ARFI Ultrasound Monitoring of Hemorrhage and Hemostasis In Vivo in Canine Von Willebrand Disease and Hemophilia

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ~2mm vein in six normal, eight naïve hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naïve VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naïve hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naïve hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders

Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII

Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Ab’s to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders

Anti‐Methamphetamine Antibody Gene Therapy Ameliorates Methamphetamine‐induced Locomotor Effects in Mice for 8 Months after a Single Treatment

There currently are no FDA‐approved pharmacological treatments to aid in treatment of methamphetamine (METH) substance use disorders. To address this, our laboratory has combined short chain variable fragments (scFvs) derived from antibodies with high binding affinity to METH with adeno‐associated viruses (AAV) to develop a long‐lasting potential therapy. These first generation gene therapies showed expression of the anti‐METH scFvs for at least 212 days but achieved an average serum concentration of less than 70.0 μg/ml in mice. We hypothesized that incorporating a constant region (Fc) derived from an IgG antibody would extend serum half‐life and increase circulating concentrations of the anti‐METH antibody fragment, thus providing greater levels of protection against METH in mice. To test this hypothesis, we cloned an anti‐METH scFv‐Fc encoding DNA sequence into custom expression plasmids and packaged into AAV serotype 8 viral vectors to make AAV‐7F9‐Fc. To test this new gene therapy, BALB/C mice were administered 1×1012 vector copies per mouse of either the AAV‐7F9‐Fc or empty AAV8 vector. Mice were allowed to reach peak AAV‐7F9‐Fc expression (~28 days, as determined by ELISA) before testing in locomotor studies. Locomotor activity was used to measure protection from the stimulant effects of METH. Briefly, after a 30‐minute baseline activity was recorded, mice were administered saline, 0.56, 1.0, 3.0, or 5.56 mg/kg METH sc, and locomotor activity was measured for 90 minutes. METH was administered consecutively with a two‐day washout period in between doses. This regimen was also performed on separate mice 7–8 months after administration of AAV‐7F9‐Fc therapy to test duration of protection. Our results show that by incorporating an IgG Fc moiety into the anti‐METH constructs, the average expression increased by over 30 fold compared to a our first generation scFvs to a peak serum concentration of 5.227.7 μg/ml, and mean of 2,116.1 μg/ml, and persisted over 239 days. The AAV‐7F9‐Fc mice showed significantly less locomotor activity (p < 0.05) than empty AAV8 treated mice at the 1.0 and 3.0 mg/kg METH doses. Furthermore, the AAV‐7F9‐Fc treated mice showed no significant activity differences from baseline at any of the METH doses administered. In a separate, long‐term study, 7–8 months after AAV‐7F9‐Fc treatment, mice showed significantly lower levels of locomotor activity at 0.56 and 1.0 mg/kg sc METH doses than the sham treated group. These data suggest our AAV‐7F9‐Fc can protect against stimulant effects of METH, and that the duration of action is at least 8 months in mice. Overall, these data suggest that 1) the addition of the IgG Fc region into the AAV‐scFv constructs resulted in dramatically higher anti‐METH antibody circulating concentrations than our first generation anti‐METH scFvs and 2) this higher antibody titer was able to provide protection against METH stimulant effects at low to moderate doses for at least 8 months in mice. Support or Funding Information Supported by NIDA R01 DA036600 & NIGMS T32 GM106999 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal

Long-term expression of murine activated factor VII is safe, but elevated levels cause premature mortality

Intravenous infusion of recombinant human activated Factor VII (FVIIa) has been used for over a decade in the successful management of bleeding episodes in patients with inhibitory antibodies to Factor VIII or Factor IX. Previously, we showed that expression of murine FVIIa (mFVIIa) from an adeno-associated viral (AAV) vector corrected abnormal hemostatic parameters in hemophilia B mice. To pursue this as a therapeutic approach, we sought to define safe and effective levels of FVIIa for continuous expression. In mice transgenic for mFVIIa or injected with AAV-mFVIIa, we analyzed survival, expression levels, in vitro and in vivo coagulation tests, and histopathology for up to 16 months after birth/mFVIIa expression. We found that continuous expression of mFVIIa at levels at or below 1.5 μg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 μg/ml or higher were associated with thrombosis and early mortality, with pathologic findings in the heart and lungs that were rescued in a low–factor X (low-FX) mouse background, suggesting a FX-mediated effect. The findings from these mouse models of continuous FVIIa expression have implications for the development of a safe gene transfer approach for hemophilia and are consistent with the possibility of thromboembolic risk of continuously elevated FVIIa levels