Expression of transgenes in peripheral blood mononuclear cells and their characterisation

Adoptivna celična terapija, ki temelji na uporabi gensko modificiranih limfocitov-T reprogramiranih za prepoznavo določenih tumor-specifičnih antigenov se je izkazala za uspešno metodo zdravljenja nekaterih vrst rakavih obolenj. Uspešnost je bila dokazana predvsem pri nekaterih terapijah B-celičnih levkemij, kot je naprimer akutna limfoblastna levkemija ter nekaterih ne-hodgkinovih limfomov. Pri tem so bili uporabljeni s himernim antigenskim receptorjem spremenjeni limfociti T (CAR-T) usmerjeni proti označevalcu CD19, značilnemu za limfocite B. V okviru magistrske naloge smo skušali s pomočjo elektrogenskega prenosa v humane enojedrne celice periferne krvi (PBMC) vstaviti zapis za anti-CD19 CAR. Določili smo optimalne pogoje za nespecifično aktivacijo in razmnoževanje gensko spremenjenih PBMC. Z uporabo plazmida z zapisom za GFP smo optimizirali pogoje elektroporacije. Celice smo okarakterizirali s pretočno citometrijo in fluorescenčno mikroskopijo. Optimalni pogoji transfekcije za sočasni vnos plazmidov z zapisom za CAR in encim transpozazo, ki je omogočila trajno spremembo genoma limfocitov T so bili pri enem pulzu napetosti 2250V in trajanju 20 ms. Prisotnost CAR smo dokazali neposredno s pretočno citometrijo in posredno z funkcijskim ELISA testom, kjer smo dokazovali prisotnost IL-2.Adoptive cell therapy based on the use of genetically modified T cells, programmed to identify specific tumor-specific antigens, has been proven to be a successful method of treating certain types of cancer. The efficacy has been proven primarily in the treatment of B-cell leukemias, such as, for example, acute lymphoblastic leukemia and some types of non-hodgkin`s lymphoma. In this case, the anti-CD19 CAR-T cells targeted against the B- lymphocyte antigen CD19 were used. In this thesis we attempted to insert anti-CD19 CAR molecular construct into human peripherial mononuclear blood cells by electroporation . First, we determined the optimum concentrations of reagents for non-specific activation and amplification of PBMC. Optimization of the electroporation conditions was carried out using the eGFP plasmid. The cells were characterized by flow cytometry and by fluorescence microscopy. Optimal transfection conditions for the simultaneous introduction of plasmids with CAR and the transposase enzyme, which allowed a permanent change in the genome of T lymphocytes, were at a pulse of 2250V and a duration of 20 ms. The presence of CAR was demonstrated in a direct manner with flow cytometry and in a indirect manner using the functional ELISA test for the IL-2 detection

Antitumor effectiveness of gene electrotransfer of plasmids encoding interleukins IL-2 and IL-12 in different murine tumor models

