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Gene therapy for the hemophilias
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T . VANDENDRIESSCHE, D. COLLEN and M. K. L . CHUAH
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology-University of Leuven, 49 Herestraat B-3000 Leuven, Belgium
Correspondence: T. VandenDriessche, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, 49 Herestraat B-3000 Leuven, Belgium.
Tel.:þ32 16 346144; fax:þ32 16 345990; e-mail: thierry.vandendriessche@ med.kuleuven.ac.be or marinee.chuah@med.kuleuven.ac.be
Summary. Significant progress has recently been made in the development of gene therapy for the treatment of hemophilia A and B. These advances parallel the development of improved gene delivery systems. Long-term therapeutic levels of factor (F) VIII and FIX can be achieved in adult FVIII- and FIXdeficient
mice and in adult hemophiliac dogs using adenoassociated viral (AAV) vectors, high-capacity adenoviral vectors
(HC-Ad) and lentiviral vectors. In mouse models, some of the highest FVIII or FIX expression levels were achieved using HC-Ad vectors with no or only limited adverse effects. Encouraging preclinical data have been obtained using AAV vectors, yielding long-term FIX levels above 10% in primates and in hemophilia B dogs, which prevented spontaneous bleeding. Non-viral ex vivo gene therapy approaches have also led to long-term therapeutic levels of coagulation factors in animal models. Nevertheless, the induction of neutralizing antibodies (inhibitors) to FVIII or FIX sometimes precludes stable phenotypic correction following gene therapy. The risk of inhibitor formation varies depending on the type of vector, vector serotype, vector dose, expression levels and promoter used,
route of administration, transduced cell type and the underlying mutation in the hemophilia model. Some studies suggest that continuous expression of clotting factors may induce immune tolerance, particularly when expressed by the liver. Several gene therapy phase I clinical trials have been initiated in patients suffering from severe hemophilia A or B. Some subjects report fewer bleeding episodes and occasionally have low levels of clotting factor activity detected. Further improvement of the various gene delivery systems is warranted to bring a permanent cure for hemophilia one step closer to reality.
Keywords: factor VIII, factor IX, gene therapy, hemophilia, immune tolerance, viral vector.

Introduction
Hemophilia A and B are congenital X-chromosome linked coagulation disorders, due to a deficiency in coagulation factor (F)VIII or FIX, respectively. Hemophilia A occurs in 1 in 10 000 individuals whereas hemophilia B affects 1 in 30 000 [1].
Hemophilia is characterized by spontaneous and prolonged bleeding that can result in disability or even death. Current
treatment consists of infusion of plasma-derived or recombinant FVIII or FIX. Although this treatment markedly improves both the life expectancy and the quality of life of patients suffering from hemophilia, they are still at risk for life-threatening bleeding episodes and chronic joint damage, especially since prophylactic treatment is restricted by the limited availability and high cost of purified FVIII and FIX. An important sideeffect of clotting factor substitution therapy is that some patients develop neutralizing antibodies against FVIII or FIX, which
render further substitution ineffective. Inhibitors occur in 10–40% of hemophilia A and in about 5% of hemophilia B patients treated by protein-replacement therapy.
Hemophilia is well suited for gene therapy since it is due to a single gene defect and the therapeutic window is relatively
broad. A slight increase in plasma FVIII or FIX levels can potentially convert severe to mild hemophilia, whereas levels as high as 150% of normal levels are not associated with any thrombotic side-effects. The concentration of FVIII in normal plasma is low (100%¼100–200 ngmL*1), but it is relatively difficult to express high levels of FVIII. FIX is easier to express, but the normal plasma level is high (100%¼5 mgmL*1). Gene therapy could provide a cure for this disease and potentially provide constant, sustained FVIII or FIX synthesis in the patient. This would obviate the risk of spontaneous bleeding, the need for repeated FVIII or FIX infusions and the risk of viral infections associated with plasma-derived FVIII or FIX. Hemophilia gene therapy requires the use of a gene delivery system that is efficient, safe, non-immunogenic and allows for long-term gene expression. Most importantly, gene therapy for hemophilia A and B must compare favorably with existing protein replacement therapies. The availability of animal models including FVIII and FIX knockout mice and hemophilia A and B dogs, which mimic the clinical symptoms of hemophilia, significantly facilitate preclinical efficacy and safety studies of gene therapy strategies.
Both viral and non-viral vectors have been considered for the development of hemophilia gene therapy (reviewed also in [2,3]). In general, viral vector-mediated gene transfer is far more efficient than non-viral gene transfer and has therefore been the method of choice. These vectors include retroviral, lentiviral, adenoviral and adeno-associated viral vectors, each with their own advantages and limitations. However, there are several reasons why a non-viral treatment would still be desirable. Non-viral vectors can be assembled in cell-free systems from well-defined components and have the potential to be less immunogenic than viral vectors. Details of each of these gene therapy strategies for hemophilia will be discussed below.

