The complex, confusing and poorly understood immune responses to AAV-mediated gene transfer in haemophilia-Is more or less immunosuppression required?

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.

Mathematical models of coagulation-are we there yet?

BACKGROUND: Mathematical models of coagulation have been developed to mirror thrombin generation in plasma, with the aim of investigating how variation in coagulation factor levels regulates hemostasis. However, current models vary in the reactions they capture and the reaction rates used, and their validation is restricted by a lack of large coherent datasets, resulting in questioning of their utility. OBJECTIVES: To address this debate, we systematically assessed current models against a large dataset, using plasma coagulation factor levels from 348 individuals with normal hemostasis to identify the causes of these variations. METHODS: We compared model predictions with measured thrombin generation, quantifying and comparing the ability of each model to predict thrombin generation, the contributions of the individual reactions, and their dependence on reaction rates. RESULTS: We found that no current model predicted the hemostatic response across the whole cohort and all produced thrombin generation curves that did not resemble those obtained experimentally. Our analysis has identified the key reactions that lead to differential model predictions, where experimental uncertainty leads to variability in predictions, and we determined reactions that have a high influence on measured thrombin generation, such as the contribution of factor XI. CONCLUSION: This systematic assessment of models of coagulation, using large dataset inputs, points to ways in which these models can be improved. A model that accurately reflects the effects of the multiple subtle variations in an individual's hemostatic profile could be used for assessing antithrombotics or as a tool for precision medicine.

Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

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.

Global coagulation assays in hemophilia A: A comparison to conventional assays.

BACKGROUND: Global assays measure the interactions of coagulants, anticoagulants, and platelets on thrombin generation and may reflect the comprehensive coagulation potential in patients with hemophilia better than conventional assays. OBJECTIVES: The objectives of the current study were to investigate the value of global assays for measuring and monitoring the coagulation potential of patients with hemophilia A (HA). PATIENTS/METHODS: Rotational thromboelastometry, thrombin generation assay (TGA), and activated partial thromboplastin time (APTT) clot waveform analysis were investigated in a cohort of patients with severe, moderate, and mild HA and compared with conventional assays. RESULTS: The maximum velocity (MaxVel) parameter of modified thromboelastometry analysis, initiated by tissue factor and in the presence of corn trypsin inhibitor (CTI), had 92% sensitivity and 95% specificity for hemophilia diagnosis. The MaxVel also strongly correlated with factor VIII (FVIII) levels of patients with HA (r = .805, P < .0001). CTI improved the sensitivity of TGA, providing more accurate results. In particular, peak height parameter of platelet-rich plasma samples with CTI had a sensitivity and specificity of 100% and 94%, respectively, in all patients with HA. APTT clot waveform analysis minimum value of first derivative (Min1) and minimum value of second derivative (Min2) parameters (representing speed and acceleration of clot formation, respectively) were sensitive and correlated more strongly with FVIII levels than APTT clotting times did (Min1: r = 0.786, P < 0.0001; Min2: r = 0.759, P < 0.0001; APTT: r = -0.513, P = 0.001). CONCLUSIONS: The sensitivity and specificity of the global assays was method dependent. Correlation between clinical end points and thrombin generation might also be valuable in the era of non-factor replacement therapy.

Factor VIII: the protein, cloning its gene, synthetic factor and now - 35 years later - gene therapy; what happened in between?

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.

Interaction Between the a3 Region of Factor VIII and the TIL'E' Domains of the von Willebrand Factor.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.

Recent advances in developing specific therapies for haemophilia.

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.

Gene Therapy for Hemophilia.

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.

Advances in Gene Therapy for Hemophilia.

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.

Thrombin generation assay identifies individual variability in responses to low molecular weight heparin in pregnancy: implications for anticoagulant monitoring.

Low molecular weight heparin (LMWH) given to inhibit coagulation and reduce the risk of thrombosis, is typically monitored by anti-Xa assay. However, anti-Xa levels may not necessarily provide an accurate measure of coagulation inhibition. Moreover, pregnancy is associated with hypercoagulability, which may compromise the efficacy of LMWH. We looked at the association between anti-Xa levels and parameters of thrombin generation assay [TGA; area under the curve (AUC), peak height (PH) and time to peak (ttP)] using samples from 41 pregnant women receiving LMWH and 40 normal pregnant women controls. TGA results confirmed the physiological hypercoagulability of normal pregnancy (mean normalised values: AUC 119%; PH 157%; ttP 72%). Although anti-Xa measures correlated with all three TGA parameters, this group correlation masked significant inter-individual variability, demonstrated by the R(2) value or coefficient of determination. Anti-Xa levels contributed to 74% of variation in AUC values, 63% of variation in PH values and only 53% of variation in ttP values. The remainder reflects the contribution of patients' intrinsic coagulation status. Hence, some patients with 'safe' anti-Xa levels may potentially be under-anticoagulated, particularly in pregnancy. Measuring coagulability directly with TGA may lower the risk of adverse events due to under-anticoagulation in selected patients.

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Solution structure of the major factor VIII binding region on von Willebrand factor.

Although much of the function of von Willebrand factor (VWF) has been revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D' region of VWF, which constitutes the major FVIII binding site. D' consists of 2 domains, trypsin-inhibitor-like (TIL') and E', of which the TIL' domain lacks extensive secondary structure, is strikingly dynamic and harbors a cluster of pathological mutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbone malleability of TIL' is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL', which is supported by the rigid scaffold of the TIL' and E' domain ß sheets. Furthermore, surface-charge mapping of the TIL'E' structure reveals a potential mechanism for the electrostatically guided, high-affinity VWF⋅FVIII interaction. Our findings provide novel insights into VWF⋅FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

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.

AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage.

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Human congenital diseases with mixed modes of inheritance have a shortage of recessive disease. A demographic scenario?

An archive of congenital human diseases is presented, aiming to contain all those where recessive (biallelic) can be compared with X-linked and/or dominant (monoallelic) inheritance. A significant deficit of recessive inheritance is evident, both in disease inheritance and in contribution to inheritance per known disease gene. The deficit contrasts with expectation derived from the cell biology of mutation, and from the importance of recessive mutation in evolution and its preponderance in N-ethyl-N-nitrosourea (ENU) mutagenesis. The deficit fits well with the standard model of demographic change since the neolithic era, and may also reflect natural selection acting on heterozygotes.