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Title: Strategies to facilitate the long-term expression of transgenes for treatment of chronic liver disease
Author: Haasteren, Joost van
ISNI:       0000 0004 8503 0400
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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The development of new gene therapy products offers ground-breaking opportunities for the treatment of human disease. To provide lasting benefit to patients, the expression of a correct copy of a non-functional gene needs to be life-long, and the maintenance of therapeutic levels of gene expression is a key challenge. Gene therapy for chronic liver disease using recombinant adeno-associated virus, is very encouraging, but as the vector exists as an extra-chromosomal episome, it can be lost during subsequent cell division, especially in paediatric liver disease. In this study, maintenance of transgene expression was investigated through repeated administration of rAAV after inducing immunogenic tolerance to rAAV capsid proteins. Using a liver-specific rAAV2/8, induction of tolerance to the non-self transgene luciferase was successfully demonstrated in mice. However, rAAV2/8 delivery of the AAV8 capsid was unable to induce humoral tolerance to the AAV8 capsid itself. Similarly, a lentiviral directed approach in the liver did not induce tolerance to AAV8 capsid. An alternate method to achieve life-long transgene expression utilised a gene editing strategy called homology-independent targeted integration (HITI), that facilitates insertion of a transgene sequence in a precise location in the genome, allowing, for example, therapeutic transgene expression under the strict control the endogenous promoter of an otherwise defective target gene. A fluorescent reporter cell line was constructed to test the feasibility of HITI and was used to confirm multiple permutations of the integration location and the sequence inserted, each of which offers a distinct therapeutic opportunity. Optimisation of the HITI donor sequence was required to prevent potential false positive expression in the analysis of in vivo HITI applications. One permutation was successful in addressing alpha-1 antitrypsin deficiency in vitro and subsequently in a disease mouse model. Two new quantitative analytical methods, Capture-Seq and CasEnrich, were used to determine the propensity for off-target integration in the models tested. While these next-generation sequencing approaches were optimised for analysis of HITI on- and off-target integration events, they can be readily applied to interrogate the genomic consequences other gene editing and gene therapy approaches. In conclusion, in the absence of a successful protocol for repeat rAAV administration in vivo, the HITI approach provides a promising platform for targeted genomic integration leading to life-long expression of therapeutic transgenes from an endogenous promoter.
Supervisor: Gill, Deborah ; Hyde, Steve Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Gene editing ; Gene therapy