Preclinical studies
Adeno-associated viral vectors
Vectors derived from the adeno-associated virus (AAV) are promising vectors for hemophilia gene therapy. AAV vectors are capable of achieving long-term transgene expression in the absence of expression of viral genes and are able to transduce non-dividing cells in vivo, including in liver and muscle. In the liver, usually only a fraction of the stably transduced genomes (typically 10%) integrate into the host chromosomes, and transgene expression is derived mainly from the non-integrated, extra-chromosomal AAV genomes [4]. While virtually all hepatocytes take up vector, only approximately 5% of hepatocytes express the transgene.

One drawback of AAV is its limited cloning capacity. To overcome the size constraints of recombinant AAV, strategies have been developed whereby the heavy and light chains of the B-domain-deleted human FVIII cDNA(hFVIIIDB) are delivered independently by using two separate vectors [5]. Alternatively, split AAV vector technology expands the packaging capacity of AAV through head-to-tail dimerization of two different AAV vectors that span either the 50 or 30 end of the FVIII expression cassette [6]. Although therapeutic FVIII levels can be achieved using these dual vector approaches, a single AAV-FVIII vector would still be preferred. Small regulatory elements designed for liver-specific transgene expression have been linked to the hFVIIIDB cDNA [7] and incorporated into an AAV vector.

Portal vein injection of AAV-FVIII [1011 vector genomes (vg) mouse*1)] into immunodeficient mice resulted in longterm expression of therapeutic FVIII levels (30%), but in C57BL/6 mice, anti-hFVIII antibodies led to the loss of FVIII expression. Unexpectedly, at 10 months after injection of the virus, hFVIII protein was detected (about 30%) which coincided with the disappearance of the anti-hFVIII inhibitory antibodies [8]. These results suggest that immune tolerance to hFVIII could be induced by sustained expression of hFVIII following gene therapy. Using a different AAV vector whereby a minimal transthyretin promoter was used to drive the canine (c) FVIIIDB cDNA, long-term therapeutic FVIII levels could be achieved in hemophilia A mouse (8% of normal canine activity; (3*1013 vg kg*1) and dog models (1.5–2.5%; 1013 vg kg*1), leading to partial phenotypic correction of the bleeding diathesis [9].

Several studies have shown that hepatic delivery of AAV-FIX vectors results in long-term therapeutic FIX levels in normal and hemophilic mice, hemophilia B dogs and primate models [10–17]. FIX expression levels vary, depending largely on the promoter used and the presence of other cis-acting elements, such as introns. One of the most potent expression cassettes contained a FIX minigene driven from a hepatocyte-specific ApoE–hAAT chimeric promoter coupled to the hepatocyte control region (HCR). Hepatic delivery of an AAV vector (1012 vg kg*1) containing this FIX expression cassette resulted in 14% of stable FIX levels in hemophilia B dogs [18]. A similar vector yielded therapeutic cFIX levels (5–12%) and sustained correction of canine hemophilia B with complete prevention of spontaneous bleeding following hepatic gene delivery of relative low AAV vector doses (1012 vg kg*1), even in the context of a FIX null mutation, which is invariably associated with a high risk of inhibitor formation in protein or gene therapy [15].

Nevertheless, one animal carrying such a null mutation developed anti-cFIX antibodies that resulted in transient FIX expression. Another potent AAV-FIX vector uses a chimeric liverspecific promoter composed of the thyroid hormone-binding globulin sequences linked to the a1-microglobulin/bikunin enhancer sequences [13,14]. Portal vein administration resulted in stable expression (>7 months) of therapeutic cFIX levels (5%) and prevention of bleeding in the hemophilia B dog model (4.6*1012 vg kg*1) [14].

