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1 Hemophilia A

1.1 Introduction

Hemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of the coagulation protein, factor (F)VIII. FVIII plays an essential role in the intrinsic pathway during blood coagulation, and the genetic defect causes a decreased and delayed generation of thrombin leading to disordered clot formation and a bleeding diathesis. The clinical abnormality is associated with bleeding episodes affecting joints, muscles, and soft tissue. Repeated hemorrhage results in chronic arthropathy finally associated with loss of joint movement. Both hemophilia A and hemophilia B (discussed below) are inherited as X-linked recessive traits; i.e., male individuals are affected and female individuals are asymptomatic or mildly affected carriers.

1.2 FVIII Protein and FVIII Gene (F8) (Fig. 9.1)

The FVIII protein was first isolated and purified over 30 years ago [1], and the human FVIII gene (F8) was initially cloned in 1984 [2, 3]. F8 is located in the most distal band (Xq28) of the long arm of the X chromosome. The gene contains 25 introns and 26 exons, 24 of which vary in length from 69 to 262 base pairs (bp), while the two larger exons 14 and 26 contain 3106 and 1958 bp, respectively. Most of exon 26 consists of a 3′ untranslated sequence, however, and exon 14, therefore, bears by far the largest exonic coding sequence, principally that of the B domain. Spliced FVIII mRNA is approximately 9 kb in length and predicts a precursor protein of 2351 amino acids.

Fig. 9.1
figure 1

Linear representation of F8, the structure of FVIII, and its cleavage site. The 26 exons (above) and domain organization (below) of FVIII based on amino acid homology. Activation of FVIII leads to release of the B and a3 domains. In the activated FVIII heterotrimer, the A1 and A3 domains retain the metal ion-mediated interaction, and the stable A1/A3-C1-C2 dimer is weakly associated with the A2 subunit through electrostatic interactions. Either spontaneous dissociation of A2 or proteolysis of FVIIIa results in loss of its function. Th thrombin

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Removal of a 19 peptide secretory leader sequence of FVIII results in the mature sequence of 2332 amino acids. The FVIII molecule consists of three homologous A domains, a B domain, and two homologous C domains structurally in the order A1-A2-B-A3-C1-C2. Prior to secretion, FVIII is processed into a series of metal ion-linked heterodimers produced by cleavage at the B-A3 junction together with a number of additional cleavages within the B domain. FVIII is highly sensitive to proteolytic processing after secretion, and only a small fraction of circulating FVIII retains a single-chain form. Most consist of heavy chains of variable length (including sequences of the A1, A2, and B domains) linked ion covalently to light chains composed of the A3, C1, and C2 domains [4]. Limited proteolysis at Arg372 and Arg1689 by thrombin or FXa generates cofactor activity (FVIIIa). Expression of recombinant (r)FVIII lacking the entire length of the B domain has confirmed, however, that this domain is dispensable for the activation and procoagulant function of the protein. The cofactor function of FVIIIa accelerates the activation of FX by activated FIX (FIXa) on a suitable phospholipid surface, thus amplifying the clotting stimulus by several orders of magnitude. Specific proteolytic cleavages between the FVIII domains both activate and inactivate the cofactor [5]. The active form (FVIIIa) cation-dependently bonds to the A3-C1-C2 light chain together with the heavy chain held by electrostatic association between the A1 and A2 domains. FVIIIa is highly unstable owing to spontaneous dissociation of the A2 subunit and proteolytic inactivation of FVIIIa through cleavage at Arg336 by activated protein C (APC) and FXa. An additional cleavage site for APC at Arg562 in the A2 subunit results in complete inactivation of FVIIIa [5].

For convenience, F8 defects associated with hemophilia A may be divided into several categories: (1) gross gene rearrangements, (2) deletions or insertions of genetic sequence of a size varying from one base pair up to the entire gene, and (3) single DNA base substitutions resulting in either amino acid replacement (“missense”), premature peptide chain termination (“nonsense” or stop mutations), or mRNA splicing defects. More than 2500 unique molecular defects in F8 have been described and are included in the worldwide Factor VIII Variant Database (http://www.factorviii-db.org). Molecular characterization of patients with hemophilia A is dependent initially upon the analysis of intron 22 and intron 1 in F8 [6, 7]. The single, clinically most important defect is a gene rearrangement (an inversion) involving intron 22 (Fig. 9.2), which is evident in approximately 50% of all severe disease cases [6]. If genetic variations in intron 1 or intron 22 are not detected, full F8 mutation screening is generally performed by direct sequencing, covering all exons, intron-exon boundaries, and the promoter region. Genetic variations have not been identified in 2–18% of patients [8]. Consistent gene defects in patients with mild and moderate FVIII deficiencies are not common, and full mutation analysis of F8 by direct sequencing is usually necessary in these patients.

