Journal of Thrombosis and Haemostasis, 13 (Suppl. 1): S133–S142

DOI: 10.1111/jth.12926

INVITED REVIEW

New approaches to gene and cell therapy for hemophilia T . O H M O R I , * H . M I Z U K A M I , † K . O Z A W A , ‡ Y . S A K A T A * and S . N I S H I M U R A * § ¶ *Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University; †Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Tochigi; ‡The Institute of Medical Science, The University of Tokyo; §Department of Cardiovascular Medicine, The University of Tokyo; and ¶Translational Systems Biology and Medicine Initiative, The University of Tokyo, Tokyo, Japan

To cite this article: Ohmori T, Mizukami H, Ozawa K, Sakata Y, Nishimura S. New approaches to gene and cell therapy for hemophilia. J Thromb Haemost 2015; 13 (Suppl. 1): S133–S42.

Summary. Hemophilia is considered suitable for gene therapy because it is caused by a single gene abnormality, and therapeutic coagulation factor levels may vary across a broad range. Recent success of hemophilia B gene therapy with an adeno-associated virus (AAV) vector in a clinical trial showed the real prospect that, through gene therapy, a cure for hemophilia may become a reality. However, AAV-mediated gene therapy is not applicable to patients with hemophilia A at present, and neutralizing antibodies against AAV reduce the efficacy of AAV-mediated strategies. Because patients that benefit from AAV treatment (hemophilia B without neutralizing antibodies) are estimated to represent only 15% of total patients with hemophilia, the development of basic technologies for hemophilia A and those that result in higher therapeutic effects are critical. In this review, we present an outline of gene therapy methods for hemophilia, including the transition of technical developments thus far and our novel techniques. Keywords: cell therapy; gene therapy; hemophilia; vectors, genetic; viruses, adeno-associated.

Introduction Hemophilia is an hereditary congenital hemorrhagic diathesis caused by mutations in blood coagulation factor VIII (FVIII) or IX (FIX) genes. Disorders with FVIII and FIX mutations are known as hemophilia A and B, respectively. Hemophilia A is the most common form, with one in 5000 males born with this disorder. The development of coagulation factor concentrates, including recombinant preparations, has made it easy to control bleeding and Correspondence: Yoichi Sakata, Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel.: +81 285 58 7397; fax: +81 285 44 7817. E-mails: [email protected] © 2015 International Society on Thrombosis and Haemostasis

significantly improves patient prognosis. The prevention of hemophilic arthropathy by prophylactic injections of a coagulation factor preparation (regular replacement therapy) initiated in childhood is now common [1]. These advances have dramatically improved the quality of life (QOL) of patients with hemophilia. However, because of the extremely short half-life of coagulation factor preparations used for current treatments, patients are required to inject the preparation as frequently as 1–3 times a week in the case of regular replacement therapy. The need for frequent intravenous injections, particularly in childhood, imposes a substantial burden on patients and their families. It is expected that some patients will have difficulty in injecting the preparation independently as they become senescent. Moreover, because of its nature as a hereditary disease, distress experienced by genetic carriers regarding marriage and childbirth is also a significant social problem. From these viewpoints, gene therapies that can provide permanent therapeutic effects with a single treatment are anticipated to resolve the problems currently faced in hemophilia care. Gene therapy could also become a cost-effective alternative. The median cost for the treatment and care of hemophilia is now estimated at $23 265–98 334 year 1 in the United States [2]. It may have an even greater impact in developing countries, where the use of factor concentrates is often limited [3,4]. Major viral vectors used in gene therapy for hemophilia Gene transfer often involves the use of viral vectors because of their high efficiency in transducing a target gene. Adeno-associated virus (AAV) vectors and lentiviral vectors are the most commonly used vectors for hemophilia [5]. AAV is a single-stranded DNA virus of the Parvovirus genus [6]. AAV vectors have many advantages over other vectors, including gene transferability to quiescent cells, no pathogenicity, high safety, and low immunogenicity. However, disadvantages include difficult purification and limited length of the carrier gene of approximately 5 kb. Diverse serotypes are known for