In macaques, FIX expression was variable following hepatic delivery of AAV-FIX, which uses a ubiquitously expressed promotor (CMV-bactin) to drive hFIX (4*1012 vg kg*1) [17]. Two macaques had long-term therapeutic levels of hFIX (4–10%), two had low levels of FIX that became undetectable after 15 weeks and one animal developed anti-hFIX inhibitors that correlated with transient FIX expression. The liver was predominately transduced, with significant lower transduction levels in spleen, and no detectable gene transfer in the testis. It appears that transduction efficiency was at least 10 times more efficient in macaques than in mice, when comparable vector
doses were used [18].

Administration of AAV-FIX vectors via the portal or tail vein produced no antibodies in mice, even when challenged subsequently with hFIX. AAV-mediated hepatic gene transfer appears to induce regulatory CD4þT-cells promoting tolerance to FIX, even in mice with a large FIX deletion [19] and may possibly involve Fas/FasL-induced apoptosis. Liver-targeted delivery seems the preferred route for administration of rAAV-FIX particles, both because of the potentially higher levels of FIX achieved compared with other tissues (e.g.muscle, see below) and the reduced immune response [16,20].

Intramuscular injection of high-titer AAV vectors (2*1011 vg per mouse) encoding CMV-driven hFIX into immuno-deficient mice resulted in therapeutic plasma levels of FIX [4–7%] [21] which could be increased 2–4-fold by using alternative promoter/ enhancers [22]. Following intramuscular injection of AAV-hFIX in immunocompetent mice, no circulating hFIX could be detected which correlated with the induction of Th2-dependent anti-hFIX neutralizing antibodies [16,20,23]. This is in contrast with the lack of antibody responses following hepatic AAV-hFIX transduction. Hence, it appears that the target tissue greatly influences the immunogenicity of the hFIX protein. Hemophilia B dogs with a null mutation invariably developed anti-cFIX antibodies following intramuscular AAVcFIX delivery (1012 vg kg*1), unless they received transient immunosuppression [24]. In most hemophilia B dogs with a missense mutation, anti-cFIX antibodies were absent or transient and therefore did not prevent sustained systemic expression of FIX. However, the risk of inhibitor development increased with increasing vector doses and correlated most strongly with increased dose per site [25–27].

Since most humans are seropositive for AAV-2, the presence of pre-existing neutralizing AAV-specific antibodies interferes with AAV transduction and would preclude efficient in vivo gene delivery in clinical trials. To circumvent this potential problem, other AAV serotypes could be used. Hepatic transduction with AAV5 serotypes resulted in higher transduction efficiencies (typically 15% of hepatocytes), leading to 3–10- fold higher FIX expression levels in mice (20% of normal FIX levels, 1011 vg) [28]. In a hemophilia B dog, high cFIX levels could be achieved (66% of normal levels) following hepatic transduction with an AAV5 vector that expresses cFIX from a chimeric liver specific promoter [29]. This dog had previously been exposed to an AAV2-cFIX vector that resulted in stable but low FIX expression levels. This further confirms that serotype switching allows for vector readministration. Perhaps the most efficient serotype for hepatic gene delivery is AAV8, which typically yields 10–100-fold higher transgene expression levels than AAV2. In particular, supranormal FIX levels could be achieved following intraportal injection in mice (about 300%, 1011 vg) [30].

Transduction of skeletal muscle with AAV serotypes 1, 3, and 5 produced 100–1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, AAV type 1 continued to express levels of cFIX on average at 1600% followed by type 5 (130%), type 3 (65%), type 4 (5%), and finally type 2 (2%) (2.5*1011 vg per mouse) [31]. In addition, long-term supraphysiologic cFIX levels could be achieved following AAV1-cFIX transduction of skeletal muscle in hemophilia B mice, in the absence of anti-cFIX antibodies [32] in contrast to AAV2 vectors, which require immunosuppressive treatment to obtain long-term cFIX expression [32]. These results indicate that the AAV serotype not only determines the efficiency of gene transfer but may also influence the immunogenicity of the FIX protein.

