Published online Feb 27, 2018. doi: 10.4254/wjh.v10.i2.277
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
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.
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.
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.
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.
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.
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.
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).