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：
8738740
负责人：
JOHN P ADELMAN
金额：
$ 67.52万
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 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8738740
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 Genomics 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 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 human disease improved in vivo innovation loss of function meetings nervous system disorder neurotransmission novel strategies 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）：目前无法在不改变内源mRNA的生理水平的情况下纠正神经系统中引起疾病的突变。这是一个严重的挑战，因为单倍不平衡或两倍的过表达通常足以引起神经系统疾病。一个例子是由MECP2基因突变引起的RETT综合征。 MECP2基因复制以及功能丧失会导致严重疾病。我们建议通过利用RNA编辑酶在内源性mRNA中特异性固定突变的自然能力来应对挑战。作为基因疗法的靶标，mRNA比DNA具有优势。 Messenger RNA是细胞质的，它是一种容易获得的底物，与DNA保持“错误”的DNA不同，mRNA CREANTOBLED，补充了治疗靶标。我们的新方法是导演的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的巨大尚未开发的潜力。通过腺苷或胞苷脱氨酸发生的RNA编辑是自然过程。当它发生在mRNA特定密码子的编码序列中时，可以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在AMPA型谷氨酸受体mRNA中编辑单个腺苷。认识到这项活动的力量，我们设计了一种混合模块化腺苷脱氨酶。当与小的反义指南RNA结合使用时，我们可以特异性地针对任何选择的腺苷。将采用类似的策略来创建以网站为导向的胞苷脱氨酶。与已建立的疗法严格专注于调节基因表达，SDRE还可以微调蛋白质功能。可以纠正因氨基酸取代或过早停止密码子引起的疾病构成的遗传突变，并且可以选择性地引入恢复功能的第二位抑制突变。我们将在神经生物学的背景下证明SDRE的力量，但重要的是，它适用于任何生物系统。