Site-directed RNA editing: a new method to correct disease causing mutations

定点RNA编辑：纠正致病突变的新方法

• 批准号: 8900376
• 负责人: JOHN P ADELMAN
• 金额: $ 68.2万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8900376
• 关键词: Adenosine; Affect; Amino Acid Sequence; Amino Acid Substitution; Animal Model; Basic Science; Biological; Blood Coagulation Factor; Brain Diseases; Cell Count; Cell-Free System; Chloride Channels; Code; Codon Nucleotides; Communities; Cystic Fibrosis; Cytidine; Cytidine Deaminase; DNA; Deamination; Disease; Engineering; Enzymes; Gene Duplication; Gene Expression; Genes; Genetic; Glutamate Receptor; Goals; Guide RNA; Health; Hemophilia A; Hybrids; In Situ; Inherited; Laboratories; Messenger RNA; Methods; Methyl-CpG-Binding Protein 2; Modeling; Mus; Mutation; Nervous system structure; Neurobiology; Neurons; Nonsense Codon; Physiological; Process; Proteins; Public Health; Puerto Rico; RNA Editing; Records; Research Personnel; Resources; Rett Syndrome; Site; Small Interfering RNA; Staging; Suppressor Mutations; System; Therapeutic; Translating; Universities; Xenopus oocyte; Zebrafish; adenosine deaminase; biological systems; disease-causing mutation; functional restoration; gain of function mutation; gene therapy; genome editing; human disease; improved; in vivo; innovation; loss of function; meetings; nervous system disorder; neurotransmission; novel strategies; protein function; recombinational repair; skills; success; therapeutic target; tool

DESCRIPTION (provided by applicant): There is currently no way to correct disease-causing mutations in the nervous system without altering the physiological level of the endogenous mRNA. This is a serious challenge because haplo-insufficiency or two-fold over-expression is often sufficient to cause neurological disorders. An example is Rett Syndrome, caused by mutations in the Mecp2 gene. Mecp2 gene duplication, as well as loss-of-function, results in severe disease. We propose to meet the challenge by harnessing the natural ability of RNA editing enzymes to site-specifically fix mutations in endogenous mRNAs. As a target for gene therapy, mRNA offers advantages over DNA. Messenger RNA is cytoplasmic, a readily available substrate, and unlike DNA in which 'mistakes' will be maintained, mRNAs turnover, replenishing the therapeutic target. Our new approach, Site Directed RNA Editing (SDRE), offers enormous untapped potential for correcting mutations, particularly those affecting the nervous system, and for exploring fundamental biological questions. RNA editing, which occurs through adenosine or cytidine deamination, is a natural process. When it occurs within the coding sequence of an mRNA specific codons can be re-coded to produce an altered amino acid sequence. For example, excitatory neurotransmission absolutely depends on the editing of a single adenosine within AMPA-type glutamate receptor mRNAs. Recognizing the power of this activity, we engineered a hybrid modular adenosine deaminase. When used in combination with a small antisense guide RNA we can site-specifically target any chosen adenosine. A similar strategy will be employed to create a site-directed cytidine deaminase. Unlike established therapies that focus strictly on regulating gene expression, SDRE can also fine-tune protein function. Inherited mutations that underlie diseases due to amino acid substitutions or premature stop codons can be corrected, and second-site suppressor mutations that restore function can be selectively introduced. We will demonstrate the power of SDRE within the context of neurobiology, but importantly, it applies to any biological system.

描述（由申请人提供）：目前没有办法在不改变内源性mRNA的生理水平的情况下纠正神经系统中的致病突变。这是一个严重的挑战，因为单倍不足或两倍过表达通常足以引起神经系统疾病。一个例子是Rett综合征，由Mecp2基因突变引起。Mecp2基因复制以及功能丧失会导致严重的疾病。我们建议通过利用RNA编辑酶的天然能力来位点特异性地固定内源性mRNA中的突变来应对挑战。作为基因治疗的靶点，mRNA提供了优于DNA的优势。信使RNA是细胞质的，是一种容易获得的底物，并且不像DNA中的“错误”将被维持，mRNA周转，补充治疗靶点。我们的新方法，定点RNA编辑（SDRE），为纠正突变，特别是那些影响神经系统的突变，以及探索基本的生物学问题提供了巨大的未开发潜力。通过腺苷或胞苷脱氨基作用发生的RNA编辑是一个自然过程。当它出现在mRNA的编码序列内时，特定密码子可以被重新编码以产生改变的氨基酸序列。例如，兴奋性神经传递完全依赖于AMPA型谷氨酸受体mRNA中单个腺苷的编辑。认识到这种活性的力量，我们设计了一种混合模块化腺苷脱氨酶。当与小的反义引导RNA组合使用时，我们可以位点特异性地靶向任何选择的腺苷。将采用类似的策略来产生定点胞苷脱氨酶。与严格专注于调节基因表达的既定疗法不同，SDRE还可以微调蛋白质功能。由于氨基酸取代或过早终止密码子导致的疾病的遗传突变可以得到纠正，并且可以选择性地引入恢复功能的第二位点抑制突变。我们将在神经生物学的背景下展示SDRE的力量，但重要的是，它适用于任何生物系统。