# FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts

## TL;DR

This study explores how Factor VIII moves through cells by using gene editing to remove specific proteins and observing the effects on its trafficking.

## Contribution

The study reveals new insights into the role of specific proteins in the trafficking of Factor VIII using CRISPR/Cas9 gene knockouts.

## Key findings

- FVIII trafficking is influenced by GABARAPs, CANX, and CALR proteins, with similar phenotypes observed in their knockouts.
- FVIII colocalizes with Rab8, Rab7, and VAMP8 in the trans-Golgi space, indicating involvement of the endomembrane system.
- Cell treatments like BFA, CQ, and glucose starvation affect FVIII distribution and secretion patterns.

## Abstract

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 GABARAPKO, CANXKO, and CALRKO 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 trans-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.