PB0729 - Development of a viral vector for hemopexin protein expression for gene therapy in disorders associated with increased levels of free extracellular heme

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 2x1012, 1x1013 and 2x1013 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.