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Background The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose. Methods In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII. Results The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration). Conclusions Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

SummaryLack of effective treatment for menorrhagia is the greatest unmet healthcare need in women with von Willebrand disease (VWD). We conducted a single-centre phase II clinical trial to determine efficacy and safety of recombinant IL-11 (rhIL-11, Neumega®) given subcutaneously for up to seven days during six consecutive menstrual cycles each in seven women with mild VWD and menorrhagia refractory to haemostatic or hormonal agents. rhIL-11 reduced menstrual bleeding severity as measured by pictorial blood assessment chart (PBAC) ≥50% (to 0.05. Platelet VWF mRNA expression by quantitative PCR increased mean four-fold (1.0–13.5). rhIL-11 was well tolerated with grade 1 or less fluid retention, flushing, conjunctival erythema, and local bruising. In summary, rhIL-11 reduces menorrhagia safely and warrants further study.

Type 3 von Willebrand disease (VWD) is a rare bleeding disorder with markedly decreased or absent von Willebrand factor (VWF) protein, accompanied by a parallel decrease in VWF function and factor VIII (FVIII) activity. The goal of this study was to describe the population of patients enrolled in the USA Centers for Disease Control Universal Data Collection (UDC) study with type 3 VWD, defined as a VWF:Ag of

Over 80% of adults with hemophilia are infected with hepatitis C (HCV) infection through exposure to clotting factor concentrates 20 or more years ago. Although liver biopsy is considered the gold standard to assess the severity of HCV liver disease, the majority of those with hemophilia do not undergo biopsy, even by the less invasive transjugular route. Little is known about the actual or perceived bleeding risks which may affect patient decisions about biopsy. Further, little is known about the minimal hemostatic dose of factor replacement which may affect physician decisions about biopsy. Of 161 hemophilic men enrolled in the multicenter HIV and HCV in Hemophilia (HHH) study which evaluates extent of and risks for HCV disease progression, 112 (69.6%) have decided against biopsy. Among the reasons, given by 75 of the latter, were fear of the procedure, in 23 (30.7%); lack of insurance, in 11 (14.7%); past biopsy, in 11 (14.7%); lack of time, in 10 (13.3%); lack of perceived indication, in 6 (8.0%); presence of an inhibitor, in 5 (6.7%); fear of bleeding, in 5 (6.7%); and other illness or uncertainty, in 4 (5.3%). The 49 (30.4%) agreeing to liver biopsy were more likely than those declining biopsy to be HIV+ (53.1% vs. 34.8%, p < 0.05) and less likely to have an inhibitor (0% vs. 13.4%, p = 0.002), but were as likely to have hemophilia A, 81.6% vs. 83.0%; be over 35 years of age, 49.0% vs. 53.1%; Caucasian, 81.6% vs. 85.7%; and HCV genotype 1, 80.8% vs. 79.5%, all p > 0.05. At the time of biopsy, the median platelet count was 185,000/ul (38,000–366,000/ul), the median PT was 12.0 seconds (9.4–15.4 sec), and the median INR was 1.0 (0.9–1.2). All patients received two doses of factor, and 25 (71.4%) received a third dose; three (8.6%) received a fourth dose for unrelated procedures. The median pre- and post-biopsy F.VIII doses were 49.5 U/kg, 26.6 U/kg at 1–4 hours and 45.0 U/kg at 24–48 hours; and the median F.IX doses were 74.0 U/kg, 44.3 U/kg, and 45.0 U/kg, respectively. No biopsy-related bleeding occurred. Minor adverse events included fever in two, liver function abnormality in one, and duodenal ulcer bleeding in one. The median Knodell, Ishak, and Metavir scores did not differ significantly by HIV status (+ vs.), type hemophilia (A vs. B), age (>35 vs ≤ 35 yr), HCV genotype (1 vs. non-1), or race (Caucasian vs. non-Caucasian). Although preliminary, these findings suggest that fear of the procedure is the most common deterrent to liver biopsy in patients with hemophilia. Yet, when performed by the transjugular route, liver biopsy appears to be safe, with excellent hemostasis achieved with a minimum of two doses of factor, including a 100% dose pre- and a 50% dose post-biopsy. These findings will require confirmation in larger numbers of subjects.

Based on studies in mice, hemophilic dogs, and non-human primates demonstrating long-term (>5 yrs) expression of Factor IX (FIX) after infusion of an AAV vector expressing FIX into the portal vein or the hepatic artery, we undertook a Phase I dose escalation study of AAV-FIX in humans with severe hemophilia B. The first two doses, 2x1011 vg/kg, and 1x1012 vg/kg, were safe but subtherapeutic. Two subjects treated at a dose of 5x1012 vg/kg showed detectable circulating levels of FIX (up to 11.8% and 3% respectively), but expression was transient and accompanied in one case (subject E) by a transient asymptomatic transaminitis. There was never evidence of a FIX inhibitor. Two differences between the large animal models and humans with the disease were hypothesized to contribute to the difference in duration of expression; long-term in hemophilic dogs, short-term in hemophilic humans. First was pre-existing immunity to wild-type AAV-2, which infects humans, but not dogs; and the other was prior exposure to viral hepatitis , found in humans but not in animals. To further assess the roles of viral hepatitis and of the immune response to AAV-2, we treated an additional subject (subject G) at a dose of 1x1012 vg/kg. This subject was 20 yrs. of age and had never been infected with hepatitis. Nevertheless, his transaminases began to rise 3 weeks after vector injection, peaked 6 weeks after injection, and resolved spontaneously as had been seen in subject E. In subject G, magnitude of the peak ALT response was 5-fold less than that found in subject E (5-fold higher dose). Both subjects had similar and low baseline anti-AAV antibody titers. Immune response to AAV-2 was assessed by ELISpot at serial time points before and after vector injection in subject G. The subject’s PBMCs were incubated with a peptide library arrayed in a matrix of 24 pools, each containing 12 peptides of 15-mers overlapping by 10 and spanning the entire VP-1 protein. There was no detectable IFN- γ secretion in response to AAV-2 peptides at baseline, although there was a strong IFN- γ response to PHA. Two weeks after vector infusion, three pools elicited IFN- γ secretion from the subject’s PBMCs. Response to the same pools of peptides, but not to other pools, was repeatedly detected over the next 6 weeks. By week 12, IFN- γ responses were no longer detectable. The matrix array allowed identification of two specific AAV-2 capsid peptides as the T cell immunoreactive epitopes. These peptides are highly conserved in AAV serotypes 1–8. Similar experiments were conducted with a FIX peptide library and demonstrated no response. These data are consistent with a model in which a T cell response to AAV capsid epitopes results in elimination of the transduced cells. This response is only briefly detectable in PBMCs, and hepatitis is not an important risk factor. These immune responses may limit use of standard serotypes of AAV for gene transfer into human liver. Transient immunomodulation may prevent these responses.