S134 T. Ohmori et al

AAV, each of which has a distinct organ/cell tropism [6]. It has also been reported that high levels of gene expression can be obtained by a technique that generates a double strand by inserting a complementary sequence to the single-stranded DNA (self-complementary AAV) [7]. Serotype 8 (AAV8) is currently being applied to clinical practise and allows gene expression in the liver, where coagulation factors are physiologically produced, even when it is injected intravenously [8]. Lentiviral vectors can efficiently and stably transduce a target gene into diverse cells ex vivo. Although lentiviral vectors are a subclass of retroviral vectors, they are more efficient at transducing quiescent cells such as hematopoietic stem cells than retroviral vectors. Compared with AAV vectors, lentiviral vectors have several advantages for gene therapy, including larger packaging capacity and a reduced probability of preexisting immunity to the vector components. The integration of proviral sequences into host genomic DNA provides a further advantage by maintaining transgene expression even after host cell proliferation and differentiation. Previously, leukemic transformation caused by integration of the retroviral vector in the vicinity of LMO-2 was problematic for human gene therapy targeting hematopoietic cells in patients with severe combined immunodeficiency [9]. Lentiviral vectors have a low risk of insertional mutagenesis because they less frequently integrate to transcription start sites compared with retroviral vectors [10]. In addition, current lentiviral vectors aimed for treatment in clinical practise are significantly safer than those used previously because they

A

B

ITR

FIX gene

Normal coagulation factors

ITR

Therapeutic gene is packaged into a AAV8

Current status of gene therapy for hemophilia The current types of gene therapy for hemophilia mainly utilize techniques to ectopically express a normal gene by a vector, while leaving the abnormal somatic gene as it is. The strategies of gene therapies for hemophilia are mainly categorized into two approaches: (i) direct injection of a vector into the body and (ii) the transplantation of cells expressing a coagulation factor after ex vivo transduction. Gene therapy involving direct vector administration

This technique involves transferring a foreign gene to target cells by directly injecting the vector itself into the body to express a coagulation factor. The main method for direct vector injection is the use of an AAV vector (Fig. 1). The initial approaches for treatment of patients with hemophilia used the first-generation serotype AAV2, and the administration methods for AAV included intramuscular and intrahepatic injection. Kay et al. [11] reported the use of an AAV vector intramuscularly administered to three patients with hemophilia B as a Phase I study. The expression level of the coagulation factor did not reach the expected treatment range [11], albeit the transgenic FIX expression persisted locally at the

C

AAV vectors

Direct delivery

self-inactivate by deleting the promoter activity of the long terminal repeat. Concerns regarding the risk of insertional mutagenesis might be further reduced by the use of integrase-defective lentiviral vectors.

Injection of the vector after blood flushing

Hepatocytes Shutting blood flow using a balloon

Neutralizing antibodies

Injection into the patient

Insertion of a catheter to portal vein

Target organ (liver) Fig. 1. Gene therapy approaches for hemophilia by direct administration of AAV8. (A) The therapeutic gene (FIX or FVIII) is packaged into AAV8 and then directly injected into the body. AAV8 can efficiently transduce hepatocytes to express coagulation factor. (B) AAV vectors enter the cells via endocytosis and then express the target gene as an episome (upper panel). Neutralizing antibodies against AAV capsid proteins interfere with AAV transduction of the cells (lower panel). (C) Techniques to minimize the inhibitory effect of neutralizing antibodies against AAV. Blood flow in the left portal vein was transiently occluded with a balloon catheter. To remove neutralizing antibodies in the blood, saline followed by AAV8 was injected sequentially through the catheter. © 2015 International Society on Thrombosis and Haemostasis

