24 research outputs found
The Endothelial Protein C Receptor (EPCR) Regulates Endogenous Factor VII Levels in Mice
Generation of Transgenic Mice Expressing High Levels of Activated Murine Coagulation Factor VII.
Lessons from Transgenic Mice Expressing Supra-Physiological Levels of Activated Murine Factor VII.
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
Rotational thromboelastometry can predict the probability of bleeding events in a translational rat model of haemophilia A following gene-based FVIIa prophylaxis
The endothelial protein C receptor enhances hemostasis of FVIIa administration in hemophilic mice in vivo
Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII
The Development and Characterization of an scFv-Fc Fusion-Based Gene Therapy to Reduce the Psychostimulant Effects of Methamphetamine Abuse
Methamphetamine (METH) continues to be among the most addictive and abused drugs in the United States. Unfortunately, there are currently no Food and Drug Administration-approved pharmacological treatments for METH-use disorder. We have previously explored the use of adeno-associated viral (AAV)-mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH single-chain variable fragment (scFv)-Fc fusion construct (termed 7F9-Fc) packaged into AAV serotype 8 vector (called AAV-scFv-Fc) and tested in vivo and ex vivo. A range of doses [1 × 10
, 1 × 10
, and 1 × 10
vector copies (vcs)/mouse] were administered to mice, eliciting a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3831 µg/ml, respectively. Expressed 7F9-Fc exhibited high-affinity METH binding, IC
= 17 nM. Between days 21 and 35 after vector administration, at both 1 × 10
vc/mouse and 1 × 10
vc/mouse doses, the AAV-7F9-Fc gene therapy significantly decreased the potency of METH in locomotor assays. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH in the serum than vehicle-treated mice, and METH concentrations in the brain were reduced by 1.2 times the value for vehicle mice. These data suggest that an AAV-delivered anti-METH Fc fusion antibody could be used to persistently reduce concentrations of METH in the central nervous system. SIGNIFICANCE STATEMENT: In this manuscript, we describe the testing of a novel antimethamphetamine (METH) single-chain variable fragment-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of significant antibody concentrations that mitigate METH's psychostimulant effects in mice over an extended time period
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The development and characterization of an scFv-Fc fusion based gene therapy to reduce the psychostimulant effects of methamphetamine abuse
Abstract Methamphetamine (METH) continues to be amongst the most addictive and abused drugs in the US. Unfortunately, there are currently no FDA approved pharmacological treatments for METH substance abuse disorder. As an alternative approach, we have previously explored the use of Adeno-associated viral (AAV) mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH scFv-Fc fusion construct (7F9-Fc), packaged into AAV serotype 8 vector (called AAV-scFv-Fc), and tested in vivo and ex vivo. A range of doses (1 × 10 10 . 1 × 10 11 , and 1 × 10 12 vector copies(vc)/mouse) were administered to mice, which exhibited a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3,831 μg/ml. The dose of 1 × 10 12 vc/mouse was further tested in METH locomotor and biodistribution studies to determine the efficacy of the antibody protection. Expressed 7F9-Fc exhibited high affinity binding, 17 nM, to METH. Between days 21 and 35 after vector administration, the 7F9-Fc gene therapy significantly reduced the effects of METH in locomotor assays following administration of moderate and high doses of subcutaneous METH, 3.1 and 9.4 mg/kg respectively. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH into the serum than vehicle mice, and METH concentrations in the brain were reduced by 1.2 times compared to vehicle mice. Taken together, these data suggest that a AAV-delivered anti-METH Fc fusion antibody could be a design for persistently reducing concentrations of METH in the CNS. Significance Statement In this manuscript, we describe the use of a novel anti-METH scFv-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of enough antibody to mitigate METH’s psychostimulant effects in mice over an extended time period