INFORMATII GENERALE

TITLU PROIECT: “Dezvoltarea unei noi tehnologii pentru a estima maturarea celulelor hepatice transdiferențiate în celule producătoare de insulină”

COD PROIECT: PN-III-P2-2.1-PED2021-3180

Nr. Contract: 629PED/2022

 Acronim: miR-DIAB

Autoritatea de finantare: Unitatea Executiva pentru finantarea Invatamantului Superior, a Cercetarii, Dezvoltarii si Inovarii (UEFISCDI) Programul 2 – Creşterea competitivităţii economiei româneşti prin cercetare, dezvoltare şi inovare, Subprogramul 2.1. Competitivitate prin cercetare, dezvoltare şi inovare – ”Proiect experimental demonstrativ” (PED)

Director de proiect: Dr. Daniela Lixandru

Coordonator: Institutul Clinic Fundeni

Durata proiectului: 24 luni, 04/07/2022 – 03/07/2024

Valoarea totala: 598.782 RON

OBIECTIV GENERAL

Proiectul miR-DIAB propune o abordare nouă pentru terapia personalizată a diabetului, prin dezvoltarea unei noi tehnologii și anume, un scor de predicție a eficienței transdiferențierii ficatului la pancreas (TD), bazat pe semnătura miARN a donatorului de ficat. În plus, utilizarea tehnologiei inovatoare de manipulare a miARN va permite creșterea numărului de celule producătoare de insulină rezultate și a capacității terapeutice ca terapie de înlocuire celulară pentru pacienții cu diabet.

 REZULTATE

ETAPA DE RAPORTARE: etapa nr.1/2022

Denumirea etapei 1: Obtinerea culturilor primare de hepatocite (1-2 culturi) (partial). Analiza rolului miRNA specifice in procesul de transdiferentiere a ficatului la pancreas (partial) Optimizare protocoale de lucru studiu animal – in vivo (partial)

Rezumat etapa 1: Pe parcursul primei etape a fost initiata si optimizata izolarea de celule hepatice (2 donatori) in vederea realizarii bancii de celule master (P0-P4). Au fost stabilite analizele de expresie genica si proteica pentru un panel reprezentativ de biomarkeri (vimentina, N si E-Caderina, CK-18, Snail) de inclus pentru cele doua tipuri de celule, de tip hepatocitar si cele transdiferenţiate, de tip β pancreatice. Experimentele de manipulare a expresiei miRNA vor fi realizate initial pentru miR-424-5p si miR-26a inaintea procesul de transdiferentiere si se vor desfasura conform unui design experimental ce implica inhibarea si supraexpresia in sistem de culturi bidimensionale, aderente, a stocurilor generate in cadrul miR-Diab de la pacienti diabetici, comparandu-se expresia acestora cu stocurile de la pacientii non-diabetici de referinta. In acelasi timp, am demarat pregatirea loturilor de animale pentru a analiza în continuare eficacitatea procesului de transdiferenţiere prin implantarea subcutanata (Sub-Q) la soarecii imunodeficienti SCID-Beige.

ETAPA DE RAPORTARE: etapa nr.2/2023

Denumirea etapei 2: Obtinere culturi primare de hepatocite (3-5 pacienti) Analiza rolului miRNA selectate in procesul de transdiferentiere a ficatului la pancreas Studiul animal in vivo – follow-up 3-4 luni (2 modele) (partial) Diseminare.

