782. Long-Term Expression of EIAV Lentivectors Pseudotyped with Influenza Virus Spike Glycoproteins after Nasal Delivery to Mice

Abstract

Top of pageAbstract There are multiple challenges for achieving long-term expression by lentivirus-mediated gene transfer. Potential problems include down-regulation or destruction of transduced cells due to an immune response or shut down of gene expression due to silencing. Cell- specific promoters may help alleviate some of these potential problems by restricting expression to a smaller subset of cells, or by promoting regulated expression in terminally differentiated cells. The purpose of this study was to determine how constructs containing various airway-specific promoters behave in an in vivo mouse model for airway gene transfer. Specifically, we were interested in the duration of expression following nasal delivery and whether there was an immune response to lentivectors based upon equine infectious anemia virus (EIAV). SIN vectors were constructed containing gfp, luciferase, or lacZ reporter genes driven by the human FOXJ1 promoter or the long terminal repeats (LTR) of Jaagsiekte Sheep Retrovirus (JSRV) or Enzootic Nasal Tumor Virus (ENTV). Control vectors contained a composite CMV/beta-actin (CB) promoter. Adult outbred mice were inoculated intranasally with luciferase vectors pseudotyped with influenza hemagglutinin (HA). We utilized a cooled-coupled device (CCD) camera to image the mice starting at week 2 post-infection. At week 12, some of the mice were infected with a second HA-pseudotyped vector that expressed GFP or beta-galactosidase. Blood was drawn from these mice prior to infection and at 1 and 14 weeks post-infection to determine if there was a neutralizing antibody response to HA pseudotyped vectors or to control vectors pseudotyped with VSV- G. Stable luciferase expression was observed through week 22 in mice receiving the FOXJ1-luciferase vector. Mice receiving the ENTV, JSRV and CB promoter constructs showed more variation in levels of expression. Luciferase expression was still observed in most of the mice at week 14 when all but three of the FOXJ1-luciferase mice were sacrificed. Analysis of heat-inactivated serum demonstrated that neutralizing antibodies directed against the HA pseudotyped vector were produced in mice that received two doses of at least 100,000 infectious vector particles. Neutralizing activity was not observed after a single dose of the vector. Experiments are in progress to determine vector expression levels following re-administration to mice that have developed neutralizing serum antibodies. In conclusion, we observed expression from the EIAV FOXJ1-luciferase vector for at least 22 weeks following a single administration to the nose. Stability of luciferase expression was dependent upon the promoter used to drive transcription. Expression from the FOXJ1 promoter showed the most consistency between mice and was more stable over time than the other promoters that were tested.