Supervisor: prof. Andres Merits (University of Tartu)
Opponent: Cristian Smerdou, PhD
Gene Therapy and Hepatology Unit of the Center For Applied Medical Research CIMA Pamplona, Spain
Alphaviruses are small positive-strand RNA viruses that make up the genus Alphavirus of the family Togaviridae. Alphaviruses infect a large variety of cell types within organisms. This property makes it possible to use these viruses to develop replication-competent vectors that may have clinical applications.
This thesis is based on three studies dedicated to the development and analysis of infectious DNA/RNA layered clone of SFV4 and a panel of vector constructs based on this clone. The first aim was identifying and subsequently eliminating the reasons for the instability of pSP6-SFV4 in bacterial cells. It was found that the instability of this plasmid was caused by the toxic effects of SFV envelope proteins that were cryptically expressed in bacterial cells. The problem was eliminated by interrupting the corresponding reading frame with an intron; this manipulation proved to have no adverse effect on the SFV4 rescued from the obtained DNA/RNA layered vector.
A central problem hampering the medical use of alphavirus vectors is concern over their safety issues associated with the fact that alphavirus-based vectors are very difficult to regulate and control. Therefore we were designed, constructed and tested DNA/RNA layered vectors, in which the rescue of replication-competent RNA genome of SFV was efficiently inhibited by aberrantly spliced introns and the delivery of splice-switch oligonucleotides completely or almost completely restored the infectivity of the vector.
Using cellular miRNAs to target the genomes of recombinant alphavirus may also represent a viable method to control rescue as well as replication and spread of the vector. It was found that repression of virus replication can be achieved by the insertion of different types of miRNA targets, including those naturally occurring in cellular mRNAs.