Development of retinal gene therapy to treat dominantly inherited disease using a novel RNA-based silencing system

遗传病现在是年轻人无法治愈的失明的最常见原因。基因疗法是一种通过操纵遗传密码来治疗疾病的方法。最近，英国国家医疗服务体系首次批准使用基因疗法治疗一种罕见的遗传性失明。这项研究的目的是开发另一种基因治疗方法，但这一次是为了一种更常见的致盲原因。被描述为“显性遗传”的遗传病会从一代传到下一代，通常是由一个基因上的缺陷引起的，这种基因产生了一种有毒蛋白质。视网膜色素变性(RP)是年轻人遗传性失明的一种不可治愈的原因，通常主要是遗传性的。它最常见的原因是视紫红质(Rho)基因突变，该基因编码视网膜中的光敏色素。只有一个Rho基因拷贝的患者可以完全清楚地看到东西，但如果第二个拷贝有突变，使视紫红质蛋白异常，那么这种突变将积累在感光细胞(光感受器)中，导致它们退化。这是一个持续数年的缓慢过程，但最终当所有的光感受器都消失时，受影响的患者就会完全失明。可悲的是，他们也会将基因突变遗传给他们的孩子，后者有50%的机会因遗传相同的突变而失明。我们提出的研究涉及使用现有的基因治疗技术来利用一种自然发生的细胞途径，这种途径用于使基因失活。当一个基因被读取时，DNA被转换成RNA，然后这个RNA被切成更小的片段，组成特定蛋白质的密码--也就是众所周知的信使RNA。然而，还有一些被称为microRNAs的较小的RNA片段可以与信使RNA结合并使其失活。这些微小的RNA分子调节基因的表达--它们是在细胞核中通过一个复杂的过程产生的，这个过程涉及在它们可以与信使RNA结合之前将它们折叠成一个环。然而，在2007年，麻省理工学院大卫·巴特尔的实验室发现，一些基因释放的RNA片段可以自发形成microRNA环，而不需要复杂的处理。这些微小的核糖核酸分子被称为“镜像”。我们提出的研究涉及使用灭活病毒(称为病毒载体)将来自Mirtron的microRNA分子直接送入光感受器细胞，目的是使突变的视紫红质失活。我们设计的病毒载体类似于英国NICE最近批准的病毒载体，因为我们知道它是安全有效的。我们已经在病毒载体中放置了两个镜像，以及一个额外的正常的Rho基因副本，该副本经过了轻微的修改，因此镜像无法使其失活。因此，当病毒载体被注射到视网膜中时，突变的Rho基因被抑制，而正常复制被增强。我们在实验室的一只小鼠身上进行了测试，这只小鼠与人类患者具有相同的Rho突变，我们可以在我们测试的剂量之一推迟小鼠的视网膜退化。这项实验代表了第一次在活体动物身上成功应用Mirtron基因疗法，我们对此感到非常兴奋，因为它具有巨大的潜力，可以治疗以遗传性为主的眼部疾病(可能还有眼睛以外的其他疾病)。尽管我们已经写下了要发表的结果，但我们热衷于将其开发为患者的治疗方法，这就是我们申请MRC DPFS资金的原因。我们只测试了一个病毒载体，尽管它起作用了，但我们意识到载体中的遗传密码可以得到大幅改进，以产生更好的效果。我们需要在另一个人类RP的小鼠模型和包含整个人类Rho基因的小鼠模型上测试该载体，这样我们就可以测量效果，并准确地计算出Rho基因应该被定位在哪里，我们需要多少个镜像。

Genetic diseases are now the most common cause of untreatable blindness in young people. Gene therapy is a method of treating a disease by manipulating the genetic code. Recently the first ever gene therapy was approved for use in the NHS and this was for a rare inherited form of blindness. The purpose of this research is to develop another genetic treatment, but this time for a more common cause of blindness.Genetic diseases which are described as 'dominantly inherited' pass from one generation to the next and are usually caused by a defect on one gene that makes a toxic protein. Retinitis pigmentosa (RP) is an incurable cause of genetic blindness in young people and it is often dominantly inherited. It is most commonly caused by mutations in the rhodopsin (RHO) gene which codes for the light sensitive pigment in the retina. Patients with only one copy of the RHO gene can see perfectly well, but if the second copy has a mutation in it that makes abnormal rhodopsin protein then this will accumulate in the light sensing cells (photoreceptors) and cause them to degenerate. This is a slow process over several years, but eventually when all the photoreceptors have gone the affected patient becomes completely blind. Sadly they also pass on the genetic mutation to their children who have a 50% chance of going blind from inheriting the same mutation.Our proposed research involves using established gene therapy techniques to take advantage of a naturally occurring cell pathway that is used to inactivate genes. When a gene is read, the DNA is converted into RNA and this RNA is then chopped up into smaller fragments that make the code for a particular protein - otherwise known as messenger RNA. There are however smaller RNA fragments known as microRNAs which can bind to the messenger RNA and inactivate it. These microRNA molecules regulate gene expression - they are made in the cell nucleus by a complicated process that involves folding them into a loop before they can bind to the messenger RNA. In 2007 however it was discovered in David Bartel's lab at the Massachusetts Institute of Technology that some genes release RNA fragments that can spontaneously form microRNA loops without the complicated processing. These microRNA molecules are known as 'mirtrons'. Our proposed research involves using an inactivated virus (known as a viral vector) to deliver microRNA molecules derived from mirtrons directly into the photoreceptor cells with the aim of inactivating the mutant rhodopsin. We have designed the viral vector to be similar to the one recently approved by NICE in England because we know it is safe and effective. We have put two mirtrons in the viral vector, together with an extra normal copy of the RHO gene which has been modified slightly so that the mirtrons cannot inactivate it. Hence when the viral vector is injected into the retina, the mutant RHO gene is suppressed and the normal copy is boosted. We tested this in a mouse in our laboratory that has the same RHO mutation as human patients and we could delay the mouse retinal degeneration at one of the doses we tested. This experiment represents the first time that mirtron gene therapy has been successfully applied in a living animal and we are extremely excited about it, because it has huge potential to treat patients with dominantly inherited eye disease (and probably other diseases outside the eye).Although we have written up the results for publication, we are keen to develop this as a treatment for patients and this is why we have applied for MRC DPFS funding. We have only tested one viral vector and although it worked, we are aware that the genetic code in the vector could be improved substantially to give an even better effect. We need to test the vector in another mouse model of human RP and one that contains the entire human RHO gene so that we can measure the effects and work out exactly where the RHO gene should be targeted and how many mirtrons we need.