Fig. 9.2
figure 2

Simplified representation of the intron 22 inversion. Recombination between homologous sequences in intron 22 and ~400 kb telomeric to the gene leads to separation of exons 1–22 from exons 23–26

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1.3 FVIII Inhibitors

The presence of neutralizing alloantibodies (inhibitors) to therapeutic FVIII is a serious complication in hemophilia A, occurring in approximately 30% of patients classed as severe [9]. In those individuals, the inhibitors usually develop during the first 20–30 days of exposure and appear to result from a multi-causal immune response involving both patient-related and treatment-related factors. A gene defect is thought to contribute to about 40% of the risk of inhibitor formation, although the immunology of inhibitor development remains to be fully understood. Large deletions and nonsense mutations seem to present the highest risk, whereas missense and splicing mutations mediate the lowest risk [10]. The most common acquired risk factor is the intensity of exposure to FVIII concentrate [11]. In non-severe hemophilia A patients and different from severe hemophilia A, inhibitors usually arise when the immune system is under intense stimulation or when exposure to FVIII concentrates is unusually high, independent of exposure days, in particular in the postoperative period [12, 13]. Mutations leading to an abnormal conformation of FVIII are associated with a high risk of inhibitor development in mild hemophilia A [13], especially in those patients with mutations in the A2 and A3C1C2 domains (e.g., Arg593Cys and Arg2150His, respectively) [14, 15].

1.4 Treatment

Comprehensive guidelines for the treatment of hemophilia were endorsed as high standard by the International Society on Thrombosis and Hemostasis (ISTH) in 2012 (https://www.isth.org/?page=Published_Guidance) and were published in detail in 2013 by the World Federation of Hemophilia (WFH) working group [16]. The primary aim of hemophilia care is to prevent and treat hemorrhage using FVIII therapeutic products. An integrated support system, called comprehensive care, is highly recommended and includes specific therapy for prophylaxis or acute bleeding and also treatment for complications and drug-related side effects [17]. As noted above, however, the development of inhibitors presents many difficulties for the use of FVIII concentrates, and long-term complications, such as joint destruction with the need for synovectomy or joint replacement, together with infections transmitted by clotting factor concentrates are also important issues for maximally effective clinical management. Treatment for hemophilia A may not be straightforward, therefore, and depends overall on the severity of the FVIII defect, the presence of inhibitors, available resources, and clinical complications.

1.4.1 Replacement Therapy

Acute bleeding should be treated immediately with FVIII concentrate. Home infusion therapy has made it possible for patients to treat themselves quickly just after bleeding (and prophylactically). In the absence of an inhibitor, each unit of intravenously infused FVIII per kilogram of body weight will raise the plasma FVIII level by approximately 2 IU/dL [17]. The half-life of FVIII is 8–12 h, and optimal doses and duration of treatment are dependent on the site of bleeding and individual patient pharmacokinetics (Table 9.1) [16].

Table 9.1 Summary of the desired plasma factor peak levels and duration of administration (modification of [16])
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Hemarthrosis is a common complication in hemophilia patients, and repeated hemorrhage often occurs into the same joint, commonly called a target joint. In addition, compartment syndrome is a painful and potentially serious condition caused by bleeding or swelling within an enclosed bundle of muscles, and this poses a particular risk in hemophilia when soft tissue hemorrhage occurs [18]. The WFH strongly recommends the use of viral-inactivated plasma-derived (pd) or recombinant (r)FVIII concentrates in preference to cryoprecipitate or fresh frozen plasma for the treatment of hemophilia A [16]. rFVIII products especially have the theoretical advantage of being free from blood-borne pathogens. Recent research has also focused on inhibitor risk in patients treated with pd-FVIII or rFVIII, but the outcomes remain controversial. The Research of Determinants of Inhibitor Development (RODIN) study group has comprehensively examined the association between FVIII products and inhibitor development, and the only significant result determined was an unexpectedly higher risk of inhibitor development using second-generation full-length recombinant products compared with third-generation rFVIII products [19]. A more recent report [20] indicated that the risk of developing an inhibitor when using rFVIII products in previously untreated patients (PUPs) was significantly higher than when using pd-FVIII concentrates that contain von Willebrand factor (VWF), although this finding was not totally consistent with the earlier RODIN findings. Further research on inhibitor development is warranted to elucidate the precise immunogenicity of rFVIII.

