Establishment of TBE virus as a vector for gene transfer

Tick-borne encephalitis (TBE) virus is a human pathogenic member of the virus family Flaviviridae, which also includes other important human pathogens such as yellow fever virus, Japanese encephalitis virus, West nile virus and hepatitis C virus. We intend to develop strategies to utilize this virus for medical purposes, such as gene therapy of hereditary disorders, anti-cancer therapy, and therapy of chronic infections such as HIV (AIDS). To achieve this, the ability of this virus to cause disease must be abolished, e.g. by destroying its capacity to form infectious particles and to spread in the body. At the same time its intrinsic property to invade certain cells and introduce genetic material in these cells should be maintained and thus utilized to efficiently transfer the genes that code for therapeutic or prophylactic proteins into the desired cells. Similar approaches have been developed for viruses of other viral families and in some cases have already reached the stage of clinical testing in humans. However, the suitability of TBE virus for gene transfer has so far not been examined, although many of its properties (e.g. a small RNA genome, neurotropism, low cytopathogenicity) suggest it to be quite advantageous for this purpose. In this project we will perform basic experiments to investigate whether in principle TBE virus could be utilzed for such medical purposes and what the specific advantages or possible drawbacks of this system may be. These studies should lay the groundwork for future developments that may finally lead to TBE virus-based therapeutic or prophylactic agents.

The overall goal of this project was to establish a molecular tool for research and medicine based on a human pathogen, tick-borne encephalitis virus (TBEV). Viruses comprise genetic information engulfed in an envelope which consists of proteins and in some cases also lipids. This envelope protects the genome inside and serves to introduce these genes in a very specific and efficient manner into appropriate host cells, which will express and multiply the genetic information and package it into new viral particles. Viruses are not alive, but rather can be considered as natural gene transfer vehicles, which evolved to very efficiently and specifically transport genetic information from cell to cell. We wanted to find ways how to utilize the particular properties of TBEV for scientific and medical purposes. In both research and clinical applications, it is often desirable to specifically introduce genes into host cells to study the products of such genes or to achieve a therapeutic effect. Gene therapy is a promising approach to hopefully cure various hereditary disorders as well as cancer or infectious diseases in the future. In the course of the project we successfully modified the TBEV genome by genetic engineering in a way to be useful for transferring a desired gene into the host cell. Specific manipulations generated a viral vehicle which could be assembled in the laboratory, but had lost its ability to spread and cause disease. Methods were developed which enabled us to activate such a vehicle on demand in the laboratory. The properties of our TBEV-based vehicle were tested side-by-side with another viral vector and this comparison indicated that our approach resulted in a more sustained response. Research on various activating mechanisms involving specific cleavage events of viral components shed light on fundamental aspects of virus assembly and revealed a previously unknown mechanism by which TBEV can invade its host cell. Research frequently opens new and unexpected avenues. In the case of this project, our work led us to the establishment of a new technology, - for which a patent application has been filed by our university - by which the efficiency of various viral gene transfer vectors can be improved.