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

使用基于 RNA 的新型沉默系统开发视网膜基因疗法来治疗显性遗传性疾病

• 批准号: MR/V027557/1
• 负责人: Robert MacLaren
• 金额: $ 85.06万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV027557%2F1
• 关键词: Development; retinal; gene; therapy; treat

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.

遗传疾病现在是年轻人不可治疗的失明的最常见原因。基因治疗是一种通过操纵遗传密码来治疗疾病的方法。最近，有史以来第一种基因疗法被批准用于NHS，这是一种罕见的遗传形式的失明形式。这项研究的目的是开发另一种遗传治疗，但这一次是出于更常见的失明原因。被称为“主要遗传”从一代人传递到第二代的遗传疾病，通常是由一种产生有毒蛋白质的基因的缺陷引起的。色素性视网膜炎（RP）是年轻人遗传失明的原因，通常是主要遗传的。它最常见的是由视紫红质（Rho）基因突变引起的，该突变编码为视网膜中的光敏色素。只有一个Rho基因拷贝的患者可以很好地看待，但是如果第二副副本具有使异常的视紫红质蛋白的突变，那么它将积聚在光感应细胞（光感受器）中，并导致它们退化。这是几年来缓慢的过程，但是最终，当所有光感受器消失时，受影响的患者变得完全失明。可悲的是，他们还将遗传突变传递给了他们的孩子，他们有50％的机会遗传相同的突变。我们提出的研究涉及使用既定的基因治疗技术来利用一种自然存在的细胞途径，该细胞途径用于失活基因。读取基因时，将DNA转换为RNA，然后将该RNA切成较小的片段，从而使特定蛋白质代码 - 否则称为Messenger RNA。但是，有较小的RNA片段称为microRNA，可以与Messenger RNA结合并灭活。这些microRNA分子调节基因表达 - 它们是通过复杂的过程在细胞核中制成的，该过程涉及将它们折叠成循环，然后它们才能结合到Messenger RNA。然而，在2007年，它是在马萨诸塞州理工学院的戴维·巴特尔（David Bartel）实验室中发现的，一些基因释放了RNA片段，这些片段可以自发形成microRNA回路而无需复杂的处理。这些microRNA分子被称为“ mirtron”。我们提出的研究涉及使用灭活病毒（称为病毒载体）将源自mirtron的microRNA分子直接传递到感光细胞中，目的是使突变型肖陶蛋白失活。我们设计的病毒载体与英格兰尼斯最近批准的媒介相似，因为我们知道它是安全有效的。我们已经将两个mirtron放在病毒载体中，以及Rho基因的额外正常副本，该副本已被稍微修饰，以使mirtron无法失活。因此，当将病毒载体注入视网膜中时，突变的Rho基因被抑制并增强正常拷贝。我们在实验室的一只小鼠中对此进行了测试，该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一种剂量下延迟小鼠视网膜变性。 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.我们仅测试了一个病毒载体，尽管它起作用，但我们知道可以改善向量中的遗传代码，从而产生更好的效果。我们需要在人类RP的另一个小鼠模型中测试矢量，其中包含整个人类Rho基因，以便我们可以测量效果并准确地算出Rho基因应靶向的位置以及我们需要多少个mirtron。