Several clinical trials have been undertaken to investigate the use of rFVIII bioengineered to extended half-life (EHL), including Fc-fusion, PEGylated, and glycoPEGylated proteins. Fc-fusion proteins are designed to be recycled into the circulation through pH-dependent binding to neonatal Fc receptors (FcRn) that delay lysosomal degradation [21], and one such preparation, rFVIIIFc, is currently available [22]. Moreover, other EHL-modified rFVIII products, including BAX 855 (PEGylation) [23] and N8-GP (glycoPEGylation) [24], have also completed Phase 3 trials. All of these novel products appeared to face to a “ceiling effect,” however, and half-lives appear to be limited to approximately 1.5 to 1.6-fold of unmodified FVIII. This limitation could be related to the absence of VWF, the career protein.

For mild or moderately severe FVIII deficient patients, desmopressin (DDAVP) is an alternative option that rapidly increases FVIII:C by three- to sixfold from baseline levels [25]. DDAVP is generally administered intravenously at a dose of 0.3 μg/kg over 20–30 min and mediates elevated FVIII in 30–60 min that persists for 6–12 h. Tachyphylaxis occurs after repeated administration, however, due to depletion of endothelial stores [26].

1.4.2 Prophylaxis

Strategies for prophylaxis replacement therapy are based on evidence that moderately severe patients who have FVIII levels more than 1 IU/dL experience many fewer spontaneous bleeding events and have much better long-term joint function than those patients classed as severe [27]. In addition, randomized control studies have also convincingly demonstrated that prophylaxis with rFVIII can prevent joint damage and decrease the frequency of joint and other hemorrhages in young boys with severe hemophilia A [28]. Furthermore, even using prophylactic regimens that resulted in trough FVIII levels <1 IU/dL, episodes of joint hemorrhage were significantly decreased [29]. A range of different prophylactic protocols have been proposed, including 10–20 IU/kg or 25–40 IU/kg per dose 2–3 times a week, once a week for very young children [16], or temporal prophylaxis just before physical activity associated with a higher risk of injury [30]. It is clear, however, that the pharmacokinetics of FVIII is diverse, and the lowest effective level of FVIII should be determined individually for each patient considered for primary or secondary prophylaxis.

1.4.3 Adjunctive Therapy

Replacement therapy is the primary hemostatic treatment in patients with hemophilia. In addition, the concept of “RICE” (rest, ice, compression, and elevation) plays an important role as a first aid for acute bleeding in muscles and joints. Antifibrinolytics that enhance clot stability, such as tranexamic acid and epsilon aminocaproic acid, are also useful for mucosal and oral bleeding including dental extraction [31]. Tranexamic acid is commonly used at 25 mg/kg per dose every 6–8 h intravenously or orally. Physical fitness and the development of strong muscles are also recommended to protect joints. An appropriate exercise lifestyle should be encouraged, especially for children, to avoid overweight or obesity [32]. Noncontact sports such as swimming should be encouraged. In the absence of thoroughly controlled prophylaxis, high-contact sports including football or high-velocity activities including skiing should be avoided due to the obvious potential for life-threatening injuries.

1.4.4 Inhibitors

1.4.4.1 Hemostatic Management

Anti-FVIII alloantibodies occur about 30% of patients with hemophilia A and remain a challenging clinical problem in congenital FVIII deficiency [9]. Acute bleeding in patients with inhibitors may be treated effectively with infusions of concentrates which “bypass” the intrinsic coagulation pathway. Two such bypassing agents are available, activated prothrombin complex concentrate (aPCC) and recombinant FVIIa (rFVIIa), and both are safe and efficient almost equally [33], despite the differences in their content, dosing, and mode of action. Preference often seems to be dependent on individual patient circumstances, and combined use of both drugs is sometimes required [34]. Prophylaxis with aPCC in both adult and pediatric patients with inhibitors has been reported to be effective in preventing joint bleeds and longer-term orthopedic damage [35]. This type of treatment could be an important alternative choice for inhibitor patients who have failed or are ineligible for ITI.

