A universal genome editing strategy to develop an airway stem cell therapy for cystic fibrosis

项目摘要 囊性纤维化（CF）是由称为囊性纤维化跨膜传导的单个基因突变引起的 调节子（CFTR），编码一个转运Cl-离子的离子通道。CF影响许多器官，但肺衰竭 由反复呼吸道感染引起的肺炎是导致死亡的主要原因。当CF首次被描述时，大多数 患者在儿童时期死亡。不能恢复CFTR功能的治疗干预仍然使患者能够 活到成年。最近开发的小分子调节剂适度恢复CFTR功能， 已经显示出减少肺部恶化。然而，这些疗法是昂贵的，有副作用- 但不能治疗所有的CF患者。CF是一种单基因遗传病，是体内多个基因作用的靶点 旨在恢复CFTR功能的治疗研究。然而，这些研究并不成功，因为 在递送和针对病毒载体介导的免疫方面的挑战。其中校正的离体基因治疗 已经提出将气道干细胞移植到患者体内。然而，这些都受到我们失败的限制 以有效地纠正容易获得的气道干细胞类型中的CFTR突变。我们已经确定了鼻窦 作为一种容易获得的窦基底干细胞来源，并优化了Cas9和腺相关病毒的使用。 病毒（AAV）对这些基底干细胞进行基因编辑。我们已经纠正了最常见的CFTR突变（F508 del >40%的等位基因中存在突变。然而，其他几种突变不能用目前的疗法治疗， 保持。此外，使用Cas9的基因编辑的安全性和编辑的基因的长期分化潜力也是值得关注的。 需要表征再生窦和支气管上皮的窦基底干细胞。在这里，我们建议 使用Cas9/AAV将具有截短的CD 19（tCD 19）富集标志物的CFTR cDNA插入到CFTR基因的外显子1， 内源性CFTR基因座，以实现CFTR突变的普遍校正。我们将描述 使用下一代测序的基因组编辑和编辑的窦基底干的分化潜力 细胞使用单细胞RNA测序和免疫组织化学。初步结果表明，我们可以得到一个 基因编辑后的tCD 19+窦基底干细胞富集群体。这些纠正的鼻窦基底干细胞 保留它们的分化潜能以产生显示>90% CFTR功能的上皮片层（对CFTR的响应）。 在Ussing室电生理学测定中，相对于野生型样品的CFTR抑制剂或激活剂）。 然而，需要进一步的工作来确定是否纠正窦基底干细胞产生所有其他气道 细胞类型。在此资助期间，我们将进一步验证该平台的安全性和有效性， 评估这些细胞的长期分化潜力，以产生存在于两种细胞中的所有细胞类型。 窦和支气管上皮。这些实验是优化自体免疫系统的重要的第一步。 移植编辑的气道干细胞以治疗CF。在这一过程中发展起来的知识可以应用于 其他呼吸道疾病。

Project Summary Cystic fibrosis (CF) is caused by mutations in a single gene called the cystic fibrosis transmembrane conductance regulator (CFTR), which codes for an ion channel that transports Cl- ions. CF affects many organs but lung failure caused by repeated respiratory infections is the leading cause of death. When CF was first described, most patients died as children. Therapeutic interventions that do not restore CFTR function still enabled patients to survive into adulthood. Recently developed small molecule modulators restore CFTR function modestly and have been shown to reduce pulmonary exacerbations. However, these therapies are expensive, have side- effects and cannot treat all CF patients. As a monogenic disease, CF has been the target of several in vivo gene therapy studies that aimed to restore CFTR function. However, these studies were unsuccessful due to challenges in delivery and immunity mediated against viral vectors. Ex-vivo gene therapy in which corrected airway stem cells are transplanted into patients have been proposed. However, these are limited by our failure to efficiently correct CFTR mutations in a readily accessible airway stem cell type. W e have identified the sinuses as a readily accessible source of sinus basal stem cells and optimized the use of Cas9 and adeno-associated virus (AAV) to gene edit these basal stem cells. We have corrected the most common CFTR mutation (F508del mutation) in >40% alleles. However, several other mutations which cannot be treated using current therapies remain. Moreover, the safety of gene editing using Cas9 and the long-term differentiation potential of edited sinus basal stem cells to regenerate the sinus and bronchial epithelia need to be characterized. Here, we propose to use Cas9/AAV to insert the CFTR cDNA with a truncated CD19 (tCD19) enrichment marker at exon 1 of the endogenous CFTR locus to achieve universal correction of CFTR mutations. We will characterize the safety of genome editing using next-generation sequencing and the differentiation potential of edited sinus basal stem cells using single-cell RNA seq and immunohistochemistry. Preliminary results show that we can obtain an enriched population of tCD19+ sinus basal stem cells after gene editing. These corrected sinus basal stem cells retain their differentiation potential to produce epithelial sheets that show >90% CFTR function (response to CFTR inhibitor or activator) relative to wild-type samples in an Ussing chamber electrophysiological assay. However, further work is needed to determine if the corrected sinus basal stem cells generate all the other airway cell types. Over the period of this grant, we will further validate the safety and efficacy of this platform and evaluate the long-term differentiation potential of these cells to generate all the cell types present in both the sinus and bronchial epithelia. These experiments are an important first step to optimize the autologous transplantation of edited airway stem cells to treat CF. The knowledge developed in this process can be applied to other airway diseases in the future.