AAV-based Gene Therapy Mediating RNA Editing as a Treatment for C9ORF72-ALS

肌萎缩侧索硬化症(ALS)是一种以上下运动神经元进行性丧失为特征的破坏性神经退行性疾病。随着欧洲老年人口的增加，肌萎缩侧索硬化症的发病率预计将大幅增加。迫切需要新的治疗方法。然而，开发有效的ALS治疗方法一直是困难的，因为ALS的发病机制早在患者被诊断之前就开始了，ALS的病程随着时间的推移而变化，而且人类遗传学表明ALS是疾病亚型的集合，这表明个性化药物可能是必要的。ALS最常见的遗传原因是C9ORF72基因第一内含子内的GGGGCC(G4C2)六核苷酸重复序列扩张(HRE)。此外，C9ORF72基因的HRE是导致额颞部痴呆的最常见原因。因此，专门针对HRE设计的治疗方法将使大量患者受益。尽管反义寡核苷酸(ASO)是C9ORF72-ALS研究最活跃的治疗策略，但ASOS只针对一条RNA链-通常是正义链，导致ALS病理的反义RNA转录本没有改变。此外，ASOS需要每四周鞘内注射一次。这是非常有侵入性的，对于ALS患者来说，这可能是技术上的挑战。此外，ASOS还与毒性有关，包括炎症效应、肾毒性和血小板减少症。使用腺相关病毒(AAV)的基因治疗在MN疾病的临床前和临床测试中显示出巨大的前景。250天后，一次向中枢神经系统注射AAV对小鼠显示出治疗效果。在这里，我们建议开发一种AAV治疗载体，在一次注射后，在很长一段时间内同时删除包含正、反义HRE的RNA转录本。我们将使用从诱导的多能干细胞分化而来的人MN来开发这种载体。一个小鼠模型将被用来评估体内的疗效，我们将专门测试在症状出现之前给药的有效性，我们认为这是最能防止MN退化的时候。DPR蛋白PolyGP已经在患者脑脊液中显示出作为C9ORF72-ALS生物标记物的重要前景，在我们的实验中将通过ELISA监测靶点参与，并将与疗效相关。由于AAV现在被临床批准用于一种MN疾病，并且多家公司在GMP条件下生产AAV载体，我们的新AAV载体可以迅速转化为临床测试。

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons (MNs). As the number of older Europeans increases, the incidence of ALS cases are expected to significantly increase. New therapeutics are urgently needed. However, developing effective treatments for ALS has been difficult because pathogenesis begins long before patients are diagnosed, ALS disease course changes over time, and human genetics suggests that ALS is a collection of disease subtypes, suggesting that personalized medicine might be necessary. The most common known genetic cause of ALS is a GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) within the first intron of the gene C9ORF72. In addition, HRE in C9ORF72 is the most common cause of frontotemporal dementia. Therefore, therapeutics designed specifically against HRE would benefit a large number of patients. Although antisense oligonucleotides (ASOs) are the most actively investigated therapeutic strategy for C9ORF72-ALS, ASOs target only one RNA strand – typically the sense strand, leaving the antisense RNA transcript, which induces ALS pathology, unaltered. Also, ASOs need to be administered by intrathecal injection every four weeks. This is very invasive, and in ALS patients it can be technically challenging. Moreover, ASOs have been associated with toxicity, including inflammatory effects, nephrotoxicity, and thrombocytopenia. Gene therapeutics using adeno-associated virus (AAV) have shown tremendous promise in pre-clinical and clinical testing for MN diseases. A single administration of AAV to the CNS showed therapeutic effects over 250 days later in mice. Here, we propose developing an AAV therapeutic vector that simultaneously deletes both the sense and antisense HRE-containing RNA transcripts over a long period of time and after only a single injection. We will develop this vector using human MNs differentiated from induced pluripotent stem cells. A mouse model will be used to evaluate efficacy in vivo, and we will specifically test the efficacy of administering before symptom onset, which we believe is when MN degeneration can be best prevented. The DPR protein polyGP, which has already shown significant promise as a C9ORF72-ALS biomarker in patient cerebrospinal fluid, will be monitored via ELISAs for target engagement in our experiments and will be correlated with efficacy. Since AAV is now clinically approved for one MN disorder and multiple companies produce AAV vectors under GMP conditions, our new AAV vectors could be rapidly translated into clinical testing.