1.4.4.2 Immune Tolerance Induction (ITI)

Immune tolerance induction (ITI) utilizing frequent and long-term administration of FVIII-containing concentrates is presently the only strategy proven to eradicate persistent and high-responding inhibitors. Retrospective cohort studies have demonstrated similarly high success rates (60–80%) with various different ITI protocols [36]. Evidence from large-scale and methodologically rigorous trials is lacking, however, and the optimal ITI regimen and predictors of ITI outcome remain to be firmly established. A number of patient-related or treatment-related features have been analyzed as predictors of ITI outcome including the time interval to success, inhibitor titer prior to ITI start, historical inhibitor peak titer, time interval between inhibitor diagnosis and ITI start, and FVIII dose regimen [36, 37]. Meta-analyses of data from both the International Immune Tolerance Registry (IITR) [38] and the North American Immune Tolerance Registry (NAITR) [39] have indicated, however, that only a historical peak inhibitor titer <50 BU/mL and an inhibitor titer <10 BU/mL at the time of ITI start are reasonable predictors of success. In this context, an international ITI study in hemophilia A inhibitor patients with good risk factors indicated that the use of both high-dose FVIII (200 IU/kg per day) and low-dose FVIII (50 IU/kg every other day) were equally effective (approximately 70%), although the low-dose group had more bleeding events during the period of study [40].

1.4.5 Future Prospective Therapy for Hemophilia A

Although the treatment of hemophilia has advanced considerably in the last quarter century, several elegant studies have been described in an attempt to resolve remaining challenges. For example, the concept of rebalancing hemostasis by moderating natural anticoagulants rather than replacing FVIII is being explored. Hence, targeting antithrombin with small RNA interference agents [41] and monoclonal antibodies against tissue factor pathway inhibitor [42] are now undergoing clinical trials. In addition a bispecific antibody (anti-FIXa/FX antibody) designed to mimic FVIIIa has been developed [43], and Phase 1/2 clinical trials using this antibody (ACE910, emicizumab) has been completed. Prophylactic treatment with weekly subcutaneous administration was well tolerated and reduced the number of bleeding episodes in hemophilia A patients both with and without inhibitors [44]. The advantages of these novel therapeutic agents include the potential for less frequent administration (once per 1–4 weeks), subcutaneous injections (preferable for younger children), eliminating the risk of developing anaphylactic inhibitors, and effectiveness regardless of the presence of inhibitors.

2 Hemophilia B

2.1 Introduction

Hemophilia B is a bleeding disorder caused by mutations in the FIX gene (F9) leading to deficient or defective FIX, a pivotal serine protease in the intrinsic coagulation cascade. Hemophilia B (one per 30,000 male births) was first distinguished from the more prevalent hemophilia A (one per 5000 male births) in 1952 when it was noted that mixing plasma samples from two patients diagnosed with “hemophilia” mutually corrected the prolonged in vitro clotting times.

2.2 FIX Gene (F9) and FIX Protein

The F9 coding sequence was partially described in the early 1980s and the full sequence was published in 1985 [45]. F9 is located in the X chromosome at Xq27.1 and spans 33 kbp, including seven introns and eight exons that are transcribed into a 2802-bp mRNA (Fig. 9.3). Exons I and II code for a prepro leader sequence and a Gla domain, whereas exons IV and V each code for EGF domains. The last three exons code for the activation peptide and the catalytic domain. The 5′ sequence includes a LINE-1 element, and a major transcription initiation site appears to be at or near an adenine nucleotide at −176. The Factor IX Variant Database (http://www.factorix.org) identifies many F9 mutations that can be attributed to changes in the CpG nucleotide (from CG to TG or CA). The majority of defects (64%) are point mutations, followed by small insertions or deletions (18%), splice site mutations (9%), large deletions or insertions (6%), and mutations in the promoter region. Large deletions, covering several tens of kb or an entire loss of total FIX, are thought to be associated with the development of anti-FIX inhibitors [46]. The F9 Leyden phenotype is distinctively characterized by a severe deficiency at birth, but with FIX levels that start to increase during the second decade of life and achieve near normality during the third decade [47]. Normal or borderline-low levels are subsequently maintained. The pathogenesis of this unique abnormality appears to center on the promoter map where one site is partly overlapped by an androgen response element which regulates transcription levels following the hormonal changes that occur during puberty.

