Ataxin-7 oligonucleotide knock-down to treat SCA7 retinal and cerebellar disease

描述（由申请人提供）：脊髓小脑性共济失调7型（SCA7）是一种常染色体显性遗传病，导致锥体杆营养不良形式的视网膜变性。由SCA7患者遗传的突变是在ataxin-7基因中CAG/ polyQ重复扩增。SCA7突变导致产生有毒的聚q扩增ataxin-7蛋白。由于有毒基因产物驱动SCA7的所有后续疾病病理，最有吸引力的治疗模式是终止突变基因产物的表达。在这里，我们建议继续进行一项转化研究计划，旨在为CAG重复扩增突变引起的CAG视网膜疾病和潜在的相关SCAs提供治疗药物。我们正在使用一种已经成功应用于减少一种有毒蛋白表达的策略：基于寡核苷酸的敲除，来追求ataxin-7“基因沉默”。为了实现这个转化研究项目的目标，我们建立了一个学术-工业合作伙伴关系。通过与ISIS制药公司（一家专门从事反义寡核苷酸（ASO）生产和单链siRNA （ss-siRNA）制造的公司）的密切合作，我们发现了显著降低ataxin-7表达的线索。基于这些体外结果，我们选择了铅进行体内验证，并在野生型小鼠中进行了玻璃体内注射试验。与注射稀释剂的眼睛相比，这些试验性的玻璃体内注射在注射抗ataxin-7 ASO的眼睛中产生了强大的（> %）ataxin-7表达下调。进一步的初步研究在SCA7敲入小鼠中发现，治疗后的眼睛视网膜感光细胞中ataxin-7蛋白聚集体显著减少。我们还在患者的成纤维细胞系中发现了CAG-repeat靶向寡核苷酸，用于等位基因选择性靶向polyq扩增的ataxin-7。因此，我们提出了两个目标：1)根据敲除效果和眼毒性参数，我们将选择一种ataxin-7 ASO，在SCA7敲入小鼠的右眼玻璃体内注射ataxin-7 ASO，并在左眼控制ASO/稀释剂，以确定ASO是否可以预防或改善SCA7视网膜变性。读数将包括ataxin-7聚集，视网膜组织学和视觉功能。一旦我们确定了有效的ataxin-7 ASO，我们将重复试验以确认疗效，比较ASO治疗组和对照治疗组的基因表达变化，确定在症状性SCA7小鼠中给予ASO是否可以阻止或逆转疾病进展，并评估ataxin-7 ASO作为SCA7小脑变性治疗的效用。2)选择CAG-repeat靶向寡核苷酸后，我们将测试寡核苷酸是否可以预防或改善SCA7敲入小鼠的视网膜变性，然后采用与ataxin-7 ASO相似的实验策略，将寡核苷酸推进验证性研究、转录组表达分析、症状干预试验和评估作为SCA7神经变性的治疗方法。在该项目结束时，我们将很好地与我们的工业合作伙伴一起进行ind支持研究，作为人类SCA7患者临床试验的前奏。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder that results in a cone-rod dystrophy form of retinal degeneration. The mutation inherited by SCA7 patients is a CAG/polyglutamine (polyQ) repeat expansion in the ataxin-7 gene. The SCA7 mutation results in production of a toxic polyQ-expanded ataxin-7 protein. Since the toxic gene product drives all subsequent disease pathology in SCA7, the most attractive therapeutic paradigm is to terminate expression of the mutant gene product. Here we propose continuation of a translational research program intended to yield therapeutic agents for SCA7 retinal disease, and potentially for related SCAs caused by CAG repeat expansion mutations. We are pursuing ataxin-7 "gene silencing" using a strategy that has already been successfully applied to reduce the expression of a toxic protein: oligonucleotide-based knock-down. To achieve the goals of this translational research program, we have created an academic-industrial partnership. In close collaboration with ISIS Pharmaceuticals, a company that specializes in anti-sense oligonucleotide (ASO) production and single-stranded siRNA (ss-siRNA) creation, we identified leads that markedly knock-down ataxin-7 expression. Based upon these in vitro results, we selected leads for in vivo validation, and performed pilot intra-vitreal injections in wild-type mice. These pilot intra-vitreal injections yielded robust (> 0%) knock-down of ataxin-7 expression for eyes injected with anti-ataxin-7 ASO, in comparison to eyes injected with diluent. Further pilot studies in SCA7 knock-in mice resulted in significant reductions in ataxin-7 protein aggregates in retinal photoreceptor cells in treated eyes. We also identified CAG-repeat targeting oligonucleotides for allele-selective targeting of polyQ-expanded ataxin-7 in fibroblast cell lines from patients. We therefore propose two aims: 1) Based upon efficacy of knock-down and ocular toxicity parameters, we will select one ataxin-7 ASO and perform intra-vitreal injections of ataxin-7 ASO in the right eye and control ASO/diluent in the let eye of SCA7 knock-in mice to determine if ASO delivery can prevent or ameliorate SCA7 retinal degeneration. Read-outs will include ataxin-7 aggregation, retinal histology, and visual function. Once we identify an efficacious ataxin-7 ASO, we will repeat the trial to confirm efficacy, compare gene expression alterations in ASO-treated and control-treated eyes, determine if ASO delivery in symptomatic SCA7 mice can stop or reverse disease progression, and evaluate the utility of the ataxin-7 ASO as a therapy for SCA7 cerebellar degeneration. 2) After we select a CAG-repeat targeting oligonucleotide, we will test if the oligonucleotide can prevent or ameliorate retinal degeneration in SCA7 knock-in mice, and then advance the oligonucleotide for confirmatory studies, transcriptome expression analysis, symptomatic intervention trials, and evaluation as a therapy for SCA7 neurodegeneration, applying similar experimental strategies as with the ataxin-7 ASO. At the conclusion of this project, we will be well positioned to proceed with IND-enabling studies with our industrial collaborator, as a prelude to a clinical trial in humn SCA7 patients.