ABSTRACT
The greater genomic conservation between humans and non-human primates (NHP) enables target validation studies for developing of therapeutic strategies for human diseases. Together with predicting activity and potential adverse clinical signs, the inclusion of NHP testing bequeaths to efficacy models for dose titration and pharmacodynamic effects. We have used lipid nanoparticle encapsulated siRNA to silence ApoB in the liver and assessed the phenotypic effects on serum lipids with various levels of hepatic ApoB mRNA knockdown in healthy lean cynomolgus monkeys. ApoB siRNA dosed animals demonstrated significant reductions of hepatic ApoB mRNA and serum APOB protein, with a substantial lowering of plasma lipid levels without obvious signs of toxicity. Microarray based assessment of ApoB siRNA mediated effects revealed a number of differentially expressed genes which mapped onto biological pathways and processes related to lipid and cholesterol metabolism. Furthermore, we identified potential targets and cellular effects that could be studied for therapeutic benchmarking of APOB mediated effects. The network of ApoB regulated genes should be of significance for the understanding and development of novel hypercholesterolemia therapies.
Subject(s)
Apolipoproteins B/genetics , Gene Expression Regulation , Liver/metabolism , RNA Interference , Animals , Biopsy , Cholesterol/metabolism , Genome , Genomics , Humans , Lipid Metabolism , Lipids/blood , Lipids/chemistry , Liver/pathology , Macaca fascicularis , Male , Nanoparticles/chemistry , Oligonucleotide Array Sequence Analysis , Protein Biosynthesis , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara-/- mice. Combining the profiles from mice treated with the PPARalpha agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARalpha regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara-/- mice. In contrast to Ppara-/- mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies.
Subject(s)
PPAR alpha/genetics , RNA Interference , Animals , Gene Expression Profiling , Injections , Liver/metabolism , Mice , Mice, Knockout , PPAR alpha/metabolism , Phenotype , RNA, Small Interfering/administration & dosage , Transcription, GeneticABSTRACT
In rodents, treatment with peroxisome proliferator-activated receptor alpha (PPARalpha) agonists results in peroxisome proliferation, hepatocellular hypertrophy, and hepatomegaly. Drugs in the fibrate class of PPARalpha agonists have also been reported to produce rare skeletal muscle toxicity. Although target-driven hepatic effects of PPARalpha treatment have been extensively studied, a characterization of the transcriptional effects of this nuclear receptor/transcription factor on skeletal muscle responses has not been reported. In this study we investigated the effects of PPARalpha agonists on skeletal muscle gene transcription in rats. Further, since statins have been reported to preferentially effect type II muscle fibers, we compared PPARalpha signaling effects between type I and type II muscles. By comparing the transcriptional responses of agonists that signal through different nuclear receptors and using a selection/deselection analytical strategy based on ANOVA, we identified a PPARalpha activation signature that is evident in type I (soleus), but not type II (quadriceps femoris), skeletal muscle fibers. The fiber-type-selective nature of this response is consistent with increased fatty acid uptake and beta-oxidation, which represent the major clinical benefits of the hypolipidemic compounds used in this study, but does not reveal any obvious off-target pathways that may drive adverse effects.
Subject(s)
Muscle Fibers, Slow-Twitch/drug effects , Muscle, Skeletal/drug effects , PPAR alpha/agonists , Animals , Bezafibrate/pharmacology , Fatty Acids/metabolism , Female , Fenofibrate/pharmacology , Gene Expression Profiling , Glucose/metabolism , Hypolipidemic Agents/pharmacology , Liver/drug effects , Liver/metabolism , Muscle Fibers, Slow-Twitch/metabolism , Muscle, Skeletal/metabolism , Pyrimidines/pharmacology , Rats , Rats, Inbred Strains , Rosiglitazone , Thiazolidinediones/pharmacology , Tretinoin/pharmacologyABSTRACT
The antiandrogen flutamide (FLU) is used primarily for prostate cancer and is an idiosyncratic hepatotoxicant that sometimes causes severe liver problems. To investigate FLU's overt hepatic effects, especially on inducible drug clearance-related gene networks, FLU's hepatic gene expression profile was examined in female Sprague-Dawley rats using approximately 22,500 oligonucleotide microarrays. Rats were dosed daily for 3 days with FLU at 500, 250, 62.5, 31.3, and 15.6 mg/kg/day, and hepatic RNA was isolated. FLU resulted in the dose-dependent regulation of approximately 350 genes. Employing a gene-response compendium, FLU was compared with three classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone, and four atypical CYP1A inducers, indole-3-carbinol (I3C), omeprazole (OME), chlorpromazine (CPZ), and clotrimazole (CLO). The FLU gene response was comparable with classical AhR ligands across a signature AhR ligand gene set that included CYP1A1 and other members of the AhR gene battery. Dose-related responses of CYP1 genes established a maximum response ceiling and discerned potency differences in atypical inducers. FLU had a sharp down-regulation of c-fos that was comparable with all the compounds except CPZ and CLO. FLU absorption, distribution, metabolism, and excretion (ADME) gene expression analysis revealed that FLU, as well as I3C and OME, induced CYP2B and CYP3A, distinguishing them from the classical AhR ligands. By using a compendium of gene expression profiles, FLU was shown to signal in rats similar to an AhR activator with additional CYP2B and CYP3A effects that most resembled the ADME gene expression pattern of the atypical CYP1A inducers I3C and OME.
