Editing of the AAT locus using novel base editing and prime editing technologies

PROJECT 2 - Project Summary/Abstract Gene editing has the potential to correct mutations and provide long-term therapeutic benefit for patients with rare monogenic diseases like alpha-1 antitrypsin deficiency (AATD). AATD is caused by mutations in the AAT (or PI) gene, which encodes a serine protease inhibitor that is made in hepatocytes and delivered to lung to neutralize neutrophil elastase. The PI*Z mutation encodes mutant Z-AAT protein that aggregates in hepatocytes, which can cause liver disease and reduces serum AAT. Reduced serum AAT causes progressive airway disease and emphysema. Gene correction would address both aspects of AATD. CRISPR-mediated homology directed repair (HDR) can be used to partially correct mutations in mouse liver. Yet, HDR is limited by the need to deliver a long DNA repair template, its inefficiency in non-dividing or slow- dividing cell types, and its generation of genotoxic double-strand breaks. To address these limitations, this proposal will develop two CRISPR-based gene correction strategies that do not require a double-strand break: prime editing and adenine base editing. Prime editor (PE) is comprised of Cas9 nickase fused to reverse transcriptase and an extended guide RNA that doubles as a template for reverse transcriptase to copy editing information into the genomic target. Adenine base editor (ABE), comprised of a Cas9 nickase fused to an adenosine deaminase, can correct G-to-A point mutations in mouse liver. The PI*Z allele results from a G-to-A mutation; and thus, is a good candidate for gene correction via ABE and PE. The goal of this project is to optimize PE and ABE tools for AAT gene correction in vivo by developing ABE and PE vectors that can be accommodated by adeno-associated virus (AAV) capsids; maximizing on-target editing and minimizing off-target editing; and determining how immune responses affect editing. Aim 1 will develop novel PE tools for in vivo AAT gene correction. A split AAV PE platform will be developed to maximize prime editing efficiency in vivo, then PE gene correction and lung phenotype will be measured in a PI*Z transgenic mouse model and a clinically-relevant AAT null/PI*Z mouse model. Aim 2 will enhance the specificity of ABE for in vivo AAT gene correction. Long-term ABE expression can induce off-target editing. Therefore, new ABE variants will be optimized to increase activity and reduce RNA editing effects, and split AAV delivery of ABE will be investigated in a PI*Z model. This Aim will also develop self-inactivating ABE to reduce off-target effects. Aim 3 will characterize and mitigate immune responses to PE and ABE, which harbor viral reverse transcriptase and bacterial TadA protein, respectively, and Cas9, a known antigen. This Aim will investigate antibody and T cell response to PE and ABE in mice, how immune response regulates editing, and whether CAR-Treg can mitigate immune response. Project 2 will benefit from extensive interactions with the other projects and cores in this P01. Completing this project will improve the efficiency and safety of PE and ABE in vivo, providing an HDR-independent gene editing blueprint for AATD, and other monogenic diseases.