Saporin As a Novel Suicide Gene in Anticancer Gene Therapy

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 and N-(2,3-dioleoyloxy-1-propyl) trimethy- plasmids in transfection experiments. lammonium methyl sulfate (DOTAP), to deliver SAP Large scale preparations of purified DNA for in vivo cDNA to rapidly growing murine melanomas. Despite the experiments were performed using EndoFree Plasmid poor delivery efficiency associated with non-viral sys- Giga Kit (Qiagen, Milano, Italy) according to the tems,27 we report here that SAP expression was very manufacture’s instructions. All plasmid preparations were effective in retarding tumor growth. tested for endotoxin contamination using the Limulus Amebocyte Lysate assay (Bio-Whittaker, Verviers, Belgium). The endotoxin content was less than 0.01 EU/mg of plasmid DNA. Materials and methods Cells and reagents Transfection assays The B16F1 murine melanoma cell line was obtained from Cells (1.6 Â 104)/well were plated in a 48-multiwell plate American Type Culture Collection (ATCC) (Manassas, and incubated for 24h. Cells were then co-transfected VA) and maintained in Rosewell Park Memorial Institute with increasing amounts of pSfiSV19-SAP or pCI-SAP, 1640, supplemented with 5% fetal calf serum (FCS), 2 mM mixed with complementary decreasing amounts of control

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al 167 vector and with a constant amount of luciferase encoding Tumor volume is presented as mean7s.e. of at least five plasmid (0.4 mg/well). Plasmid DNA (total 0.8 mg/well) animals per group. Statistical significance was assessed 30 and 100 mM polyethylenimine (PEI) were diluted using unpaired-sample Student’s t-test. All data were separately in 0.9% sodium chloride solution and then considered statistically significant at Po0.05. ANOVA mixed at the amino to phosphate group ratio (N/P) 9:1. one-way post-test statistical analysis (GraphPad Prism After 20 min, the mixture was added to the cells and software) was also used when three groups could be incubated for 4h; the supernatant was then removed and compared, confirming the statistical significance of our replaced with fresh medium. in vivo data. Twenty-four hours after transfection, cells were washed once with phosphate-buffered saline (PBS), resuspended in 100 ml/well of lysis buffer (125 mM Tris-HCl (pH 8), 10 mM dithiothreitol, 10 mM ethylenediaminetetraacetic Results acid (pH 8), 50% Glycerol, 5% Triton X-100) and Expression of SAP-encoding constructs in vitro incubated at 41C for 30 min with shaking. Luciferase The possibility of using SAP cDNA as a novel suicide activity in cell lysates was measured using the Luciferase gene in cancer gene therapy was first investigated in vitro, Assay System (Promega). Results were normalized by assessing the effects of two different SAP-encoding relative to protein content, measured using the Bradford constructs, before testing the efficacy of the SAP gene in protein assay method. Protein concentrations were killing B16 tumor cells in vivo. To achieve this goal, we calculated from a bovine serum albumin standard curve engineered the mature SAP coding sequence-316 into the and luciferase activity was expressed in relative light units in vitro amplification mammalian expression vectors28,29 per microgram of protein and presented as a percent of to allow the cytosolic expression of SAP under either the that estimated in the control transfections using only CMV or the SV40 promoter, generating respectively pCI- luciferase pCMVLuc, as in.31 Results of representative SAP and pSfiSV19-SAP constructs. This would allow experiments are expressed as means7s.d. of at least three toxin expression