Sci. Transl. Med. ︱前沿！ASOs特异性靶向突变HTT基因，可以改善亨廷顿舞蹈症(HD)的认知缺陷和行为障碍

In 1872 George Huntington described the disorder in his first paper "On Chorea" at the age of 22. Credit: Wikipedia

亨廷顿氏舞蹈症（HD）是一种渐进性、且常染色体为显性的神经退行性疾病，由HTT（Huntingtin）基因内部CAG序列的重复扩增所引起。换言之，在HTT基因的第一个外显子中有重复的CAG三联密码子，通常当CAG三联子重复次数超过35次时，便会诱发HD【1】。

HD主要特征包括：逐渐丧失的自主运动控制能力、精神障碍、以及认知能力的下降【2】。虽然已研发了一些针对HD症状的治疗方法，且有一定的效果，但是，到目前为止，还没有任何一种疾病修饰治疗方法可以来治愈HD【3】。

就目前的研究来看，由异常HTT基因编码的突变亨廷顿蛋白（muHTT）是HD发病机制的唯一原因。但总的来说，HD具有很复杂的发病机制，很难进行靶向治疗【4】。不过，可喜的是，包括反义寡核苷酸（ASOs）技术在内的基因抑制技术可以靶向muHTT蛋白，进而调控HTT基因表达【5-6】。

ASOs通常指进行了某些化学修饰的短链核酸（约15-25个核苷酸组成），它的碱基顺序排列与特定的靶标RNA序列互补，进入细胞后可按照Watson-Crick碱基互补配对的原则与靶标序列形成双链结构【7】。

ASOs的具体机制是：(i). ASOs与靶标mRNA结合形成DNA-RNA杂合分子，进而激活核糖核酸酶H（RNase H），而该酶可以特异性地切割杂合分子中的RNA链，进而致使靶标mRNA降解。(ii). 而对于不能激活RNAase H活性的ASOs，则可以通过空间位阻效应阻止核糖体结合来抑制靶mRNA的翻译；另外还可以通过封闭剪接位点有选择的促进蛋白某个可变剪接体的表达，从而纠正错误的剪接【7】。

ASOs可以进入哺乳动物的脑脊液（CSF），并且自由分布在神经元、神经胶质细胞和室管膜细胞内；同时，因为ASOs在大脑中有很长的半衰期，所以ASOs很适合于慢性中枢神经系统紊乱性疾病【5, 8】。

Michael R. Hayden等人在先前的研究中就已经成功揭示出HTT基因的单核苷酸多态性（SNPs）和muHTT有着微妙的关系，且ASOs可以靶向HD-SNPs，进而可以选择性的抑制muHTT蛋白。也就是说，ASOs特异性靶向HD-SNPs可以作为一种等位基因-特异性muHTT基因沉默疗法（allele-specific muHTT gene silencing therapy）为80-90%的HD患者提供一定治疗(Table 1)【9-11】。

ASOs对于预防muHHT蛋白很有潜力，然而，等位基因-特异性ASOs（allele-specific ASOs）改善HD等疾病的具体机制仍然不清楚。

尽管，HD被认为是一种运动功能障碍性疾病，但是认知能力下降和精神障碍也是这种疾病的显著特征【12】。任何HD治疗的确可以改善HD患者的精神障碍、缓解认知能力下降，然而，大多数临床前工作却主要集中在对运动功能障碍的治疗上，却忽视了HD的其他病症和病因，包括情绪和认知等等，而进行全面的病症和病因了解对于研发潜在的HD疾病修饰疗法尤其重要的。

2018年10月3日，来自（加拿大）英属哥伦比亚大学儿童与家庭研究所、（美国）Ionis制药公司及（美国）爱荷华大学Carver医学院的联合研究团队，将他们的一项前沿性研究成果以Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease为题在线发表在Science Translational Medicine上（2018 IF=16.710）。

研究表明：ASOs可以特异性地靶向突变的HTT基因，进而对HD患者的认知障碍产生良好的治疗效果【13】。

在此研究中，研究者们将综合total HTT-suppressing ASO和allele-specific muHTT ASOs两种策略，对HD小鼠模型（Hu97/18、Hu18/18）的表型进行特异性改变，继而阐明ASOs对H的D精神病学的影响，以及ASOs是否能改善认知能力的下降。

Fig. 1  muHTT suppression rescues cognitive deficits. 

