Efficient inhibition of beta-secretase gene expression in HEK293 cells by tRNAVal-driven and CTE-helicase associated hammerhead ribozymes.
The beta-amyloid peptide (Abeta) is a major component of toxic amyloid plaques found in the brains of patients with Alzheimer's disease. Abeta is liberated by sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The level of Abeta depends directly on the hydrolytic activity of beta-secretase. Therefore, beta-secretase is an excellent target for drug design. An approach based on RNA-cleaving ribozymes was developed to control expression of beta-secretase. Two sites of mRNA coding beta-site APP cleaving enzyme were chosen as target sequences for endogenously delivered ribozymes. The ribozyme cassette was designed to constitute a catalytic hammerhead core and substrate recognition arms, flanked at the 5'-terminus by tRNAVal and at the 3'-terminus by constitutive transport element sequences. Ribozyme cassettes were cloned into a pUC19 plasmid and used for transient transfection of HEK293 cells. We demonstrate that such ribozymes efficiently inhibit beta-secretase gene expression at both the mRNA (up to 95%) and the protein (up to 90%) levels. Inhibition of beta-site APP cleaving enzyme activity directly influences the intra- and extracellular population of Abeta peptide. Therefore, such ribozymes may be considered as molecular tools for silencing the beta-secretase activity, and further, as therapeutic agents for anti-amyloid treatment.
D016229:Amyloid beta-Peptides / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012366:RNA, Transfer, Val / Q000235:genetics / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D016337:RNA, Catalytic; D012366:RNA, Transfer, Val; C427204:hammerhead ribozyme; D016282:Aspartic Acid Endopeptidases; D020365:RNA Helicases