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

项目摘要 人类和雪貂的气道在其上下呼吸道的解剖特性方面具有生理相似性。 呼吸道和肺生理学。虽然雪貂对大流行性流感的易感性是已知的， 近世纪来，随着雪貂囊性纤维化（CF）模型的建立和 针对呼吸道感染的被动免疫，雪貂已经成为一种有吸引力的哺乳动物模型， 临床前研究，以评估人类肺部疾病的治疗和预防方法。 重组腺相关病毒（rAAV）-在小鼠和雪貂气道中表达中和抗体， 已被证明能有效预防流感病毒感染。rAAV2.5T经定向筛选， AAV 2和AAV 5改组衣壳基因文库在极化的人气道上皮细胞中的进化 体外空气-液体界面（HAE-ALI）。它被认为是一种有希望的体内基因传递载体 从顶腔进入人体气道。然而，研究其在雪貂气道中的体内转导概况发现， 不希望的载体沉积在肺泡中，而不是在气管和肺传导气道中， 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，当使用雪貂模型来检查CF基因治疗的功效时， 流感和COVID-19预防/治疗在临床前研究中是有利的，目前有一个显着的 缺乏一种理想的rAAV载体，可以将人和雪貂上皮细胞都接种在传导气道上。 人和雪貂的传导气道主要表达α2-6 N-连接唾液酸（SA），与之相反， α2-3 N-连接的SA，其是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 基因已经证明了它在选择新的rAAV载体方面取得了巨大的成功，这些载体具有对有利细胞的改变的向性。 类型我们建议通过以下途径将AAV2.5T衣壳从α2-3 N连接的SA嗜性进化为α2-6 N连接的SA嗜性： 从AAV2.5T衣壳基因文库中选择。我们将利用雪貂的进化来引导气道 在体内与生产性转导报告子结合。因此，我们的研究将创造一种新的rAAV载体， 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，以检查基于rAAV的基因转移用于表达中和抗体的功效。 抗体和CF肺病的基因治疗。使用新药物的临床前研究的结果 rAAV载体和雪貂模型可以顺利地转化为人类的治疗方法。

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.