370. F/HN Pseudotyped Lentivirus Generates Therapeutically Relevant and Long-Lasting Alpha-1-Antitrypsin Expression in Mouse Lung

Abstract

A protease/anti-protease imbalance is a characteristic feature of inflammatory lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). However, alpha-1-antitrypsin (AAT) enzyme replacement therapy (ERT) trials have not shown conclusive evidence of a therapeutic benefit. This may be due to inefficient protein delivery to the lung and/or the half-life of the protein. The UK Cystic Fibrosis Gene Therapy Consortium (GTC) has pseudotyped a simian immunodeficiency viral vector with the Sendai virus F and HN proteins (rSIV.F/HN) and selected an internal synthetic promoter/enhancer consisting of the elongation factor 1α promoter and the CMV enhancer (hCEF) for efficient transduction of airway epithelial cells. Here, we assessed whether transduction of murine lungs with rSIV.F/HN with a human AAT transgene generates therapeutic levels of AAT. Mice were transduced with rSIV.F/HN-hCEF-AAT (2e8 TU/mouse) by nasal instillation and culled 10 days after transduction. AAT levels in lung homogenate and broncho-alveolar lavage fluid (BALF) were at least 3 logs above controls (p<0.05). A urea assay was used to determine that lavage diluted the epithelial lining fluid (ELF) by ≈40-fold, which allowed us to calculate the AAT concentration in ELF to be 92±28 mg/ml (= 1.4 μμ). In the context of AAT deficiency, an ELF concentration of 70 mg/ml (1.1 μM) AAT is of therapeutic relevance. For comparison transfection of mouse lung with the cationic lipid GL67A complexed to pCMV-AAT only led to 0.4±0.1 mg/ml (n=6) of AAT in ELF. A neutrophil elastase (NE) activity assay showed that the recombinant AAT successfully neutralised NE activity (p<0.05). In a separate experiment, mice were treated with rSIV.F/HN-hCEF-AAT (4e7 TU/mouse) and quantification of AAT 7 and 90 days post-transduction showed that expression was stable over this period. We also transduced mice with rSIV.F/HN-hCEF carrying a secreted Gaussia luciferase reporter gene (1e7 TU/mouse) and showed that stable expression in lung and BALF persisted for at least 12 months. Here, we also demonstrate for the first time that rSIV.F/HN transduction of lung generates significant (p<0.05) levels of GLux and AAT recombinant proteins in serum. AAT enzyme replacement therapy is currently being assessed for the treatment of a range of diseases including diabetes and graft-vs-host disease, but shortage of human plasma-derived protein, as well as high costs, may limit the application. In conclusion, rSIV.F/HN produces therapeutically relevant and long lasting levels of AAT in murine lung. In addition, we showed that AAT escapes from the lung into the circulation, suggesting that gene therapy may help to overcome some of the current bottlenecks of ERT.