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Principal Investigator: Sriram Vaidyanathan
Organization: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
Fiscal Year: 2024
Award: $249,000
Funding agency: National Heart Lung and Blood Institute
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.
Terms: <0-11 years old><21+ years old><Accessory Sinuses><Address><Adeno-Associated Viruses><Adult><Adult Human><Affect><Agar><Airway Disease><Airway infections><Allele Frequency><Alleles><Allelomorphs><Anti-Oncogenes><Antioncogene Protein p53><Antioncogenes><Assay><Autograft><Autologous Transplantation><Autotransplant><Base Pairing><Bioassay><Biological Assay><C-K-RAS><CD19><CD19 gene><CF lung disease><CF patients><CFTR><CFTR Protein><Cancer Suppressor Genes><Cause of Death><Cell Body><Cell Therapy><Cells><Cellular Tumor Antigen P53><Cellular biology><Child><Child Youth><Children (0-21)><Code><Coding System><Complementary DNA><Cystic Fibrosis><Cystic Fibrosis Transmembrane Conductance Regulator><DNA><DNA Therapy><Delta F508 mutation><Deoxyribonucleic Acid><Dependoparvovirus><Dependovirus><Development><Disease><Disorder><EGF Receptor><EGFR><ERBB Protein><Electrophysiology><Electrophysiology (science)><Emerogenes><Engineering><Epidermal Growth Factor Receptor><Epidermal Growth Factor Receptor Kinase><Epidermal Growth Factor Receptor Protein-Tyrosine Kinase><Epidermal Growth Factor-Urogastrone Receptors><Epithelium><Exons><F508 deletion><F508 mutation><F508-del><F508del><Failure><Frequencies><Future><Gene Frequency><Gene Transfer Clinical><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Grant><Guide RNA><HER1><Immunity><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><Immunosuppressed Host><Implant><In Vitro><Infection><Initiation Codon><Initiator Codon><Ion Channel><Ionic Channels><Ions><K-RAS2A><K-RAS2B><K-Ras><K-Ras 2A><K-Ras-2 Oncogene><KRAS><KRAS2><KRAS2 gene><Ki-RAS><Knowledge><Length><Life Expectancy><Lung><Lung Grafting><Lung Respiratory System><Lung Transplantation><Mediating><Membrane Channels><Mendelian disease><Mendelian disorder><Mendelian genetic disorder><Mice><Mice Mammals><Mucoviscidosis><Murine><Mus><Mutation><NGS Method><NGS system><Nasal Sinuses><Nasal cavity/Paranasal><Nasal cavity/Paranasal sinuses><Natural regeneration><Neurophysiology / Electrophysiology><Onco-Suppressor Genes><Oncogene K-Ras><Oncogenes-Tumor Suppressors><Oncogenic><Oncoprotein p53><Organ><P53><Paranasal Sinuses><Patients><Phosphoprotein P53><Phosphoprotein pp53><Population><Position><Positioning Attribute><Process><Production><Progenitor Cell Transplantation><Progenitor Cells><Protein TP53><Pulmonary Cystic Fibrosis><Pulmonary Graft><Pulmonary Transplant><Pulmonary Transplantation><RASK2><Recessive Oncogenes><Recurrence><Recurrent><Regeneration><Reporting><Respiratory Infections><Respiratory Tract Infections><Safety><Sampling><Sinus><Solid Neoplasm><Solid Tumor><Source><Start Codon><Stem Cell Transplantation><Stem cell transplant><TGF-alpha Receptor><TP53><TP53 gene><TRP53><Techniques><Testing><Therapeutic><Therapeutic Intervention><Trachea><Trachea Proper><Transforming Growth Factor alpha Receptor><Transplantation><Tumor Protein p53><Tumor Protein p53 Gene><Tumor Suppressing Genes><Tumor Suppressor Genes><Tumorigenicity><Urogastrone Receptor><Viral Vector><Work><adeno associated virus group><adulthood><allelic frequency><autologous graft><autosome><autotransplantation><bronchial epithelium><c-erbB-1><c-erbB-1 Protein><cDNA><cell biology><cell mediated therapies><cell transduction><cell type><cell-based therapeutic><cell-based therapy><cellular therapeutic><cellular therapy><cellular transduction><clinical applicability><clinical application><cystic fibrosis lung><cystic fibrosis lung disease><cystic fibrosis patients><cystic fibrosis transmembrane regulator><developmental><electrophysiological><erbB-1><erbB-1 Proto-Oncogene Protein><erbBl><experiment><experimental research><experimental study><experiments><gRNA><gene corrected><gene correction><gene editing method><gene editing methodology><gene editing platform><gene editing strategy><gene editing system><gene editing techniques><gene editing technology><gene editing tools><gene repair therapy><gene therapy><gene-based therapy><gene-editing approach><gene-editing toolkit><genetic therapy><genome editing><genome mutation><genomic correction><genomic editing><genomic therapy><immunosuppressed patient><improved><in vivo><indel><individuals with CF><individuals with cystic fibrosis><inhibitor><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><intervention therapy><kids><lung failure><lung function><lung health><lung transplant><monogenic disease><monogenic disorder><mortality><mutation correction><next gen sequencing><next generation sequencing><nextgen sequencing><oncosuppressor gene><organ rejection><organ transplant rejection><p53 Antigen><p53 Genes><p53 Tumor Suppressor><pathogen><patients with CF><patients with cystic fibrosis><progenitor cell based therapy><progenitor cell therapy><progenitor cell treatment><progenitor therapy><progenitor transplantation><progenitor treatment><protein p53><proto-oncogene protein c-erbB-1><pulmonary><pulmonary failure><pulmonary function><pulmonary health><regenerate><response><restoration><scRNA-seq><side effect><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><single-gene disease><single-gene disorder><small molecule><stem and progenitor cell therapy><stem and progenitor cell transplantations><stem cell based therapy><stem cell mediated therapy><stem cell therapeutics><stem cell therapy><stem cell treatment><stem cell-based therapeutic><stem cell-based treatment><stem cells><transduced cells><transplant><v-Ki-RAS2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog><windpipe><youngster><ΔF508>