Progress in the molecular biology of inherited bleeding disorders (2008)

Since the advent of recombinant DNA technology in the early 1980s, the application of molecular biological approaches to haemophilia treatment has shown progressive advancement. More recently, the addition of various forms of cellular therapies to complement gene and protein-based therapeutics has further enhanced the potential for innovative treatments of these conditions. In this chapter, we have summarized the recent trends pertaining to several aspects of novel protein, gene, and cell-based therapeutic strategies for haemophilia. The ability to produce biological ‘facsimiles’ of plasma-derived coagulation factors within fermenting mammalian cell culture systems promised a supply of haemophilia replacement products liberated from the uncertainties of securing source plasma (a critical driver of this technology during the era of HIV and hepatitis contamination of blood derivatives) and a potentially limitless supply of replacement products that would lead to reduced costs of therapy. This would in turn facilitate an expanded application of prophylaxis strategies and open up therapy to the developing world. However, efficient production of recombinant clotting factors required overcoming significant challenges because of the complexity of both their protein structure and post-translational modifications that are critical for their hemostatic activity. Although they have proven their efficacy in clinical practice, therapeutic costs remain high and such biologicals have not greatly impacted the developing world where as many as 80% of the world’s haemophiliacs do not receive adequate therapy. Nevertheless, recombinant DNA technology remains a promising platform to reduce the costs of therapy, increase the availability to the developing world and further enhance the quality of life of patients with haemophilia. Following the initial successful expression of recombinant factor VIII (rFVIII) and IX (rFIX), ongoing research has provided detailed structural and functional characterizations for each phase of their life cycle: biosynthesis, macromolecular interactions, activation/inactivation, and clearance. This has come through insights from the study of haemophilia mutations, site-directed mutagenesis, detailed structural models and an expanded repertoire of animal models through molecular biology advances. This has opened up new frontiers for bioengineering strategies to overcome some of the remaining limitations inherent to current clotting factor concentrates (Fig. 1). Novel coagulation proteins. Basic science studies have provided insights into why the expression of rFVIII in heterologous mammalian cell lines is so poor [1]. There is an inefficient expression of mRNA, inefficient folding of the primary translation product, and a requirement for a facilitated transport mechanism from the endoplasmic reticulum to the Golgi apparatus. Targeted bioengineering strategies to overcome these limitations have led to novel FVIII molecules with enhanced secretion efficiency up to 30-fold higher secretion rates compared with that of wild-type FVIII. Such high efficiency expression molecules can now be partnered with novel recombinant production systems such as enhanced mammalian expression systems and transgenic animals. This brings the possibility of producing rFVIII at a significantly lower cost. The higher yields may even open up research into alternative therapeutic strategies such as oral therapy. Partnering such technology with gene therapy has the potential to yield plasma levels that are several fold higher than those achieved in clinical trials to date. Elucidation of the mechanisms involved in the activation and inactivation of FVIII has led to targeted strategies to increase its potency and stability through bioengineering. FVIII molecules that are resistant to inactivation are in preclinical evaluation and have demonstrated increased thrombin generation potential and effective hemostasis in animal models despite lower protein doses. Such molecules could provide effective hemostasis in gene therapy strategies even when expression is limited. Technologies, such as the addition of polyethylene glycol (PEGylation) or polysialic acid polymer conjugates to prolong the half-life of biological proteins has been applied to a number of therapeutics with success [2]. However, the complexities of clotting factor proteins have limited this application for haemophilia replacement products. New technological advancements with more targeted conjugation techniques have increased the enthusiasm for such strategies. Direct chemical modification of FVIII by such techniques is being investigated in preclinical trials. However, indirect strategies have also been explored. Chemical modification of FVIII’s carrier partner in plasma, von Willebrand factor (VWF) could extend the half-life of VWF and in turn extend the half-life of co-infused FVIII. This would avoid many of the limitations of this technology whereby direct chemical modification