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

Abstract

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.

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

“…For instance, apolipoprotein B knockdown by a scAAV-delivered shRNA in murine liver resulted in a specific reduction of LDL cholesterol in diet-induced dyslipidemic mice, whereas high-density lipoprotein (HDL) cholesterol remained unaffected. 138 An additional option for treatment stems from retrospective profiling studies, which link aberrant microRNA expression to disease development and progression. A recent study showed that microRNA-122 inhibition by scAAV-delivered Tough Decoy RNA (a recently developed, compact, RNA polymerase III-driven microR-NA decoy) 139 lowers both HDL and LDL in healthy mice.…”

Systemic RNAi Delivery to the Muscles of ROSA26 Mice Reduces lacZ Expression

“…Previous studies showing liver toxicity with a variety of vectors carrying shRNA sequences 21–25 nucleotides also showed that shorter shRNA sequences often displayed no toxicity [17], [22]. We therefore developed a 19-mer shRNA sequence against β-gal and delivered it to ROSA26 mice using tail vein injection of 2×10 12 vg of rAAV6 (Figure 1).…”

Therapeutic gene targeting approaches for the treatment of dyslipidemias and atherosclerosis

“…Liverdirected ApoB siRNAs decreased total lipid levels by 70%, when the ApoB was decreased by 95% in the liver of mice [10]. Furthermore, AAV-mediated shRNA knockdown of ApoB reduced cholesterol in mice 8 weeks after a single injection of AAV-shApoB [27]. However, fat accumulation within the liver was detected [27].…”

“…Furthermore, AAV-mediated shRNA knockdown of ApoB reduced cholesterol in mice 8 weeks after a single injection of AAV-shApoB [27]. However, fat accumulation within the liver was detected [27]. In nonhuman primates, ApoB siRNAs encapsulated to stable nucleic acid lipid particles (SNALPs) lowered total cholesterol levels up to 62% with 2.5 mg/kg [28].…”