Development of a Human in Vitro Cell Model for Haemophilia a
Authors: Toenges Rosa , Hodroj Nadin , Kurrle Nina , Miesbach Wolfgang , Schnuetgen Frank , Serve Hubert 
Published: November 2019

Aim
Haemophilia A is an X-chromosome linked hereditary bleeding disorder caused by loss-of-function mutations in the clotting factor VIII (FVIII) gene resulting in either a deficit or a total lack of the corresponding activity. Although the quality of the standard replacement therapy has improved significantly over the last decades, patients still require FVIII administration at regular intervals and frequently develop an immune response against the exogenous protein that interferes with FVIII function. FVIII gene therapy could provide a less intrusive and perhaps safer alternative to current FVIII replacement therapy.
Here, we aimed to develop a human based cell model to facilitate future effective development and evaluation of therapeutic gene therapy approaches for haemophilia A. In order to closely model key challenges for such a development, we chose to introduce patient-specific FVIII mutations into immortalized human hepatic sinusoidal endothelial cells, which are the cells that naturally secret FVIII.
Methods
First, we immortalized primary human hepatic sinusoidal endothelial cells (HHSEC), the natural cell of FVIII synthesis in humans, by lentiviral transduction of a doxycycline-inducible SV40-Large T oncogene. After establishment of the HHSEC cell line, we chose from the FVIII gene variant database (European Association for Haemophilia and allied disorders) five different FVIII mutations resulting in a severe form of haemophilia A. All of the selected variants were small deletions resulting in frameshift mutations, leading to a loss-of-function FVIII gene product. The FVIII mutations were introduced by lentiviral transduction of sgRNAs causing DNA double strand breaks at the respective positions together with the RNA-guided CRISPR/Cas9 (saCas9) endonuclease. Characterization and verification of immortalized HHSEC with and without FVIII mutation were performed by sequencing, immunofluorescence and aPTT-based FVIII activity assays.
Results
Our first experiments resulted in the successful immortalization of primary HHSEC cells. Since the cells were conditionally immortalized, they returned to the primary cell state upon removal of doxycycline. By western blotting we could demonstrate SV40-Large T oncogene expression under doxycycline conditioning, and a decrease in expression after doxycycline withdrawal. Additionally, using cumulative proliferation assays (cell counting assay), we could estimate a doubling time of almost 2 days for the immortalized HHSEC under doxycycline treatment and strong proliferative disadvantage in absence of doxycycline. Subsequently, we examined the FVIII activityof immortalized HHSEC using immunofluorescence microscopy and an aPTT-based FVIII clotting assay. Immunohistochemical staining for FVIII in the immortalized HHSEC revealed a strong signal resembling vesicular structures as expected. Using the established aPTT-based FVIII clotting assay, which is also used in the clinic to estimate FVIII residual activities, we could demonstrate functional FVIII activity of 75% up to 99% of normal human plasma. FVIII activity was measured in medium supernatant after 24h of incubation, at least at three independent days. The FVIII activity showed always similar values and stayed constant over the time.
We successfully introduced two out of five haemophilia A patient-specific mutations into the FVIII gene of the HHSEC. Sequence analysis revealed for one mutant cell line a predominant deletion of seven base pairs and for the other cell line a mixed indel generation both resulting in frameshift mutations. FVIII clotting assays showed a reduction of FVIII activity to 40%. Likewise, the immunohistochemical staining showed reduced intensity. In ongoing experiments, we currently generate a full FVIII knock-out cell line by subcloning, in order to get cell lines strongly resembling FVIII activity levels that are seen in severe haemophilia A patients (&lt;1%).
Conclusion
By immortalization and transduction of HHSECs we generated a human haemophilia A cell line model based on mutagenesis of the endogenous genetic FVIII locus in the cell line, which mainly accounts for FVIII production in humans. This cell model will be used to study FVIII synthesis, signaling and processing and, in addition, aids in the development of new strategies for haemophilia A gene therapy or new FVIII preparations.

Disclosures
Miesbach: Bayer, Chugai, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire, UniQure: Speakers Bureau; Bayer, BioMarin, CSL Behring, Chugai, Freeline, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda/Shire, UniQure: Consultancy; Bayer, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire: Research Funding.