of FVIII compromises its macromolecular interactions and reduces its specific activity. An alternative indirect strategy is the application of PEGylated liposomes as an alternative plasma carrier for FVIII. With this technique, FVIII is associated with synthetic lipid bilayer spheres that have been chemically modified to extend their half-life in vivo, and designated sterically stabilized liposomes. These have shown promise in small animal models with enhanced duration of hemostatic activity. Clinical trials with this FVIII carrier system have provided some evidence for enhanced duration of haemostatic activity in patients with haemophilia even though classical pharmacokinetic parameters were not enhanced. The efficacy of this strategy will be investigated further in a phase II trial. These strategies, however, will only impact FVIII half-life as an infusate. Extended half-life molecules for gene therapy purposes will require insights into the mechanism of clearance of FVIII from plasma. Experimental evidence has suggested that FVIII interacts initially with heparan sulphate proteoglycans on the cell surface and then interacts with at least two receptors of the low-density lipoprotein receptor family – low-density receptor-related protein (LRP) and low-density lipoprotein receptor (LDLR). Receptor blockade in animal models is effective at enhancing FVIII half-life up to five-fold. However, these receptors have affinities for a number of plasma proteins that may preclude using this strategy in humans. Potential LRP and LDLR receptor binding sites have been identified within FVIII. These regions of FVIII may be targeted for site-directed mutagenesis to reduce receptor affinity. This has been challenging as these regions overlap with sites critical for FVIII functional interactions. If a FVIII mutant can be identified with limited compromise of FVIII function yet reduced receptor-mediated clearance, this could enhance gene therapy strategies by yielding higher plasma levels. With up to a quarter of patients with severe haemophilia A at risk for inhibitor development to FVIII, this is an important area to apply bioengineering strategies. Human FVIII inhibitor antibodies have limited cross-reactivity with porcine FVIII. A recombinant form of porcine FVIII [3] is being investigated in clinical trials and promises to be an effective hemostatic agent in patients with FVIII inhibitors. Ongoing research has identified critical regions of FVIII that contribute to its immunogenicity. Bioengineering strategies have included the substitution of major inhibitory epitopes in human FVIII with porcine sequences resulting in recombinant porcine/human hybrids with markedly reduced reactivity with inhibitor antibodies. Clinical questions remain as to whether such molecules would also reduce the risk for inhibitor formation in certain individuals with severe haemophilia A. Expression of FIX is significantly more efficient than FVIII. Preclinical studies have demonstrated long-term expression of FIX at therapeutic levels within haemophilia B animal models with current gene therapy strategies [1]. However, expression has been limited and transient in early human trials. While new vector strategies are being explored, investigators are also testing FIX molecules that have been modified to enhance mRNA levels, increase potency, and extend plasma half-life. These enhanced FIX molecules may also allow gene therapy protocols to proceed at lower vector dosages, thereby limiting vector-related toxicities. Direct modification of rFIX to extend its half-life as an infusate is also a key area of investigation. While chemical modifications such as PEGylation, as described for FVIII are possible, other novel technologies are being explored. In one example, FIX is fused with the Fc portion of an antibody to greatly prolong the plasma half-life. In addition, Fc-fusion proteins have also been utilized to facilitate alternative delivery strategies such as oral and intrapulmonary routes [4]. Recombinant factor VIIa (rFVIIa) has been an important addition to the therapeutic portfolio for haemophiliacs with inhibitors to either FVIII or FIX. However, its clinical application is compromised in part because of a short half-life and high costs for therapy. PEGylation strategies are being applied to extend its plasma half-life. Recently, a rFVII-albumin fusion protein was generated in mammalian cell lines and exhibited a 6- to 9-fold longer plasma half-life in rats and rabbits. In addition, site-directed mutagenesis has yielded a rFVIIa molecule with greatly enhanced potency [5]. Clinical trials with these agents are now being launched. Finally, rFVIIa has also been explored within gene therapy strategies to potentially enhance hemostasis in the presence of FVIII or IX inhibitors [6]. If therapeutic expression levels prove elusive in preclinical trials, a rFVIIa bioengineered for higher potency may be useful. One of the most compelling concepts in molecular medicine is gene therapy (Fig. 2). Its ultimate goal is to edit a