Haemophilia, the journey in search of a cure. 1960–2020

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

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver

Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA.

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression. Stable and persistent human FIX (hFIX) expression (> 22 weeks) was observed from 4 vectors after injection into the portal circulation of immunodeficient mice. The level of expression was dependent on promoter with the highest expression, 10% of physiologic levels, observed with a vector containing the cytomegalovirus (CMV) enhancer/beta-actin promoter complex (CAGG). The kinetics of expression after injection of vector particles into muscle, tail vein, or portal vein were similar with hFIX detectable at 2 weeks and reaching a plateau by 8 weeks. For a given dose, intraportal administration of rAAV CAGG-FIX resulted in a 1.5-fold or 4-fold higher level of hFIX compared to tail vein or intramuscular injections, respectively. Polymerase chain reaction analysis demonstrated predominant localization of the rAAV FIX genome in liver and spleen after tail vein injection with a higher proportion in liver after portal vein injection. Therapeutic levels of hFIX were detected in the majority of immunocompetent mice (21 of 22) following intravenous administration of rAAV vector without the development of anti-hFIX antibodies, but hFIX was not detected in 14 immunocompetent mice following intramuscular administration, irrespective of strain. Instead, neutralizing anti-hFIX antibodies were detected in all the mice. These observations may have important implications for hemophilia B gene therapy with rAAV vectors

Advances in Gene Therapy for Haemophilia.

Gene therapy provides hope for a cure for patients with haemophilia by establishing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene copy to replace the haemophilic patient's own defective gene. Haemophilia 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 haemophilia B using adeno-associated viral (AAV) vectors was reported by our group in 2011. This has been followed by unprecedented activity in this area with the commencement of 7 new early Phase trials involving over 55 patients with haemophilia A or haemophilia 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 authorisation. In this review, we discuss the data from our studies and emerging results from other gene therapy trials in both haemophilia A and B

Gene Therapy for Hemophilia

The X-linked bleeding disorder hemophilia causes frequent and exaggerated bleeding that can be life-threatening if untreated. Conventional therapy requires frequent intravenous infusions of the missing coagulation protein (factor VIII [FVIII] for hemophilia A and factor IX [FIX] for hemophilia B). However, a lasting cure through gene therapy has long been sought. After a series of successes in small and large animal models, this goal has finally been achieved in humans by in vivo gene transfer to the liver using adeno-associated viral (AAV) vectors. In fact, multiple recent clinical trials have shown therapeutic, and in some cases curative, expression. At the same time, cellular immune responses against the virus have emerged as an obstacle in humans, potentially resulting in loss of expression. Transient immune suppression protocols have been developed to blunt these responses. Here, we provide an overview of the clinical development of AAV gene transfer for hemophilia, as well as an outlook on future directions

Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B. © 2002 by The American Society of Hematology

Mbd3 Promotes Reprogramming of Primary Human Fibroblasts

Mbd3 (Methyl-CpG binding domain protein), a core member of NuRD (nucleosome remodelling and deacetylation) is essential for embryogenesis. However, its role in reprogramming of somatic cells into induced pluripotent stem cells (iPSC) remains controversial. Some reports suggest that Mbd3 inhibits pluripotency, whilst others show that it greatly enhances reprogramming efficiency. Our study is the first to assess the role of Mbd3 on reprogramming of primary human fibroblasts using Yamanaka episomal plasmids (Reprogramming factors (RF) under feeder-free conditions. We showed that shRNA-mediated partial depletion of Mbd3 resulted in >5-fold reduction in the efficiency of reprogramming of primary human fibroblasts. Furthermore, iPSC that emerged after knock-down of Mbd3 were incapable of trilineage differentiation even though they expressed all markers of pluripotency. In contrast, over-expression of the Mbd3b isoform along with the Yamanaka episomal plasmids increased the number of fibroblast derived iPSC colonies by at least two-fold. The resulting colonies were capable of trilineage differentiation. Our results, therefore, suggest that Mbd3 appears to play an important role in reprogramming of primary human fibroblasts, which provides further insight into the biology of reprogramming but also has direct implication for translation of iPSC to clinic

Polonium-210 poisoning: a first-hand account

Background: Polonium-210 (210Po) gained widespread notoriety after the poisoning and subsequent death of Mr Alexander Litvinenko in London, UK, in 2006. Exposure to 210Po resulted initially in a clinical course that was indistinguishable from infection or exposure to chemical toxins, such as thallium. Methods: A 43-year-old man presented to his local hospital with acute abdominal pain, diarrhoea, and vomiting, and was admitted to the hospital because of dehydration and persistent gastrointestinal symptoms. He was initially diagnosed with gastroenteritis and treated with antibiotics. Clostridium difficile toxin was subsequently detected in his stools, which is when he first raised the possibility of being poisoned and revealed his background and former identity, having been admitted under a new identity with which he had been provided on being granted asylum in the UK. Within 6 days, the patient had developed thrombocytopenia and neutropenia, which was initially thought to be drug induced. By 2 weeks, in addition to bone marrow failure, he had evidence of alopecia and mucositis. Thallium poisoning was suspected and investigated but ultimately dismissed because blood levels of thallium, although raised, were lower than toxic concentrations. The patient continued to deteriorate and within 3 weeks had developed multiple organ failure requiring ventilation, haemofiltration, and cardiac support, associated with a drop in consciousness. On the 23rd day after he first became ill, he suffered a pulseless electrical activity cardiorespiratory arrest from which he could not be resuscitated and was pronounced dead. Findings: Urine analysis using gamma-ray spectroscopy on day 22 showed a characteristic 803 keV photon emission, raising the possibility of 210Po poisoning. Results of confirmatory analysis that became available after the patient's death established the presence of 210Po at concentrations about 109-times higher than normal background levels. Post-mortem tissue analyses showed autolysis and retention of 210Po at lethal doses in several organs. On the basis of the measured amounts and tissue distribution of 210Po, it was estimated that the patient had ingested several 1000 million becquerels (a few GBq), probably as a soluble salt (eg, chloride), which delivered very high and fatal radiation doses over a period of a few days. Interpretation: Early symptoms of 210Po poisoning are indistinguishable from those of a wide range of chemical toxins. Hence, the diagnosis can be delayed and even missed without a high degree of suspicion. Although body surface scanning with a standard Geiger counter was unable to detect the radiation emitted by 210Po, an atypical clinical course prompted active consideration of poisoning with radioactive material, with the diagnosis ultimately being made with gamma-ray spectroscopy of a urine sample