directly in the same compartment where wells per condition/sample. Statistical significance was the target ribosomes are located. Recombinant SAP, assessed using unpaired-sample Student’s t-test (Graph- when expressed and purified from E.coli, was previously Pad Prism software). All data were considered statistically shown to be extremely active both in cell-free translation significant at Po0.05. Analysis of variance (ANOVA) systems, derived from rabbit reticulocytes16 and in cell one-way post-test statistical analysis (GraphPad Prism killing assays.31 software) was also used to confirm the statistical To examine SAP-encoding constructs activity, transient significance of the data. co-transfection experiments were performed with increasing amounts of either pCI-SAP or pSfiSV19-SAP and a MTT staining fixed amount of the luciferase reporter gene. Either Vero B16F1 melanoma cells were seeded and transfected as cells, derived from African green monkey kidney epithe- described above. After 24h of incubation at 37 1C and 5% lium, a well characterized model cell line to study toxin 32–34 CO2, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium delivery or B16-F1 cells, a subclone of an aggressive bromide (MTT) (5 mg/ml in PBS) was added (10 ml/well) mouse melanoma that is widely used in antitumor and the plate was placed at 371C and 5% CO2. After 3 h, screening procedures, were transfected and the cell lysates supernatants were removed and 200 ml/well of dimethyl- assayed for luciferase expression. The results are ex- sulfoxide were added to dissolve the crystals formed. Cell pressed as a percentage of the luciferase activity estimated viability was assessed by reading the absorbance at in control transfections using only luciferase cDNA. 570 nm. Figure 1 shows that the luciferase relative activity is progressively inhibited when increasing amounts of SAP- In vivo studies encoding plasmid are used during transfection, reflecting All studies on animal models were approved by the a total protein synthesis inhibition in the transfected cells. Ethical Committee of the San Raffaele H Scientific Both constructs displayed a very high activity (Figure 1) Institute and performed according to its guidelines. with an effect on protein synthesis found to be dependent C57BL/6 female mice (Charles River Laboratories, Calco, on the specific promoter driving SAP expression and on Italy) weighing 18–20 g were injected subcutaneously with the cell type examined. SAP expression driven by the 5 Â 104 B16F1 cells in the left flank. About 8–10 days CMV promoter was apparently much higher than the one after inoculation, when mice developed a 5 mm diameter driven by the SV40 promoter. The activity of the tumor, selected groups of animals received a direct pSfiSV19-SAP construct was similar in B16 and Vero intratumoral injection of pCI-SAP or control plasmid cells, showing a 70% reduction of luciferase expression (50 mg per animal) complexed with lipofectamine or following transfection with the lowest concentration of DOTAP dissolved in 20 mM N-2-hydroxyethylpipera- SAP plasmid (100 ng). Conversely, the effects by the zine-N0-2-ethanesulphonic acid at ratio (N/P) 1:6.7 and CMV promoter-driven construct exceeded by 15- to 700- 1:4.8, respectively. The effect of single or repeated folds that of pSfiSV19-SAP. Notably, the effect of pCI- administrations (2 and 5 days after the first injection) SAP transfections were particularly prominent in B16 on tumor growth was monitored with callipers. Animals cells. Therefore, we titrated the minimal amount of SAP were killed before the tumor reached a diameter of 1.5 cm. cDNA needed to inhibit luciferase expression/activity; the

