Evaluating the impact of coagulation factors on gene delivery using pseudotyped adenoviruses: receptor usage bioavailability and immunogenicity

The use of viruses to delivery genetic therapy has been widely tested in the pre-clinical setting and some clinical progress has been made. Adenoviruses, a very commonly used type of virus for this procedure, shows excellent potential since it can achieve very efficient gene delivery to cells in cell culture systems but in the body it shows some toxicity that limits its application, particularly when delivered through the bloodstream. The most commonly used adenovirus is derived from serotype 5 but there are many more serotypes that are currently under evaluation. The use of adenoviruses derived from other serotypes is useful for delivery of cells to tissues that are refractory to gene transfer mediated by the traditional serotype 5 form. Of the virus proteins on the surface of the virus (the virus capsid) the fiber protein (that projects from the capsid surface) is the main determinant of how a virus interacts with receptors on target cells. Many serotypes bind different receptors therefore one can exploit this profile to engineer gene delivery systems for specific applications e.g.in cancer, cardiovascular disease, or for vaccination. When delivered via the bloodstream adenovirus serotype 5 vectors predominantly infect liver and spleen and we have recently identified a new pathway that dictates this infectivity profile - essentially the virus binds very tightly to a distinct family of coagulation factors that circulate in the blood and this interaction targets the virus to specific receptors in the liver and spleen. This interaction is mediated by the fiber protein. We now show that this is a very common pathway in that many other fibers from different serotypes of adenovirus also bind these coagulation factors. This has broad implications for the use of these alternate adenoviruses as gene delivery vectors in gene therapy applications and characterisation of this is the central theme of our current grant application. We have worked with Crucell, a biotechnology company based in the Netherlands, to assess the influence of the coagulation pathway on adenovirus infectivity on viruses being developed for cancer, cardiovasccular disease and vaccination (adenoviruses derived from subgroups B and D mainly but also from A and F) and have preliminary data showing that (A) coagulation factors bind directly to each adenovirus, that (B) this influences in vitro infectivity of liver-derived cells lines and (C) pilot data in vivo to show that modulating coagulation factor levels in the blood strongly influences the distribution of the viruses when delivered intravenously. In this study we therefore wish to characterise fully the influence of this pathway on the biology, infectivity and toxicology of these alternate adenoviruses using a range of in vitro and in vivo experiments. Description of this will broadly impact researchers and clinicians working on the use of these viruses in gene therapy and vaccination procedures and further our understanding of adenovirus-host interactions that strongly dictates the use of these viruses in the clinical setting. Ultimately, we believe that modulating the coagulation factor:virus interaction will improve gene delivery profiles in vivo and severely reduce the dose limiting toxicity observed to date in some adenovirus clinical trials using systemic delivery of virus. In turn, this will make the adenovirus vector a more attractive virus for clinical applications.

Adenovirus vectors are very powerful tools for in vitro and in vivo gene delivery. Their use in vivo in the clinical setting is limited by dose-limiting toxicity and a lack of knowledge of how the virus interacts with the host. We recently showed (Parker et al., Blood Oct 15th 2006) that coagulation factors VII, IX, X and protein C, all with a homologous domain structure (gla-EGF-EGF-SP) can mediate an increase in hepatocyte transduction by serotype 5 adenoviruses in vitro. We also showed that FX plays a fundamental role in liver cell infection following intravenous administration of virus. Adenovirus pseudotyping (producing Ad5 vectors with fibers from alternate serotypes) is being utilised to target adenoviruses to other cellular receptors and improve delivery to vascular tissue and cancer cells, for example, as well as for vaccination programs. How these viruses interact with the host in vivo is therefore very important for development of these viruses through to clinical usage. We provide extensive supporting data for this grant to show that all viruses with alternate fibers (from groups A, B, D and F) bind directly to FX and that FX binding shows a strong influence on cell infection in vitro and kinetics of the respective adenovirus in vivo following systemic administration into mice. We therefore believe that these coagulation factors (especially FX) play a pivotal role in the host interactions and cell infectivity of these pseudotyped adenoviruses. In this grant we wish to fully characterise this since it has very important implications for how these alternate adenoviruses are used in the clinical setting. We will use a range of in vitro studies to assess cell infectivity in the presence and absence of coagulation factors and the influence on targeting of each virus to their primary receptors. In vivo we will assess this using warfarinised mice and determine the influence of blood clearance, toxicity, inflammation and interactions with blood cells. Joint with BB/E020844/1 and BB/E021301/1