Directed evolution of artificial viruses

It is long known that viruses can selectively deliver their genetic payload to cells. This has spawned a huge number of investigations into the use of viruses as vectors for gene therapy. Even though some marginal successes have been achieved, a lot of hurdles still have to be overcome before gene therapy can reach its full potential. In the past researchers have tried to tackle these problems through the rational design of such carriers; an approach that as of now has not been very successful. Instead of rationally altering the viral coat protein to achieve changes in e.g. their tropism, we used high-throughput combinatorial techniques to create a very large library (>109 clones) of random mutants. By applying selective pressure on this library, only those clones which possess desired properties remain. This way, the process resembles natural evolution, with the exception that we can choose what to select for. We give a proof of principle for this method using polyomavirus VP1 virus-like particles (VLPs) as model delivery system. VLPs are aggregates of viral structural proteins. They resemble the native viral capsid in structure, tropism and transduction efficiency, but do not contain any viral genetic material. One class of promising VLPs is those derived from polyomaviruses. The polyomavirus coat protein VP1 is one of the three structural proteins of the virus. After overexpression, VP1 proteins self-assemble to form VLPs. These VLPs are able to encapsidate double-stranded DNA in a sequence independent fashion and transfect mammalian cells, show good immune evasion, and have a high insert capacity for peptides. This makes them a good starting point for directed evolution. To combine the beneficial properties of different polyomaviruses, we recombined the VP1 DNA sequences of different polyomavirus species through a technique called DNA shuffling to form novel polyomavirus VP1 hybrids. A vast library of hybrid VP1 genes was created, with an average of 5.7 crossovers per VP1 gene. These libraries are expressed in eukaryotic 293TT cells while supplying SV40 VP2/3 and large T antigen in trans. From these cells hybrid VLPs are purified. These VLPs have been subjected to selections based on packaging and transfection. As a proof of principle we demonstrate an enrichment of the wild-type VP1 gene from an excess of non-functional mutants, showing the potential of this system.