Monoallelic repair of expanded huntingtin by trans-splicing

通过反式剪接对扩展的亨廷顿蛋白进行单等位基因修复

• 批准号: 8129437
• 负责人: Christian L. Lorson
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8129437
• 关键词: Ablation; Adolescent; Affect; African; Age of Onset; Alleles; Amino Acids; Animal Model; Antisense Oligonucleotides; Asians; Atrophic; Brain; CAG repeat; Cause of Death; Cell physiology; Cells; Cessation of life; Complex; Corpus striatum structure; Dentatorubral-Pallidoluysian Atrophies; Development; Diagnosis; Disease; Dominant Genes; Effectiveness; Embryo; Event; Exons; Fibroblasts; Frequencies; Future; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genetic screening method; Glutamine; Human; Huntington Disease; Impaired cognition; Incidence; Individual; Inherited; Knowledge; Lead; Left; Length; Mediating; Messenger RNA; Mind; Modality; Molecular; Motor Neurons; Movement Disorders; Mus; Mutate; Mutation; Neurodegenerative Disorders; Neurons; Onset of illness; Patients; Personality; Population; Production; Proteins; Public Health; RNA; RNA Interference; RNA Sequences; RNA Splicing; Regimen; Rodent; Screening procedure; Sequence Homology; Series; Signal Transduction; Specificity; Spinobulbar Muscular Atrophy; Spliceosomes; Stretching; Symptoms; System; Technology; Testing; Testis; Therapeutic; Time; Trans-Splicing; Transcriptional Regulation; Transfection; Translating; Trinucleotide Repeats; Type 1 Spinocerebellar Ataxia; Work; base; design; effective therapy; gain of function; gain of function mutation; gene therapy; human Huntingtin protein; induced pluripotent stem cell; interest; loss of function; mRNA Precursor; mutant; novel; novel strategies; polyglutamine; public health relevance; putamen; repaired; research study; restoration; scaffold

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder which is caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). It is characterized by progressive movement disorder, cognitive decline and psychiatric disturbances. Death ensues approximately 20 years after onset of the disease. No effective therapy currently exists for HD. HTT is a soluble protein of ~3,144 amino acids that has no sequence homology with other proteins. Increasing evidence indicates that HTT functions as a molecular scaffold that is able to organize a variety of signaling complexes. It is expressed ubiquitously in humans and rodents, with the highest levels found in CNS neurons and the testes. Because of the involvement of a single causal gene and simple genetic testing, HD and other diseases caused by CAG-repeat expansions offer unique therapeutic opportunities. The HD mutation is considered to be a "gain of function" mutation. Therefore, therapeutic approaches that are based on reducing mutant HTT expression such as RNA interference are currently considered to be promising strategies for HD treatment. It is likely that these agents will cause inactivation or impair normal function of both mutant and wild type HTT alleles. Therefore, monoallelic therapies targeting only the pathological allele are of particular interest. We propose to conduct proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is an efficient means to repair specifically the expanded, pathological allele, without functionally affecting the normal allele. This technology will restore not only the expression of the corrected mRNA from the dominant gene but also the production of the corresponding protein. Advantages of this technology include the possibility of utilizing small corrective RNA sequences that target exonic sequences within the mutated gene with high specificity as well as the natural regulation of gene expression. In specific aim 1 we will generate and functionally optimize trans-splicing constructs. The efficiency of these constructs will be tested using a binary cell transfection system, consisting of the trans-splicing module and an HTT minigene. In specific aim 2, selected optimized trans-splicing constructs will be further tested for the ability to repair endogenous HTT pre-mRNA. In addition, we will assess restoration of cellular functions as a result of successful HTT repair. Future work will be aimed at transferring the most efficient constructs into HD animal models. The knowledge gained in this project might eventually lead to a novel gene therapy for HD. PUBLIC HEALTH RELEVANCE: Huntington's Disease is a progressive and deadly neurodegenerative disease for which there is no cure. An inherited mutation causes the death of neurons in the brain. This project explores the possibility of repairing the mutation and to restore normal cellular functions.

描述（由申请人提供）：亨廷顿病（HD）是一种常染色体显性神经退行性疾病，由亨廷顿蛋白（HTT）氨基末端的聚谷氨酰胺扩张引起。其特点是进行性运动障碍、认知能力下降和精神障碍。发病后约 20 年死亡。目前尚无针对 HD 的有效疗法。 HTT 是一种约 3,144 个氨基酸的可溶性蛋白质，与其他蛋白质没有序列同源性。越来越多的证据表明，HTT 作为分子支架发挥作用，能够组织多种信号复合物。它在人类和啮齿动物中普遍表达，其中中枢神经系统神经元和睾丸中的表达水平最高。由于涉及单一致病基因和简单的基因检测，HD 和由 CAG 重复扩增引起的其他疾病提供了独特的治疗机会。 HD突变被认为是“功能获得”突变。因此，基于减少突变型 HTT 表达（例如 RNA 干扰）的治疗方法目前被认为是 HD 治疗的有前途的策略。这些药物可能会导致突变型和野生型 HTT 等位基因失活或损害其正常功能。因此，仅针对病理等位基因的单等位基因疗法特别令人感兴趣。 我们建议进行概念验证研究，以证明剪接体介导的反式剪接是特异性修复扩展的病理性等位基因的有效方法，而不会对正常等位基因产生功能性影响。这项技术不仅可以恢复显性基因校正后的 mRNA 的表达，还可以恢复相应蛋白质的产生。该技术的优点包括可以利用小校正RNA序列，以高特异性靶向突变基因内的外显子序列，以及基因表达的自然调节。在具体目标 1 中，我们将生成并在功能上优化反式剪接构建体。这些构建体的效率将使用二元细胞转染系统进行测试，该系统由转拼模块和 HTT 小基因组成。在具体目标 2 中，将进一步测试选定的优化反式剪接构建体修复内源 HTT 前 mRNA 的能力。此外，我们将评估 HTT 修复成功后细胞功能的恢复情况。未来的工作将旨在将最有效​​的结构转移到 HD 动物模型中。在这个项目中获得的知识最终可能会导致一种新的亨廷顿病基因疗法。 公共卫生相关性：亨廷顿氏舞蹈病是一种进行性且致命的神经退行性疾病，无法治愈。遗传性突变会导致大脑中神经元的死亡。该项目探索修复突变并恢复正常细胞功能的可能性。

项目成果

Replacement of huntingtin exon 1 by trans-splicing.

通过反式剪接替换亨廷顿蛋白外显子 1。

• 发表时间: 2012-12
• 作者: Rindt, Hansjörg;Yen, Pei;Thebeau, Christina N;Peterson, Troy S;Weisman, Gary A;Lorson, Christian L
• 通讯作者: Lorson, Christian L

Optimization of trans-Splicing for Huntington's Disease RNA Therapy.

亨廷顿病 RNA 治疗反式剪接的优化。

• 发表时间: 2017
• 作者: Rindt, Hansjörg;Tom, Colton M;Lorson, Christian L;Mattis, Virginia B
• 通讯作者: Mattis, Virginia B