A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy

Abstract

The gibbon ape leukaemia virus envelope fusogenic membrane glycoprotein (GALV FMG) is a highly potent cytotoxic gene with great potential for use in cancer gene therapy. Here, we show that production of a VSV-G pseudotyped lentiviral vector expressing GALV FMG reconciles the requirements of viral production with the cytotoxic effects of GALV in human cells and has high titres on both dividing and quiescent tumour cells. Direct intratumoral injection of these stocks eradicated progressively growing human tumour xen-

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

“…7 However, when we cloned the cDNA of the GALV FMG 2,4 downstream of the TDE-SV40 element with the aim of generating melanoma-specific cell killing in vitro, although there was a clear lag period in the formation of syncytia compared with CMV-GALV, significant amounts of cell fusion subsequently developed after 72-96 h (data not shown). Since GALV has such a potent bystander killing effect 2,3 it was clear that it would be necessary to improve the specificity of the transcriptional elements that are used to direct its expression. Therefore, we investigated whether it would be possible to identify an element within the human tyrosinase promoter 8 that would be transcriptionally silent in non-melanoma cells.…”

“…Previously, we have shown that tumour cells dying through GALV-mediated cell fusion induce hsp70 expression. 2,3 Hsp70 is transcriptionally activated by binding of the HSF-1 transcription 11 to the HSE, a well-defined transcriptional element upstream of a variety of heat shock genes. 9,10 We initially reasoned that by placing one or more HSE upstream of the weak Tyr-300 element, small levels of GALVmediated cell killing might induce endogenous HSF-1 as part of the stress response which would amplify expression from the HSE-Tyr-300 element.…”

“…[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.…”

“…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.…”

“…2 In addition, expression of viral FMG is also highly immunostimulatory as judged by the immunogenicity of FMG-expressing vaccines and by the induction of stress-related proteins such as inducible heat shock proteins during the killing process. 2,3 These dual properties of high local killing capacity and immunostimulatory activity make FMG attractive transgenes for use in gene therapy of cancer.…”