Apolipoprotein B Knockdown by AAV-delivered shRNA Lowers Plasma Cholesterol in Mice
Authors: Annemart Koornneef, Piotr Maczuga, Richard van Logtenstein, Florie Borel, Bas Blits, Tita Ritsema, Sander van Deventer, Harald Petry, Pavlina Konstantinova
Journal: Molecular Therapy (2011)

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. 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. IntroductionElevated levels of serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) are major risk factors and contributors to the development of atherosclerosis and cardiovascular disease. Currently, hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) are the preferred pharmacologic agents for hypercholesterolemia treatment due to their superior efficacy in lowering LDL-C, proven ability to reduce cardiovascular disease morbidity and mortality, and a low incidence of adverse events. However, statins are not uniformly effective in all patients and some patients can be relatively refractory to treatment due to absent or diminished LDL-C receptor activity.1Rader DJ Cohen J Hobbs HH Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J Clin Invest. 2003; 111: 1795-1803Crossref PubMed Scopus (468) Google Scholar Furthermore, 10–15% of patients are unable to take statins due to intolerable muscle symptoms like myalgias, myositis, or mitochondrial injury.2Kashani A Phillips CO Foody JM Wang Y Mangalmurti S Ko DT et al.Risks associated with statin therapy: a systematic overview of randomized clinical trials.Circulation. 2006; 114: 2788-2797Crossref PubMed Scopus (408) Google Scholar Thus, effective therapeutic alternatives with a different mechanism of action are needed.Apolipoprotein B (ApoB) is required for the assembly of lipoprotein particles and is a constituent of all classes of lipoproteins that are considered atherogenic.3Young SG Recent progress in understanding apolipoprotein B.Circulation. 1990; 82: 1574-1594Crossref PubMed Scopus (328) Google Scholar ApoB exists in two isoforms, ApoB100 and ApoB48, which are transcribed from a single gene. ApoB100 is the full-length ApoB protein and is synthesized in the liver. It forms the structural protein in very LDL-C (VLDL-C) and LDL-C particles, which are essential for the transport and metabolism of cholesterol. ApoB48 contains the amino-terminal 2,152 amino acids of ApoB100 as a result of ApoB mRNA editing which yields an early stop codon. In humans, ApoB is edited solely in the small intestine where ApoB48 plays a role in dietary fat absorption as part of chylomicrons. By contrast, some species such as mice and rats also edit ApoB in their liver and hence synthesize both ApoB100 and ApoB48.4Greeve J Altkemper I Dieterich JH Greten H Windler E Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins.J Lipid Res. 1993; 34: 1367-1383Abstract Full Text PDF PubMed Google ScholarElevated ApoB levels correlate with increased risk of coronary artery disease.5Zambon A Brown BG Deeb SS Brunzell JD Genetics of apolipoprotein B and apolipoprotein AI and premature coronary artery disease.J Intern Med. 2006; 259: 473-480Crossref PubMed Scopus (30) Google Scholar As a large, lipid-associated protein, ApoB belongs to the so-called "non-druggable" targets, which are not amenable to conventional therapies. However, ApoB is a highly validated target for treatment of increased LDL-C levels and subsequent cardiovascular disease.6Raal FJ Santos RD Blom DJ Marais AD Charng MJ Cromwell WC et al.Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial.Lancet. 2010; 375: 998-1006Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar An approach targeting ApoB with second-generation antisense oligonucleotides has progressed into phase III clinical trials for treatment of homozygous familial hypercholesterolemia.6Raal FJ Santos RD Blom DJ Marais AD Charng MJ Cromwell WC et al.Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial.Lancet. 