Optimizing Surgical Transplant of CFTR Gene-Corrected Human Basal Stem Cells to the Upper Airway

项目总结/摘要 囊性纤维化（CF）是一种常染色体隐性单基因疾病， 跨膜传导调节因子（CFTR）基因。最常见的突变，被称为IF 508，发生在 约85%的CF患者。由于CFTR基因编码阴离子转运蛋白，CFTR突变改变了 电解质和水的运输，导致致密，病理，粘液和其他分泌物。最 CFTR功能障碍的有害影响发生在呼吸系统中，伴随复发性感染， 上下呼吸道的炎症。尽管医学治疗取得了重大进展， 对于CF的改进的、持久的疗法仍然存在巨大的未满足的需求。 在过去的3年里，我们的互补科学家和医生的合作小组已经决定， 为患有CF的患者开发一种新的基于干细胞的治疗策略。对于几个关键的 出于这些原因，我们将我们的努力导向CF上呼吸道疾病的基于细胞的治疗， 原代人气道基底干细胞，称为ABC。第一个重要的里程碑是利用CRISPR/Cas9 基因组编辑技术，以有效纠正CF患者培养的ABC中的BNF 508突变 接受鼻窦手术这种基因校正方法导致氯阴离子的显著恢复 从0-3%运输到ABC中的30-40%。这一令人鼓舞的、新公布的发展现在提供了 将CFTR基因校正的ABC作为干细胞重新引入体内环境的临床前路线图 替代疗法 在这项提案中，我们将严格确定最有效的生物材料平台， 人ABC的移植和植入，并在实验室中评估基因校正的ABC的行为， 活体动物模型，使用显微外科模型系统的上气道移植，我们有 开发为了将这种方法转化为CF，所概述的实验是必要的临床前步骤 为CF患者提供一种创新的、可能是变革性的治疗方法， 用于人类气道疾病的基于细胞的疗法。

PROJECT SUMMARY/ABSTRACT Cystic fibrosis (CF) is an autosomal recessive single-gene disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, termed ∆F508, occurs in ~85% of CF patients. Because the CFTR gene encodes for an anion transport protein, CFTR mutations alter electrolyte and water transport, resulting in dense, pathologic, mucous and other secretions. The most harmful effects of CFTR dysfunction occur in the respiratory system, with recurrent infections and inflammation of the upper and lower airways. Despite substantial progress with medical therapies, there remains a tremendous unmet need for improved, durable therapies for CF. For the past 3 years, our collaborative group of complementary scientists and physicians has determined to develop a novel, stem cell-based treatment strategy for patients suffering from CF. For several critical reasons, we directed our efforts to cell-based therapy of CF upper airway disease using ex vivo-expanded, primary human airway basal stem cells, termed ABCs. The first major milestone was to utilize CRISPR/Cas9 genome editing technology to efficiently correct the ∆F508 mutation in ABCs cultured from CF patients undergoing sinus surgery. This gene correction approach has led to significant restoration of chloride anion transport from 0-3% to 30-40% in ABCs. This encouraging, and newly published, development now provides a pre-clinical roadmap for re-introducing CFTR gene-corrected ABCs into in vivo contexts as a stem cell replacement therapy. In this proposal, we will rigorously determine the most efficient, biomaterial platform for ex vivo-to-in vivo transplant and engraftment of human ABCs, and assess the behavior of gene-corrected ABCs in the lab and live animal model using a microsurgical model system of upper airway transplantation that we have developed. The experiments outlined are essential pre-clinical steps in order to translate this approach to CF patients to generate an innovative and possibly transformative therapy for patients with CF, and the first stem cell-based therapy for human airway disease.