Advancing lentiviral gene therapy for cystic fibrosis.

推进囊性纤维化的慢病毒基因治疗。

基本信息

批准号：
10319911
负责人：
Laura Isabel Marquez Loza
金额：
$ 3.69万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-10 至 2023-12-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10319911
关键词：
Address Adverse effects Anions Apical Basal Cell Bicarbonates Bronchi Carrying Capacities Cause of Death Cell Death Cell Line Cells Cellular Stress Clinical Data Clinical Trials Codon Nucleotides Complement Complementary DNA Coupled Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Dose Endogenous Retroviruses Ensure Epithelial Epithelial Cells FDA approved Family suidae Flow Cytometry Future Gap Junctions Gene Expression Genes Genetic Diseases Genome Glycoproteins Goals Human Immunohistochemistry In Vitro Infection Interphase Cell Lead Lentivirus Vector Life Luciferases Lung Measures Mediating Messenger RNA Mutation Newborn Infant Papio Pharmaceutical Preparations Pharmacotherapy Phenotype Pulmonary Cystic Fibrosis Regulator Genes Surface System Testing Therapeutic Tissues Trachea Tropism Viral Virus Work airway epithelium airway surface liquid body system cell type cellular transduction cystic fibrosis airway cystic fibrosis airway epithelia cystic fibrosis patients design disease-causing mutation effective therapy experimental study gene therapy gene therapy clinical trial improved in vitro testing in vivo in vivo Model porcine model pre-clinical progenitor protein function receptor respiratory transduction efficiency transgene expression vector

项目摘要

Project Summary Cystic fibrosis (CF) is a common, life-shortening genetic disease. Mutations in the cystic fibrosis conductance regulator (CFTR) gene lead to defective or absent CFTR that is unable to effectively transport anions in many tissues, but pulmonary complications are the most common cause of death. Recent advances in CF treatment include drugs able to rescue protein function. However, these treatments are mutation-class specific and are ineffective for some mutations. Thus, there is a persistent need to develop mutation agnostic treatments that can benefit all people with CF, such as gene addition. Lentiviral vectors are well suited for CF gene therapy because they have sufficient carrying capacity, and their ability to integrate into the host’s genome ensures long-term benefits. Currently available vectors, however, produce low vector titers or inefficiently transduce airway epithelial cells from the apical surface. Despite these limitations, treatment of newborn CF pigs with a lentiviral vector carrying a functional copy of CFTR partially rescued CF phenotypes, suggesting that achieving therapeutic CFTR expression is within reach. My long-term goal is to design a lentiviral vector suitable for the effective treatment of pulmonary CF in humans. The overall objective of his proposal is to evaluate two independent but complementary strategies, to improve lentiviral delivery of CFTR to human airway epithelial cells. Lentiviral vectors can accommodate envelopes from other viruses, termed pseudotyping, which can lead them to preferentially transduce specific cell types. For the first strategy, I screened seven viral envelopes and identified two derived from baboon endogenous retrovirus (BaEV) that produce high vector titers and efficiently transduce primary human airway epithelial cells from the apical surface. The second strategy is to increase CFTR expression in transduced cells. Because airway epithelial cells are electrochemically coupled through gap junctions, a few cells expressing high levels of CFTR may perform sufficient CFTR-mediated anion transport to correct CF phenotypes. Increased gene expression can be achieved through codon optimization. I compared three codon optimized CFTR (coCFTR) sequences and identified one that significantly increases CFTR-mediated transepithelial Cl- transport. My central hypothesis is that increasing transduction efficiency and transgene expression will be sufficient to achieve wild type levels of CFTR-mediated anion transport. To test my hypothesis, I propose the following Specific Aims: 1) Determine if BaEV pseudotyped lentiviral vectors can efficiently deliver CFTR to primary human airway epithelial cells, and 2) Determine if coCFTR delivery reduces the proportion of complemented cells needed to achieve wild type levels of CFTR-mediated anion transport. These strategies will be tested in vitro using human CF donor-derived airway epithelia, and in vivo using the CF pig model. These experiments will provide valuable preclinical data that will guide vector design and inform future clinical trials using lentiviral vectors to treat CF.

项目概要囊性纤维化 (CF) 是一种常见的、缩短寿命的遗传性疾病。调节 (CFTR) 基因导致 CFTR 有缺陷或缺失，从而无法在许多细胞中有效转运阴离子组织，但肺部并发症是 CF 治疗的最新进展。包括能够拯救蛋白质功能的药物，但是，这些治疗是针对突变类别的，并且是特定的。因此，一直需要开发能够与突变无关的治疗方法。使所有患有CF的人受益，例如基因添加。慢病毒载体非常适合CF基因治疗，因为它们具有足够的承载能力，并且其然而，目前可用的载体能够整合到宿主基因组中，从而确保长期利益。尽管如此，载体滴度较低或从心尖表面转导气道上皮细胞效率较低。局限性，用部分携带 CFTR 功能拷贝的慢病毒载体治疗新生 CF 猪拯救了 CF 表型，表明实现治疗性 CFTR 表达是可以实现的。我的长期目标是设计一种适合有效治疗人类肺CF的慢病毒载体。他提案的总体目标是评估两种独立但互补的策略，以改进将 CFTR 慢病毒递送至人气道上皮细胞慢病毒载体可以容纳来自的包膜。其他病毒，称为假型，可以导致它们优先转导特定的细胞类型。第一个策略，我筛选了七个病毒包膜，并鉴定出两个源自狒狒内源性逆转录病毒 (BaEV)，产生高载体滴度并有效转导来自原代人气道上皮细胞第二种策略是增加转导细胞中的 CFTR 表达。上皮细胞通过间隙连接进行电化学耦合，少数细胞表达高水平的 CFTR 可以进行足够的 CFTR 介导的阴离子转运来纠正 CF 表型的增加。可以通过密码子优化来实现，我比较了三个密码子优化的CFTR（coCFTR）序列。并确定了一种可以显着增加 CFTR 介导的跨上皮 Cl- 转运的方法。提高转导效率和转基因表达足以达到野生型水平为了检验我的假设，我提出以下具体目标：1）确定 BaEV假型慢病毒载体是否可以有效地将CFTR传递至原代人气道上皮细胞，以及 2) 确定 coCFTR 递送是否减少了实现野生型所需的互补细胞的比例 CFTR 介导的阴离子转运水平将使用人类 CF 供体来源进行体外测试。气道上皮细胞，以及使用 CF 猪模型进行体内实验，这些实验将提供有价值的临床前数据。这将指导载体设计并为未来使用慢病毒载体治疗 CF 的临床试验提供信息。