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al 168 1000 a 1000 Vero pSfiSV19-SAP a pCI-SAP 100 * * 100 10 * **

g prot.% ** µ ** g prot.% 10 ** 1 ** µ ** *** RLU/ *** RLU/ 0.1 1

0.01 0 0.1 0.2 0.3 0.4 0.1 Saporin cDNA (µg) 0 0.01 0.025 0.5 0.1 Saporin cDNA (µg) b 1000 pSfiSV19-SAP B16 b 120 pCI-SAP 100 100 * * 10 * 80

g prot.% ** ** µ 60 ** 1 ** Abs 570 % RLU/ 40 0.1 *** *** *** *** 20 0.01 0 0.1 0.2 0.3 0.4 0 Saporin cDNA (µg) 0 0.02 0.05 0.1 0.2 Saporin cDNA (µg) Figure 1 Effects of SAP-encoding constructs in vitro. Vero (a)or B16 (b) cells were co-transfected with increasing amounts of Figure 2 Titration of pCI-SAP construct on B16 cells. (a) B16 cells pSfiSV19-SAP (white bars) or pCI-SAP (black bars) constructs and were co-transfected with decreasing amounts pCI-SAP (starting with a constant amount of the luciferase encoding plasmid using 0.1 mg/well) and a constant amount of luciferase encoding (pCMVLuc, 0.4 mg/well). Twenty-four hours after transfection inhibi- plasmid (pCMVLuc, 0.4 mg/well). Twenty-four hours after transfection tion of luciferase expression was evaluated. Luciferase activity was inhibition of luciferase expression was evaluated. Luciferase activity expressed in RLU per microgram of protein and presented as a was expressed in RLU per microgram of protein and presented as a percent of that estimated in the control transfections using pCMVLuc percent of that estimated in the control transfections using pCMVLuc alone. The data presented are representative of a set of at least alone. (b) B16 cells transfected with increasing amounts of pCI-SAP three independent experiments. The results are expressed as were stained with MTT 24 h after transfection and absorbance at mean7s.d. of 4 points per group. *Po0.05; **Po0.01 and 570 nm was measured. The data presented are representative of a ***Po0.001 relative to control-transfected cells (point ‘0’). set of at least three independent experiments. The results are expressed as mean7s.d. of 4 points per group. **Po0.01 and ***Po0.001 relative to control-transfected cells (point ‘0’). experiments revealed that as few as 10 ng of the plasmid per 1.6 Â 104 B16 cells were sufficient to drastically reduce the luciferase activity to 18% with respect to control- in the melanoma cell line, we next tested the effect of treated cells (Figure 2a). pCI-SAP delivery on tumor growth in vivo. These transfection assays indicate that melanoma cells are extremely sensitive to SAP action, showing luciferase Injection of a SAP-encoding construct induces a marked synthesis inhibition, even when minute amounts of SAP- antitumor effect in B16 melanoma-bearing mice encoding construct were used. The co-transfection meth- The potential of pCI-SAP to promote tumor cell killing in od has proven to be very useful in evaluating the effects of vivo was evaluated by direct injection of the plasmid SAP cDNA delivery as it is not limited by cell transfection complexed with either DOTAP or lipofectamine in B16F1 efficiency as classical cytotoxicity assays. Cytotoxicity subcutaneous tumors in C57 mice. These two cationic observed by MTT staining of cells treated with increasing lipids were selected as the most efficient ones in our hands amount of SAP encoding plasmids 24h after transfection for delivering the luciferase reporter gene to the tumor ranged from 42 to 56% only (Figure 2b). These results are tissue upon direct intratumoral administration of DNA- consistent with the expected efficiency of PEI-mediated lipoplexes (Figure 3). A single administration of 50 mgof transfections that we previously estimated to be around pCI-SAP 10 days after tumor cell implantation, when 50% (data not shown). tumors reached a diameter of about 5 mm, was sufficient As we need a promoter that induces a burst of SAP to significantly slow down the rate of tumor growth expression sufficient for the rapid killing of cancer cells, (Figure 4a and b). Injection of control plasmid complexed and the CMV promoter proved to be more effective than to the same lipid vectors exerted only a modest effect on the SV40 one for expression of cytosolic SAP cDNA tumor growth (such a weak inhibition is often observed

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al

35 169 1000 when similar lipids are used to deliver DNA in vivo ). To improve the efficiency and duration of the suicide gene expression, repeated administrations of the SAP gene 100 were performed. The antitumor effect was, as expected, more evident in mice that received repeated intratumoral g prot.

µ injections of pCI-SAP/DOTAP complexes at days 2 and 5 from the first treatment (Figure 4c). In this case we 10 RLU/ observed an 8-day delay of tumor growth in pCI-SAP- treated animals with respect to untreated animals and 6 days with respect to those that received control injections. 1 Most, but not all, of the mice treated with pCI-SAP in AP RI developed ulcers like lesions at the injection site. Apart Naked DOT PE-I25 PEI-22 DM Lipofect from this intriguing observation, the SAP antitumor effect