Novel gene and cell therapy for hemophilia S135

injection site for at least 3.7 years after initiation of the treatment [12]. In 2006, the direct injection of AAV2 into a hepatic artery was performed in seven patients [13]. In two patients receiving the high-dose vector (2 9 1012 vector genomes [vg] kg 1), the peak factor IX expression reached 3.11% but was transient (up to 8 weeks) and accompanied by an increase in liver enzymes. The reason for this was considered to be exclusion of the transduced liver cells because of cellular immunity to the AAV capsid antigen [14]. However, the discovery of several different AAV serotypes provided further approaches for efficient hemophilia gene therapy. A new serotype (AAV8) discovered in monkeys allowed the highly specific expression of transgenes into the liver [15]. Comparison of diverse serotypes (AAV2, AAV5, AAV7, AAV8) showed that AAV8 was superior for gene transfer to hemophilia A mice, and its therapeutic effect was similar by intraportal or intravenous administration [16]. Moreover, successful treatments with AAV8 injections in hemophilia B dogs and monkeys were reported [17–19]. Finally, Nathwani et al. [20] reported a successful Phase I study of human hemophilia B using AAV8, in which a treatment range of coagulation factor activity was obtained in the high-dose group (2 9 1012 vg kg 1). The major advantage of using AAV8 is that only a single administration in a peripheral vein allows the production of coagulation factor in the liver. During a follow-up period of up to 3 years, no toxic side effects were reported and the therapy was shown to have long-term efficacy [21]. We confirmed the maintenance of a treatment range of coagulation factor levels for over 7 years in monkeys using the same technique (unpublished data). Single gene therapy with AAV8 can decrease the need for regular replacement therapy, thus remarkably improving the QOL of patients over a long period. An online search of ClinicalTrials.gov using hemophilia and AAV as keywords generated three hits, all of which are current clinical trials under recruitment, all focusing on the treatment of hemophilia B using AAV8 (Table 1). A certain level of success has been achieved in therapy for hemophilia using AAV8, but there are also several problems. One of these is the presence of neutralizing antibodies to AAV in some individuals, probably because

of previous infection with AAV. The neutralizing antibodies prevent vector transduction and therefore inhibit the potential therapeutic effect of AAV-mediated gene therapy [22,23] (Fig. 1). We developed a highly sensitive assay system to measure neutralizing antibodies to AAV and examined the rate of carriers in healthy individuals and patients with hemophilia [24]. The percentage of individuals carrying AAV8 neutralizing antibodies who were healthy was 32.9–37.6% and 28.8–35.6% in those with hemophilia [24]. The young population (under 40 years of age) tended to have a lower prevalence of AAV-neutralizing antibodies [24]. These data suggest that a beneficial effect of simple gene therapy by AAV8 can be expected in 60–70% of patients with hemophilia, while the remaining 30–40%, particularly patients aged ≥ 40 years, will benefit only minimally from this technique. We therefore developed a new injection procedure using macaques to investigate how this gene therapy method can also be applied to neutralizing antibody-positive patients (Fig. 1) [22]. To minimize interactions between neutralizing antibodies in blood and the vector, we inserted a catheter to the portal vein anterogradely from the superior mesenteric vein, after temporarily shutting the portal vein flow using a balloon, flushed the blood with saline, and injected the AAV8 vector. Using this technique, an increase in coagulation factor levels within the treatment range was observed even in monkeys that were positive for neutralizing antibodies to AAV8 [22]. The success of gene therapy for hemophilia B in humans has been a very promising topic during the past decade. However, the increase in coagulation factor levels remains insufficient, with only slight improvements observed (severe cases become moderate or mild cases) [20]. Although there will be no need for regular replacement therapy, a perioperative period and bleeding-related replacements will remain as requirements. Increasing the potency of gene transfer vectors is critical for achieving a higher therapeutic effect at lower vector doses. One way to improve vector potency is by altering the coding sequence to increase protein expression (codon optimization). To enhance the biological activity of the coagulation factor itself, a naturally occurring gain-of-function

Table 1 Summary of current gene therapy clinical trials for hemophilia that are under recruitment

Identifier

Type of AAV

Promoter

Transgene

Phase

Ages

Method

Sponsor

Locations

NCT00979238

scAAV8

LP1

hFIXco

1

NCT01687608

scAAV8

TTR

1/2

ssAAV8

HCRhAAT

Peripheral vein infusion Peripheral vein infusion Peripheral vein infusion

St. Jude Children’s Research Hospital Baxter Healthcare Corp. Spark Therapeutics (Pfizer Inc.)

