Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo.

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb-/- mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

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.CSF2RAcoop), 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 ß subunit and a dysfunctional α subunit (due to a function-altering CSF2RAG196R mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RAcoop transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RAcoop transduction of healthy primary CD34+ cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RAcoop 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.