Search

Attempts to achieve a functional cure or amelioration of the severe X linked bleeding disorders haemophilia A (factor VIII deficiency) and haemophilia B (factor IX deficiency) using AAV-based vectors have been frustrated by immune responses that limit efficacy and durability. The immune responses include adaptive and innate pathways as well as cytokine mediated inflammation, especially of the target organ cells-hepatocytes. Immune suppression has only been partly effective in clinical trials at ameliorating the immune response and the lack of good animal models has delayed progress in identifying mechanisms and developing more effective approaches to controlling these effects of AAV gene transfer. Here we discuss the arguments for and against more potent immunosuppression to improve factor expression after AAV-mediated gene therapy., (© 2024 The Authors. Journal of Viral Hepatitis published by John Wiley & Sons Ltd.)

The single most important step on the path to our modern understanding of blood coagulation and haemophilia in the 20th century was taken by British pathologist Robert Gwyn Macfarlane with his 1964 publication 'An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier'. In the same year, Ratnoff and Davie in the USA reached the same conclusion. Macfarlane and Rosemary Biggs had previously, in 1952, discovered factor IX as the factor deficient in haemophilia B. In 1973, Arthur Bloom defined the distinct role of Factor VIII and von Willebrand factor in haemophilia A and von Willebrand's disease respectively. This inspired the efforts of Tuddenham and his group towards the purification of Factor VIII which reached homogeneity in 1982, leading to the cloning of the Factor VIII gene in 1984 in collaboration with US scientists at Genentech, which in turn enabled development of safe recombinant factor concentrates for patients with haemophilia. Brownlee cloned the factor IX gene in 1982 at the Sir William Dunn Institute of Pathology in Oxford. This led eventually to the first successful trial of gene therapy for haemophilia B in 2011 by the Nathwani group at UCL, which built on pioneering work of US groups and was partnered with St Jude in Memphis where Nathwani started the project. This trial has fuelled the current quest for a functional cure of haemophilia A and B. The UK has, therefore, made a rich contribution to advances in haemostasis over the last 60 years, often in partnership with other groups across the world., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

The foundation of haemophilia A therapy in the last 35 years has been critically dependent on isolation of the Factor VIII (FVIII) protein and discovery of the cDNA sequence of the FVIII gene, published in 1984. Identification of the FVIII sequence resulted in a new era of recombinant concentrates and led to significant improvements in safety, set against the tragedy of widespread HIV and hepatitis infections in haemophilia patients from contaminated plasma-based products. We chronicle the scientific methods and race leading up to the publication of the FVIII DNA sequence and the legacy that follows through to revolutionary gene therapy treatment in clinical trials today., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Haemophilia therapy has undergone very rapid evolution in the last 10 years. The major limitation of current replacement therapy is the short half-life of factors VIII and IX. These half-lives have been extended by the addition of various moieties, allowing less frequent infusion regimens. Entirely novel approaches have also entered the clinic, including a bispecific antibody that mimics factor VIII and strategies that rebalance the haemostatic mechanism by reducing antithrombin through inhibition of synthesis. These two treatments are available by subcutaneous injection at infrequent intervals and both can be used in patients with neutralising antibodies (inhibitors). Finally, a cure may be on the horizon with preliminary evidence of success for gene therapy in haemophilia B and A., (© 2018 John Wiley & Sons Ltd.)

Gene therapy provides hope for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene copy to replace the hemophilic patient's own defective gene. Hemophilia may be considered a "low-hanging fruit" for gene therapy because a small increment in blood factor levels (≥2% of normal) significantly improves the bleeding tendency from severe to moderate, eliminating most spontaneous bleeds. After decades of research, the first trial to provide clear evidence of efficiency after gene transfer in patients with hemophilia B using adeno-associated virus vectors was reported by the authors' group in 2011. This has been followed by unprecedented activity in this area, with the commencement of seven new early-phase trials involving >55 patients with hemophilia A or hemophilia B. These studies have, in large part, generated promising clinical data that lay a strong foundation for gene therapy to move forward rapidly to market authorization. This review discusses the data from the authors' studies and emerging results from other gene therapy trials in both hemophilia A and B.

The best currently available treatments for hemophilia A and B (factor VIII or factor IX deficiency, respectively) require frequent intravenous infusion of highly expensive proteins that have short half-lives. Factor levels follow a saw-tooth pattern that is seldom in the normal range and falls so low that breakthrough bleeding occurs. Most hemophiliacs worldwide do not have access to even this level of care. In stark contrast, gene therapy holds out the hope of a cure by inducing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene to replace the hemophilic patient's own defective gene., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Combined deficiency of factor V and factor VIII (F5F8D) is caused by mutations in one of 2 genes, either LMAN1 or MCFD2. Here we report the identification of mutations for 11 additional F5F8D families, including 4 novel mutations, 2 in MCFD2 and 2 in LMAN1. We show that a novel MCFD2 missense mutation identified here (D81Y) and 2 previously reported mutations (D89A and D122V) abolish MCFD2 binding to LMAN1. Measurement of platelet factor V (FV) levels in 7 F5F8D patients (4 with LMAN1 and 3 with MCFD2 mutations) demonstrated similar reductions to those observed for plasma FV. Combining the current data together with all previous published reports, we performed a genotype-phenotype analysis comparing patients with MCFD2 mutations with those with LMAN1 mutations. A previously unappreciated difference is observed between these 2 classes of patients in the distribution of plasma levels for FV and factor VIII (FVIII). Although there is considerable overlap, the mean levels of plasma FV and FVIII in patients with MCFD2 mutations are significantly lower than the corresponding levels in patients with LMAN1 mutations. No differences in distribution of factor levels are observed by sex. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the ER, with the impact of LMAN1 mediated indirectly through its interaction with MCFD2.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

Haemophilia A is an X-linked, recessive, inherited bleeding disorder which affects 1 in 5000 males born worldwide. It is caused by mutations in the FactorVIII (F8) gene on chromosome Xq28. We describe for the first time two mutation specific, single cell protocols for pre-implantation genetic diagnosis (PGD) of haemophilia. A that enable the selection of both male and female unaffected embryos. This approach offers an alternative to sexing, frequently used for X-linked disorders, that results in the discarding of all male embryos including the 50% that would have been normal. Two families with a history of severe haemophilia. A requested carrier diagnosis and subsequently proceeded to PGD. The mutation in family 1 is a single nucleotide substitution c.5953C > T, R1966X in exon 18 and in family 2, c.5122C > T, R1689C in exon 14 of the F8 gene. Amplification efficiency was compared between distilled water and SDS/proteinase K cell lysis (98.0%, 96/98 and 80%, 112/140 respectively) using 238 single lymphocytes. Blastomeres from spare IVF cleavage-stage embryos donated for research showed amplification efficiencies of 83.3% (45/54) for the R1966X and 92.9% (13/14) for the R1689C mutations. The rate of allele dropout (ADO) on heterozygous lymphocytes was 1.1% (1/93) for R1966X and 5.94% (6/101) for R1689C mutations. A single PGD treatment cycle for family 1 resulted in two embryos for transfer but these failed to implant. However, with family 2, two embryos were transferred to the uterus on day 4 resulting in a successful singleton pregnancy and subsequent live birth of a normal non-carrier female.