¹Department of Biological and Technological Research and Cancer Immunotherapy and Gene Therapy Program, San Raffaele H Scientific Institute, Milan, Italy

Correspondence: Dr MS Fabbrini, Istituto Biologia Biotecnologia Agraria, IBBA, National research council, CNR, via Bassini, Milano 15 20132, Italy. E-mail: fabbrini@ibba.cnr.it

²These authors contributed equally to this work.

³Current address: Istituto di Genetica Molecolare, CNR, Pavia, Italy

⁴Current address: Istituto per la Ricerca Biomedica, Bellinzona, Switzerland

⁵Current address: Telethon Onlus, Scientific Office, Milan, Italy

⁶Current address: Istituto Biologia e Biotecnologia Agraria, IBBA, CNR, via Bassini, Milan 15 20133, Italy

Received 28 April 2006; Revised 3 July 2006; Accepted 19 August 2006; Published online 29 September 2006.

Abstract

We used a non-viral gene delivery approach to explore the potential of the plant saporin (SAP) gene as an alternative to the currently employed suicide genes in cancer therapy. Plasmids expressing cytosolic SAP were generated by placing the region encoding the mature plant ribosome-inactivating protein under the control of cytomegalovirus (CMV) or simian virus 40 (SV40) promoters. Their ability to inhibit protein synthesis was first tested in cultured tumor cells co-transfected with a luciferase reporter gene. In particular, SAP expression driven by CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6 10⁴ B16 cells drastically reduced luciferase activity to 18% of that in control cells. Direct intratumoral injection of pCI-SAP complexed with either lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) in B16 melanoma-bearing mice resulted in a noteworthy attenuation of tumor growth. This antitumor effect was increased in mice that received repeated intratumoral injections. A SAP catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect demonstrating that this was specifically owing to the SAP N-glycosidase activity. Our overall data strongly suggest that the gene encoding SAP, owing to its rapid and effective action and its independence from the proliferative state of target cells might become a suitable candidate suicide gene for oncologic applications.