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. 2014 Feb;21(2):123-30.
doi: 10.1038/gt.2013.62. Epub 2013 Nov 21.

A Novel Gene Delivery Method Transduces Porcine Pancreatic Duct Epithelial Cells

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Free PMC article

A Novel Gene Delivery Method Transduces Porcine Pancreatic Duct Epithelial Cells

M A Griffin et al. Gene Ther. .
Free PMC article

Abstract

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

Conflict of interest statement

CONFLICTS OF INTERESTS

Authors have no conflicts of interests.

Figures

Figure 1
Figure 1. Celiac artery catheterization via umbilical arteries
(A) In newborns, celiac artery can be reached by placing a catheter (arrow, green) into the umbilical arteries, which connect to the aorta. The catheter is then advanced to the celiac artery. (1) aorta; (2) right iliac artery; (3) umbilical cord (1 vein and two arteries); (4) pancreas; (5) hepatic artery; (6) splenic artery; (7) celiac artery with catheter; (8) umbilical artery; (9) left femoral artery. (B) Angiography confirming cannulation of the celiac artery (arrows). Vector or vehicle was injected and the catheter was flushed with normal saline.
Figure 2
Figure 2. AAV9 gene delivery does not induce pancreatic inflammation in pigs
Thirty (A, B) and ninety days (C, D) after the celiac artery injection of AAV9CMV.sceGFP (2.4×1012 vg per animal), pancreas sections were obtained. Pancreas had a lobular architecture, with ducts (arrow), acini (*) and islet cells (block arrow). There were no inflammatory cells (H&E stain). A, C x10 mag, bar = 50 µm; B, D ×60 mag, bar = 20 µm.
Figure 3
Figure 3. AAV9 transduces porcine ductal epithelial cells
Pancreas sections 30 days after newborn pigs received AAV9CMV.sceGFP (A, C, D, E, F) (2.4×1012 vg per animal) or vehicle (B) into the celiac artery. Immunofluorescence (A, B) and immunohistochemistry (C–F) images are shown. Arrows point to intralobular (larger) ducts, arrowheads point to intercalated (smaller) ducts. C and D; E and F are serial sections from the same animal, primary antibody is omitted in D and F. A, B ×20 mag; C, D ×10 mag, scale bar = 100 µm; E, F ×60 mag, scale bar = 20 µm. Green: GFP, Blue: DAPI nuclei.
Fig. 4
Fig. 4. AAV9 transduces ductal epithelial cells-time and dose response
Ten random pancreatic fields (20× mag) were assessed per animal (immunofluorescence). % GFP positive cells were calculated by counting GFP expressing divided by the total number of cells in the field (n=1 for all time points and doses except, n=2 for 6.1×1012 vg at 1 month, and n=7 for 2.4×1012 vg at 1 month). Circles: 1 month; Squares: 3 months.
Fig. 5
Fig. 5. Celiac artery delivery of AAV9 vector leads to GFP expression in pancreas and several other tissues
(A) One and three months after delivery of AAV9CMV.sceGFP (2.4×1012 vg per animal) to the celiac artery in the newborn period, RNA was isolated from pancreas and end-point PCR was used to detect GFP mRNA. The results are representative of n=7 for one month exposure and n=1 for three-month exposure. Lane 1=ladder; lane 2=negative control; lane 3=positive control (10 ng GFP plasmid); lane 4=pancreas one month after delivery; lane 5=pancreas three months after delivery. (B). End-point PCR of tissues 30 days after injecting 2.4 × 1012 vg AAV9CMV.sceGFP to the celiac artery of newborn pigs. MW: molecular weight ladder; (−) ctrl: negative control (sham animal); (+) ctrl: positive control (plasmid eGFP). Organs that receive arterial supply from celiac artery are in bold. The stomach and duodenum, two organs that receive blood supply from celiac artery were not transduced.
Fig. 6
Fig. 6. AAV9 vector expression of GFP in CFTR-expressing duct cells
Immunofluorescent images of pancreas from pigs, 30 days after receiving 2.4×1012 vg AAV9CMV.sceGFP in the newborn period. (A) anti-CFTR antibody (red) for pancreatic ducts; (B) anti-amylase (red, arrowheads) for acinar cells; (C) anti-insulin (red, arrowheads) for β cells; (D) anti-glucagon (red, arrowheads) for α cells; (E) anti-somatostatin (SS) (red, arrowheads); (F) anti-pancreatic polypeptide (PP) (red-yellow indicating colocalization with eGFP, arrows); DAPI (blue) for nuclei. AAV9-GFP (green, arrows) was transduced in the cells that were expressing CFTR (red, arrowhead) on the apical side, A ×40 mag; B, C, D, E, F= ×20 mag. A, B, C, D, E= cells expressing GFP are shown with arrows.

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