Apolipoprotein B Knockdown by AAV-delivered shRNA Lowers Plasma Cholesterol in Mice

• Published in: Molecular therapy Vol. 19; no. 4; pp. 731 - 740
• Main Authors: Koornneef, Annemart, Maczuga, Piotr, van Logtenstein, Richard, Borel, Florie, Blits, Bas, Ritsema, Tita, van Deventer, Sander, Petry, Harald, Konstantinova, Pavlina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2011; Elsevier Limited; Nature Publishing Group
• Subjects: Adeno-associated virus; Animals; Apolipoproteins; Apolipoproteins B - genetics; Apolipoproteins B - metabolism; Blotting, Western; Cardiovascular disease; Cell Line; Cell Line, Tumor; Cholesterol; Cholesterol - blood; Cholesterol - genetics; Dependovirus - genetics; Gastroenterology; Gene therapy; Genetic Vectors - genetics; Hepatology; High density lipoprotein; Humans; Lipoproteins; Liver; Macaca; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Original; Proteins; R&D; Research & development; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering - genetics; Statins; Toxicity; Netherlands
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2011.6

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.