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

基于蛋白质的疗法，用于治疗与异常基因表达相关的核苷酸扩增疾病

• 批准号: 10086917
• 负责人: JOSEPH C. RUIZ
• 金额: $ 23.1万
• 依托单位: ENZERNA BIOSCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10086917
• 关键词: Affect; Alleles; Animal Model; Antisense Oligonucleotides; Awareness; Binding; Bioenergetics; Biological Assay; Cardiac; Cells; Clinical; Clinical Management; Code; Collaborations; Data; Defect; Development; Diabetes Mellitus; Disease; Engineering; Epigenetic Process; FDA approved; Family; Fibroblasts; Friedreich Ataxia; Functional disorder; Galactose; Gene Delivery; Gene Expression; Gene Silencing; Genes; Genetic Diseases; Genetic Transcription; Genome; Huntington Disease; Hydrogen Peroxide; Impairment; Individual; Inherited Spinocerebellar Degenerations; Institutes; Introns; Iron; Lead; Legal patent; Measures; Mediating; Messenger RNA; Metabolic stress; Metabolism; Mitochondria; Movement Disorders; Muscle Weakness; Mutate; Mutation; Myotonic Dystrophy; Neurodegenerative Disorders; Neurologic; Nuclear; Nucleotides; Oxygen Consumption; Pathogenicity; Patients; Pharmaceutical Preparations; Phase; Phenotype; Production; Proteins; Protocols documentation; RNA; RNA Sequences; RNA-Binding Proteins; Resistance; Respiration; Reverse Transcription; Route; Site; Stress; Stress Tests; Symptoms; Technology; Therapeutic; Trinucleotide Repeats; Triplet Multiple Birth; Untranslated RNA; Virginia; Visual; base; body system; cellular transduction; curative treatments; design; efficacy study; expression vector; frataxin; gallium arsenide; innovation; mitochondrial dysfunction; novel therapeutics; palliative; phase 2 study; protein expression; research and development; respiratory; restoration; safety study; success; symptom management; targeted treatment; therapeutic gene; transcriptome sequencing; triplex DNA; vector

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.

核苷酸扩增障碍由一组遗传性疾病组成，分为两类 组取决于重复序列（例如 CCG、CCCCGG、GAA、CTG 和 CAG）是否位于 基因组的编码区或非编码区。核苷酸扩增障碍没有治愈方法； 只能提供姑息措施来控制临床症状。超过 25 种核苷酸紊乱， 主要与神经退行性疾病相关，包括亨廷顿病， 强直性肌营养不良和弗里德赖希共济失调。弗里德赖希共济失调 (FA)，与扩大的 GAA 重复相关 frataxin 基因第一个内含子中的阵列（最多 1700 个重复；正常等位基因有 10-66 个重复），是唯一 核苷酸扩增障碍是一种隐性突变。扩展的 GAA 重复序列导致沉默 frataxin (FXN) 表达，可能是由于 RNA:DNA 三链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关联 线粒体铁处理受损，使细胞对 ROS 介导的生物能高度敏感 功能障碍。弗里德赖希共济失调的临床表现发生在各个器官系统中，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。目前尚无 FDA 批准的 FA 疾病缓解药物。直接治疗策略 靶向 FXN mRNA 中扩展的重复序列，例如反义寡核苷酸 (ASO)，已经产生了有希望的 结果。然而，ASO 递送的困难以及 ASO 治疗剂终生给药的需求仍然存在 基于 ASO 的治疗的限制因素。我们寻求开发一种基于蛋白质的 FA 治疗方法 基于RNA结合蛋白的PUF结构域家族设计RNA结合蛋白，其识别 GAA 在突变的 FXN mRNA 中重复 [PUF(GAA)] 并评估 PUF(GAA) 逆转突变的能力 FXN 表达的转录沉默。 FXN 表达恢复至 FXN 表达水平达到正常水平的 35-50%，并挽救与 FA 相关的两种线粒体缺陷： 在半乳糖中繁殖时对代谢应激的抵抗力；和对 H2O2 诱导的抑制的抵抗力 线粒体呼吸。 一旦可行性得到证实，第二阶段将重点开发研究级腺病毒 组成型表达核靶向 PUF (GAA) 以开发基因递送的相关载体 (AAV) 方案以及在 FA 动物模型中进行初步功效和安全性研究，然后再进行生产 用于 IND 的临床级 AAV 能够对这种创新的治疗性基因疗法进行安全性和有效性研究 FA。从长远来看，与基因递送载体相结合，我们的创新基因治疗方法可能会 为破坏基因表达的核苷酸扩增疾病的靶向治疗提供了新途径。