DOTAP/DOPE Lipofectamine was accompanied by no evidence of systemic toxicity (i.e. animal death, loss of body weight, other tissue damage or Figure 3 In vivo comparison of non-viral vectors in luciferase gene changes in behavior or aspect). delivery upon direct intratumoral injections. Subcutaneous B16 These results suggest that intratumoral administration tumors with a diameter of 5 mm were injected with 30 mg of pCMVLuc complexed with cationic vectors at the following N/P equivalents of pCI-SAP may induce local production of toxin that is ratio: 4.8 for DOTAP and DOTAP/DOPE, 9 for PEI-22 and PEI-25, able to significantly attenuate the growth of an aggressive 3.3 for DMRI, 10 for lipofectin and 6.7 for lipofectamine. Naked DNA tumor such as B16 melanoma. or lipoplexes were dissolved in saline (except for DOTAP containing complexes that are dissolved in HEPES 20 mM). Luciferase expres- The catalytic activity of SAP is mainly responsible for sion in tumor homogenates after 24 h is expressed in RLU per the observed antitumor effects microgram of total protein. The results are presented as mean7s.e. To confirm that the observed antitumor effect relies on of five animals per group. the expression of the SAP gene within tumor cells and is

untreated untreated pCI/DOTAP pCI/DOTAP pCISAP/DOTAP pCISAP/DOTAP a 1500 b 1500 ) 3 )

3 ^ 1000 ^ 1000 ∗ ∗ ^ ^ ∗ 500 500 ^^ ^^ Tumor volume (mm Tumor volume (mm ^

0 0 031 2 45678 03456781 2 days after injection days after injection

c 1500 ) 3

1000

500 ^ ∗∗ Tumor volume (mm ∗ ^^ 0 034567812 9 10 11 12 days after injection Figure 4 Non-viral DNA complexes encoding cytosolic SAP injected into B16 melanoma-bearing mice are able to induce an antitumor effect. Subcutaneous B16 tumors with a diameter of 5 mm were injected with 50 mg/animal of pCI (control) or pCI-SAP complexed with lipofectamine (a) or DOTAP (b and c). The effect of single (a and b) or repeated (c) administrations was monitored. The time of injection is indicated by arrows. Results are presented as mean7s.e. of five animals per group. ^Po0.05 relative to untreated; *Po0.05 relative to control plasmid; ^^Po0.01 relative to untreated; **Po0.01 relative to control plasmid.

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al 170 1000 a pCI-SAPKQ untreated pCI/DOTAP 100 pCISAPKQ/DOTAP * * 1500 pCISAP/DOTAP 10 ) 1 3 1000 0.1 g prot.% µ b 1000 *

RLU/ pCI-SAP 500 ^^

100 volume (mm Tumor

** 10 ^ *** *** 0 *** 03456781 2109 1 *** *** days upon injection

0.1 0 0.4 0.1 0.2 0.05 0.01 0.025 µg plasmid DNA

Figure 5 SAP-KQ, a SAP active site mutant with drastically reduced RIP activity is not effective both in vitro and in vivo. B16 cells were co- transfected with increasing amounts of (a) pCI-SAPKQ or (b) pCI-SAP and with a constant amount of luciferase encoding plasmid (pCMVLuc, 0.4 mg/well) and 24 h after transfection the inhibition of luciferase expression was evaluated. Luciferase activity was expressed in RLU per microgram of protein and presented as a percent of that estimated in the control transfections using pCMVLuc alone. The data presented are representative of a set of three independent experiments. The results are presented as mean7s.d. of four points per group. *Po0.05, **Po0.01 and ***Po0.001 relative to control plasmid transfected cells. (c) B16 melanoma-bearing mice with subcutaneous tumors of 5 mm diameter were injected with 50 mg/animal of pCI (control), pCI-SAP or pCI-SAPKQ. Time of injection is indicated by the arrow. The results are presented as mean7s.e. of five animals per group. ^Po0.05 relative to untreated; *Po0.05 relative to control plasmid, ^^Po0.01 relative to untreated.

