Optimized Gene Replacement for AAT deficiency and Modeling of Clinical Outcomes in small and large animal models

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Project Summary (Project 1) Alpha-1 antitrypsin deficiency (AATD) is a common single gene disorder caused by mutations in the SERPINA1 gene, which normally encodes a very abundant serum antiprotease, whose primary function is to protect the interstitial elastin matrix of the lung from degradation by neutrophil elastase (NE). The E242K (PI*Z) mutant allele is very common among those of European ancestry, and E342K homozygotes encode a protein with impaired secretion, resulting in deficient serum levels, leading to unrestrained NE activity, degrading pulmonary elastin. This process triggers lung inflammation and leads to loss of lung elastic recoil and airways obstruction, creating the clinical picture of emphysema, which is the life-limiting effect of AATD in most patients. Current AATD therapy consists of weekly IV infusion of AAT, but this therapy has never been proven to benefit lung function in patients in prospective studies, leading to questions about whether the previously identified serum target level (11 µM) is sufficient, or whether gene therapy to restore such a level would be efficacious. Our laboratory has previously developed rAAV gene therapy vectors and used them in clinical trials. Those trials have failed to achieve the target, achieving levels just over 0.5 µM but being sustained over 5 years in patients. We have also use CRISPR-based technology to produce the first SERPINA1-knockout mice and have begun a collaboration with the University of Iowa group, who have created both SERPINA1-knockout and SERPINA1-E342K ferret models. In the current proposal, improved and optimized vectors will be tested in both mice and ferret models. In the ferrets, they will be tested alongside sophisticated conditional transgenic reconstitution to determine whether the optimal target for therapy is 11 µM vs. 25 µM. These studies will pave the way for better rAAV-AAT vectors appropriate for future clinical product development. In this project, we propose to study genetic emphysema due to alpha-1 antitrypsin deficiency (AATD). AATD is both fairly common as a genetic disease and is a model of much more common causes of chronic obstructive pulmonary disease (COPD). In the proposal, we will use advanced gene editing tools to create genetically defined animal models of AATD (known as transgenic animals), both in mice and in ferrets, which are a good model to study lung diseases. In the course of the study, we will use both additional transgenic approaches and modified recombinant viruses (rAAV) to genetically treat AATD in these models and examine how we might design evaluate the effectiveness of future gene therapies for genetic emphysema.