Gene Therapy in Hemophilia A and B

In the last decade, considerable progress has been made in the management of hemophiliacs so that today these patients can expect a near normal life span. Yet, this can be achieved only by the repeated infusion of factor concentrates. The ultimate goal, of course, should be the eradication of this disease, and this appears to be possible, at least in the foreseeable future, by gene therapy strategies. Hemophilia is well suited for gene therapy strategies because the expression of factor VIII and factor IX genes is not tightly controlled, already low plasma levels of these factors improve the clinical presentation greatly, and excellent animal models of hemophilia exist that closely resemble the human disease. The goal of hemophilia gene therapy is a long-lasting production of the factors without serious complications, especially the development of immune inhibitors. The success largely depends on finding suitable gene delivery vehicles.

This issue of Seminars in Thrombosis and Hemostasis comprehensively reviews the present status of gene therapy for both hemophilia A and B. Extensive animal experiments have provided direction toward pilot phase I clinical trials. From the available results so far obtained it appears that still major (but not necessarily insurmountable) hurdles have to be overcome to make gene therapy for hemophilia a safe and viable alternative to the present treatment modalities.

In the first article, Couto reviews the preclinical experience so far gained with gene therapy for hemophilia. The contribution summarizes the studies using recombinant adeno-associated virus (AAV) vectors to achieve expression of clotting factors. Several animal models have been used, and different modes of vector administration have been explored. Until recently, most work involved the expression of factor IX, but the development of factor VIII expression cassettes has also stimulated research on hemophilia A. The factor expression varies with the route of administration of the vector, but good dose-dependent and time-dependent distribution to most tissues has been observed. Toxicity studies are very favorable; however, the formation of antibodies remains a problem. The work reviewed serves as an excellent starting point for potential successful gene therapy for both types of hemophilia.

In the next contribution, Thorrez and coworkers summarize the progress made in developing appropriate adenoviral vectors for hemophilia gene therapy. Early-generation adenoviral vectors yielded elevated factor VIII and IX levels in hemophilic dog and mouse models, but this was associated with significant acute and chronic toxicity and inflammatory responses. Subsequently, newer versions of adenoviral vectors were developed, known as high-capacity (HC) vectors. These have a better safety profile and lead to a more prolonged expression of factors VIII and IX. However, differences were noted between animal species, especially dogs and mice. The reasons for this are at this time incompletely understood; prediction of how these HC vectors might perform in clinical trials is uncertain. In addition, the transgene expression gradually declines in mice, dogs, and baboons, so that further research is needed to improve this technology. Despite these difficulties, tremendous progress has been made to find the optimal vectors for gene therapy for hemophilia A and B.

Next, Van Damme and coworkers describe the status of using oncoviral and lentiviral vectors for gene therapy. These vectors can potentially lead to long-term factor expression because they integrate in the target cell genome. Oncoviral vectors only transduce dividing cells, whereas lentiviral vectors transduce a wide spectrum of cells, irrespective if their cell division status. When both vectors were used, in vivo preclinical animal studies showed therapeutic factor VIII and IX levels when genes were transduced into hepatocytes. Ex vivo experiments were hampered by low and only transient factor expression. The immune response (i.e., antibody formation) also presents a problem with these therapeutic approaches.

Gómez-Vargas and Hortelano next review the status of nonviral gene therapy in hemophilia. Nonviral vectors have potential advantages over viral vectors, and may avoid risks of mutagenesis, carcinogenesis, and immune responses. In addition, nonviral vectors can be produced in large quantities. The in vivo injection of naked DNA is one of the possibilities discussed. Although considerable gene transfer to the liver was achieved, naked DNA is rapidly degraded by nucleases in plasma. Various ways to transfer naked DNA are described and several organ systems, in addition to the liver, may be targeted. Some of the problems encountered with naked DNA can be circumvented by coupling the DNA to a variety of compounds, and good results have been observed with these techniques. Nonviral vectors also have been employed for ex vivo gene transfer using several different cell lines. There still are many problems that need to be solved before this promising technology can be recommended for clinical trials in humans.