2010; 375: 998-1006Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar Another therapeutic approach that holds great promise for silencing ApoB is based on RNA interference (RNAi).RNAi is an evolutionary conserved sequence-specific post-transcriptional gene-silencing mechanism in eukaryotes that results in the degradation of mRNA and subsequently in decrease of protein synthesis.7Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11581) Google Scholar Therapeutic RNAi can be induced by synthetic small interfering RNAs (siRNAs) or by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs) that target a disease-causing gene.8Soutschek J Akinc A Bramlage B Charisse K Constien R Donoghue M et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1889) Google Scholar,9Grimm D Small silencing RNAs: state-of-the-art.Adv Drug Deliv Rev. 2009; 61: 672-703Crossref PubMed Scopus (155) Google Scholar,10Liu YP Haasnoot J ter Brake O Berkhout B Konstantinova P Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron.Nucleic Acids Res. 2008; 36: 2811-2824Crossref PubMed Scopus (176) Google Scholar shRNAs are typically expressed from a constitutive strong polymerase III (pol III) promoter, and are processed intracellularly by Dicer, rendering a mature siRNA which interacts with the cytosolic multiprotein RNA-induced silencing complex (RISC). The RISC-incorporated siRNA binds to the complementary mRNA sequence and mediates its sequence-specific degradation.11Zamore PD Tuschl T Sharp PA Bartel DP RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google ScholarThe largest obstacle in using synthetic siRNAs as therapeutics in vivo is the need for efficient and specific delivery to target cells and organs. Chemical modifications and conjugations resulting in lower effective dose and longer half-life of the siRNA have improved their efficacy.12Love KT Mahon KP Levins CG Whitehead KA Querbes W Dorkin JR et al.Lipid-like materials for low-dose, in vivo gene silencing.Proc Natl Acad Sci USA. 2010; 107: 1864-1869Crossref PubMed Scopus (635) Google Scholar,13Semple SC Akinc A Chen J Sandhu AP Mui BL Cho CK et al.Rational design of cationic lipids for siRNA delivery.Nat Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1097) Google Scholar Chemically modified ApoB-specific siRNAs, when delivered systemically, can silence ApoB expression up to 90% in murine liver.8Soutschek J Akinc A Bramlage B Charisse K Constien R Donoghue M et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1889) Google Scholar,14Rozema DB Lewis DL Wakefield DH Wong SC Klein JJ Roesch PL et al.Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.Proc Natl Acad Sci USA. 2007; 104: 12982-12987Crossref PubMed Scopus (562) Google Scholar,15Wolfrum C Shi S Jayaprakash KN Jayaraman M Wang G Pandey RK et al.Mechanisms and optimization of in vivo delivery of lipophilic siRNAs.Nat Biotechnol. 2007; 25: 1149-1157Crossref PubMed Scopus (743) Google Scholar Inclusion of siApoB in liposomal formulations is another highly effective approach for ApoB silencing in the liver. Systemic delivery of these molecules in nonhuman primates showed immediate, potent, and lasting ApoB and cholesterol reduction, which exceeded the results obtained with rodents in terms of degree and persistence of ApoB silencing.16Zimmermann TS Lee AC Akinc A Bramlage B Bumcrot D Fedoruk MN et al.RNAi-mediated gene silencing in non-human primates.Nature. 2006; 441: 111-114Crossref PubMed Scopus (1175) Google Scholar However, even with current improvements, siRNAs need to be delivered monthly or bimonthly, which is not achievable for many diseases, especially in the brain, eye, heart, or other internal organs. An alternative for delivery of small RNAs is administration of a single dose of recombinant viral vectors based on lentivirus or adeno-associated virus (AAV), which yield long-term expression.17Manjunath N Wu H Subramanya S Shankar P Lentiviral delivery of short hairpin RNAs.Adv Drug Deliv Rev. 2009; 61: 732-745Crossref PubMed Scopus (135) Google Scholar,18Grimm D Pandey K Kay MA Adeno-associated virus vectors for short hairpin RNA expression.Meth Enzymol. 