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1.  Physical Approaches for Nucleic Acid Delivery to Liver 
The AAPS Journal  2008;10(4):589-595.
The liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on the availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward the development of physical methods for nucleic acid delivery to the liver. Emphasis is placed on the mechanism of action, pros, and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to the liver.
doi:10.1208/s12248-008-9067-y
PMCID: PMC2628207  PMID: 19083101
gene delivery; liver; nonviral vectors; physical method; transfection
2.  Physical Approaches for Nucleic Acid Delivery to Liver 
The AAPS journal  2008;10(4):589-595.
Liver is a key organ for numerous metabolic pathways and involves many inherited diseases that, although being different in their pathology, are often caused by lack or overproduction of a critical gene product in the diseased cells. In principle, a straightforward method to fix such problem is to introduce into these cells with a gene-coding sequence to provide the missing gene product, or with the nucleic acid sequence to inhibit production of the excessive gene product. Practically, however, success of nucleic acid-based pharmaceutics is dependent on availability of a method capable of delivering nucleic acid sequence in the form of DNA or RNA to liver cells. In this review, we will summarize the progress toward development of physical methods for nucleic acid delivery to liver. Emphasis is placed on the mechanism of action, pros and cons of each method developed so far. We hope the information provided will encourage new endeavor to improve the current methodologies or develop new strategies that will lead to safe and effective delivery of nucleic acids to liver.
doi:10.1208/s12248-008-9067-y
PMCID: PMC2628207  PMID: 19083101
Gene delivery; non-viral vectors; physical method; liver; transfection
3.  Intraperitoneal Injection of Clodronate Liposomes Eliminates Visceral Adipose Macrophages and Blocks High-fat Diet-induced Weight Gain and Development of Insulin Resistance 
The AAPS Journal  2013;15(4):1001-1011.
Macrophage infiltration in adipose tissue is strongly correlated with obesity. The exact role of macrophage in the development of obesity, however, has not been fully understood. In this study, using intraperitoneal injection of clodronate liposomes, we tissue-specifically depleted visceral adipose tissue macrophages (VATMs) and explored their roles in initiation and progression of obesity. Two sets of experiments were conducted, using mice on a high-fat diet as the animal model. Mice were injected with clodronate liposomes at the beginning of high-fat diet feeding to investigate the role of VATMs in the initiation of obesity. Treatment starting on week 5 was designed to explore the function of VATMs in the progression of weight gain. The results show that intraperitoneal injection of clodronate liposomes effectively depleted VATMs, which blocked high-fat diet-induced weight gain, fat accumulation, insulin resistance, and hepatic steatosis. Similarly, clodronate liposomes suppressed progression of weight gain in mice after being fed with a high-fat diet for 4 weeks and improved insulin sensitivity. Gene expression analysis showed that depletion of VATMs was associated with downregulation of the expression of genes involved in lipogenesis and gluconeogenesis including acc1, fas, scd1, and pepck, decreased expression of genes involved in chronic inflammation including mcp1 and tnfα, and suppressed expression of macrophage specific marker genes of f4/80 and cd11c in adipose tissue. Depletion of VATMs was associated with prevention of the formation of crown-like structures in white adipose tissue and the maintenance of a low level of blood TNF-α. Collectively, these data demonstrate that VATMs appeared to play a crucial role in the development of obesity and obesity-associated diseases and suggest that adipose tissue macrophages could be regarded as a potential target for drug development in prevention and therapy of obesity and obesity-associated complications.
doi:10.1208/s12248-013-9501-7
PMCID: PMC3787235  PMID: 23821353
high-fat diet-induced obesity; inflammation; insulin resistance; liposomes; visceral adipose tissue macrophage
4.  Resveratrol Suppresses T0901317-Induced Hepatic Fat Accumulation in Mice 
The AAPS Journal  2013;15(3):744-752.
