Using genetic modifiers to identify and target pathogenic mechanisms in Huntington's disease

亨廷顿舞蹈病（HD）是近50种遗传性“重复扩展疾病”之一，已知由类似的短重复DNA片段引起。这些疾病中有许多涉及神经系统的进行性退化和痴呆。不幸的是，没有一种有效的治疗方法。在这个项目中，我将专注于HD的治疗靶点识别，但进展可能与所有重复扩张疾病相关。HD患病率约为1 / 8000，通常始于中年，尽管有很多差异。它涉及运动控制、心理健康、行为和记忆的逐渐恶化，直到10-30年后过早死亡。HD是由一种名为亨廷顿蛋白（HTT）的基因中的C-A-G重复扩增引起的。基因是一段很短的DNA，使用4个字母的化学代码（A、G、C或T）作为在细胞中制造蛋白质的指导手册。HTT编码一种大型蛋白质，在组织细胞中具有许多功能。它通常在DNA编码中包含大约20个重复的CAG三联体（即CAGCAGCAG…），但在HD中，该部分扩展到至少36个CAG。使用这种错误代码生成的HTT蛋白对大脑中的神经细胞（神经元）是有毒的，最终导致它们的退化和HD症状。虽然HTT中的CAG重复序列导致HD，但我们最近发现细胞中还有其他基因可以改变HD症状开始的年龄。其中一些“基因修饰剂”可以改变发病多年，这意味着如果我们能够针对它们使用药物，我们就可以看到显著的临床效果。我的目标是开发一个管道来推进基因修饰物作为HD的新治疗靶点。我的出发点是一个14个潜在修饰基因的列表，这些基因是我们和其他人在HD患者的大型遗传研究中发现的。为了发现这些基因是否以及如何影响HD，我将首先在两个不同但互补的实验系统中筛选它们：Q109人类干细胞/神经元和果蝇。这些干细胞是由一名有109个CAG重复序列的HD患者捐献的，因为它们可以在实验室中转化为大脑神经元，所以特别有用。当我们在实验室培养这些细胞时，CAG重复随着时间的推移而扩大，这一过程被认为会导致大脑的神经变性。我将从Q109细胞中删除潜在的修饰基因，以测试它们是否影响CAG扩增率和/或神经元功能和存活。HD果蝇提供了一种在整个生物体中筛选潜在修饰基因的替代系统。这些果蝇被改造成有128个cag的HTT基因，导致它们出现行走问题、神经退化和寿命缩短。我将使用基因工具来测试降低这些果蝇体内潜在修饰因子水平的影响。接下来，我将选择那些影响最大和最有治疗潜力的修饰剂（即减少修饰剂可改善HD病理）进行进一步研究。我已经确定了一种与刺激CAG扩张相关的基因，从而导致HD的早期发病。这将是第一个用于详细分子研究的修饰剂，以弄清楚它究竟是如何影响HD病理的，以及我们是否可以用药物靶向它。本项目中确定的其他改性剂也将采用类似的工艺。然后，我将与我的学术和商业伙伴一起，将有希望的药物目标推进药物发现计划。最后，我的目标是通过比较极早或极晚发病HD患者的神经元生物学来发现HD病理学的新驱动因素。我将使用一种叫做RNA测序的尖端技术来测量神经元中所有2万个人类基因的表达水平，然后分析是否存在与早发性或晚发性疾病有关的差异。这个实验可以确定导致神经变性的新途径，也可以为我的管道测试提供新的修饰基因。

Huntington's disease (HD) is one of almost 50 inherited 'repeat expansion disorders' known to be caused by similar short repeated sections of DNA. Many of these diseases involve progressive degeneration of the nervous system and dementia. Unfortunately, none has an effective treatment. In this project I will focus on therapeutic target identification in HD, but advances could be relevant to all repeat expansion disorders.HD affects around 1 in 8000 and usually starts in middle age, although there is much variation. It involves progressive worsening of movement control, mental health, behaviour and memory until premature death 10-30 years later. HD is caused by a C-A-G repeat expansion in one gene called Huntingtin, or HTT. A gene is a short stretch of DNA that uses a 4 letter chemical code (A, G, C or T) as an instruction manual for making proteins in cells. HTT codes for a large protein with many jobs in organising cells. It normally contains a short run of about 20 repeated CAG triplets in its DNA code (ie CAGCAGCAG...) but in HD this section is expanded to at least 36 CAGs in a row. HTT proteins made using this faulty code are toxic to nerve cells (neurons) in the brain, ultimately leading to their degeneration and symptoms of HD. Although the CAG repeat in HTT causes HD, we have recently discovered that there are other genes in cells that can change the age at which HD symptoms start. Some of these 'genetic modifiers' can change onset by many years meaning that if we can target them with drugs we could see a dramatic clinical effect.I aim to develop a pipeline for advancing genetic modifiers as new therapeutic targets in HD. My starting point is a list of 14 potential modifier genes that we and others have identified in large genetic studies of HD patients. To discover whether and how these genes impact on HD I will first screen them in two different but complementary experimental systems: Q109 human stem cells/neurons and fruit flies. The stem cells were donated by an HD patient who had 109 CAG repeats and are particularly useful because they can be turned into brain neurons in the lab. When we grow these cells in the lab the CAG repeat expands over time, a process thought to drive neurodegeneration in the brain. I will delete potential modifier genes from Q109 cells to test whether they affect CAG expansion rates and/or neuronal function and survival. The HD fruit flies provide an alternative system in which to screen potential modifier genes in the context of a whole organism. These flies are engineered to have a HTT gene with 128 CAGs that causes them to develop walking problems, neurodegeneration and reduced lifespan. I will use genetic tools to test the impact of reducing the levels of potential modifiers in these flies.Next, I will select those modifiers with the biggest impact and the most therapeutic potential (i.e. where reduction of a modifier improves HD pathology) for further investigation. I have already identified one gene that is associated with stimulating CAG expansion and hence earlier onset of HD. This will be the first modifier taken forward for detailed molecular studies to work out how exactly it is affecting HD pathology and whether we can target it with drugs. A similar process will be taken with other modifiers identified in this project. I will then take promising drug targets forward to drug discovery programmes with my academic and commercial partners.Finally, I aim to discover new drivers of HD pathology by comparing the biology of neurons made from patients with very early or very late onset HD. I will use cutting-edge technology called RNA sequencing to measure the levels of expression of all 20,000 human genes in the neurons and then analyse whether there are differences that can be linked to early or late onset disease. This experiment could identify new pathways that lead to neurodegeneration and also suggest new modifier genes for testing in my pipeline.