Administration of mRNA alleviates disease biomarkers in a mouse model of pulmonary alveolar proteinosis (PAP)

Autoimmune (a)PAP is a lung disease caused by accumulation of surfactants in the alveoli. Autoantibodies against GM-CSF prevent macrophage clearing surfactants. Lentiviral vector-mediated GM-CSF expression can correct biomarkers in GM-CSF knockout mice. However, the toxicity: efficacy window was narrow due to prolonged over-expression of GM-CSF. Transient expression of GM-CSF through mRNA delivery may improve this window. The cationic lipid GL67A has been safely used in clinical trials. We established the optimal ratio of mRNA to GL67A in in vitro experiments using firefly luciferase mRNA. Molar charge ratios of 1:1, 3:1 and 4:1 (mRNA:lipid) led to high-level gene expression and were subsequently assessed in mouse lung in vivo. Mice (n=5/group) were transfected with 10µg RNA encoding secreted Gaussian luciferase (GLux) and culled 24hr post-treatment. Control animals were treated with 80µg plasmid carrying the GLux cDNA complexed to GL67A using our established formulation of 4:1 (pDNA:lipid). A molar charge ratio of 3:1(mRNA:GL67A) was well tolerated and GLux expression following mRNA transfer was similar to pDNA in bronchoalveolar lavage fluid (BALF) (mRNA:1817±107,pDNA:2727±846 RLU/μl BALF). We next administered GM-CSF knockout mice with GM-CSF mRNA or GLux mRNA (n=8/group) complexed to GL67A(5 doses at weekly intervals, 10μg/dose). The mice recapitulate biomarkers of PAP disease including BALF turbidity. 4 days after the last dose, mice treated with GM-CSF mRNA had reduced (p<0.05)BALF turbidity (GM-CSF: OD₆₀₀ₙₘ5.74±0.33, control: OD₆₀₀ₙₘ2.93±0.29). In summary, mRNA/GL67A complexes transduce mouse lung efficiently and ameliorate a disease biomarker in PAP mice. Future studies will focus on analysis of additional biomarkers and assessment of toxicity.