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Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein

Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein
Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein
Background: In a strategy termed Protease Targeting, retroviral vectors carrying an EGF infectivity-blocking domain fused to the N-terminus of the envelope SU via a MMP (matrix metalloproteinase)-cleavable linker were successfully used to target gene delivery to EGF receptor-(EGF-R-)positive tumour cells over-expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune-stimulatory properties of GALV via local, MMP-mediated release human granulocyte-macrophage colony stimulating factor (GM-CSF).
Methods: We generated GALV envelope expression constructs displaying EGF or GM-CSF blocking ligands at the N-terminus of GALV envelope SU via a non-cleavable, Factor Xa protease or MMP-cleavable linker and investigated their cytotoxicity on MMP-positive and negative cell lines.
Results: The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non-cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease-cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the blocking domain were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.
Conclusions: Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell-surface MMPs. In addition, shedding of the SU-fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell-cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus-cell fusion.
gibbon ape leukaemia virus (GALV), envelope, matrix metalloproteinase (MMP), cancer gene therapy, protease targeting
1099-498X
592-600
Kirkham, Lucy A.
f75d2539-08a0-4775-8b4e-32605b1a096d
Bateman, Andrew R.
a851558d-8b9b-4020-b148-a239c2b26815
Melcher, Alan A.
00d1a334-30ea-49e9-b47b-f1792325a835
Vile, Richard G.
5e0f62aa-ef75-47cc-9adb-a95b5a87e806
Fielding, Adele K.
f1a331a0-d040-4dae-84ca-5234cc0f9375
Kirkham, Lucy A.
f75d2539-08a0-4775-8b4e-32605b1a096d
Bateman, Andrew R.
a851558d-8b9b-4020-b148-a239c2b26815
Melcher, Alan A.
00d1a334-30ea-49e9-b47b-f1792325a835
Vile, Richard G.
5e0f62aa-ef75-47cc-9adb-a95b5a87e806
Fielding, Adele K.
f1a331a0-d040-4dae-84ca-5234cc0f9375

Kirkham, Lucy A., Bateman, Andrew R., Melcher, Alan A., Vile, Richard G. and Fielding, Adele K. (2002) Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein. The Journal of Gene Medicine, 4 (6), 592-600. (doi:10.1002/jgm.306).

Record type: Article

Abstract

Background: In a strategy termed Protease Targeting, retroviral vectors carrying an EGF infectivity-blocking domain fused to the N-terminus of the envelope SU via a MMP (matrix metalloproteinase)-cleavable linker were successfully used to target gene delivery to EGF receptor-(EGF-R-)positive tumour cells over-expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune-stimulatory properties of GALV via local, MMP-mediated release human granulocyte-macrophage colony stimulating factor (GM-CSF).
Methods: We generated GALV envelope expression constructs displaying EGF or GM-CSF blocking ligands at the N-terminus of GALV envelope SU via a non-cleavable, Factor Xa protease or MMP-cleavable linker and investigated their cytotoxicity on MMP-positive and negative cell lines.
Results: The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non-cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease-cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the blocking domain were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.
Conclusions: Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell-surface MMPs. In addition, shedding of the SU-fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell-cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus-cell fusion.

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More information

Published date: 2002
Additional Information: Research Article
Keywords: gibbon ape leukaemia virus (GALV), envelope, matrix metalloproteinase (MMP), cancer gene therapy, protease targeting

Identifiers

Local EPrints ID: 26427
URI: http://eprints.soton.ac.uk/id/eprint/26427
ISSN: 1099-498X
PURE UUID: 55b48629-51a4-4ea0-862f-b6dff4764768

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Date deposited: 21 Apr 2006
Last modified: 15 Jul 2019 19:14

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