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: 10615250
• 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/10615250
• 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; Epithelial; Exons; Failure; Frequencies; Future; Gene Frequency; Genes; Grant; Guide RNA; Immunity; Immunocompromised Host; Immunohistochemistry; Implant; In Vitro; 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 Failure; Respiratory Tract Infections; Safety; Sampling; Sinus; Solid Neoplasm; Source; Stem cell transplant; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic Intervention; Transplantation; Tumor Suppressor Genes; Tumorigenicity; Viral Vector; Work; 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 health; mortality; mutation correction; next generation; 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电导的单个基因中的突变引起的 调节剂（CFTR），该调节器编码用于传输CL离子的离子通道。 CF影响许多器官，但肺部失败 由反复的呼吸道感染引起的是死亡的主要原因。当CF首次描述时，大多数 患者小时候死亡。无法恢复CFTR功能的治疗干预措施仍然使患者能够 生存到成年。最近开发的小分子调节剂适度恢复了CFTR功能 已显示可减少肺部恶化。但是，这些疗法很昂贵，有侧面 影响，无法治疗所有CF患者。作为一种单基因疾病，CF一直是几个体内基因的靶标 旨在恢复CFTR功能的治疗研究。但是，由于 针对病毒向量介导的递送和免疫力的挑战。校正的前体基因疗法 已经提出了气道干细胞将其移植到患者中。但是，这些受到我们的失败的限制 为了有效纠正易于访问的气道干细胞类型中的CFTR突变。我们已经确定了鼻窦 作为鼻窦基底干细胞的容易获得的来源，并优化了CAS9的使用并与腺相关 病毒（AAV）以基因编辑这些基础干细胞。我们纠正了最常见的CFTR突变（F508DEL 突变）> 40％等位基因。但是，其他几个无法使用当前疗法进行治疗的突变 保持。此外，使用CAS9的基因编辑的安全性和编辑的长期分化潜力 需要表征鼻窦基底干细胞再生窦和支气管上皮。在这里，我们建议 使用CAS9/AAV用截短的CD19（TCD19）富集标记在外显子1处插入CFTR cDNA 内源性CFTR基因座以实现CFTR突变的通用校正。我们将表征 使用下一代测序的基因组编辑和编辑的基底茎的分化潜力 使用单细胞RNA SEQ和免疫组织化学的细胞。初步结果表明，我们可以获得 基因编辑后，TCD19+基底干细胞的富集群体。这些校正了鼻窦基底干细胞 保留其差异化潜力，以产生表现> 90％CFTR功能的上皮床单（响应 CFTR抑制剂或激活剂）相对于使用室内电生理测定法中的野生型样品。 但是，需要进一步的工作来确定校正后的基底干细胞是否产生所有其他气道 细胞类型。在这笔赠款期间，我们将进一步验证该平台的安全性和功效 评估这些细胞的长期分化潜力，以生成两者中存在的所有细胞类型 窦和支气管上皮。这些实验是优化自体的重要第一步 移植编辑的气道干细胞以治疗CF。可以应用此过程中发展的知识 将来到其他气道疾病。