defective gene sequence in situ to achieve complete reversion of a disease phenotype for the lifetime of the individual. Despite recent successes in site-specific correction of defective gene sequences in the mammalian genome most current gene therapy strategies rely on gene addition rather than gene correction methodologies. Gene addition strategies use vectors as delivery vehicles to provide a wild-type copy of the defective gene to a physiologically relevant target tissue. The most efficient vectors are engineered from viruses which have evolved mechanisms for entering eukaryotic cells and harnessing their synthetic machinery to produce foreign proteins. Haemophilia gene therapy. Despite a strong record of success in animal models, a range of problems has limited the scope of successful application of gene therapy in humans to a few severe immunodeficiency disorders. Clinical studies over the past decade have identified several major obstacles to gene therapy, including: (i) gene silencing; (ii) insertional mutagenesis; (iii) phenotoxicity caused by overexpression or ectopic expression of the donated gene; (iv) immunotoxicity, i.e. harmful immune responses to either vector or transgene product; (v) risks of horizontal transmission of the donated DNA; (vi) risks of vertical transmission, i.e. inadvertent germline transmission of the donated DNA. Typically, for each specific combination of vector, transgene, and target tissue, one or two of these problems predominate as the major clinical obstacle(s). Haemophilia is a disorder for which gene therapy could prove extremely useful. Patients with factor levels ≥5% normal are only mildly affected, demonstrating that for severe haemophilia patients, increasing factor levels by a relatively small increment would substantially improve disease phenotype [7]. These observations, combined with the high disease prevalence, the width of the therapeutic window, the ability to accommodate the wild-type cDNA sequence in most gene transfer vectors, the reliability and availability of animal models of the disease and the continued lack of access to treatment for 80% of the world’s haemophilia population have generated substantial interest in developing gene therapy approaches for haemophilia. For genetic disease, where long-term expression of the donated gene is the goal, two potential strategies can be pursued. The first is to introduce the transgene into a stem cell via an integrating vector so that all progeny carry and express the donated gene. Because integration occurs throughout the genome, the major obstacle that has arisen in this setting is insertional mutagenesis and resulting malignant transformation [8]. The second strategy is to introduce the gene of interest into a long-lived postmitotic cell, such as cardiac or skeletal muscle, nerve cells, or hepatocytes. In these settings, for obvious reasons, gene transfer must occur in vivo, but because the cell has a long lifespan, expression can be achieved for prolonged periods even if the vector does not integrate into the host genome. Both types of strategies have been pursued for haemophilia; trials have included i.v. infusion of retroviral vectors [9], plasmid transfection of autologous fibroblasts [10], infusion of adenoviral vectors [11], and the use of adeno-associated viral (AAV) vectors. Our work has focussed on AAV vectors because they mediate high-level stable transduction of both skeletal muscle and liver following a single administration in small and large animals [12], and have a strong safety profile among viral vectors. They are also the simplest of all viral vectors containing only the transgene expression cassette flanked by two non-coding viral inverted terminal repeats enclosed in a capsid composed of three structural proteins, VP1, 2, and 3. Wild-type AAV is not associated with any disease pathology in humans and requires a helper virus such as adenovirus to replicate. Moreover, the vector is completely devoid of viral coding sequences, reducing any risk of immune response to transduced cells on that basis. A wealth of experience with these vectors in animals and in over 500 human subjects has failed to show any association with oncogenesis, save in a single mouse disease model [13, 14]. Finally, expression is mediated primarily through episomally stabilized transgene copies further reducing risks related to insertional mutagenesis. Multiple serotypes of AAV have been isolated with differences among capsid sequences giving rise to distinct tissue tropisms for each serotype. Majority of clinical gene transfer experience has been with AAV serotype 2. In haemophilia, the option to use skeletal muscle as the target tissue for gene delivery is critical for the substantial numbers of adult patients whose liver disease renders them ineligible for liver-directed gene therapy. Based on preclinical studies demonstrating the safety and efficacy of direct i.m. injection of an AAV vector expressing FIX, a clinical trial of parenterally administered AAV vectors was carried out [15]. The