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INDUCIBLE GENETIC MOUSE MODEL OF INTRADUCTAL PAPILLARY MUCINOUS NEOPLASM USING IN VIVO ADENO-ASSOCIATED VIRUS WITH CRISPR-CAS9
Hannah N. Rinehardt
*1, Alexander Kolodychak
2, Alexis Martyn
1, Vinitha Dhamotharan
1, Alexander Kreger
3, Madison Thomas
1, Lydia Liszewski
1, Andrew Kolodychak
4, Ting Zhang
1, George Gittes
11Pediatric Surgery, Children's Hospital of Pittsburgh John G Rangos Sr Research Center, Pittsburgh, PA; 2Brown University Warren Alpert Medical School, Providence, RI; 3UH Cleveland Medical Center, Cleveland, OH; 4University of Notre Dame, Notre Dame, IN
Purpose: Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic neoplasm of the pancreas. IPMN can harbor dysplasia or pancreatic ductal adenocarcinoma (PDAC) and is a frequent indication for partial pancreatic resection. IPMN is an active area of research as recent data have demonstrated that IPMN is not a simple, localized cyst, but rather represents a complex, carcinogenic process of the entire pancreas. Traditional genetically engineered mouse models (GEMMs) of IPMN require breeding of pancreas-specific inducible-Cre mice and multiple oncogenes, which suffer from laborious breeding and embryonic lethality. A recent publication described an inducible model of PDAC by injection of adeno-associated virus (AAV) into the pancreas of Cas9-expressing mice. We aimed to develop a similar method to induce IPMN in Cas9-expressing mice.
Method: Our lab has developed a novel method of targeting the ductal epithelium of the pancreas through laparotomy, duodenotomy, and ampullary catheter insertion for viral infusion. We purchased commercially available Pdx-Cre (whole pancreas), Ptf1a-Cre (exocrine pancreas), and Rosa26-LSL-Cas9 (cre-inducible Cas9) mice for breeding. Mice expressing pancreas-specific Cas9 were infused with the AAV at 8-14 weeks old. We utilized AAV6 due to its tropism for pancreatic ductal epithelium. We obtained a commercially available plasmid containing guide RNAs (sgRNAs) targeting KRAS G12D, p53, and Lkb1 (KPL). We modified this plasmid to remove the sgRNA targeting p53, so the sgRNA for KRAS and Lkb1 remained (KL). Animals were sacrificed 4-8 weeks post-operatively for histologic analysis.
Results: 4 weeks after infusion of AAV6 with KPL and KL we identified histologic evidence of IPMN throughout the pancreata. This was evident by H&E staining and by staining with Alcian Blue (Figure 1). The KPL cohort demonstrated advanced disease with tumor invasion of adjacent organs and hemorrhagic ascites at sacrifice. The KL cohort demonstrated mild disease with no evidence of invasion nor ascites. The KPL cohort had evidence of predominantly high-grade dysplasia in tumors and no evidence of normal pancreatic parenchyma. The KL cohort had evidence of predominantly low-grade dysplasia in tumors and areas of normal pancreatic parenchyma were present (Figure 2). Glucose tolerance testing at 4 weeks post-infusion demonstrated normal glucose homeostasis in both cohorts of mice.
Conclusion: We successfully developed an inducible-model of IPMN by infusing AAV6 with sgRNAs targeting KRAS, Lkb1, plus or minus p53 into mice with pancreas-specific Cre-inducible Cas9 expression. This model could be used in the future to investigate pathophysiology of disease and test therapeutics. The inducible nature of our model facilitates investigation of IPMN by avoiding the pitfalls of congenital GEMMs.
Alcian blue staining demonstrates low-grade IPMN in KL cohort mouse 4 weeks post-infusion of AAV6 sgRNA KRAS G12D Lkb1.
Hematoxylin and eosin staining demonstrates low-grade IPMN burden in the head of the pancreas, with adjacent normal pancreatic histology in KL cohort mouse 4 weeks-post infusion of AAV6 sgRNA KRAS G12D Lkb1.
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