Gene therapy, bioengineered clotting factors and novel technologies for hemophilia treatment

The World Federation of Hemophilia estimates that of the 400,000 individuals worldwide with hemophilia, 300,000 receive either no, or very sporadic, treatment. Thus, considerable innovation will be required to provide cost-effective therapies/cures for all affected individuals. The high cost of prophylactic regimens hampers their widespread use, which further justifies the search for novel cost-effective therapies and ultimately a cure. Five gene transfer phase I clinical trials have been conducted using either direct in vivo gene delivery with viral vectors or ex vivo plasmid transfections and reimplantation of gene-engineered cells. Although there was evidence of gene transfer and therapeutic effects in some of these trials, stable expression of therapeutic factor VIII or FIX levels has not yet been obtained. Further improvements of the vectors and a better understanding of the immune consequences of gene transfer is warranted, as new trials are being initiated. Bioengineered clotting factors with increased stability and/or activity are being validated further in preclinical studies. Novel clotting factor formulations based on PEGylated liposomes with prolonged activities are being tested in the clinic, and are yielding encouraging results.     

Immune tolerance therapy for factor VIII inhibitors: moving from empiricism to an evidence-based approach

Summary.  Currently, the only proven strategy for achieving antigen-specific tolerance to factor VIII (FVIII) is immune tolerance induction (ITI) therapy. This paper discusses our current knowledge of the host and treatment factors, as well as supportive care initiatives, known or suspected to influence the outcome of ITI in the treatment of inhibitors arising in patients with severe hemophilia A. Among these, questions surrounding the choice of therapeutic product and/or dosing regimen generate the most controversy, given the lack of a definitive evidence-based approach to either. Furthermore, the potential for central venous access device (CVAD) and intercurrent bleeding complications to impact the ultimate success of ITI remains unclear. The ongoing clinical trials designed to further clarify several of these polarizing issues are reviewed. This paper also explores the current and future role of immune modulation in possible salvage, ancillary or primary alternative tolerance induction strategies. The special cases of low titer/ responding inhibitors and inhibitors developing in mild hemophilia A patients are considered. Finally, this paper summarizes the currently recommended approach to ITI and makes the case for a move from empiric therapeutics to a risk-stratified evidence-based approach to FVIII inhibitor eradication.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Hemophilia: treatment options in the twenty-first century

Summary.  In the last three decades, hemophilia has moved from the status of a neglected and often fatal hereditary disorder to that of a fully defined group of molecular-pathological entities for which safe and effective treatment is available. Hemophilia is likely to be the first widespread severe genetic condition to be cured by gene therapy in the third millennium. In the socio-economic arena it remains a challenge to humanity to know that four-fifths of the world's hemophiliacs still receive no treatment at all. Production of factor (F) VIII and IX in the milk of transgenic farmyard animals could provide a source of less expensive replacement therapy for developing countries. Affordable gene transfer will be the ultimate solution for hemophilia in the third world as in the first. Thus it may be confidently predicted that the early new millennium will see an end to this ancient scourge.     

Gene therapy for treatment of inherited haematological disorders

Gene therapy, a molecular medicine based on vector-mediated transfer of therapeutic genes, holds promise for a cure of monogenetic inherited diseases. In recent years, tremendous progress has been reported in the treatment of haematological disorders: clinical trials in severe combined immune deficiencies have been successful by using retroviral vectors to express target genes in haematopoietic stem cells, which after transplantation efficiently reconstituted the immune system concomitant with substantial improvement in the clinical status of patients. Conversely, unexpected adverse events were also encountered. In other work, progress towards clinical studies on ex vivo gene transfer for Fanconi anaemia and haemoglobinopathies has been made. Each approach features a unique treatment strategy and also faces various impediments to success. In the case of the X-linked bleeding disorder haemophilia, several Phase I/II clinical trials were conducted, including in vivo administration of viral vectors to skeletal muscle and liver. Adeno-associated viral gene transfer of coagulation Factor IX has been documented in human subjects, reaching therapeutic levels after infusion into a hepatic blood vessel. However, sustained expression of therapeutic levels (as shown in large animal models of haemophilia) has not yet been achieved in humans. In general, long-term follow-up will be important for assessment of the safety of all existing gene therapy strategies.     

Role of regulatory T cells in tolerance to coagulation factors

Summary.  The immune response to coagulation factors VIII or IX, in particular formation of inhibitory antibodies, complicates treatment of hemophilia. Therefore, a number of recent studies in animal models have explored novel approaches toward induction of immune tolerance in protein or gene replacement therapy. Strong evidence has emerged that regulatory T cells (Treg) are an important component of the mechanism by which tolerance is maintained and inhibitor formation, a T help dependent response, is prevented. Limited data in patients also support this concept. In particular, CD4⁺ CD25⁺ FoxP3⁺ Treg, whether naturally occurring or induced, have been invoked in suppression of antibody and of cytotoxic T lymphocyte responses to the therapeutic clotting factor. This review summarizes the data on this emerging concept of Treg-mediated regulation of the immune response in treatment of hemophilia, strategies and mechanisms of Treg induction and function, and the implications for development of immune tolerance protocols.     

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain

Summary. Background: The development of anti‐factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. Objective: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. Methods: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII‐C2) to FVIII‐deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII‐C2, followed by a subcutaneous challenge of either FVIII‐C2 or FVIII. The development of anti‐FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. Results and Conclusions: Mucosal administration of FVIII‐C2 decreases the titer of anti‐FVIII‐C2 inhibitors after FVIII‐C2 challenge, and decreases the percentage of FVIII‐C2 specific antibodies after challenge with full‐length FVIII. Tolerance induction to FVIII‐C2 is associated with increased IL‐10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII‐C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII‐specific mucosal tolerance induction as an immunotherapy treatment for anti‐FVIII inhibitor development warrants further investigation.     

