Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues.
Hemopexin (HPX) is a circulating
protein responsible for removing free
heme from the circulation, whose levels can be severely depleted in conditions such as
sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of
heme in these conditions. Gene transfer of liver-produced
proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in
hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by
enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the
biological activity of transgenic hHPX was confirmed using two different models of
heme challenge consisting of serial
heme injections or
phenylhydrazine-induced
hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels 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
heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of
heme overload. Additional studies are needed to explore the effect of transgenic HPX
protein in animal models of chronic
hemolysis.