Scot A. Fahs

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A.

Summary.  Background: Previous studies from our laboratory have demonstrated that lineage‐targeted synthesis of factor VIII (FVIII) under the direction of the platelet‐specific integrin αIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. Objective: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII‐deficient (FVIIInull) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)‐based gene transfer cassette encoding 2bF8. Results: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV‐transduced BM survived tail clipping and we did not detected inhibitory or non‐inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIIInull secondary recipients demonstrated sustained platelet‐FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long‐term repopulating haematopoietic stem cells. Conclusions: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus‐mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.

Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity

Although genetic induction of factor VIII (FVIII) expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of preexisting FVIII inhibitory antibodies to determine whether such antibodies would affect therapeutic engraftment. We generated a line of transgenic mice (2bF8) that express FVIII only in platelets using the platelet-specific alphaIIb promoter and bred this 2bF8 transgene into a FVIII(null) background. Bone marrow (BM) from heterozygous 2bF8 transgenic (2bF8(tg+/-)) mice was transplanted into immunized FVIII(null) mice after lethal or sublethal irradiation. After BM reconstitution, 85% of recipients survived tail clipping when the 1100-cGy (myeloablative) regimen was used, 85.7% of recipients survived when 660-cGy (nonmyeloablative) regimens were used, and 60% of recipients survived when the recipients were conditioned with 440 cGy. Our further studies showed that transplantation with 1% to 5% 2bF8(tg+/-) 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 negate engraftment of 2bF8 genetically modified hematopoietic stem cells, and transplantation of these hematopoietic stem cells can efficiently restore hemostasis to hemophilic mice with preexisting inhibitory antibodies under either myeloablative or nonmyeloablative regimens.

Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

The natural cell type(s) that synthesize and release factor VIII (FVIII) into the circulation are still not known with certainty. In vitro studies indicate that artificial expression of FVIII in endothelial cells produces an intracellular pool of FVIII that can be mobilized together with its carrier protein, von Willebrand factor (VWF), by agonists. Here, we show that expression of human B-domain deleted FVIII (hFVIII) in the vascular endothelium of otherwise FVIII-deficient mice results in costorage of FVIII and VWF in endothelial Weibel-Palade bodies and restores normal levels and activity of FVIII in plasma. Stored FVIII was mobilized into the circulation by subcutaneous administration of epinephrine. Human FVIII activity in plasma was strictly dependent on the presence of VWF. Endothelial-specific expression of hFVIII rescued the bleeding diathesis of hemophilic mice lacking endogenous FVIII. This hemostatic function of endothelial cell-derived hFVIII was suppressed in the presence of anti-FVIII inhibitory antibodies. These results suggest that targeting FVIII expression to endothelial cells may establish a releasable pool of FVIII and normalize plasma FVIII level and activity in hemophilia A, but does not prevent the inhibitory effect of anti-FVIII antibodies on the hemostatic function of transgene-derived hFVIII as is seen with platelet-derived FVIII expression.

Lentivirus‐mediated platelet gene therapy of murine hemophilia A with pre‐existing anti‐factor VIII immunity

Summary.  Background:  The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof‐of‐principle that platelet‐specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies.

A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII

The cellular source of coagulation factor VIII (FVIII) remains controversial. Like many coagulation proteins, FVIII is produced in the liver, and FVIII synthesis has long been associated with hepatocytes. But extrahepatic synthesis also occurs, and mounting evidence suggests that hepatocytes are not responsible for FVIII production. To determine the tissue that synthesizes FVIII, we developed a Cre/lox-dependent conditional knockout (KO) model in which exons 17 and 18 of the murine factor VIII gene (F8) are flanked by loxP sites, or floxed (F8(F)). In cells expressing Cre-recombinase, the floxed sequence is deleted, resulting in F8(F→KO) gene inactivation. When F8(F) mice were crossed with various tissue-specific Cre strains, we found that hepatocyte-specific F8-KO mice are indistinguishable from controls, whereas efficient endothelial-KO models display a severe hemophilic phenotype with no detectable plasma FVIII activity. A hematopoietic Cre model was more equivocal, so experimental bone marrow transplantation was used to examine hematopoietic FVIII synthesis. FVIII(null) mice that received bone marrow transplants from wild-type donors were still devoid of plasma FVIII activity after hematopoietic donor cell engraftment. Our results indicate that endothelial cells are the predominant, and possibly exclusive, source of plasma FVIII.