2005; 392: 381-405Crossref PubMed Scopus (56) Google Scholar The latter holds a unique set of advantages over other vector systems, including the complete lack of pathogenicity and low immunogenicity.19Daya S Berns KI Gene therapy using adeno-associated virus vectors.Clin Microbiol Rev. 2008; 21: 583-593Crossref PubMed Scopus (625) Google Scholar AAV induces efficient and long-term transduction of nondividing cells, an important facet for liver-based gene therapeutic strategies.20Vaessen SF Veldman RJ Comijn EM Snapper J Sierts JA van den Oever K et al.AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency.J Gene Med. 2009; 11: 697-707Crossref PubMed Scopus (16) Google Scholar Unlike the use of synthetic siRNA molecules, AAV gene therapy allows long-term efficacy with a single dose.In this study, we evaluated the potential of AAV-delivered shRNA for long-term silencing of ApoB in murine liver. A single intravenous injection of AAV-shA10 resulted in prolonged ApoB knockdown with no obvious signs of toxicity, concomitant with a reduction in plasma cholesterol levels. In addition, in mice fed on an atherogenic diet there was a significant reduction of LDL-C, whereas high-density lipoprotein cholesterol (HDL-C) levels remained unchanged.ResultsDesign and validation of shRNAs targeting conserved ApoB sequencesThree conserved regions in the last exon of the human, macaque, and mouse ApoB mRNA sequences were selected to design 19 anti-ApoB shRNAs. shApoB expression plasmids (A1–A19) were constructed with the H1 pol III promoter based on a 19-nucleotide target sequence (Supplementary Table S1). Some of the shRNAs were targeting partly overlapping sequences. For example, shRNAs A9–A14 were designed by sliding the ApoB target sequence with one nucleotide. All shRNA sequences had identical five-nucleotide loop sequences and the top two basepairs of the pSuper system, and ended with a 3′ UU overhang derived from the pol III termination signal.To evaluate the suppressive activities of the 19 shApoB constructs in vitro, we co-transfected them in human embryonic kidney 293T (HEK293T) cells with renilla luciferase reporter constructs containing the last ApoB exon in the 3′ UTR (Luc-ApoB). An shRNA construct targeting green fluorescent protein (GFP) was included as a negative control and firefly luciferase expression was measured to control for transfection efficiency. Firefly and renilla luciferase expression were measured 2 days post-transfection and their ratio was used to calculate the relative luciferase activity. The renilla/firefly ratio in the presence of shGFP was set at 100% (Figure 1a). Four shApoB constructs (A9, A10, A14, and A15) were highly effective and inhibited relative Luc-ApoB expression more than 75%. Ten shApoB constructs (A4, A5, A6, A7, A11, A12, A16, A17, A18, and A19) showed moderate activity and induced 50–75% inhibition of Luc-ApoB. The remaining shApoB constructs were not active. Interestingly, A13 was inactive, whereas the following shRNA construct A14 was highly active, indicating that a single-nucleotide shift can render a shRNA ineffective. In total, one out of five tested shApoB constructs was highly active against the Luc-ApoB reporter.The ability of several shApoB constructs to knockdown endogenous ApoB was assessed in vitro. The mouse hepatoma Hepa 1–6 and human hepatoma Huh7 cell lines were transfected with the highly active shApoB constructs A9, A10, A14, and the intermediately active A4, A5, A17, and A18 constructs. As a negative control the shGFP construct was transfected. The three highly active shApoB constructs inhibited murine endogenous ApoB mRNA by ~60% while the others were less effective (Figure 1b). Similar results were obtained for human ApoB knockdown in the Huh7 cell line (data not shown). Generally, the inhibitory effect of the shApoB constructs on endogenous ApoB showed a similar trend to the Luc-ApoB knockdown results.The highly active shApoB construct A10 (shA10) was selected for further analysis, and siRNA expression from this construct (siA10) was determined in vitro. HEK293T cells were transfected with increasing amounts of shA10 plasmid, and 2 days post-transfection total RNA was isolated and siA10-specific small RNA Taqman assay was performed (Figure 1c). To calculate the copy number per cell, we used a standard line based on serial dilutions of the guide strand from a synthetic siA10 RNA oligo. Increasing the amount of transfected shA10 construct clearly correlated with an increase in expression of siA10 (Figure 1c). For example, transfection with 5 and 100 ng shA10 construct resulted in ~125 and 970 siA10 copies/cell, respectively. Having established a good correlation between shA10 activity and siA10 expression in vitro, we proceeded to test the in vivo efficacy of this shRNA construct.Efficient shRNA-mediated knockdown of ApoB and decrease of cholesterol