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

描述(申请人提供)：目前没有办法在不改变内源性信使核糖核酸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，因为单倍体功能不全或双重过度表达通常足以导致神经功能障碍。一个例子是雷特综合症，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编辑酶的自然能力来定点固定内源mRNAs中的突变来应对这一挑战。作为基因治疗的靶点，信使核糖核酸具有比DNA更好的优势。信使RNA是细胞质，是一种容易获得的底物，与DNA不同，信使RNA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和探索基本的生物学问题提供了巨大的尚未开发的潜力。通过腺苷或胞苷脱氨基发生的RNA编辑是一个自然的过程。当它出现在信使核糖核酸的编码序列中时，特定的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mRNAs中单个腺苷的编辑。认识到这种活动的力量，我们设计了一种混合模块化腺苷脱氨酶。当与小的反义引导RNA结合使用时，我们可以定点靶向任何选定的腺苷。类似的策略将被用来创造一种定点定向的胞苷脱氨酶。与严格专注于调控基因表达的现有疗法不同，SDRE还可以微调蛋白质功能。由于氨基酸替换或过早停止密码子导致的疾病背后的遗传突变可以得到纠正，可以选择性地引入恢复功能的第二位点抑制基因突变。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。

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.