Contribution of platelet vs. endothelial VWF to platelet adhesion and hemostasis

Summary.  Background:  von Willebrand factor (VWF) is a glycoprotein that plays an important role in primary hemostasis. VWF is synthesized and stored in endothelial cells (ECs) and megakaryocytes/platelets. Plasma VWF is primarily derived from ECs and is generally believed to be essential for hemostasis. VWF synthesized in megakaryocytes is stored in platelet α‐granules, from which it is released following platelet activation. The relative contribution of VWF stored in ECs or megakaryocytes/platelets or present in plasma to hemostasis is not clear.

Factor IX ectopically expressed in platelets can be stored in α-granules and corrects the phenotype of hemophilia B mice

We developed 2bF9 transgenic mice in a hemophilia B mouse model with the expression of human factor IX (FIX) under control of the platelet-specific integrin alphaIIb promoter, to determine whether ectopically expressing FIX in megakaryocytes can enable the storage of FIX in platelet alpha-granules and corrects the murine hemophilia B phenotype. FIX was detected in the platelets and plasma of 2bF9 transgenic mice by both antigen and activity assays. Approximately 90% of total FIX in blood was stored in platelets, most of which is releasable on activation of platelets. Immunostaining demonstrated that FIX was expressed in platelets and megakaryocytes and stored in alpha-granules. All 2bF9 transgenic mice survived tail clipping, suggesting that platelet-derived FIX normalizes hemostasis in the hemophilia B mouse model. This protection can be transferred by bone marrow transplantation or platelet transfusion. However, unlike our experience with platelet FVIII, the efficacy of platelet-derived FIX was limited in the presence of anti-FIX inhibitory antibodies. These results demonstrate that releasable FIX can be expressed and stored in platelet alpha-granules and that platelet-derived FIX can correct the bleeding phenotype in hemophilia B mice. Our studies suggest that targeting FIX expression to platelets could be a new gene therapy strategy for hemophilia B.

Expression of human factor VIII under control of the platelet-specific αIIb promoter in megakaryocytic cell line as well as storage together with VWF

Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.

Targeting platelet GPIbα transgene expression to human megakaryocytes and forming a complete complex with endogenous GPIbβ and GPIX

Summary.  Bernard–Soulier Syndrome (BSS) is a severe congenital platelet disorder that results from a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex that is composed of four subunits (GPIbα, GPIbβ, GPIX, and GPV). Mutations in either GPIbα, GPIbβ, or GPIX can result in BSS with many of the known mutations occurring in GPIbα. In this study, we have developed a gene therapy strategy to express hemagglutinin (HA)‐tagged GPIbα in megakaryocytes and potentially correct a hereditary deficiency. To direct GPIbα expression in megakaryocytic lineage cells, we designed a GPIbα cassette where human GPIbα cDNA was placed under control of the megakaryocytic/platelet‐specific αIIb promoter and inserted into a lentiviral vector. Human CD34+ peripheral blood cells (PBC) and Dami cells were transduced with αIIb‐HA‐GPIbα‐WPT virus. Flow cytometry analysis demonstrated that 50.1% of the megakaryocytes derived from CD34+ stem cells and 97.3% of Dami cells were transduced and expressed transgene GPIbα protein. Immunoprecipitation with Western blot analysis demonstrated that transgene protein associated with endogenous GPIbβ and GPIX proteins. To address further the lineage‐specific expression of the αIIb‐HA‐GPIbα construct, three cell lines, Dami, AtT‐20 and HepG2, were transfected with GPIbα expression plasmids and analyzed by confocal microscopy. The results demonstrated that among these three cell lines, the tissue‐specific αIIb promoter was active only in Dami cells. Thus, GPIbα can be efficiently and specifically expressed in the megakaryocytic compartment of hematopoietic cells and the transgene product associates with endogenous GPIbβ and GPIX forming a complete complex. This strategy could potentially be utilized for gene therapy of BSS.