Znanstveno izhodišče: V zadnjih letih so se z razvojem novih tarčnih terapij posledično spremenili tudi načini terapije raka. V ospredje prihajajo različni pristopi na področju imunoterapije, kot so zaviralci imunskih kontrolnih točk, celična terapija, protitumorske vakcine in genska terapija z uporabo različnih citotoksičnih in imunomodulatornih molekul. Kot dostavni sistem pri genski terapiji raka lahko uporabimo tudi genski elektroprenos, kjer z dovajanjem električnih pulzov omogočamo lokalni vnos plazmidne DNA v celice različnih tkiv. Tako lahko dosežemo sočasni vnos dveh plazmidov z zapisom za interlevkina 2 (IL-2) in 12 (IL-12) v tumorje, ki se nato izražata v transfeciranih celicah in kot vnetna interlevkina sprožita imunski odziv. Protitumorska učinkovitost genskega elektroprenosa posameznega plazmida IL-2 ali plazmida IL-12 je bila dokazana že v številnih predkliničnih in tudi kliničnih študijah. Pri bolnikih, ki slabše odgovorijo na terapijo, bi lahko genski elektroprenos interlevkinov v kombinaciji z drugimi citokini ali z drugimi imunoterapevtskimi pristopi (npr. zaviralci imunskih kontrolnih točk) bistveno prispeval k celokupni učinkovitosti terapije. Kombinacija IL-2 in IL-12 temelji na njunem sinergističnem mehanizmu delovanja. Tako bi lahko imunski sistem bolje aktivirali ter s tem povečali protitumorsko učinkovitost v primerjavi s samostojnim elektroprenosom plazmida IL-2 ali IL-12. Namen doktorske naloge je bil določiti protitumorsko in antiangiogeno delovanje intratumorskega genskega elektroprenosa plazmidov z zapisom za IL-2 in IL-12 na dveh različnih mišjih tumorskih modelih. Raziskali smo učinek genskega elektroprenosa plazmidov na tumorske celice in vitro, protitumorsko in antiangiogeno delovanje kombinacije v primerjavi s samostojno terapijo, vpliv na izražanje različnih citokinov po terapiji ter prisotnost različnih imunskih celičnih populacij in vivo. Z navedenimi raziskavami smo želeli preveriti učinkovitost te terapije in poglobiti razumevanje o mehanizmih delovanja kombinacije IL-2 in IL-12 v tumorju. Metode: Najprej smo določili vpliv genskega elektroprenosa plazmidne DNA na preživetje in izražanje genov v dveh mišjih tumorskih celičnih linijah, melanoma B16F10 in raka debelega črevesja CT26, in vitro. Testirali smo dva različna pulzna protokola. Učinkovitost transfekcije 48 ur po genskem elektroprenosu pEGFP in pDsRed smo določili z uporabo fluorescentne mikroskopije v realnem času (večfunkcijski čitalec Cytation 1). Za učinkovitost transfekcije 48 ur po genskem elektroprenosu plazmidov IL-2 in IL-12 pa smo celicam izolirali RNA in določili raven izražanja interlevkinov IL-2 in IL-12 z metodo kvantitativnega PCR v realnem času (qRT-PCR) in testom ELISA. Poleg učinkovitosti transfekcije smo po genskem elektroprenosu določili še preživetje celic s pomočjo reagenta PrestoBlue?. V naslednjem sklopu smo določili učinkovitost transfekcije genskega elektroprenosa plazmidne DNA z zapisom za EGFP in DsRed in vivo. Tumorje smo analizirali s pomočjo pretočnega citometra BD FacsCantoTM in fluorescentne mikroskopije (mikroskop Olympus BX-51). V nadaljevanju smo nato določili protitumorski učinek kombinirane terapije na mišjih tumorskih modelih B16F10 in CT26. Rast tumorjev smo spremljali z merjenjem treh pravokotnih premerov tumorjev s kljunastim merilom. Pri miših, pri katerih smo opazili popolni odgovor (100 dni po terapiji brez otipljivega tumorja), smo tumor ponovno nasadili na levi bok in rast spremljali še dodatnih 100 dni. Odziv na terapijo oz. mehanizme protitumorskega učinka po genskem elektroprenosu smo dodatno ovrednotili z določevanjem infiltracije imunskih celic v tumor z imunohistokemičnim barvanjem in določevanjem izražanja izbranih citokinov s testoma ELISA in Magpix v tumorskih lizatih. Tumorske rezine smo imunohistokemično pobarvali s primarnimi oz. sekundarnimi protitelesi proti površinskim markerjem celic T pomagalk (CD4+), celic T ubijalk (CD8+), makrofagov (F4/80+ in MHC II+), dendritskih celic (CD11+) in endotelijskih celic (CD31+). Slike rezin smo zajeli s pomočjo svetlobne mikroskopije (mikroskop Olympus BX-51) pod 400-kratno povečavo. Optimizacijo terapije smo izvedli tako, da smo tumorjem 24 ur pred terapijo injicirali 50 µl kolagenaze (s koncentracijo 250 IU/ml) ter hialuronidazo (s koncentracijo 10.000 IU/ml) 2 uri pred terapijo. V zadnjem sklopu smo na CT26 tumorskem modelu določili še