两篇Nature证实基因沉默药物有望治疗两种致命的神经疾病
生物谷 · 2017/04/19
4月12日,《Nature》期刊在线发表两篇文章,揭示一种经设计抵抗导致脊髓小脑共济失调2型(spinocerebellar ataxia type 2, SCA2)的基因突变的药物可能也被用来治疗肌萎缩性脊髓侧索硬化症(Amyotrophic lateral sclerosis, ALS)。


在两项针对小鼠的研究中,研究人员证实一种经设计抵抗导致脊髓小脑共济失调2型(spinocerebellar ataxia type 2, SCA2)的基因突变的药物可能也被用来治疗肌萎缩性脊髓侧索硬化症(Amyotrophic lateral sclerosis, ALS)。这两项研究均于2017年4月12日在线发表在Nature期刊上,论文标题分别为“Antisense oligonucleotide therapy for spinocerebellar ataxia type 2”(记为第一项研究)和“Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice”(记为第二项研究)。

第一项研究的共同通信作者、美国犹他大学神经学系教授Stefan M. Pulst博士说,“我们的结果为我们可能有朝一日能够治疗这些破坏性的疾病提供希望。”在1996年,Pulst博士和其他的研究人员已发现ATXN2基因发生的突变会导致SCA2疾病。这种疾病是一种致命性的遗传疾病,主要破坏大脑中的小脑,导致病人出现平衡、协调、行走和眼球运动方面的问题。

在第一项研究中,Pulst团队发现他们能够通过将一种经编程沉默ATXN2基因的药物注射到SCA2模式小鼠大脑中来降低这些问题的产生。在第二项研究中,美国斯坦福大学医学院遗传学系副教授Aaron Gitler博士及其团队证实注射相同类型的药物到ALS模式小鼠的大脑中能够阻止较早的死亡和与ALS相关的神经问题。ALS是一种瘫痪性的而且经常是致命性的疾病。

Gitler博士说,“令人吃惊的是,ATXN2基因可能是开发治疗ALS和其他神经疾病的方法的关键。”2010年,Gitler博士和同事们已发现ATXN2基因突变与ALS之间存在关联。

他们使用的这类药物被称作反义寡核苷酸(antisense oligonucleotide)。这类药物是短的DNA序列,能够结合到携带着基因指令的mRNA分子上。这就阻止细胞制造蛋白,这一过程被称作基因沉默。

第一项研究的共同通信作者、犹他大学神经学系研究员Daniel R. Scoles博士说,“我们的反义寡核苷酸阻止细胞读取ATXN2基因的蓝图。”

一种反义寡核苷酸药物已被美国食品药品管理局(FDA)批准用来治疗脊髓性肌肉萎缩症,即一种导致儿童的手臂和腿部肌肉无力和恶化的遗传疾病。研究人员正在开展早期临床试验以便探究治疗几种神经疾病(包括亨廷顿氏舞蹈病和遗传性ALS)的基因沉默药物的安全性高和疗效。

美国国家卫生研究院(NIH)属下的国家神经性疾病与中风研究所(National Institute of Neurological Disorders and Stroke, NINDS)项目主任Amelie Gubitz博士说,“反义寡核苷酸给研究人员提供一种有前景的工具来研究很多疾病的内在原因和开发基因靶向疗法。”

与SCA2相关联的ATXN2基因突变导致这种基因发生多聚谷氨酰胺扩增(polyglutamine expansion),即一串重复的三碱基密码子CAG拷贝(密码子CAG在翻译时对应着谷氨酰胺)。一般而言,对ATXN2基因具有更长的一串CAG密码子的SCA2病人而言,症状出现得越早而且更加严重。ATXN2基因仅具有27~33个CAG重复序列的人不会患上SCA2,但是具有增加的ALS风险。

Pulst团队与一家制药公司合作开发沉默ATXN2基因而不是CAG重复序列的反义寡核苷酸。他们随后在两种经基因改造(具体而言就是小脑中的神经元经编程后表达ATXN2蛋白突变体)后产生与SCA2相关联的问题的小鼠品系体内测试了这种反义寡核苷酸。

在这两种小鼠品系中,这种反义寡核苷酸似乎都是有效的。相比于接受安慰剂注射的小鼠而言,接受这种药物注射的小鼠能够在旋转杆上更长地行走。电学记录表明这种药物让小脑中的神经元放电模式恢复到正常水平。除了降低ATXN2表达水平之外,Pulst团队发现这种药物也恢复了似乎受到ATXN2突变体抑制的几种基因的表达水平。

与此同时,Gitler团队利用不同的小鼠测试了通过沉默ATXN2基因来抵抗ALS的想法。这些小鼠经过基因改造产生较高水平的人TDP-43版本。TDP-43是一种在正常情形下调节基因的蛋白。鉴于ALS病人经常含有毒性的TDP-43团块,该团队研究了这些小鼠。这些小鼠快速地产生行走问题,而且较早地死亡。Gitler团队和他的合作者们之前针对酵母和果蝇的研究已提示着ATXN2突变体可能控制着TDP-43的毒性。

与安慰剂相比,将反义寡核苷酸注射到这些新生小鼠的神经系统中会把它们的平均寿命延长了35%,改善它们的行走能力,同时降低它们的大脑和脊髓中的ATXN2基因表达水平。

当Gitler团队让这些表达人TDP-43的小鼠与经基因编程不含有ATXN2基因的小鼠进行杂交繁育时,他们观察到类似的结果。相比于表达人TDP-43的小鼠,它们的后代活得更长,而且行走得更好。而且相比于表达人TDP-43的小鼠,这些后代的大脑也具有更少的毒性TDP-43团块。

NINDS项目主任Daniel Miller博士说,“针对酵母和果蝇的很多研究为这些激动人心的结果打下基础。它们证实对简单的疾病模型开展大量研究能够产生深刻的认识,从而有助我们理解和潜在地治疗看似不可能治疗的疾病。”

Pulst博士和Gitler博士赞成,在他们使用的反义寡核苷酸能够在病人体内使用之前,还需要开展更多的研究。这两家实验室当前正通过开展进一步的临床前实验而采取下一步行动。

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  • Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

    There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies1 in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease2. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function3, 4. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.

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  • Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2–5 years after disease onset1. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords2, and rare mutations in the gene encoding TDP-43 can cause ALS3. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases4. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression5. However, as SOD1 mutations account for only around 2–5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions1, 6. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies7, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS7, 8. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy9. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS6, targeting ataxin-2 could represent a broadly effective therapeutic strategy.

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