Just as Sisyphus in Greek mythology was condemned to repeatedly roll a large stone to the summit of a mountain, only to have it roll down again, efforts to achieve the pinnacle of hemophilia gene transfer success— sustained transgene expression—continue despite continuing obstacles. Indeed, despite those who think gene transfer is a myth, recent advances in adeno-associated viral (AAV) gene transfer in hemophilia B, the most successful approach to date, are encouraging. Hemophilia is an X-linked congenital bleeding disorder caused by deficient and/or defective coagulation factor (F)-VIII (hemophilia A) or FIX (hemophilia B). It is considered a model disease for gene therapy. Although recombinant F replacement therapy has been available for the treatment of individuals with hemophilia for over a decade, current therapy continues to be suboptimal. In fact, the complications of past and current therapies have been worse than the disease itself. Thus, safety is the essential requisite for implementation of hemophilia gene transfer.
To achieve gene transfer in hemophilia, the gene for FVIII or FIX must be delivered by viral vector, plasmid, or naked DNA into cells, integrate into host cells, and result in transgene expression and measurable circulating FVIII or IX levels of greater than 1% activity to prevent bleeding. To date, five clinical hemophilia human gene transfer trials have been initiated. The purpose of this article is to provide a brief background and clinical update of hemophilia gene transfer with particular emphasis on the most successful application: AAV-mediated gene transfer.
Hemophilia and Limitations of Current Therapy
Hemophilia A and B are congenital bleeding disorders that affect one in 5,000 males. Clinically indistinguishable, these diseases are characterized by spontaneous and traumatic bleeding into muscles, joints, and body cavities, including intracranial and retroperitoneal hemorrhage. The rare inherited bleeding disorder was recognized as early as the third century. Writings in the ancient Talmud warned against circumcision after several affected males died after the procedure. The untimely deaths of relatives of the famous carrier Queen Victoria, young affected males, changed the course of history in 19th century Europe and Russia.
Although significant progress has been made in hemophilia treatment, many challenges remain. Recombinant clotting F is costly, subject to shortages, and unavailable to the majority affected worldwide.1 For those who use plasma-derived products, there is potential transmissible agent risk, including HIV and hepatitis C (HC) infection,2–4 and new agents.5 Furthermore, inhibitor formation, which may develop after treatment with either plasma-derived or recombinant therapy, continues to be one of the most serious complications of hemophilia, with high morbidity and mortality.6 Moreover, current treatment—whether plasma-derived or recombinant—is invasive, requiring intravenous infusions to treat acute bleeding or several-times-weekly infusion to prevent bleeding (prophylaxis). Invasive treatment is particularly difficult in young children, whose small veins limit venous access and in whom the use of intravenous access devices is frequently complicated by infections and thrombosis.7–9 To summarize, not only the disease characteristics but also the significant past complications of the disease and lack of available treatment worldwide argue that hemophilia is a model disease for gene transfer.
Hemophilia Gene Transfer
The recent unequivocal demonstration that prophylaxis, i.e. F infusion three to four times weekly, prevents bleeding into joints and associated joint damage and disability has for the first time established the importance of maintaining an uninterrupted measurable level of FVIII or IX clotting activity.10 At a minimum F level of 1%, spontaneous bleeding is eliminated and, with it, most of the morbidity of the disease (see Table 1). This observation underscores the potential benefit of hemophilia gene transfer, which could potentially more easily achieve uninterrupted F levels than current frequent intravenous F concentrate infusions. Moreover, as precise regulation of expression is not required and the location for transduction is not critical, safe and effective hemophilia gene transfer could potentially provide a significantly more simple, better lifestyle for affected individuals.
Hemophilia Clinical Gene Transfer Trials
It has been suggested that the ideal hemophilia gene transfer is non-toxic, non-infectious, non-immmunogenic, safe at all ages, effective, and available in a single, non-invasive dose.11 To date, there has been a wide variety of vectors, including: retroviral, lentiviral, adenoviral, AAV, and plasmids; and cell types, including: bone marrow stromal cells, platelets, epidermis, skeletal muscle, and hepatocytes studied in animal models. Following the demonstration of safety and efficacy in gene transfer studies in hemophilia animal models, five clinical human gene transfer studies have enrolled over 40 subjects (see Table 2). These trials have evaluated FVIII delivery by plasmid,12 FVIII gene delivery by retroviral FVIII,13 FIX delivery by AAV vector,14,15 and adenoviral vector delivery of FVIII.16 The latter trial was abandoned for safety reasons. In the other four gene transfer trials, transgene expression was successful but short-lived.
Among the first human gene transfer approaches, Roth et al. implanted dermal fibroblasts transfected ex vivo with a plasmid carrying a B-domain-deleted (BDD) human F (hF)-VIII gene by omental injection under laparoscopy in 12 men with severe hemophilia A.12 Following implantation of the transduced cells, there was transient FVIII expression for up to 10 months, with FVIII levels up to 5%. This was associated with decreased use of exogenous clotting F concentrate in some of the subjects. In a second human gene transfer trial, Powell et al. administered a retroviral vector carrying a BDD hFVIII gene by peripheral intravenous infusion in 13 subjects with hemophilia A.3 Transgene expression lasted for up to one year with FVIII levels up to 19%, although this may have been due to intermittent factor infusion rather than transgenes, as the target hepatocytes are non-dividing cells. The increase in FVIII levels was associated with a decrease in bleed frequency in five of the subjects, and was associated with increased half-life after treatment with FVIII concentrate. By far the most successful human hemophilia gene transfer approach has been the use of the AAV vector to carry the hFIX gene.