Rezumat etapa 2/2023: Pe parcursul etapei 2 a fost continuată activitatea de includere in studiu de pacienţi noi, respectiv de izolare celule hepatice, fiind realizată banca de celule master (P0-P4) pentru 4 donatori in vederea realizării experimentelor propuse. S-a realizat clonarea factorilor transcriptionali PDX1, NeuroD1, MAFA si FOXA2, miR-ul mimic si inhibitor (miR-424-3p si miR-424-5p) in vectorul de expresie pAdTrack-CMV. De asemenea, s-au construit adenovirusurile corespunzatoare pentru fiecare construct, prin impachetare si amplificare in celulele Ad-HEK-293. Folosind cromatografia, adenovirusurile amplificate au fost purificate si apoi titrate pentru a determina numarul particulelor virale infectioase pentru a fi folosite in etapa urmatoare la transdiferentierea celulelor hepatice in celule β-pancreatice secretoare de insulina. Experimentele de manipulare a expresiei miRNA au fost realizate initial pentru miR-424-5p si miR-26a folosind reactivul de transfecţie RNAiMax inaintea procesul de transdiferentiere cu 24, 48 si 96 de ore (n=3). In acelasi timp, a fost finalizat un studiu pentru stabilirea importantei factorului VEGF-A in vivo pe soarecii SCID-Beige (n=75) si am demarat pregatirea loturilor pentru cele 2 modele propuse pentru a analiza în continuare eficacitatea procesului de transdiferenţiere prin implantarea subcutanata (Sub-Q) a celulelor netransdiferentiate (NT), transdiferenţiate (TD), TD cu miR supraexprimat (TDmiR+), si TD cu miR inhibat (TDmiR-) impreuna cu celule care exprima VEGF-A (n=128).

PUBLICATII

Articole ISI publicate in 2022:

• miRNAs as Biomarkers in Diabetes: Moving towards Precision Medicine (Review) Maria Alexandra Angelescu, Octavian Andronic, Simona Olimpia Dima, Irinel Popescu, Irit Meivar-Levy, Sarah Ferber, Daniela Lixandru Int. J. Mol. Sci., 23 (21), 12843, 2022, IF:6.208 https://doi.org/10.3390/ijms232112843

Articole ISI publicate in 2023:

• Alina-Veronica Ghionescu, Andrei Sorop, Simona Dima The pivotal role of EMT-related noncoding RNAs regulatory axes in hepatocellular carcinoma Pharmacol., 11 September 2023, Sec. Pharmacology of Anti-Cancer Drugs, Volume 14 – 2023 | https://doi.org/10.3389/fphar.2023.1270425

Prezentari la conferinte nationale/internationale în 2023:

• Ekaterini Linioudaki, Ioana Raluca Florea, Andrei Sorop, Veronica Madalina Ilie, Marton Fogarasi, Irit Meivar-Levy, Simona Olimpia Dima, Irinel Popescu, Sarah Ferber, Daniela Lixandru „Unraveling the role of miRNAs in liver to pancreas transdifferentiation”, Congresul Universitaţii de Medicină şi Farmacie „Carol Davila”, al XI-lea Congres Științific cu participare internațională, Perspective Interdisciplinare, 26-28 octombrie 2023, (prezentare orala);

Lucrarea prezentata a luat premiul pentru “Cea mai buna prezentare” din cadrul sesiunii “Tanărul cercetător” pentru specialităţi preclinice.

EN – GENERAL INFORMATION

Project TITLE “Development of a new technology to estimate the maturation of liver cells transdifferentiated in insulin-producing cells”

Project COD PN-III-P2-2.1-PED2021-3180

Contract No. 629PED/2022

Acronim: miR-DIAB

Contracting Authority: Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI)

Project Leader: Dr. Daniela Lixandru

Coordinator: Fundeni Clinical Institute

Project Duration: 24 months, 04/07/2022 – 03/07/2024

Total budget: 598.782 RON

MAIN OBJECTIVE

miR-DIAB project  propose a new approach for personalized therapy of diabetes, by developing a new technology as a prediction score of the liver to pancreas transdifferentiation (TD) efficiency, based on miRNA signature of the liver donor. Furthermore, using the innovative technology of miRNA manipulation will allow to increase the number of the resulted insulin producing cells and therapeutic capacity as cell replacement therapy for patients with diabetes.

