Baculovirus surface modifications for enhanced gene delivery and biodistribution imaging (Bakulovirusten pintaproteiinien muokkaaminen geeniterapian tehostamista ja kulkeutumisen kuvantamista varten)
Year: 2006
Type: dissertation
Abstract: As gene therapy continues to evolve to clinical use, more interested is directed towards developing new and alternative gene delivery tools to compensate for the deficiencies of major vectors.During the last decade the research interest has turned to baculoviruses, which are ubiquitous viruses with high species specificity to intervertebrates.In this work baculoviruses were modified to extend their properties as gene delivery vectors.A versatile avidin-displaying baculovirus, Baavi, was created by inserting avidin into a baculovirus native envelope glycoprotein, gp64.Together with the strong interaction of avidin and biotin, various ligands could then be used to further modify the properties of baculovirus.We were able to show that possibly due to the positive charge of the avidin, Baavi had increased transduction efficiency as compared to unmodified baculovirus in various cell lines.Cell biotinylation increased the transduction efficiency a further 100-150 %.As the use of avidin display enables coating of the viral surface, we then utilized magnetic microspheres to physically target cells in vitro and showed increased binding with epidermal growth factor-ligand.The non-invasive imaging of gene delivery vector biodistribution and kinetics in vivo after administration are essential for clinical gene therapy.Traditional biodistribution imaging, based on the transduction pattern, does not always result in accurate presentation of the viral biodistribution as baculovirus may be unable to express the transgene in all penetrated cells.Magnetic resonance imaging has previously been utilized to image viral transgene expression, but by using 50 nm iron particles it was possible for the first time to image viral particle accumulation to rat choroid plexus cells with different time points.We were able to image the iron related signal loss up to two weeks and confirm the viral transgene expression to the same cells.We concluded that the payload could be transported to choroid plexus cells, together with the baculoviral gene delivery.By using biotinylated chelates with a technetium-label it was possible to use a microSPECT/CT device to image viral kinetics and biodistribution in vivo.Viral accumulation in different organs was analyzed after different administration routes using planar and 3D imaging as a function of time.We showed that intraperitoneal administration of Baavi resulted in signal accumulation in the spleen and kidneys and transgene expression at later time points.We also studied the truncated version of the G-protein from Vesicular Stomatitis virus, VSV-GED and were able to show that by including it to the envelope with gp64, a significant increase in the transduction efficiency resulted in nearly all the studied cell lines.The mechanism was studied by inhibiting endosome maturation with ammonium chloride and monensin.Possibly the mechanism of action was by aiding gp64-potentiated membrane fusion.In conclusion, the novel baculoviruses developed in this thesis result in increased transduction efficiency in vertebrate cells and provide more versatile tools to further develop gene therapy vectors for clinical use.
Author: J K Räty
Language: Finnish
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3096458556