Adenoviral vectors Adenoviral vectors can transduce a broad range of dividing as well as non-dividing cells. The adenoviral genome remains episomal in the transduced cells, implying that there is virtually no risk for neoplastic transformation due to insertional mutagenesis.
However, dividing cells will gradually lose the adenoviral vector along with its potentially therapeutic gene. Since
most humans have been naturally infected with adenovirus, it is possible that the presence of adenovirus-specific antibodies interferes with adenoviral transduction in vivo. To circumvent this potential problem and to allow for repeated administration, other adenoviral serotypes or non-human adenoviral vectors could be used.
Early generation adenoviral vectors are rendered replicationdeficient by removal of at least one essential viral regulatory gene (e.g. E1 gene). Therapeutic FVIII or FIX levels were achieved with this vector in various animal models, which have been reviewed previously [34]. However, these vectors still contain residual viral genes that can be expressed at low levels even in the absence of E1, presumably as a result of activation by cellular E1-like proteins. Inflammatory responses and toxicity have been observed in animal models and in clinical trials, which prompted the development of high-capacity adenoviral (HC-Ad) vectors that are devoid of all viral genes.

Injection of HC-Ad vectors encoding full-length hFVIII cDNA under the control of the human albumin promoter into FVIII-deficient mice resulted in long-term therapeutic FVIII expression (50–400%, 2*1011 vector particles [vp]) and phenotypic correction of the bleeding phenotype [36]. In some animals, however, expression was short-lived due to the induction of neutralizing anti-hFVIII antibodies. No significant histopathologic findings or toxicities were observed to be associated with the vector. At a 10-fold lower vector dose, no FVIII could be detected, suggesting a non-linear threshold effect, which is probably at least partly due to uptake and degradation of viral particles by Kupffer cells. Even in the absence of antibodies, FVIII expression gradually declined over several months.

We have recently generated improved HC-Ad vectors, which yield at least 20-fold higher FVIII expression levels in mice [35], using comparable vector doses to those described in the previous studies [34]. These HC-Ad vectors expressed hFVIIIDB or cFVIIIDB under the control of different liverspecific promoters. Intravenous administration of these vectors into hemophilic SCID mice at a dose of 5*109 infectious units (IU) (about 1.5*1011 vp) resulted in unprecedented, efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 1500%), correcting the bleeding diathesis. Injection of 100-fold lower doses still resulted in therapeutic FVIII levels. In immunocompetent hemophilic mice, FVIII expression levels peaked at 7500% but declined thereafter due to neutralizing anti-FVIII antibodies and a cellular immune response. Vector administration did not result in thrombocytopenia, anemia nor elevation of the pro-inflammatory cytokine IL-6, and caused no or only transient elevations in serum transaminases. Following transient in vivo depletion of tissue macrophages (particularly Kupffer cells) prior to gene transfer, significantly higher and stable FVIII expression levels were observed in both hemophilic and hemophilic-SCID mice.

This suggests that the therapeutic window of HC-Ad vectors could be improved further by minimizing their interaction with the innate immune system. Intravenous injection of an HC-Ad vector into a hemophilia A dog at a dose of 4.3*109 IU kg*1

VandenDriessche et a led to transient therapeutic cFVIII levels that partially corrected the whole blood clotting time. Inhibitory antibodies to cFVIII could not be detected and there were no signs of hepatotoxicity or of hematologic abnormalities. In a separate study, signifi- cantly higher (10-fold) and more prolonged FVIII expression levels (100%, 6*1010 vp) with less toxicity were achieved in hemophilia A mice with HC-Ad vectors encoding hFVIIIDB from a modified albumin promoter than when early generation E1E2aE3-deleted vectors were used [36]. Although no anti- FVIII antibodies were detected, expression slowly declined to less than 10% of initial levels over a 40-week interval. Supraphysiologic serum levels of hFIX were obtained (maximum levels of 800%; 2*109 IU per mouse) following injection of HC-Ad vectors in hemophilia B and normal mice [37].

