Adenovirus and coagulation factor interactions and the impact on virus stability and utility for gene therapy

Adenoviruses are a family of related viruses which cause mild infections in people and which have been converted into gene transfer vectors by blocking their ability to replicate in cells. This is achieved by crippling the ability of the virus to cause infection by deleting some if its DNA. The most common member of the adenovirus family used is adenovirus serotype 5 (Ad5). This allows the adenoviral vector to be used for gene therapy (the use of genes to treat human disease). In fact, adenoviruses are one of the most common vectors in use in gene therapy clinical trials because they are easy to work with, safe and are efficient at delivering genes to cells and tissues in the body. Depsite their common use we are still discovering new properties relating to this virus which highlights how important it is to understand it better in order that gene therapies can be made safer and more efficient (i.e. to optimise their use to treat patients). We made a recent discovery that when injected into the bloodstream adenovirus serotype 5 becomes coated in one of our own proteins in the blood called a coagulation factor. Interestingly, the coated virus then only delivers genes to the liver via the interaction with the coagulation factor and not a direct interaction between the liver and the viru (i.e. the coagulation factor acts as a bridge between the cell and the virus). The coagulation factor "sticks" to a specific protein on the coat of the virus called the hexon. Some other members of the adenovirus family also do this while others do not and the reason why this interaction has arisen is not well understood. Other groups have also suggested that either the host coats the virus with FX in order to help the immune system recognise it and remove it, while others have suggested that the virus itself "picks up" the coagulation factor to protect itself from the immune system. In this project we are going to investigate this important pathway further. We want to know whether the interaction is protective or not for the virus and if it is, whether the protection can be conferred to other viruses which do not bind to the coagulation factor. This will lead us to an understanding of how the virus and coagulation factor interact with different parts of our immune system. We also want to know exactly how the coagulation factor delivers the virus to the liver and whether modifying this pathway can lead to the development of gene therapies which are more efficient at delivering to other tissues in the body other than the liver, and this will broaden the use of the virus for treating patients through gene therapy. We will investigate these areas of adenovirus research by making changes to different parts of the virus's coat in order to allow or block the interactions and we will use genetic engineering approaches to achieve these important modifications. These experiments will help our further understanding of adenoviruses and ultimately lead to more optimal and safe vectors for all adenoviral gene therapy approaches.

Adenoviral vectors, particularly human serotype 5 (of 58 individual serotypes), are widely used for gene therapy due to versatile tropism, large transgene capacity and ability to achieve efficient transgene without risk of insertional mutagenesis. Efficient delivery of the virus is the major factor restricting further progress of many important new adenoviral gene therapies, largely because certain properties of the vector have not been optimised for gene transfer. Host interactions govern infectivity of the virus, and subsequent responses dictate efficiency of gene therapy and safety. The major Ad5: host interaction is with coagulation factor X which binds Ad5 hexon protein hypervariable regions (HVRs) leading to liver transduction following intravascular delivery. While many adenoviral serotypes interact with FX, others do not and the reason for the evolution of this interaction is unclear. Controversially, it has subsequently been suggested that Ad5: FX interaction can either protect from or prime virus for innate immune system-mediated clearance. Here we propose to investigate and dissect the true role for Ad5: FX interaction. We will study biodistribution and transduction of Ad5 and Ad5 ablated for FX binding in both immunocompetent and immunocompromised mice. To fully understand this interaction we will assess the role of other capsid proteins, fiber and penton base by using a range of chimeric adenoviral vectors. By generating truncated chimeric FX molecules we will pinpoint the exact liver transduction mechanism and test whether a "mini FX" molecule can be fused to a targeting ligand to redirect Ad tropism while preventing innate immune interactions. We will also assess whether engineering Ad5: FX interacting hexon HVRs into a non-FX binding serotype modulates immune responses. This work will lead to definitive understanding of the Ad5: FX interaction and highlight whether it can be harnessed to improve adenoviral gene therapy efficiency and safety.

The beneficiaries are far ranging. First, the adenovirus research community, both basic (working on basic mechanisms of adenovirus or gene therapy/ vaccination) and clinical (immunologists treating dissemnated adenovirus infections as well as those involved in gene therapy/ vaccination clinical trials) will benefit from increased knowledge regarding the interactions of adenovirus with the host, in particular the immune system. The work will further our basic knowledge of adenovirus and provide optimal vectors for future gene therapy applications. This may well be through a direct translational route partnered with clinical researchers in the NHS as adenoviral gene therapy is already in the clinic.

The PDRA working on the grant will gain broad skills directly related to many areas of modern research including molecular, virological, biochemical and in vivo skills. Their attendance and presentation at conferences will directly benefit their core skills in presentation, communication and collaboration. Working within a individual project within a research group will foster their skills in both independent and team working. Since the PDRA will be expected to design experiments and plan their work (in liaison with the PI and other co-Inv) they will develop time management and leadership skills. In liaison with the PI the PDRA will also be expected to be mindful of designing and delivering the project within a defined budget and timescale, clearly developing their project management and financial control skills.

Second, commercial enterprises exploring adenovirus for gene therapy and vaccination (e.g Crucell) may be interested in the medium term in exploration of the technology generated in this grant to improve future applications of adenovirus.

In the long term this research has the potential to lead to studies which directly impacts patients' lives. This could be envisaged to be on two levels. It may lead to a better understanding of how adenovirus interacts with the host and this may help in the setting of treating disseminated adenoviral infections. Second, commercial companies may be interested in new optimised adenoviral vectors. As gene therapy is growing more such areas may be of interest to both large pharmaceutical companies and small to medium size biotechnology companies.