In vivo delivery of engineered tRNAs for suppression of nonsense mutations

体内递送工程化 tRNA 以抑制无义突变

基本信息

批准号：
10390358
负责人：
John Lueck
金额：
$ 61.03万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-10 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10390358
关键词：
Accounting Affect Amino Acids Aminoglycosides Anticodon Bacterial Infections Bar Codes Biological Availability Cell Culture Techniques Cells Charge Chloride Channels Clinical Codon Nucleotides Complementary DNA Cultured Cells Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA DNA cassette Data Set Disease Effectiveness Electrophysiology (science)Electroporation Engineering Epithelial Cells Exhibits Family suidae Fluorescent in Situ Hybridization Gene Delivery Genes Genetic Diseases Genetic Transcription Genome Genomics Goals Hereditary Disease Homeostasis Human Human Genetics Immunofluorescence Immunologic In Vitro Inbred CFTR Mice Infection Lead Length Libraries Liquid substance Lung Maps Measures Mediating Methods Modeling Mucous body substance Mus Mutant Strains Mice Mutation Nonsense Mutation Nonsense-Mediated Decay Nucleotides Persons Pharmaceutical Preparations Phenotype Physiologic pulse Plasmids Positioning Attribute Proteins Quantitative Reverse Transcriptase PCR Reporter Reporter Genes Safety Site Specificity Structure Technology Terminator Codon Therapeutic Tissues Toxic effect Transcript Transfer RNA Translating Translation Process United States Western Blotting Wild Type Mouse Work airway epithelium cell type design disease-causing mutation effective therapy effectiveness evaluation efficacy testing electric field expression vector gene therapy improved in vivo loss of function mRNA Expression mRNA Transcript Degradation mutant nanoparticle nephrotoxicity novel ototoxicity porcine model premature protein complex protein function ribosome profiling small molecule trafficking transcriptome transgene expression vector

项目摘要

Abstract: Nonsense mutations change an amino acid codon to a premature termination codon (PTC), resulting in a defective truncated protein and severe forms of disease. Nonsense mutations account for greater than ten percent of all genetic diseases, accounting for nearly 1,000 genetic human disorders, including cystic fibrosis (CF). Indeed, 10% of people with CF have nonsense mutations (Type 1 mutations) that lead to premature truncation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and significant loss of transcripts due to nonsense mediated decay (NMD). The most common nonsense mutations include G542X, R553X, R1162X and W1282X, which account for 10% of all CF nonsense mutations, result in loss of CFTR function and the most severe CF phenotypes. The CFTR protein is a chloride channel whose absence in CF alters normal homeostasis of lung lining fluid, resulting in highly viscous mucus that allows bacterial infection and ultimately lethal infections. Correction of between 10 and 15% of the mutant CFTR in the lung is predicted to be the threshold for effective treatment of the disease. While aminoglycosides and non-aminoglycoside small- molecules have been developed and allow readthrough of PTCs, ototoxicity and nephrotoxicity with extended use in the case of aminoglycosides and poor activity for the other drugs has restricted their use clinically. We have recently developed a library of Anti-Codon Edited (ACE)-tRNAs that recognize and promote read-through of all `in-frame' PTCs. Each of the ACE-tRNAs has a single site mutation that recognizes the PTC but the ACE- tRNA is charged with an amino acid to readthrough the PTC. We have taken advantage of the small tRNA expression cassette (~72 bp), and generated several compact DNA vectors, we call minivectors. With a library of >500 ACE-tRNAs, we can insert any desired amino acid into any PTC. We have shown that this approach works with high efficiency both in vitro and in vivo in rescuing PTC-containing luminescent reporter genes, as well as G542X-, R1162X- and W1282X-CFTR within the genomic context in CRISPRed 16HBE14o- cells leading to correction of both NMD and CFTR protein function. Our goal is to test the efficacy, persistence of action and safety of ACE-tRNAs delivered as minivectors for PTC readthrough and correction in human airway epithelial PTC cell culture models and in vivo in lung of pig and CFTR PTC mutant mice. To deliver these ACE-tRNA to the lungs of mice and pigs, we will use transthoracic electroporation which has been shown to be safe, simple, and highly efficient at gene delivery to the lung, including the airways. The overarching goal is to determine the therapeutic promise of ACE-tRNAs for treatment of nonsense associated diseases.

摘要：废话突变将氨基酸密码子更改为过早终止密码子（PTC），结果在有缺陷的截短蛋白质和严重形式的疾病中。废话突变占十多个所有遗传疾病的百分比，占近1,000种遗传性疾病，包括囊性纤维化（CF）。实际上，有10％的CF患者具有胡说八道的突变（1型突变），导致过早囊性纤维化跨膜电导调节剂（CFTR）蛋白的截断和明显的损失由于介导的衰减（NMD）而引起的转录本。最常见的胡说突变包括G542X， R553X，R1162X和W1282X，占所有CF废话突变的10％，导致CFTR的损失功能和最严重的CF表型。 CFTR蛋白是氯化物通道，其在CF中的缺失改变肺衬里液体的正常稳态，导致高粘性粘液，使细菌感染并最终致命感染。预测肺中10％至15％的突变体CFTR的纠正被预测成为有效治疗疾病的阈值。而氨基糖苷和非氨基糖苷小已经开发了分子并允许读取PTC，耳毒性和肾毒性具有扩展在氨基糖苷的情况下，其他药物的活性不佳已限制了它们在临床上的使用。我们最近，已经开发了一个反密码子编辑（ACE）-trnas的库，该图书馆识别和促进通读在所有“框架内” PTC中。每个ACE-TRNA都有一个单个位点突变，可以识别PTC，但ACE- TRNA充满氨基酸，以读取PTC。我们已经利用了小trna 表达式盒（〜72 bp），并产生了几个紧凑的DNA矢量，我们称为小型。与图书馆在> 500个ACE-TRNA中，我们可以将任何所需的氨基酸插入任何PTC中。我们已经证明了这种方法在拯救含PTC的发光记者基因的体外和体内效率高效率以及G542X-，R1162X-和W1282X-CFTR在CRISPRED 16HBE14O细胞中的基因组环境中校正NMD和CFTR蛋白功能。我们的目标是测试效力，行动的持久性和 Ace-trnas作为PTC读取和校正的小型ace-trnas的安全性 PTC细胞培养模型和猪肺和CFTR PTC突变小鼠的体内。将这些Ace-tRNA传递到小鼠和猪的肺，我们将使用经胸电穿孔，这已被证明是安全，简单，并且在包括气道在内的肺部递送基因方面高效。总体目标是确定 ACE-TRNA治疗胡说八道相关疾病的治疗承诺。