Rawle and Lillicrap evaluate the usefulness and the limitations of animal models to study gene therapy approaches in hemophilia. At this time, mouse and dog models have been most widely used. The hemophilic mice were created by manipulation of the mouse genome, whereas the dogs are spontaneously hemophilic and appropriately bred. Both hemophilia A and B are represented. The hemophilic mouse model lends itself ideally for phenotypic correction of the bleeding defect and for obtaining detailed knowledge of the immune system. Three hemophilic large dog models exist in North America; all have hemophilia A and two also have hemophilia B animals. The experiences obtained with these models are reviewed and very encouraging results have evolved that are of considerable help for potential use of this technology in humans. It is pointed out, however, that major differences exist between animal species and even within some species, depending on the nature of the hemophilic gene defect. Thus, caution must be exercised when extrapolating data from animal experiments to humans.

Herzog and Dobrzynski review the immune responses to gene therapy for hemophilia. After an extensive description of our present knowledge of inhibitor formation, the authors elaborate on the risks of an immune response during gene therapy. Many factors apparently determine this complication, and vector design and choice, vector dose, and route of administration are important considerations. In addition, the type of mutation that underlies hemophilia is a major determinant. All of these factors are extensively reviewed, primarily on the basis of animal models, and much research needs to be performed to improve safety of these approaches before going to clinical trials. Research into the role of immune suppression and immune tolerance may ameliorate these difficulties.

Pipe discusses means to improve the properties of factors VIII and IX for gene transfer. An understanding of the biosynthetic pathway and structure/function analysis of these factors has opened the possibility of developing mutants that could improve gene transfer. It has long been recognized that, for example, the removal of the B domain from the large factor VIII molecule does not impair the biological activity and does not increase the risk of inhibitor formation. Presently available data on increasing the biosynthesis and secretion of factor VIII are comprehensively reviewed, and many possibilities exist to improve the functional activity of factor VIII. In addition, improvement of plasma half-life of factor VIII could aid in a better gene transfer. Similar strategies have been pursued to improve the factor IX molecule. The various pathways are summarized expertly and comprehensively. The data emphasize the tremendous progress that is being made in addressing the possibilities of a safe gene transfer for hemophiliacs ultimately to eliminate the disease.

In the next contribution, Ragni discusses the important question of why gene transfer therapy should be used, when conventional factor replacement therapy appears to be efficacious and safe. The advantages and disadvantages of both treatment options are examined. Although replacement therapy with recombinant factor concentrates has virtually normalized the life expectancy of hemophiliacs with both hemophilia A and B, inhibitor development, shortages of supply, accessibility, and costs are still major issues. Gene transfer potentially eliminates the disease, but safety is still of great concern. As research progresses, many of these outstanding issues will be addressed and gene transfer will eventually become safe and technologically feasible.

Chuah and coworkers review the present experience with gene transfer in hemophilia A patients. At this time, three phase I clinical trials have been conducted. The first used ex vivo transfected dermal fibroblasts expressing a modified factor VIII molecule. The treatment was well tolerated with no serious side effects, but only very low levels of factor VIII were expressed. In a second trial, oncoviral vectors expressing factor VIII were administered intravenously. This procedure also was safe, but again no sustained expression was achieved. In a third trial, an HC adenoviral vector was used. This patient expressed some factor VIII for several months, but a transient inflammatory response was encountered. It becomes clear that improvements are needed in gene delivery technology, and that results obtained in animal models cannot necessarily be repeated in humans.

In the last article, High reviews the status of gene transfer for patients with hemophilia B. In some ways hemophilia B is a more straightforward model than hemophilia A. The cDNA is smaller and large animal models exist with a greater range of molecular defects for this disease. Gene transfer studies have been rather successful in animal models, leading to attempts at gene transfer in hemophilia B patients. Two phase I trials are ongoing. Both use AAV vectors as transfer vehicles. In the first study the vector was introduced into skeletal muscle of the upper and lower extremities. This approach was found to be safe and free of major side effects and circulating factor IX levels were low, although transfer and expression could be demonstrated over several months in muscle tissue. On the basis of the safety of this vector, the second trial is still ongoing, which introduces the vector systemically to target the liver. Although data on success are not yet available, this approach also is apparently safe and the trial proceeds with caution. Some findings in humans were not seen before in various animal models, again indicating that extensive clinical trials are needed, regardless of data obtained from preclinical animal models.

Great thanks and appreciation are expressed to all authors for their excellent contributions; this issue represents a valuable source of information for all interested in gene therapy for hemophilia. Special thanks go to Drs. VandenDriessche and Chuah for inviting the experts, assembling this issue, and making this issue a most informative learning experience.