Adeno-associated Virus Hemophilia Gene Transfer
AAV is an ideal vector for gene transfer as it is a non-pathogenic and replication-defective parvovirus, has low immunogenicity, does not integrate, and has a high transduction efficiency.17 Following the safe long-term expression of FIX in hemophilic mice and dogs following intramuscular AAV FIX gene delivery,18,19 the first human AAV clinical gene transfer trial was initiated. Reported by Manno et al., the AAV-hFIX construct was administered by multiple intramuscular injections in eight men with severe hemophilia B with good tolerance.14 Persistence of the vector in muscle tissue was confirmed for up to 3.5 years by Southern blot and immunohistochemical staining, although FIX levels achieved were <2%.
As hepatocytes play a central role in synthesis of clotting factors and are transduced with nearly 100% efficiency by AAV vector,17 the second AAV-FIX gene transfer trial was initiated once animal studies showed that administration via portal vein was safe and resulted in long-term FIX production.20–23 In the human trial, the AAV-2-hFIX construct was administered by hepatic artery infusion in seven men with severe hemophilia B with good tolerance.15 Peak FIX levels up to 11% were achieved, although only for up to eight weeks and were unexpectedly accompanied by an asymptomatic, reversible transaminitis beginning three weeks after vector infusion.15 The short-lived response was subsequently shown to be caused by a host T-cell CD8+ T-lymphocyte response directed against AAV2-capsid,15,23,24 leading to the destruction of the AAV2-hFIX16-transduced hepatocytes24 and elimination of the transgene and circulating FIX levels.
Because subsequent animal studies showed that the host CD8+ T cells proliferated not only in the presence of AAV-2 but also with other AAV serotypes,24 the concern was raised that use of an alternate AAV serotype would not provide an easy solution to the problem, although this hypothesis will be tested in a trial of AAV-9-FIX to begin in 2008. For this reason, suppression of the CD8+ response seemed a more viable approach. Indeed, Mingozzi demonstrated in macaques that sirolimus (Rapamune®) and mycophenolate mofetil (Cellcept®) given transiently beginning one week before and for up to 10 weeks after AAV FIX hepatic gene transfer did not alter the characteristics of AAV transduction and was safe when co-administered with an AAV vector.25 A further finding was that sirolimus, which also induces T regulatory T cells,26,27 prevented an inhibitor response to factor IX, as T regulatory cells play a critical role in the prevention of inhibitor response.28
Based on these findings, a human trial is planned to test whether transient immunosuppression at the time of AAV2-hFIX gene transfer via hepatic arteries into the liver will suppress host CD8+ T-cell response to allow long-term transgene expression. With evidence that immunosuppression is safe in hemophilic men, including the use of cyclophosphamide in the treatment of inhibitors29,30 and sirolimus and mycophenolate mofetil in hemophilia liver transplantation,4,31 and based on safety in macaques,25 the human trial will evaluate sirolimus and mycophenolate mofetil given for eight to 10 weeks beginning at the time of AAV2-hFIX gene transfer. Although both drugs may cause bone marrow suppression, the short period during which they will be given to suppress the immune response during AAV2-hFIX gene transfer should greatly reduce toxicity and infection risk.
Hemophilia Gene Transfer—Potential Risks
The development of an immune response directed against the vector, which occurred in the most recent AAV trial,15 is one of a number of potential risks of gene transfer. Others include the risk for inflammatory reactions,32,33 inhibitor formation,34–36 insertional mutagenesis,37,38 germline transmission,39 vector replication and shedding,39 thrombosis,40,41 and tumorigenesis.42 Inflammatory reactions have occurred with adenoviral vector gene delivery and with induction of a cytokine cascade.32,33 Inhibitor formation is another potential risk, as the transgene could represent a ‘danger signal’34 similar to inhibitor antibody formation to infused F concentrate in up to 15–20% of hemophilia A patients.6
Based on gene transfer studies in animals, the risk of inhibitor formation may be related to the underlying hemophilia mutation or the vector type used in gene transfer. Following AAV gene transfer in hemophilia B dogs, anti-IX inhibitor antibodies developed exclusively in dogs with a null mutation, but not in those with a mis-sense mutation.35,36 Furthermore, lentiviral transduction of hepatocytes in mice resulted in long-term anti- IX formation.43 Another risk, insertional mutagenesis, has occurred with lentiviral gene therapy in animals42 and in children with X-linked severe combined immunodeficiency (SCID) treated with a retroviral vector.38 In the latter, leukemia resulted when the therapeutic gene inserted adjacent to a transcription coactivator locus for leukemic cells. As there is no selection advantage for gene-corrected cells in hemophilia, this risk is considered to be low in hemophilia gene transfer.
Vector shedding, another potential risk, has occurred following AAV gene transfer in animals39 and in humans,14 but there were no vector sequences detected in any tissue and no evidence of genetic integration based on extensive studies in animals and humans.14,39 Thrombosis is another potential risk given the epidemiological findings that elevated FVIII and IX levels are associated with cardiovascular risk.40,41
Hemophilia Gene Transfer—The Future
In conclusion, the results of human gene transfer trials, particularly those utilizing AAV delivery systems, are of great interest. The prospects for future hemophilia gene transfer appear optimistic. Whether this or a subsequent approach is successful, it seems likely that more than one approach for hemophilia gene transfer approach will be required. For example, targeting hepatocytes might be avoided in individuals with hemophilia and chronic hepatitis C liver disease, and retroviral vectors might be avoided in those with HIV infection. ■