Hemophilia A and hemophilia B: focus on arthropathy and variables affecting bleeding severity and prophylaxis

Hemophilia A (HA) and hemophilia B (HB) are X‐linked, recessive disorders. Although their clinical manifestations are essentially indistinguishable, it has been suggested that bleeding episodes in patients with HA are generally more severe and occur at higher frequency than in patients with HB. Nevertheless, considerable debate remains regarding the relative severity of HA and HB. Based on the relative risk of undergoing joint arthroplasty, it appears that patients with HA have more severe joint deterioration compared with patients with HB. Although it is difficult to speculate on the factors that might modify bleeding severity in patients with hemophilia, recent observations indicate that other coagulation proteins, such as tissue factor pathway inhibitor or polymorphisms in coagulation factor genes and genetic defects associated with hypercoagulability may account for the variability in clinical phenotype among patients with hemophilia. Numerous studies have provided evidence supporting the clinical and social benefits of administration of clotting factor in prophylaxis. However, it is still unclear why this approach is more commonly utilized in patients with HA than in those with HB.     

重组狗凝血因子Ⅷ的慢病毒介导体外表达的研究

本研究的目的是制备携带狗凝血因子Ⅷ（cFⅧ）基因的慢病毒载体,探讨慢病毒载体能否介导cFⅧ在体外的有效表达。用构建携带cFⅧ基因的慢病毒载体pTK161（含PUB启动子）和pTK162（含2OH1启动子）,同时构建含绿色荧光蛋白（GFP）的慢病毒载体pTK161′（含PUB启动子）和pTK162′（含2OH1启动子）的方法,分别与包装质粒ΔNRF、包膜蛋白质粒VSV-G共转染293T包装细胞,将包装好的病毒颗粒再感染293T细胞,检测病毒滴度和培养细胞上清中cFⅧ活性。结果显示：经限制性酶切鉴定,成功构建了pTK161、pTK162、pTK161′和pTK162′正向连接载体;pTK161′和pTK162′的病毒滴度分别为1.54×10^6U/ml和2.83×106U/ml;pTK161、pTK162感染靶细胞后24小时细胞上清中即可检测到cFⅧ的表达,72小时表达量达高峰。pTK162载体cFⅧ表达活性接近正常狗血浆FⅧ活性,而且明显高于pTK161（p〈0.05）,6周后cFⅧ表达活性仍达最高值的1/4。结论：构建的自身失活慢病毒载体可携带较大片段的cFⅧ基因,并在体外可获得有效表达;本研究为慢病毒载体介导的血友病A的基因治疗研究提供实验依据。     

Sustained Human Factor VIII Expression in Hemophilia A Mice Following Systemic Delivery of a Gutless Adenoviral Vector

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.     

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.     

Laboratory testing in hemophilia: Impact of factor and non‐factor replacement therapy on coagulation assays

The advent of extended half‐life (EHL) recombinant clotting factors and innovative non‐factor replacement therapeutics, such as emicizumab, offers several advantages over existing products for the prophylactic treatment of people living with hemophilia (PwH). These include low annual bleeding rates with less frequent dosing, higher trough plasma concentrations, and a more convenient route of administration. However, increasing use of these therapies poses challenges to clinicians and coagulation laboratories due to the lack of standardized assays for monitoring of hemostatic parameters, and the potential for misinterpretation of test results, which may jeopardize patient safety. Definitive diagnosis of hemophilia and treatment monitoring is reliant on demonstrating factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B) deficiency using a functional coagulation assay. The most frequently used assays are based on activated partial thromboplastin time, using a one‐stage or two‐stage process. While one‐stage and chromogenic assays have performed well with human‐derived FVIII and FIX and full‐length recombinant products, EHL recombinant factors are heterogeneous in structure and mode of action and therefore show wide variation in activity levels between different one‐stage assays, and between one‐stage and chromogenic assays. In the context of the recommended stepwise approach for laboratory diagnosis of hemophilia, we examine the diagnostic challenges associated with the use of EHL factors and novel non‐factor therapeutics and consider the optimal diagnostic approach in PwH who are receiving these treatments. Ultimately, accurate diagnostic solutions are a prerequisite for personalized therapy to minimize treatment burden and improve quality of life in PwH.     

Immune tolerance induction for the eradication of inhibitors in patients with hemophilia A

Background: Both genetic and environmental factors contribute to the formation of alloantibodies that bind to functional domains on the Factor VIII (FVIII) molecule and inhibit its function. Patients with hemophilia A who develop high-titer inhibitors are at increased risk for serious hemorrhage and disability, particularly arthropathy, because bleeding events do not respond to standard therapy. Immune tolerance induction (ITI) is usually attempted to eradicate newly diagnosed inhibitors, restore replacement FVIII pharmacokinetics, and improve bleed management and quality of life. Objective: This paper summarizes regimens used for ITI, predictors of success and failure, and adjunctive therapies for patients failing ITI therapy. Methods: This is a systematic review of published reports on ITI regimens, data from registries capturing response rates and predictors of success, and reports of adjunctive treatments used to enhance ITI therapy. Results/conclusion: Many issues remain unresolved, chief among them optimal dose and dosing regimen, choice of FVIII product, and the role of adjunctive therapy. Resolution of these issues, as well as new approaches to inhibitor management, may come from ongoing basic science research and clinical trials.     

The future of recombinant coagulation factors

Summary.  Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.