Development of rAAV-mediated gene therapy for a severe paediatric metabolic liver disease: Ornithine Transcarbamylase deficiency.

The urea cycle is an essential pathway in the liver which is necessary for the detoxification of ammonia, produced by the breakdown of proteins. Ammonia is highly neurotoxic, and defects in any of the urea cycle enzymes leads to high rates of mortality and morbidity. Ornithine transcarbamylase (OTC) deficiency is the most common of the urea cycle defects. In the most severe forms of the disease, symptoms present early in the newborn period progressing to coma and death if untreated. Patients are managed by pharmacological and dietary intervention but ultimately require a liver transplant for long-term survival. However, liver transplantation is associated with its own risks - mortality, morbidity and life-long need for immunosuppression. Gene therapy offers an attractive alternative to liver transplantation, as the patient's own cells are repaired by transfer of a functional gene. Adeno-associated viruses (AAV) are emerging as a highly effective gene delivery system, and the ability to alter the outer coat (capsid) of the virus allows a variety of cells to be targeted. AAV8 is currently yielding promising results in the liver in a clinical trial for Haemophilia B (Factor IX deficiency), led by UCL. While exciting, this success is made possible by the low number of liver cells that need to be genetically repaired in this condition, as the Factor IX protein is released (secreted) into the bloodstream with as little as 1-2% of normal levels providing therapeutic benefit. In many metabolic conditions involving the liver, such as OTC deficiency, where the gene product is not secreted, a much higher proportion of cells must be modified for clinical success. Prof. Alexander has successfully cured OTC-deficient mice using an AAV8-based gene therapy approach, however, AAV8 is far less effective for gene delivery to human liver cells. An exciting collaboration with colleagues at Stanford University has led to the development of a novel AAV, referred to as LK03, which targets human liver cells with a much higher efficiency that AAV8 (12 times). We therefore propose a first-in-man use of an AAV gene delivery system based on AAV/LK03 for the treatment of OTC deficiency in paediatric patients. This application describes pivotal preclinical studies required to support authorisation of a phase I/II UK-based AAV/LK03-mediated clinical trial for OTC deficiency, and builds on already funded studies seeking to refine AAV/LK03 for clinical trial use. The proposed preclinical studies include immunological investigation of the target paediatric population, quality control tests (potency, safety and purity) on clinical-grade reagent, and toxicology and biodistribution studies in non-human primates. Non-human primates are the chosen animal model for these studies as the gene therapy delivery system is highly specific for human liver cells, with very little ability to function in mouse liver cells. Initially, a survey of the immunological status (seroprevalence) will be carried out on the target paediatric population. The presence of antibodies (humoral immunity) to AAV/LK03 can impede the effectiveness of the gene delivery. Given that previous surveys have shown that humoral immunity to AAV does not generally develop before the age of two, and the majority of trial participants will be younger than this, this is unlikely to be a significant problem. The gene therapy reagent will be produced in the UCL Vector Core Facility and subjected to standard safety, purity and sterility testing. The reagent will then be tested in Cynologmus macaques for toxicology and biodistribution. At the conclusion of the pre-clinical testing, the results will be presented to the Medicines and Healthcare Products Regulatory Agency (MHRA) in the form of an Investigational Medicinal Product Dossier (IMPD) to request Clinical Trial Authorisation. These results will also support an Orphan Designation application of the gene therapy reagent at the European Medicine Agency.