not only owing to an unspecific protein production, we approaches. Toxins have been widely used in anticancer repeated the experiments using a SAP catalytic active site therapy as components of immunotoxins or recombinant mutant (SAP-KQ), which has a significantly reduced RIP chimeras in which the catalytic moieties (endowed by activity (Fabbrini and Ceriotti, unpublished results). poor selectivity and permeability) were linked to tumor- Co-transfection assays performed in B16 cells comparing targeting ligands or antibodies that mediate their binding pCI-SAP and pCI-SAP-KQ plasmids activity (Figure 5a) and internalization by malignant cells.36 Such protein confirmed that pCI-SAP drastically reduced luciferase toxins include bacterial toxins like Pseudomonas aerugi- activity in transfected cells in a dose-dependent manner, nosa exotoxin A and diphtheria toxin37 or plant toxins whereas no dose–response could be detected when like ricin and SAP.38 In particular, native seed extracted pCI-SAP-KQ plasmid was used. SAP has been conjugated to specific monoclonal anti- Accordingly, when the wild-type and mutant genes bodies (anti-CD19, -CD22, and -CD38) producing a were complexed with DOTAP and injected into B16 cocktail of immunotoxins that resulted curative in animal tumors, pCI-SAP/DOTAP administration markedly af- models of B-cell lymphoma.21 Also SAP-based chimeric fected tumor growth, whereas pCI-SAP-KQ/DOTAP toxins targeting either receptors highly expressed on the failed to exert a significant antitumor effect, showing surface of cancer cells like those for basic fibroblast only a modest delay in tumor growth, comparable to that growth factor (bFGF) or for human urokinase or specific observed in control-treated animals (Figure 5b). prostate antigen expressing cells exerted high specific We conclude that SAP-specific catalytic activity is cytotoxic activity.17,19,25,26 responsible for the observed cell killing effects, therefore However, the cytotoxicity of all these compounds excluding that this effect can be merely owing to the depends not only on tumor accessibility and penetration exogenous expression of a heterologous plant protein. but also on an efficient internalization, intracellular sorting and release of catalytically active toxin domains into the cytoplasm. Another common drawback of such Discussion an approach is the resistance acquired by cancer cells caused by either loss or downregulation of specific In this study, we investigated the potential use of the plant receptors by autocrine ligands which, thus, reduce binding toxin SAP cDNA as a suicide gene in cancer gene therapy and internalization of the recombinant toxin.39

