In the Presence of Pre-Existing Factor VIII (FVIII) Immunity, Hematopoietic Stem Cells (HSC) That Are Genetically Modified To Express FVIII in Platelets Were Successfully Transplanted into Hemophilic Mice under Myeloablative and Various Non-Myeloablative Conditions.
Authors: Shi Qizhen , Fahs Scot A. , Wilcox David A. , Kuether Erin L. , Weiler Hartmut , Montgomery Robert R. 
Published: October 2019

Abstract
While genetic induction of FVIII expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of pre-existing FVIII inhibitory antibodies, and whether such antibodies would affect therapeutic engraftment. The development of inhibitors to exogenous FVIII is considered a severe and important complication of FVIII infusion in hemophilia A patients. The clinical hallmark of inhibitor development in hemophilia A patients is failure to response to routine replacement therapy for bleeding episodes. In this study, we wanted to explore whether syngeneic transplantation of HSC that are genetically modified to express FVIII in platelets would restore hemostasis to hemophilic mice with pre-existing FVIII immunity. We generated a line of transgenic mice that express FVIII only in platelets using the platelet-specific αIIb promoter (2bF8) and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from immunized or non-immunized heterozygous 2bF8 transgenic mice was transplanted into immunized FVIIInull mice following lethal or sub-lethal irradiation. The recipients were analyzed by PCR, quantitative real-time PCR, platelet-lysate FVIII activity assay, inhibitor assay, and tail clip survival test to assess engraftment and phenotypic correction. Our results demonstrated that pre-existing anti-FVIII inhibitory antibodies in recipients did not inhibit successful engraftment of BM cells with 2bF8 genetic modification into hemophilia A mice. When we looked at the effect of inhibitor titer, following one month BM reconstitution, all recipients with inhibitor titers of 50 – 400 BU/ml (n = 9) survived tail clipping and 80% of recipients with a titer of 401 – 20,000 BU/ml (n = 25) also survived tail clipping when 1100 R (myeloablative) or 660 R (non-myeloablative) regimens were employed. When comparing conditioning regimens, in immunized recipients conditioned with 1100 R that received BM from 2bF8 donors the tail clip survival rate was 85% (n = 20). In the group of recipients in which 660R was employed 85.7% (n = 14) recipients survived tail clipping. When immunized recipients were conditioned with 440 R, 60% (n = 5) survived. In the group of recipients in which 220 R was employed, none (n = 4) survived tail clipping. The tail clip survival rate was not significantly different between the groups that received BM from immunized 2bF8 donors and from non-immunized donors. Different proportions of mixed BM from 2bF8 transgenic mice and FVIIInull mice were transplanted into lethally irradiated FVIIInull recipients to address what fraction of platelets would need to express FVIII in order to restore hemostasis to hemophilic mice with inhibitors. Our results showed that transplantation with only 5% 2bF8 BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not alter engraftment of 2bF8 genetically modified HSC and transplantation of these HSC can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies under either myeloablative or non-myeloablative regimens.