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Of mice and men
S120 Gene therapy for pulmonary alveolar proteinosis
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  1. N Atsumi1,
  2. A Pilou1,
  3. I Pringle2,
  4. RC Ashworth2,
  5. C Meng3,
  6. M Chan3,
  7. DR Gill2,
  8. S Hyde2,
  9. C Morgan4,
  10. EWFW Alton1,
  11. U Griesenbach3
  1. 1Department of Gene Therapy, NHLI, Imperial College London and UK Gene Therapy Consortium, London, UK
  2. 2Radcliffe Department of Medicine, Oxford University and Gene Therapy Consortium, London, UK
  3. 3Department of Gene Therapy, NHLI, Imperial College London and Radcliffe Department of Medicine, Oxford University, London, UK
  4. 4Royal Brompton Hospital, London, UK

Abstract

Introduction Pulmonary 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 Results To 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).

Conclusion rSIV.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 1
Abstract S120 Figure 1

Comparison 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).

https://doi.org/10.1136/thoraxjnl-2017-210983.126

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