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疾病组成，其被分类为两种遗传疾病： 组取决于是否重复（例如，CCG、CCCCGG、GAA、CTG和CAG）位于 基因组的编码区或非编码区。对于核苷酸扩增障碍没有治愈性疗法; 仅可能提供缓和措施来控制临床症状。超过25种核苷酸紊乱， 主要与神经变性疾病相关，包括亨廷顿氏病， 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GAA重复扩大相关 在frataxin基因的第一个内含子中的一个阵列（高达1700个重复;正常等位基因具有10-66个重复），是唯一的 一种隐性突变的核苷酸扩增障碍。扩增的GAA重复序列导致沉默， 这可能是由于RNA：DNA三链体的形成而导致转录停止。 FA影响1：50，000的个体，使其成为遗传性共济失调的最常见形式。FA相关 受损的线粒体铁处理，使细胞对ROS介导的生物能 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 并发症没有FDA批准的用于FA的疾病修饰药物。治疗策略直接 靶向FXN mRNA中的扩增重复序列，如反义寡核苷酸（阿索）， 结果然而，阿索递送的困难和需要终身施用阿索治疗剂仍然存在 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， GAA重复序列[PUF（GAA）]在突变的FXN mRNA中的表达，并评估PUF（GAA）逆转突变的FXN mRNA的能力。 FXN表达的转录沉默。FXN表达的恢复将指示成功， 35-50%的FXN表达正常水平，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抗性;以及对H2 O2诱导的 线粒体呼吸 一旦可行性得到证明，第二阶段将集中于研究级腺病毒的开发， 组成型表达核靶向PUF（GAA）的相关载体（AAV）， 研究方案，并在FA动物模型中进行初步有效性和安全性研究，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治疗药物的安全性和有效性研究成为可能， 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 为靶向治疗破坏基因表达的核苷酸扩增疾病提供了新途径。