Abstract Number: PB0729
Meeting: ISTH 2022 Congress
Theme: Hemostatic Systems in Cancer, Inflammation and Immunity » Coagulation Proteins Beyond Hemostasis
Background: Extracellular heme induces tissue factor-dependent coagulation activation and tissue damage. Hemopexin (HPX), responsible for removing free heme from the circulation, has been shown to be depleted in hemolytic diseases. Gene therapy with adeno-associated viral vectors (AAV) has been applied to replenish circulating proteins in hemophilia with promising results
Aims: Development of an adeno-associated virus-based vector for HPX expression
Methods: An AAV-8 vector was produced containing the full cDNA sequence of human HPX (hHPX). Mice were injected with escalating doses of 2×1012, 1×1013 and 2×1013 vector genomes/kg, while a control group received an empty vector. Blood was obtained every two weeks for HPX ELISA. Expression was also confirmed in liver samples obtained after 6 weeks, and functional activity assessed by heme challenge and phenylhydrazine-induced hemolysis.
Results: Liver hHPX expression was confirmed by RT-PCR and western blot. Sustained expression of hHPX were observed up to 58 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. Levels of hHPX were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short term studies of heme challenge.
Conclusion(s): Long-term expression of hHPX can be achieved with AAV vectors in mice. Additional studies are required to confirm the biological effect of the transgenic protein and its effect in animal models of chronic hemolysis.
Image
-a- mRNA expression of hHPX in liver from mice treated with intravenous injections of rAAV-HPX or with an empty vector -n=6 per group; organs were obtained 6 weeks after vector infusion-; -b- hHPX expression is demonstrated by western blot of liver samples obtained 6 weeks after vector infusion -n=3 per group-; -c- serial determination of hHPX levels in plasma form C57Bl/6 mice up to 46 weeks of vector infusion measured by ELISA with an antibody that does not cross-react with murine HPX; -d- kinetics of hHPX levels before -basal- and after -day+7- the infusion of 70 µmol/kg heme -i.p.- as determined by ELISA.
To cite this abstract in AMA style:
Lima F, Hounkpe B, Peachazepi de Moraes C, Borba I, Costa F, de Paula E. Development of a viral vector for hemopexin protein expression for gene therapy in disorders associated with increased levels of free extracellular heme [abstract]. https://abstracts.isth.org/abstract/development-of-a-viral-vector-for-hemopexin-protein-expression-for-gene-therapy-in-disorders-associated-with-increased-levels-of-free-extracellular-heme/. Accessed November 29, 2023.« Back to ISTH 2022 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/development-of-a-viral-vector-for-hemopexin-protein-expression-for-gene-therapy-in-disorders-associated-with-increased-levels-of-free-extracellular-heme/