Towards the directed evolution of virus-like particles derived from polyomaviruses

Abstract

Virus-like particles (VLPs) are assemblies of viral structural proteins. These particles resemble the native viral capsid in structure, tropism, and transduction efficiency, but do not contain any viral genetic material. This makes them a safer alternative to viral vectors for gene therapy, and enables them to be used for vaccination. However, several issues, such as a limited natural tropism and pre-existing immunity, prevent most wild-type VLPs from being used clinically. Therefore, we set out to modify VLPs to improve their characteristics for gene therapy. The goal of this project was to develop an evolution-based method for the improvement of the characteristics of VLPs for gene delivery. Directed evolution provides an attractive alternative to rational design for the creation of novel vectors for gene therapy. Instead of rationally altering the viral coat protein to achieve changes in e.g. their tropism, high-throughput combinatorial techniques are used to create a very large library 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 define the selection criteria. We chose to use the VLPs derived from polyomaviruses – in particular the hamster polyomavirus (HaPyV) – as model vectors for our studies. The plan was to create vast libraries of mutant capsid genes, and from these libraries, select clones that possess improved properties for gene delivery. The expression of the libraries would be performed using a cell-free protein synthesis system compartmentalized into small, micrometer-scale emulsion droplets with on average one gene per droplet; a technique called in vitro compartmentalization (IVC). However, over the course of our research it proved to be impossible to synthesize sufficient quantities of correctly-assembled VLPs using this technique. During these studies, we investigated several issues regarding the reproducibility of cell-free expression and showed that, by normalizing the cellular extract used for cell-free expression, this variability can be reduced significantly. Despite the many conditions we tested, none of these conditions led to VLP assembly sufficient for in vitro compartmentalization. On the other hand, we discovered that the assembly of VLPs after expression in Escherichia coli already takes place inside the bacteria. However, this did not lead to DNA packaging. Therefore, we shifted our focus towards the production of VLPs in eukaryotic cells. First, we created vast libraries of hybrid VP1 genes using DNA shuffling. Next, we aimed to prove that polyomavirus-derived VLPs demonstrate genotype-phenotype linkage, i.e. package their own coding DNA, a necessity for directed evolution. As a proof of principle for genotype-phenotype linkage we demonstrated the enrichment of a wild-type VP1 gene from an excess of non-functional mutants. As the VLPs form, they package available genetic material compartmentalized within the same cell. We observed a 10-fold enrichment after one single selection step, showing the potential of this system. This thesis shows that hybrid VP1 genes can be created and that VLPs derived from polyomaviruses are able to package their own coding DNA, and can thus be used for directed evolution.