RESULTS

Stage no. 1/2022

Name stage 1: Obtaining primary cultures of hepatocytes (1-2 cultures) (partially). Analysis of the role of specific miRNAs in the transdifferentiation process of liver to pancreas (partially) Optimization of work protocols animal study – in vivo (partially)

Abstract: During the first stage, the isolation of liver cells (2 donors) was initiated and optimized in order to create the master cell bank (P0-P4). Gene and protein expression analyzes were established for a representative panel of biomarkers (vimentin, N and E-Cadherin, CK-18, Snail) to be included for the two types of cells, hepatocytic and transdifferentiated, pancreatic β type. The miRNA expression manipulation experiments will be initially performed for miR-424-5p and miR-26a before the transdifferentiation process and will be carried out according to an experimental design that involves inhibition and overexpression in a two-dimensional, adherent culture system, of the stocks generated within miR-Diab from diabetic patients, comparing their expression with stocks from non-diabetic reference patients. At the same time, we started preparing batches of animals to further analyze the effectiveness of the transdifferentiation process by subcutaneous implantation (Sub-Q) in immunodeficient SCID-Beige mice.

Stage no. 2/2023

Name stage 2: Obtaining primary cultures of hepatocytes (3-5 patients). Analysis of the role of selected miRNAs in the transdifferentiation process of liver to pancreas Animal study in vivo – follow-up 3-4 months (2 models) (partially) Dissemination.

Abstract: During stage 2, the activity of including new patients in the study was continued, respectively of liver cell isolation, and the bank of master cells (P0-P4) was created for 4 donors in order to carry out the proposed experiments. The transcriptional factors PDX1, NeuroD1, MAFA and FOXA2, the miRs mimic and inhibitory (miR-424-3p and miR-424-5p) were cloned in the pAdTrack-CMV expression vector. Also, the corresponding adenoviruses were constructed for each construct, by packaging and amplification in Ad-HEK-293 cells. Using chromatography, the amplified adenoviruses were purified and then titrated to determine the number of infectious viral particles to be used in the next step to transdifferentiate liver cells into insulin-secreting pancreatic β-cells. The miRNA expression manipulation experiments were initially performed for miR-424-5p and miR-26a using the RNAiMax transfection reagent before the transdifferentiation process with 24, 48 and 96 hours (n=3). At the same time, a study was completed to establish the importance of the VEGF-A factor in vivo on SCID-Beige mice (n=75) and we started preparing the batches for the 2 proposed models to further analyze the effectiveness of the transdifferentiation process through subcutaneous implantation (Sub-Q) of untransdifferentiated (NT), transdifferentiated (TD), miR-overexpressed TD (TDmiR+), and miR-inhibited TD (TDmiR-) cells together with cells co-expressing VEGF-A (n=128).

PUBLICATIONS

 ISI article published in 2022:

• miRNAs as Biomarkers in Diabetes: Moving towards Precision Medicine (Review) Maria Alexandra Angelescu, Octavian Andronic, Simona Olimpia Dima, Irinel Popescu, Irit Meivar-Levy, Sarah Ferber, Daniela Lixandru Int. J. Mol. Sci., 23 (21), 12843, 2022, IF:6.208 https://doi.org/10.3390/ijms232112843

ISI article published in 2023:

• Alina-Veronica Ghionescu, Andrei Sorop, Simona Dima The pivotal role of EMT-related noncoding RNAs regulatory axes in hepatocellular carcinoma Pharmacol., 11 September 2023, Sec. Pharmacology of Anti-Cancer Drugs, Volume 14, 2023, IF:5.988 | https://doi.org/10.3389/fphar.2023.1270425

Scientific Congresses 2023:

• Ekaterini Linioudaki, Ioana Raluca Florea, Andrei Sorop, Veronica Madalina Ilie, Marton Fogarasi, Irit Meivar-Levy, Simona Olimpia Dima, Irinel Popescu, Sarah Ferber, Daniela Lixandru „Unraveling the role of miRNAs in liver to pancreas transdifferentiation”, “Carol Davila” University of Medicine and Pharmacy Congress, XI Scientific Congress with international participation, Interdisciplinary Perspectives, 26-28 October 2023, (oral presentation);

The presented work won the award for “Best Presentation” from the “Young Researcher” session for preclinical specialties.