Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RA-Deficiency.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP. To facilitate the translation of PMT therapy to human hPAP patients, a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human CSF2RA-cDNA driven from an EF1α short promoter (Lv.EFS.CSF2RA^coop), and a series of nonclinical efficacy and safety studies were performed in cultured macrophage cell lines and primary human cells. Studies in cytokine-dependent Ba/F3 cells demonstrated efficient transduction, vector-derived CD116 expression proportional to vector copy number, and GM-CSF-dependent cell survival and proliferation. Using a novel cell line constructed to express a normal GM-CSF receptor β subunit and a dysfunctional α subunit (due to a function-altering CSF2RA^G196R mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RA^coop transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RA^coop transduction of healthy primary CD34⁺ cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RA^coop reconstituted GM-CSF receptor α expression, restoring GM-CSF signaling in hPAP macrophages, and had no adverse effects in the intended target cells, thus supporting testing of PMT therapy of hPAP in humans.