Liver X receptor (LXR) has been identified as a potential target for treatment of atherosclerosis and diabetes. Activation of LXR, however, is associated with increased lipogenesis and fat accumulation in the liver. The objective of the current study was to examine the effect of resveratrol on LXR activator-induced fat accumulation in liver using mice as an animal model. Three groups of C57BL/6 mice were studied. Animals in group 1 were treated with T0901317, a potent activator of LXR in mice. Animals in group 2 served as the control and were treated with carrier solution and those in group 3 were treated with T0901317/resveratrol combination. Using histochemical and biochemical methods, we demonstrate that resveratrol treatment significantly suppressed fat accumulation in the liver induced by T0901317. In addition, resveratrol completely blocked elevation of blood levels of triglyceride and cholesterol and reduced blood glucose level. Quantitative PCR analysis revealed that resveratrol treatment did not change the mRNA levels of abca1, abcg1, cyp7a1, srebp-1c, chrebp, and acc genes compared to that of animals treated with T0901317 alone but reduced pepck and g6p gene expressions. Immunohistochemistry and Western blot analyses show resveratrol treatment activated AMP-activated protein kinase (AMPK) and increased phosphorylation of acetyl-CoA carboxylase. Treatment with T0901317 on hepatocytes increased intracellular fat accumulation and this increase was suppressed by resveratrol; the suppressive effect of resveratrol was greatly repressed by Compound C which is an inhibitor of AMPK. Collectively, these data suggest that resveratrol blocks T0901317-induced lipid accumulation in the liver and can be considered for inclusion into the treatment of diseases involving activation of liver X receptor.
doi:10.1208/s12248-013-9473-7
PMCID: PMC3691433  PMID: 23591747
AMPK; fatty liver; gluconeogenesis; lipogenesis; liver X receptor; resveratrol; T0901317
5.  The Liver X Receptor Agonist T0901317 Protects Mice from High Fat Diet-Induced Obesity and Insulin Resistance 
The AAPS Journal  2012;15(1):258-266.
The effect of activation of liver X receptor by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl]phenyl] benzenesulfonamide (T0901317) on high fat diet (HFD)-induced obesity and insulin resistance was examined in C57BL/6 mice. When on HFD continuously for 10 weeks, C57BL/6 mice became obese with an average body weight of 42 g, insulin resistant, and glucose intolerant. Twice weekly intraperitoneal injections of T0901317 at 50 mg/kg in animals on the same diet completely blocked obesity development, obesity-associated insulin resistance, and glucose intolerance. Quantitative real-time PCR analysis showed that T0901317-treated animals had significantly higher mRNA levels of genes involved in energy metabolism, including Ucp-1, Pgc1a, Pgc1b, Cpt1a, Cpt1b, Acadm, Acadl, Aox, and Ehhadh. Transcription activation of Cyp7a1, Srebp-1c, Fas, Scd-1, and Acc-1 genes was also seen in T0901317-treated animals. T0901317 treatment induced reversible aggregation of lipids in the liver. These results suggest that liver X receptor could be a potential target for prevention of obesity and obesity-associated insulin resistance.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9429-3) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9429-3
PMCID: PMC3535091  PMID: 23180161
diabetes; high fat diet-induced obesity; liver X receptor; nuclear receptor; T0901317
6.  Glucocorticoid Receptor-Mediated Transcriptional Regulation of N-acetyltransferase 1 Gene Through Distal Promoter 
The AAPS Journal  2012;14(3):581-590.
