Miselli, Giuseppina (2020) The jurona rhabdovirus as a viral vector platform. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: The jurona rhabdovirus as a viral vector platform
Miselli, GiuseppinaUNSPECIFIED
Date: 2020
Number of Pages: 79
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Dottorato: Medicina molecolare e biotecnologie mediche
Ciclo di dottorato: 32
Coordinatore del Corso di dottorato:
Avvedimento, Vittorio Enricoavvedim@unina.it
Nicosia, AlfredoUNSPECIFIED
Date: 2020
Number of Pages: 79
Keywords: Rhabdoviruses, viral vectors
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Date Deposited: 25 Mar 2020 12:02
Last Modified: 05 Nov 2021 13:03
URI: http://www.fedoa.unina.it/id/eprint/13216

Collection description

Recombinant viral vectors represent a powerful emerging technology for the development of novel classes of biologics to prevent or treat human diseases. Replication competent viral vectors with natural or engineered tumor selectivity (Oncolytic Viruses, OV) have reached different levels of clinical development with one product (Talimogene Laherparepvec) being approved for the treatment of Melanoma. Viral vectors have also been shown to mediate efficient in vivo transduction of therapeutic genes, thus paving the way to the development of a number of gene therapy products or genetic vaccines aimed at eliciting specific immune responses against the encoded proteins. In this PhD project, we aimed at generating a new recombinant viral vector platform from a virus of the Rhabdoviridae family that could be used as oncolytic virus, or as a vectored vaccine as well as being amenable to large scale manufacturing using an industrial process. For this purpose, we screened five Rhabdoviruses from the American Type Culture Collection (ATCC) for their growing properties on Vero cells. Our screening identified the Jurona virus as the best candidate for vector construction. We established a reverse genetic system based on the T7 RNA polymerase (T7 RNAP) expression helper virus-free method to recover Jurona recombinant viral particles from a cDNA clone of the viral genome. We designed and cloned five plasmids by using various cloning methods: the full Jurona anti-genome, the T7 RNAP, and three helper viral proteins (Nucleocapsid (N), Phosphoprotein (P), and Large polymerase (L)). Following initial failure to rescue the Jurona vector, we used an end-joining-RT PCR sequencing strategy to identify a previously unknown leader sequence in the Jurona genome. By introducing this new sequence in our Jurona full-length constructs, we successfully rescued the Jurona vectors using the reverse genetic system. We showed that Jurona is capable of efficient expression of a heterologous gene upon infection of target cells and of potent oncolytic activity in vitro. Finally, we demonstrated that the Jurona vector could be efficiently produced in Vero cells, a validated cell line for vaccine production, and that it could be purified without losing infectivity. Thus, the Jurona vector platform may represent a novel valuable option for oncolytic virus and genetic vaccine development, to be used alone or in combination with other compounds for the development of highly effective immunotherapeutic treatments.


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