in vivoTo test the in vivo efficacy of shA10, self-complementary AAV serotype 8 (scAAV8) viral vectors were produced encoding anti-ApoB shA10 or a control shScramble (shScr). C57BL/6 mice were injected via the tail vein with scAAV8-shA10 (AAV-shA10) or the control vector scAAV8-shScr (AAV-shScr) at four different concentrations: 108, 109, 1010, and 1011 genome copies (gc) per animal. Two weeks after injection, mice were sacrificed and liver and plasma were analyzed for ApoB knockdown. A dose-dependent increase in AAV transduction efficiency was demonstrated by monitoring GFP fluorescence intensity and GFP mRNA expression in the liver (Figure 2a and Supplementary Figure S1), and this correlated with a dose-dependent increase in siA10 and siScr expression (Figure 2b and data not shown). ApoB mRNA expression in the liver was strongly reduced by administration of increasing doses of AAV-shA10 and reached up to 95% knockdown compared to phosphate-buffered solution (PBS)-injected controls at the highest administered AAV dose (Figure 2c). In contrast, the AAV-shScr control vector did not affect ApoB mRNA levels, apart from an unexpected inhibitory effect at the highest dose of 1011 gc.Figure 2AAV-delivered shA10 strongly inhibits ApoB and plasma cholesterol levels 2 weeks post-transduction. Mice were intravenously injected with 108, 109, 1010, and 1011 gc AAV-shA10 or control AAV-shScr per animal. Two weeks after transduction, animals were sacrificed and liver and plasma were collected for analysis. (a) Representative image of GFP fluorescence in picric-acid fixed liver, which was transduced with 108, 109, 1010, or 1011 gc AAV-shA10 per animal, coexpressing GFP under control of the liver-specific LP-1 promoter. Nuclei were visualized in blue by staining with Hoechst. Photos were taken using the same exposure and magnification settings. Bar = 10 µm. (b) Small RNA TaqMan detection of siA10 expression from liver transduced with 108, 109, 1010, or 1011 gc AAV-shA10 per animal. Total RNA was isolated from snap-frozen livers and siA10-specific TaqMan was performed. (c) Relative ApoB mRNA expression in transduced liver. Total RNA was isolated from snap-frozen liver tissue and qRT-PCR was performed with ApoB- and actin-specific primers. ApoB mRNA levels were calculated relative to actin mRNA and the PBS-treated group was set at 100%. (d) Western blot analysis of secreted ApoB100 and ApoB48 protein in 0.1 µl plasma. ApoB protein was detected using a polyclonal anti-ApoB antibody, and antibody binding was visualized by a chemiluminescence detection kit. (e) Total cholesterol levels in plasma collected from fasted mice. Cholesterol levels were analyzed on the automated clinical chemistry analyzer Modular Analytics P800. (f) Plasma cholesterol distribution over lipoprotein subclasses in FPLC-separated pooled plasma from mice injected with 1011 gc AAV-shA10 or AAV-shScr per animal. Data are represented as mean + SE, treatment groups are n = 5. AAV-shA10, adeno-associated virus expressing short hairpin RNA against apolipoprotein B100; AAV-shScr, adeno-associated virus expressing short hairpin RNA scramble; FPLC, fast protein liquid chromatography; gc, genome copies; GFP, green fluorescent protein; LP-1, liver-specific regulatory element; siA10, small interfering RNA against apolipoprotein B100.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To compare the effect of AAV-shA10 on ApoB mRNA with changes in ApoB protein levels, we assayed plasma and liver ApoB protein levels by western blot at 2 weeks post-transduction. AAV-shA10 strongly reduced secreted ApoB100 in a dose-dependent manner, whereas AAV-shScr did not affect ApoB100 protein expression (Figure 2d). Similar results were observed for liver ApoB100 protein knockdown (data not shown). In mice, unlike humans, 60–70% of hepatic ApoB mRNA is edited to form truncated ApoB48. Similar to ApoB100, we found that ApoB48 protein levels in plasma and liver were reduced (Figure 2d and data not shown).These results indicate that a single AAV-shA10 dose achieves a potent knockdown of ApoB mRNA and protein in murine liver and plasma. Furthermore, knockdown of ApoB was associated with changes in total cholesterol levels. Being the structural protein of LDL-C, lowering of ApoB is expected to reduce cholesterol