ABSTRACT
Human arylamine N-acetyltransferase 1, (HUMAN)NAT1, is a phase II xenobiotic-metabolizing enzyme that plays an important role in drug and carcinogen biotransformation and cancer development. Its gene expression has been shown to be regulated by environmental factors. The purpose of the current study is to determine the involvement of nuclear receptors in transcriptional regulation of (HUMAN)NAT1 gene. We show that among the nuclear receptors examined, including the glucocorticoid receptor, retinoid acid receptor-related orphan receptor alpha, constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and retinoic acid receptor, the glucocorticoid receptor plays a dominant role in regulating (HUMAN)NAT1 gene expression through distal promoter (P3). The involvement of the glucocorticoid receptor in transcription regulation of (HUMAN)NAT1 gene expression was demonstrated by dexamethasone treatment, reporter assay using plasmid-containing 3 kbp of 5′-end region of promoter 3, and treatment of anti-glucocorticoid RU486 in primary culture of human hepatocytes and transfected HepG2 cells. In addition, translation inhibition did not affect dexamethasone-induced gene expression through P3, suggesting that dexamethasone effect is directly mediated by glucocorticoid receptor activation. Furthermore, deletion analysis revealed the presence of multiple responsive elements within the 3 kbp fragment of P3. Transfection assays in mice using hydrodynamics-based procedure and reporter gene assay in a mouse cell line revealed that glucocorticoid-induced NAT gene expression is species dependent. Dexamethasone treatment of transfected mice and mouse cell line decreased (MOUSE)Nat2 gene expression, (HUMAN)NAT1 homologue. These results suggest that glucocorticoids serve as a modulator for (HUMAN)NAT1 gene expression via the P3-containing 5′-flanking region.
doi:10.1208/s12248-012-9370-5
PMCID: PMC3385828  PMID: 22644701
arylamine N-acetyltransferases; glucocorticoids; phase-II enzymes; promoter analysis; regulation of gene expression; transcriptional regulation
7.  Intracellular Gene Transfer in Rats by Tail Vein Injection of Plasmid DNA 
The AAPS Journal  2010;12(4):692-698.
In this study, we examined the effect of various factors on gene delivery efficiency of tail vein injection of plasmid DNA into rats. We measured the level of reporter gene expression in the internal organs including the lung, heart, spleen, kidney, and liver as function of injection volume, injection time, and DNA dose. Persistency of reporter gene expression in transfected animals was also examined. We demonstrated that plasmid delivery to rats by the tail vein is effective as long as the volume of injected DNA solution is adjusted to 7–8% of body weight with an injection time of less than 10 s. With the exception of a short-term increase in serum concentration of alanine aminotransferase and transient irregularity in cardiac function during and soon after the injection, the procedure is well tolerated. Lac Z staining of the liver from transfected animals showed approximately 5–10% positive cells. Persistency test for transgene expression in animals using plasmid carrying cDNA of human alpha 1 antitrypsin gene driven by chicken beta actin gene promoter with CMV enhancers showed peak level of transgene product 1 day after the injection followed by a gradual decline with time. Peak level was regained by a second injection performed on day 38 after the first injection. These results show that tail vein injection is an effective means for introducing plasmid DNA into liver cells in rats. We believe that this procedure will be extremely useful for gene function studies in the context of whole animal in rats.
doi:10.1208/s12248-010-9231-z
PMCID: PMC2976992  PMID: 20859713
gene delivery; gene therapy; hydrodynamic gene delivery; nonviral vectors; siRNA delivery
8.  Nonviral gene delivery: What we know and what is next 
The AAPS Journal  2007;9(1):E92-E104.
Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer and gene therapy. In the past, the primary focus has been on application of physical, chemical, and biological principles to development of a safe and efficient method that delivers a transgene into target cells for appropriate expression. This review summarizes the current status of the most commonly used nonviral methods, with an emphasis on their mechanism of action for gene delivery, and their advantages and limitations for gene therapy applications. The technical aspects of each delivery system are also reviewed, with a focus on how to achieve optimal delivery efficiency. A brief discussion of future development and further improvement of the current systems is intended to stimulate new ideas and encourage rapid advancement in this new and promising field.
doi:10.1208/aapsj0901009
PMCID: PMC2751307  PMID: 17408239
Gene delivery; gene therapy; nonviral vectors; transfection

Results 1-8 (8)