The HC-Ad vectors expressed hFIX from a ApoE/AAT promotor/ enhancer promoter [38] and contained a stuffer fragment composed of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. While remaining in the therapeutic range, serum FIX concentrations slowly declined by 95% over a period of 1 year, possibly due to gradual loss of vector genomes. This could be overcome by using transposition technology [39]. At this dose, IL-6 and TNF-a serum concentrations were elevated in animals that received the first-generation but not the HC-Ad vector. Both first-generation and HC-Ad adenovirus-treated animals demonstrated
a threshold effect.
MoMLV-based retroviral vectors Moloney murine leukemia virus (MoMLV)-based retroviral vectors offer the potential for long-term gene expression by virtue of their stable chromosomal integration and lack of viral gene expression. However, cell division is required for stable transduction. Gene therapy for hemophilia with MoMLV-based vectors would require either direct in vivo transduction of cells that are naturally proliferating or induced to proliferate, or ex vivo expansion and transduction of target cells followed by their readministration.

We have previously shown that hemophilia A mice can be cured by in vivo gene therapy using retroviral vectors. High-titer MoMLV-based retroviral vectors expressing hFVIIIDB were pseudotyped with the vesicular stomatitis virus (VSV-G) andwere injected intravenously (0.8–1.5*108 IU per mouse) into newborn, FVIII-deficient mice [40]. High-levels of functional hFVIII production could be detected in about 50% of the recipients, some of which expressed stable (>14 months) physiologic or supranormal levels (up to 1250%), which corrected the bleeding diathesis. However, the remaining mice did not have their condition corrected, which correlated with the induction of hFVIII-specific neutralizing antibodies and cellular immune responses. Efficient gene transfer occurred into liver,
spleen and lungs but not in other organs, including the testes,and predominant FVIII mRNA expression occurred in the liver.

To our knowledge, this was the first demonstration that hemophilia A could be cured by gene therapy in an animal model that mimics the cognate human disease. The efficient hepatic gene transfer in neonatal mice could be due primarily to the higher hepatocyte turnover rate in newborn vs. adult animals in conjunction with the use of high-titer vectors. In a separate study, but using a similar retroviral construct, FVIII levels varied between 10 and 75% of the normal human levels in juvenile and adult rabbits [41]. The exact reason for the unexpected efficient gene transfer in adult rabbits is not fully understood, but is at least partly due to the use of high-titer vector preparations.
Similarly, direct infusion of MoMLV retroviral vectors containing the cFIX cDNA into neonatal C57Bl/6 or hemophilia B mice resulted in stable therapeutic FIX levels (150% after 10 months; 107 IU per mouse), which correlated with efficient hepatic transduction [42]. Treatment of one newborn hemophilia B dog (1.3*1010 IU kg*1) resulted in therapeutic levels of cFIX (10%), and expression was stable over at least 5 months follow-up [43]. Although in vivo retroviral vector-based approaches may have implications for the treatment of hemophilia in pediatric patients, the use of this approach in adults is limited due to the paucity of proliferating hepatocytes. In adult hemophilia
B dogs, artificial induction of hepatocyte proliferation (by partial hepatectomy) was required to obtain a therapeutic effect [43].

Attempts at achieving long-term FVIII or FIX expression by ex vivo gene therapy using a variety of retrovirally transduced primary cells have shown that the implantation site, the target cell type and/or the vector design (particularly the strength of the expression cassette) are critically important for obtaining detectable FVIII or FIX levels in the circulation (reviewed also in [2,33]). We and others have shown that therapeutic hFVIII levels could be obtained in mice following transplantation of retrovirally transduced cells. However, long-term FVIII expression has not yet been achieved by ex vivo retroviral gene therapy. The disappearance of functional FVIII could be attributed to the loss of transduced cells and/or to transcriptional repression. Similarly, therapeutic but transient FIX in vivo expression levels were achieved following transplantation of cells transduced with FIX retroviral vectors. However, the use of alternative promoters and modifications in vector design has overcome this limitation [44]. For instance, sustained systemic expression of FIX can be achieved by ex vivo gene therapy following transduction of myoblasts with MoMLV-FIX retroviral vectors expressing FIX, using muscle creatinin enhancer/ promoter elements [45]. Prolonged FIX expression can also be achieved using retrovirally transduced fibroblasts in a rabbit model [46].

