| Popis: |
Gene therapy with human adenovirus type S (AdS) has been extensively explored for the treatment of diseases resistant to traditional therapies. Following intravenous administration, Ad is rapidly cleared from systemic blood circulation with a half life of 2 minutes, and more than 99 % of the injected dose is sequestered in the liver. The resulting innate and adaptive immune responses dramatically affect the kinetics and toxicity profile of the vector. These issues currently restrict the use of Ad-based vectors, particularly for clinical gene therapy protocols that involve systemic administration. We propose that such limitations can be improved by engineering artificial lipid envelopes around Ad. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid. Zwitterionic and cationic lipid formulations can efficiently envelop Ad, however this resulted in a significant reduction of gene expression in vitro due to the inadequate escape of the enveloped virus from the endosomal compartment. In this thesis, pH-sensitive lipid-envelopes to enhance the virus release from the endosome following endocytosis were explored. Also, different viral envelopment methodologies (sonication and extrusion) were compared in terms of percentage of virion envelopment and efficiency of gene expression. The artificially enveloped Ad were characterised physicochemically by dot blots, dynamic light scattering and atomic force microscopy. Biologically, the gene expression of enveloped Ad in different pH-sensitive enveloped Ad was studied in vitro and in vivo. The critical role of blood components during systemic administration of Ad was investigated by looking at the interaction of enveloped Ad in cationic, non-pH-sensitive (DOTAP:Chol) or anionic, pH-sensitive (DOPE:CHEMS) lipid bilayers with several different blood components. When Ad was enveloped by cationic bilayers, significantly high levels of viral uptake in HepG2 cells were achieved, independent of any blood coagulation factor, whereas, the levels of cellular uptake and gene expression were similar to naked Ad vectors when an anionic lipid envelope was used. In vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration into mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction compared to anionic envelopes. AL T / AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. Furthermore, envelopment in DOTAP:Chollipid bilayers significantly increased lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. These results suggest that artificial envelopes for Ad significantly alter the interactions with blood components and divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity. Finally, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within lipid bilayers. Such a dual-activity vector can possibly offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, ribo-green assays and dot blots showed that siRNA and Ad can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. In summary, the studies in this thesis contribute to greater understanding of the mechanisms, obstacles and opportunities offered by artificial lipid envelopment of Ad and how these affect the biological activity of these promissing gene therapy vectors in vitro and in vivo. |