study showed long-term (>3 years) expression of human FIX, as evidenced by immunofluorescence staining of biopsied injected muscle, but the circulating levels of FIX were not adequate to improve disease phenotype. To improve circulating levels of FIX and to address the issue of immune response to FIX, we worked in the haemophilic dog model to devise methods of intravascular delivery to large areas of skeletal muscle and achieved long-term high-level (>10%) FIX expression in these animals [16]. Multiple groups have reported long-term expression (>3 years) at therapeutic levels (6–8% of normal factor levels) in haemophilic dogs after infusing a single dose of AAV into the portal vein [17, 18]. A dose escalation clinical study of hepatic delivery of an AAV-2 vector expressing human FIX from a liver-specific promoter has been carried out [19]. This was the first and currently the only trial of AAV infusion into liver in human (a second clinical trial is now undergoing regulatory review). The most important discovery arising from our liver study was the identification of human-specific immune responses to AAV-transduced hepatocytes. This represents a challenge to the goal of using recombinant viruses to introduce new genetic material into the liver. Analysis of T cell responses in lymphocytes from one of the subjects in the trial demonstrated a concomitant increase in circulating AAV capsid-specific T cells [20]. Subsequent mapping of the immunodominant epitope within the AAV capsid allowed direct quantitation of a capsid-specific CD8+ T cell population, which was shown to fluctuate with a time course that closely matched the rise and fall of serum transaminases [21]. The most parsimonious hypothesis to account for these observations is a CD8+ T cell response to capsid that recognized and destroyed the transduced hepatocytes. The clinical protocol for the upcoming trial was therefore amended to include a short course of immunosuppression to block the T cell response to vector capsid until it can be degraded and cleared from the transduced cell. As an alternate approach, Nienhuis et al. have developed a high efficiency double-stranded AAV-8 vector expressing FIX [21]. Based on data suggesting that AAV-8 uncoats rapidly and thus may be degraded more quickly, and that AAV-8 is less likely to trigger an immune response because of decreased binding to dendritic cells, this vector will be administered without accompanying immunosuppression. Long-term expression with either of these vectors would offer a new possibility for treatment of individuals with haemophilia. Because the hepatocyte is the primary cell type for blood clotting factor productions, hepatocyte-based therapies have been considered as an attractive possibility for congenital bleeding disorders including haemophilia. A pilot transplantation study of hepatocytes from non-haemophilic has shown an increase in the clotting in the FIX In this application of the hepatocyte transplantation in an rise of the coagulation factor suggesting that therapeutic levels could be to a specific on the number of cells and In the hepatocyte transplantation has been applied to three patients from congenital by the of and In each of the three patients, the hepatocyte transplantation provided significant and prolonged therapeutic which has been to the coagulation factor production from the In of the of this in animals and humans to bleeding is a on the number of hepatocytes that can be at a single time into the liver. do not within the liver as long as cells functional for the clotting factor For these reasons, alternative that the hepatocyte to be this liver tissue technologies have been developed tissue is an in which a functional liver system is at ectopic sites in using isolated hepatocytes other type of One of the major in this has been a transient time in which the engineered liver have been To this functional could be in studies where hepatocytes with and type were the The engineered activity for at activity levels in the in response to several liver This data suggested that hepatocytes with cells could be developed within the engineered which would be important for the expression of liver-specific protein Moreover, liver at sites in was to of clotting have also been developed for functional liver at The of as a platform for tissue by hepatocyte transplantation in engineered liver that could FVIII and FIX mRNA expression levels in the engineered were to the More recently, a novel technology to a hepatic cell in culture has been developed To this type of novel hepatic cell the utilized a culture on which a was A hepatic cell including and was the of the hepatic cell into the in the of a functional liver system within the As the methods to functional liver to this technology would be to genetic modification with other types of cells and would not be to hepatocytes which would provide a of cell for cell-based therapy for haemophilia. therapy has the of being a less form of treatment for haemophilia. 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