首先，为了确定ASOs介导的HTT蛋白抑制是否可以改变认知障碍和神经病学病症，研究者们随即检测了HD小鼠模型中的allele-specific ASOs，结果发现在ASOs治疗早期和晚期，HD小鼠的认知缺陷(Fig.1)和行为障碍均能获得改善(Fig. 2-3)。

Fig. 2  muHTT suppression normalizes anxiety behavior. 

Fig. 3  muHTT suppression may ameliorate depressive behavior.

在临床上，通过腰椎穿刺（lumbar puncture，LP）可以将HTT ASOs送入到HD患者体内。所以，紧接着，在LP操作下，研究者们将HTT ASOs成功注入到食蟹猴模型体内，以确定在灵长类动物的大脑中，ASOs是否依旧对认知缺陷和精神病病理相关的障碍起到改善效果。

研究者们检测了食蟹猴大脑中有关认知和精神功能的区域，即检测了皮质和边缘系统中的ASOs浓度，揭示出ASOs会强烈抑制HTT蛋白的表达，进而说明在灵长类动物大脑中，ASOs也可以改善认知缺陷和行为障碍(Fig.4)。

Fig. 4  HTT suppression in cortical and limbic structures in the NHP brain. 

总的来说，ASOs会特异性靶向HTT突变基因，从而抑制muHTT蛋白表达，最终恢复HD小鼠和食蟹猴模型的认知能力缺陷、改善神经病学上的障碍，如焦虑、抑郁等。

此外，在上个月（9月16日），逻辑神经科学报道了同样发表在Science Translational Medicine上，由(英国)伦敦大学等多家研究单位共同完成的一项突破性成果中（详情见补充阅读）。在该项研究中，Byrne等人阐明了通过对生物流体中的突变HTT蛋白（muHTT）浓度和神经丝光蛋白（NfL）浓度的共同检测，且综合临床评估、MRI等其他临床检测手段，将大大提高对早期HD预测的准确度和效率。而Southwell和Hayden等人的这项研究更加具有纵向性和未来延展空间。

补充阅读

通讯作者Dr.Hayden简介

Michael R. Hayden

Principal Investigator

Senior Scientist, CMMT, BCCHR, UBC
Canada Research Chair in Human Genetics and Molecular Medicine
University Killam Professor, Department of Medical Genetics, UBC

Email: [e-mail]

Lab Research

Researchers in the Hayden laboratory have found a critical pathway in the development of juvenile forms of Huntington’s disease. Blocking the action of caspase-6 prevents the progression of the disease in mice. This finding could lay the groundwork for an effective approach to therapy for Huntington disease.

Current Research Projects 

Silencing the gene that causes Huntington disease– Mutant huntingtin protein is the cause of Huntington disease (HD) and engages in a variety of aberrant interactions in neurons. Preventing generation of this toxic protein by gene silencing, the process of switching off a gene, should prevent all subsequent pathology and prevent or delay the onset of HD. Everyone has two copies of the huntingtin gene. In HD, one of these copies carries the mutation while the other copy is normal. The normal huntingtin protein is important for maintaining neuronal health and long-term reduction of this protein may not be well-tolerated. We are developing a strategy of silencing only the mutant copy of a patient’s huntingtin gene using antisense oligonucleotides targeted to HD mutation-associated single nucleotide variants as a treatment for HD.

