Interrogation of the adenovirus hexon: A new paradigm for virus biology and application to therapeutic gene delivery

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Gene therapy is rapidly developing into an effective method to cure or halt the progression of many human diseases. The first gene therapy products have been licensed recently in China (for cancer therapy). Therefore, the use of genes to treat human disease is a realistic opportunity to provide efficient and lasting therapies, especially those diseases that lack optimal drug-based therapies. An absolutely critical aspect of gene therapy is gene delivery, i.e. the method that is used to deliver the therapeutic cargo to the correct cells and tissues in the body. Gene delivery is achieved by a 'vector' that has the capability of transferring the therapeutic DNA into cells - these vectors can either be non-viral or viral-based. Adenovirus vectors are commonly used as a viral-based delivery system and are currently utilised in some 25% of all ongoing clinical trials in gene therapy. The advantages of these viruses is that they have the ability to transfer genes to a broad range of cells, hence this makes them suitable for a wide range of applications to gene therapy. However, despite the extensive use of this virus in the clinic, basic mechanims that define how the virus actually infects cells have not been unravelled. In cell culture the virus binds to a defined receptor (called the coxsackie and adenovirus receptor, CAR) and enters the cell by interaction with proteins called integrins. However, when injected into the bloodstream we recently showed that the virus actually uses proteins from the blood to infect cells rather than a direct interaction of the virus with the target cells in the liver. The viruses recruit coagulation factor FX (FX) to a protein on the virus surface called the hexon and this virus:FX complex is responsible for cell binding through a domain on the FX protein (the serine protease domain). This is a critical new concept for adenovirus biology and the application of this virus to gene therapy in humans. The interaction of FX with the hexon was not expected and has assigned an important new function to this virus protein in mediating infectivity of the virus. Our study is therefore designed to fully document this novel interaction by using high resolution techniques (crystallography) and through the molecular analysis of hexon mutants to generate viruses that are devoid of FX binding capacity. We will also interrogate the interaction with FX. One of the major issues with adenovirus vectors is that many humans have been exposed to the virus in nature and thus have pre-existing immunity to the virus. A substantial proportion of this is against the hexon protein and hence we will assess the impact of FX and the adenovirus hexon mutants on the ability of neutralising antibodies to block infection. Our aim is to create vectors devoid of FX binding and devoid of neutralising capacity. This will create a new generation of adenovirus vectors for wide ranging applications to human gene therapy.