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.

遗传性疾病现在是导致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，第一个基因疗法被批准在 NHS 中使用，用于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，但这次是针对一种更常见的失明原因。被描述为“显性遗传”的遗传疾病从一代传到下一代，通常是由一个基因缺陷引起的产生有毒蛋白质。色素性视网膜炎（RP）是导致年轻人遗传性失明的无法治愈的原因，并且通常是显性遗传的。它最常见的是由编码视网膜光敏色素的视紫红质 (RHO) 基因突变引起的。只有一个 RHO 基因拷贝的患者可以看得很清楚，但如果第二个拷贝发生突变，产生异常的视紫红质蛋白，那么这种蛋白就会在光传感细胞（感光细胞）中积累并导致它们退化。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，他们还将基因突变遗传给了他们的孩子，他们有 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已建立的基因治疗技术来利用自然发生的细胞途径，该途径用于灭活基因。当读取基因时，DNA 会转化为 RNA，然后该 RNA 会被切成更小的片段，从而生成特定蛋白质的代码 - 也称为信使 RNA。然而，还有一些更小的 RNA 片段，称为 microRNA，可以与信使 RNA 结合并使其失活。这些 microRNA 分子调节基因表达——它们是在细胞核中通过复杂的过程产生的，其中包括将它们折叠成环，然后才能与信使 RNA 结合。然而，2007 年，麻省理工学院 David Bartel 的实验室发现，一些基因释放的 RNA 片段可以自发形成 microRNA 环，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）将来自 mirtron 的 microRNA 分子直接传递到感光细胞中，目的是灭活突变视紫红质。我们设计的病毒载体与英国 NICE 最近批准的病毒载体类似，因为我们知道它是安全有效的。我们在病毒载体中放入了两个 mirtron，以及 RHO 基因的额外正常副本，该基因已被稍微修改，以便 mirtron 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，而正常的拷贝被增强。我们在实验室的一只小鼠身上进行了测试，该小鼠与人类患者具有相同的 RHO 突变，并且我们可以在我们测试的一种剂量下延迟小鼠视网膜变性。这个实验代表了 mirtron 基因疗法首次成功应用于活体动物，我们对此感到非常兴奋，因为它在治疗显性遗传性眼病（可能还有其他眼外疾病）患者方面具有巨大的潜力。我们已经撰写了结果以供发表，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。我们只测试了一种病毒载体，尽管它有效，但我们知道载体中的遗传密码可以大幅改进，以产生更好的效果。我们需要在另一种人类 RP 小鼠模型和包含整个人类 RHO 基因的小鼠模型中测试该载体，以便我们可以测量效果并准确计算出 RHO 基因应该靶向的位置以及我们需要多少个 mirtron。