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：通过靶向硒代半胱氨酸重新编码的引起疾病的废话突变的读取 项目摘要 废话（停止）突变建立了约10％的引起疾病的突变，并且在Duchenne中很常见 肌肉营养不良，囊性纤维化和癌症。这些突变导致翻译和铅的早期终止 到非功能蛋白。可以使这些过早终止密码子读取的疗法已经 寻求多年的成功。现有方法受到缺乏特异性，低效率， 或需要传递小基因。 氨基酸硒代半胱氨酸（SEC），有时称为21氨基酸，被掺入人 蛋白质通过蛋白石（UGA）停止密码子的重新编码。这种重新编码机制被A的存在激活 在3'未翻译区域（3'UTR）中的SEC掺入序列元件（SECI）。该元素被证明 足以刺激硒代半胱氨酸掺入，即使远离UGA密码子也很活跃。 在这里，我们将开发一种新的方法，用于通过SEC诱导蛋白石胡说八道突变。 重新编码。这将通过使用短杂交寡核苷酸来实现 靠近secis元素的mRNA，以基因特异性诱导读取，并恢复 功能蛋白。在AIM 1中，我们将筛选与目标mRNA和一个杂交的寡核苷酸 内源性含有SECIS的转录本。在AIM 2中，我们将开发一个基于高通量细胞分类的测定法， 允许在组合大量可能的可能中鉴定最佳寡核苷酸 设计。我们将筛选多种可能的设计，包括含有杂交部分和A的寡核苷酸 （可能缩写）secis元素。我们将使用两个与疾病相关的基因证明我们的方法 （DMD和CFTR）并验证疾病细胞模型中鉴定出的寡核苷酸。 拟议的方法比当前可用的治疗方法具有多个重要优势 废话突变。首先，寡核苷酸是针对靶向基因的特异性的，减少了靶向的关注点 效果。其次，相同的寡核苷酸可能可能用于同一基因中的任何胡说八道突变， 降低开发成本并解决非常罕见的突变的患者。最后，安全有效的交付 已经证明了几个组织的短寡核苷酸。在一起，特异性，广泛 可用性和使用验证的交付技术，使我们的方法对治疗特别有吸引力 目的。 该项目与尼比布的利益保持一致，将开发一种适用于广泛的平台技术 疾病和疾病范围。如果成功，该项目将对 因胡说八道突变和癌症引起的遗传疾病的人的生活。