Development of baculovirus display strategies towards targeting to tumor vasculature

Abstract

Matilaisen väitöstutkimuksen tavoitteena oli kehittää bakulovirusta käytettäväksi geeniterapiaan syövän hoitoon. Bakulovirus on hyönteisvirus ja siten turvallisempi vaihtoehto kokeellisessa geeniterapiassa tällä hetkellä käytettäville patogeenisille viruksille tai niiden muunnelmille.Matilainen tutki myös virusten ja käytettyjen kohdennuspeptidien sisääntulomekanismeja ihmisen syöpäsoluihin. Tutkimuksessa löydettiin mahdollisesti bakuloviruksen uusi vaihtoehtoinen sisääntuloreitti. Virusten ja kohdennusmolekyylien reseptoreiden sisääntulomekanismien tutkimus ja tunteminen on edellytyksenä onnistuneiden geeninsiirtojen aikaansaamiselle ja täten myös onnistuneelle geeniterapialle.Matilainen käytti tutkimusmenetelminä mm. konfokaalimikroskopiaa, jossa solusta ja siellä olevista viruksista saadaan kolmiulotteinen kuva sekä elektronimikroskopiaa, jonka avulla pystytään näkemään yksittäisiä bakuloviruksia soluissa.

Targeting of viral vectors to specific cells has received an increasing interest in the area of therapeutic gene transfer. However, specific targeting to tumors, especially to tumor vasculature has been impeded by the limitations of present vector systems and lack of selective markers. Therefore, peptides that home to specific sites in the tumor vasculature or tumors, are attractive as targeting moieties for gene therapy purposes. Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype of the Baculoviridae family, is a promising new vector candidate for gene therapy. Moreover, baculovirus display -technology enables the presentation of targeting moieties on the viral surface. The goal of this study was to develop baculovirus vectors for targeted and enhanced gene transfer to cancer cells. To this end, specific tumor and tumor vasculature targeting peptides, such as RGD, RKK, LyP-1, F3 and CGKRK, were successfully displayed on the baculoviral surface. Most importantly, the recombinant viruses showed enhanced binding and gene delivery to their target cells in vitro. In order to develop a functional targeted virus vector, the entry mechanism of baculovirus was elucidated. Baculovirus was shown to enter human hepatocarcinoma cells using the endosomal entry route, possibly via clathrin coated vesicles. In addition, the use of macropinocytosis was suggested. Additionally, to study the receptor of RKK targeting motif, α2β1 integrin, echovirus 1 (EV1) entry was studied. The α2β1 integrin was shown to internalize in concert with EV1 using caveolae endocytosis. Together, the results of this thesis demonstrate the feasibility of baculovirus display of targeting peptides to enhance gene delivery to target cells and, thus, suggests baculovirus to possess potential for targeted tumor therapy