Interstitial Lung Diseases (ILDs), represent a large group of chronic pulmonary disorders that are common causes of morbidity and mortality of both children and adults worldwide. Alveolar dysfunction, pulmonary fibrosis, and vascular remodeling associated with chronic ILDs lead to progressive respiratory failure for which they are few effective therapies. Both genetic and environmental factors underlie the pathogenesis of ILDs; including smoking, air pollution, and chronic inflammation and genetic disorders. Mutations in genes regulating surfactant homeostasis or alveolar type 2 (AT2) cell function or survival includes ABCA3, SFTPB, SFTPC, SFTPA, Telomerase (and related genes) that cause respiratory failure in neonates, children, and older individuals. Our PCTC Consortium seeks to develop novel strategies designed to use CRISPR/CAS9 gene editing for lung progenitor cells for correction of a prototypic Childhood Interstitial Lung Diseases (CHILD) disorder that disrupts pulmonary surfactant homeostasis (ABCA3 deficiency) that leads to fatal infantile lung disease. Mutations in in the ABCA3 gene disrupts surfactant lipid and protein production gene causing severe respiratory dysfunction after birth or chronic lung disease in infancy. We will apply CRISPR/CAS9 mediated gene editing to correct ABCA3 in alveolar progenitor cells as disease targets applicable to other genetic and acquired disorders affecting AT2 cells and their progenitors. The identification, targeting and gene editing of alveolar AT2 cells and their progenitors will be widely applicable for the treatment of both genetic and acquired diseases of the peripheral lung in the future.
TARGETING LUNG EPITHELIAL PROGENITOR CELLS TO CORRECT LETHAL ABCA3 DEFICIENCY Jeffrey A. Whitsett, M.D. (CCHMC), Edward E. Morrisey, Ph.D. (U Penn), Darrell N. Kotton, M.D. (Boston U), Lawrence M. Nogee, M.D. (Johns Hopkins), Sessions F. Cole, and Jennifer Wambach (St. Louis Children's Hospital, Washington University) Overview: Mutations in ABCA3, an AT2 cell selective lipid transporter, cause lethal acute and chronic interstitial lung diseases in infants and children. Defects in ABCA3 cause surfactant deficiency and AT2 cell injury resulting in respiratory distress and interstitial lung disease. Our Consortium will develop novel strategies to model and correct ABCA3 deficiency in alveolar progenitor cells, seeking to permanently correct the surfactant deficiency and accompanying lung injury caused by ABCA3 mutations. A unique subset of Axin2+ AT2 progenitor cells will be identified for gene correction; patient specific IPSCs will be produced, differentiated into alveolar progenitors, phenotyped, and used for study of gene editing. A milestone-driven program, will utilize patient-specific IPSC cells, isolated AT2 cells from patients with ABCA3 deficiency, and conditional Abca3flox/flox mice to model the disease and develop corrective strategies in vitro and in vivo. Effects of ABCA3 mutations on the routing, processing, stability, and function of the ABCA3 protein, surfactant proteins, and lipid homeostasis will be modeled in human IPSCs and AT2 progenitor cells. ABCA3 sufficient and patient-specific IPSCs will be differentiated in vitro to produce AT2 cells that will be used for phenotyping and optimization of gene editing via CRISPR/CAS. AT2 progenitor cells will be characterized and provided to the research community for future use in pharmacological approaches to treat the disease. The biological effects of ABCA3 dysfunction and correction will be identified in human cells in vitro. Transgenic mice in which the murine Abca3 gene has been conditionally deleted in AT2 cells will be used in concert with mice expressing Cas9 to test delivery of guide RNAs and AT2 cells via viral and direct mRNA delivery in vivo. We will identify and selectively target unique Wnt responsive Axin2+ (AEPs) AT2 progenitor cells to test strategies to correct ABCA3 in vitro and in vivo by gene editing. We will develop efficient strategies for in vivo gene editing to AT2 cell progenitor cells, with the long term goal of using CRISPR/CAS9 our new Cas9 variants to correct human ABCA3 alleles in vivo. Viral and direct RNA delivery of CRISPR/Cas9 and guide RNA to AT2 progenitor cells will be optimized, and the biological effects of gene correction determined in vivo and in vitro. Our PCTC consortium will develop precise milestones needed to move the project from in vitro and in vivo studies in the mouse towards clinical studies in which we will seek to correct ABCA3 mutations in the clinic for prevention or amelioration of ABCA3-related lung disease. Development of gene editing strategies for ABCA3 deficiency serves as a prototype to assess feasibility of gene editing in lung progenitor cells that will be broadly applicable for treatment of genetic diseases affecting the alveolar epithelium.
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