izventarčno in sistemsko učinkovitost kombinirane terapije (abskopalni učinek) ter ocenili aktivnost splenocitov po kombinirani terapiji. Raziskave na živalih so potekale v skladu z navodili in dovoljenjem Ministrstva za kmetijstvo, gozdarstvo in prehrano Republike Slovenije (št. dovoljenja U34401-1/2015/17 in U34401-3/2022/11). Za obdelavo podatkov smo uporabili program GraphPad, s pomočjo katerega smo ugotavljali razlike med skupinami z uporabo različnih statističnih testov. Rezultati: Za naše poskuse smo izbrali dve mišji celični linijimelanoma B16F10 in raka debelega črevesja CT26. V in vitro poskusu smo tri dni po genskem elektroprenosu plazmidov z EGFP, DsRed ter njune kombinacije in IL-2, IL-12 in njune kombinacije določili učinkovitost transfekcije in preživetje po elektroprenosu. Učinkovitost transfekcije je bila višja po elektroporaciji z EP1 protokolom in višja pri celicah B16F10 kot pri CT26. To smo dokazali tako pri rezultatih izražanja fluorescentnih proteinov kot tudi obeh interlevkinov. Genski elektroprenos plazmidov EGFP in DsRed ter njune kombinacije smo izvedli tudi v oba tumorska modela in vivo. Podatki pretočne citometrije in mikroskopije so pokazali, da je bila v tumorskem modelu B16F10 povprečna učinkovitost transfekcije po elektroporaciji z EP1 protokolom pri vseh skupinah večja kot po EP2, vendar razlike niso statistično pomembne. Nasprotno pa v CT26 nismo zaznali transfeciranih celic pri nobeni izmed dveh metod. Proučevali smo tudi protitumorski učinek genskega elektroprenosa kombinacije plazmidov IL-2 in IL-12 na obeh mišjih tumorskih modelih. Pri B16F10 modelu smo v skupini, ki je bila tretirana s kombinacijo plazmidov, opazili največji zaostanek v rasti tumorja v primerjavi z ostalimi skupinami. V tej skupini smo opazili tudi popolne odgovore in preživetje po ponovnem injiciranju tumorskih celic. Enak poskus smo ponovili pri tumorskem modelu CT26, kjer smo v skupini, tretirani s kombinacijo plazmidov, opazili zaostanek v rasti tumorjev, vendar pa nismo dosegli popolnih odgovorov, zato smo na tem tumorskem modelu izvedli optimizacijo terapije, tako da smo učinkovitost transfekcije izboljšali s predhodno obdelavo tumorjev s kolagenazo in hialuronidazo. Po optimizaciji smo opazili večji zaostanek v rasti v vseh skupinah, tretiranih s plazmidoma IL-2 in IL-12 posamezno ter njuno kombinacijo, v skupini s kombinacijo pa tudi popolne odgovore in preživetje po ponovnem injiciranju tumorskih celic. Pri CT26 tumorskem modelu smo določili še izventarčno in sistemsko učinkovitost optimizirane kombinirane terapije. Največji abskopalni efekt in citotoksično aktivnost splenocitov smo opazili pri tumorjih, tretiranih s kombinacijo. Določili smo tudi mehanizme protitumorskega učinka po genskem elektroprenosu in vivo na tumorskem modelu B16F10 in CT26. Za oceno učinkovitosti terapije smo opravili histološko analizo ter določili koncentracijo 32 vnetnih citokinov v vzorcih tumorja in seruma. Pri obeh modelih je bila infiltracija različnih imunskih celic najvišja pri skupini, tretirani s kombinacijo plazmidov. Opazili smo tudi povišanje različnih vnetnih citokinov v vseh skupinah, tretiranih z elektroporacijo, v skupini s kombinacijo plazmidov pa je bila močno povišana koncentracija IL-12, IFN? in TNF?, za katere je značilna izrazita protitumorska učinkovitost. Zaključki: Naši podatki kažejo na uspešnost genskega elektroprenosa kombinacije plazmidov z zapisom za IL-2 in IL-12 na dveh tumorskih modelih z različnim imunskim statusom. Kombinacija IL-2 in IL-12, ki se izražata v tumorskem okolju v zadostni količini, povzroči migracijo in stimulacijo imunskih celic na mesto tumorja, ki skupaj z antiangiogenim delovanjem IL-12 povzročijo uničenje tumorskih celic. Poleg tega je naš kombinirani pristop privedel tudi do protitumorskega imunskega spomina. V raziskavi smo tako dokazali, da je genska terapija s kombinacijo plazmidov IL-2 in IL-12 učinkovita za lokalno in tudi sistemsko terapijo tumorjev. Kljub dobrim rezultatom na obeh tumorskih modelih pa bi lahko terapijo na modelu CT26 še dalje optimizirali, da bi dosegli primerljivo učinkovitost z modelom B16F10. Rezultati doktorske naloge so doprinesli k razumevanju delovanja IL-2 in IL-12 na ravneh in vitro ter in vivo. Nenazadnje bodo rezultati doktorske naloge prav tako v pomoč pri potencialnem vpeljevanju terapije s kombinacijo teh dveh interlevkinov v klinično prakso.Scientific