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Saporin As a Novel Suicide Gene in Anticancer Gene Therapy

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Saporin As a Novel Suicide Gene in Anticancer Gene Therapy Cancer Gene Therapy (2007) 14, 165–173 r 2007 Nature Publishing Group All rights reserved 0929-1903/07 $30.00 www.nature.com/cgt ORIGINAL ARTICLE Saporin as a novel suicide gene in anticancer gene therapy N Zarovni1, R Vago1,2, T Solda`3, L Monaco4 and MS Fabbrini5 Department of Biological and Technological Research and Cancer Immunotherapy and Gene Therapy Program, San Raffaele H Scientific Institute, Milan, Italy 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 Â 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. Cancer Gene Therapy (2007) 14, 165–173. doi:10.1038/sj.cgt.7700998; published online 29 September 2006 Keywords: plant ribosome-inactivating protein; melanoma; suicide gene expression; non-viral gene delivery; anticancer gene therapy Introduction Strategies based on suicide genes that have been explored for the treatment of various cancer types mostly Gene therapy has been investigated as an alternative or rely on gene-directed enzyme prodrug therapy. The gene complementary strategy to overcome the limitations of encoding an enzyme can be delivered to tumor cells, conventional anticancer therapy approaches. Rather than followed by the administration of a prodrug, which is in correcting specific genetic defects within cancer cells, then converted locally into a cytotoxin by this specific one current promise of cancer gene therapy stands in enzymatic activity. Several such systems already under- eradicating the tumor by the transfer of so-called suicide went clinical trials: Herpes simplex virus thymidine genes, or by the delivery of immunomodulatory genes that kinase/gancyclovir, bacterial cytosine deaminase/5-fluoro- promote tumor rejection by the activation of the immune cytosine, bacterial nitroreductase/CB1954and cytochrome 5 system.1,2 Additional gene therapy strategies are designed P450/cyclophosphamide. to target tumor stroma, aimed in particular at inhibiting As an alternative, recently it has been suggested that 6,7 neoangiogenesis or destroying tumoral vascular supply.3,4 some bacterial or plant toxins such as diphtheria toxin Pseudomonas aeruginosa exotoxin A8,9 or ricin9,10 would be suitable candidates for cancer gene therapies. In Correspondence: Dr MS Fabbrini, Istituto Biologia Biotecnologia contrast to other suicide genes that act by disrupting Agraria, IBBA, National research council, CNR, via Bassini, DNA synthesis, thus targeting only rapidly dividing cells, Milano 15 20132, Italy. these toxins are potent inhibitors of protein synthesis, E-mail: [email protected] which acts in a cell cycle-independent manner, killing 1These authors contributed equally to this work. both quiescent and rapidly dividing tumor cells. Thus, 2 Current address: Istituto di Genetica Molecolare, CNR, Pavia, such toxins can be an effective therapy for aggressively Italy 3 growing tumors as well as for other tumor types that grow Current address: Istituto per la Ricerca Biomedica, Bellinzona, more slowly. Switzerland 4Current address: Telethon Onlus, Scientific Office, Milan, Italy Another plant toxin potentially interesting for cancer 5Current address: Istituto Biologia e Biotecnologia Agraria, IBBA, gene therapy approaches is saporin (SAP), a monomeric CNR, via Bassini, Milan 15 20133, Italy plant ribosome-inactivating protein (RIP) accumulated in 11 Received 28 April 2006; revised 3 July 2006; accepted 19 August seeds and leaves of the soapwort, Saponaria officinalis. 