Targeted correction of the human CFTR gene

 描述（由申请人提供）：囊性纤维化影响高加索人群中2，000 - 3，000例活产婴儿中的1例，相当于美国约30，000人。囊性纤维化（CF）是由囊性纤维化跨膜传导调节蛋白（CFTR）缺陷引起的常染色体隐性疾病，CFTR是介导上皮氯转运的蛋白激酶A激活的氯通道。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，但它是最常导致死亡的肺部疾病。CF的肺部表现的唯一治愈性治疗是移植，但20%的CF患者可能永远不会移植由于急性器官短缺。CF患者的平均寿命只有37岁。 自1989年CFTR基因测序以来，已有超过20项基因治疗试验试图治愈肺CF。然而，基因治疗仍然具有挑战性，因为在体内将外源基因递送到肺中存在困难。含有外源CFTR的病毒质粒通常仅瞬时表达，而慢病毒和逆转录病毒等整合载体会导致非特异性整合，并伴随着风险。此外，宿主免疫反应通常会清除用于基因递送的病毒载体，特别是在肺中。作为 结果，除了在英国正在进行的使用包含在脂质体载体中的雾化pGM 169/GL 67 A的试验外，基因治疗试验几乎停止了对这种疾病的治疗。与基因治疗相反，基因编辑的最新进展可能使CF突变的体内校正成为可能。位点特异性基因编辑可以在其内源位点校正CFTR基因，从而导致在正常调控控制下的永久基因修饰。本申请的目的是开发一种新的治疗CF肺部表现的方法。我们已经开发了新的肽核酸（PNA），可以形成一个三重螺旋结构，特别是在CFTR基因。三链体形成诱导天然细胞宿主DNA修复-重组途径，使得能够在同时提供供体DNA时校正CFTR基因中的F508 del突变。这些途径是无错误的，因此可以用于以极低的脱靶率编辑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，用于封装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，优化和测试含有肽核酸和互补DNA（PNA/DNA）的新型纳米颗粒，以实现肺中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。由于F508 del CFTR突变是CF最常见的原因，因此我们在本申请中针对该缺陷。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。

 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.