1. MitomiRs: their roles in mitochondria and importance in cancer cell metabolismAndrej Renčelj, Nada Gvozdenović, Maja Čemažar, 2021, review article Abstract: Background. MicroRNAs (miRNAs) are short non-coding RNAs that play important roles in almost all biological path- ways. They regulate post-transcriptional gene expression by binding to the 3%untranslated region (3%UTR) of messenger RNAs (mRNAs). MitomiRs are miRNAs of nuclear or mitochondrial origin that are localized in mitochondria and have a crucial role in regulation of mitochondrial function and metabolism. In eukaryotes, mitochondria are the major sites of oxidative metabolism of sugars, lipids, amino acids, and other bio-macromolecules. They are also the main sites of adenosine triphosphate (ATP) production. Conclusions. In the review, we discuss the role of mitomiRs in mitochondria and introduce currently well studied mito- miRs, their target genes and functions. We also discuss their role in cancer initiation and progression through the regu- lation of mRNA expression in mitochondria. MitomiRs directly target key molecules such as transporters or enzymes in cell metabolism and regulate several oncogenic signaling pathways. They also play an important role in the Warburg effect, which is vital for cancer cells to maintain their proliferative potential. In addition, we discuss how they indirectly upregulate hexokinase 2 (HK2), an enzyme involved in glucose phosphorylation, and thus may affect energy metabo- lism in breast cancer cells. In tumor tissues such as breast cancer and head and neck tumors, the expression of one of the mitomiRs (miR-210) correlates with hypoxia gene signatures, suggesting a direct link between mitomiR expression and hypoxia in cancer. The miR-17/92 cluster has been shown to act as a key factor in metabolic reprogramming of tumors by regulating glycolytic and mitochondrial metabolism. This cluster is deregulated in B-cell lymphomas, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, and T-cell lymphomas, and is particularly overexpressed in several other cancers. Based on the current knowledge, we can conclude that there is a large number of miRNAs present in mitochondria, termed mitomiR, and that they are important regulators of mitochondrial function. Therefore, mitomiRs are important players in the metabolism of cancer cells, which need to be further investigated in order to develop a potential new therapies for cancer. Keywords: microRNAs, mitomiR, mitochondria, cancer cell metabolism Published in DiRROS: 22.07.2024; Views: 148; Downloads: 48
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2. Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologuesUrška 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: 760; Downloads: 374
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3. Non-clinical in vitro evaluation of antibiotic resistance gene-free plasmids encoding human or murine IL-12 intended for first-in-human clinical studyŠpela Kos, Maša Omerzel, Tanja Jesenko, Boštjan Markelc, Urška Kamenšek, Katarina Žnidar, Urška Matkovič, Andrej Renčelj, Gregor Serša, Rosana Hudej, Aneja Tuljak, Matjaž Peterka, Maja Čemažar, 2021, original scientific article Abstract: Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans. Keywords: interleukin 12, gene electrotransfer, antibiotic resistance, plasmids Published in DiRROS: 07.09.2022; Views: 715; Downloads: 381
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5. Genska terapija v onkologiji, prvi razvojni koraki v SlovenijiMaja Čemažar, Tanja Jesenko, Maša Omerzel, Boštjan Markelc, Urška Kamenšek, Simona Kranjc Brezar, Špela Kos, Urša Lampreht Tratar, Katarina Žnidar, Andrej Renčelj, Urška Matkovič, Teja Valant, Kristina Levpušček, Živa Pišljar, Tilen Komel, Tim Božič, Urša Kešar, Barbara Starešinič, Katja Uršič Valentinuzzi, Monika Savarin, Primož Strojan, Gorana Gašljević, Maja Ota, Aleš Grošelj, Črt Jamšek, Rosana Hudej, Matjaž Peterka, Franc Smrekar, Barbara Hubad, Marjan Hosta, Jaka Kužnik, Alojz Hosta, Damijan Miklavčič, Matej Reberšek, Aleksandra Cvetkoska, Anja Zajc, Janja Dermol-Černe, Nataša Tozon, Nina Milevoj, Alenka Nemec Svete, Gregor Serša, 2022, professional article Abstract: Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija. Keywords: genska terapija, interlevkin-12, plazmidna DNA, elektroprenos genov, rak kože Published in DiRROS: 01.07.2022; Views: 1535; Downloads: 296
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