A Hyperfusogenic Gibbon Ape Leukemia Envelope Glycoprotein: Targeting of a Cytotoxic Gene by Ligand Display

Abstract

An important goal in cancer gene therapy is the development of novel targeted cytotoxic genes. The observation that transfection of a GaLV envelope glycoprotein lacking an R peptide into human cells results in considerable cell-cell fusion and subsequent cell death prompted us to explore the potential for using this fusogenic membrane glycoprotein (FMG) as a targeted cytotoxic gene. As proof of principle, we therefore displayed epidermal growth factor (EGF) on the N terminus of GaLV envelope glycoproteins both with and without an R peptide (GaLV R+ and GaLV R-). Transfection of the GaLVR+ envelope expression plasmids did not cause cell-cell fusion. The GaLV R+ envelopes were incorporated into retroviral vectors whose infectivity was investigated on EGF receptor-positive and -negative cells. The vector incorporating an N-terminally unmodified envelope was able to infect all human cell lines tested. Infectivity of the vector incorporating an envelope on which EGF was displayed was restricted on EGF receptor-positive cells (but not on EGF receptor-negative cells) and could be restored by protease cleavage of the displayed domain or competition with exogenous ligand. The cell-cell fusion capacity of the GaLV R- envelope glycoproteins (N-terminally unmodified and with N-terminal display of both EGF and insulin-like growth factor I [IGF-I]) was investigated by plasmid DNA transfection. While the N-terminally unmodified GaLV R- fused all human cell types tested, fusogenicity of GaLV R- on which EGF or IGF-I was displayed was considerably restricted on receptor-positive cells. "Reciprocal" competition experiments showed that fusogenicity could be restored by competition only with the relevant exogenous ligand. Thus the specificity of cell-cell fusion by a hyperfusogenic GaLV envelope glycoprotein can be regulated by N-terminal display of growth factor ligands. There is therefore significant potential for further development of the targeting of the cell-killing capability of this fusogenic viral glycoprotein by using strategies similar to those we have developed for the targeting of retroviral vectors.

Fusogenic membrane glycoproteins induce syncytia formation and death in vitro and in vivo: a potential therapy agent for lung cancer

“…[1][2][3][4][7][8][9][10][11][12][13][14][15] In this study, we demonstrate that HERV-W FMGs' cytotoxicity resulting from the formation of gigantic cells is sufficient to induce tumor regression, with a very strong bystander effect, after non-viral, intratumoral gene delivery as previously described for GALV.…”

“…Interestingly, after being transiently transfected, an FMG-positive cell can also fuse with its neighbors, leading to the formation of multinucleated syncytia. As these syncytia are not viable and will eventually die within a few days, [1][2][3] FMG transfection has rapidly been identified as a potential antitumor treatment. The recruitment of FMG-negative neighboring cells is responsible for a powerful bystander effect superior to that of suicide genes such as herpes simplex virus thymidine kinase-1 (HSV-TK).…”

A transcriptional feedback loop for tissue-specific expression of highly cytotoxic genes which incorporates an immunostimulatory component

“…[2][3][4] However, the FMG are so potent that expression of only a few molecules is sufficient to initiate fusion. Hence, even low levels of leakiness from the TDE-SV40 melanoma-specific promoter 7 were sufficient to cause marked cytotoxicity to a range of non-melanoma cell lines that we tested.…”

“…9,10 To assess promoter/enhancer strengths, plasmids were constructed using standard techniques such that different promoters and promoter/enhancer fragments were placed upstream either of the chloramphenicol acetyl transferase (CAT) gene, 7 the human GM-CSF gene 27 or the cDNA of the gibbon ape leukaemia virus fusogenic membrane glycoprotein (GALV-FMG). 2,4 Levels of gene expression were assayed using either CAT assays, 7 by measuring levels of human GM-CSF secreted from the plasmids by ELISA (Pharmingen, San Diego, CA, USA) or by semi-quantitative rtPCR as described below.…”

“…To this end, we have been developing a new class of genes with both direct cytotoxic and immunostimulatory properties, called the fusogenic membrane glycoproteins (FMG). [2][3][4] FMG kill tumour cells by causing fusion between cells expressing the FMG and neighbouring cells which express the receptor for the viral fusogen. The fusogenicity of these proteins means that large local bystander effects can be achieved, where non-expressing cells can be recruited into large multi-nucleated syncytia Tyr-300 initiated expression of both the cytotoxic and the HSF-1 genes in melanoma cells.…”