Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model.

INTRODUCTION: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. METHODS: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. RESULTS: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. CONCLUSIONS: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Functional regulation of von Willebrand factor ameliorates acute ischemia-reperfusion kidney injury in mice.

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and may become fatal. In addition to its hemostatic functions, von Willebrand factor (VWF) is known to play a role in cross-talk between inflammation and thrombosis. We hypothesized that VWF may be involved in the pathophysiology of AKI, major causes of which include insufficient renal circulation or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the role of VWF in AKI using a mouse model of acute ischemia-reperfusion (I/R) kidney injury. We analyzed renal function and blood flow in VWF-gene deleted (knock-out; KO) mice. The functional regulation of VWF by ADAMTS13 or a function-blocking anti-VWF antibody was also evaluated in this pathological condition. Greater renal blood flow and lower serum creatinine were observed after reperfusion in VWF-KO mice compared with wild-type (WT) mice. Histological analysis also revealed a significantly lower degree of tubular damage and neutrophil infiltration in kidney tissues of VWF-KO mice. Both human recombinant ADAMTS13 and a function-blocking anti-VWF antibody significantly improved renal blood flow, renal function and histological findings in WT mice. Our results indicate that VWF plays a role in the pathogenesis of AKI. Proper functional regulation of VWF may improve the microcirculation and vessel function in the kidney, suggesting a novel therapeutic option against AKI.

Scavenger Receptor A-Mediated Targeting of Carboxylated Polyrotaxanes to Macrophages and the Impacts of Supramolecular Structure.

Because macrophages are involved in the pathology of many diseases, targeting delivery of therapeutic molecules to macrophages is important issue. Polyrotaxanes (PRXs) composed of multiple cyclodextrins threaded with a linear polymer were utilized as a therapeutic agent for metabolic disease and for regulating cellular metabolism. For targeting delivery of PRXs to macrophages, carboxyethyl ether group-modified PRXs (CEE-PRXs) are designed for promoting interaction to macrophage scavenger receptor class A (SR-A). The cellular internalization of anionic CEE-PRXs in SR-A-positive macrophage-like cells (RAW264.7) is remarkably higher than that of nonionic PRX, whereas the cellular internalization efficiency in SR-A-negative cells is comparable between anionic and nonionic PRX. Furthermore, the molecular weight of axle polymer and the number of CEE groups modified on PRX are found to be the predominant factors governing cellular internalization efficiency in SR-A-positive RAW264.7 cells. Thus, CEE-PRXs are a promising design for targeting delivery of PRXs to macrophages.

Von Willebrand Factor Aggravates Hepatic Ischemia-Reperfusion Injury by Promoting Neutrophil Recruitment in Mice.

Hepatic ischaemia-reperfusion (I/R) injury is a serious liver damage that critically influences the clinical outcome of liver surgery or transplantation. Since recent studies indicated the critical involvement of von Willebrand factor (VWF) in reperfusion injuries of brain and myocardium, we hypothesized that VWF-dependent thrombotic or inflammatory responses also play a role in hepatic I/R injury. Using a mouse model of hepatic I/R injury, we explored the functional relevance of the VWF-ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) axis in this pathologic condition. Time-course studies during hepatic I/R revealed significantly lower alanine aminotransferase (ALT) values, as well as greater hepatic blood flow, in VWF gene-deleted (KO) mice in comparison with wild-type (WT) mice. Histological analysis revealed a significantly lesser extent of neutrophil infiltration and hepatocellular necrosis in liver tissues of VWF-KO mice. Human recombinant ADAMTS13 significantly improved the impairment in ALT values and hepatic blood flow and decreased neutrophil infiltration within the liver tissue of WT mice. Real-time intravital imaging successfully visualized significantly reduced leukocyte-vessel wall interactions in I/R liver of VWF-KO mice. Taken together, our results indicate that VWF promotes neutrophil recruitment in ischaemic mouse liver, critically aggravating reperfusion injury, and suggest that functional regulation of VWF by ADAMTS13 represents a promising therapeutic option for hepatic I/R injury.

Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

A novel cell-sheet technology that achieves durable factor VIII delivery in a mouse model of hemophilia A.

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

Coagulation potential of immobilised factor VIII in flow-dependent fibrin generation on platelet surfaces.

