Faculty Bibliography

In a phase I study, administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and FIX expression for at least 10 months after vector injection, the last time point assessed by muscle biopsy. In hemophilia B dogs we have demonstrated FIX in both muscle biopsies and circulation >4 years following AAV2-FIX injection. Because circulating FIX levels remained less than 1% of normal in human subjects from the study, the duration of AAV2-mediated transgene expression in humans is unknown. We sought to determine if FIX gene transfer and expression persisted locally at injection sites. Muscle biopsies were obtained from one subject 3.7 years following treatment and revealed transgene FIX DNA and protein by quantitative PCR, DNA fluorescence in situ hybridization, and immunohistochemistry for FIX. These results demonstrate, for the first time, multiyear FIX expression by AAV2 vector in humans and suggest that improved muscle delivery provides effective treatment for protein deficiencies or muscle-specific diseases

OBJECTIVE: To evaluate risk factors for recurrent thrombosis in pediatric patients. STUDY DESIGN: This prospective observational cohort study enrolled 120 patients with acute venous thromboembolism from January 2003 to April 2005. Data collection included medical and family history, radiologic and laboratory studies, therapy, and follow-up. RESULTS: The overall prevalence of recurrent thrombosis in our cohort was 19/120 (15.8%). Patients with recurrence were older, with a median age of 14.8 years (range 2 weeks-23.6 years), compared with 10.1 years (range newborn 23.4 years) in patients without recurrence (P = .03). Six of the 19 patients with recurrent thrombosis had cystic fibrosis (CF), compared with 0/101 without recurrence (P < .001). Five of these 6 patients were colonized with Burkholderia cepacia in their sputum. Central venous catheters were associated with most, but not all, of the thromboses in patients with CF. CONCLUSIONS: In this study, patients with CF had a high risk of recurrent venous thrombosis, as well as a high prevalence of colonization with B cepacia. The cause of this risk has not been defined. This observation may have important implications for thromboprophylaxis, particularly in the setting of central venous catheters

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression

Many issues affect hemophilia care providers when managing bleeding episodes in individuals with hemophilia. A diverse group of hemophilia providers from the United States met at two workshops to discuss the issues influencing treatment choices in the current millennium and to learn about current advances in treatment of hemophilia. This paper provides a summary of the discussions at these workshops. Despite the progress made in the management of patients with hemophilia, the workshop highlighted the fact that there were still many unanswered questions

OBJECTIVE: To describe the outcome and treatment of two patients with recombinant factor VIIa (rFVIIa) for severe hemorrhage associated with extracorporeal membrane oxygenation (ECMO). DESIGN: Case report. SETTING: A 38-bed pediatric intensive care unit and 20-bed pediatric cardiac intensive care unit at a tertiary care children's hospital. Patient: Two patients with life-threatening hemorrhagic complications associated with ECMO requiring massive transfusion of blood products. INTERVENTIONS: Administration of repeated doses of rFVIIa at 90 microg/kg/dose. MEASUREMENT AND MAIN RESULTS: Patient 1 was an 11-yr-old male with a dilated cardiomyopathy who had undergone an orthotopic heart transplant treated with venoarterial ECMO postoperatively for right ventricular dysfunction. Patient 2 was a 13-yr-old male treated with venoarterial ECMO for cardiopulmonary failure from necrotizing staphylococcal pneumonia. Both patients had severe hemorrhage from the cannulation sites and thoracostomy tubes requiring massive transfusion to maintain intravascular blood volume and replace clotting factors. Both patients were treated with rFVIIa every 2-4 hrs and attained hemostasis. Patient 1 was administered three doses and Patient 2 was administered ten doses. No evidence of abnormal thrombus formation was noted in their respective ECMO circuits. CONCLUSIONS: The efficacy of rFVIIa in reducing intractable bleeding postcardiac surgery and in other coagulopathic states is being investigated. Despite theoretical concerns of thrombosis, these cases illustrate that there may be a role for the cautious use of rFVIIa in treating severe and intractable hemorrhage associated with ECMO

