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

尿素循环是肝脏中必不可少的途径，它是蛋白质分解产生的氨解毒所必需的。氨具有高度的神经毒性，任何尿素循环酶的缺陷都会导致高死亡率和发病率。鸟氨酸转氨基甲酰基酶缺乏症是尿素循环缺陷中最常见的一种。在最严重的疾病形式中，如果不治疗，新生儿早期就会出现症状，进展为昏迷和死亡。患者通过药物和饮食干预进行治疗，但最终需要肝移植才能长期生存。然而，肝移植有其自身的风险——死亡率、发病率和终生需要免疫抑制。基因治疗是肝移植的另一种有吸引力的选择，因为患者自身的细胞可以通过功能基因的转移来修复。腺相关病毒（AAV）正在成为一种高效的基因传递系统，其改变病毒外壳（衣壳）的能力使多种细胞成为靶标。目前，AAV8在由伦敦大学学院领导的血友病B（因子IX缺乏症）肝脏临床试验中取得了令人鼓舞的结果。虽然令人兴奋，但这一成功是由于在这种情况下需要进行基因修复的肝细胞数量较少，因为因子IX蛋白被释放（分泌）到血液中，仅为正常水平的1-2%，从而提供了治疗效果。在许多涉及肝脏的代谢条件下，如OTC缺乏症，基因产物不分泌，为了临床成功，必须对更高比例的细胞进行修饰。Alexander教授使用基于AAV8的基因治疗方法成功治愈了otc缺陷小鼠，然而，AAV8对于将基因传递到人类肝细胞的效果要差得多。与斯坦福大学的同事进行了令人兴奋的合作，开发了一种名为LK03的新型AAV，它以比AAV8高得多的效率（12倍）靶向人类肝细胞。因此，我们建议首次使用基于AAV/LK03的AAV基因递送系统来治疗儿科患者的OTC缺乏症。该申请描述了支持英国AAV/LK03介导的OTC缺陷I/II期临床试验授权所需的关键临床前研究，并建立在已经资助的研究基础上，旨在改进AAV/LK03用于临床试验。拟议的临床前研究包括目标儿科人群的免疫学调查，临床级试剂的质量控制测试（效力、安全性和纯度），以及非人灵长类动物的毒理学和生物分布研究。这些研究选择了非人类灵长类动物作为动物模型，因为基因治疗传递系统对人类肝细胞具有高度特异性，而对小鼠肝细胞的作用很小。最初，将对目标儿科人群进行免疫状况（血清阳性率）调查。针对AAV/LK03的抗体（体液免疫）的存在会阻碍基因传递的有效性。鉴于先前的调查显示，对AAV的体液免疫通常在两岁之前不会形成，而且大多数试验参与者将小于2岁，这不太可能是一个重大问题。基因治疗试剂将在伦敦大学学院载体核心设施生产，并经过标准的安全性，纯度和无菌测试。然后，该试剂将在猕猴身上进行毒理学和生物分布测试。在临床前测试结束时，结果将以研究药品档案（IMPD）的形式提交给药品和保健产品监管机构（MHRA），以请求临床试验授权。这些结果也将支持该基因治疗试剂在欧洲医药管理局的孤儿指定申请。

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.