background: In recent years, the development of new targeted therapies has led to changes in the treatment of cancer. Various immunotherapy approaches such as immune checkpoint inhibitors, cell therapy, antitumour vaccines and gene therapy with various cytotoxic and immunomodulatory molecules have come to the fore. Gene electrotransfer can be used as a delivery strategy for gene therapy in cancer, whereby electrical pulses allow the local introduction of plasmid DNA into the cells of various tissues. In this way, two plasmids with transcripts for interleukin 2 (IL -2) and interleukin 12 (IL -12) can be introduced simultaneously into tumours, which are then expressed in the transfected cells and trigger an immune response as inflammatory interleukins. The antitumour efficacy of genetic electroporation of individual plasmid IL-2 or IL-12 has been demonstrated in numerous preclinical and clinical studies. In patients who respond poorly to therapy, gene transfer of interleukins in combination with other cytokines or other immunotherapeutic approaches (e.g. immune checkpoint inhibitors) could contribute significantly to the overall efficacy of therapy. The combination of IL-2 and IL-12 is based on their synergistic mechanism of action. In this way, the immune system could be better activated, increasing antitumour efficacy compared to plasmid IL-2 or IL-12 alone. The aim of this dissertation was to determine the antitumour and antiangiogenic activity of intratumoural gene transfer plasmids transcribed for IL-2 and IL-12 in two different mouse tumour models. We investigated the effect of the gene transfer plasmids on tumour cells in vitro, the antitumour and antiangiogenic activity of the combination compared to single therapy, the effect on the expression of different cytokines after therapy and the presence of different immune cell populations in vivo. These studies were conducted to test the efficacy of this therapy and to further our understanding of the mechanisms of action of the combination of IL-2 and IL-12 in tumours. Methods: We first investigated the effects of plasmid DNA gene transfer on survival and gene expression in two mouse tumour cell lines, melanoma B16F10 and colon cancer CT26, in vitro. Two different pulse protocols were tested. Transfection efficiency 48 hours after gene electroporation of pEGFP and pDsRed was determined using real-time fluorescence microscopy (Cytation 1 multifunctional reader). For transfection efficiency 48 hours after gene transfer of plasmids IL-2 and IL-12, RNA isolated from the cells was used to determine the expression levels of interleukins IL -2 and IL -12 by quantitative real-time PCR (qRT-PCR) and ELISA. In addition to transfection efficiency, we also determined cell survival after gene transfer using PrestoBlue〢 reagent. Next, we determined the transfection efficiency of electro-transfection of plasmid DNA transcribed for EGFP and DsRed in vivo. Tumours were analysed using a BD FacsCantoTM flow cytometer and fluorescence microscopy (Olympus BX -51 microscope). In another work, we then determined the antitumour effect of the combination treatment in the B16F10 and CT26 mouse tumour models. Tumour growth was monitored by measuring three rectangular diameters of the tumours with callipers. In mice in which a complete response was observed (100 days after treatment with no palpable tumour), the tumour was reinserted on the left flank and growth was monitored for an additional 100 days. Response to therapy or mechanisms of antitumour action after gene transfer were further investigated by determining infiltration of immune cells into the tumour by immunohistochemical staining and expression of selected cytokines by ELISA and Magpix in tumour lysates. Tumour sections were immunohistochemically stained with primary and secondary antibodies against surface markers of T helper cells (CD4+), T killer cells (CD8+), macrophages (F4/80+ and MHC II +), dendritic cells (CD11+) and endothelial cells (CD31+). Images of the sections were taken with a light microscope (Olympus BX -51 microscope) under 400x magnification. To optimise the therapy, 50 µL collagenase (250 IU/mL) was injected into the tumours 24 hours before therapy and hyaluronidase (10,000 IU/mL) was injected 2 hours before therapy. In a final step, we determined the extracellular and systemic efficacy of the combination therapy (abscope