levels. Indeed, dose-dependent ApoB knockdown correlated tightly with reduction in total cholesterol levels, resulting in a maximal cholesterol decrease of 79% compared to PBS-treated controls (Figure 2e). Profiling of cholesterol distribution over the lipoprotein subclasses revealed a major reduction in HDL-C by AAV-shA10, concomitant with a decrease in VLDL-C and LDL-C levels (Figure 2f).AAV-shA10 administration does not cause severe toxicity in miceIt has been reported that high levels of shRNA expression can lead to severe liver toxicity symptoms like weight loss, activation of liver-specific transaminases, changes in liver morphology, or oversaturation of the cellular miRNA machinery.21Grimm D Streetz KL Jopling CL Storm TA Pandey K Davis CR et al.Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1352) Google Scholar We monitored alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels 2 weeks post-transduction with AAV-shA10 and AAV-shScr and observed no significant increase in liver transaminases for the lower viral doses of 108, 109, and 1010 gc per animal. However, the group injected with 1011 gc AAV-shScr per animal showed a 10–30-fold increase in ALT and AST levels, indicating liver damage (Figure 3a). AAV-shA10 injection at a dose of 1011 gc per animal gave rise to a two- to fivefold increase in liver transaminases (Figure 3a). Histological analysis of liver sections revealed no gross changes in liver morphology except for the 1011 gc AAV-shScr dose where abnormal morphology was observed (Supplementary Figure S2).Figure 3Effect of AAV-delivered shA10 on liver transaminases, cellular miRNA expression, and hepatic fat accumulation. Mice were intravenously injected with 108, 109, 1010, and 1011 gc AAV-shA10 or control AAV-shScr per animal. Two weeks after transduction, animals were sacrificed and liver and plasma were collected for analysis. (a) Liver transaminases ALT and AST at 2 weeks postinjection. Transaminases were analyzed on the automated clinical chemistry analyzer Modular Analytics P800. (b) Relative expression of liver-specific mir-122, let-7a, and mir-29a. Total RNA was isolated from snap-frozen livers and miRNA-specific TaqMans were performed. miRNA expression levels were calculated relative to actin mRNA and the PBS-treated group was set at 100%. Data are represented as mean + SE, treatment groups are n = 5. (c) Representative image of hepatic lipid accumulation. Frozen liver sections were stained with Oil Red O for lipids, and nuclei were counterstained with hematoxylin solution. Fat accumulation was visible starting at a dose of 109 gc AAV-shA10. Photos were taken using the same exposure and magnification settings. Bar = 10 µm. AAV-shA10, adeno-associated virus expressing short hairpin RNA against apolipoprotein B100; AAV-shScr, adeno-associated virus expressing short hairpin RNA scramble; ALT, alanine aminotransferase; AST, aspartate aminotransferase; gc, genome copies; let-7a, microRNA let-7a; mir-122, microRNA 122; mir-29a, microRNA 29a; miRNA, microRNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)shRNAs and miRNAs share the same processing pathway and nuclear export machinery and overexpression of shRNAs can modify the processing of cellular miRNA transcripts.22Kim VN Small RNAs: classification, biogenesis, and function.Mol Cells. 2005; 19: 1-15Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar We investigated whether high expression of shRNA in the liver competes with the intracellular processing of miRNAs. mir-122 was selected because it represents 70% of all liver miRNAs and is expressed almost exclusively in the liver.23Lagos-Quintana M Rauhut R Yalcin A Meyer J Lendeckel W Tuschl T Identification of tissue-specific microRNAs from mouse.Curr Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2718) Google Scholar mir-122-specific TaqMan analysis was performed on RNA samples isolated from mice 2 weeks after administration of increasing doses of AAV-shScr and AAV-shA10. Expression profiles revealed that there was no significant decrease in mir-122 expression except for the group transduced with 1011 gc AAV-shScr per animal (Figure 3b). In this group, mir-122 expression decreased >50%. Therefore, we analyzed for increased expression levels of AldoA, Bckdk, and Ndrg3, because these genes have been reported as target genes of mir-122.24Elmén J Lindow M Silahtaroglu A Bak M Christensen M Lind-Thomsen A et al.Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.Nucleic Acids Res. 2008; 36: 1153-1162Crossref PubMed Scopus (575) Google Scholar Gene expression was not significantly affected in murine liver transduced with 1011 gc AAV-shScr or AAV-shA10 (Supplementary Figure S3). We further monitored the expression of the less abundant liver miRNAs let-7a and mir-29a (Figure 3b). The expression profile of these miRNAs was similar to mir-122, indicating that there were no severe toxic effects induced by AAV-shA10 or AAV-shScr. Finally, the level of induction of markers of the interferon response (STAT1, TNF-α, INF-β, INF-γ) was examined by quantitative reverse transcriptase-PCR (qRT-PCR) on RNA isolated from murine white blood cells 1 week after viral administration. No significant induction of the interferon-pathway genes was observed upon viral administration indicating lack of shRNA toxicity at this time-point (data not shown).ApoB knockdown results in fat accumulation in murine liverApoB suppression results in decreased cholesterol levels due to impairment of VLDL-C and LDL-C assembly, but can also lead to hepatic triglyceride accumulation. Triglycerides are normally incorporated into VLDL-C, and hepatic accumulation of these lipids can potentially cause steatosis.14Rozema DB Lewis DL Wakefield DH Wong SC Klein JJ Roesch PL et al.Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.Proc Natl Acad Sci USA. 2007; 104: 12982-12987Crossref PubMed Scopus (562) Google Scholar To assess the effect of ApoB knockdown on hepatic lipid accumulation, we performed Oil red O staining on liver sections obtained from mice injected with 108, 109, 1010, or 1011 gc AAV-shScr and AAV-shA10 per animal at 2 weeks postadministration (Figure 3c). Analysis of the liver sections revealed increased accumulation of hepatic lipid content at the effective 109, 1010, and 1011 gc AAV-shA10 doses compared with PBS- and AAV-shScr-injected mice (Figure 3c). In addition, plasma triglyceride levels were also decreased after AAV-shA10 transduction, demonstrating diminished hepatic triglyceride export capacity in these mice (data not shown). These results indicate that a single AAV-shA10 administration in mice results in a dramatically decreased ApoB expression in murine liver with expected phenotypic side effects.Long-term efficacy of AAV-shA10 in murine liverIn order to monitor the viral persistence and robustness of ApoB knockdown by AAV-shA10, a second group of mice was injected with 109 and 1011 gc AAV-shA10 or control AAV-shScr and sacrificed at 8 weeks postinjection. At this time-point, we observed a significant reduction in ApoB mRNA for both AAV-shA10 viral doses confirming stable transduction of scAAV8 in murine liver (Figure 4a). However, maximum ApoB mRNA knockdown was slightly less than at 2 weeks post-transduction (Figures 2c and 4a). Similarly, GFP and siA10 expression was lower at the 8-week time-point, compared to 2 weeks postinjection. This effect was most pronounced in the group of animals injected with the 1011 dose (Supplementary Figure S4a,b). Nevertheless, AAV-shA10 was still effective at lowering ApoB100 and ApoB48 protein and cholesterol levels (Figure 4b and Supplementary Figure S4c). AAV-shA10 injection at a dose of 1011 gc per animal gave rise to a two- to fivefold increase in liver transaminases, which was most pronounced for ALT levels at 3 weeks postinjection and transaminases were still above baseline after 8 weeks (Figure 4c). During the 8-week period the highest dose of 1011 gc AAV-shScr did not induce a dramatic increase in ALT and AST levels, as was observed in the group of animals sacrificed at 2 weeks postinjection (Figures 3b and 4c). We next tested for altered expression of mir-122, mir-29a, let-7a, and mir-122 target genes at 8 weeks postinjection. No significant changes were measured in liver miRNA or mir-122 target gene expression, although a trend was observed for lower miRNA levels in the animals injected with 1011 gc AAV-shA10 (Supplementary Figure S4d,e).Figure 4ApoB mRNA and cholesterol decrease 8 weeks post-transduction with AAV-shA10. Mice were intravenously injected with 109 and 1011 gc AAV-shA10 or control AAV-shScr per animal. Eight