Protein-based Therapeutics to treat nucleotide expansion disorders associated with aberrant gene expression

核苷酸扩张性疾病由一组遗传性疾病组成，分为两类 根据重复序列(例如，CCG、CCCCGG、GAA、CTG和CAG)是否位于 基因组的编码或非编码区。没有针对核苷酸扩张性疾病的根治疗法； 只有提供姑息措施才能控制临床症状。超过25种核苷酸紊乱， 主要与神经退行性疾病有关的疾病已被发现，包括亨廷顿氏病， 强直性肌营养不良和弗里德里希共济失调。与扩增的GAA重复相关的Friedreich‘s共济失调(FA) 在Frataxin基因的第一个内含子中排列(最多1700个重复；正常等位基因有10-66个重复)，是唯一的 核苷酸扩张性疾病是一种隐性突变。扩大的GAA重复导致沉默 Frataxin(FXN)的表达，推测是由于RNA：DNA三联体的形成导致转录停止。 FA影响1：50,000人，使其成为遗传性共济失调最常见的形式。FA已关联 线粒体铁处理受损，使细胞对ROS介导的生物能量高度敏感 功能障碍。Friedreich‘s共济失调的临床表现跨器官系统，包括肌肉 虚弱、运动障碍、神经发育和功能不良、糖尿病和心脏病 并发症。目前还没有FDA批准的针对FA的疾病修饰药物。直接治疗策略 靶向FXN mRNA中的扩展重复序列，如反义寡核苷酸(ASO)，已经产生了很有希望的结果 结果。然而，ASO交付的困难和终生服用ASO治疗的需要仍然存在 ASO治疗的限制因素。我们寻求开发一种基于蛋白质的FA治疗方法 设计基于RNA结合蛋白PUF结构域家族的RNA结合蛋白，识别 GAA在突变的FXN mRNA中重复[PUF(GAA)]，并评估PUF(GAA)逆转 转录沉默的FXN表达。通过将FXN表达恢复为 FXN正常表达水平的35-50%和与FA相关的两种线粒体缺陷的修复： 在半乳糖中繁殖时对代谢胁迫的抗性；以及对过氧化氢诱导的抑制的抗性 线粒体呼吸作用。 一旦可行性被证明，第二阶段将专注于研究级腺病毒的开发 组成性表达核靶向PUF(GAA)的相关载体(AAV)发展基因传递 用于FA在动物模型中的初步疗效和安全性研究，然后进行生产 用于IND的临床级AAV使这种创新的治疗性基因疗法能够进行安全性和有效性研究 FA.从长远来看，结合基因传递载体，我们创新的基因治疗方法可能会 为干扰基因表达的核苷酸扩张性疾病提供了一条新的靶向治疗途径。

Nucleotide expansion disorders are comprised of a group of genetic diseases that are classified in two groups depending on whether the repeats (e.g., CCG, CCCCGG, GAA, CTG, and CAG) are located within a coding or non-coding region of the genome. There are no curative therapies for nucleotide expansion disorders; it is only possible to provide palliative measures to manage the clinical symptoms. Over 25 nucleotide disorders, mainly associated with neurodegenerative diseases have been identified, including Huntington’s disease, myotonic dystrophy and Friedreich’s ataxia. Friedreich’s Ataxia (FA), associated with an expanded GAA repeat array (up to 1700 repeats; normal alleles have 10-66 repeats) in the first intron of the frataxin gene, is the only nucleotide expansion disorder that is a recessive mutation. The expanded GAA repeat leads to silencing of frataxin (FXN) expression, presumably due to the formation of a RNA:DNA triplex that halts transcription. FA affects 1:50,000 individuals, making it the most common form of hereditary ataxia. FA is associated with impaired mitochondrial iron handling and makes cells highly susceptible to ROS-mediated bioenergetic dysfunction. Clinical manifestations of Friedreich’s Ataxia occur across organ systems, and include muscle weakness, movement disorders, poor neurological development and function, diabetes, and cardiac complications. There are no FDA approved disease modifying drugs for FA. Therapeutic strategies directly targeting expanded repeats in FXN mRNA, such as antisense oligonucleotides (ASO), have produced promising results. However, difficulties in ASO delivery and need for lifelong administration of the ASO therapeutic remain limiting factors for ASO-based therapies. We seek to develop a protein-based therapeutic approach for FA by designing RNA binding proteins, based on the PUF domain family of RNA binding protein, that recognize GAA repeats [PUF(GAA)] in mutated FXN mRNA and assess the ability of PUF(GAA) to reverse the transcriptional silencing of FXN expression. Sucess will be indicated by restoration of FXN expression to 35-50% of normal levels of FXN expression and rescue of two mitochondrial defects associated with FA: resistance to metabolic stress when propagated in galactose; and resistance to H2O2-induced inhibition of mitochondrial respiration. Once feasibility is demonstrated, Phase II will focus on the development of research grade adenoviral associated vectors (AAV) that constitutively express nuclear targeted PUF(GAA) to develop gene delivery protocols and for initial efficacy and safety studies in animal models of FA before progressing to production of clinical grade AAV for IND enabling safety and efficacy studies of this innovative curative gene therapeutic for FA. In the long term, combined with gene delivery vectors, our innovative gene therapeutic approach may provide a new route for targeted therapy for nucleotide expansion disorders that disrupt gene expression.