Development of allele-specific protein-based therapeutic targeting the pathogenic RNA associated with Spinocerebellar ataxia type 3

摘要 多聚谷氨酰胺(PolyQ)病是一种较为常见的遗传性神经退行性疾病 疾病。它们是由扩展的CAG重复序列引起的，该重复序列编码了 疾病蛋白。在九种已知的多项智力障碍中，脊髓小脑性共济失调3型(SCA3)，也被称为 马查多-约瑟夫病(MJD)是美国第二常见的疾病，也是世界上最常见的疾病。 SCA3也是最常见的遗传性共济失调，变性主要影响小脑， 脑干、黑质、丘脑和脊髓。在SCA3中，ATXN3编码内的CAG重复 通常含有12到44个CAG的序列被扩展到60到87个三联体。目前，只有姑息疗法 管理症状的治疗方法是可用的。直接针对扩展的SCA3的治疗策略 反义寡核苷酸(ASO)等mRNAs已经产生了令人振奋的结果。最近两家公司的停工 亨廷顿病的多Q障碍临床试验中基于ASO的疗法表明，需要 用于制定和评估一套不同的治疗模式。 在这项提案中，我们建议使用我们的人工站点特异性RNA内切酶(Asres)技术来 设计CAG重复序列特异性RNA内切酶，以破坏已扩增的致病SCA3RNA。Asres包含 从PUF蛋白中分离的RNA结合域，它由一系列~36个氨基酸模块组成，这些模块 识别一种特定的核糖核酸。在概念验证研究中，我们设计了Asres帽子似乎 优先靶向扩增的ATXN3 RNA。在此FastTrack提案中，我们将寻求进一步提高 SCA3的治疗选择。在第一阶段，我们寻求评估工程资产的可行性，以期 位于编码序列内的两个SNP，它们频繁地共分离扩展的等位基因以携带 基于ASO的疗法对这些SNPs显示出令人振奋的疗效结果的观察优势。 这些研究将使Enzerna能够建立一个基因治疗组合，提供精确的可能性 适合患者携带的特定SCA3疾病等位基因的药物方法。在第二阶段，在 将进行体外和体内研究，以评估SCA3相关表型异常的抢救 AAV介导的候选AS治疗药物的交付。 从长远来看，与基因传递载体相结合，Asres为选择 致病ATXN3转录本的降解。通过瞄准SCA3(MJD)的基本基础，Asres提供了 对这种无法治愈的人类疾病进行预防性和/或根治性治疗的可能性。 。

ABSTRACT Polyglutamine (polyQ) diseases represent one of the more common classes of inherited neurodegenerative diseases. They are caused by expanded CAG repeats that encode abnormally long glutamine stretches in the disease proteins. Of the nine known polyQ disorders, Spinocerebellar Ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is the second most common in the US and the most common in the world. SCA3 is also the most common dominantly inherited ataxia with degeneration primarily affecting the cerebellum, brainstem, substantia nigra, thalamus and spinal cord. In SCA3, a CAG repeat within the ATXN3 coding sequences which normally harbors 12 to 44 CAGs is expanded to 60 to 87 triplets. Currently, only palliative therapeutics to manage symptoms are available. Therapeutic strategies directly targeting expanded SCA3 mRNAs, such as antisense oligonucleotides (ASO), have produced promising results. The recent halt of two ASO-based therapeutics in clinical trials for the polyQ disorder, Huntington’s Disease demonstrates, the need for the development and assessment of a diverse set of treatment modalities. In this proposal, we propose to use our Artificial SiteSpecific RNA Endonucleases (ASREs) technology to design CAG repeat specific RNA endonuclease to destroy expanded pathogenic SCA3 RNAs. ASREs contain RNA binding domains isolated from PUF proteins, which consist of a series of ~36 amino acid modules that recognize one specific ribonucleotide. In proof-of-concept studies, we have designed ASREs hat appear to preferentially target the expanded ATXN3 RNA. In this FastTrack proposal, we will seek to further increase therapeutic options for SCA3. In Phase I, we seek to assess the feasibility of engineering ASREs that can target two SNPs, located within the coding sequences, that frequently co-segregate the expanded allele to take advantage of the observations that ASO based therapeutics to these SNPs show promising efficacy results. These studies will enable Enzerna to build a portfolio of gene therapeutics that provide the possibility of precision medicine approaches appropriate for the specific SCA3 disease allele carried by the patient. In Phase, II, in vitro and in vivo studies will be conducted to assess rescue of SCA3-associated phenotypic anomalies after AAV-mediated delivery of the candidate ASRE therapeutics. In the long term, combined with gene delivery vectors, ASREs provide a new strategy for selective degradation of pathogenic ATXN3 transcripts. By targeting the underlying basis of SCA3 (MJD), ASREs provide the possibility of a preventative and/or curative therapy for this incurable class of human diseases. .