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Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma
Tilen Komel, Maša Bošnjak, Simona Kranjc Brezar, Mariangela De Robertis, M. Mastrodonato, G. Scillitani, G. Pesole, Emanuella Signori, Gregor Serša, Maja Čemažar, 2021, original scientific article

Abstract: Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 %s, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma.
Keywords: gene therapy, gene electrotransfer, IL-12, immunotherapy, melanoma
Published in DiRROS: 23.09.2022; Views: 27; Downloads: 16
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Biological factors of the tumour response to electrochemotherapy : review of the evidence and a research roadmap
Gregor Serša, Katja Uršič Valentinuzzi, Maja Čemažar, Richard Heller, Maša Bošnjak, Luca Giovanni Campana, 2021, review article

Abstract: The beneficial effects of electrochemotherapy (ECT) for superficial tumours and, more recently, deepseated malignancies in terms of local control and quality of life are widely accepted. However, the variability in responses across histotypes needs to be explored. Currently, patient selection for ECT is based on clinical factors (tumour size, histotype, and exposure to previous oncological treatments), whereas there are no biomarkers to predict the response to treatment. In this field, two major areas of investigation can be identified, i.e., tumour cell characteristics and the tumour microenvironment (vasculature, extracellular matrix, and immune infiltrate). For each of these areas, we describe the current knowledge and discuss how to foster further investigation. This review aims to provide a summary of the currently used guiding clinical factors and delineates a research roadmap for future studies to identify putative biomarkers of response to ECT. These biomarkers may allow researchers to improve ECT practice by customising treatment parameters, manipulating the tumour and its microenvironment, and exploring novel therapeutic combinations.
Keywords: biological factors, biomarkers, electrochemotherapy, bleomycin, cisplatin
Published in DiRROS: 23.09.2022; Views: 22; Downloads: 10
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Electrochemotherapy in mucosal cancer of the head and neck : a systematic review
Primož Strojan, Aleš Grošelj, Gregor Serša, Christina Caroline Plaschke, Jan B. Vermorken, Sandra Nuyts, Remco De Bree, Avraham Eisbruch, William M. Mendenhall, Robert Smee, Alfio Ferlito, 2021, review article

Abstract: Electrochemotherapy (ECT) is a local ablative treatment that is based on the reversible electroporation and intracellular accumulation of hydrophilic drug molecules, which greatly increases their cytotoxicity. In mucosal head and neck cancer (HNC), experience with ECT is limited due to the poor accessibility of tumors. In order to review the experience with ECT in mucosal HNC, we undertook a systematic review of the literature. In 22 articles, published between 1998 and 2020, 16 studies with 164 patients were described. Curative and palliative intent treatment were given to 36 (22%) and 128 patients (78%), respectively. The majority of tumors were squamous cell carcinomas (79.3%) and located in the oral cavity (62.8%). In the curative intent group, complete response after one ECT treatment was achieved in 80.5% of the patients, and in the palliative intent group, the objective (complete and partial) response rate was 73.1% (31.2% and 41.9%). No serious adverse events were reported during or soon after ECT and late effects were rare (19 events in 17 patients). The quality-of-life assessments did not show a significant deterioration at 12 months post-ECT. Provided these preliminary data are confirmed in randomized controlled trials, ECT may be an interesting treatment option in selected patients with HNC not amenable to standard local treatment.
Keywords: electrochemotherapy, head and neck cancer, mucosal cancer
Published in DiRROS: 23.09.2022; Views: 26; Downloads: 6
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Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues
Urška Kamenšek, Andrej Renčelj, Tanja Jesenko, Tinkara Remic, Gregor Serša, Maja Čemažar, 2022, original scientific article

