Lentivirus-Mediated Platelet-Specific Gene Therapy of Hemophilia A.
Authors: Shi Qizhen , Wilcox D.A. , Fahs S.A. , Fang J. , Johnson B.D. , Weiler H. , Montgomery R.R. 
Published: October 2019

A variety of approaches have been utilized for the gene therapy of FVIII deficiency in mice, dogs, and humans. In humans results have been suboptimal. This has prompted exploration of other strategies to achieve clinical efficacy. Previously we demonstrated with a FVIII-expression vector that FVIII is synthesized and can be stored in endothelial cells (EC), Dami cells, and megakaryocytes in Weibel-Palade bodies and α-granules as well as storage granules in AtT-20 cells. In ECs and AtT-20 cells FVIII storage is VWF-dependent although in α-granules that VWF-dependency is not definitively established. Hematopoietic stem cells are an attractive target for gene therapy, as they can both self-renew and differentiate into all blood lineages where expression can be controlled through lineage-specific promoters. Targeting the FVIII synthesis to megakaryocytes will direct the storage of FVIII together with VWF in platelet α-granules. This pool of FVIII (and VWF) is protected from inhibitors and capable of regulated release at the site of vascular injury. In our study, the platelet-specific glycoprotein IIb (αIIb) promoter was used to direct human B-domain deleted FVIII (hB-FVIII) expression to platelets in FVIIInull mice. Two strategies were used to introduce FVIII expression in platelets of FVIII deficient mice - 1) lentivirus-mediated bone marrow transduction and syngeneic transplantation and 2) transgenic expression of platelet FVIII in mice developed through ES cell-mediated transgenic targeting. Functional FVIII was quantitated by a modified chromogenic assay of plasma and platelet lysate or releasate. Confocal intracellular immuno-colocalization of FVIII and VWF was studied. FVIII inhibitors were studied by ELISA. Phenotypic characterization was demonstrated by “tail-clip” survival test. Our results demonstrated that FVIII was undetectable in the plasma of mice obtained by either of our two strategies. In contrast, platelet lysates from these models demonstrated FVIII activity with the levels of FVIII being 1) 0.72 ± 0.22 mU/108 platelets following bone marrow transduction or 2) 0.74 ± 0.09 mU/108 by transgenic expression. Functional FVIII could be released from platelets in both groups by platelet agonist stimulation. Both groups of mice survived tail-clip and demonstrated phenotypic correction. In controls, no FVIII was detected in platelet lysate or releasate from either wild-type or FVIIInull mice. To date no inhibitory antibodies have been demonstrated in our bone marrow recipients. Confocal microscopy showed that FVIII protein was colocalized with VWF in platelets with both strategies. To demonstrate the stable long-term expression of FVIII in lentivirus-mediated bone marrow transduction transplantation, we did sequential bone marrow transplantation from one of our primary recipients 5 months after transplantation and the results demonstrated that 2° recipients also have phenotype correction and similar levels of platelet FVIII expression. Marrow from the 2° recipients was successfully transplanted into 3° recipients. These data demonstrate that FVIII can be specifically expressed and stored in platelets by lentivirus-mediated bone marrow transduction/transplantation and can correct the murine hemophilia A phenotype even in the absence of measurable plasma FVIII. This expression of platelet FVIII appears to be stable following sequential transplantation and suggests this as a promising novel strategy for gene therapy of hemophilia A patients - even for patients with FVIII inhibitors.