171. Production of Therapeutically Relevant Levels of FVIII After Transduction of Lungs With F/HN-Pseudotyped Lentivirus

Abstract

We have previously shown that the lung can be used as “factory” for production of secreted proteins, which are released into the circulation, using Sendai virus-mediated gene transfer. However, gene expression was transient and repeated administration of the vector was not feasible due to induction of immune responses. We have developed a lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN) to allow efficient transduction of the airways. The unique features of rSIV.F/HN, namely stable expression for >20 months after a single dose and efficient transduction after repeated administration, make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV.F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n=5-6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p<0.01) levels of GLux in serum (274±72 RLU/ul, control: 41±6 RLU/ul) which persisted for at least 12 months further supporting the hypothesis that the lung is a suitable, non-invasive factory for production of secreted proteins. Gene therapy strategies for haemophilia have focussed on intravenous or intramuscular delivery of the gene transfer agent. Here, we treated the murine lung with rSIV.F/HN carrying the FVIII cDNA (1.6e8-3.4e8 TU/mouse, n=3-4/group) or placebo and assessed whether therapeutically relevant levels of FVIII can be produced. Significant (p<0.05) and dose-related levels of FVIII were detectable in lungs and BALF 10 and 28 days post-transduction. Dose-related levels of FVIII were also detectable in plasma, which reached a therapeutically relevant level of 3.0% of normal 1 month after gene transfer. These data support the concept that rSIV.F/HN-mediated transduction of lungs can produce therapeutically relevant and persistent levels of recombinant protein in blood.