IntroductionPulmonary alveolar proteinosis (PAP) is characterised by accumulation of surfactant in the terminal airways. Granulocyte-Macrophage Colony-Stimulating-Factor (GM-CSF) stimulates alveolar macrophages to clear surfactant. The presence of GM-CSF autoantibodies in autoimmune PAP (aPAP) leads to surfactant build-up and impaired gas exchange. This causes respiratory symptoms and can ultimately be fatal due to hypoxaemic respiratory failure. We hypothesise that lentivirus-mediated gene transfer of GM-CSF may be suitable to treat aPAP and propose to assess efficacy of GM-CSF gene transfer in GM-CSF knockout mice, which recapitulate aPAP lung disease. The murine GM-CSF (mGM-CSF) cDNA was cloned into a lentiviral vector, which was pseudotyped with the F and HN proteins from Sendai virus to enable efficient lung transduction (rSIV.F/HN-mGM-CSF).Methods and ResultsTo confirm if the vector produces mGM-CSF we first transduced A549 cells with multiplicity of infection (MOI) of 0.1–100 (n=6/group). 48 hours after transduction dose-related mGM-CSF expression was confirmed in the medium. We next assessed whether the mGM-CSF produced after gene transfer was biologically active by comparing the proliferation rate of FDC-P1 cells, a mGM-CSF-dependent mouse myeloid progenitor cell line, in the presence of gene therapy- produced mGM-CSF (0.001–10 ng/ml) and purchased recombinant mGM-CSF protein (n=6/group). The dose-related proliferation rates in both conditions were similar (figure 1ss). In preliminary experiments, we next assessed whether gene transfer led to GM-CSF production in vivo. rSIV.F/HN-mGM-CSF (1e7 transduction units (TU)/mouse) was administered to wild-type mice by nasal “sniffing”. Control mice remained untransduced (n=3/group). mGM-CSF levels were quantified in lung tissue homogenate and broncho-alveolar lavage fluid (BALF) 14 days after gene transfer. mGM-CSF levels in untreated mice were below the limit of detection of the ELISA, but high levels of mGM-CFS were detectable in lung tissue (median 825 (range 460–3790) pg/mg) and BALF (median: 3330 (range 2307–7958) pg/ml).ConclusionrSIV.F/HN-mGM-CSF produced mGM-CSF in vitro and in vivo. The biological function of the protein was confirmed in vitro and evaluation of mGM-CSF gene transfer efficacy in murine aPAP model is ongoing.Abstract S120 Figure 1Comparison of biological function of murine (m) GM-CSF produced after lentiviral-gene transfer and purchased purified protein (red: mGM-CSF produced through gene transfer, black: purchased mGM-CSF protein).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.