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Free keywords:
TPH1 - serotonin - thrombosis
Abstract:
Serotonin is not only a neurotransmitter in the central nervous system, but also a ubiquitous hormone in the periphery involved in vasoconstriction and platelet function. Tryptophan hydroxylase is the rate-limiting enzyme in serotonin biosynthesis. By gene targeting, we have shown that serotonin is synthesized independently by two different tryptophan hydroxylase isoenzymes in peripheral tissues and neurons and identified a neuronal tryptophan hydroxylase isoform. Mice deficient in peripheral tryptophan hydroxylase (TPH1) and serotonin exhibit a reduced risk of thrombosis and thromboembolism. Therefore, we designed several antitph1 hammerhead miniribozymes and tested their cleavage activity against short synthetic Tph1 RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of Tph1 mRNA that was dependent on substrate/miniribozyme ratio and duration of exposure to miniribozyme. Interestingly, we detected different in vitro cleavage rates after we had cloned the miniribozymes into tRNA expression constructs, and found one with a high cleavage rate. We also demonstrated that this active tRNA–miniribozyme chimera is capable of selectively cleaving native Tph1 mRNA in vivo, with concomitant downregulation of the serotonin biosynthesis. Therefore, this Tph1-specific miniribozyme may provide a novel and effective form of gene therapy that may be applicable to a variety of thrombotic diseases.