Modulating mHTT post-translational modifications (PTMs) to enhance its clearance – Huntingtin (HTT) undergoes a myriad of post-translational modifications (PTMs) including phosphorylation, proteolytic cleavages and fatty acylation that influence the protein function, localization and clearance. Those PTMs are essential for neuronal viability, but are altered in HD. We have shown that promoting or preventing specific HTT PTMs can either dramatically improve or exacerbate HD symptoms. There is also evidence that HTT PTMs work in concert and may regulate one another. However, the interactions between the networks of HTT PTMs remain mostly unstudied. Our objectives are therefore to identify new rate-limiting PTMs, characterize the interrelationship of the HTT PTM network in vivo and understand how it relates to HTT function, stability and clearance. This project will allow us to determine and validate molecular targets for therapeutic strategies that could be used in synergy with HTT gene silencing.

Discovery of novel therapeutic targets for neuroprotection in Huntington Disease – Glutamate excitotoxicity and mitochondrial dysfunction are critical, closely-linked pathogenic mechanisms in several acute and neurodegenerative brain disorders, including HD. Together, these processes contribute to altered intracellular calcium dynamics, bioenergetic defects, cell death signaling, and synaptic instability. We are investigating novel therapeutic targets involved in these pathways with the goal of improving mitochondrial health and normalizing synaptic function in HD.

Population genetics and epidemiology of the Huntington disease mutation – The HD mutation is associated with specific sets of genetic variants in the surrounding HTT gene, known as haplotypes. We are performing detailed investigations of haplotypes HD mutation in different populations around the world. Haplotypes of the HD mutation allow for identification of new targets for therapeutic gene silencing and offer insight into the origin of the HD mutation in different ethnic groups. We additionally study how many people have the HD mutation, how often this mutation results in HD symptoms, and how often unstable new mutations for HD occur in the general population.

(Credit: Hayden Lab)

参考文献

【1】M. E. MacDonald, et al. A novel gene containing a trinucleotide repeat that isexpanded and unstable on Huntington’s disease chromosomes. Cell 72, 971–983 (1993).

【2】R. Ghosh, S. Tabrizi, Clinical aspects of Huntington’s Disease, in Current Topics in Behavioral Neuroscience, H. H. P. Nguyen, M. A. Cenci, Eds. (Springer Berlin Heidelberg, 2013), pp. 1–29.

【3】A. Videnovic, Treatment of Huntington disease. Curr. Treat. Options Neurol.15, 424–438 (2013).

【4】C. Zuccato, M. Valenza, E. Cattaneo, Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol. Rev. 90, 905–981 (2010).

【5】H. B. Kordasiewicz, et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron 74, 1031–1044 (2012).

【6】J. L. McBride, et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi. Proc. Natl. Acad. Sci. U.S.A. 105, 5868–5873 (2008).

【7】C. F. Bennett, E. E. Swayze, RNA targeting therapeutics: Molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol. 50, 259–293 (2010).

【8】F. Rigo, et al. Pharmacology of a central nervous system delivered 2’-O-methoxyethyl–modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. J. Pharmacol. Exp. Ther. 350, 46–55 (2014).

【9】S. C. Warby, et al. CAG expansion in the Huntington disease gene is associated with a specific and targetable predisposing haplogroup. Am. J. Hum. Genet. 84, 351–366 (2009).

【10】C. Kay, et al. Huntingtin haplotypes provide prioritized target panels for allele-specific silencing in Huntington disease patients of European ancestry. Mol. Ther.23, 1759–1771 (2015).

【11】S. C. Warby, et al. HTT haplotypes contribute to differences in Huntington disease prevalence between Europe and East Asia. Eur. J. Hum. Genet. 19, 561–566 (2011).

【12】D. I. Helder, et al. Impact of Huntington’s disease on quality of life. Mov. Disord. 16, 325–330 (2001).

【13】Amber L. Southwell, et al. Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease. Sci. Tansl. Med.10, eaar3959 (2018)

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