Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Optimization of pre-analytical heat treatment for inhibitor detection in haemophilia A.

INTRODUCTION: Factor VIII (FVIII) antibody formation is the greatest clinical and laboratory challenge within the haemophilia centre. The Nijmegen-Bethesda assay (NBA) is the gold standard for inhibitor quantification, but affected by pre-analytical variables including a patient's FVIII activity (FVIII:C). Pre-analytical heat treatment (PHT) provides a methodology for inhibitor testing when measurable FVIII:C is present. METHODS: We evaluated the effect of different PHT conditions (time/temperature) on FVIII:C as well as on potency of inhibitory activity in samples containing FVIII:C (endogenous pooled plasma and exogenous recombinant FVIII (rFL-FVIII) concentrate) or FVIII inhibitor. RESULTS: PHT of endogenous FVIII at 37°C, 47°C and 52°C resulted in declining measurable FVIII:C at 120 minutes (69%, 57% and 13% of the original FVIII:C, respectively). Incubation at 56°C resulted in FVIII:C ≤ 1IU/dL after 60 minutes for endogenous FVIII and 120 minutes for rFL-FVIII. Incubation at 58°C resulted in FVIII:C < 1IU/dL at 15-30 minutes for endogenous FVIII and at 30-60 minutes for rFL-FVIII. No difference was seen for inhibitor detection following PHT (56°C or 58°C) by NBA or anti-FVIII IgG ELISA. CONCLUSION: PHT at 58°C for 30 minutes demonstrated consistent reduction in FVIII:C < 1IU/dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.

Ongoing risk of thrombosis with factor XI concentrate: 5 years experience in two centres.

INTRODUCTION: Factor XI (FXI) deficiency is the commonest of the rare bleeding disorders, affecting 2079 individuals in the United Kingdom. Treatment options for bleeding or surgery include antifibrinolytics, fresh frozen plasma or plasma-derived (pd) FXI concentrates. There were a number of reports of thrombosis following treatment with FXI concentrates prior to changes in their manufacturing processes made in the mid-1990's. AIMS: The aim of the study was to determine the occurrence of adverse events (haemorrhagic and thrombotic) following usage of pd-FXI concentrates at two large UK haemophilia centres. Retrospective chart review of all consecutively treated patients with BPL Factor XI(®) or Hemoleven(®) over a 5-year period (11/06-11/11) was performed. RESULTS: Twenty-nine patients (median age = 57.1 years) received treatment over 64 treatment episodes (surgery = 56, bleeding = 5, other = 3), using 126 000 U of concentrate. Median baseline FXI:C was 9 U dL(-1) (range = <1-51), with 21 having severe and eight partial deficiency. BPL Factor XI(®) was used in 39 episodes (79 110 U) and Hemoleven(®) 25 episodes (46 890 U). There were six clinically significant bleeding events, managed either with a single additional dose of FXI concentrate (n = 4) or requiring no further intervention (n = 2). One patient required blood transfusion and one oral iron replacement. Two thrombotic events (transient ischaemic attack and pulmonary emboli), occurred in two patients with severe FXI deficiency, despite cautious FXI concentrate usage in the perioperative period. CONCLUSIONS: FXI concentrate use is efficacious and safe in the majority of cases although physicians should remain mindful of the possibility of thrombotic complications.

Wilate use in 47 children with von Willebrand disease: the North London paediatric haemophilia network experience.

Children with von Willebrand disease (VWD) in whom DDAVP is ineffective or contraindicated require treatment with a coagulation factor concentrate containing von Willebrand factor (VWF) and factor VIII (FVIII). The aim of this study was to monitor the safety, efficacy and tolerability of Wilate(®) (a VWF:FVIII concentrate with a 1:1 ratio) used across the North London Paediatric Haemophilia Network since May 2010. In total, 47 children (aged 0.0-17.0 years) with type 1 (n = 28), type 2 (n = 7), type 3 (n = 10) and acquired VWS (n = 2) have been treated for bleeds, surgery and/or prophylaxis using 260 000 IU Wilate(®). Analysis of dose and frequency of treatment show expected responses to treatment with mean doses of 55, 50 and 50 IU kg(-1) for bleeds, surgery and prophylaxis respectively. Most bleeds responded to a single treatment. Surgical procedures were covered with clinician approved dosing schedules with 95% (39/41) reported as having excellent or good efficacy. There was no accumulation of FVIII or VWF and no thromboembolic events. This case series confirms the efficacy, safety and tolerability of Wilate(®) in neonates, children and adolescents when used on-demand, prophylactically and in the surgical setting.

