IPS derived endothelial cells from Hemophilia A patients with nonsense mutations contain endogenous FVIII and the high risk inhibitor mutation R1941X shows abnormal intracellular trafficking of the FVIII heavy chain

H. Singer, N. Nuesgen, J. Oldenburg, O. El-Maarri (Bonn, Germany)

Date: 16.02.2017,
Time: 11:00 - 12:00

Objective: In about 20% of patients with severe Haemophilia A (HA), treatment with replacement FVIII is complicated due to the development of inhibitory antibodies against FVIII. The mutation type plays a pivotal role in the risk of inhibitor development. F8 nonsense mutations are grouped into the category of null mutations, which are believed to provoke higher immunogenicity against replacement FVIII by lacking specific endogenous cross reactive material (CRM negative) like F8 mRNA or protein. We therefore developed an IPS cell-based model for HA-patients to investigate the CRM-status of nonsense mutations in differentiated vascular endothelial cells (vECs) in relation to their inhibitor risk.

Methods: PBMCs from three HA-patients (R363X/A1, R431X/A2, R1941X/A3) and one healthy donor were expanded for erythroid progenitor cells (EPCs). EPC reprogramming was implemented using non-integrative episomal vectors containing the five reprogramming factors (Oct4, Sox2, Lin28, Klf4 and L-Myc). Stable IPS clones were differentiated into generic vascular endothelial cells using small molecule CHIR, Bmp4 and VEGF-A to analyze F8 mRNA (RT-PCR) and protein (FVIII:Ag & immunostaining).

Results: Differentiated endothelial cells from all three reprogrammed HA-patients contain full-length F8 mRNA without abnormal splicing. Furthermore, in all three patients intracellular FVIII protein was detectable with a polyclonal antibody (FVIII:Ag) and with different domain-specific monoclonal antibodies against FVIII (immunostaining). Patient-derived vECs with a nonsense mutation in the heavy chain (R363X/A1 & R431X/A2) showed normal intracellular trafficking of FVIII. However, patient-derived vECs harboring the high-risk inhibitor mutation R1941X/A3 represent an abnormal intracellular trafficking of the heavy chain by not showing any co-localization with vWF, ER or Golgi when stained with an A2-domain specific antibody.

Conclusion: This IPS-based cellular model enables us for the first time to track native FVIII in vascular endothelial cells. Our data clearly demonstrates a positive intracellular CRM-status in three HA-patients with different nonsense mutations, showing abnormal intracellular trafficking for the high risk inhibitor mutation R1941X, indicating that the high immunogenicity of specific nonsense mutations is not attributable to a negative CRM.