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

项目摘要/摘要 囊性纤维化是一种常染色体隐性遗传性单基因疾病，由囊性纤维化基因突变引起 跨膜电导调节因子(CFTR)基因。最常见的突变，称为∆F508，发生在 约85%的CF患者。由于cftr基因编码一种阴离子运输蛋白，cftr突变会改变 电解质和水的运输，导致致密、病理性、粘液等分泌物。最多的 CFTR功能障碍的有害影响发生在呼吸系统，反复感染和 上呼吸道和下呼吸道发炎。尽管医学疗法取得了实质性的进展，但 对于改进的、持久的CF治疗方法，仍然是一个巨大的未得到满足的需求。 在过去的3年里，我们由互补的科学家和医生组成的合作小组决心 开发一种新的、基于干细胞的治疗策略，用于治疗CF患者。对于几个关键的 因此，我们将精力集中在基于细胞的治疗上，使用的是体外扩增的 原代人呼吸道基底干细胞，称为ABC。第一个重要的里程碑是利用CRISPR/Cas9 基因组编辑技术有效纠正CF患者外周血中∆F508突变 正在接受鼻窦手术。这种基因校正方法已经显著恢复了氯离子。 在ABC中从0-3%到30-40%的传输。这一令人鼓舞的、新公布的发展现在提供了 将经CFTR基因修正的ABCs作为干细胞重新引入体内的临床前路线图 替代疗法。 在这份提案中，我们将严格确定体外到体内最有效的生物材料平台 移植和植入人类ABC，并评估经过基因校正的ABC在实验室和 使用我们已有的上呼吸道移植显微外科模型系统建立的活体动物模型 发展起来的。概述的实验是将这种方法转化为CF所必需的临床前步骤 为CF患者产生一种创新的、可能具有变革性的治疗方法，以及第一个干细胞 以细胞为基础的人类呼吸道疾病治疗。

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.