411. Trapping of Adenovirus Vectors in Blood

Abstract

In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (|[sim]|2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.