474. Deletion of All Membranous Genes of Recombinant Sendai Virus Results in the Dramatic Reduction of Toxicity for Pulmonary Gene Transfer in Neonatal Mice

Abstract

Top of pageAbstract Background and Aim: We previously demonstrated the dramatically high efficiency of recombinant Sendai virus (SeV) for pulmonary gene transfer, 1,000-100,000 fold over that via adenoviruses, and suggested that its possible application for gene therapy of cystic fibrosis (CF) (Yonemitsu Y et al. Nature Biotechnol 2000). As widely known, however, experimental inoculation of SeV exhibits inflammatory and immune responses in rodent lung, frequently resulted in the death of animals. For future directions of pulmonary gene therapy for monogenic diseases, including CF, neonatal gene transfer is ideal, because the lung disease may be in reversible. We recently developed a new and advanced design of SeVs for clinical application, namely F-deficient SeV (SeV/dF), temperature-sensitive SeV/dF (tsSeV/dF), and SeV deleted with all membranous genes (SeV/dFdMdHN). Here we characterized these new constructs for pulmonary gene transfer in neonatal mice, especially referring to toxicity. Method and Results: Recombinant SeVs expressing GFP (2.5 |[times]| 10|[and]|6 pfu in 10|[mu]|l PBS /neonate or 2.5 |[times]| 10|[and]|7 pfu in 100|[mu]|l PBS /adult) was placed into the nasal cavity and the solution was sniffed into the lungs of neonate (day 0) or adult (8 weeks old, female) of ICR (CD-1) mice. The efficiency of gene transfer and the level of gene expression were also monitored using same amount of SeVs expressing luciferase. The peak of gene expression in lung of neonatal mice was at 48 h and decreased gradually, and then almost disappeared at 14 days after infection in all constructs, similar to the finding found in adult mice. Increase of the body weight of neonates transfected with tsSeV/dF or SeV/dFdMdHN, however, was significantly larger than those of animals transfected with wild-SeV or SeV/dF, and the effect was dramatic in use of SeV/dFdMdHN. Mortality rate was dramatically diminished in the mice translated SeV/dFdMdHN (survival rate=100%, n=43) compared with wild-SeV (25%, n=36) or tsSeV/dF (75%, n=44). Assessments of immunological parameters are now underway. Discussion and Conclusion: Our current study revealed that deletion of all membranous genes of recombinant SeV dramatically reduced the toxicity for pulmonary gene transfer in neonatal mice. In conclusion, the potential of pulmonary gene thansfer using SeV/dFdMdHN warrants for further intervention to develop more efficient therapy of neonatal lung diseases including cystic fibrosis.