ABSTRACT The main objective of this grant is to use genetically engineered mouse models to determine the mechanisms by which trypsinogen (PRSS1) mutations in humans cause hereditary pancreatitis. The majority of non-alcoholic cases of chronic pancreatitis develop on the basis of genetic susceptibility, driven by mutations in risk genes that encode digestive enzymes or their inhibitor, such as cationic trypsinogen (protease serine 1, PRSS1), chymotrypsin C (CTRC) or the pancreatic secretory trypsin inhibitor (serine protease inhibitor Kazal type 1, SPINK1). Our previous studies defined a trypsin- dependent pathological pathway associated with mutations in these risk genes that promote intra- pancreatic trypsinogen autoactivation and result in increased ectopic trypsin activity. In the present proposal, we will validate this model in vivo. To this end, we developed novel mouse lines T7 D23A and T7 K24R that carry mutations in the activation peptide of the native mouse cationic trypsinogen (isoform T7). In vitro the D23A mutation causes a dramatic 50-fold increase in trypsinogen autoactivation, while mutation K24R increases autoactivation by 4-fold. Thus, the models can provide information on how different trypsin levels determine pancreatitis responses and pathology. We hypothesize that mice with trypsinogen mutations that stimulate autoactivation will develop spontaneous pancreatitis or exhibit heightened pancreatitis responses when challenged with hyperstimulation insults. In our specific aims we will study the spontaneous pancreatitis in the T7 D23A mouse; evaluate the increased sensitivity to secretagogue induced pancreatitis in the T7 K24R mouse and investigate the protective role of trypsin inhibition against pancreatitis by altering Spink3 (ortholog of human SPINK1) expression levels in these models. Successful completion of these aims will firmly establish that increased trypsinogen autoactivation leading to elevated intra-pancreatic trypsin activity is a relevant disease-mechanism in pancreatitis and should be the focus of future therapeutic strategies.