A novel double-targeted bacteriophage vector for systemic cancer gene therapy

摘要

A successful cancer gene therapy requires systemic gene delivery vectors capable of efficient and selective gene transfer to tumour sites. Two main approaches have been undertaken for targeted cancer gene therapy: i) restriction of transgene expression in tumour cells by using tumour specific promoters and ii) selective delivery of vectors of gene therapy to tumours by ligand targeting. Amongst many characterised tumour specific promoters, the stress-inducible promoter of the Glucose-regulated protein 78 (Grp78) gene holds great potential to improve the practice of cancer gene therapy as its activity is highly induced under conditions often found in the tumour microenvironment such as acidic pH, glucose deprivation and chronic anoxia. However, the clinical application of the Grp78 promoter in cancer gene therapy has been limited by lack of delivery strategies via the systemic routes. In 2006, an efficient tumour targeted vector, adeno-associated virus/phage termed AAV/Phage or AAVP, was reported. In this AAVP vector, the M13 bacteriophage (phage) was used as a vehicle to target αv integrin receptors, which are selectively overexpressed in tumours, and to deliver the AAV transgene cassette driving transgene expression from a cytomegalovirus (CMV) promoter. Systemic administration of the targeted AAVP viral phage vector has been shown to successfully mediate therapeutic transgene expression in several preclinical tumour models. Thus, the AAVP particle provides a suitable vector candidate for double targeting of cancer after systemic administration. Herein, I combined ligand-directed targeting of AAVP vector with transcriptional targeting using the Grp78 promoter in one single platform. In the first half of the thesis I show that the double-targeted vector provides sustained and long-term transgene expression in transduced tumour cells in vitro. Moreover, compared to the conventional phage carrying the CMV promoter, the new hybrid phage provided a significant tumour killing both in vitro and in vivo using the HSVtk gene and ganciclovir suicide therapy. More importantly in chapter 5, I describe a novel mechanism of Grp78 promoter activation by HSVtk and ganciclovir suicide therapy. In chapter 6, in addition to the vector safety, I show the superior tumour transduction in vivo after systemic administration of double-targeted AAVP vector over the original AAVP carrying the CMV promoter. In Chapter 7, I demonstrate strong dose dependent chemotherapeutic induction of the Grp78 promoter in the context of AAVP vector. Together, all data presented in this thesis validate the great potential of the Grp78 promoter in targeted systemic cancer gene therapy and report the efficacy of the double-targeted AAVP vector which proves useful for translation into clinical applications.