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Background and Aims: Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAVs) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to redose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models., Methods: We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP and a power injector to supply hydrodynamic force. Human factor IX (hFIX), deficient in hemophilia B, was used as a model gene therapy., Results: Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by polymerase chain reaction. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7%-51.9% vs 18.9%, P < .0001)., Conclusions: Biliary hydrodynamic injection using ERCP can achieve higher transfection efficiency into hepatocytes compared with AAVs at magnitudes of less cost in a clinically relevant human-sized large animal. This technology may serve as a platform for gene therapy of human liver diseases., (Copyright © 2021 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

The biliary system is routinely accessed for clinical purposes via endoscopic retrograde cholangiopancreatography (ERCP). We previously pioneered ERCP-mediated hydrodynamic injection in large animal models as an innovative gene delivery approach for monogenic liver diseases. However, the procedure poses potential safety concerns related mainly to liver or biliary tree injury. Here, we sought to further define biliary hydrodynamic injection parameters that are well-tolerated in a human-sized animal model. ERCP was performed in pigs, and hydrodynamic injection carried out using a novel protocol to reduce duct wall stress. Each pig was subjected to multiple repeated injections to expedite testing and judge tolerability. Different injection parameters (volume, flow rate) and injection port diameters were tested. Vital signs were monitored throughout the procedure, and liver enzyme panels were collected pre- and post-procedure. Pigs tolerated repeated biliary hydrodynamic injections with only occasional, mild, isolated elevation in aspartate aminotransferase (AST), which returned to normal levels within one day post-injection. All other liver tests remained unchanged. No upper limit of volume tolerance was reached, which suggests the biliary tree can readily transmit fluid into the vascular space. Flow rates up to 10 mL/sec were also tolerated with minimal disturbance to vital signs and no anatomic rupture of bile ducts. Measured intrabiliary pressure was up to 150 mmHg, and fluid-filled vesicles were induced in liver histology at high flow rates, mimicking the changes in histology observed in mouse liver after hydrodynamic tail vein injection. Overall, our investigations in a human-sized pig liver using standard clinical equipment suggest that ERCP-guided hydrodynamic injection will be safely tolerated in patients. Future investigations will interrogate if higher flow rates and pressure mediate higher DNA delivery efficiencies., Competing Interests: Conflict of Interest: R.L.K, Y.H., V.K., and F.M.S. have a patent application filed on the work. All other authors do not have conflict of interests. There are no other licensing or consulting interests; this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism. [ABSTRACT FROM AUTHOR]