Fig. 9.3
figure 3

Structure of F9 and FIX peptide

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FIX is a single-chain glycoprotein composed of 415 amino acids [48]. It is a vitamin K-dependent protein containing 12 residues of γ-carboxyglutamic acid (Gla domain) located in the amino-terminal region of the molecule. This region of the protein contains two potential epidermal growth factor (EGF)-like domains, an activated peptide and a catalytic domain. Cotranslational translocation into the lumen of the endoplasmic reticulum (ER) occurs concomitantly with signal peptide cleavage. The addition of core high-mannose oligosaccharides to the polypeptide in the ER is followed by glucose trimming of the N-linked oligosaccharide structures and γ-carboxylation of the 12 amino-terminal glutamic acid residues. Upon transit into the Golgi compartment, additional modifications occur, which include (1) complex modification of N-linked oligosaccharides; (2) tyrosine sulfation at Tyr155; (3) Ser/Thr glycosylation at residues Ser61 and Ser53, as well as several Thr residues within the activation peptide; and (4) cleavage of the propeptide. In addition, FIX isolated from human plasma is phosphorylated at Ser158 within the activation peptide. A majority of the modifications within FIX occur within the activation peptide and may regulate activation of FIX. Appropriate γ-carboxylation and propeptide cleavage are essential for the functional secretion and activity of secreted FIX. During the coagulation cascade, FIX is cleaved at two internal peptide bonds (Ala145-Ala, Arg180-Val), resulting in the formation of a light chain and a heavy chain, held together by a disulfide bond. During the conversion to FIXa, an activation glycopeptide of 35 amino acid residues is released.

2.3 FIX Inhibitor

FIX inhibitors are relatively uncommon, being detected in 1–3% of patients with hemophilia B. Genetic factors play a major role, and specific mutations in F9 appear to be associated with an increased incidence of inhibitor development. Large deletions and frameshift mutations leading to the loss of coding information are much more likely to be associated with inhibitor development. It has been reported that large deletions account for only 1–3% of all hemophilia B patients but account for 50% of inhibitor patients [49]. Thorland et al. [50] genotyped eight unrelated hemophilia B patients who had anaphylactic reactions to FlX-containing products, as well as an inhibitor, and compared their gene mutations with those of 550 other hemophilia B patients recorded in the hemophilia B database. Individuals with complete gene deletions were found to be at greatest risk for anaphylaxis. Anaphylaxis occurred more frequently in families with null mutations (large deletions, frameshift, or nonsense mutations) than in those with missense mutations. In addition, Astermark et al. [51] have recently reviewed the potential role of immune response genes, environmental factors, and other concurrent immune system challenges, noting especially the occurrence of a microsatellite polymorphism in the promoter region of the IL-10 gene which was previously shown to be highly associated with inhibitor formation in hemophilia A.

2.4 Treatment

The basic treatment principles described in the hemophilia A section above, including RICE and adjuvant therapy, can be also applied to hemophilia B. The only difference is that DDAVP does not affect FIX levels and has no value for the treatment of hemophilia B. As in hemophilia A, the main treatment for hemophilia B is replacement therapy, using FIX products either on demand or prophylactically. A number of new agents are being developed and are described below. The World Federation of Hemophilia Treatment working group has published detailed recommendations of an up-to-date list of various products currently available (see http://www.wfh.org).

2.4.1 FIX Products

Clotting factor concentrates are administrated intravenously to correct the FIX deficiency. Guidelines for target peak and trough levels are the same as in hemophilia A. Available FIX products include specific FIX concentrates that may be plasma derived or recombinant and multifactor concentrates that contain FII, FVII, FIX, and FX, known as prothrombin complex concentrates. The use of pure FIX products is always preferred. Similar to the novel FVIII proteins discussed above, extended half-life (EHL) products including fusion and PEGylated FIX have been recently introduced. The mean terminal half-life of the fusion proteins, rFIX-Fc and FIX-albumin (rIX-FP), appeared to be extended to 82.1 h [52] and 91.5 h [53], respectively, approximately 3–4 times that of conventional FIX therapy. In addition, the site-directed glycoPEGylated protein (N9-GP) that covalently binds to FIX demonstrated a mean half-life of 93 h [54]. The Fc-fusion FIX protein is now commercially available, and the other EHL proteins are completing Phase 3 clinical trials.

