Development of an AAV vector for treatment of inherited retinal dystrophy caused by RPE65 deficiency

Leber先天性黑色素沉着症是一种严重的遗传性视网膜营养不良症，人群频率约为1/50,000，其特征是在儿童时期丧失视力。大约5%的LCA是由RPE65基因突变引起的，RPE65基因编码一种视网膜色素上皮(RPE)特异性蛋白，对维甲酸循环到感光细胞是必不可少的。我们的试验和美国的两个类似试验的结果首次显示了这种新疗法对一种以前无法治疗的疾病的影响。虽然所有试验中的一些受试者都从这种新的干预措施中受益，但人类受试者的反应程度与具有相同基因缺陷的小鼠和狗的改善程度不同。我们的数据表明，这种差异反映了人类对RPE65的需求比啮齿动物和狗更大，而当前版本的载体并不完全满足这一要求。该项目的目的是通过一些特定的修饰来优化RPE65结构，从而提高LCA2的基因治疗效果。由于我们已经达到了这些受试者的最大耐受载体剂量，我们打算通过一些特定的可申请专利的修改来优化构建，以提高转基因表达水平的效率。通过提高d蛋白生产的效率，我们的目标是在疾病的动物模型中实现最佳治疗所需的媒介剂量减少100倍以上。从改进的载体中获得的RPE65蛋白水平将在体外进行初步测试。一种或两种已被证明有能力在体外推动更高水平表达的结构将被用于动物研究，首先在小鼠身上，然后在狗身上，以测试较低剂量的新载体是否可以实现同等的视网膜功能和视力挽救。该项目的成功完成将使我们能够开发一种用于治疗LCA2的改进的AAV载体的临床试验。更有效的构建应该使我们能够开发出一种高效的基因治疗载体，它有可能成为世界上第一批获得临床应用许可的基因治疗产品之一。这将对患者的生活质量产生相当大的影响，好处包括改善夜视、提高视力和随着时间的推移保持视力，具体取决于治疗时视网膜的状况。尽管正在进行的临床试验表明，20岁以下的患者可以从治疗中受益，但由于预期在生命早期接受治疗后的最佳结果，最常见的最终用户将是十几岁的LCA2患者。受试者的年轻意味着最佳的成功治疗结果可能会导致多年的视力提高和/或更宽的视野，随之而来的是自给自足能力的改善和潜在的其他经济利益。LCA是一种相对罕见的疾病，人口频率约为1/50,000。在英国，大约5%的LCA是由RPE65突变引起的，因此AAV-RPE65基因治疗的潜在最终用户。只有20岁以下的患者才可能从治疗中受益；然而，在这一群体中，预期几乎完全采用既定的治疗方法。视网膜营养不良是一组高度多样化的疾病，可由多达200个不同基因的突变引起。这项应用中使用的核心技术-携带转基因的AAV载体视网膜下注射-将适用于为大多数视网膜营养不良患者开发基因治疗方案。AAV介导的基因治疗10种形式的遗传性视网膜变性已经报道了强有力的概念验证研究，我们已经报告了5种。本项目所取得的成果也将促进这些疗法的临床应用。

Leber congenital amaurosis (LCA) is a severe inherited retinal dystrophy with a population frequency around 1/50,000, characterised by loss of vision in childhood. Approximately 5% of LCA is caused by mutations in the RPE65 gene, which encodes a retinal pigment epithelium (RPE) specific protein essential for recycling of retinoids to the photoreceptor cells. The results of our trial, and 2 similar trials in the US, show for the first time the impact of this novel therapy on a condition that was previously untreatable. Whilst some subjects in all the trials have benefited from this new intervention, the extent of response in human subjects does not match the improvements in mice and dogs with the same genetic defect. Our data indicates that this difference reflects a greater requirement for RPE65 in humans than in rodents and dogs, and this is not fully met with the current version of the vector. The aim of this project is to improve the efficacy of gene therapy for LCA2 by optimising the RPE65 construct through a number of specific modifications. Since we have reached the maximal tolerated vector dose in these subjects, we intend to improve the efficiency of transgene expression levels by optimising the construct through a number of specific patentable modifications. By improving the efficiency of d protein production we aim to achieve a more than 100-fold reduction in the vector dose required for the optimal treatment in animal models of the disease. The RPE65 protein levels obtained from the improved vector will be tested initially in vitro. One or two constructs with a proven ability to drive higher levels of expression in vitro will be used in animal studies, first in mice and subsequently in dogs in order to test whether lower doses of the new vector can achieve equivalent rescue of retinal function and rescue of vision. Successful completion of this project will enable us to develop a clinical trial of an improved AAV vector for the treatment of LCA2. A more effective construct should enable us to develop a highly effective gene therapy vector that has the potential to be one of the world's first first licenced gene therapy products for clinical application. This would have considerable impact on the quality of life of the patients, with benefits ranging from improved nightvision to improved visual acuity and preservation of vision over time, depending on the condition of the retina at the time of treatment. As the best outcome is expected after treatment early in life, most commonly the end users would be pre-teen LCA2 patients, although ongoing clinical trials have shown that patients up to twenty years of age can benefit from treatment. The young age of the subjects means that a optimally successful treatment outcome could result in years of increased visual acuity and/or a wider visual field, with concomitant improvements in self-sufficiency and potentially other economic benefits. LCA is a relatively rare condition with a population frequency of approximately 1/50,000. In the UK around 5% of all forms of LCA are caused by RPE65 mutations and thus potential end-users for AAV-RPE65 gene therapy. Only patients under the age of 20 are likely to benefit from treatment; however, in this group a near-complete take-up of an established therapy is expected. Retinal dystrophy is a highly diverse group of disorders that can be caused by mutations in as many as 200 different genes. The core technology used in this application - subretinal injection of AAV vectors carrying a transgene - will be suitable for the development of gene therapy protocols for the majority of retinal dystrophies. Robust proof-of-concept studies have been reported for AAV-mediated gene therapy for ten forms of inherited retinal degeneration, of which we have reported five. The results obtained in this project will also facilitate the progression of these therapies to clinical application.