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

开发囊性纤维化气道干细胞疗法的通用基因组编辑策略

• 批准号: 10683742
• 负责人: Sriram Vaidyanathan
• 金额: $ 24.9万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683742
• 关键词: Address; Adult; Affect; Agar; Airway Disease; Alleles; Autologous Transplantation; Base Pairing; Biological Assay; CD19 gene; Cause of Death; Cell Therapy; Cells; Cellular biology; Child; Code; Complementary DNA; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Delta F508 mutation; Dependovirus; Development; Disease; Electrophysiology (science); Engineering; Epidermal Growth Factor Receptor; Epithelium; Exons; Failure; Frequencies; Future; Gene Frequency; Genes; Grant; Guide RNA; Immunity; Immunocompromised Host; Immunohistochemistry; Implant; In Vitro; Infection; Initiator Codon; Ion Channel; Ions; KRAS2 gene; Knowledge; Length; Life Expectancy; Lung; Lung Transplantation; Mediating; Mendelian disorder; Mus; Mutation; Natural regeneration; Oncogenic; Organ; Patients; Population; Positioning Attribute; Process; Production; Pulmonary Cystic Fibrosis; Recurrence; Reporting; Respiratory Tract Infections; Safety; Sampling; Sinus; Solid Neoplasm; Source; Stem cell transplant; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic Intervention; Trachea; Transplantation; Tumor Suppressor Genes; Tumorigenicity; Viral Vector; Work; autosome; bronchial epithelium; cell type; cellular transduction; clinical application; cystic fibrosis patients; experimental study; gene correction; gene therapy; genome editing; improved; in vivo; inhibitor; insertion/deletion mutation; lung failure; lung health; mortality; mutation correction; next generation sequencing; organ transplant rejection; pathogen; pulmonary function; response; restoration; side effect; single-cell RNA sequencing; small molecule; stem cell therapy; stem cells; tool

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.

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一基因突变引起的 调节器(CFTR)，用于传输氯离子的离子通道的代码。慢性阻塞性肺疾病影响多个器官，但肺衰竭 反复呼吸道感染是导致死亡的主要原因。当CF首次被描述时，大多数 患者在孩提时代就去世了。不能恢复CFTR功能的治疗干预仍然使患者能够 活到成年。新近开发的小分子调节剂适度恢复CFTR功能和 已被证明可以减少肺部恶化。然而，这些疗法价格昂贵，而且有副作用- 但不能治疗所有的慢性萎缩性胃炎患者。作为一种单基因疾病，CF已成为体内多个基因的靶点 旨在恢复CFTR功能的治疗研究。然而，这些研究没有成功，原因是 在传递和免疫方面的挑战，以对抗病毒载体。体外基因治疗，在其中纠正了 将呼吸道干细胞移植到患者体内已被提出。然而，这些都受到我们失败的限制 有效地纠正一种容易获得的呼吸道干细胞类型中的CFTR突变。我们已经确定了鼻窦 作为一种容易获得的鼻窦基础干细胞来源，并优化了Cas9和腺相关基因的使用 病毒(AAV)对这些基础干细胞进行基因编辑。我们已经纠正了最常见的CFTR突变(F508del 突变)在&gt；40%等位基因中。然而，其他几种不能用现有疗法治疗的突变 留下来。此外，使用Cas9进行基因编辑的安全性和编辑后的长期分化潜力 窦基底干细胞再生窦和支气管上皮需要确定其特征。在这里，我们建议 利用Cas9/AAV将CD19(TCD19)截短的CD19(TCD19)富集区的cDNAc DNA插入到 内源性cftr基因座，实现对cftr突变的普遍纠正。我们将描述其安全性 利用下一代测序进行基因组编辑和编辑后的窦基底部干的分化潜能 细胞采用单细胞RNA序列和免疫组织化学方法。初步结果表明，我们可以获得一个 基因编辑后tCD19+窦基质干细胞的富集群。这些矫正后的鼻窦基底干细胞 保留其分化潜能，以产生显示90%CFTR功能的上皮片(响应 在Ussing小室电生理测试中，相对于野生型样品，可使用CFTR抑制剂或激活剂)。 然而，还需要进一步的工作来确定校正后的鼻窦基底干细胞是否会产生所有其他的呼吸道。 单元类型。在此授权期内，我们将进一步验证该平台的安全性和有效性，并 评估这些细胞的长期分化潜力，以生成两种细胞中存在的所有细胞类型 鼻窦和支气管上皮。这些实验是优化自体的重要的第一步 经编辑的呼吸道干细胞移植治疗慢性阻塞性肺疾病在这个过程中发展起来的知识可以被应用 对未来其他呼吸道疾病的影响。