1. Αποθετήριο Παραδοτέων Υποτρόφων Δράσεων ΕΣΠΑ Τριτοβάθμιας Εκπαίδευσης 2017-2023
2. Όλο το Αποθετήριο
3. Ερευνητικές ομάδες

Τύπος:	Ανακοίνωση σε συνέδριο
Τίτλος:	An novel PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of oral cancer
Συγγραφέας:	[EL] Μηλιώτου, Ανδρούλλα[EN] Miliotou, Androulla [EL] Ντέντη, Χαρίκλεια[EN] Ntenti, Charikleia [EL] Παππάς, Ιωάννης[EN] Pappas, Ioannis [EL] Παπαδοπούλου, Λευκοθέα[EN] Papadopoulou, Lefkothea
Ομιλητής:	[EL] Γεωργίου, Σοφία[EN] Georgiou, Sofia
Ημερομηνία:	28/05/2022
Περίληψη:	Chimeric Antigen Receptor (CAR) technology is a promising cell-based cancer immunotherapy, employing T-cells or Natural Killer (NK) cells, genetically engineered to express CAR receptors against tumor-associated antigens. CAR is a fusion protein consisting of a single chain fragment variant (scFv) from a monoclonal antibody and T-cells’ intracellular signaling domains. Different generations of CAR technology exist, depending on the intracellular domains, where 2nd generation CARs, as the ones employed here, have the 4-1BB or CD28 intracellular domains fused to CD3z. This study aims at the development of a CAR engineered NK-92 cell approach towards oral cancer immunotherapy. Oral cancer is highly resistant to conventional therapies and characterized by over-expression of ErbB proteins. ErbB receptors recognition via the ErbB ligand, T1E, fused to a CD28 plus CD3ζ endodomain, results in immune cell-mediated cytotoxicity in various types of cancer. Our group developed a new delivery platform, using in vitro transcribed (IVT)-mRNAs in combination with PTD technology, via a novel, patented conjugation reaction (Greek patent ΔΕ1010063; International PCT/GR2020/000059). Towards our goal, we constructed two different IVT-mRNAs for CAR, harboring T1E scFv and fused to CD28 or 4-1BB intracellular signaling sequences. Designed CAR sequences were cloned into pGEM vector for the in vitro transcription and the produced IVT-mRNAs were covalently conjugated to PTD to offer stability and to be transduced into NK-92 cells. The produced PTD-IVT-mRNAs were found to be relatively stable and protected in serum and in RNA-enriched environment. qPCR analysis has shown that PTD-IVT-mRNA of CAR sequences were efficiently transduced into NK-92 cells, compared to the corresponding naked IVT-mRNA, while time course analysis has shown that PTD-IVT-mRNA was transduced into NK-92 cells after 2h of incubation via a biphasic curve and remained intracellularly for up to at least 120 h. Western blot analysis against the CD3ζ epitope showed that PTD-IVT-mRNAs were expressed into the corresponding CAR molecules relatively rapid, persisted thereafter. Subcellular fractionation depicted that CAR molecules located to cellular membranes and cytoplasm, as well. NK-92 cells were initially checked for their well-known cytotoxicity to K562 cells. Trypan blue exclusion assay at the pool of cells showed marked cytotoxicity. Then, the CAR-engineered-NK-92 cells were co-incubated with HSC-3 cancer cells at a ratio 10/1 or 20/1 for different time points. Cell death was then assessed both at the supernatant as well among attached cells, after trypsinization. Co-incubation of NK-92 cells alone caused cell death to HSC-3 cells. Co-incubation of CAR-engineered NK-92 cells increased cell death of HSC-3 cells by almost 2-folds, compared to untransfected NK-92 cells. In conclusion, PTD technology was successfully exploited for the rapid, efficient transduction and translation of the stable IVT-mRNA, encoding the CAR molecule into NK-92 cells. CAR-engineered NK-92 cells have shown efficient cytotoxicity over HSC-3 cancer cells, leading to encouraging results for the application of our novel PTD-IVT-mRNA delivery platform for CAR immunotherapy.
Γλώσσα:	Ελληνικά
Τόπος δημοσίευσης:	Thessaloniki, Greece
Σελίδες:	1
Θεματική κατηγορία:	[EL] Φαρμακολογία και Φαρμακευτική[EN] Pharmacology and Pharmacy
Κάτοχος πνευματικών δικαιωμάτων:	© The Author(s) 2022
Ηλεκτρονική διεύθυνση του τεκμηρίου στον εκδότη:	https://www.vitacongress.gr/datafiles//Program_5-gene-therapy_Finals_Spreads(1).pdf
Όνομα εκδήλωσης:	5ο Πανελλήνιο Συνέδριο Γονιδιακής Θεραπείας και Αναγεννητικής Ιατρικής
Τοποθεσία εκδήλωσης:	Θεσσαλονίκη, Ελλάδα
Ημ/νία έναρξης εκδήλωσης:	27/05/2022
Ημ/νία λήξης εκδήλωσης:	29/05/2022
Σημειώσεις:	This research is co-funded by Greece and the European Union (European Social Fund- ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning 2014- 2020» in the context of the project “Development of a novel approach of CAR technology towards immunotherapy of oral cancer” (MIS 5070970)
Εμφανίζεται στις συλλογές:	Ερευνητικές ομάδες