The formation of a stable blood clot is essential to maintain hemostasis and is dependent on the generation of insoluble fibrin polymers, through a complex interplay of coagulation factors in conjunction with platelet aggregation. Consequently, genetic bleeding disorders can result from either coagulation factor deficiencies as in the case of hemophilia A or B, lack of von Willebrand’s factor expression (type 3 von Willebrand’s disease)  or from platelet abnormalities (e.g. hereditray thrombocytopenia due to GATA-1 deficiency). Over the past 15 years, hemophilia had become one of the most studied disease models for gene therapy since it is due to a single gene defect and since only a slight increase in plasma FVIII or FIX levels can already convert a severe to a moderate phenotype which significantly improves the patients’ quality of life. We were the first to demonstrate that hemophilia A could be cured by gene therapy in hemophilic animal models. Recently, phase I/II clinical gene therapy studies have shown that therapeutic FIX levels (more than 10%) could be achieved in patients suffering from hemophilia using viral vectors derived from human adeno-associated virus serotype 2 (AAV2). Though this is a major achievement in the field, FIX expression slowly declined to basal levels in the course of a month due to the elimination of FIX-expressing hepatocytes by an AAV2-specific cytotoxic CD8+ T-cell response (CTL). This leaves gene therapists with one final hurdle to overcome which is to prolong clotting factor  expression at therapeutic levels. If this challenge can be overcome, gene therapy has the potential to provide life-long correction of the hemophilias. This requires the use of a gene delivery system that is efficient, safe and non-immunogenic. Therefore, there is still a need to improve gene therapy approaches for hemophilia A and B. If successful, it will not only be important for hemophilia but also for a wide range of disorders due to serum protein deficiencies. In order to pin down the most promising vector in view of moving towards a possible phase I clinical trial for hemophilia, we had compared the potential of different vector technologies including AAV, retroviral, lentiviral vectors, high-capacity adenoviral vectors and hepatocyte-specific nanoparticles for hemophilia. We concluded that adeno-associated viral vectors (AAV) based on alterantive serotypes (AAV8 and AAV9) and lentiviral vectors (LV) are currently the most promising vectors for gene therapy of hemophilia and we are actively pursuing the use of these vectors for preclinical studies in hemophilic mice, dogs and non-human primates in anticipation of clinical trials in patients with severe hemophilia (see Figure 1)

Another bleeding disorder that could potentially by treated by gene therapy is X-linked thrombocytopenia due to a mutation in the GATA-1 transcription factor, which is essential for normal erythropoiesis and megakaryocyte differentiation. Frequent platelet transfusions can offer a temporary solution for patients suffering from this disease but eventually this treatment option becomes ineffective due to immune responses directed against the allogeneic platelets. Although bone marrow transplantation (BMT) may provide a cure for these patients, the lack of histocompatible donors and the potential severe complications associated with BMT often precludes this form of treatment and consequently most patients die from their disease at relatively young age, primarily due to uncontrolled, potentially life-threatening hemorrhagic episodes. There have been several successful gene therapy clinical trials showing long-term correction of several lethal hematopietic disorders including severe combined immune deficiency (SCID-X1, SCID-ADA) more recently chronic granulomatous disease (CGD) using retrovirally transduced hematopoietic stem/progenitor cells (HSC). This justifies the use of genetically engineered HSC for gene therapy of hereditary thrombocytopenia due to GATA-1 deficiency.  Gene therapy for GATA-1 deficiency requires the use of an efficient gene delivery approach, while minimizing the risk of insertional oncogenesis. Self-inactivating (SIN) lentiviral vectors are tehrefore being explored that express GATA-1 from a megakaryocyte-specific promoter. This restricts GATA-1 expression to the appropriate lineage and prevent expression in ectopic lineages yielding increased platelet numbers in GATA-1 deficient mice after transplantation of gene-modified hematopoietic stem cells. These studies are being continued to further analyze the consequences of GATA-1 expresison on the correction of platelet dysfunction.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder in humans, caused by a defective (type 1 and 3 VWD) or dysfunctional (type 2 VWD) von Willebrand factor (VWF) protein, an adhesive multimeric glycoprotein that plays an important role in primary and secondary hemostasis. In primary hemostasis, VWF functions as a bridge between subendothelial structures, such as collagen, and platelets, allowing them to adhere to sites of vascular injury in high-shear conditions. In secondary hemostasis, VWF functions as a carrier protein for coagulation factor VIII (FVIII). The abolition of these 2 functions in VWD results in mild to severe (type 3) bleeding problems such as postoperative bleedings, epistaxis, and menorrhagia. To our knowledge, there are  no published reports on gene therapy for VWD using clinically relevant approaches or target cells. Development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing, including glycosylation and multimerization. Because VWF is normally expressed by endothelial cells (in addition to megakaryocytes), they constitute an attractive target cell type for gene therapy of VWD. Endothelial cells can be readily isolated and expanded from human blood (so-called blood outgrowth endothelial cells or BOECs), as opposed to vascular endothelial cells, which facilitates their use in gene therapy applications. A lentiviral vector was therefore constructed, encoding the complete human VWF protein, that was used to transduce BOECs isolated from dogs with VWD type 3. Whereas VWD BOECs obtained from an established canine model of type 3 VWD failed to express VWF, high levels of VWF proteins accumulated in the cytoplasm and in Weibel-Palade bodies of transduced BOECs. Moreover, transgene-encoded VWF was secreted by the transduced cells and was functional, as evidenced by its efficient binding to collagen and to GPIb and by the presence of a broad range of multimers. The results establish proof of concept that BOECs are attractive target cells for gene therapy of VWD and are therefore equipped with the necessary posttranslational machinery to generate functional VWF. In vivo studies addressing the therapeutic potential of VWD-transduced BOECs in VWD mice are currently in progress.

