Thesis supervisor: Professor Ülo Langel, Professor Andres Merits (University of Tartu)
Opponent: PhD Kenneth Herbert Lundstrom
PanTherapeutics, Rue des Remparts 4, CH-1095 Lutry (Lausanne), Switzerland
The current state of tumour treatment is not perfect. The existing treatment procedures rely heavily on surgery, as a result of which the malignant mass is removed but still has a risk of recurrence. In addition, radiation or chemotherapy approaches can be applied, however both of these approaches often result in unwanted side effects in the patient without ensuring the complete neutralisation of the tumour cells. This is the reason why it is important to continue with the development of novel anti-tumour approaches which would enable to offer the patients fresh solutions to their health problems, allowing for higher treatment rate and the possibility of reduction in unwanted symptoms.
The work at hand has approached the issue of tumour therapy by exploring three systems which have been linked with each other through the potential practical application which involves the creation of an efficient and safe tumour inhibition system with a perspective for future clinical applications. In the first study, a control mechanism was proposed for alphavirus-based DNA/RNA-layered vectors, which would allow the system to be controlled via the introduction of one or more defective intron sequences placed in region corresponding to the non-structural ORF of virus genome. The rescue of infectious RNAs from these constructs was successfully inhibited to a great extent with the possibility to reverse the situation by the introduction of SCOs, which would allow to correct the aberrant splicing profile. As the intron-containing sequence must be based on a DNA/RNA-layered vector, rather than on RNA genomes, packed into a virus particle, an efficient method for transfection using these constructs is required. This issue was addressed in a study, where a promising class of delivery agents, CPPs (exemplified by the novel PepFect6 reagent), was evaluated for such application. The main goal for this research was to determine the properties of the peptide-based delivery system when in interaction with virus-based sequences. It was demonstrated that the application of CPPs for the delivery of virus-based constructs has significant potential, as the reagent managed to induce high levels of intracellular delivery together with the lack of detectable interference with the infection process or without the suppression of replication capabilities of the virus vectors and virus particles. Based on this knowledge, a CPP-based tumour-specific delivery strategy was devised for large plasmid delivery into malignant tissues. This approach entailed constructing a peptide which would be completely inactivated by a shielding molecule PEG, connected to the CPP through a MMP2-cleavable linker. This allows the transfection complexes to lay dormant upon entering the blood circulation and only be activated once encountering the specific protease, MMP2, which is greatly upregulated in tumour cells, effectively creating a tissue-specific delivery system.
Based on this research, a safe and tumour-selective approach for chemotherapy can be proposed, consisting of a controllable DNA virus and/or DNA/RNA-layered replicon vector, expressing a therapeutic transgene, which would be selectively delivered into tumour cells by the tumour-targeting CPP complexes together with the SCOs required for the activation of the vectors. Such system would enable the tissue-specific induction of cell apoptosis; in addition, enhanced resistance or inhibitory effect towards the tumour tissues can be exerted by the expression of therapeutic proteins, which will be released to organism after the termination of the transfected/infected tumour cells.