Lentiviral vectors
Lentiviral vectors have been derived from primate (HIV, SIV) and non-primate lentiviruses (FIV, EIAV) and contain the transgene of interest along with the cis-acting elements necessary for stable transduction [47]. Unlike onco-retroviral vectors, lentiviral vectors can transduce both dividing and non-dividing cells Initial studies suggested that lentiviral vectors were toxic to hepatocytes and that non-cycling hepatocytes were relatively refractory to lentiviral transduction, yielding only low levels of FIX or FVIII [48,49]. However, subsequent studies showed that the hepatotoxicity of lentiviral vectors was limited and transient, and that quiescent hepatocytes could be transduced with lentiviral vectors [50–52]. Differences in vector purity and vector design may at least partly account for these different
results. In particular, incorporation of cis-acting sequences from the HIV-1 pol gene (central polypurine tract or cPPT) into the lentiviral vector-facilitated hepatic gene transfer possibly by facilitating intranuclear transport of the lentiviral preintegration complexes, resulting in higher FIX expression levels [50,52–54] (T.VandenDriessche et al., unpublished observations).

Stable therapeutic levels of FIX (2–4%) could be achieved in adult immunodeficient mice requiring lower vector doses (1.5*109 IU per mouse) and obviating the need for partial hepatectomy [54,55]. Similarly, variable therapeutic FVIII levels (up to 75%, 4*107 IU mouse*1) could be achieved when 5-week-old hemophilia A mice were injected with FIVbased lentiviral vectors encoding hFVIIIDB [56].

Lentiviral-FIX gene transfer resulted in induction of anti-FIX neutralizing antibodies in immunocompetent mice, which curtailed long-term FIX gene expression [55]. We and others have shown that lentiviral vectors can efficiently transduce antigenpresenting cells (APCs), particularly splenic macrophages, Kupffer cells and B-cells [52,54,55]. Since inadvertent gene expression in APCs increases the risk of inducing neutralizing antibodies against the transgene product [57], the robust humoral response against FIX may at least be partly due to the efficient inadvertent lentiviral transduction of APCs. The use of hepatocyte-specific instead of ubiquitous promoters is warranted to alleviate this potential concern [54]. In addition, to further augment the specificity and efficiency of gene transfer into hepatocytes, lentiviral vectors could be pseudotyped with alternative envelopes [58,59]. Studies in larger animal models and continued efforts to improve the vector design are warranted to explore the full potential of lentiviral vectors for hemophilia gene therapy.

Other approaches
The use of non-viral vectors for gene therapy has been hampered by low efficiency and/or transient expression of FVIII or FIX. Recently, long-term therapeutic FIX expression (10–40%) has been achieved using optimized FIX expression constructs [39] following hydrodynamic transfection, which involves infusion of plasmids in large volumes of aqueous solutions [60]. In addition, integrating non-viral systems based on transposase or recombinase-mediated integration have been used, which gave rise to stable therapeutic FIX expression levels [61,62].
However, the potential immune and cellular consequences of expressing these foreign proteins remain to be addressed.

Furthermore, clinically acceptable and efficient non-viral transfection methods would need to be developed before these approaches can realize their full potential for in vivo gene therapy. Nevertheless, non-viral approaches have been used for ex vivo transfection. For instance, human endothelial cells obtained from blood could be transfected using non-viral methods with a hFVIIIDB expression plasmid and a selectable marker gene [63]. After selective enrichment of the stably transfected cells in selection medium, engineered cells were injected into immunodeficient mice. This resulted in long-term therapeutic and even supraphysiologic FVIII expression levels (600%). The transfected cells accumulated only in bone marrow and spleen. However, the transplanted cells seem to proliferate in vivo, which raises new safety concerns.