Safety and efficacy of a von Willebrand factor/factor VIII concentrate (Wilate®): a single centre experience.

von Willebrand disease (VWD) is the commonest inherited bleeding disorder. Management of major surgery or bleeding often requires treatment with a plasma-derived (pd) VWF/FVIII containing concentrate. Wilate® is a dual-virally inactivated pd-concentrate, produced specifically for the treatment of VWD, containing physiological (1:1) ratios of VWF: FVIII. We reviewed efficacy and safety of Wilate® usage (2007-2012) at our centre including 2 years following product switching the majority of patients. Clinical and laboratory data of all adult patients treated with Wilate® during the study period were evaluated. Fifty four patients used 3 972 150 IU of Wilate® (1378 infusions) between 1/3/07 and 1/5/12. Efficacy was rated as being excellent or good in 94% of surgical episodes (n = 70; 34 patients) and 98% of bleeding/traumatic episodes (n = 46; 25 patients). Eight patients (2 636 100 IU) were managed on home treatment regimens. Two patients switched to Wilate® prophylaxis in the evaluation period, demonstrating similar efficacy to a previous product. Incremental recoveries (n = 37) were 2.24 IU dL(-1) per IU kg(-1) for FVIII:C, 2.39 IU dL(-1) per IU kg(-1) for VWF:Ag and 1.88 IU dL(-1) per IU kg(-1) for VWF:RCo. Six adverse events occurred in six patients (11.1% patients) over 1378 infusions (0.44%). Half of these were retrospectively felt to be infusion speed related. No notable accumulation of FVIII was seen in patients treated for ≥3 days. There was no treatment failure, thrombosis, transfusion transmitted infection or inhibitory VWF antibodies seen. Our findings confirm safety and efficacy of Wilate® in an adult VWD population with lack of notable FVIII accumulation.

New thrombopoietin receptor agonists for platelet disorders.

Since thrombopoietin (TPO) was cloned in 1994, TPO receptor (TPO-R) agonists have been developed which have shown significant clinical activity in various conditions characterized by thrombocytopenia. First-generation TPO-R agonists were recombinant forms of human TPO. The clinical development of these molecules was discontinued after one of them, pegylated recombinant human megakaryocyte growth and development factor, was associated with the development of neutralizing autoantibodies cross-reacting with endogenous TPO. Second-generation TPO-R agonists are now available, which present no sequence homology to endogenous TPO. Two of these new agents, romiplostim and eltrombopag, have been granted marketing authorization for use in patients with primary immune thrombocytopenia unresponsive to conventional treatments. Clinical trials with TPO-R agonists are also ongoing in other thrombocytopenias, such as hepatitis C virus-related thrombocytopenia and the myelodysplastic syndromes.

Ankle sprain: an unexpected complication.

Blunt arterial injury is usually caused by high velocity trauma and can result in intimal dissection. We present a case of a professional footballer who sustained an intimal tear of the posterior tibial artery following a minor eversion injury of the ankle. The injury was noticed because of the physical demands of this patient's profession. This was confirmed by an arteriogram and was treated with bypass surgery using an arm vein. Arterial intimal injury has not been reported previously with this type of injury. A high index of suspicion is needed to diagnose these injuries and revascularisation either by primary anastomosis or vein interposition graft is suggested.

Catheter induced pulmonary arterial perforation during open heart surgery.

Four cases of the pulmonary artery perforation induced by the flow directed pulmonary artery catheter (Swan-Ganz Catheter) are reported. These cases were experienced in approximately 1500 major cardiac procedures over a four year period in the cardiothoracic surgical service at a community cardiac center. Their clinical manifestations, management and eventual outcomes are discussed. Based on this clinical experience and review of the literature, current management plans are discussed. We suggest preventative measures to avoid similar complications.

Freezing preservation of adult mammalian heart at high subzero temperatures.

