8.1 Pancreatic tissue structure
Islet cells are spatially adjacent to exocrine and ductal cells, and expression in tissue homogenates can easily mask cell-type off-targeting.
8.2 AAV serotype selection
AAV6/8/9 can be used as comparison objects; retrograde administration through the pancreatic duct often increases local exposure, but the technical requirements are higher.
8.3 Pancreas-specific promoter
INS1/2 is used for β cells, GCG is used for α cells, and PDX1 needs to be interpreted in conjunction with development and disease states.
8.4 Administration method and dosage reference
Retrograde perfusion of the pancreatic duct: targeting the exocrine part of the pancreas. Mice 2×10^11-2×10^12 vg/mouse (AAV6), 100 μL, intubated through duodenal papilla/common bile duct, peristaltic pump 10 μL/min×10 min perfusion; the proximal hepatic end of the common bile duct is clamped to prevent viruses from entering the liver; AAV6 has the highest transduction of acinar cells (about 37% acinar expression), ductal cells are also transduced, and islet cell transduction is limited; the Ela promoter is acinar-specific and can be detected in 4-6 weeks [8-1,8-5].
Systemic administration: 1×10^11-1×10^12 vg/animal. It is important to note that the liver and acinar cells are off-target. Acute pancreatitis can be induced if the perfusion rate is too fast (>50 μL/min) or the pressure is too high, and the peristaltic pump must be accurately controlled [8-4].
8.5 Key research evidence
Quirin et al. established an AAV retrograde pancreatic duct gene delivery protocol [8-1]; Orabi et al. targeted inhibition of calcineurin in pancreatic acinar cells to prevent post-ERCP pancreatitis [8-4]; Xiao et al. established a Nat Protoc protocol for pancreatic duct infusion of AAV and cell labeling dyes for pancreatic cell lineage tracing and genetic manipulation [8-5]. It is recommended to verify islet co-staining, hormone secretion and blood glucose phenotypes.
8.6 References for this chapter
[8-1] Quirin KA, et al. AAV retrograde pancreatic ductal gene delivery. Mol Ther Methods Clin Dev. 2018;8:8-20. [H]
[8-2] Chen M, et al. Capsid-optimized AAV8 delivery to pancreas and pancreatic tumors. Hum Gene Ther Methods. 2017;28:49. [H]
[8-3] Ramzy A, et al. AAV8 Ins1-Cre recombination and off-target considerations. Sci Rep. 2020;10:10518. [H]
[8-4] Orabi AI, et al. Targeted inhibition of pancreatic acinar cell calcineurin prevents post-ERCP pancreatitis. Cell Mol Gastroenterol Hepatol. 2017;4:247-262. DOI:10.1016/j.jcmgh.2017.04.008. [H]
[8-5] Xiao X, Guo P, Prasadan K. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of AAV vectors and/or cell-tagging dyes. Nat Protoc. 2014;9:2719-2724. DOI:10.1038/nprot.2014.183. [F]