Gene therapy for heart disease: Duchenne muscular dystrophy

Heart disease remains one of the most prominent health challenges affecting millions worldwide despite many breakthroughs in cardiovascular medicine. Gene therapy may provide an alternative treatment to attain functional correction of the damaged heart. However, gene delivery into the heart has been particularly challenging and was hampered by the inability to express the therapeutic gene in a sufficient number of cells to achieve therapeutic efficacy. We have recently developed a novel means to deliver genes to the heart using a novel human AAV9 serotype resulting in highly efficient and widespread cardiac gene transfer superior to any previously described gene therapy approach, without any apparent side-effects (see Figure 2).

This advancement may overcome the current challenges that hamper progress to treat genetic or acquired heart disease including myocardial ischemia, cardiomyopathy, cardiac hypertrophy and muscular dystrophy. Moroever, the use of AAV9 may serve as a potential platform technology to (i) dissect the molecular processes involved in heart disease; (ii) screen or validate promising angiogenic or stem cell recruitment factors and (iii) rapid generation of animal models of human heart disorders that could be used to validate new pharmacologic agents. To further validate the AAV9 technology for cardiac gene delivery, several heart conditions will be targeted: Duchenne’s muscular dystrophy and  cardiac hypertrophy/ventricular remodelling after myocardial infarction (see PROJECT 4). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in a gene that encodes dystrophin, a large cytoskeletal protein that complexes with other partners at the sarcolemma and is essential for membrane integrity of the muscle fiber. As a consequence of the modular structure of dystrophin, internally truncated proteins missing some of the repeats can be fully functional or at least partly active as seen in patients with mild (Becker) forms of DMD. It has been shown recently, that a single administration of an AAV1 vector expressing antisense sequences linked to a modified U7 small nuclear RNA results in persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA of the mdx mouse. Sustained production of functional dystrophin at physiological levels in entire groups of skeletal muscles had been achieved resulting in the partial correction of the muscular dystrophy. Unfortunately, the heart was relatively refractory to AAV1 transduction follwing systemic vector injection, which hampers cardiac correction. Since cardiac dysfunction is the main cause of death of patients suffering from DMD, it is important to evaluate whether the use of AAV9 for cardiac delivery of antisense RNA overcomes this limitation.

