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: 10707223
• 负责人: Oded Regev
• 金额: $ 24.2万
• 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/10707223
• 关键词: 3' Untranslated Regions; Abbreviations; Address; Adenosine; Affect; Amino Acids; Antisense Oligonucleotides; Binding; Biological Assay; Catalytic RNA; Cell Line; Cell Separation; Cell model; Chemicals; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; 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; Inosine; Lead; Length; Luciferases; Malignant Neoplasms; Messenger RNA; Mutate; Mutation; National Institute of Biomedical Imaging and Bioengineering; Nonsense Codon; Nonsense Mutation; Oligonucleotides; Patients; Persons; Production; Protein Truncation; 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; 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; protein expression; reconstitution; recruit; small molecule; success; technology platform; usability

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：通过靶向硒半胱氨酸重新编码通读致病无义突变 项目总结 无义(STOP)突变约占致病突变的10%，在杜兴很常见 肌肉营养不良、囊性纤维化和癌症。这些突变会导致翻译和销售线索提前终止 到无功能的蛋白质。能够通读这些提前终止密码子的治疗方法已经被 多年来一直在寻找，但收效甚微。现有的方法受到缺乏特异性、效率低、 或者是需要传递小基因。 氨基酸硒半胱氨酸(SEC)，有时被称为第21个氨基酸，被结合到人类 蛋白质蛋白(UGA)终止密码子的重新编码。此重新编码机制通过存在 3‘非翻译区(3’UTR)中的SEC掺入序列元件(SECIS)。该元素被显示为 足以刺激硒半胱氨酸的掺入，即使在远离UGA密码子的地方也是活跃的。 在这里，我们将开发一种新的方法，通过SEC诱导蛋白无义突变的通读 重新编码。这将通过使用短杂交寡核苷酸来实现，该寡核苷酸将带来靶向 接近SECIS元件的mRNA，以基因特异性的方式诱导通读，并恢复 功能蛋白质。在目标1中，我们将筛选与靶mRNA和AN杂交的寡核苷酸。 内源性SECIS转录本。在目标2中，我们将开发一种基于细胞分选的高通量分析方法， 允许在大量可能的组合中识别最佳的寡核苷酸 设计。我们将筛选多种可能的设计，包括含有杂交部分的寡核苷酸和 (可能缩写)SECIS元素。我们将使用两个与疾病相关的基因来演示我们的方法 (DMD和CFTR)，并在疾病细胞模型中验证已识别的寡核苷酸。 与目前可用的治疗方法相比，建议的方法有几个重要的优点 无意义的突变。首先，寡核苷酸是针对目标基因的，减少了对脱靶的担忧 效果。其次，相同的寡核苷酸有可能用于同一基因中的任何无意义突变， 降低开发成本并解决具有非常罕见的突变的患者。最后，安全高效的交付 短链寡核苷酸对几种组织的作用已经被证明。总而言之，特定性，广泛性 可用性和成熟的交付技术的使用，使我们的方法对治疗性特别有吸引力 目的。 结合NIBIB的利益，该项目将开发一种适用于广泛的 障碍和疾病的谱系。如果项目成功，将对项目的质量产生巨大影响 患有由无义突变引起的遗传病和癌症的人的生命。