Hammerhead ribozyme as a therapeutic agent for hyperlipidemia: production of truncated apolipoprotein B and hypolipidemic effects in a dyslipidemia murine model.
In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we used adenovirus-mediated vector to target hammerhead ribozyme at GUA(6679) downward arrow of apoB mRNA (designated AvRB15) in the liver of a dyslipidemic mouse model that is deficient in apoB mRNA editing enzyme and overexpresses human apoB100. In this study, we delivered approximately 4 x 10(11) virus particles of AvRB15 (active ribozyme) or AvRB15-mutant (inactive ribozyme) to the animals. Using Southern blot analysis, we readily detected RB15 DNA in the mouse liver as long as day 35 after injection. This result was correlated with the RNA expression of RB15 by RNase protection assay. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site was confirmed by sequencing. Importantly, the levels of human and mouse apoB mRNA decreased approximately 80% after AvRB15 transduction. There was a marked decrease in plasma cholesterol, triglyceride, and human apoB of 42, 51, and 62%, respectively, when compared with the inactive ribozyme-treated group. Moreover, ribozyme cleavage of apoB mRNA generated a truncated protein of the expected size (apoB48.1), which was associated with lipoprotein particles in the very low density, low density, and high density lipoprotein fractions. Taken together, these results indicate that apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.
Enjoji
M
M
Departments of Medicine and Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Wang
F
F
Nakamuta
M
M
Chan
L
L
Teng
B B
BB
D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000097:blood / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D002784:Cholesterol / Q000097:blood; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood / Q000628:therapy*; D008074:Lipoproteins / Q000097:blood; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction / Q000379:methods; D016337:RNA, Catalytic / Q000235:genetics / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D014280:Triglycerides / Q000097:blood
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008074:Lipoproteins; D016337:RNA, Catalytic; D011994:Recombinant Proteins; D014280:Triglycerides; C427204:hammerhead ribozyme; D002784:Cholesterol