United States United Kingdom United States

NCT01620801

hFIX Padua (R338L) co hFIXco

18 years or older 18–75 years

1/2

18 years or older

United States Australia

sc, self-complementary; ss, single stranded; LP1, apolipoprotein hepatic control region and human alpha-1-antitrypsin promoter with a modified SV40 intron; TTR, transthyretin promoter; HCRhAAT, alpha-1-antitrypsin promoter coupled to apolipoprotein hepatic control region; co, codon optimized; hFIX, human coagulation factor IX. © 2015 International Society on Thrombosis and Haemostasis

S136 T. Ohmori et al

mutation of FIX (FIX Padua [R338L]) has been used for gene therapy in animal models and a human clinical trial of patients with hemophilia B [25–28]. The expression of FIX Padua by AAV or lentiviral vectors significantly increased FIX activity in the plasma without detectable adverse effects, including thrombosis and immunogenicity [25–28]. Finding new AAV serotypes with a high specificity to human liver is imperative [29] because the therapeutic effect of this technique remains unsatisfactory in human compared with beneficial levels in the treatment range achievable in mice. Advances in human gene therapies for hemophilia using AAV8 have not been applied to patients with hemophilia A despite its significantly larger patient population. One reason is that the FVIII gene is large (7.0 kbp), generally exceeding the allowable range for insertion into AAVs. For that reason, a method to insert FVIII cDNA that lacks the B domain instead of the fulllength gene is generally used. It is also difficult to obtain a sufficiently high treatment range for FVIII levels even using the same amount of AAV vector, compared with FIX, despite the blood molarity level of FVIII being lower than that of FIX. McIntosh et al. [30] recently reported the successful application of AAV8 technology to hemophilia A in monkeys. They obtained a treatment range of coagulation factor expression using a codonoptimized FVIII gene with 6 glycosylation triplets from the B domain rather than a wild-type gene [30]. The same group plans to initiate another clinical trial in patients with hemophilia A in 2015 [31]. Good manufacturing practise production of AAV8 for hemophilia B clinical trials reportedly requires a total of 432 independent 10stack culture chambers to produce a total of ~2 9 1015 vg [32]. As the length of the FVIII gene compared with the FIX gene significantly impairs the efficacy of vector production (unpublished data), an efficient high-yield vector production system to allow gene therapy for hemophilia A is warranted. GlyberaÒ, the first commercially available AAV vector, has already been approved by the European Commission to treat lipoprotein lipase deficiency [33]. GlyberaÒ is produced using a baculovirus expression system. The production system is particularly appropriate for the development of gene therapies to treat diseases requiring larger vector doses, including hemophilia [34]. Lentiviral vectors might be an alternative for transducing coagulation factor genes into hepatocytes by the direct injection of the vector. Off-target expression in hematopoietic cells, which lead to cellular immune responses, could be abrogated by the incorporation of target sequences against miR-142-3p, a microRNA for hematopoietic cells. Liver-directed gene therapy using either integration-competent or even integration-defective lentiviral vectors containing miR-142-3p target sequences sustained gene expression in hemophilia B mice [35,36]. Pseudotyping of the vector by the GP64 envelope glyco-

protein further improved the restricted expression of the transgene in the liver, thereby resulting in stable FVIII expression in hemophilia A mice [37]. Cell therapy

