STRATEGIES FOR ENHANCING VIRAL GENE TRANSFER AND THE THERMOSTABILITY OF VIRAL VECTORS IN VACCINE APPLICATIONS
Tesi di Dottorato
Data di Pubblicazione:
2015
Citazione:
STRATEGIES FOR ENHANCING VIRAL GENE TRANSFER AND THE THERMOSTABILITY OF VIRAL VECTORS IN VACCINE APPLICATIONS / M. Pelliccia ; added supervisor: S. Krol ; internal advisor: P. Milani ; external advisor: S. Molly ; supervisor: F. Stellacci. Università degli Studi di Milano, 2015 Mar 18. 25. ciclo, Anno Accademico 2013. [10.13130/pelliccia-maria_phd2015-03-18].
Abstract:
At the most basic level viruses are biological nano-containers constituted by genetic material enclosed in a protein shell, capsid. A peculiar feature of viruses, both bacterial and some eukaryotic viruses, lies in the high packaging density of the genome in order to fit itself in the small capsid and hence the high internal osmotic pressure. Virus is a relatively stable particle equipped with fascinating mechanical properties of the capsid that are crucial for the virus lifecycle. Viruses have only one purpose: infect a host cell for reproducing themselves in order to generate new viral progeny (Roos et al. 2007). Therefore, the first and foremost consideration arising from the concept of virus reflects its pathogenesis and virulence that can ultimately result in many important infectious diseases such as common cold, influenza, hepatitis, rabies, measles, cancer and AIDS. As a consequence, pathogenic viruses represent a heavy hurdle for the global health and there is a strong need for developing robust strategies such as vaccines or antiviral drugs against virus infections (Baram- Pinto et al. 2010). On the other hand, viruses in the course of evolution have become efficient specialized gene delivery agents. Therefore they represent powerful tools in biomedicine for gene therapy and vaccine purposes (Schaffer et al. 2008). For successful gene therapy and immunization programs, the efficiency and stability of viral vectors are fundamental aspects (Jorio et al. 2006).
To address this challenge, in the present research project we have investigated the interaction between viruses and nanomaterials. In the last years materials on the nanoscale for their unique properties have provided a broad range of potential biomedical uses (Verma et al. 2008) and for that reason we decided to explore their application with viruses. More specifically, we have examined three types of sulfonate- functionalized gold nanoparticles (AuNPs), namely, MUS:OT, MUS and MUS:brOT NPs, which are less than 5 nm in size, negatively charged and poorly cytotoxic (Verma et al. 2008). The NPs are coated with self-assembled monolayer (SAM) of thiolated organic molecules and one of the ligand is a sulfonated molecule, MUS (Verma et al. 2008). The MUS ligand itself was tested in our experiments as well. As virus models we focused on human recombinant adenovirus type 5 (Ad), one of the most promising viral vector as vaccine and gene therapy carrier and two picornaviruses of the genus enterovirus, namely, EV1 and CVB3, important human pathogens associated with several infectious diseases (e.g. myocarditis, aseptic meningitis, encephalitis, paralysis)(Kossila et al. 2002)(Marjomäki et al. 2014a). In spite of their medical impact, there are no therapeutic treatments available against picornavirus infections and the only vaccine products are against three types of poliovirus and hepatitis A virus (Merilahti et al. 2012).
Two sets of experiments were carried out: (1) Short-term incubation of Ad with nanomaterials for 1 h at 37°C prior
transducing HeLa cells or before in vivo administration in zebrafish and mice. The results demonstrated that Ad shortly pre-treated with nanomaterials showed a significant increase in the gene expression in vitro and in vivo The NPs’enhanced adenovirus transduction aims to reduce Ad vector doses in vivo thereby minimizing the adverse reactions of the immune response due to high vector dosage; (2) Long-term thermostabilization studies of Ad, EV1 and CVB3 in vitro in the presence and in the absence of our nanomaterials and other substances such as sugars (sucrose, glucose, glycerol) and Polyethylene glycol (PEG) molecules at 37°C or room temperature for extensive periods of time. Our results showed the capability of the nanomaterials and
Tipologia IRIS:
Tesi di dottorato
Keywords:
Viruses, osmotic pressure; gene therapy; vaccine; cold chain; flow cytometry; gene transfer, viral genome; adenovirus; enterovirus; thermal stability; enhancement; sucrose; functionalized gold nanoparticles; electrostatic interactions; hydrophobic interactions; viscosity
Elenco autori:
M. Pelliccia
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