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Hepatocyte transfection in small pigs after weaning by hydrodynamic intraportal injection of naked DNA/minicircle vectors


Stoller, Fabienne; Schlegel, Andrea; Viecelli, Hiu Man; Rüfenacht, Véronique; Cesarovic, Nikola; Viecelli, Claudio; Deplazes, Sereina; Bettschart-Wolfensberger, Regula; Hurter, Karin; Schmierer, Philipp A; Sidler, Xaver; Kron, Philipp; Dutkowski, Philipp; Graf, Rolf; Thöny, Beat; Häberle, Johannes (2015). Hepatocyte transfection in small pigs after weaning by hydrodynamic intraportal injection of naked DNA/minicircle vectors. Human Gene Therapy. Methods, 26(5):181-192.

Abstract

Liver is an attractive organ for gene delivery in order to correct various genetic (metabolic) diseases. Hydrodynamic vein injection of naked DNA/minicircles devoid of viral or plasmid backbones was demonstrated in, for example, murine phenylketonuria to allow sustained therapeutic transduction of hepatocytes. Here we show successful hepatocyte transfusion in domestic small pigs immediately after weaning upon portal vein catheterization and hydrodynamic injection of naked DNA/minicircle vectors expressing the luciferase gene from the CMV or a liver-specific promoter. First, we established a surgical method allowing hydrodynamic portal vein pressurization up to 120 mmHg and infusion of naked DNA in pigs (n = 5) with long-term survival. No acute adverse effects such as changes in liver transaminases or signs of liver cell damage were observed. We then showed efficiency of stable hepatocyte transfection at 10 and 28 days in single experiments (n = 7) where we found that up to 60% of samples (45/75) were polymerase chain reaction (PCR)-positive for minicircle-DNA. Of these samples, 13% of the positive specimen (6/45) showed low but stable luciferase expression when driven by a liver-specific promoter, as well as appropriate copy numbers per diploid genome. In conclusion, we accomplished a safe procedure for stable transfection of liver cells upon hydrodynamic gene delivery using minicircle vectors in small pigs as a prerequisite to potentially treat infants with genetic liver diseases.

Abstract

Liver is an attractive organ for gene delivery in order to correct various genetic (metabolic) diseases. Hydrodynamic vein injection of naked DNA/minicircles devoid of viral or plasmid backbones was demonstrated in, for example, murine phenylketonuria to allow sustained therapeutic transduction of hepatocytes. Here we show successful hepatocyte transfusion in domestic small pigs immediately after weaning upon portal vein catheterization and hydrodynamic injection of naked DNA/minicircle vectors expressing the luciferase gene from the CMV or a liver-specific promoter. First, we established a surgical method allowing hydrodynamic portal vein pressurization up to 120 mmHg and infusion of naked DNA in pigs (n = 5) with long-term survival. No acute adverse effects such as changes in liver transaminases or signs of liver cell damage were observed. We then showed efficiency of stable hepatocyte transfection at 10 and 28 days in single experiments (n = 7) where we found that up to 60% of samples (45/75) were polymerase chain reaction (PCR)-positive for minicircle-DNA. Of these samples, 13% of the positive specimen (6/45) showed low but stable luciferase expression when driven by a liver-specific promoter, as well as appropriate copy numbers per diploid genome. In conclusion, we accomplished a safe procedure for stable transfection of liver cells upon hydrodynamic gene delivery using minicircle vectors in small pigs as a prerequisite to potentially treat infants with genetic liver diseases.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
04 Faculty of Medicine > Center for Integrative Human Physiology
05 Vetsuisse Faculty > Veterinary Clinic > Equine Department
05 Vetsuisse Faculty > Veterinary Clinic > Department of Farm Animals
05 Vetsuisse Faculty > Veterinary Clinic > Department of Small Animals
Dewey Decimal Classification:570 Life sciences; biology
630 Agriculture
Language:English
Date:2015
Deposited On:13 Nov 2015 10:47
Last Modified:08 Dec 2017 14:44
Publisher:Mary Ann Liebert
ISSN:1946-6536
Additional Information:This is a copy of an article published in the Human Gene Therapy. Methods © 2015 [copyright Mary Ann Liebert, Inc.]; Human Gene Therapy. Methods is available online at: http://online.liebertpub.com
Publisher DOI:https://doi.org/10.1089/hgtb.2014.140
PubMed ID:26398117

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