Targeted correction of the human CFTR gene

人类CFTR基因的靶向校正

• 批准号: 8962446
• 负责人: Marie E Egan
• 金额: $ 55.49万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8962446
• 关键词: Acute; Affect; Alleles; Automobile Driving; Binding; Biocompatible; Caucasians; Cause of Death; Cells; Cessation of life; Chemistry; Chloride Channels; Chloride Ion; Chlorides; Codon Nucleotides; Complementary DNA; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Repair; Defect; Disease; Drug Formulations; Encapsulated; Epithelial; Epithelial Cells; Epithelium; FDA approved; Frequencies; Gastrointestinal tract structure; Gene Delivery; Gene Proteins; Gene Targeting; Gene therapy trial; Gene-Modified; Genes; Genetic Recombination; Genomics; Glycolates; Goals; Grant; Human; Immune response; Individual; Ion Transport; Life Expectancy; Liposomes; Live Birth; Liver; Longevity; Lung; Lung diseases; Mediating; Modeling; Modification; Mus; Mutation; Nose; Nucleotide Excision Repair; Nucleotides; Obstructive Lung Diseases; Oligonucleotides; Organ; Pancreas; Pathway interactions; Peptide Nucleic Acids; Plague; Plasmids; Polymers; Population; Post-Translational Protein Processing; Proteins; Protocols documentation; Pulmonary Cystic Fibrosis; Reagent; Reporting; Respiratory Failure; Retroviridae; Risk; Safety; Site; Site-Directed Mutagenesis; Specificity; Structure; Subfamily lentivirinae; Sweat Glands; System; Testing; Toxic effect; Transplantation; Vertebral column; Viral; Viral Vector; Work; Zinc Fingers; airway epithelium; base; cystic fibrosis mouse; cystic fibrosis patients; deep sequencing; design; gastrointestinal; gene correction; gene replacement therapy; gene therapy; genotoxicity; homologous recombination; improved; in vivo; in vivo Model; interest; mutant; nanoparticle; novel; nuclease; premature; programs; public health relevance; reproductive; respiratory; targeted delivery; vector

 DESCRIPTION (provided by applicant): Cystic fibrosis affects 1 in 2,000-3,000 live births in the Caucasian population, corresponding to approximately 30,000 people in the US. Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the cystic fibrosis transmembrane conductance regulator protein (CFTR), a protein kinase A- activated chloride channel that mediates epithelial chloride transport. Though CF is a multisystem disease affecting sweat glands, the reproductive tract, the pancreas, gastrointestinal tract, and liver, itis lung disease that most commonly leads to fatality. The only curative therapy for lung manifestations of CF is transplantation, but 20% of CF patients may never be transplanted due to acute organ shortage. The mean lifespan of CF patients is only 37 years. Since the CFTR gene was sequenced in 1989, there have been over 20 trials in gene therapy in an attempt to cure pulmonary CF. Gene therapy, however, has remained challenging, because of difficulties with in vivo delivery of exogenous genes to the lung. Viral plasmids containing exogenous CFTR are often only transiently expressed, and integrating vectors such as lenti- and retroviruses result in non-specific integration with attendant risks. In addition, the host immune response often clears viral vectors that are used for gene delivery, particularly in the lung. As a result, gene therapy trials have all but ceased in this disease, with the exception of an ongoing trial in the UK that is using nebulized pGM169/GL67A contained in a liposomal carrier. Recent advances in gene editing - as opposed to gene therapy - may make in vivo correction of the CF mutation possible. Site-specific gene editing could correct the CFTR gene at its endogenous site, resulting in permanent gene modification that is under normal regulatory control. The goal of this application is to develop a novel therapy for the pulmonary manifestations of CF. We have developed novel peptide nucleic acids (PNA) that can form a triple helical structure specifically within the CFTR gene. The triplex formation induces natural cellular host DNA repair-recombination pathways enabling correction of the F508del mutation in the CFTR gene when a donor DNA is supplied alongside. These pathways are error- free and can thus be used to edit the CFTR gene with extremely low off-target rates. We propose to use biocompatible nanoparticles made from the FDA-approved polymer PLGA for encapsulating the PNA and donor DNA molecules for in vivo delivery and gene editing of the CFTR gene in vivo. We will develop, optimize, and test novel nanoparticles containing peptide nucleic acids and complementary DNA (PNA/DNA) to effect CFTR gene correction in the lung. This therapy has the potential to ameliorate and/or cure the pulmonary manifestations of cystic fibrosis. Because the F508del CFTR mutation is the most common cause of CF, we are targeting that defect in this application. The overall goal of this program is to evaluate feasibility, reliability, safety nd efficacy of nanoparticle-based gene editing for treatment of CF.

 描述(申请人提供)：囊性纤维化影响高加索人口中2,000-3,000活产儿中的1例，相当于美国约30,000人。囊性纤维化(CF)是一种常染色体隐性遗传病，由囊性纤维化跨膜电导调节蛋白(CFTR)缺陷引起，CFTR是一种蛋白激酶A激活的氯通道，介导上皮氯的运输。虽然CF是一种影响汗腺、生殖道、胰腺、胃肠道和肝脏的多系统疾病，但最常导致死亡的是肺部疾病。对于肺部表现的CF，唯一的治疗方法是移植，但由于急性器官短缺，20%的CF患者可能永远不会移植。CF患者的平均寿命只有37岁。自1989年CFTR基因测序以来，已有20多项基因治疗试验试图治愈肺纤维化。然而，基因治疗仍然具有挑战性，因为在体内将外源基因输送到肺部存在困难。含有外源CFTR的病毒质粒通常只能瞬时表达，整合载体如Lenti-和逆转录病毒会导致非特异性整合，并伴随着风险。此外，宿主免疫反应经常清除用于基因传递的病毒载体，特别是在肺部。作为一名 结果，这种疾病的基因治疗试验几乎停止了，除了在英国正在进行的一项试验，该试验使用的是脂质体载体中包含的雾化pGM169/GL67A。基因编辑的最新进展--与基因治疗相反--可能使体内纠正Cf突变成为可能。定点基因编辑可以在其内源位置纠正CFTR基因，导致在正常调控下的永久性基因修饰。这一应用的目标是开发一种治疗CF的肺部表现的新方法。我们已经开发出一种新型的多肽核酸(PNA)，它可以在CFTR基因内形成特殊的三螺旋结构。当供体DNA与供体DNA一起供应时，三链形成诱导了自然的细胞宿主DNA修复-重组途径，使得能够纠正CFTR基因中的F508del突变。这些途径是无错误的，因此可以用来以极低的脱靶率编辑CFTR基因。我们建议使用FDA批准的聚合物PLGA制成的生物相容纳米颗粒来包裹PNA和供体DNA分子，用于体内递送和体内CFTR基因的基因编辑。我们将开发、优化和测试包含多肽核酸和互补DNA(PNA/DNA)的新型纳米颗粒，以实现肺内CFTR基因的校正。这种疗法有可能改善和/或治愈囊性纤维化的肺部表现。因为F508del CFTR突变是导致CF的最常见原因，所以我们在这项应用中针对这一缺陷。该计划的总体目标是评估基于纳米颗粒的基因编辑治疗CF的可行性、可靠性、安全性和有效性。