Readthrough of disease-causing nonsense mutations by targeted selenocysteine recoding

通过靶向硒代半胱氨酸重新编码通读引起疾病的无义突变

• 批准号: 10575981
• 负责人: Oded Regev
• 金额: $ 20.11万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575981
• 关键词: 3' Untranslated Regions; Address; Adenosine; Affect; Amino Acids; Binding; Biological Assay; Catalytic RNA; Cell Line; Cell Separation; Cell model; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Development; Disease; Drug Delivery Systems; Duchenne muscular dystrophy; Elements; Exhibits; Fluorescence; Gene Delivery; Genes; Genetic Diseases; Goals; Hereditary Disease; Human; Hybrids; Inosine; Lead; Length; Luciferases; Malignant Neoplasms; Messenger RNA; Mutate; Mutation; National Institute of Biomedical Imaging and Bioengineering; Nonsense Mutation; Oligonucleotides; Patients; Persons; Production; Proteins; Quality of life; RNA; RNA Editing; Reporter; Screening Result; Selenocysteine; Specificity; Structure; System; Technology; Terminator Codon; Testing; Therapeutic; Tissues; Toxic effect; Transcript; Translations; Uridine; Virus; base; combinatorial; cost; design; disease-causing mutation; exon skipping; gene therapy; high reward; high risk; high throughput screening; interest; novel; novel strategies; novel therapeutic intervention; premature; reconstitution; recruit; small molecule; success; usability; yeast two hybrid system

TITLE: Readthrough of disease-causing nonsense mutations by targeted selenocysteine recoding PROJECT SUMMARY Nonsense (stop) mutations comprise about 10% of disease-causing mutations, and are common in Duchenne muscular dystrophy, cystic fibrosis, and cancer. These mutations cause early termination of translation and lead to non-functional proteins. Therapies that enable readthrough of these premature termination codons have been sought for many years with limited success. Existing approaches are limited by lack of specificity, low efficiency, or the need to deliver small genes. The amino acid selenocysteine (Sec), sometimes known as the 21st amino acid, is incorporated into human proteins via recoding of opal (UGA) stop codons. This recoding mechanism is activated by the presence of a Sec incorporation sequence element (SECIS) in the 3’ untranslated region (3’UTR). This element was shown to be sufficient to stimulate selenocysteine incorporation and is active even when located far from the UGA codon. Here we will develop a novel approach for inducing readthrough of opal nonsense mutations through Sec recoding. This will be achieved through the use of short hybridizing oligonucleotides that bring the targeted mRNA in proximity to a SECIS element, inducing readthrough in a gene-specific manner, and restoring a functional protein. In Aim 1, we will screen oligonucleotides that hybridize to both the target mRNA and an endogenous SECIS-containing transcript. In Aim 2, we will develop a high-throughput cell sorting-based assay, allowing the identification of optimal oligonucleotides among the combinatorially large number of possible designs. We will screen multiple possible designs, including oligonucleotides containing a hybridizing part and a (possibly abbreviated) SECIS element. We will demonstrate our approach using two disease-associated genes (DMD and CFTR) and validate the identified oligonucleotides in disease cell models. The proposed approach has several important advantages over currently available therapeutic approaches to nonsense mutations. First, the oligonucleotides are specific to the targeted gene, reducing concerns of off-target effects. Second, the same oligonucleotide can potentially be used for any nonsense mutation in the same gene, reducing development cost and addressing patients with very rare mutations. Finally, safe and efficient delivery of short oligonucleotides to several tissues has already been demonstrated. Together, the specificity, broad usability, and use of proven delivery technologies, make our approach particularly attractive for therapeutic purposes. Aligned with NIBIB’s interests, this project will develop a platform technology that is applicable to a broad spectrum of disorders and diseases. If successful, the project will have a tremendous impact on the quality of life of people suffering from genetic diseases caused by nonsense mutations and from cancer.

标题：通过靶向硒代半胱氨酸重新编码的致病无义突变通读 项目摘要 无义（终止）突变约占致病突变的10%，在杜氏症中很常见。 肌肉萎缩症、囊性纤维化和癌症。这些突变导致翻译提前终止， 非功能性蛋白质。能够通读这些提前终止密码子的疗法已经被广泛应用。 多年来一直在努力，但收效甚微。现有的方法受到缺乏特异性、效率低、 或者是传递小基因的需要。 硒代半胱氨酸（Sec），有时被称为第21位氨基酸，被掺入人体内。 蛋白质通过蛋白质蛋白石（UGA）终止密码子的重新编码。这种重新编码机制是由一种 在3'非翻译区（3'UTR）中的Sec掺入序列元件（SECIS）。该元素被证明是 足以刺激硒代半胱氨酸掺入，并且即使在远离UGA密码子时也是有活性的。 在这里，我们将开发一种新的方法，通过SEC诱导蛋白石无义突变的通读 重新编码这将通过使用短杂交寡核苷酸来实现，所述短杂交寡核苷酸将靶向的寡核苷酸与靶向的寡核苷酸结合。 mRNA在SECIS元件附近，以基因特异性方式诱导通读，并恢复转录水平。 功能蛋白在目标1中，我们将筛选既与靶mRNA杂交又与靶mRNA杂交的寡核苷酸。 内源性含SECIS的转录物。在目标2中，我们将开发一种基于高通量细胞分选的检测方法， 允许在组合的大量可能的寡核苷酸中鉴定最佳寡核苷酸， 的设计.我们将筛选多种可能的设计，包括含有杂交部分和寡核苷酸。 （可能缩写）SECIS元素。我们将使用两个疾病相关基因来证明我们的方法 (DMD和CFTR）并验证疾病细胞模型中鉴定的寡核苷酸。 与目前可用的治疗方法相比，所提出的方法具有几个重要的优点， 无义突变首先，寡核苷酸对靶基因具有特异性，减少了脱靶的担忧 方面的影响.第二，相同的寡核苷酸可以潜在地用于相同基因中的任何无义突变， 降低开发成本并解决具有非常罕见突变的患者。最后，安全高效的交付 已经证明了短寡核苷酸对几种组织的作用。合在一起，特异性，广泛性 可用性和使用经过验证的输送技术，使我们的方法对治疗特别有吸引力。 目的 与NIBIB的利益一致，该项目将开发一种适用于广泛应用的平台技术。 一系列紊乱和疾病。如果成功，该项目将对质量产生巨大影响。 生活的人患有无意义突变和癌症引起的遗传疾病。