Developing rhodopsin gene therapy to treat dominant retinitis pigmentosa

视网膜色素变性（RP）影响大约1/4000的人口，是发达国家内不可逆视力丧失的最常见遗传原因。患有RP的个体出生时具有正常功能的眼睛，但继续发展以夜盲症形式的视力逐渐丧失，周边视力丧失（导致“隧道视力”），并且在更严重的情况下，降低中央清晰度和/或视力。这是一种高度可变的疾病，在不同的个体中以不同的速度进展：有些人在儿童时期发展出明显的功能性视力障碍，而对于其他人来说，这可能直到生命的后期才变得明显。这种变异性反映了大量的基因突变（遗传密码中的“错误”），这些突变可能导致RP的临床综合征。到目前为止，导致RP的最常见的基因突变是那些干扰蛋白质视紫红质的基因突变。这种蛋白质在视杆光感受器细胞中起着核心作用，视杆光感受器细胞是视网膜的功能单位或“像素”（眼睛后部的感光层）。这些细胞将光转化为有意义的信息传达给大脑，从而调节视觉。在“显性”RP（这是最常见的形式）中的视紫红质基因中的突变导致个体的双眼的所有视杆细胞内产生缺陷的视紫红质蛋白。这种异常蛋白质的存在会导致视网膜变性和视力逐渐丧失。本博士的目的是为显性RP开发一种新的治疗策略。建议的方法包括将额外的健康视紫红质基因插入到有缺陷的视杆细胞中。该基因编码正常功能的视紫红质蛋白。预计通过这种基因转移，视杆细胞将产生更大量的健康蛋白质，这将克服突变视紫红质的有害影响，从而保护视杆细胞并维持视力。将基因传递到视网膜将用含有健康视紫红质基因的特殊工程病毒（AAV）进行。这种病毒没有复制能力，本身就能引起疾病，它将通过注射到视网膜下的视杆细胞中。这一策略已被证明是成功的恢复视力在一些小鼠模型的视网膜退行性疾病和早期人类临床试验。在我的博士项目中，我将致力于开发一种新的AAV载体，以优化健康的视紫红质在小鼠模型中的人类显性RP的交付。这是重要的，因为有效的病毒载体将允许递送更少的病毒颗粒（从而最小化任何潜在的副作用），同时允许高水平的健康视紫红质，这可能是在显性RP中拯救退化的视网膜所需的。在开发出优化的AAV后，我将首先在正常小鼠中测试其效果以确定安全剂量范围，并在缺乏视紫红质的小鼠中证明插入基因的功能功效。然后，我计划在人类视网膜组织中以其优化剂量测试新病毒（来自同意接受视网膜手术的患者），并在显性RP的小鼠模型中评估这种策略在保留视网膜结构和功能方面的成功。我希望这项工作将成为在具有显性RP的人类受试者中进行临床试验的基础，并且我可以在博士学位后的几年中积极监督这一转变。因此，该项目的最终目标是开发一种基因技术，可以减缓甚至阻止这种迄今无法治疗和使人衰弱的疾病的进展。它有可能为世界各地成千上万的RP患者带来生活质量和工作能力的显着改善。

Affecting approximately 1 in 4000 of the population, retinitis pigmentosa (RP) is the most common genetic cause of irreversible sight loss within the developed world. Individuals with RP are born with normally functioning eyes but go on to develop progressive loss of vision in the form of night-blindness, loss of peripheral vision (resulting in 'tunnel vision') and in more severe cases, reduced central clarity and/or sight. This is a highly variable condition that progresses at different rates within different individuals: some develop functionally apparent visual impairment in childhood whilst for others, this may not become manifest until later in life. This variability reflects the large number of genetic mutations ('mistakes' within the genetic code) that can result in the clinical syndrome of RP. By far the most common set of gene mutations to cause RP are those which interfere with the protein rhodopsin. This protein plays a central role within rod photorecepor cells, the functional units or 'pixels' of the retina (the light sensitive layer at the back of the eye). These cells convert light into a meaningful message to convey to the brain thereby mediating the sense of sight. Mutations in the rhodopsin gene in 'dominant' RP (which is the most frequently occurring form) lead to the production of a defective rhodopsin protein within all rod cells of both eyes of the individual. The presence of this abnormal protein causes a degeneration of the retina and gradual sight loss.The aim of this PhD is to develop a new treatment strategy for dominant RP. The suggested approach involves inserting additional healthy rhodopsin genes into the defective rods. This gene encodes the normally functioning rhodopsin protein. It is anticipated that through this gene transfer, rod cells will produce a greater quantity of healthy protein which should overcome the deleterious effects of the mutant rhodopsin, thus preserving rods and maintaining vision. Delivery of the gene to the retina will be performed with a specially engineered virus (AAV) containing the healthy rhodopsin gene. The virus, which has no capacity to replicate and in itself cause disease, will be delivered to rod cells through an injection underneath the retina. This strategy has previously proved successful in restoring vision in a number of mouse models of retinal degenerative disease and in early human clinical trials.In my PhD project, I will aim to develop a new AAV vector to optimise the delivery of healthy rhodopsin in mouse models of human dominant RP. This is important as an efficient viral vector will allow fewer viral particles to be delivered (thus minimising any potential side-effects), whilst allowing for the high levels of healthy rhodopsin that are likely to be required to rescue the degenerating retina in dominant RP. Having developed the optimised AAV, I will first test its effect in normal mice to establish the safe dose range, and in mice lacking rhodopsin to demonstrate functional efficacy of the inserted gene. I then plan to test the new virus at its optimised dose in human retinal tissue (from consenting patients who have undergone retinal surgery for unrelated reasons), and in a mouse model of dominant RP to assess the success of this strategy in preserving retinal structure and function. It is my hope that this work will form the basis for a clinical trial in human subjects with dominant RP and that I can be active in overseeing this transition in the years following my PhD. The ultimate goal of this project is thus to develop a genetic technology which may slow or even halt the progression of this hitherto untreatable and debilitating disease. It has the potential to impart a significant improvement in quality of life and ability to work for many thousands of individuals with RP across the world.