Lily M. Du

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.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

Platelet gene therapy improves hemostatic function for integrin αIIbβ3-deficient dogs

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3’s major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

C560Rβ3 caused platelet integrin αIIbβ3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality

β3‐Deficient megakaryocytes were modified by human β3‐lentivirus transduction and transplantation to express sufficient levels of a C560Rβ3 amino acid substitution, for investigation of how an activated αIIbβ3 conformation affects platelets in vivo in mice.

919. Gene therapy for platelet disorders: targeting transgene expression in canine platelets

Platelets mediate the primary response to vascular injury; as such molecular defects in some platelet proteins can disrupt normal hemostasis and lead to uncontrolled hemorrhage. To develop a large animal model to examine lineage-specific gene therapy for disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a self-inactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human integrin αIIb gene promoter. This construct, αIIb-GFP-WPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to analyze transgene expression in the progeny of transduced cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, Elongation Factor-1, which drives constitutively active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from granulocyte-colony stimulating factor mobilized PBC, which were collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and c-mpl ligand), transduced for 2 days and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL-11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP was megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP under control of the αIIb-promoter were megakaryocytes. When αIIb-GFP transduced PBC were xenotransplanted into immune-compromised (NOD/SCID) mice, flow cytometric analysis demonstrated that GFP-positive platelets were circulating within the vasculature and could be isolated at the site of a tail vein injury at two weeks post-transplant of canine PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1-promoters ability to drive lineage-specific transgene expression. Furthermore, transduced PBC are capable of forming megakaryocytes and platelet progeny expressing transgene products in vivo. This work suggests that there is potential feasibility for using a lineage-specific αIIb gene promoter to develop gene therapy for disorders affecting platelets.