Coagulation factor VIII (FVIII) plays an essential role in haemostasis. To date, physiologic activity of FVIII circulating in the bloodstream (S-FVIII) is evaluated by classic coagulation assays. However, the functional relevance of FVIII (-von Willebrand factor complex) immobilised on thrombogenic surfaces (I-FVIII) remains unclear. We used an in vitro perfusion chamber system to evaluate the function of I-FVIII in the process of mural thrombus formation under whole blood flow conditions. In perfusion of either control or synthetic haemophilic blood, the intra-thrombus fibrin generation on platelet surfaces significantly increased as a function of I-FVIII, independent of S-FVIII, under high shear rate conditions. This I-FVIII effect was unvarying regardless of anti-FVIII inhibitor levels in synthetic haemophilic blood. Thus, our results illustrate coagulation potentials of immobilised clotting factors, distinct from those in the bloodstream, under physiologic flow conditions and may give a clue for novel therapeutic approaches for haemophilic patients with anti-FVIII inhibitors.

Ratio of von Willebrand factor propeptide to ADAMTS13 is associated with severity of sepsis.

Von Willebrand factor (VWF)-cleaving protease (ADAMTS13) cleaves ultralarge VWF (ULVWF) secreted from endothelium and by which is regulating its physiologic function. An imbalance between ULVWF secretion and ADAMTS13 level occurs in sepsis and may cause multiple organ dysfunction. We evaluated the association between the VWF-propeptide (VWF-pp)/ADAMTS13 ratio and disease severity in patients with severe sepsis or septic shock. In 27 patients with severe sepsis or septic shock and platelet count less than 120,000/µL, we measured plasma VWF, VWF-pp, and ADAMTS13 levels on hospital days 1, 3, 5, and 7. The VWF-pp/ADAMTS13 ratio was increased greater than 12-fold in patients with severe sepsis or septic shock on day 1 and remained markedly high on days 3, 5, and 7 compared with normal control subjects. The VWF-pp/ADAMTS13 ratio significantly correlated with Acute Physiology and Chronic Health Evaluation II score on days 1 and 5; Sepsis-related Organ Failure Assessment score on days 1, 3, and 5; maximum Sepsis-related Organ Failure Assessment score and tumor necrosis factor α level on days 1, 3, 5, and 7; and creatinine level on days 1, 5, and 7. Patients with greater than stage 1 acute kidney injury had significantly higher VWF-pp/ADAMTS13 ratio than patients without acute kidney injury. In summary, the VWF-pp/ADAMTS13 ratio was associated with disease severity in patients with severe sepsis or septic shock and may help identify patients at risk for multiple organ dysfunction by detecting severe imbalance between ULVWF secretion and ADAMTS13 level.

Endothelial progenitor cell-based therapy for hemophilia A.

As shown by the results of both pre-clinical and clinical studies reported in past decades, the goal of establishing an effective and successful gene therapy for hemophilia A remains feasible and realistic. However, at this time, no single approach has been shown to be clearly superior, and a number of recurring challenges remain to be overcome. Given the persistent problems presented by the host immune response to systemic in vivo gene delivery, and the additional obstacles of inadequate transgene delivery and expression, we propose a re-evaluation of an ex vivo gene transfer approach that utilizes a genetically modified stem cell population. In this strategy, autologous blood outgrowth endothelial progenitor cells are obtained from hemophilic animals, into which a normal copy of the factor VIII gene is introduced via an engineered virus. Cell numbers are expanded in culture prior to their re-implantation under the skin of the hemophilic animals in an artificially developed supporting environment. Follow-up assessment of the treatment involves the general evaluation of clotting activity, the specific measurement of factor VIII levels in the blood, and clinical observation.

A microRNA-regulated and GP64-pseudotyped lentiviral vector mediates stable expression of FVIII in a murine model of Hemophilia A.

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.

Antithrombotic properties of pravastatin reducing intra-thrombus fibrin deposition under high shear blood flow conditions.

