Developing gene therapy to treat blindness caused by Stargardt Disease

Stargardt病导致年轻人失明，原因是眼球后部视网膜上排列着光感受器的感光细胞丢失。Stargardt病是由一种名为ABCA4的蛋白质缺乏引起的，ABCA4代表“ATP结合盒A4亚型”。这种蛋白质对光敏感光器所需的视觉色素的循环利用非常重要。当ABCA4缺乏时，光敏色素会在视网膜中积累，从而导致毒性损伤和最终细胞死亡。Stargardt病通常出现在儿童时期，然后在许多情况下伴随着几年的渐进性失明和失明。因此，在确诊后的几年内，有机会通过某种形式的治疗进行干预，以防止视力丧失。基因疗法是一种新技术，已被证明在将缺陷基因重新引入视网膜方面非常有效。基因疗法于2007年在三个临床试验中首次以这种方式使用，以治疗由被称为RPE65的精氨酸基因缺陷引起的遗传性失明。最近，我们在牛津开始了一种新的基因疗法，用于治疗一种名为脉络膜血症的光感受器疾病，到目前为止，已有几名患者接受了治疗，但没有出现不良反应。在这些基因治疗试验中，我们使用了一种被称为腺相关病毒或AAV的小病毒颗粒。这种病毒颗粒是已知的最小的生命形式之一，它与人类的任何疾病无关。病毒通过保持休眠和不被免疫系统检测来生存。我们利用了AAV的优势，移除了病毒基因，并用特定视网膜疾病缺失的基因取而代之。病毒逃避免疫系统的能力非常有帮助，因为没有炎症意味着病毒在注射到眼睛中时不会损害视网膜。然而，AAV颗粒太小，无法携带在某些类型的视网膜退化中缺失的许多基因。不幸的是，治疗Stargardt病需要替换的ABCA4基因太大了，不适合AAV。这个项目的目的是通过探索最近发现的与AAV有关的新特性来解决这个问题。最近发现，一个大的基因可以被分成两个片段，每个片段都由一个AAV颗粒携带，这些颗粒能够在感染神经细胞后将基因重组回全长。在这个项目中，我们的目标是进行一系列实验，优化ABCA4基因的两个片段的重组过程，以便将其成功地输送到光感受器中。我们将在一只基因工程小鼠身上验证该基因的功能，这只小鼠也缺乏相同的基因，在视网膜检查中具有类似Stargardt病的特征。如果我们能够利用我们的新载体纠正甚至改善ABCA4的不足，那么我们就有了理想的背景信息来支持新的临床试验。

Stargardt disease leads to blindness in young people due to loss of the light sensing cells known as photoreceptors that line the retina at the back of the eye. Stargardt disease is caused by the deficiency of a protein known as ABCA4 which stands for "ATP Binding Cassette subtype A4". This protein is very important for recycling the visual pigments necessary for the light-sensitive photoreceptors. When there is a deficiency of ABCA4, the light-sensitive pigments accumulate in the retina which leads to toxic damage and eventual cell death. Stargardt disease usually presents in childhood and there then follows a period of several years of progressive loss of sight and blindness in many cases. There is therefore a window of opportunity of several years after diagnosis in which to intervene with some form of treatment to prevent sight loss.Gene therapy is a new technique which has been shown to be highly effective at reintroducing defective genes into the retina. Gene therapy was first used in this manner in 2007 in three clinical trials to treat inheriuted blindess caused by defiency of anothe gene known as RPE65. More recently we have started a new gene therapy treatment in Oxford to treat a photoreceptor disease known as choroideraemia and several patients have been treated so far without adverse effects. In these gene therapy trials we are using a small viral particle known as adeno associated virus, or AAV. This viral particle is one of the smallest lifeforms known and it is not associated with any disease in humans. The virus survives by remaining dormant and undetected by the immune system. We have taken advantage of AAV by removing the viral genes and replacing them with the gene that is missing for the particular retinal disease. The ability of the virus to evade the immune system is very helpful because the lack of inflammation means that the virus does not damage the retina when injected into the eye. The AAV particle is however too small to carry many genes that are missing in certain types of retinal degeneration. Unfortunately the ABCA4 gene, which would need to be replaced to cure Stargardt disease, is just too large to fit into AAV.The purpose of this project is to solve this problem by exploring a new property which has recently been identified in relation to AAV. It has recently been discovered that a large gene can be broken into two segments each of which is carried by an AAV particle and these particles are able to recombine the gene back into full length after infecting nerve cells. In this project we aim to perform a series of experiments whereby we optimise the process of recombining two fragments of the ABCA4 gene in order to deliver its successfully into photoreceptors. We will validate the function of the gene in a genetically engineered mouse, which is also deficient of the same gene and has features similar to Stargardt disease on retinal examination. If we can correct or even improve the deficiency of ABCA4 using our new vector then we would have the ideal background information to support a new clinical trial.