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

• 批准号: 10207977
• 负责人: John Lueck
• 金额: $ 63.41万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 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/10207977
• 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; 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

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：无义突变将氨基酸密码子改变为提前终止密码子（PTC），导致 有缺陷的截短蛋白质和严重的疾病。无义突变占十余种 占所有遗传病的百分比，占近 1,000 种人类遗传性疾病，包括囊性纤维化 （CF）。事实上，10% 的 CF 患者存在无义突变（1 型突变），这些突变会导致早产 囊性纤维化跨膜电导调节蛋白（CFTR）的截短和显着丧失 由于无义介导的衰变（NMD）而导致的转录本。最常见的无义突变包括 G542X、 R553X、R1162X 和 W1282X 占所有 CF 无义突变的 10%，导致 CFTR 丢失 功能和最严重的 CF 表型。 CFTR 蛋白是一种氯离子通道，CF 中不存在该蛋白 改变肺内壁液体的正常稳态，导致粘液高度粘稠，导致细菌感染 并最终导致致命的感染。预计肺中 10% 至 15% 的突变 CFTR 会得到纠正 是有效治疗该疾病的阈值。而氨基糖苷类和非氨基糖苷类小 分子已经开发出来，可以读取 PTC、耳毒性和肾毒性，并具有扩展 氨基糖苷类药物的使用以及其他药物的活性较差限制了其临床使用。我们 最近开发了一个反密码子编辑 (ACE)-tRNA 库，可以识别并促进通读 所有“框架内”PTC。每个 ACE-tRNA 都有一个单位点突变，可以识别 PTC，但 ACE-tRNA 可以识别 PTC。 tRNA 带有氨基酸，可读取 PTC。我们利用了小 tRNA 表达盒（~72 bp），并生成了几个紧凑的 DNA 载体，我们称之为微型载体。有图书馆 超过 500 个 ACE-tRNA，我们可以将任何所需的氨基酸插入到任何 PTC 中。我们已经证明这种方法 在体外和体内均能高效地拯救含有 PTC 的发光报告基因，如 以及 CRISPRed 16HBE14o- 细胞基因组背景中的 G542X-、R1162X- 和 W1282X-CFTR 校正 NMD 和 CFTR 蛋白功能。我们的目标是测试行动的有效性、持久性和 以微型载体形式提供的 ACE-tRNA 用于人气道上皮细胞 PTC 通读和校正的安全性 猪和 CFTR PTC 突变小鼠肺中的 PTC 细胞培养模型和体内。将这些 ACE-tRNA 递送至 对于小鼠和猪的肺部，我们将使用经胸腔电穿孔，这已被证明是安全、简单的， 并且能够高效地将基因递送至肺部（包括气道）。总体目标是确定 ACE-tRNA 在治疗无意义相关疾病方面的治疗前景。