In Contrast to Full-Length Factor VIII, the Calnexin (ER Chaperone) and LMAN1 (ERGIC transporter) Knockouts Have Minimal Effect on the Secretion of B-Domain-Deleted Factor VIII: Implication for Gene Therapy

 

Introduction: The B-domain of factor VIII (FVIII) is a large domain that encompasses approximately 40% of its size and harbors the majority of its glycosylation sites. This domain does not play a direct role in the coagulation activity of FVIII. Therefore, B-domain-deleted (BDD)-FVIII is being used successfully in recombinant replacement therapy. However, the efficiency of BDD-FVIII in gene therapy protocols looks less than optimal in comparison to factor IX gene therapy. The route taken by BDD-FVIII leading to higher secretion levels is still not well understood and remains to be elucidated. The B-domain is the site of interaction with chaperones and cargo proteins (endoplasmic reticulum [ER]: CANX and CALR; COPII: LMAN1 and MCFD2), which constitute the classical conventional secretion pathway. In the absence of B-domain, these interactions are disrupted, and FVIII may traverse an alternative pathway. Therefore, a detailed molecular analysis is required to illustrate the differences in intracellular trafficking caused by the deletion of the B-domain. Such an analysis would be done by studying the knockout (KO) effect of landmark transporter proteins on the intracellular trafficking of full-length (FL) as well as BDD-FVIII protein.

Materials and Methods: We used two cell lines: HEK293 cells overexpressing FL-FVIII (HEK28) and HEK293 cells overexpressing BDD-FVIII (HEKBDD-2). These two cell lines were used to generate a CRISPR-based KO of the CANX and LMAN1 genes.

Our KOs were verified by Western blot analysis and immunofluorescence staining using antibodies against CANX and LMAN1 proteins. The fluorescence images were acquired using a Zeiss Apotome microscope.

After verification of the KOs, we measured FVIII activity. We compared the FVIII activity in our KO cells to the activity in the wild-type cells. The media were clarified by centrifugation and assayed for FVIII activity using the Chromogenix Coamatic Factor VIII assay.

Results and Conclusions: Our results show that the KOs caused a relatively small effect on BDD-FVIII secretion in comparison to FL-FVIII. The Calnexin KO caused an increase of 80% and a decrease of about 22% for FL-FVIII and BDD-FVIII respectively. However, the LMAN1 KO caused a decrease of about 75 and 40% for FL-FVIII and BDD-FVIII, respectively. These results suggest that at any time 75% of FL and only 40% of BDD-FVIII leave the cell through the conventional secretion pathway. In addition, Calnexin's absence does not appear to affect BDD-FVIII secretion. This also suggests that CANX does not interact with BDD-FVIII, hence it is not held in the ER by the quality control function of CANX. Therefore, BDD-FVIII appears to exit the ER easily. In conclusion, these results clearly show different effects of KOs on the FL-FVIII and BDD-FVIII, which is caused by differential intracellular interactions of both proteins with the secretion machinery. This has an implication on vector design for hemophilia A gene therapy.

Publication History

Article published online:
26 October 2022

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