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记录通读引起疾病的无义突变 项目概要 无义（终止）突变约占致病突变的 10%，在 Duchenne 中很常见 这些突变会导致翻译提前终止并导致肌肉萎缩症、囊性纤维化和癌症。 能够读取这些过早终止密码子的疗法已经被研究出来。 多年来一直在寻求，但成效有限，现有方法因缺乏特异性、效率低而受到限制。 或者需要传递小基因。 氨基酸硒代半胱氨酸（Sec），有时被称为第 21 个氨基酸，被纳入人体 通过记录蛋白石 (UGA) 终止密码子来记录蛋白质 这种记录机制是通过存在一个密码子来激活的。 3’非翻译区 (3’UTR) 中的 Sec 掺入序列元件 (SECIS) 该元件显示出 足以刺激硒代半胱氨酸的掺入，并且即使位于远离 UGA 密码子时也具有活性。 在这里，我们将开发一种新方法，通过 Sec 诱导蛋白石无义突变的通读 这将通过使用带来目标的短杂交寡核苷酸来实现。 mRNA 靠近 SECIS 元件，以基因特异性方式诱导通读，并恢复 在目标 1 中，我们将筛选与目标 mRNA 和功能蛋白杂交的寡核苷酸。 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， 允许在大量可能的组合中鉴定最佳寡核苷酸 我们将筛选多种可能的设计，包括含有杂交部分和 （可能缩写）SECIS 元素我们将使用两个疾病相关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，所提出的方法具有几个重要的优点 首先，寡核苷酸对目标基因具有特异性，减少了脱靶的担忧。 其次，相同的寡核苷酸可能用于同一基因中的任何无义突变。 降低开发成本并解决患有非常罕见突变的患者最后，安全有效的交付。 短寡核苷酸对多种组织的特异性和广泛性已经得到证实。 可用性以及经过验证的递送技术的使用使我们的方法对治疗特别有吸引力 目的。 与 NIBIB 的利益相一致，该项目将开发一种适用于广泛领域的平台技术 如果成功，该项目将对疾病的质量产生巨大影响。 患有由无义突变引起的遗传疾病和癌症的人们的生活。