Developing rhodopsin gene therapy to treat dominant retinitis pigmentosa

在发达国家，大约每4000人中就有1人患有视网膜色素变性(RP)，这是导致不可逆性视力丧失的最常见遗传原因。患有RP的人出生时眼睛功能正常，但后来发展为进行性视力丧失，表现为夜盲、周边视力丧失(导致隧道视力)，在更严重的情况下，中央清晰度和/或视力下降。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：一些人在童年时出现明显的功能性视力障碍，而另一些人则可能要到晚年才会显现出来。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，最常见的一组引起视紫红质的基因突变是那些干扰视紫质蛋白的基因突变。这种蛋白质在视杆感光细胞、视网膜的功能单位或“像素”(位于眼睛后部的光敏层)中起着中心作用。这些细胞将光转化为有意义的信息，传递给大脑，从而调节视觉。视紫红质基因在显性的RP(这是最常见的形式)中的突变导致个体双眼的所有视杆细胞中产生有缺陷的视紫红质蛋白。这种异常蛋白的存在会导致视网膜的退化和渐进性的视力丧失。该PHD的目的是为显性RP开发一种新的治疗策略。建议的方法包括将更多健康的视紫红质基因插入有缺陷的视紫红质视杆中。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，视杆细胞将产生更多的健康蛋白质，这些蛋白质应该可以克服突变视紫红质的有害影响，从而保护视杆并维持视力。将基因输送到视网膜将通过一种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，本身就会导致疾病，它将通过在视网膜下注射将病毒传递给视杆细胞。在我的博士项目中，我的目标是开发一种新的AAV载体，以优化在人类占主导地位的RP小鼠模型中健康视紫红质的输送。这一点很重要，因为高效的病毒载体将允许更少的病毒颗粒被传递(从而将任何潜在的副作用降至最低)，同时允许高水平的健康视紫红质，这可能是拯救主要的RP中退化的视网膜所必需的。在开发出优化的AAV后，我将首先在正常小鼠身上测试其效果，以确定安全剂量范围，并在缺乏视紫红质的小鼠上测试其效果，以证明插入的基因的功能有效性。然后，我计划在人类视网膜组织(来自出于无关原因接受视网膜手术的患者)和优势RP小鼠模型中，以其最佳剂量测试新病毒，以评估这一策略在保护视网膜结构和功能方面的成功。我希望这项工作将为显性RP受试者的临床试验奠定基础，并希望我能在获得博士学位后的几年里积极监督这一转变。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愈的虚弱疾病的发展。它有可能极大地提高生活质量和为世界各地成千上万的RP患者工作的能力。

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.