After acute myocardial infarction (AMI), left ventricular (LV) remodeling occurs as an adaptive process by which the myocardium changes shape, size, and function in response to increased mechanical and neurohumoral stress. These adaptations include scar maturation and hypertrophy of remote myocardium to compensate for myocardial loss and increase in wall stress. In case of insufficient compensation, LV remodeling becomes maladaptive leading to ventricular dilatation, overt heart failure, and compromised survival. The molecular mechanisms of cardiac hypertrophy are not fully understood. It is widely acknowledged that microRNAs (miRNAs) play a key role in regulating stem cell fates and cellular differentiation in a variety of processes that are relevant for health and disease, particularly in cancer, immunology, developmental biology and organogenesis. miRNAs are tiny RNA molecules that are phylogenetically conserved and down-regulate protein production, either by promoting the degradation of messenger (m)RNA or by inhibiting translation of protein from mRNA. Recently, it has been demonstrated that miRNA regulation plays an important role in heart development in mouse embryos. Understanding the role of miRNAs in cardiac hypertrophy and heart failure has important fundamental and translational implications, and would provide important new and unique insights into the pathophysiological mechanisms of heart failure. Moreover, it may pave the way towards modulating heart function by interfering with miRNA expression, which would be a conceptually novel approach. Using AAV9 we will over-express different miRNAs and antagomirs and assess their role in cardiac remodelling after AMI.

Several studies, including our own, demonstrated the remarkable potential of viral vectors to obtain long-term therapeutic effects with no or only limited side-effects, at least in animal models. However, immune responses to the vector particles, the virally transduced cells and even the therapeutic proteins still poses a limitation to their clinical applications. Current non-viral methods provide an attractive alternative to viral vectors sicne they are typically easier and safer to use than viruses. Advantages of non-viral systems include their reduced immunogenicity, no strict limitation of the size of therapeutic expression cassette and improved safety/toxicity profiles. However, the efficiency of naked DNA delivery is generally low, and the currently used non-viral systems are not equipped to promote integration into chromosomes resulting in only transient gene expression. As a result, stable gene transfer frequencies using non-viral systems have typically been very low. To develop a clinically relevant non-viral vector approach, the most important challenge is to design a system that simultaneously achieves high efficiency, prolonged gene expression, and low toxicity.

To achieve this, we have been exploring the use of non-viral transfection approaches in conjunction with transposon-based gene transfer vectors equipped with an integrating feature resulting in more efficient and long-term therapeutic gene expression than currently used non-viral gene transfer approaches. Transposons are discrete segments of DNA that have the distinctive ability to move a defined DNA segment from one genetic location to another in a genome Transposons are not infectious and their integration into chromosomes provides the basis for long term, or possibly permanent, transgene expression in genetically modified cells and organisms. Transposons belonging to the Tc1/mariner superfamily of eukaryotic elements, such as Sleeping Beauty (SB), are able to transpose in human cells, thus they can be considered as natural, non-viral gene delivery vehicles. The development of efficient and safe non-viral vectors would greatly facilitate clinical implementation of ex vivo and in vivo gene therapies using stem cells and other primary target cells. However, even with the most robust transposons available to date, stable gene transfer efficiencies remain low. To overcome these hurdles, we have explored the use of novel engineered hyperactive transposases derived from Sleeping Beauty obatined by in vitro evolution. These novel transposases resulted in unprecedented and robust gene transfer in CD34+ HSCs, muscle and mesenchymal stem progenitors and liver in vivo paving the way towards a viable non-viral alternative for integrating viral vectors.

Establishing long-term antigen-specific unresponsiveness in a fully immunocompetent host is required to effectively to prevent or suppress cellular and/or humoral immune responses following allogeneic transplantation and gene therapy and to treat autoimmune diseases and allergy. Maintenance of antigen-specificity typically requires presentation of the antigen within the context of major histocompatibility complex class II determinants (MHC-II) on antigen-presenting cells (APC). However, exposure of APC to exogenous antigens often results in the concomitant expression of co-stimulatory signals and ultimately in T cell activation, which, consequently, precludes induction of immune tolerance. Conversely, the lack of co-stimulatory signals on APCs may confer antigen-specific unresponsiveness. This could possibly be achieved by using genetically modified APC that present the antigenic peptide in association with MHC-II, while preventing expression of such co-stimulatory molecules. Dendritic cells are known to induce antigen-specific unresponsiveness or boost an immune response, depending on their state of maturation, which makes them particularly attractive for genetic modification. However, genetic modification of dendritic cells often results in the upregulation of costimulatory molecules and MHC-II with a concomitant increase in their immune stimulatory properties. Moreover, their life-span is limited in vivo since myeloid cells turnover relatively rapidly compared to lymphoid cells.   

