Targeting ALS-linked FUS with allele-specific antisense oligonucleotides

项目摘要： 肌萎缩侧索硬化症（ALS）是最常见的运动神经元疾病，是一种致命的神经退行性疾病， 从症状发作起的中位生存时间为2-3年。目前只有两个批准 ALS的治疗方法，通道调节剂AmutekR，以及最近的神经保护性抗氧化剂RadicavaR。 这两种疗法都不能阻止疾病进展，ALS仍然是一种毁灭性的，致命的疾病和主要疾病。 未满足的医疗需求。近年来，ALS致病基因的鉴定取得了重大进展。 这一进展有助于促进对受疾病影响的运动神经元过程的理解。 尽管取得了这些进展，但许多已鉴定的ALS基因并不编码传统的“可药物治疗”靶点， 将这些进展转化为治疗ALS的新的小分子药物一直是一个挑战。然而，一些ALS患者 基因是新的、基于基因的治疗方法的良好候选者，例如基因治疗或反义寡核苷酸。 (ASO)疗法一个ALS基因，其中有强大的理由为基础的基因治疗方法被称为 FUS（融合在肉瘤中）。FUS的突变于2009年首次在患有ALS的家庭中被描述。以来 然后，FUS突变被确定为青少年发作ALS的最常见危险因素之一， 特别是侵袭性进展，通常在发病后一年内导致死亡。有强有力 FUS突变导致有毒基因产物的证据。这些证据表明， 可以降低突变的、有毒的FUS产物水平的方法构成了治疗FUS的令人信服的基础。 FUS突变的ALS患者。这种治疗机制可以通过设计成 特异性减少由FUS基因的突变拷贝制备的FUS基因产物。ASO很短，是人工合成的 一系列经过修饰的遗传物质，可以被设计用来识别和击倒特定的基因产物。 最近批准的阿索治疗药物SpinrazaR治疗脊髓性肌萎缩症为阿索铺平了道路 退行性神经系统疾病的治疗方法这些阿索药物直接施用到中枢神经系统中。 通过注射到脊髓周围的液体中来治疗神经系统。虽然靶向这两种药物的治疗性ASO 基因拷贝在亨廷顿病和SOD 1-ALS的临床试验中取得了进展， 来设计特异性针对突变基因拷贝的ASO。这种“等位基因特异性”的ASO将是理想的 许多严重遗传性神经疾病的治疗候选者。在这项研究计划中，我们将验证 一种新的等位基因特异性ASO设计方法。我们将针对多个靶点设计和测试等位基因特异性ASO 在FUS基因中，能够特异性敲低许多ALS患者的突变型FUS基因产物， FUS突变。我们将证明，ASO实现了高度的等位基因特异性，并能够 在基于细胞的FUS-ALS模型中拯救疾病特征。在研究计划的后续阶段， 这些候选ASO将被优化并最终开发为ALS伴FUS患者的治疗方法 突变。经验证的等位基因特异性阿索技术具有重要的医学和商业价值。 最终，拟议的研究将使具有FUS突变的ALS患者群体受益， 目前患有毁灭性的，无法治愈的和致命的疾病，以及患有其他严重疾病的患者， 具有毒性功能获得性或显性阴性疾病机制的遗传性疾病。

Project Summary: Amyotrophic Lateral Sclerosis (ALS), the most common motor neuron disorder, is a fatal, neurodegenerative disease with a median survival time of 2-3 years from symptom onset. There are currently only two approved therapies for ALS, RilutekR, a channel modulator, and more recently, RadicavaR, a neuroprotective antioxidant. Neither of these therapies can halt disease progression, and ALS remains a devastating, fatal disease and major area of unmet medical need. Recently, there has been substantial progress in identifying ALS disease genes. This progress has helped promote understanding of processes in motor neurons that are affected by the disease. Despite these advances, many identified ALS genes do not encode traditional “druggable” targets, and it has been challenging to translate these advances into new small molecule drugs for ALS. However, some of the ALS genes are good candidates for new, gene-based treatments, such as gene therapy or antisense oligonucleotide (ASO) therapy. One ALS gene for which there is strong rationale for a gene-based therapeutic approach is called FUS (Fused in Sarcoma). Mutations in FUS were first described in families afflicted with ALS in 2009. Since then, mutations in FUS were identified as one of the most common risk factors for juvenile onset ALS with particularly aggressive progression that typically results in death within one year of onset. There is strong evidence that mutations in FUS result in a toxic gene product. This evidence suggests that therapeutic approaches that can reduce the level of the mutant, toxic FUS product comprise a compelling basis for treating ALS patients with FUS mutations. This therapeutic mechanism could be achieved with an ASO designed to specifically reduce the FUS gene product made from the mutant copy of the FUS gene. ASOs are short, synthetic stretches of modified genetic material that can be designed to recognize and knockdown specific gene products. The recent approval of the ASO therapeutic, SpinrazaR, for spinal muscular atrophy has paved the way for ASO therapeutics for degenerative, neurological disorders. These ASO drugs are administered directly into the central nervous system by injection into the fluid surrounding the spinal cord. While therapeutic ASOs that target both gene copies are advancing in clinical trials for Huntington’s disease and SOD1-ALS, it has been more challenging to design ASOs that specifically target the mutant gene copies. Such “allele-specific” ASOs would be ideal therapeutic candidates for many severe genetic neurological diseases. In this research program, we will validate a new design method for allele-specific ASOs. We will design and test allele-specific ASOs for multiple targets in the FUS gene that would enable specific knockdown of mutant FUS gene product for many ALS patients with FUS mutations. We will demonstrate that the ASOs achieve a high degree of allele specificity and are able to rescue a disease signature in a cell-based model of FUS-ALS. In subsequent phases of the research program, these candidate ASOs will be optimized and ultimately developed as treatments for ALS patients with FUS mutations. The validated allele-specific ASO technology would have significant medical and commercial value. Ultimately, the proposed research stands to benefit the population of ALS patients with FUS mutations, who currently suffer from a devastating, incurable and lethal condition, as well as patients suffering from other severe genetic diseases with toxic gain-of-function or dominant-negative disease mechanisms.