Targeted correction of the human CFTR gene

人类CFTR基因的靶向校正

基本信息

批准号：
9272950
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
项目类别：
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/9272950
关键词：
Acute Affect Alleles Automobile Driving Binding Caucasians Cause of Death Cells Cessation of life Chemistry Chloride Channels Chlorides Codon Nucleotides Complementary DNA Cyclic AMP-Dependent Protein Kinases Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA DNA Repair Defect Disease Encapsulated Epithelial Epithelial Cells Epithelium FDA approved Formulation 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 Modification Mus Mutation Nebulizer Nose Nucleotide Excision Repair Nucleotides Obstructive Lung Diseases Oligonucleotides Organ Pancreas Pathway interactions Peptide Nucleic Acids Plasmids Polymers Population Post-Translational Protein Processing Proteins Protocols documentation Pulmonary Cystic Fibrosis Reagent Reporting Respiratory Failure Respiratory System Respiratory tract structure Retroviridae Risk Safety Site Site-Directed Mutagenesis Specificity Structure Sweat Glands Testing Toxic effect Transplantation Vertebral column Viral Viral Vector Work airway epithelium base biomaterial compatibility curative treatments cystic fibrosis mouse cystic fibrosis patients deep sequencing design gene correction gene replacement therapy gene therapy genotoxicity homologous recombination improved in vivo in vivo Model interest liposome vector mouse model multidisciplinary mutant nanoparticle novel novel therapeutics nuclease premature programs public health relevance recombinational repair reproductive tract targeted delivery vector zinc finger nuclease

项目摘要

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（由适用提供）：囊性纤维化影响高加索人群中2,000-3,000名活产的1分，对应于美国约30,000人。囊性纤维化（CF）是由囊性纤维化跨膜电导调节蛋白（CFTR）缺陷引起的常染色体隐性疾病，这是一种蛋白激酶A-活化的氯化物通道，介导上皮氯化物。尽管CF是一种影响汗腺的多系统疾病，但生殖道，胰腺，胃肠道和肝脏，但最常见的ITIS疾病，最常见的是死亡。 CF肺肺表现的唯一治疗疗法是移植，但由于急性器官短缺，可能永远不会移植20％的CF患者。 CF患者的平均寿命仅为37年。由于CFTR基因于1989年进行了测序，因此在基因治疗中进行了20多次试验，以治愈肺CF。然而，由于在体内递送外源基因向肺部递送的困难，基因疗法一直持挑战。含有外源CFTR的病毒质粒通常只能瞬时表达，而整合矢量（如扁豆和逆转录病毒）会导致非特异性整合与随之而来的风险。此外，宿主免疫反应通常会清除用于基因递送的病毒载体，尤其是在肺中。作为结果，基因治疗试验几乎在该疾病中都停止了，除了在英国正在进行的试验中，该试验正在使用脂质体载体中含有雾化的PGM169/GL67A。与基因治疗相反的基因编辑的最新进展可能会使CF突变的体内校正成为可能。位点特异性基因编辑可以纠正其内源性位点的CFTR基因，从而导致正常调节控制的永久基因修饰。该应用的目的是为CF的肺部表现开发一种新的疗法。我们已经开发了新型的肽核酸（PNA），该肽可以在CFTR基因内专门形成三重螺旋结构。当与供体DNA与旁边提供供体DNA时，三层形成可诱导天然细胞宿主DNA修复途径，从而实现CFTR基因中F508DEL突变的校正。这些途径是不误差的，因此可以用来以极低的脱靶速率来编辑CFTR基因。我们建议使用由FDA批准的聚合物PLGA制成的生物相容性纳米颗粒来封装PNA和供体DNA分子在体内递送和基因编辑中，对CFTR基因在体内进行了编辑。我们将开发，优化和测试含有肽核酸和完整DNA（PNA/DNA）的新型纳米颗粒，以影响肺中的CFTR基因校正。该疗法有可能改善和/或治愈囊性纤维化的肺表现。由于F508DEL CFTR突变是CF的最常见原因，因此我们将其靶向本应用程序中的缺陷。该计划的总体目标是评估基于纳米颗粒的基因编辑对CF的可行性，可靠性，安全性ND的效率。