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

基本信息

批准号：
9325591
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9325591
关键词：
Adenosine Affect Amino Acid Sequence Amino Acid Substitution Animal Model Basic Science Biological Blood Coagulation Factor Brain Diseases Cell Count Cells Chloride Channels Code Codon Nucleotides Communities Cystic Fibrosis Cytidine Cytidine Deaminase DNA Deamination Disease Engineering Enzymes Gene Duplication Gene Expression Gene Targeting Genes Genetic Glutamate Receptor Goals Guide RNA 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 Suppressor Mutations System Therapeutic Translating Universities Xenopus oocyte Zebrafish adenosine deaminase biological systems disease-causing mutation duplicate genes functional restoration gain of function mutation gene therapy genome editing human disease improved in vivo innovation loss of function nervous system disorder neurotransmission novel strategies overexpression protein function public health relevance recombinational repair skills success therapeutic target tool

项目摘要

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mRNA的生理水平的情况下纠正神经系统中引起疾病的突变。这是一个严峻的挑战，因为单倍体不足或两倍过度表达通常足以引起神经系统疾病。一个例子是雷特综合症，由 Mecp2 基因突变引起。 Mecp2 基因重复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定点修复内源 mRNA 中的突变来应对这一挑战。作为基因治疗的靶标，mRNA 比 DNA 具有优势。信使RNA是细胞质的，是一种容易获得的底物，与DNA不同的是，DNA中会保留“错误”，mRNA会更新，补充治疗靶点。我们的新方法，定点 RNA 编辑 (SDRE)，为纠正突变（尤其是影响神经系统的突变）和探索基本生物学问题提供了巨大的未开发潜力。 RNA 编辑是通过腺苷或胞苷脱氨基发生的，是一个自然过程。当它出现在 mRNA 的编码序列中时，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，兴奋性神经传递绝对取决于 AMPA 型谷氨酸受体 mRNA 内单个腺苷的编辑。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。当与小反义引导 RNA 结合使用时，我们可以位点特异性地靶向任何选定的腺苷。将采用类似的策略来创建定点胞苷脱氨酶。与严格关注基因表达的现有疗法不同，SDRE 还可以微调蛋白质功能。可以纠正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，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背景下展示 SDRE 的力量，但重要的是，它适用于任何生物系统。