rAAV system of regulatable Cas9/sgRNA

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Project Summary/Abstract – Project 3: The ability to correct disease gene mutations in vivo has broad potential utility for both therapy and basic research. CRISPR/Cas9 is a powerful RNA-guided tool for genome editing. Our recent discovery that CRISPR/Cas9 delivery can cure genetic disease in adult mouse liver provided proof-of-concept of gene correction therapy. This subproject will interact synergistically with all other Projects and Cores of this tPPG to develop new rAAV CRISPR tools to treat alpha- 1 antitrypsin deficiency (AATD). The main goal of this proposal is to establish a pre-clinical rAAV paradigm for CRISPR-mediated correction of AAT deficiency in mouse models carrying the mutant human AAT gene. The impact of this project is to develop somatic gene correction using rAAV systems to (1) maximize efficiency and safety of CRISPR delivery, (2) maximize the rate of homologous recombination for gene correction, and (3) efficiently correct AAT mutation in the liver to treat lung disease in mice. The development of safe and effective delivery vehicles and genome editing tools to correct AAT deficiency will guide future clinical trials for CRISPR- mediated gene therapy for AAT lung disease. Because AAV serotypes can target a wide range of tissues, our approach has broad basic research and clinical applications beyond AATD. Project 3 has three Aims that focus on different aspects of liver-directed somatic AAT correction: Aim 1: Develop liver-directed rAAV vehicles to maximize efficiency and precision of Z-AAT genome-editing in mice. Aim 2: Investigate rAAV HDR templates to correct Z-AAT mutation in mouse liver. Aim 3: Explore Z-AAT correction in mouse models in vivo to treat the lung disease.