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.).

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.

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.

[Clinical application of the activated partial thromboplastin time (APTT) with modified incubation time for the diagnosis of lupus anticoagulant (LA)]. / Aplicación del tiempo de tromboplastina parcial activado (TTPA) con tiempo de incubación modificado en el diagnóstico del anticoagulante lúpico (AL).

BACKGROUND: Although several tests are used to screen for the presence of LA, none detects all its types. The shortening of APT observed when the pre-incubation period is prolonged, proved to be a sensitive test for the presence of LA. MATERIAL AND METHODS: We determined the APTT, performed with a 4 or 15 min preincubation period (APETs and APTT15 respectively), in 22 healthy subjects, 3 commercial positive controls for LA, 16 patients with a previous diagnosis of LA and 54 patients with recurrent fetal loss and/or infertility. Evidence of LA was established by a positive Staclot-LA test. RESULTS: APTTs and APTT15 were 31.5 +/- 4.7 and 28.4 +/- 4.5 seconds respectively in samples from 22 normal subjects. The figures in samples with LA, were 71.5 +/- 20.3 s and 58.6 +/- 18 s respectively. The difference between the two APTTs performed on an individual sample was defined as the APTT 4-15 and was 2.6 +/- 2.0 in normal subjects 2.5 +/- 2.8 in 13 patients anticoagulated with warfarin, -10.0 +/- 6.5 in 13 patients receiving heparin, and 13.2 +/- 4.9 in 15 patients with LA. The test values for LA patients were significantly higher than those for normal subjects (p < 0.0001). For values over 5, the APTT 4-15 had 93.3% sensitivity and 100% specificity. In one patient with recurrent fetal loss or infertility, who was LA positive, the APTT 4-15 was positive with a value of 14. CONCLUSIONS: This modified TTPA is easy to perform, and provides a reasonably discriminatory value for the presence of LA. Therefore, we recommend the TTPA 4-15 to screen for LA.

Aplicación del tiempo de tromboplastina parcial activado (TTPA) con tiempo de incubación modificado en el diagnóstico del anticoagulante lúpico (AL) / Clinical application of the activated partial thromboplastin time (APTT) with modified incubation time for the diagnosis of lupus anticoagulant (LA)

Background: Although several tests are used to screen for the presence of LA, none detects all its types. The shortening of APTT observed when the pre-incubation period is prolonged, proved to be a sensitive test for the presence of LA. Material and methods: We determined the APTT, performed with a 4 or 15 min preincubation period (APTTs and APTT15 respectively), in 22 healthy subjects, 3 commercial positive controls for LA, 16 patients with a previous diagnosis of LA and 54 patients with recurrent fetal loss and/or infertility. Evidence of LA was established by a positive Staclot-LA test. Results: APTTs and APTT15 were 31.5±4.7 and 28.4±4.5 seconds respectively in samples from 22 normal subjects. The figures in samples with LA, were 71.5±20.3 s and 58.6±18 s respectively. The difference between the two APTTs performed on an individual sample was defined as the APTT 4-15 and was 2.6±2.0 in normal subjects 2.5±2.8 in 13 patients anticoagulated with warfarin, -10.0±6.5 in 13 patients receiving heparin, and 13.2±4.9 in 15 patients with LA. The test values for LA patients were significantly higher than those for normal subjects (p <0.0001). For values over 5, the APTT 4-15 had 93.3 per cent sensitivity and 100 per cent specificity. In one patient with recurrent fetal loss or infertility, who was LA positive, the APTT 4-15 was positive with a value of 14. Conclusions: This modified TTPA is easy to perform, and provides a reasonably discriminatory value for the presence of LA. Therefore, we recommend the TTPA 4-15 to screen for LA.