https://hdl.handle.net/20.500.11811/876 2026-04-10T18:23:58Z https://hdl.handle.net/20.500.11811/13786 FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts El Hazzouri, Salime; Al-Rifai, Rawya; Surges, Nicole; Rath, Melanie; Singer, Heike; Oldenburg, Johannes; El-Maarri, Osman Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the <em>trans</em>-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. 2025-07-01T00:00:00Z https://hdl.handle.net/20.500.11811/13024 The role of microRNAs in defining LSECs cellular identity and in regulating <em>F8</em> gene expression Jamil, Muhammad Ahmer; Al-Rifai, Rawya; Nuesgen, Nicole; Altmüller, Janine; Oldenburg, Johannes; El-Maarri, Osman <strong>Introduction:</strong> Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.<br /> <strong>Methods:</strong> Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.<br /> <strong>Results:</strong> The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at <em>p</em> < 0.05) and other endothelial cells (72 microRNAs at <em>p</em> < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver.<br /> <strong>Discussion:</strong> Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in <em>F8</em> regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with <em>F8</em> expression within LSECs. This microRNA cohort emerges as a crucial modulator of <em>F8</em>, both directly through suppression and indirect effects on established <em>F8</em>-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients. 2024-02-19T00:00:00Z