Development of a Novel rAAV Vector Without Cross-species Barrier to Transduce Human and Ferret Conducting Airways

开发一种无跨物种障碍的新型 rAAV 载体来转换人类和雪貂的气道

基本信息

批准号：
10301711
负责人：
Jianming Qiu
金额：
$ 24.48万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10301711
关键词：
2019-nCoV Air Alveolar Cell Anatomy Animal Model Apical Bacterial Infections Binding Bronchioles COVID-19 COVID-19 prevention Capsid Cause of Death Cell surface Cells Cessation of life Chickens Chronic Clinical Clinical Trials Communicable Diseases Coronavirus Cystic Fibrosis Deposition Development Directed Molecular Evolution Disease Disease Progression Drug or chemical Tissue Distribution Ensure Enterobacteria phage P1 Cre recombinase Epithelial Epithelial Cells Evolution Ferrets Future Gangliosides Gene Delivery Gene Library Gene Transfer Genes Genetic Diseases Genome Glycoproteins Goals Human In Vitro Infection Inflammation Influenza Influenza prevention Libraries Link Liquid substance Lower respiratory tract structure Lung Lung diseases Modeling Morphologic artifacts Mus Mutation Natural regeneration Organ Outcome Parents Passive Immunization Patients Pattern Physiological Physiology Polysaccharides Predisposition Property Pulmonary Cystic Fibrosis Pulmonary Fibrosis Recombinant adeno-associated virus (rAAV)Regulator Genes Reporter Respiratory Tract Infections SARS coronavirus Severe Acute Respiratory Syndrome Sialic Acids Site Testing Therapeutic Trachea Transduction Gene Transgenes Translating Treatment Efficacy Tropism United States Variant Viral Virus Xenograft procedure adeno-associated viral vector airway epithelium base cell type clinical application efficacy evaluation experimental study gene therapy in vivo influenza infection influenzavirus neutralizing antibody next generation novel pandemic influenza preclinical study prophylactic receptor respiratory infection virus respiratory virus screening success tissue tropism vector

项目摘要

PROJECT SUMMARY Human and ferret airways share physiologic similarities in the anatomic properties of their upper and lower respiratory tracts and lung physiology. While the susceptibility of ferrets to pandemic influenza has been known for almost a century, with the recent establishment of cystic fibrosis (CF) ferret models and the advance of passive immunization against respiratory infections, ferrets have become an attractive mammalian model in preclinical studies to evaluate the therapeutic and prophylactic approaches for human pulmonary diseases. Recombinant adeno-associated virus (rAAV)-expression of neutralizing antibody in mice and ferret airways has been proven to elicit efficient protection against influenza virus infections. rAAV2.5T was selected by directed evolution of an AAV2 and AAV5 shuffled capsid gene library in polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI) in vitro. It was thought to be a hopeful candidate vector for in vivo gene delivery to human airways from apical lumen. However, studies of its transduction profile in ferret airways in vivo found undesired vector deposition in alveoli, but not in the trachea and lung conducting airways which are the predominant targets for CF gene therapy and also the primary sites where infection of influenza virus and SARS- CoV-2 naturally occurs. Thus, while using ferret models to examine the efficacies of CF gene therapy and influenza and COVID-19 prevention/treatment is favorable in preclinical studies, currently there is a significant lack of an ideal rAAV vector that can transduce both human and ferret epithelial cells on the conducting airways. Both human and ferret conducting airways predominantly express α2-6 N-linked sialic acid (SA), in contrast to the α2-3 N-linked SA that is the primary attachment receptor of the rAAV2.5T vector. The cell surface glycan molecules largely determine the tissue tropism of rAAV vectors, and the directed evolution of the AAV capsid gene has demonstrated its great success in selecting novel rAAV vectors with an altered tropism for favored cell types. We propose to evolve the AAV2.5T capsid from α2-3 N-linked SA tropic to α2-6 N-linked SA tropic through the selections from the AAV2.5T capsid gene libraries. We will employ the evolution in ferret conducting airways in vivo with a productive transduction reporter. Thus, our study will create a novel rAAV vector that can transduce both the conducting airways of ferrets and humans, which will increase the ferret models’ applicability in preclinical studies to examine the efficacies of the rAAV-based gene transfer for the expression of neutralizing antibody and the gene therapy of CF lung disease. The outcomes from preclinical studies utilizing the novel rAAV vector and ferret models can then be smoothly translated to developing therapeutics in humans.

项目摘要人类和雪貂气道在其上和下部的解剖学特性中具有生理相似性呼吸道和肺部生理学。尽管已知雪貂对大流行影响的敏感性已知近一个世纪以来，随着最近建立囊性纤维化（CF）雪貂模型和进步针对呼吸道感染的被动免疫抑制，雪貂已成为一种有吸引力的哺乳动物模型临床前研究评估人类肺部疾病的治疗和预防方法。重组腺相关病毒（RAAV） - 小鼠和雪貂气道中和抗体的表达被证明可以有效保护影响病毒感染。 RAAV2.5T是通过指示选择的 AAV2和AAV5在两极分化的人类气道上皮中培养的capsid基因库的演变体外空气界面（HAE-ALI）。人们认为它是体内基因传递的有希望的候选媒介从根尖管腔的人类气道。但是，发现其体内雪貂气道中其翻译曲线的研究发现肺泡中的脱离载体沉积，但在气管和肺部进行气道中没有 CF基因疗法的主要靶标，也是影响造成影响和SARS-影响影响的主要部位 COV-2自然发生。在使用雪貂模型来检查CF基因治疗的有效性和流感和COVID-19预防/治疗在临床前研究中是有利的，目前有重要的缺乏理想的RAAV载体，可以将人类和雪貂上皮细胞转导在导电道上。与人类和雪貂进行通风主要表达α2-6n连接的唾液酸（SA），与 α2-3N连接的SA是RAAV2.5T载体的主要附着受体。细胞表面聚糖分子在很大程度上确定了RAAV载体的组织向量，以及AAV CAPSID的定向演化吉恩（Gene 类型。我们建议将AAV2.5T的capsid从α2-3n连接的SA热带转化为α2-6n连接的SA热带来自AAV2.5T CAPSID基因库的选择。我们将利用雪貂进行呼吸道的进化带有生产转移记者的体内。这是我们的研究将创建一个新颖的RAAV矢量，可以翻译雪貂和人类的导电通风道，这将增加雪貂模型的适用性临床前研究检查基于RAAV的基因转移对中和表达的效率 CF肺部疾病的抗体和基因治疗。使用小说的临床前研究的结果然后可以将RAAV载体和雪貂模型顺利地转化为在人类中发展疗法。