B lymphocytes offer an attractive alternative, due to their ability to survive for long periods of time after adoptive transfer into a recipient host. Moreover, they efficiently interact with T cells within lymph nodes and splenic germinal centers. However, the use of genetically modified B cells for immuno-modulation has initially been hampered by relatively poor gene transfer efficiencies  and by their ability to tolerize only naïve but not primed T cells.The low gene transfer efficiencies in primary B cells could be overcome using Moloney murine leukemia virus (MLV)-based retroviral vectors instead. Indeed, we and others have shown that relatively high stable transduction efficiencies (>50%) could be achieved, following retroviral transduction of primary murine B cells {Janssens, 2003 #652}. In collaboration with J.M. Saint-Remy (CMVB, KU Leuven), we have contributed to the design of a novel strategy for the induction and maintenance of antigen-specific unresponsiveness based on type 1 regulatory T cells (Tr1).  B cells were transduced with a retroviral vector encoding a major T cell epitope from a common allergen (Der p2)-derived peptide fused to an endosomal targeting sequence (gp75) to redirect the peptide to MHC class II and ensure efficient antigen-presentation to CD4+ T cells. Long-lasting and robust antigen-specific immune unresponsiveness was achieved, following adoptive transfer of the genetically modified B cells into mice and immunization with the cognate allergenic peptide, in prophylactic and therapeutic settings. T cells from recipient mice had matured towards Tr1-like regulatory cells.  Induction of antigen-specific immune tolerance required IL-10 production by the transduced B cells, since adoptive transfer of B cells from IL-10 deficient mice prevented immune tolerance induction. Hence, in vivo induction of Tr1-like cells represents a novel and robust mechanism by which long-term immune tolerance can be established following gene transfer.  The seemingly exquisite specificity of our strategy, and the fact that a single injection of retrovirally-transduced B cells is sufficient for long term prevention but also suppression of antibody production, hold much promise for clinical applications. The mechanisms of immune tolerance are being unraveled which led to exciting new insight in the role of Toll-like receptor signalling (TLR2) in immune tolerance induction Moreover, the general validity of this immune tolerance induction paradigm is being tested used other clinically relevant antigens, including clotting factors.

Understanding the mechanisms that contribute to the development of neutralizing antibodies specific for clotting factors (i.e. “inhibitors”) following protein substitution therapy (PST) or gene therapy (GT) represents  one of the key issues that are critical to improve the outcome of  current and future hemophilia therapies and which may ultimately lead to improved treatment strategies with reduced risks of inhibitor development. The development of antibodies to non-self antigens in general and,  in particular,  inhibitors to FVIII following PST or GT, depends on effective T-B cell collaboration and requires not only the interaction of the T-cell receptor (TCR) with the antigenic peptides presented on MHC (i.e. signal 1), but also co-stimulatory signals (i.e. signal 2) and possibly also  immune “danger” signals  (i.e. signal 0). The lack of signal 2 and/or signal 0 may result in T-cell unresponsiveness. In particular,  signal 2 is provided by the interaction between  B7 (CD80/86) molecules on B cells or other antigen-presenting cells (APCs)  (e.g. macrophages, dendritic cells) and CD28 on T helper cells. Another co-stimulatory factor on B cells is CD40 which interacts with its cognate CD4 ligand (CD40L) on T helper cells. In turn, naive B cell activation is dependent on the sequential integration of B cell receptor (BCR) cross-linking by antigen, followed by cognate interaction with T helper cells through this immunological synapse. In the absence of this interaction between these co-stimulatory molecules and their corresponding receptors on T helper cells, a non-productive T-B synapse  is formed and the effector T cells cannot be activated. Instead, this abortive interaction results in antigen-specific immune tolerance either through T cell apoptosis or anergy or through the generation of regulatory (suppressor) T cells. In addition to these co-stimulatory molecules, it may be possible to achieve antigen-specific immune tolerance by interfering with Toll-like receptor  (TLR) signalling. TLRs serve as an important link between adaptive and innate immunity. It has been proposed by Janeway and colleagues that these TLRs (alias signal 0) may be important for controlling or inducing signal 2, which in turn determines whether lymphocytes will react against  or tolerate a specific antigen. We are currently developing and validating novel alternative approaches to induce antigen-specific immune tolerance to clotting factors by modulating T-B cell collaboration, in particular by exploring novel approaches that result in down-regulation of the co-stimulatory signals (signal 0, 1 & 2) using RNA interference.