Clinical trials
Several gene therapy trials have been initiated for hemophilia A and B, which were reviewed previously [64]. The first clinical trial in hemophilia was initiated in China and involved the use of autologous skin fibroblasts transduced with a FIX retroviral vector, which were transplanted into two brothers suffering from hemophilia B, with baseline FIX levels of 2%. FIX levels increased 2-fold and persisted for over a year. In the first gene therapy trial for hemophilia A, a non-viral ex vivo transfection approach was being explored for patients with severe hemophilia A [65]. Dermal fibroblasts obtained from each patient by skin biopsy were grown in culture and transfected by electroporation with a plasmid containing sequences of the gene that encodes FVIII. Cells that produced FVIII were selected, cloned, and propagated in vitro. The cloned cells were then harvested and administered to the patients by laparoscopic injection into the omentum. The patients were followed for 12 months after the implantation of the genetically altered cells. There were no serious adverse events related to the use of FVIII-producing fibroblasts or the implantation procedure. No long-term complications developed, and no inhibitors of FVIII were detected.
In four of the six patients, plasma levels of FVIII activity rose above the levels observed before the procedure. FVIII levels corresponded to 1–2% of normal, with a maximum of 4%. The increase in FVIII activity coincided with a decrease in bleeding, a reduction in the use of exogenous FVIII, or both. In the patient with the highest level of FVIII activity, the clinical changes lasted approximately 10 months. Nevertheless, FVIII expression eventually declined to undetectable levels.
An adeno-associated viral vector encoding FIX driven from the CMV promoter has been given intramuscularly in severe hemophilia B patients with no evidence of antibodies to FIX, inflammatory responses or inadvertent germline transmission [66]. FIX gene transfer and expression was confirmed in biopsied tissues. Modest changes in clinical endpoints were observed, including circulating levels of FIX and decreased frequency of FIX protein infusion. In a separate trial, AAV vector was administered via the hepatic artery [18]. The vector expresses a hFIX minigene using a hepatocyte-specific hAAT/ ApoE promoter-enhancer and HCR elements. Blood counts and liver enzymes were normal and FIX levels remained unchanged in the low dose group (2*1011 vg kg*1).

Small amounts of vector DNA were detected in body fluids, including semen, for up to 10–12 weeks postinjection, contrary to preclinical studies in mice and dogs. This unexpected finding, prompted additional. VandenDriessche et al studies in rabbits, which revealed a dose-dependent increase in the likelihood of finding vector sequences in semen DNA [67].
Clearing of vector sequences from semen DNA eventually occurs but takes longer when high vector doses are used.
Additional studies are warranted to further evaluate and minimize the risk of inadvertent germline gene transfer.
In the high dose cohort patients received 2*1012 vg kg*1. In one patient, circulating FIX levels in the range of 5–12% were obtained, first detected 2 weeks after vector infusion and detectable over the ensuing few weeks. However, 6 weeks after vector infusion, levels fell to 2.7%. Beginning at 4 weeks after vector infusion and preceding the drop in FIX levels, there was a rise in liver transaminases that peaked at 9-fold above normal levels during the 4th week and subsequently returned to baseline.
A second patient showed no toxicity for at least 12 weeks post-infusion of the AAV-FIX vector and had maximum 3% FIX levels that gradually declined to basal levels.

In a third trial, a retroviral vector expressing B-domain deleted FVIII has been administered intravenously to severe hemophilia A patients with no evidence of adverse effects. The vector persisted in peripheral blood mononuclear cells for at least 6 months. While no subject had persistent levels >1%, some patients showed measurable FVIII levels and decreased bleeding frequency compared with historical rates. Finally, one patient suffering from hemophilia A has received HC-Ad vectors encoding full-length FVIII. This resulted in a FVIII level above 1%. However, due to transient adverse events (hepatotoxicity), the trial has been put on hold.General discussion and perspectives
The recent advances in gene transfer technology have expedited the development of gene therapy for the treatment of hemophilia A and B. Stable therapeutic levels of FVIII or FIX can now be achieved either in normal or hemophilic mice and even in hemophilic dogs. Some of these encouraging preclinical studies culminated in several phase I clinical trials for hemophilia A and B that were initiated in 1999. Several confounding variables may influence the overall efficacy of the gene therapy procedure in hemophilia patients, including the type of vector used, the purity of the vector preparation, the promoter used to drive FVIII or FIX expression, the site of administration and the transduced cell types (APCs). Some of these variables may also influence the incidence of inhibitor formation.

