RECOMBINANT ALPHAVIRAL VECTORS
In order to create a viral vector from alphaviruses, the structural genes are replaced with the therapeutic transgene. As a consequence, during vector production the structural genes have to be provided in trans by co-transfection with a helper plasmid (see diagram two).
Diagram Two: Alphavirus Vector
One of the limiting factors in alphaviral vector production is that the viral replicase inhibits the activity of RNA polymerase II, hence the viral RNA genome is first transcribed in vitro prior to its introduction into producer cells by electroporation. Typically, both the recombinant vector genome expressing the therapeutic gene and the helper virus are transcribed using SP6 polymerase and transfected into BHK21 cells. In order to enhance safety, the helper virus contains a mutation in a viral glycoprotein so that recombinant viral particles require activation by an external protease prior to their use. Alphaviruses are very efficient at taking over the machinery that controls host protein synthesis, and as such over 90% of the protein translated in an infected cell is viral. The result is that cells infected with recombinant alphaviral vectors die after three to four days, thus limiting their application in gene therapy. Despite of this alphaviruses can be successfully applied in the production of recombinant protein from mammalian cells in vitro, often generating enough protein for x-ray crystallography studies.