Abstract: Interleukin 12 (IL-12) is a cytokine used as a therapeutic molecule in cancer immunotherapy. Gene electrotransfer mediated delivery of IL-12 gene has reached clinical evaluation in the USA using a plasmid that in addition to IL- 12 gene also carry an antibiotic resistance gene needed for its production in bacteria. In Europe however, Eu- ropean Medicines Agency recommends against the use of antibiotics during the production of clinical grade plasmids. We have prepared several antibiotic resistance gene-free plasmids using an antibiotic-free selection strategy called operator-repressor titration, including plasmids encoding mouse, canine and human IL-12 orthologues. The aim of this study was to evaluate the maintenance of these plasmids in bacterial culture and test their transfection efficiency using gene electrotransfer. Plasmid maintenance was evaluated by determining plasmid yields and topologies after subculturing transformed bacteria. Transfection efficiency was evaluated by determining the plasmid copy number, expression and cytotoxicity after gene electrotransfer to mouse, canine and human melanoma cells. The results demonstrated that our IL-12 plasmids without an antibiotic resistance gene are stably maintained in bacteria and provide sufficient IL-12 expression after in vitro gene electrotransfer; therefore, they have the potential to proceed to further in vivo evaluation studies.
Keywords: electrotransfer, interleukin-12, immunotherapy, mammals
Published in DiRROS: 23.09.2022; Views: 24; Downloads: 12
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Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status
Katja Uršič Valentinuzzi, Špela Kos, Urška Kamenšek, Maja Čemažar, Simona Miceska, Boštjan Markelc, Simon Buček, Barbara Starešinič, Veronika Kloboves-Prevodnik, Richard Heller, Gregor Serša, 2021, original scientific article

Abstract: Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4%T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.
Keywords: electrochemotherapy, gene electrotransfer, interleukin-12
Published in DiRROS: 21.09.2022; Views: 44; Downloads: 16
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Evaluation of a novel plasmid for simultaneous gene electrotransfer-mediated silencing of CD105 and CD146 in combination with irradiation
Monika Savarin, Urška Kamenšek, Katarina Žnidar, Vesna Todorović, Gregor Serša, Maja Čemažar, 2021, original scientific article

Abstract: Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.
Keywords: CD105, CD146, plasmid, gene electrotransfer
Published in DiRROS: 21.09.2022; Views: 37; Downloads: 19
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PARP inhibitor olaparib has a potential to increase the effectiveness of electrochemotherapy in BRCA1 mutated breast cancer in mice
Maša Bošnjak, Tanja Jesenko, Boštjan Markelc, Larisa Janžič, Maja Čemažar, Gregor Serša, 2021, original scientific article

Abstract: Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast can-cer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to com-bine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin inducedDNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied inBRCA1mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines.Therapeutic effectiveness was studied in 2D and 3D cell cultures andin vivoon subcutaneousHCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined withthe evaluation ofcH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated theeffectiveness of ECT inBRCA1mutated HCC1937, but not in non-mutated HCC1143 cells. The combinedtherapy had a synergistic effect, which was due to the increased number of DNA double strand breaks.Addition of olaparib to ECT with bleomycinin vivoin HCC1937 tumor model had only minimal effect,indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repar inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin.
Keywords: electrochemotherapy, breast cancer, olaparib, bleomycin
Published in DiRROS: 21.09.2022; Views: 27; Downloads: 17
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Mutational burden, MHC-I expression and immune infiltration as limiting factors for in situ vaccination by TNF[alfa] and IL-12 gene electrotransfer
Urška Kamenšek, Katja Uršič Valentinuzzi, Boštjan Markelc, Maja Čemažar, Vita Šetrajčič Dragoš, Gregor Serša, 2021, original scientific article

Abstract: In situ vaccination is a promising immunotherapeutic approach, where various local ablative therapies are used to induce an immune response against tumor antigens that are released from the therapy-killed tumor cells. We recently proposed using intratumoral gene electrotransfer for concomitant transfection of a cytotoxic cytokine tumor necrosis factor-% (TNF%) to induce in situ vaccination, and an immunostimulatory cytokine interleukin 12 (IL-12) to boost the primed immune response. Here, our aim was to test the local and systemic effectiveness of the approach in tree syngeneic mouse tumor models and associate it with tumor immune profiles, characterized by tumor mutational burden, immune infiltration and expression of PD-L1 and MHC-I on tumor cells. While none of the tested characteristic proved predictive for local effectiveness, high tumor mutational burden, immune infiltration and MHC-I expression were associated with higher abscopal effectiveness. Hence, we have confirmed that both the abundance and presentation of tumor antigens as well as the absence of immunosuppressive mechanisms are important for effective in situ vaccination. These findings provide important indications for future development of in situ vaccination based treatments, and for the selection of tumor types that will most likely benefit from it.
Keywords: in situ vaccination, gene electrotransfer, interleukin 12, tumor necrosis factor [alfa]
Published in DiRROS: 19.09.2022; Views: 47; Downloads: 15
.pdf Full text (1,78 MB)

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