Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Feb 27, 2018; 10(2): 277-286
Published online Feb 27, 2018. doi: 10.4254/wjh.v10.i2.277
Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model
Norman Junge, Qinggong Yuan, Thu Huong Vu, Simon Krooss, Christien Bednarski, Asha Balakrishnan, Toni Cathomen, Michael P Manns, Ulrich Baumann, Amar Deep Sharma, Michael Ott
Norman Junge, Thu Huong Vu, Ulrich Baumann, Department of Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover 30625, Germany
Qinggong Yuan, Asha Balakrishnan, Michael P Manns, Amar Deep Sharma, Michael Ott, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover 30625, Germany
Qinggong Yuan, Simon Krooss, Asha Balakrishnan, Michael Ott, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover 30625, Germany
Christien Bednarski, Toni Cathomen, Medical Center, University of Freiburg, Institute for Cell and Gene Therapy, Freiburg 79108, Germany
Amar Deep Sharma, Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover 30625, Germany
Author contributions: Junge N was involved in conception and design of the research, performed the majority of the experiments, analysed the data and wrote the manuscript; Yuan QG performed mouse surgery and immunostaining and revised the work critically for important intellectual content; Huong Vu T had substantial contributions to the experiments, animal care and analysis and interpretation of data for the work; Krooss S, Bednarski C, Balakrishnan A and Cathomen T helped with the experiments and design of the research and revised the work critically for important intellectual content; Manns MP and Baumann U revised the work critically for important intellectual content; Sharma AD and Ott M were initiator and supervisor of the work, the developed initial concept and design of the research and conducted important preliminary studies.
Institutional animal care and use committee statement: All experiments were approved and performed according to guidelines and ethical regulations from Hannover Medical School and local government.
Conflict-of-interest statement: There are no conflicts of interest for any of the authors regarding this work.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author (
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Michael Ott, MD, Full Professor, TWINCORE, Centre for Experimental and Clinical Infection Research, Feodor-Lynen-Str 7, Hannover 30625, Germany.
Telephone: +49-511-220027120 Fax: +49-511-220027178
Received: December 7, 2017
Peer-review started: December 8, 2017
First decision: December 18, 2017
Revised: February 1, 2018
Accepted: February 23, 2018
Article in press: February 23, 2018
Published online: February 27, 2018
Research background

We describe an important proof of concept in the field of AAV gene therapy for liver based metabolic diseases (LBMD). First gene therapy studies in humans are done (Hemophilia B) or very ready to start (Crigler-Najjar Syndrome); even an EMA approved drug for AAV gene therapy (Glybera) exists already. But all these approaches have a major weakness, the missing permanence of the gene therapy effect, especially in young children. But they are the main target group for gene therapy in LBMD, since early therapy could avoid irreversible damage to the organs of the patient. In these patients the advantage of recombinant AAV gene therapy, the almost missing integration into the host genome turns into a disadvantage since donor cDNA will be lost during cell turn over.

Research motivation

Targeted integration into safe harbors like the ROSA26 locus could overcome the problem of diminishing donor-cDNA in rAAV gene therapy. There are studies, showing proof of concept for targeted integration with nucleases like zinc fingers or CRISP/CAS9, but these approaches contain also new potential sources of side effects. However in our study only natural appearing cellular repair mechanism has been used to generate a targeted integration.

Research objectives

Up to know it was assumed that the efficiency of gene addition by targeted integration into a safe harbor mediated by homologous recombination would be to low for phenotypic correction of liver based metabolic diseases (LBMD) in growing livers. But we could show in a disease model for LBMD with selection advantage of corrected hepatocytes that this is not the case. This could be transferred to other diseases like the group of familial intrahepatic cholestasis or Wilson disease or even to diseases with less selection advantage.

Research methods

C57BL/6 Fah∆exon5 mice served as an animal model for human tyrosinaemia type 1 in our study. We treated these mice with a rAAV Vector containing human Fah cDNA, a liver specific promotor (TTR) and homologous arms for ROSA26 locus. We compared this group to mice treated with a vector without homologous arms. Hepatocyte proliferation was induced by partial hepatectomy and serial hepatocyte transplantation. Survival of mice without NTBC and existence of FAH positive cell cluster at immunohistochemistry staining on liver tissue of the mice were the main endpoints.

Research results

We could show for the first time proof of concept for phenotypic correction of a LBMD in a mouse model under conditions of extensive hepatocyte proliferation with rAAV mediated gene addition by targeted integration at a safe harbor without the use of nucleases or gene repair. Further studies have to show if this concept is transferable to LBMD with less section advantage of corrected hepatocytes.

Research conclusions

Our study shows that phenotypic correction of a LBMD by rAAV gene therapy under conditions of extensive hepatocyte proliferation is possible with homologous recombination (HR) alone and does not necessarily have the need for nucleases. In conclusion we showed that HR-mediated rAAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/- mice with superior long-term efficacy compared to episomal rAAV8 therapy in proliferating livers. In opposite to approaches with the aim of point mutation repair on genes of LBMD our system with gene addition into a safe harbour can be easily transferred to other LBMDs and is not mutation specific.

Research perspectives

Our results are an important step into the solution of a main clinical problem for gene therapy of LBMD, since mostly this therapy is mandatory in growing children, where episomal gene therapy is not lasting. In opposite to studies with nucleases our study focus on a natural mechanism for targeted integration which avoids potential side effects of nucleases. A very important question for following studies would be if these results could also be observed in LBMD with less selection advantage for corrected hepatocytes (e.g., Crigler-Najjar Syndrom).