In addition to lowering cholesterol, the 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have a range of pleiotropic effects that help reduce the risk of adverse cardiovascular events. We sought to understand the molecular mechanisms by which statins could exert anti-platelet actions under physiologic whole blood flow conditions. Using an in vitro perfusion chamber system, we examined the anti-platelet effects of pravastatin under whole blood flow conditions with high or low shear rates. We determined that pravastatin significantly suppressed platelet activation-dependent procoagulant activity, decreasing P-selectin membrane expression, tissue factor accumulation, and thrombin binding within platelet thrombi generated on a von Willebrand factor-surface under high shear rate conditions. These effects resulted in reductions of intra-thrombus fibrin deposition. These antithrombotic properties of pravastatin, which were comparable to those of atorvastatin, could be abrogated by mevalonate. Our experimental approach revealed a novel mechanism mediating the anti-platelet action of statins. Shear rate-dependent antithrombotic activity may explain the favourable effect of pravastatin on the reduction in cardiovascular events that typically occur in vivo under whole blood flow conditions with high shear rates.

Characterization of viability and proliferation of alginate-poly-L-lysine-alginate encapsulated myoblasts using flow cytometry.

Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells.

Under certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice. We used lentiviral vectors to deliver the canine FVIII transgene to neonatal hemophilic mice and demonstrated that induction of long-term FVIII tolerance could be achieved. Hemophilia A mice are capable of mounting a robust immune response to FVIII after neonatal gene transfer, and tolerance induction is dependent on the route of delivery and type of promoter used. High-level expression of FVIII was not required for tolerance induction and, indeed, tolerance developed in some animals without evidence of detectable plasma FVIII. Tolerance to FVIII could be adoptively transferred to naive hemophilia recipient mice, and FVIII-stimulated splenocytes isolated from tolerized mice expressed increased levels of interleukin-10 and decreased levels of interleukin-6 and interferon-gamma. Finally, induction of FVIII tolerance mediated by this protocol is associated with a FVIII-expandable population of CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Visual evaluation of blood coagulation during mural thrombogenesis under high shear blood flow.

Mural thrombus generation at sites of damaged vessel walls is essential for both physiological haemostasis and pathological intravascular thrombosis. While thrombi are established by the concerted action of platelet aggregation and blood coagulation, most previous in vitro coagulation assays have evaluated fibrin clot formation in a closed stirring situation that lacks blood cells including platelets. We describe here a modified flow chamber system, established originally for platelet functional studies, that enables real-time observation of intra-thrombus fibrin accumulation during platelet thrombogenesis under flow conditions. Analysis by confocal laser scanning microscopy during perfusion of whole blood anticoagulated to various extents revealed that the size and shape of mural thrombi can depend on the intra-thrombus fibrin development under high shear rate conditions. These observations were confirmed by perfusion of heparinized blood or blood from haemophilia patients with or without addition of activated factor VII. Thus, our experimental system provides visual evidence supporting the concept of "cell-based coagulation under whole blood flow", which might be the most physiologically relevant model of comprehensive thrombogenicity in vivo to date. This system promises to help formulate strategies for haemostatic management of congenital coagulation disorders as well as for antithrombotic therapy targeting fatal arterial thrombosis.

Ex vivo gene therapy for hemophilia A that enhances safe delivery and sustained in vivo factor VIII expression from lentivirally engineered endothelial progenitors.

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A. Disclosure of potential conflicts of interest is found at the end of this article.

Mural thrombus generation in type 2A and 2B von Willebrand disease under flow conditions.

To explore the mechanisms that underlie the bleeding tendency in type 2A and 2B von Willebrand disease (VWD), we analyzed the mural thrombus generation process on a collagen surface under physiologic blood flow in a perfusion chamber using whole blood from these VWD patients. At a low shear rate (50 s(-1)), thrombus generation in all type 2A and 2B VWD patients was comparable to that of healthy controls. At a high shear rate (1500 s(-1)), thrombus generation was impaired in all type 2A patients, whereas that in type 2B VWD patients varied from normal to significantly defective, as judged by epifluorescence microscopy of thrombus surface coverage. However, in type 2B patients who showed normal thrombus generation at 1500 s(-1), the height and volume of thrombi was significantly reduced, albeit with the normal surface coverage, compared with control thrombi, and von Willebrand factor (VWF) was poorly distributed within the type 2B thrombus mass when analyzed in detail by confocal laser scanning microscopy. Addition of purified VWF to patient blood completely reversed the defective spatial thrombus growth in type 2B VWD. Thus, our results confirm the impaired thrombus generation in type 2B VWD, which has never been demonstrable in previous in vitro soluble-phase platelet aggregation assays, and point to the critical function of larger VWF multimers in the proper spatial growth of mural thrombi under high shear rate conditions.