Cell therapy refers to a specific hemophilia treatment that involves the induction of cells to express a coagulation factor ex vivo, followed by transplantation of the transduced cells. An advantage of cell therapy is the avoidance of systemic dissemination of vectors thereby alleviating the risk for vector-associated side effects. Technologies have been established for inducing a variety of cells, including fibroblasts, adipocytes, mesenchymal stem cells (MSCs), endothelial cells, and hepatocytes, to express a coagulation factor using a viral vector or a plasmid vector. Several studies have detected an increase in coagulation factor levels after injection of cells into various animal models. Roth et al. [38] reported the first clinical trial for cell therapy using human hemophilia patients, in which fibroblasts expressing the FVIII gene using a plasmid vector were transplanted to the omentum. In that study, 1–4 9 108 cells were transplanted to each of six patients with severe hemophilia A. In the high-dose group, FVIII activity increased by around 1–5%, but no long-term expression was detected [38]. Problems in cell transfer include the difficulty in maintaining coagulation factor-expressing transplanted cells for an extended period of time. Therefore, efforts have focused on identifying the optimal cell type and developing cell transplant techniques for cell therapies. From the perspective of the long-term maintenance of cells, the use of somatic stem cells may also show some potential. Previous studies showed that transplanting hematopoietic stem cells induced to express FVIII or FIX by retroviral or lentiviral vector gene transfer could express coagulation factors in blood cells for long periods and improve the phenotype of hemophilia mice [39–41]. In addition, peripheral blood cells such as platelets and red blood cells have also gained attention as coagulation factor-producing cells [42–44]. Platelets exerted pleiotropic functions in assisting and modulating thrombosis, vascular integrity, and inflammatory reaction by a release reaction triggered by activation during a primary hemostatic thrombus. Ectopically expressed FVIII in platelets in transgenic mice was localized within a-granules and was released at the site of vascular injury [44,45]. Because platelet-derived FVIII appears to be resistant to the presence of circulating inhibitors [45,46], it might be an attractive strategy for the treatment of hemophilia A. We transduced hematopoietic stem cells with a lentiviral vector that expressed a target protein under the control of a platelet-/megakaryocyte-specific promoter and transplanted the cells into recipient mice [47,48]. The induction of platelets to express FVIII or activated coagulation factor VII using this system improved the bleeding tendency © 2015 International Society on Thrombosis and Haemostasis

Novel gene and cell therapy for hemophilia S137

in hemophilia A mice [48–51]. This technique reportedly improved the bleeding tendency of a canine model of hemophilia A [52]. However, this approach may be less effective compared with the intravenous infusion of FVIII for complete hemostasis [53] and requires preconditioning using potentially toxic agents for transplantation. Because FVIII is stored in Weibel–Palade bodies of endothelial cells in a manner dependent on von Willebrand factor [54], and these cells are likely to physiologically produce FVIII [55], various studies have used endothelial cells as FVIII-producing cells. Early investigations indicated that cell transplantation to the omentum or the intraportal transplant of non-gene-transferred liver sinusoidal endothelial cells improved the phenotype of hemophilia A mice [56,57]. Breeding with a transgenic mouse expressing FVIII downstream of the Tie-2 promoter also increased FVIII activity and improved the bleeding tendency of hemophilia A mice [58]. With regard to their application for gene therapy, peripheral blood outgrowth endothelial cells (BOECs) have a number of potential advantages for gene transfer. BOECs can be obtained from peripheral blood cells and can be expanded as autologous cells without limit. The systemic administration of BOECs expressing FVIII achieved therapeutic levels of FVIII in NOD/SCID mice [59]. The subcutaneous implantation of BOECs transduced with a lentiviral vector also sustained therapeutic levels of FVIII in hemophilia A mice [60,61]. Recently, Ozelo et al. [62] reported that the implantation of transduced BOECs expressing FVIII into the omentum resulted in a significant increase in FVIII antigen for up to 1 year in a canine model of hemophilia A. Recent advances in induced pluripotent stem (iPS) cell research have also gained attention in the field of hemophilia therapy. iPS cells are an attractive cell source for cell therapy because the number of genetically identical autologous cells that would prevent immune rejection is effectively propagated. Xu et al. [63] reported the first evidence of hemophilia treatment by iPS cells; intraportal injection of iPS cells after their differentiation into endothelial cells improved the bleeding tendency of hemophilia A mice. We successfully transduced iPS cells to produce functional FVIII using a lentiviral vector and subcutaneous transplantation of these iPS cells to nude mice resulted in a treatment range for FVIII levels according to teratoma formation [64]. A gene-editing technique created a chromosomal inversion of the F8 gene in human iPS cells and further reversed the mutation using the same technique [65]. However, iPS cell-based therapy might not be a realistic therapeutic option at present, because more conventional gene and cell therapy approaches have been developed. The development of both cell types and cell transfer technology has remarkably improved the duration of cellular expression and longevity of the cells after transplantation. MSCs were maintained for an extended period of © 2015 International Society on Thrombosis and Haemostasis