Whether gene therapy would increase or decrease the likelihood of inhibitor formation in hemophilia patients compared with protein replacement therapy is one of the important questions that warrants further investigation. Some studies suggest that FVIII protein replacement therapy may be more immunogenic than if FVIII is produced in vivo following gene therapy [68]. Continuous production of high levels FVIII or FIX in situ following gene therapy may actually induce immune tolerance, as was recently shown following AAV-FVIII gene transfer [8]. In the case of AAV at least, the target tissue, the serotype and the underlying mutation in the FIX gene have profound consequences on the immunogenicity of FIX. A major concern is that the use of viral vectors expressing coagulation factors, or impurities in the vector preparations, may provide immunologic danger signals that may facilitate inhibitor formation [69,70]. In addition, gene transfer may result in the presentation of endogenously synthesized FVIII or FIX-derived peptides in the context of MHC class I molecules, potentially resulting in cytotoxic T-cell responses that could eliminate the FVIII- or FIX-engineered target cells [35]. The secreted FVIII protein that is produced in vivo may also be presented in the
context ofMHCclass II as in the case of infused clotting factors.

It is not clear whether gene therapy could break tolerance in patients that are tolerant to FVIII and whether inhibitors can be suppressed once they occur following gene therapy. Since many adult hemophilia patients have an infectious or inflammatory disease, complex interactions can influence the therapeutic efficacy of the gene therapy procedure or the propensity for inhibitor formation, and caution is warranted to avoid exacerbating these underlying conditions.

Integrating vectors can potentially give rise to life-long expression of FVIII or FIX. However, random chromosomal integration by integrating vectors increases the risk of insertional oncogenesis and malignant transformation, due to oncogene activation or inactivation of tumor-suppressor genes. In addition, integrating vectors pose a greater risk for permanent germline modification, which varies depending on the biodistribution and tropism of the vector. To minimize these risks, non-integrating vectors could be used instead, such as HC-Ad vectors. However, it is not certain whether these vectors can give rise to life-long expression of FVIII or FIX, since several studies demonstrated a slow but significant decline in transgene expression when this type of vector is used [34–37]. This decline could be overcome when HC-Ad vectors are used in conjunction with transposon technology [39,61], but this approach does not circumvent the risks associated with random integration. Alternatively, the risk of insertional oncogenesis could be minimized by using vectors that integrate site-speci- fically into the genome [62]. It is not certain that life-long expression of FVIII or FIX could be achieved with AAV vectors, since expression is mainly derived from the nonintegrated, extrachromosomal AAV genomes [4].

In a recent preclinical study based on retroviral marking of hematopoietic stem cells (HSCs) and in a phase I clinical trial for SCID-X1 based on retroviral transduction of HSCs, complications related to the development of a monoclonal T-cell leukemia (or leukemia-like condition) have been observed, which were possibly associated with oncogene activation due to retroviral integration [71,72]. Although it is likely that other events may have contributed to the oncogenic transformation observed in these studies, it raises new concerns regarding the actual risks of oncogenesis associated with the use of integrating (retroviral, lentiviral and AAV) vs. non-integrating vectors (HC-Ad) for hemophilia gene therapy. In most cases, no more than approximately 10% of stably transduced AAV genomes
integrate into host chromosomes in vivo, which decreases the risk of insertional oncogenesis. However, the integration of AAV vectors was found to be associated with deletions, insertions and even chromosomal translocations [73]. Although tumors have been found in mice that received AAV vectors, they are probably not formed by an insertional oncogenic event due to AAV integration, and additional studies are necessary to identify the precise cause of these tumors [74]. Accurate assessment of the potential oncogenic risk of these different vectors remains difficult and requires additional preclinical and clinical studies, especially since other confounding variables, such as the transduced cell type, the transgene used and perhaps underlying infections (e.g. HCV) or genetic factors, may influence this risk.

Despite the tremendous progress in the field over the past few years many questions remain largely unexplored. Extensive gene therapy studies in preclinical hemophilia models are needed to anticipate the possible outcome in patients and to increase the overall efficiency of the various gene therapy strategies while further improving their safety. The development of hemophilia gene therapy will undoubtedly continue to contribute to a better understanding of vector–host interactions that will benefit the entire field of gene therapy. The results from the extensive preclinical studies in normal and hemophilic animal models and encouraging preliminary clinical data indicate that the simultaneous development of different strategies is likely to bring a permanent cure for hemophilia one step closer to reality.
Acknowledgements
We thank Dr Kay, Dr Herzog and Dr Chao for their comments< and Dr Vermylen for reviewing the manuscript. Part of the work presented in this review was supported by the Fund for Scientific Research (FWO).
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