Recessive mutations in CSF2RA, the gene encoding the 1-chain of the granulocyte/macrophage-colony stimulating factor receptor (GM-R), were recently reported by the applicant as a newly-identified genetic disorder causing pulmonary alveolar proteinosis (PAP) in children. Recognizing increased serum GM-CSF as a disease biomarker, the applicant identified a cohort of affected individuals and defined the pathogenesis, presentation, diagnosis, molecular pathophysiology, and therapy response of hereditary PAP. The disease is characterized by the progressive accumulation of surfactant in alveolar macrophages (AM) and alveoli, resulting in respiratory insufficiency and, in severe cases, respiratory failure. Current therapy is whole lung lavage, a procedure performed under general anesthesia in which one lung is mechanical ventilated while the other is repeatedly filled with warmed saline, mechanically percussed to emulsify the accumulated surfactant, and then drained to physically remove it. The procedure is repeated as required, which in some children is every two months. PAP occurs in genetically modified mice deficient in the [unreadable]-chain of the GM-R (GM-R[unreadable]KO mice) or in GM-CSF (GMKO mice), and in humans with neutralizing GM-CSF autoantibodies (autoimmune PAP), all of which result in pulmonary histopathology and molecular pathology similar to hereditary PAP in children. Without stimulation by GM-CSF, surfactant catabolism in AM is impaired and results in reduced pulmonary clearance and progressive accumulation of pulmonary surfactant. Bone marrow transplantation (BMT) is a therapeutic option for these children with PAP and was attempted in one child who died of a lung infection before engraftment was complete. The accumulated surfactant in PAP likely increases the infection risk associated with myeloablation, which is required for BMT. PAP in GM-R2KO mice was 'cured'by the applicant by autologous transplantation of bone marrow after retrovirus-mediated gene transduction to correct GM-R function. The central hypothesis of this proposal is that ex-vivo lentiviral vector-mediated restoration of functional GM-R expression in autologous monocytes or G-CSF-mobilized CD34+ cells followed by intrapulmonary administration into non-myeloablated recipients will be safe, well-tolerated, and effective therapy of hPAP. This hypothesis will be tested in 3 Specific Aims: (1) macrophage-mediated cell therapy of hPAP in mice;(2) macrophage-mediated gene therapy of hPAP in mice;(3) preclinical correction of CSF2RA expression and surfactant catabolism in macrophages from children with hPAP, evaluated in vivo in the lungs of non-human primates. A novel approach avoiding known impediments to lung gene therapy will take advantage of the natural survival advantage of GM-R gene-corrected cells conferred by the elevated levels of GM-CSF, a potent growth factor for both human and murine AM. This approach gives hereditary PAP an outstanding chance of being the first human lung disease to be successfully treated by gene therapy and provides a feasible and potential therapeutic alternative for a devastating disease in children. PUBLIC HEALTH RELEVANCE: The applicant recently identified a new lung disease (hereditary PAP) in children caused by accumulation of the natural oily substance (surfactant) required for the lungs to function normally. The disease is caused by mutations in a gene whose protein product (GM-CSF receptor) is required by lung cells (alveolar macrophages) to remove surfactant. Current therapy involves repeatedly washing the lungs with large volumes of salt water to physically remove surfactant while the patient is unconscious and breathing with a machine. The proposed studies will test a new method of gene therapy in a mouse model of the disease and also correction of defective cells from affected children by virus-mediated correction with a normal gene followed by instillation into the lungs of non-human primates. Anticipated preclinical results are needed for regulatory approval for a future clinical trial of gene therapy of hereditary PAP in children.
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