effect) in the CT26 tumour model and assessed splenocyte activity after combination therapy. The animal experiments were performed in accordance with the instructions and approval of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (approval numbers U34401-1/2015/17 and U34401-3/2022/11). GraphPad was used to process the data and to identify differences between the groups using various statistical tests. Results: For our experiments, we selected two mouse cell lines: melanoma B16F10 and colon cancer CT26. In an in vitro experiment, we transfected EGFP, DsRed and their combination, and IL-2, IL-12 and their combination plasmids for three days after gene electroporation and determined transfection efficiency and survival after electroporation. Transfection efficiency was higher after electroporation with the EP1 protocol and higher in B16F10 cells than in CT26 cells. This was demonstrated for both fluorescent protein expression results and for both interleukins. Genetic electroporation of pEGFP and pDsRed and their combination was also performed in both tumour models in vivo. Flow cytometry and microscopy data showed that in the B16F10 tumour model, the mean transfection efficiency was higher after electroporation with the EP1 protocol than after EP2 in all groups, but the differences were not statistically significant. In contrast, we were unable to detect transfected cells in CT26 using either method. We also investigated the antitumour effect of gene transfer of the combination of IL-2 and IL-12 plasmids in both mouse tumour models. In the B16F10 model, the group treated with the plasmid combination showed the greatest slowing of tumour growth compared to the other groups. In this group, we also observed a complete response and survival after reinjection of tumour cells. We repeated the same experiment in the CT26 tumour model, where we observed a delay in tumour growth in the group treated with the plasmid combination, but no complete responses. Therefore, we optimised the therapy in this tumour model by improving the transfection efficiency by pretreating the tumours with collagenase and hyaluronidase. After optimisation, we observed greater growth retardation in all groups treated with plasmids IL-2 and IL-12 alone and in their combination, and complete response and survival after reinjection of tumour cells in the combination group. In the CT26 tumour model, we also determined the extraventricular and systemic efficacy of the optimised combination therapy. The greatest abscopal effect and splenocyte cytotoxic activity was observed in tumours treated with the combination. We also investigated the mechanisms of antitumour action by in vivo gene transfer in the B16F10 and CT26 tumour models. To assess the efficacy of the therapy in the tumour, we performed histological analyses and determined the concentrations of 32 inflammatory cytokines in tumour and serum samples. In both models, infiltration of various immune cells was highest in the group treated with the plasmid combination. We also observed an increase in various inflammatory cytokines in all electroporation-treated groups. However, the combination plasmid group showed a significant increase in IL -12, IFNγ and TNFα, which are characterised by their pronounced anti-tumour activity. Conclusions: Our data show successful gene transfer of a combination of IL-2 and IL-12 transporter plasmids in two tumour models with different immune status. The combination of IL-2 and IL-12 expressed in sufficient amounts in the tumour environment induces migration and stimulation of immune cells to the tumour site, which together with the anti-angiogenic effect of IL-12 leads to tumour cell killing. In addition, our combined approach also resulted in anti-tumour immune memory. In this study, we have shown that gene therapy using a combination of IL-2 and IL-12 plasmids is effective for both local and systemic treatment of tumours. Despite the good results in both tumour models, the therapy in the CT26 model could be further optimised to achieve a comparable efficacy as in the B16F10 model. The results of this PhD thesis have contributed to the understanding of the effect of IL-2 and IL-12 at the in vitro and in vivo levels. Finally, the results of the PhD thesis will also be helpful for the possible introduction of a combination therapy with these two interleukins into clinical practice

