REPORTING STAGE no.1/2024 (01.04.2024 – 30.06.2024)
Summary stage no. 1/2024: The approval for the implementation of the study was obtained, no. 32744/08.07.2024 by the Ethics Committee of the Fundeni Clinical Institute. The inclusion of patients in the NEXTGenED project will be done in compliance with the national and international ethical norms in force according to the freely expressed consent. The procedure for receiving and processing samples (Biobanking) will include specific procedures for tissue and blood banking. For all samples received for registration and biobanking, a biological sample reception form (study inclusion form) was established. During the reporting period, work protocols and procedures were developed, thus the work protocols were drawn up for 1) isolation of liver cells 2) dissociation of liver cells and 3) Seeding of cells in matrigel to obtain organoids 4) passage of organoids and 5) in vitro differentiation of hepatocytes. Development of protocols and standard operating procedures for Stage 2 activities was initiated, focusing on the construction of sgRNAs targeting the demethylation of the CDKN2A promoter, as well as the production and purification of adeno-associated viruses (AAVs), and lentiviruses. The following steps were carried out: (1) primer design, (2) optimization of the protocol for AAV production and purification, and (3) optimization of the protocol for lentiviral production and purification.
REPORTING STAGE no.2/2024 (01.07.2024 – 31.12.2024)
Summary stage no. 2/2024 During stage 2, the activity of including patients in the study was initiated, respectively isolating primary liver cells with sample banking for 19 patients, while the master cell bank (P0-P4) was created for 9 donors in order to carry out the proposed experiments. At the same time, the amplification of the plasmids necessary for demethylation of the CDKN2A promoter was carried out. For this purpose, competent DH5 α cells were prepared and transformed with the constructs necessary for demethylation, and then the plasmid DNA was isolated. The process of cloning sgRNAs specific for the CDKN2A promoter was initiated. Before actually cloning the sgRNA, it is necessary to construct 2 vectors: one being represented by the cloning vector, and the other vector that will serve as a template for generating the 8 sgRNAs. Thus, for the generation of the 2 constructs, the vector and two fragments were amplified followed by Gibson assembly. In order to determine the methylation status of the CDKN2A promoter, genomic DNA was isolated from HepG2 and Huh7 cell lines using a kit, followed by bisulfite treatment to convert unmethylated cytosine to uracil, thus determining the methylation status of the promoter. Total RNA was also isolated from HepG2, Huh7 and Hep3B cell lines in order to clone the TP53, CTNNB1, LRP1B, ARID1A, AXIN1, ARID2 genes and determine the presence of point mutations and indel mutations. In this context, the plasmids necessary for the correction of point and indel mutations were amplified and isolated, and then the identity of the isolated plasmids was verified by digestion with restriction enzymes with unique cleavage specificity in the plasmid sequence.
REPORTING STAGE no.3/2025 (01.01.2025 – 30.06.2025)
Summary stage no. 3/2025 During phase 3, the activity of including patients in the study continued, respectively isolating liver primary cells, with sample banking being carried out for 17 patients while the master cell bank for patients derived organoids (PDO) (P0-P4) was created for 6 normal and 4 tumoral in order to carry out the proposed experiments. The patients were classified according to their conditions as follows: 9 patients with hepatocellular carcinoma (HCC), 2 patients with metastatic adenocarcinoma (ADK), 1 patient with cholangiocarcinoma and 5 patients are undergoing pathological diagnosis. The group is represented by 65% men and 35% women, with an average age of 65 years. At the same time, two distinct cell types were identified based on their phenotypes, defined as epithelial and mesenchymal, respectively. For this purpose, the expression levels of the markers albumin (ALB), cytokeratin-18 (KRT-18), cytokeratin-19 (KRT-19), Nanog, SOX2 and vimentin (VIM) were assessed in primary tumor cell cultures compared to the corresponding original tumor tissue using qRT-PCR technology. The results confirmed the establishment of two distinct types of primary cell cultures, characterized by an epithelial morphology supported by the presence of ALB and KRT-18 markers and a mesenchymal morphology indicated by the expression of KRT-19 and VIM markers. All biospecimens collected and processed from the 17 patients were included in the NEXTgenED project biobank database, patients derived organoids (PDO) included.
In order todemethylate the CDKN2A promoter, the Casilio-ME targeted demethylation system based on CRISPR-Cas9 was adopted. Cloning the eight sgRNAs of interest required the construction of two intermediate vectors: a cloning vector and a template vector containing the sequence required for their amplification. For the production and purification of adeno-associated virus, a protocol of co-transfection of HEK293T17 cells with 3 plasmids was used: pAAV2/8 (encodes the AAV8 capsid), pAdDeltaF6 (encodes essential helper genes for AAV production, of adenoviral origin), pAAV-GFP (encodes the system CRISPR/Cas9). The transduction efficiency of AAV containing the genes for base editing or primary editing will be assessed using an anti-dCas9 antibody.
Finally, an integrated database of DNA and RNA samples extracted from tumor and non-tumor tissues was created for both the prospective NEXTgenED cohort and the retrospective cohort proposed in the previous stage (53 patients, 35 HCC and 18 CCA), allowing the investigation of gene expression specific to liver tumors and facilitating the identification of new biomarkers for diagnosis and potential therapeutic targets, in correlation with the clinicopathological and molecular data of the patients.
REPORTING STAGE no.4/2025 (01.07.2025 – 31.12.2025)
Summary stage no. 4/2025: During this stage, eleven patients diagnosed with hepatocellular carcinoma (HCC) and mixed primary liver tumors (HCC combined with cholangiocarcinoma) were enrolled in the study. Primary two-dimensional (2D) cultures and three-dimensional (3D) organoid models were successfully established from both tumoral and matched non-tumoral liver tissues.
During the reporting period, the biobank was expanded to include non-tumoral (n = 5) and tumoral primary 2D cultures (n = 5), as well as tumoral (n = 7) and non-tumoral organoids (n = 8). In parallel, the immunohistochemistry protocol for 3D cultures was optimized, enabling detailed phenotypic and molecular characterization of the organoid models.
A patient with HCC exhibiting CDKN2A promoter hypermethylation was identified, as part of activities performed for objective OB1: Reactivation of pathologically hypermethylated genes implicated in hepatocarcinogenesis in organoid models through CRISPR/Cas9-mediated genome editing. These epigenetic alterations were further validated using immortalized hepatic cancer cell lines, with Huh7-D12 being found to be hypermethylated at the CDKN2A promoter compared to HepG2 (non-hypermethylated control). To support the completion of this objective, five multiplex CRISPR/Cas9 plasmid vectors were generated, each incorporating 2, 3, 4, 5, or 6 guide RNA (gRNA) sequences.
For OB2 and OB3, whole-genome sequencing (WGS) data from paired tumoral and adjacent non-tumoral tissue samples of three HCC patients included in the project were analyzed, allowing comprehensive identification and profiling of somatic mutations. Analysis of the sequencing data identified two known missense point mutations in a patient with HCC, both localized within a hotspot region. Using CRISPR/Cas9-based prime editing, patient-derived cells and organoids will be employed to correct these mutations and restore the wild-type amino acid sequence. Furthermore, whole-transcriptome sequencing was performed to quantify the expression levels of genes harboring the identified mutations. Transcriptomic findings were subsequently validated by real-time quantitative PCR (RT-qPCR) analysis, ensuring the robustness and reliability of the results.