The present study adapted the overwintering strategy employed by freeze-tolerant amphibians and reptiles to freeze-preserve the isolated rat heart. The heart was flushed with a cardioplegic solution and supercooled to -1.2 and -3 degrees C. Then freezing was induced by inoculation of ice crystal. The viability of the heart explant was assessed after reanimation by the isolated working heart perfusion. There was no recovery of function in hearts flushed with solution containing 0.28 mM CaCl2. Lowering the concentration of CaCl2 to 0.15 mM, however, rendered good functional return. Furthermore, inclusion of 50 mM glycerol in the flush solution dramatically improved functional preservation. Under the best conditions defined here, the recoveries of aortic flow, coronary flow, cardiac output, systolic pressure, and work in hearts stored at -1.2 degrees C for 3 h were 72.8 +/- 6.8, 87.2 +/- 4.2, 77.6 +/- 5.4, 83.4 +/- 2.8, and 66.6 +/- 5.9% (mean +/- SEM, n = 8) of the unstored control levels, respectively. The myocardial ice content was 18.6 +/- 5.4% (n = 5) of tissue water. Prolonging the storage time to 5 h increased the ice content to 45.3 +/- 8.1% and reduced the recovery of cardiac output to 23 +/- 11% of the control value (mean +/- SEM, n = 5). Hearts frozen at -3 degrees C for 1.5 h showed 29.4 +/- 8.7% (n = 3) of control cardiac output during reperfusion. This novel approach may provide an opportunity to advance our knowledge about freezing preservation of not only the heart but other solid organs as well.

Long-term hypothermic storage of the cardiac explant. Comparison of four solutions.

This study compared the capabilities of 4 storage solutions in protecting the cardiac explant. Isolated rat heart was flushed with and stored in one of the storage solutions at 0 degrees C for 7 hours. The recovery of function was assessed using the working heart perfusion. Cardiac output returned to 34 +/- 6, 59 +/- 3 76 +/- 5, and 75 +/- 8% (mean +/- SE) of the control level in hearts stored in Euro-Collins, St. Thomas' Hospital cardioplegic, a modified University of Wisconsin (MUW) solutions, and a solution developed by us (CP-8), respectively. Therefore, MUW and CP-8 were superior to the other two solutions. During post-storage reperfusion, the coronary flow correlated directly with the aortic flow (r = 0.98), suggesting that preservation of coronary perfusion may be crucial to the recovery of function. There was also a good correlation between myocardial ATP levels and the cardiac output (r = 0.81). Thus, measurements towards the enhancement of ATP preservation and regeneration may be beneficial to the stored cardiac explant.

Optimal osmolality for cold storage of the cardiac explant.

Cell swelling is a major problem of cardiac preservation. This study evaluated the effect of storage solution osmolality on long-term preservation of cardiac function. Isolated rat hearts were flushed and stored in solutions of 260 to 350 mOsm/kg water at 0 degrees C for 9 hr. Cardiac performance was assessed using the isolated working heart reperfusion. Function of fresh unstored hearts served as the control. The stored hearts had normal heart rate after reperfusion. Other hemodynamic functions recovered to various levels of the control value and showed biphasic responses to solution osmolality. Hearts stored in hyposmotic (260 and 270 mOsm) and hyperosmotic (310, 330, and 350 mOsm) solutions performed poorly. Those stored in 280 to 290 mOsm solutions showed superior recovery in cardiac function. Among all parameters, coronary flow correlated linearly with aortic flow, cardiac output, systolic pressure, work, oxygen consumption, and coronary vascular resistance. Solutions of 280, 290, and 310 mOsm caused 6 to 9% increases in tissue water (TW) over the control hearts during 9 hr storage, whereas 350 mOsm solution maintained TW at the control level. After reperfusion, all stored hearts gained water (25 to 38%) compared to hearts with no reperfusion. Postreperfusion myocardial ATP content was only 70-79% of control level and did not correlate to function recovery. In conclusion, (1) the optimal osmolality of our preservation fluid is 280 to 290 mOsm and moderate or severe hyperosmolality is detrimental to cardiac function, and (2) preservation of coronary function may be crucial to future improvement of storage solution.

A case of acute measles meningoencephalitis with virus isolation.

A case of measles meningoencephalitis is described. Measles virus was isolated from the cerebrospinal fluid cells, in which virus antigen was also detected by indirect immunofluorescence.