Development of antithrombotic miniribozymes that target peripheral tryptophan hydroxylase.
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.
Peter
Jens-Uwe
JU
Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195, Berlin, Germany.
Alenina
Natalia
N
Bader
Michael
M
Walther
Diego J
DJ
D000818:Animals; D001483:Base Sequence; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D013816:Thermodynamics; D014365:Tryptophan Hydroxylase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D005343:Fibrinolytic Agents; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012343:RNA, Transfer; D014365:Tryptophan Hydroxylase