2.4.2 Dosing (Table 9.1)

In vivo recovery of FIX (post-dose activity levels) is not as good as with FVIII products. FIX reversibly binds to endothelial cells and diffuses into extravascular tissues due to the smaller size of the molecule compared to FVIII. In principle, therefore, although 1 IU/kg of infused FIX could be expected to raise circulating levels by 1 IU/dL, the lower in vivo recovery moderates the response, and 1 IU/kg of FIX generally increases plasma FIX activity by approximately 0.7–0.8 IU/dL [55]. Both pd-FIX and rFIX have a longer half-life (16–17 h) [56] than FVIII (8–12 h) [57], however, and the dosing interval for FIX therapy is commonly 24 h with laboratory monitoring of FIX:C. EHL FIX products have three- to sixfold longer half-lives than unmodified materials, potentially reducing the frequency of the dosing. Peak levels and trough levels should be carefully assessed, however, to ensure the maintenance of adequate hemostasis in each individual’s circumstance. EHL products may not always be consistent in this respect, and further studies are required to prepare guidelines of the most appropriate use of these modern therapies.

2.4.3 Prophylaxis

In common with hemophilia A, prophylaxis with the regular infusions of clotting factor to prevent bleeding and minimize the risk of joint destruction in advance is a highly effective treatment in patients with hemophilia B. The original concept in both hemophilia A and hemophilia B was to maintain levels of FVIII:C or FIX:C above 1 IU/dL, in order to ameliorate the phenotype from severe to moderate, and in particular to preserve joint function. Most prophylactic regimens use fixed doses, commonly administration of 25–40 IU/kg of FX twice a week. This approach is now being adapted, however, and specific dosing protocols are being adjusted to the requirements of individual patients.

2.4.4 Anaphylaxis and Inhibitors

Anaphylaxis and other allergic reactions are closely associated with FIX inhibitor development. Up to 50% of hemophilia B patients with inhibitors may have severe allergic reactions to FIX administration, including anaphylaxis. No evidence is available, however, to implicate any particular type of product as the primary cause of anaphylaxis and inhibitor development. These clinically severe events that occur after very few exposures to FIX have led to the important recommendation that all infants and small children with severe hemophilia B, particularly those with a family history and/or with genetic defects predisposed to inhibitor development, should be closely monitored over their initial 10–20 exposure day to FIX concentrates in a facility equipped to treat anaphylactic shock. Reactions can occur later, but may be less severe [58, 59]. As discussed above, genotyping can provide vital data to identify any defect in F9 (e.g., large gene deletions) known to be associated with a high risk of anaphylaxis and inhibitor development. Patients with an inhibitor of FIX and a history of anaphylaxis to FIX should be treated with rFVIIa.

2.4.5 ITI Therapy

ITI therapy is successful in the majority of patients with hemophilia A with inhibitor (approximately 70–80%), but the success rate is much lower (approximately 40%) in patients with hemophilia B. In addition, ITI in hemophilia B inhibitor patients with a history of severe allergic reactions to FIX may be associated with the development of nephrotic syndrome [38]. Those who develop nephrotic syndrome are often steroid resistant and ITI therapy is discontinued. Alternatively, ITI in conjunction with immunosuppression has been reported to be successful [60].

2.4.6 Future Prospective Therapy

Hemophilia B appears to offer an ideal model for gene therapy. The clinical disorder is a consequence of defects in the well-characterized FIX gene, with minimal influence from other genetic modifiers. In this context, a novel serotype 8 pseudotyped, self-complementary AAV (AAV8) vector expressing a codon-optimized factor IX transgene (scAAV2/8-LP1-hFIXco) was reported to be successful [61]. In a follow-up study of ten hemophilia B patients [62], FIX levels were shown to be consistently elevated to 5–7% of the normal value for 1–4.5 years. Other Phase 1/2 clinical trials on different strategies using AAV vectors are ongoing.