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

项目总结 人类和雪貂的上呼吸道和下呼吸道在解剖特性上有相似的生理特征。 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，随着囊性纤维化(CF)雪貂模型的建立和血管内皮细胞生长因子的研究进展 对于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 评价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)在小鼠和雪貂呼吸道中的表达 已被证明可有效预防流感病毒感染。RAAV2.5T是由DIRECTED AAV2和AAV5洗牌衣壳基因文库在体外培养的极化人呼吸道上皮细胞中的进化 体外形成气液界面(HAE-ALI)。它被认为是体内基因传递的一个有希望的候选载体。 从根尖腔到人的呼吸道。然而，对其在体内雪貂呼吸道的转导情况的研究发现 不受欢迎的病媒沉积在肺泡，但不在气管和肺传导气道，这是 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病毒是自然发生的。因此，在使用雪貂模型来检验CF基因治疗和 流感和新冠肺炎防治在临床前研究中是有利的，目前有显著的 缺乏理想的rAAV载体，可以同时转导人和雪貂的呼吸道上皮细胞。 人和雪貂的传导呼吸道都主要表达α2-6 N-连接唾液酸(SA)，而不是 α2-3 N-连锁SA是rAAV2.5T载体的主要附着受体。细胞表面多聚糖 分子在很大程度上决定了rAAV载体的组织趋向性和AAV衣壳的定向进化。 基因已经证明它在选择新的rAAV载体方面取得了巨大的成功，并且改变了对偏爱细胞的趋向性 类型。我们建议将AAV2.5T衣壳从α2-3 N-连锁SA嗜性进化为α2-6 N-连锁SA嗜性 从AAV2.5T衣壳基因文库中选择。我们将把这种进化应用于雪貂导气管。 在体内有一位多产的转导记者。因此，我们的研究将创建一种新型的rAAV载体，它可以转导 雪貂和人类的传导气道，这将增加雪貂模型在 检测以rAAV为基础的基因转移对中和表达的有效性的临床前研究 抗体与慢性肺病的基因治疗利用该新药进行的临床前研究的结果 RAAV载体和雪貂模型随后可以顺利地转化为在人类身上开发的治疗学。

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.