Epitope targeted AAVS for improved airway delivery

描述（由申请人提供）： 囊性纤维化（CF）是白种人最常见的致死性遗传病。由于CFTR基因突变，大多数CF患者死于进行性肺病，目前尚无治愈方法。CFTR cDNA在少至6- 10%的呼吸道上皮中的表达可以纠正阴离子转运缺陷，因此基因治疗对这种常染色体隐性遗传病具有很大的希望。病毒载体如腺相关病毒（AAV）是可用于将矫正货物递送至气道的最安全工具之一，然而，低效的递送继续限制该领域。我们建议通过使用新的肽表位来克服这一限制，所述肽表位有效地结合到来自人类和来自新的CF猪模型的分化良好的原发性CF气道上皮细胞的表面。我们的总体假设是，与CF气道上皮细胞的顶端表面具有亲和力的肽基序可以是 通过噬菌体淘选鉴定，并掺入AAV衣壳中以改善载体对气道的向性。新工程化的载体将用于通过在分化良好的上皮细胞中添加基因来校正CF表型。这项合作计划将CF和气道上皮细胞生物学（McCray和Zabner博士）的专业知识与噬菌体淘选和载体工程（Davidson和Zabner博士）的专业知识相结合。这些PI在基因治疗方面也有相当多的专业知识。该建议包括两个目标，在目标1中，我们将使用噬菌体展示文库的淘选来鉴定对分化良好的CF气道上皮细胞的粘膜表面具有亲和力的肽基序。我们将测试将这些基序插入通过衣壳改组策略出现的新AAV衣壳中，并显示出改善的转导谱，以改善对CF气道上皮细胞顶端表面的靶向。在目标2中，我们将使用肽配体修饰的AAV来纠正CF气道上皮中CFTR阴离子转运和宿主防御缺陷。 公共卫生相关性： CF是最常见的遗传性疾病之一，但基于对该疾病的分子理解的这种复发性遗传性疾病的新疗法进展缓慢。一种有希望的方法是将突变CFTR的基因替换到主要的破坏部位，即肺。然而，不幸的是，将校正的基因产物递送到排列在肺中的气道中的细胞的方法是低效的。在这项工作中，我们提出了高风险，但高影响的研究，将确定新的方法，实现有效的交付，他完整的气道上皮。我们的创新方法也可以应用于更广泛的气道疾病，从而加强我们研究结果的整体影响。

DESCRIPTION (provided by applicant): Abstract Cystic fibrosis (CF) is the most common lethal genetic disorder among Caucasians. As a consequence of mutations in the CFTR gene, most CF patients die from progressive lung disease for which there is no curative treatment. Expression of the CFTR cDNA in as few as 6- 10% of respiratory epithelia can correct the anion transport defect, and therefore gene therapy holds great promise for this autosomal recessive disease. Viral vectors such as adeno- associated virus (AAV) are among the safest tools available to deliver a corrective cargo to the airways, however, inefficient delivery continues to limit the field. We propose to overcome this limitation by using novel peptide epitopes that bind efficiently to the surface of well-differentiaed primary CF airway epithelia from humans and from a novel CF pig model. Our overall hypothesis is that peptide motifs with affinity to the apical surface of CF airway epithelia can be identified via phage panning, and incorporated into an AAV capsid to improve vector tropism for the airways. The newly engineered vectors will be used to correct the CF phenotype by gene addition in well-differentiated epithelia. This collaborative proposal combines expertise in CF and airway epithelial cell biology (Drs. McCray and Zabner) with expertise in phage panning and vector engineering (Drs. Davidson and Zabner). These PIs also have considerable expertise in gene therapy. This proposal encompasses two aims, In Aim 1, we will use panning with a phage display library to identify peptide motifs with affinity to the mucosal surface of well-differentiaed CF airway epithelia. We will test insert those motifs into new AAV capsids that emerged through capsid shuffling strategies and show improved transduction profiles, for improved targeting to the apical surface of CF airway epithelia. In aim 2, we will use the peptide ligand-modified AAVs to correct the CFTR anion transport and host defense defects in CF airway epithelia. PUBLIC HEALTH RELEVANCE: Project Narrative CF is one of the most common genetic diseases, yet new therapies for this recessively inherited disorder based on a molecular understanding of the disease have been slow to advance. One promising approach is gene replacement of the mutant CFTR to the major site of destruction, the lung. Unfortunately however, methods to deliver the corrected gene product to cell that line the airways in the lung are inefficient. In this work, we propose hih risk, yet high impact studies that will identify novel methods for achieving efficient delivery to he intact airway epithelia. Our innovative methods could also be applied to the broader spectrum of airway diseases, thus strengthening the overall impact of our findings.