Expression of GFP and DsRed fluorescent proteins after gene electrotransfer of tumour cells in vitro

Fluorescent reporter genes are widely used to study the transfection of various types of primary cells and cell lines. The aim of our research was to investigate the expression dynamics of GFP and DsRed reporter genes individually and combined after gene electrotransfer of plasmids with two different electroporation protocols in B16F10 and CT26 cells in vitro. The cytotoxicity after gene electrotransfer of both plasmids was first determined. Second, the intensity of fluorescence and the percentage of cells transfected with both plasmids individually and in combination were monitored in real time. The results show that the percentage of viability after gene electrotransfer of plasmids using the EP2 pulses was significantly higher compared to the EP1 pulses. In contrast, the percentage of transfected cells and fluorescence intensity were higher after gene electrotransfer with the EP1 pulse protocol. Moreover, the percentage of transfected cells was higher and started earlier in the B16F10 cell line than in the CT26 cell line. However, fluorescence intensity was higher in CT26 cells. Co-expression of fluorescent proteins was achieved only in a small number of cells. In conclusion, this study elucidated some of the dynamics of reporter gene expression in cancer cell lines after gene electrotransfer

Tumor Radiosensitization by Gene Electrotransfer-Mediated Double Targeting of Tumor Vasculature

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor β co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors

Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma

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

Gene Immunotherapy of Colon Carcinoma with IL-2 and IL-12 Using Gene Electrotransfer

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

Genska terapija v onkologiji, prvi razvojni koraki v Sloveniji

Gene therapy is also attracting interest in oncology. Probably the most interesting approach is immunostimulation. Plasmid DNA can be constructed, which is coding for a specific immunostimulatory molecule, which is then delivered into the cells, either in tumour or normal tissue. The transfected tissue then becomes the producer of the molecules encoded in the plasmid. The product is then released from the cells, either locally or systemically into the bloodstream. Since plasmids have hampered transport through the plasma membrane, delivery systems are needed that are either viral or nonviral. In our studies we predominantly use the non-viral transfection system, based on electroporation of the cells.Interleukin 12 (IL-12) is a cytokine with well-known anti-tumour and anti-angiogenic function. Therefore, in the SmartGene.si project we wanted to construct a plasmid DNA which is coding for IL-12 (plasmid phIL12), and perform all the necessary testing and prepare the documentation for its clinical testing in the treatment of skin tumours. The SmartGene.si consortium comprises partners from academia and industry. In the project it was necessary to prepare the plasmid according to the European Medicinal Agency (EMA) recommendations. For the application for the study approval submitted to the Agency for Medical Products and Medical Devices of the Republic of Slovenia (JAZMP), it was necessary to perform pharmacological, pharmacokinetic, and efficiency testing of phIL12. Thereafter, we had to develop the process and the facility, and prepare the drug.During the last three years, we have achieved all the goals and obtained the approval of the JAZMP for clinical testing of the product phIL12 in humans. We also obtained the approval of the National Ethics Committee. Currently, we are testing phIL-12 in a Phase I clinical protocol on head and neck skin tumours, with the aim to test the safety and feasibility of intratumoral gene electrotransfer of the plasmid phIL12. Another goal of the study is to determine a suitable dose of plasmid that could be used in future studies as adjuvant treatment to ablative therapies such as radiotherapy or electrochemotherapy.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

Gene therapy in oncology, first steps of development in Slovenia

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.Gene therapy is also attracting interest in oncology. Probably the most interesting approach is immunostimulation. Plasmid DNA can be constructed, which is coding for a specific immunostimulatory molecule, which is then delivered into the cells, either in tumour or normal tissue. The transfected tissue then becomes the producer of the molecules encoded in the plasmid. The product is then released from the cells, either locally or systemically into the bloodstream. Since plasmids have hampered transport through the plasma membrane, delivery systems are needed that are either viral or nonviral. In our studies we predominantly use the non-viral transfection system, based on electroporation of the cells. Interleukin 12 (IL-12) is a cytokine with well-known anti-tumour and anti-angiogenic function. Therefore, in the SmartGene.si project we wanted to construct a plasmid DNA which is coding for IL-12 (plasmid phIL12), and perform all the necessary testing and prepare the documentation for its clinical testing in the treatment of skin tumours. The SmartGene.si consortium comprises partners from academia and industry. In the project it was necessary to prepare the plasmid according to the European Medicinal Agency (EMA) recommendations. For the application for the study approval submitted to the Agency for Medical Products and Medical Devices of the Republic of Slovenia (JAZMP), it was necessary to perform pharmacological, pharmacokinetic, and efficiency testing of phIL12. Thereafter, we had to develop the process and the facility, and prepare the drug. During the last three years, we have achieved all the goals and obtained the approval of the JAZMP for clinical testing of the product phIL12 in humans. We also obtained the approval of the National Ethics Committee. Currently, we are testing phIL-12 in a Phase I clinical protocol on head and neck skin tumours, with the aim to test the safety and feasibility of intratumoral gene electrotransfer of the plasmid phIL12. Another goal of the study is to determine a suitable dose of plasmid that could be used in future studies as adjuvant treatment to ablative therapies such as radiotherapy or electrochemotherapy