Therapeutic strategies for microsatellite expansion diseases using RNA targeting

项目摘要 微卫星又称简单重复序列或短串联重复序列（STR），是一种长度为2-10 bp的串联重复序列 在整个人类基因组中出现的重复序列。这些重复序列的生殖系扩增超出了 临界阈值与50多种神经系统、神经退行性和神经肌肉相关 疾病，包括亨廷顿病，C9 orf 72-连锁的肌萎缩性侧索硬化/额颞叶痴呆， 几种类型的脊髓小脑共济失调，以及1型和2型肌强直性营养不良（DM 1/2）。虽然 与这些疾病相关的分子特征的完整范围在这些病症之间变化（并且通常 细胞和小鼠模型证明RNA介导的毒性是一个主要因素。因此，在本发明中， 预期突变RNA水平的抑制可解决所有相关的病理。虽然有许多策略 存在通过靶向破坏RNA来减弱基因表达，所有这些都具有与其表达模式相关的局限性。 施用和组织靶向（例如反义寡核苷酸，siRNA），或具有引起毒性的风险 通过对治疗剂的适应性免疫应答或预先存在的免疫（基于系统的 RNA靶向Cas蛋白）。在这里，我们开发了新的非免疫原性RNA靶向系统， 通过支持安全和持久表达的重组腺相关病毒载体（rAAV）递送。 我们的平台是基于人类剪接体RNA，在初步数据中，我们显示可以通过工程改造， 靶向并降解含STR转录物。作为该系统治疗潜力的原理证明， 我们专注于DM 1，最常见的成人型肌营养不良症。我们 已经开发出一种新的基于人类干细胞的类器官模型，首次提供了对 与DM 1相关的严重神经认知缺陷的分子基础。我们将使用该模型来测试 我们的RNA靶向系统的安全性和有效性与我们生成的新型小鼠模型相结合， 我们希望它能概括DM 1的多组织病理学。如果成功，我们的工作将提供一个灵活的 用于治疗STR相关疾病的基于治疗性RNA靶向的平台。

PROJECT SUMMARY Microsatellites, also known as simple sequence repeats or short tandem repeats (STRs), are 2-10 bp tandem sequence repeats that occur throughout the human genome. Germ-line expansions of these repeats beyond a critical threshold are associated with more than 50 neurological, neurodegenerative and neuromuscular disorders, including Huntington disease, C9orf72-linked amyotrophic lateral sclerosis/ frontotemporal dementia, several types of spinocerebellar ataxias, as well as myotonic dystrophy types 1 and 2 (DM1/2). Although the complete range of molecular features associated with these diseases varies among these conditions (and often are unknown), cellular and mouse models demonstrate that RNA-mediated toxicity is a major factor. Thus, suppression of mutant RNA levels is expected to address all associated pathologies. While numerous strategies exist to attenuate gene expression via targeted destruction of RNA, all have limitations relating to their mode of administration and tissue targeting (e.g. antisense oligonucleotides, siRNAs), or carry the risk of toxicity caused by adaptive immune responses or pre-existing immunity to the therapeutic agent (systems based delivery of RNA-targeting Cas proteins). Here we develop novel non-immunogenic RNA targeting system compatible with delivery by recombinant adeno-associated viral vectors (rAAVs) that support safe and long-lasting expression. Our platform is based on human spliceosomal RNAs, which in preliminary data we show can be engineered to target and degrade STR-containing transcripts. As a proof-of principle for the therapeutic potential of this system, we focus on DM1, the most common form of adult-onset most common adult-onset muscular dystrophy. We have developed a novel human stem cell based organoid model that, for the first time, provides insight into the molecular basis of the severe neurocognitive deficits associated with DM1. We will use this model to test the safety and efficacy of our RNA-targeting systems in conjunction with a novel mouse model that we generate and that we hope will recapitulate the multi-tissue pathology of DM1. If successful, our work will provide a flexible therapeutic RNA-targeting based platform for treatment of STR-associated diseases.