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of disrupted lymphocyte homeostasis. Clinical manifestations of ALPS vary but typically include autoimmune cytopenias, organomegaly, lymphadenopathy, and increased risk of malignancies. A similar spectrum of symptoms may be seen in some patients with Evans syndrome (ES), a hematologic disorder defined by autoimmune destruction of at least 2 hematologic cell types. We hypothesized that a subset of patients diagnosed with ES may have ALPS. We screened 12 children with ES by flow cytometric analysis for CD4-/CD8- (double negative) T cells (DNTs) and with the definitive test for ALPS, defective in vitro Fas-mediated apoptosis. Six of the patients had elevated DNTs, suggestive of ALPS and also had defective Fas-mediated apoptosis. The other 6 patients displayed normal T-cell apoptosis; 5 of whom had normal DNTs, and 1 had a borderline result. Thus, 7 (58%) of 12 patients with ES had elevated DNTs suggestive of ALPS, with functional confirmation in 6 of 7. This suggests that analysis of DNTs may be a sensitive first-line screening test, serving as a marker of patients who should undergo definitive testing for ALPS. Our data further suggest that a number of patients with ES may have ALPS, a novel finding with important therapeutic implications

Diagnosis and management of congenital and acquired bleeding disorders in children requires not only an understanding of the unique characteristics of pediatric hemostasis but also the natural course of bleeding disorders in children, which may differ substantially from the course observed in adult patients. In this article, three bleeding disorders of great importance to the pediatric hematologist are reviewed: neonatal alloimmune thrombocytopenia (NAIT), hemophilia and immune-mediated thrombocytopenic purpura (ITP). Current aspects of management are outlined. The unique physiology of transplacental transfer of maternally derived anti-platelet antibodies can result in neonatal immune thrombocytopenia, a significant cause of morbidity and mortality from bleeding in affected infants. For patients with hemophilia, approaches to treatment have shifted over the past decade from on-demand therapy to prophylaxis, either primary of secondary, resulting in delay of onset or complete avoidance of hemophilic arthropathy. Hemophilic inhibitors often develop in young children, prompting the need for a thorough understanding of the use of bypassing agents as well as immune tolerance induction in the young child. Finally, although several management strategies for ITP of childhood have been shown to improve the platelet count, side effects associated with corticosteroids, IVIg, anti-D and splenectomy force the practitioner to also consider the option of carefully observing, but not treating, the child with ITP

During the past two decades, the improvement of therapeutic agents for the management of haemophilia has created the opportunity for individuals with haemophilia to live normal lives. However, in some instances, the progress made has been accompanied by emergence of unexpected risks and other new complications. A number of viruses have either emerged, or become greater risks to people with haemophilia. In addition, the drive of many countries towards self-sufficiency in blood products may in fact be endangering people with haemophilia by restricting blood donation to a pool of donors with high infection risk, discouraging commercial interests from developing safer products, and discouraging use of 'foreign' products even where that may be the safer option. Gene therapy for haemophilia, although an encouraging new treatment, has brought with it a number of adverse events, including risk of virus infection and development of carcinomas. The risk of inhibitors is still the most important problem for people with haemophilia, and a recent report showed that the type of factor concentrate does not impact significantly on this risk. Despite the advent of new and promising treatments for haemophilia, heathcare providers must be alert to new risks posed by them

Autoimmune hemolytic anemia is rare in children. It is generally diagnosed in relation to a viral or bacterial infection but has also been described in association with drugs, autoimmune disease, malignancy, and immunodeficiency. The authors describe a 5-month-old infant who presented with severe autoimmune hemolytic anemia and disseminated intravascular coagulation as his initial manifestation of HIV infection

Recombinant factor VIII and IX products have well-established efficacy and safety records. However, concerns about the possibility of viral transmission have prompted efforts to develop recombinant products that are free of added human and animal proteins. The currently licensed second-generation recombinant factor VIII concentrates were introduced in 2000. Two new third-generation products, manufactured without any human- or animal-derived materials, are currently in development and clinical testing. As an alternative to exogenous factor replacement, gene therapy is under investigation for use in the treatment of hemophilia. Gene therapy involves the stable insertion of a functional gene for long-term expression and secretion of endogenous factor VIII or IX protein. Methods used to date have been based on retroviral, adenoviral, and adeno-associated viral vectors, as well as nonviral electroporation. Three phase I trials using these approaches have been completed as of 2002, and one more is ongoing. This article reviews the results of recent clinical studies investigating third-generation recombinant products and gene-based approaches to hemophilia treatment