Joerg Schuettrumpf

Factor V Leiden improves in vivo hemostasis in murine hemophilia models

Summary.  The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real‐time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser‐induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.

Corrigendum to “Inadvertent Germline Transmission of AAV2 Vector: Findings in a Rabbit Model Correlate with Those in a Human Clinical Trial”

The name of Jurg Sommer was incorrectly listed as Jurg Summer in the author line. The correct author line appears above.

750. AAV-2 Vectors and Protease-Activated Receptors: A Potential Interface between Coagulation and Gene Transfer

Phase I clinical trials demonstrated that AAV based on serotype 2 transduce human skeletal muscle and hepatocytes in vivo. AAV-2 is safe and well tolerated at doses tested, and an ongoing liver-directed study resulted in therapeutic FIX levels for hemophilia B. From these studies it is important to determine the minimum safe dose required for therapeutic efficacy. To this end, factors that increase or decrease expression by even modest amounts may become critical in the development of this novel therapeutic. We have uncovered evidence for an unexpected role of specific blood proteases in insuring optimal AAV-2 but not for AAV-5 or AAV-8 vectors. The use of specific inhibitors to thrombin or FXa (common anticoagulant drugs) prior to vector injection in mice resulted in low transgene expression (Blood 102:794a). Proteases trigger cellular responses at least in part through protease-activated receptors (PAR). Recent data show that PAR activation by thrombin enhances αVβ5-dependent (co-receptor for AAV-2) cellular function. Using PAR-1 and-2 knockout mice we sought to determine whether PAR or β5 integrin is required for AAV-2 transduction. Experiments were carried out in adult male C57Bl/6 mice homozygous (−/−) or heterozygous (+/−) for the null alleles of PAR-1, PAR-2 or β5 and compared to wild-type littermate as controls (+/+). A rAAV-2 encoding F.IX gene under the control of liver-specific promoter (hAAT/ApoE) was injected by tail vein at dose of 1 × 1012 vg/kg and circulating FIX levels were monitored for at least 10 weeks following vector injection. Injection of β5 deficient mice (n=6) resulted in FIX levels of 514 ng/ml which were lower than 3,837 ± 1,2 ng/ml among 12 controls (p < .0005). These results suggest that β5 co-receptor properties on AAV-2 transduction correlate well with previous in vitro observations (Nat Med 5:78). To test whether PARs have any role independently of β5, we further injected 19 mice of PAR-1 (−/−) or PAR-1 (+/−) genotype, and F.IX levels were 1,330 ± 376 and 1,782 ± 280 ng/ml, respectively. Among controls (n=10) levels of F.IX were 4,017 ± 1521 ng/ml. These differences were statistically significant when controls were compared to PAR-1 (−/−) or PAR-1 (+/−) at p < .005 and p <.05, respectively. Similarly, among 20 animals of PAR-2 mice (−/−) or (+/−) genotypes, FIX levels were 717 ± 222 ng/ml and 1,197 ± 648 ng/ml, respectively. Injection of controls (n=11) resulted in 3,540 ± 332 ng/ml. Next we injected AAV-2 vector in wild-type animals (n=4/group) with 10 μM/kg of specific activation peptides of PAR-1, PAR-2, or both PAR-1/-2 and compared with injection of scramble peptide as control (LSIGRL). Following simultaneous activation of PAR-1/-2 by SFLLRN, FIX levels were higher when compared to controls (2,654 vs. 1,248 ng/ml; p < 0.005). HepG2 cells, as a model for human hepatocytes, were transduced with AAV-2 following PAR-1/-2 activation and again F.IX secreted in the conditioned media was ~2-fold higher compared to control peptide. Together these data suggest a novel role of PARs and coagulation on gene transfer by viral vectors. PARs modulation may serve as an alternative strategy to improve viral vectors-cell interaction.