2006; published online 29 September 2006 SAP depurinates the 23/25/28 S ribosomal RNA, thus Saporin non-viral gene expression in B16 melanomas N Zarovni et al 166 blocking peptidyl-transfer RNA translocation during L-glutammine, 100 U/ml penicillin, 100 mg/ml streptomycin, protein synthesis12 and leading to an irreversible inactiva- 0.25 mg/ml amphotericin. The African green monkey tion of ribosomes. Moreover, a number of studies have kidney Vero cell line was from ATCC and was grown in demonstrated specific DNAse-like activity of RIPs, high-glucose Dulbecco’s Modified Eagle’s Medium sup- 13,14 including SAP, as evidenced by DNA fragmentation. plemented with 5% of FCS, 2 mML-glutammine, 100 U/ Such DNA damage, characterized by micronuclei forma- ml penicillin, 100 mg/ml streptomycin. FCS and culture tion and the appearance of cytosolic oligonucleosomes, media were from Gibco BRL (Milano, Italy). The might play a role in the induction of apoptosis upon cell pCMVLuc plasmid and Luciferase Kit were purchased exposure to the toxin and contribute to target cell killing. from Promega (Promega-Italia, Milano, Italy). All SAP is a single-chain (type I) RIP and lacks the restriction enzymes used were from New England Biolabs membrane binding domain found in the prototype ricin (Cellbio, Milano, Italy). The other chemicals were AB. Three-dimensional structure of the catalytic ricin A purchased from Sigma (Sigma-Aldrich s.r.l., Milano, chain (RTA) is superimposable with that of SAP despite Italy), unless reported otherwise. the fact that their amino-acid identity is lower than 25%. RTA has a very low lysine content and a slightly acidic pI Construction of SAP-encoding plasmids whereas 10% of the amino acids in SAP are lysine The pCI-SAP plasmid was prepared by inserting the residues, which confer also an extremely high pI (almost sequence encoding mature SAP-316 into the pCISfiRsrT 10) to this polypeptide. In addition, SAP can be vector.28 This vector has the pCI plasmid backbone internalized through members of the low-density lipopro- (Promega), featuring a cytomegalovirus (CMV) promo- tein-related receptor family15,16 and has a much higher ter, an artificial intron and splice site, a multiple cloning cell-free RIP activity, compared to RTA.15,16 Acting site and a transcription terminator placed downstream the catalytically, SAP has very high intrinsic activity as it has polyadenylation signal for improved transcription effi- been established that only a few molecules of toxin ciency. The pSfiSV19-SAP plasmid was prepared by reaching the cytosolic compartment are sufficient to subcloning the SAP sequence into the pSfiSV19 under inhibit protein synthesis and cause cell death.17 the control of the early simian virus 40 (SV40) promo- Seed-extracted SAP has been widely used to develop ter.29 The SAP encoding sequence was obtained from the mouse models selectively impaired in central nervous pET-11d-SAP3 plasmid16 digested with NcoI and EcoRI; system functions.11 and in anticancer therapy, to con- protruding ends were filled in with Klenow DNA struct immunotoxins or ligand toxins18–21 and SAP polymerase. The SAP fragment was then ligated into complementary DNA (cDNA) has been used for expres- SmaI-linearized and dephosphorylated pCIsfiRsrT and sing targeted recombinant chimeras.17,22–25 pSfiSV19 vectors. When a SAP-based secretory chimera was expressed in The pCI-SAPKQ construct was prepared by in vitro Xenopus laevis oocytes, total inhibition of cellular protein oligonucleotide mismatch site directed mutagenesis on the synthesis was observed that could be restored only by pCI-SAP plasmid template, using Pfu Turbo polymerase supplementing neutralizing anti-SAP antibodies into the (Promega-Italia, Milano, Italy). The oligonucleotides oocyte cytosol,26 indicating that, although almost all of used were: forward 50 CT ATT CAA ATG ACA GCT the chimera was secreted, the few molecules escaping from AAG GTA GCA CAG TTT AGG TAC ATT C 30; the secretory pathway were sufficient to inactivate protein reverse 50 GAA TGT ACC TAA ACT GTG CTA CCT synthesis in the host cells. TAG CTG TCA TTT GAA TAG 30 (mutated nucleotides Therefore, we decided to investigate the potential of the are shown in bold). In this way two mutations were SAP native gene in cancer gene therapy approaches. In inserted: 176 Glu to Lys and 179 Arg to Gln. Polymerase this study, we report that we developed suitable SAP chain reaction conditions were: hot start at 941C 1 min, cDNA constructs and tested them both in vitro and in vivo followed by 20 cycles 951C 1 min, 551C30s,681C 10 min. for efficient intratumoral cell killing. To avoid the side The template was digested with DpnI. effects associated to viral vectors, we used cationic lipids, pCIsfiRsrT or pSfiSV19 vectors were used as control lipofectamine
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