Developing a novel adenine base editor tool for in vivo alpha-1 antitrypsin deficiency gene correction. - PROJECT SUMMARY Alpha-1 antitrypsin disease (AATD) is caused by mutations in the SERPINA1 gene, which encodes AAT protein. AAT is produced in liver and delivered via serum to lungs, where it inhibits neutrophil elastase. The most common AATD allele—called PI*Z—is a G-to-A mutation that produces a dysfunctional misfolded protein, Z-AAT, that aggregates in hepatocytes, which can cause liver disease; reduced serum AAT causes pulmonary emphysema. Currently, the only approved therapy for AATD emphysema is costly, weekly infusions of purified AAT for life. CRISPR/Cas9-mediated homology directed repair (HDR) can correct PI*Z in the liver and partially restore serum AAT levels in an AATD mouse model. Yet, HDR is limited by the need to deliver a DNA repair template, its inefficiency in non- and slow-dividing cells, and its generation of genotoxic double-strand breaks. By contrast, CRISPR-mediated adenine base editors (ABEs) support precise editing without requiring a DNA donor or double- strand breaks. ABE consists of adenine deaminase (TadA) conjugated to Cas9 nickase. When directed by a guide RNA to a specific sequence, ABE deaminates adenine in a defined editing window. The resulting inosine is read as guanosine, thereby converting A to G. Thus, ABE is a good candidate for PI*Z correction. Preliminary evidence shows that viral delivery of a compact ABE, utilizing an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C ABE), to PI*Z transgenic mice leads to efficient editing of PI*Z in hepatocytes to significantly reduce liver disease. Yet, eNme2-C ABE deaminates not only the target adenine but also “bystander” adenines in the designated editing window, leading to mutations of unknown consequence. Moreover, the level of base editing needed to rescue lung disease is undetermined. This project seeks to optimize ABE precision for PI*Z correction and assess the therapeutic potential of ABE in treating emphysema in a mouse model of AATD. Aim 1 will characterize ABE off-target and bystander edits for PI*Z correction. TadA variants with distinct editing windows have been developed, including ABE8e and ABE9e. eNme2-C ABE8e and ABE9e edit both the target adenine and bystander adenines at the PI*Z target locus in PI*Z reporter cells. Off-target editing events by each variant will be detected and validated in PI*Z reporter cells and liver cells by deep sequencing and RNA sequencing. Bystander alleles generated by each variant will be identified, then in vitro approaches will be used to analyze the secretion and activity of each AAT bystander mutant. Aim 2 will characterize ABE-mediated PI*Z correction and lung function in AAT-null PI*Z mice, which exhibit both lung and liver disease. eNme-2 ABE will be delivered by AAV to AAT-null PI*Z mice, and pulmonary mechanics will be measured over 10 weeks. At endpoint, serum, liver, and lung tissue will be collected to measure serum AAT level, PI*Z correction and hepatocyte AAT aggregates, and alveolar morphometry. This proposal will inform the development of base editing strategies to treat AATD and provide the fellow with training in therapeutic genome editing and genetic disease biology.