time using 3D scaffolds [66]. In addition, the transplantation of MSCs expressing a coagulation factor using cell sheet technology allowed long-term gene expression and cell maintenance [67], and transplantation of a BOEC sheet expressing FVIII to hemophilia A mice maintained a treatment range of coagulation factor for nearly 1 year [60]. It is important to develop less invasive transplantation procedures for hemophilia cell therapy, because efficient replacement therapy has been established. Despite advances in treatment and the delivery of comprehensive care, the presence of hemophilic arthropathy caused by repetitive intra-articular bleeding is one of the most important factors for QOL in patients with severe hemophilia. In addition, we occasionally experience that an increase in coagulation factor levels fails to suppress joint bleeding in cases of developed hemophilic arthropathy. We have developed a new cell therapy technique in which MSCs expressing FVIII using a lentiviral vector are injected to the knee joint cavity by arthrocentesis (Fig. 2) [68]. This procedure suppressed acute joint bleeding and resultant hemophilic arthropathy in FVIII-deficient mice [68]. It was also reported that the direct injection of an AAV vector expressing FIX into the joint space improved hemophilic arthropathy in FIX-deficient mice [69]. There are several advantages of cell-based therapy compared with the direct injection of vector for the treatment of hemophilic arthropathy. MSCs can differentiate into chondrocytes and osteoblasts, modulate immune responses, and produce a number of bioactive mediators (Fig 2). These pleiotropic functions of MSCs can be exploited therapeutically to repair degenerative joints. Because intra-articular injection is a minimally invasive procedure, this procedure should become a clinically applicable approach to treat blood-induced joint disease in patients with hemophilia. To develop gene and cell therapies for hemophilia, we have used several animal models, including gene-deficient mice by gene targeting and spontaneous disease animals. Although studies using non-human primates are essential to confirm safety, we cannot analyze the actual therapeutic effect of the therapy because non-human primate models of hemophilia have not been discovered (or developed). Spontaneous hemophilia models of dogs and sheep have been reported [70,71] and employed for validation of therapeutic effects as a species between mice and humans. We successfully produced hemophilia A pigs using gene targeting and cloning technologies [72]. Hemophilia pigs could be an attractive model for human disease because the porcine blood coagulation system is very similar to that in humans [73], and porcine coagulation factor preparations have been used for hemophilia with inhibitors [74]. Hemophilia A pigs, which differ from hemophilia dogs, develop remarkable arthropathy because of repeated joint bleeding just after birth [72]. Although we have not bred them because of the severe hemorrhagic

S138 T. Ohmori et al A

B

LTR

FVIII gene

LTR FVIII expression Mesenchymal stem cell (MSC)

Lentiviral vector

Surface markers CD45–,CD34– CD44, CD51, CD105, CD29 c-Kit, Thy-1, Stro-1

Self-renewal MSC expressing Adipocyte

Osteoblasts

coagulation factor

Osteochondro progenitor cell

Myogenic precursors Hemostasis Regeneration

Arthrocentesis

Chondroblast Osteocyte

Immune modulation Chondrocyte Fig. 2. Local cell-based therapy for hemophilic arthropathy by MSCs expressing coagulation factor. (A) The therapeutic gene (FVIII or FIX) is packaged into a lentiviral vector that efficiently transduces MSCs to produce FVIII or FIX. MSC can self-renew and differentiate into a number of lineage-specific cells. MSCs expressing coagulation factor have the significant potential to treat hemophilic arthropathy (expression of coagulation factor, regenerative effects, and modulation of immune responses and inflammation). (B) Transduced MSCs expressing coagulation factor are transplanted into the joint space by arthrocentesis.

