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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.

项目概要人类和雪貂气道的上部和下部的解剖学特性具有生理相似性呼吸道和肺生理学。虽然雪貂对大流行性流感的易感性已为人所知近一个世纪以来，随着最近囊性纤维化（CF）雪貂模型的建立以及雪貂对呼吸道感染进行被动免疫，已成为一种有吸引力的哺乳动物模型评估人类肺部疾病的治疗和预防方法的临床前研究。重组腺相关病毒（rAAV）在小鼠和雪貂气道中表达中和抗体已被证明可有效预防流感病毒感染。 rAAV2.5T 是由定向筛选的极化人气道上皮中 AAV2 和 AAV5 改组衣壳基因库的进化体外气液界面（HAE-ALI）。它被认为是有希望的体内基因传递候选载体从心尖腔到人类呼吸道。然而，对其体内雪貂气道转导谱的研究发现不需要的载体沉积在肺泡中，但不会沉积在气管和肺传导气道中，而气管和肺传导气道是 CF基因治疗的主要靶点，也是流感病毒和SARS感染的主要部位 CoV-2 自然发生。因此，在使用雪貂模型检验 CF 基因治疗和流感和 COVID-19 的预防/治疗在临床前研究中是有利的，目前有显着的进展缺乏可以转导人类和雪貂传导气道上皮细胞的理想 rAAV 载体。人类和雪貂的气道均主要表达 α2-6 N 连接唾液酸 (SA)，而 α2-3 N-连接 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决定了rAAV载体的组织向性以及AAV衣壳的定向进化基因已证明其在选择新型 rAAV 载体方面取得了巨大成功，该载体对偏爱的细胞具有改变的趋向性类型。我们建议通过以下方式将 AAV2.5T 衣壳从 α2-3 N-linked SA tropic 进化为 α2-6 N-linked SA tropic 来自 AAV2.5T 衣壳基因库的选择。我们将利用雪貂传导气道的进化体内具有高效转导报告基因。因此，我们的研究将创建一种新的 rAAV 载体，可以转导雪貂和人类的传导气道，这将增加雪貂模型的适用性临床前研究旨在检验基于 rAAV 的基因转移对于中和蛋白表达的功效 CF肺病的抗体和基因治疗。利用新颖的临床前研究的结果然后，rAAV 载体和雪貂模型可以顺利转化为开发人类疗法。