333. Simultaneous Disruption of Five SerpinA1 Genes in Mice Using CRISPR/Cas9 to Generate the First Animal Model of Alpha-1 Antitrypsin Deficiency

Abstract

Alpha-1 antitrypsin (AAT) deficiency is a common autosomal co-dominant genetic disorder. This condition affects up to 1:2500 individuals of European ancestry, leading to the development of lung and in certain cases liver disease. AAT, as a member of the serpin family, is a protease inhibitor predominantly synthesized in the liver. Upon secretion into the blood stream, AAT's principle physiological role occurs in the lungs where it inactivates excess neutrophil elastase, thereby preventing potential damage to the alveoli. In humans, a glutamate to lysine change at position 342 of the protein, known as a PiZ mutation, accounts for over 90% of cases. This and other mutations lead to reduced serum levels of AAT in addition to reduced protein function. With time, the decreased levels of AAT allows for unopposed neutrophil elastase activity, causing the eventual breakdown of alveolar elastin thus resulting in emphysema. Currently, a serum deficient genetic animal model simulating the lung condition does not exist, which severely limits the development of innovative therapeutics.Our goal is to use CRISPR/Cas9 technology to generate an AAT knock-out mouse to study the pulmonary disease. Unlike humans, mice have five highly conserved tandem copies of the serpinA1 gene. This phenomenon has made it difficult to create knockout mice using traditional approaches. With the advent of novel genome editing techniques, this problem has reached a feasible threshold. To this end, we designed four guide RNA (gRNA) constructs to target conserved regions within all 5 copies of the serpinA genes. The gRNA constructs were initially evaluated using a single strand annealing assay. By co-transfecting a gRNA/Cas9 complex with appropriate reporters, the intensity of resulting GFP measured target efficiency. Afterwards, embryonic stem cell transfection and zygote microinjection were used to deliver the genome editing complex for in vitro and in vivo studies.During the screening process, 200 embryonic stem cell samples and 40 transgenic mice were evaluated by quantifying AAT serum levels and sequencing specific target regions within the genome. Upon completion, several knock-outs were identified and founder lines were established.Ongoing studies aim to determine the susceptibility of developing emphysema after challenging the knockout mice with porcine pancreatic elastase. We expect the mice will display a distinguishable pulmonary phenotype, aside from decreased levels of AAT as determined by ELISA, which will allow for the characterization of airway pathophysiology. This mouse model is of particular clinical significance as it presents the first opportunity to test current and novel therapeutics aimed at augmenting serum levels of AAT.