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al 171 The use of appropriate DNA constructs encoding ing cells. Therefore, we assume that the SAP toxicity is toxins that can be directly expressed in the cytosol of confined to the transfected cells. the target cells6–10 may overcome such problems. Cell However, in addition to the marked antitumor effect killing activity of a SAP gene in vitro resulted comparable following local gene delivery in vivo, we did observe some to that of the Herpes simplex gene encoding thymidine ulcer-like lesions at the tumor/injection site. This could kinase followed by ganciclovir treatment.40 SAP is almost possibly be due to some local inflammatory/immune four to five times more potent than plant ricin A chain reaction. Although toxins themselves might be highly over rabbit retyculocyte ribosomes showing IC50 in the immunogenic molecules or may elicit a vascular leak picomolar range.16,31 We demonstrate here that SAP syndrome,43 we did not observe such lesions when cDNA expression is highly toxic both in vitro and in vivo: injecting mutated SAP–KQ complexes. We therefore as few as 10 ng of pCI-SAP per 1.6 Â 104 melanoma cells hypothesize that such a response could be directed to were sufficient to drastically inhibit protein synthesis and some components of the intoxicated cells. Further consequently to kill the transfected cells. In addition, in investigation is needed to establish whether SAP action the B16 mouse melanoma model, direct intratumoral might be potentiated by immunomodulatory effects. An injection of pCI-SAP plasmid significantly attenuated immunomediated bystander effect was suggested in tumor growth, with an effect that was clearly improved glioblastoma treated by retrovirus expressing the ricin upon repeated administrations. The antitumor activity toxic A chain (RTA) gene, where the rejection of tumors observed was specifically owing to the intrinsic activity of was more efficient in the brain of immunocompetent SAP and not to simple expression of a heterologous plant animals than in severe-combined immunodeficient mice.9 protein, as plasmid encoding a catalytic inactive mutant Working at the DNA level avoids costly and time- SAP-KQ, generated by mutation of two key active site consuming production and purification of the recombi- residues (E176K and R179Q in mature SAP), could nant chimeric proteins, whereas allowing direct screening neither inhibit luciferase expression nor delay tumor of a wide variety of DNA constructs, for example, growth in mice. employing diverse promoters and transcription regulatory Classical suicide genes, like herpes simplex virus elements, as well as exploring different delivery modes.44 thymidine kinase, act by disrupting DNA synthesis and In the case of systemic delivery, to prevent detrimental this limit their action to tumors characterized by rapidly expression of toxin in inappropriate tissues, SAP con- dividing cells. Toxin genes act in a cell cycle independent structs could easily be improved with tissue and/or tumor- way, so that they can target quiescent and rapidly dividing specific promoters.7,9,45–49 In addition, pharmacologically tumor cells. This property makes them suited both to controlled gene expression could be ensured using contrast aggressively growing tumors (e.g., melanoma) inducible promoters such as the Tet-on system, which and for other tumor types that grow more slowly (e.g., exhibits the best features for applications in patients prostate tumors). because it permits rapid gene induction/silencing switch SAP has been shown to induce programmed cell death, in vivo in a dose-dependent manner and its inducer presumably as a consequence of protein synthesis inhibi- doxycycline is widely used as an antibiotic and well tion41,42 and may have a direct DNA fragmentation tolerated in humans (Zabala et al.50 and references activity.14 Similarly to other RIPs, one molecule of SAP therein). reaching the cytosol is thought to be sufficient for cell To test the activity of the native SAP gene in vivo we killing.17 Despite a marked decrease in cell number upon employed direct intratumoral injection of plasmid DNA transfection with SAP cDNA, no SAP polypeptide could complexed with cationic lipids in an aggressive melanoma be detected using antiSAP antibodies, as previously model. Such an approach proved to be simple, reprodu- reported also for a SAP mammalian codon optimized cible and less toxic and immunogenic compared to viral gene transfected via bFGF receptors.40 This suggests that delivery systems. Besides, low and transient transgene a brief and even low expression of the SAP native gene expression, generally considered a limitation in non-viral (below biochemical detection) is sufficient to kill virtually gene therapy, does not compromise SAP efficiency, owing each transfected cell, a desirable end point of a suicide to its high intrinsic activity and rapid mode of action. gene therapy. Non-viral vectors are increasingly being used as an Cytosolic expression of SAP ensures that it rapidly and alternative to viral vectors and photochemical internaliza- directly targets intracellular ribosomes. Although SAP tion of non-viral vectors has been reported to yield high expression would also inhibit its own translation, this gene transfer efficiency.51 does not seem to compromise its efficacy, given that The direct delivery approach in the context of either picomolar amounts of toxin are sufficient to inhibit viral or non-viral vectors is in general applicable to cellular protein synthesis. accessible solid tumors, thus limiting the field of action to In contrast, the concentrations of SAP requested for a few malignancies, for example, melanoma, head and cell killing following internalization are typically in the neck tumors, mesothelioma and so on. However, some of micromolar–nanomolar range depending on the cell these malignancies are resistant to conventional therapies, type.31 As we could not detect SAP polypeptides in cell and gene therapy trials are warranted, to delineate new cultures upon transfection, we also do not expect that potential treatments. SAP-mediated gene therapy can SAP released from dying cells would reach a sufficient thus contribute to eradicate tumor mass in combination amount to allow efficient internalization by the neighbor- with surgery or classical radio- or chemotherapy.

Cancer Gene Therapy Saporin non-viral gene expression in B16 melanomas N Zarovni et al 172 Overall, we conclude that the features of the SAP gene, 13 Roncuzzi L, Gasperi-Campani A. DNA-nuclease activity of such as its rapid and efficient action, the independence the single-chain ribosome-inactivating proteins Dianthin 30, from proliferative state of the cell, the fact that no Saporin 6 and Gelonin. FEBS Lett 1996; 392: 16–20. sustained gene expression is required for its efficacy, as 14Bagga S, Seth D, Batra JK. The cytotoxic activity of well as low leakage of the protein released from ribosome-inactivating protein saporin-6 is attributed to its intoxicated cells make it a very appealing candidate as a rRNA N-glycosidase and internucleosomal DNA fragmen- suicide gene for oncologic applications. tation activities. J Biol Chem 2003; 278: 4813–4820. 15 Cavallaro U, Nykjaer A, Nielsen M, Soria MR. 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