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1.
Gene immunotherapy of colon carcinoma with IL-2 and IL-12 using gene electrotransfer
Tilen Komel, Maša Omerzel, Urška Kamenšek, Katarina Žnidar, Urša Lampreht Tratar, Simona Kranjc Brezar, Klemen Dolinar, Sergej Pirkmajer, Gregor Serša, Maja Čemažar, 2023, izvirni znanstveni članek

Povzetek: Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.
Ključne besede: colon carcinoma, gene electrotransfer, gene immunotherapy
Objavljeno v DiRROS: 21.03.2024; Ogledov: 72; Prenosov: 43
.pdf Celotno besedilo (6,92 MB)
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2.
Radiosenzibilizacija tumorjev z elektroprenosom plazmida za dve antiangiogeni tarči
Monika Savarin, Katarina Žnidar, Gregor Serša, Tilen Komel, Maja Čemažar, Urška Kamenšek, 2023, objavljeni povzetek znanstvenega prispevka na konferenci

Ključne besede: miši, elektroprenos genov, gensko zdravljenje
Objavljeno v DiRROS: 19.06.2023; Ogledov: 307; Prenosov: 119
.pdf Celotno besedilo (310,08 KB)
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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, izvirni znanstveni članek

Povzetek: 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.
Ključne besede: CD105, CD146, plasmid, gene electrotransfer
Objavljeno v DiRROS: 21.09.2022; Ogledov: 490; Prenosov: 284
.pdf Celotno besedilo (4,74 MB)
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5.
Non-clinical in vitro evaluation of antibiotic resistance gene-free plasmids encoding human or murine IL-12 intended for first-in-human clinical study
Špela 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, izvirni znanstveni članek

Povzetek: 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.
Ključne besede: interleukin 12, gene electrotransfer, antibiotic resistance, plasmids
Objavljeno v DiRROS: 07.09.2022; Ogledov: 479; Prenosov: 283
.pdf Celotno besedilo (4,73 MB)
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6.
In vitro and in vivo correlation of skin and cellular responses to nucleic acid delivery
Maša Omerzel, Katarina Žnidar, A. Sales Conniff, Tanja Jesenko, Boštjan Markelc, Jared Tur, Nina Semenova, Kristopher Kohena, Simona Kranjc Brezar, Loree C. Heller, Maja Čemažar, 2022, izvirni znanstveni članek

Povzetek: Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immuno- genicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines.
Ključne besede: DNA sensors, cytokines, electrotransfer, skin
Objavljeno v DiRROS: 06.09.2022; Ogledov: 542; Prenosov: 285
.pdf Celotno besedilo (7,72 MB)
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7.
Genska terapija v onkologiji, prvi razvojni koraki v Sloveniji
Maja 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 Modic, 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, strokovni članek

Povzetek: 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.
Ključne besede: genska terapija, interlevkin-12, plazmidna DNA, elektroprenos genov, rak kože
Objavljeno v DiRROS: 01.07.2022; Ogledov: 1139; Prenosov: 229
.pdf Celotno besedilo (420,40 KB)

8.
Aktivacija citosolnih senzorjev DNA po obsevanju
Tanja Jesenko, Maša Omerzel, Boštjan Markelc, Gregor Serša, Katarina Žnidar, Loree C. Heller, Maja Čemažar, 2021, objavljeni znanstveni prispevek na konferenci

Ključne besede: citosolni senzorji, eksperimentalna onkologija, obsevanje
Objavljeno v DiRROS: 01.04.2022; Ogledov: 569; Prenosov: 186
.pdf Celotno besedilo (301,58 KB)

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