symptoms, we intend to establish a strategy to examine the effects of gene therapies and cell therapies using hemophilia A pigs prior to human clinical trials. Disadvantages of coagulation factor inhibitors Coagulation factor concentrates are recognized as foreign substances in patients with severe hemophilia, and ~25% of hemophilia A patients treated with replacement therapy develop neutralizing antibodies (inhibitors). It is often difficult to manage bleeding complications because the therapeutic effect of the preparation is then negated. The occurrence of inhibitors against coagulation factors is considered a serious barrier for various gene and cell therapies to hemophilia in a number of animal models. The use of viral vectors and cell matrix may act as an adjuvant to initiate anti-FVIII immune responses [62,75]. Transient immunosuppression or pretreatment with FVIII might avoid anti-FVIII immune responses in mice but was not observed in a canine model of hemophilia [62]. On the other hand, induction of the liver by AAV8 with a liver-specific promoter decreased the emergence of inhibitors [8,76]. Interestingly, AAV8-mediated gene transfer to hemophilia B mice with preexisting inhibitors caused a rapid elimination of inhibitors [77]. High levels of FIX protein suppressed memory B cells and increased regulatory T-cell induction, suggesting the presence of a variety of mechanisms to inhibit inhibitor production [77]. Immune tolerance was also confirmed in hemophilia B dogs treated with an AAV8 expressing FIX Padua [27]. In addition, the lentiviral vector-mediated liver expression

of FIX effectively eradicated FIX inhibitors in hemophilia B mice [78]. In clinical practise, regular injection of a preparation called immune tolerance induction (ITI) eliminated inhibitors developed by patients with hemophilia. Gene therapies targeting the liver would be even more interesting as new therapies replacing ITI, if they have similar inhibitor elimination effects in human clinical trials. Future types of gene therapy for hemophilia An ideal hemophilia gene therapy would ‘repair’ the abnormal coagulation factor gene at the genome DNA level. As genome-editing technologies such as the zinc finger nuclease, TALEN, and CRISPR/Cas9 systems have been significantly improved, future applications of these technologies are anticipated. AAV8-mediated expression of zinc finger nuclease corrected genetic abnormalities of hemophilia B mice and improved the bleeding tendency [79,80]. The expression of nuclease may induce unnecessary immune reactions and off-target dsDNA breaks. Barzel et al. [81] recently reported site-specific genome editing by an AAV8 vector without the expression of nucleases. FIX cDNA was successfully integrated into ~0.5% of Alb alleles in hepatocytes [81]. The genome in iPS cells from a hemophilic model was repaired at the cellular level using TALEN [65]. However, gene transfer is likely to be preferable to express high levels of a target protein at present, because the target protein in gene-editing cells could be derived from one or two copies of the gene. It should also be noted that the target protein can only be © 2015 International Society on Thrombosis and Haemostasis

Novel gene and cell therapy for hemophilia S139

produced if the gene-corrected cells physiologically produce the target protein. The efficacy of genome editing in vivo and methods to obtain high numbers of cells that physiologically express coagulation factors from genomeediting stem cells must be improved.

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Hemophilia has long been considered to be an attractive model for gene therapy because small amounts of the deficient protein can improve the bleeding phenotype. Improvements made to vectors and administration methods by researchers over approximately 20 years have at long last brought us to the point of being able to actually provide treatments to patients with hemophilia. To improve the QOL for the majority of patients with hemophilia, the development of gene therapies for hemophilia A and novel techniques that result in higher therapeutic effects are imperative. Furthermore, we should identify how to effectively translate new results from the laboratory bench to the clinic, while constantly taking safety, invasiveness, economic justifications, and therapeutic effects into consideration.

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Acknowledgements We would like to thank Drs. J. Mimuro, Y. Kashiwakura, A. Ishiwata, A. Yasumoto, A. Sakata, S. Hishikawa, T. Ikemoto, S. Madoiwa, and S. Muramatsu (Jichi Medical University) for their dedicated support for the experiments and helpful discussions. We also thank T. Nakamikawa, T. Aoki, M. Kishimoto, Y. Sutoh, N. Ito, and M. Ito (Jichi Medical University) for their excellent technical assistance. This study was supported by a Grant from Health, Labour and Science for Research on HIV/ AIDS and Grant-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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Disclosure of Conflict of Interests T. Ohmori, K. Ozawa, H. Mizukami, and Y. Sakata report grants from The Ministry of Health, Labour and Science during the conduct of the study. T. Ohmori reports grants from Bayer LCC outside the submitted work. H. Mizukami reports grants from Ministry of Education, Culture, Sports, Science and Technology during the conduct of the study. S. Nishimura has nothing to disclose.

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