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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要囊性纤维化 (CF) 是由称为囊性纤维化跨膜电导的单个基因突变引起的调节器 (CFTR)，编码传输 Cl- 离子的离子通道。 CF影响许多器官，但肺衰竭反复呼吸道感染引起的死亡是主要原因。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可适度恢复 CFTR 功能已被证明可以减少肺部症状的加重。然而，这些疗法价格昂贵，并且有副作用影响，并不能治疗所有 CF 患者。作为一种单基因疾病，CF已成为多种体内基因的靶标旨在恢复 CFTR 功能的治疗研究。然而，这些研究均未成功，原因是针对病毒载体的传递和免疫介导的挑战。离体基因治疗纠正了已经有人提出将气道干细胞移植到患者体内。然而，这些都因我们的失败而受到限制有效纠正易于获取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易于获取的来源，并优化了 Cas9 和腺相关的使用病毒（AAV）对这些基底干细胞进行基因编辑。我们纠正了最常见的 CFTR 突变 (F508del 突变）>40％的等位基因。然而，目前的疗法无法治疗其他几种突变保持。此外，使用Cas9进行基因编辑的安全性以及编辑后的长期分化潜力需要对用于再生鼻窦和支气管上皮的鼻窦基底干细胞进行表征。在此，我们建议使用 Cas9/AAV 在 CFTR cDNA 的外显子 1 处插入带有截短的 CD19 (tCD19) 富集标记内源性CFTR位点，实现CFTR突变的通用校正。我们将描述其安全性使用下一代测序进行基因组编辑以及编辑后的窦基干的分化潜力使用单细胞 RNA 测序和免疫组织化学对细胞进行分析。初步结果表明我们可以得到基因编辑后富集的 tCD19+ 鼻窦基底干细胞群。这些矫正的鼻窦基底干细胞保留其分化潜力以产生显示 >90% CFTR 功能的上皮片（对 CFTR 抑制剂或激活剂）在 Ussing 室电生理测定中相对于野生型样品。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气道细胞类型。在本次资助期间，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，以产生两种细胞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细胞的重要的第一步移植编辑的气道干细胞来治疗 CF。在此过程中开发的知识可以应用以免日后发生其他呼吸道疾病。