Type 2 Diabetes Mellitus: Limitations of Conventional Therapies and

Review pubs.acs.org/CR

Type 2 Diabetes Mellitus: Limitations of Conventional Therapies and Intervention with Nucleic Acid-Based Therapeutics Ganesh R. Kokil,† Rakesh N. Veedu,*,‡,§,∥ Grant A. Ramm,⊥,# Johannes B. Prins,∇ and Harendra S. Parekh*,† †

School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Brisbane, QLD 4102, Australia Center for Comparative Genomics, Murdoch University, 90 South Street, Murdoch, WA 6150, Australia ∥ Western Australian Neuroscience Research Institute, Perth, WA 6150, Australia ‡ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072 Australia ⊥ The Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia # Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, QLD 4006, Australia ∇ Mater Research Institute, The University of Queensland, Brisbane, QLD 4101, Australia §

4.5. Challenges in siRNA Delivery 5. Concluding Remarks and Outlook Author Information Corresponding Authors Author Contributions Notes Biographies Acknowledgments Abbreviations Used References

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CONTENTS 1. Introduction and Scope of Review 2. Type 2 Diabetes Mellitus 2.1. The Root Cause 2.1.1. Insulin Resistance 2.1.2. Pancreatic β-Cell Dysfunction 2.2. Fate of Glucose in T2DM 2.3. Clinical Consequences 2.3.1. Misfolded β-Amyloid Protein Accumulation 2.3.2. Advanced Glycation End Products 3. Oral Hypoglycemic Agents 3.1. Mechanism of Action 3.2. Conventional Therapies and Their Limitations 4. RNAi-Based Therapeutics 4.1. Targeting the Root Cause of T2DM 4.2. Normalizing Hyperglycemia 4.3. Targeting Vascular Complications of T2DM 4.4. Engineering siRNA’s for Enhanced Target Specificity and Bio/Thermostability 4.4.1. Nucleobase Modifications 4.4.2. Sugar Modifications 4.4.3. Internucleotidic-Linkage (Phosphate) Modifications 4.4.4. siRNA Design Type Modifications 4.4.5. siRNA Overhangs and Termini Modifications 4.4.6. siRNA Modification through Chemical Conjugations © XXXX American Chemical Society

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1. INTRODUCTION AND SCOPE OF REVIEW Figures released in 2013 by the International Diabetes Federation (IDF) state that over 382 million individuals worldwide are affected by diabetes mellitus (DM).1 The global prevalence of DM has increased more than 13-fold since the early 1980s, and following projected trends, the case burden is likely to approach 600 million by 2030 (Figure 1).1,2 Despite the large panel of pharmacological agents at the disposal of practitioners, none have shown promise in halting the underlying causes of DM, namely, insulin resistance and the progressive decline of pancreatic β-cell function (Figure 1).3 The mortality rates for the disease are nothing short of alarming, with DM and its associated complications attributing to one death every ∼6 s. DM places immense financial pressures on already overstretched healthcare budgets in the developed as well as developing world, putting estimated global expenditure at a staggering US$548 billion in 2013 alone.1 DM is a metabolic disorder characterized by insufficient secretion or inefficient processing of hormonal insulin, broadly classified into two typestype 1 (T1DM) and type 2 DM (T2DM)with the latter accounting for ∼90% of cases, and it is therefore the focus of this review.4 Oral hypoglycemic agent regulatory approvals have grown significantly over the past 2 decades, yet such plasma glucose-lowering interventions are still considered far from “ideal” by clinicians and researchers alike.5 Of all the pharmacological agents on the market globally used

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Received: May 29, 2014

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DOI: 10.1021/cr5002832 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Timeline of breakthroughs in antidiabetic drug discovery shown alongside the actual and projected global burden of DM.1−3

dysfunction are the key drivers of the disease,9 with a complex interrelationship responsible for initiating its pathogenesis.10 A progressive decline in β-cell function with reduced glucose uptake into various tissues (liver, muscle, adipose, and brain) due to IR is accompanied by excessive hepatic glucose output (i.e., gluconeogenesis and glycogenolysis). These drivers lead to persistent hyperglycemia and are therefore considered to be the major pathophysiological defects in T2DM.11 Additionally, genetic predisposition, increasing age, and obesity12 are three leading and well-reported exacerbators of IR and pancreatic βcell dysfunction.13 Adipocyte transformation into insulin-resistant species increases levels of circulating free fatty acids, causing lipotoxicity, which in turn impairs β-cell function. The hyperglycemia (glucotoxicity) intensifies IR and further impairs insulin secretion.14 In the latter stages of T2DM, pancreatic βcell activity is further compromised through deposition of misfolded β-amyloid protein, which ultimately depletes insulin production.15 Gluco- and lipotoxicity are also responsible for depletion of the pancreatic β-cell population, thus propagating an overall reduction in insulin secretion levels.14 Combining these pathways of insulin misregulation one can surmise that there exist two major pathological pathways at play in T2DM, inefficient processing of insulin and insufficient insulin production, both of which lead the patients toward the development of various life-threatening micro- and macrovascular complications (Figure 2).

to manage T2DM, few are known to ameliorate insulin sensitivity, while none to-date demonstrate an ability to halt insulin resistance and/or progressive decline of pancreatic β-cell function.6 A compounding factor with current oral hypoglycemic agent use is that they are not only limited in their efficacy but also suffer from an adverse effect profile that severely impacts patient compliance.5 To this end, nucleic acid-based therapeutics is gaining significant interest and momentum as an alternative mode of therapy, paving the way toward targeted treatment strategies that aim to “switch-off” expression of the causative gene(s) that promotes hyperglycemia specifically, with the primary objective of ameliorating the symptoms and so the clinical consequences of T2DM.7 Small interfering RNAs (siRNAs) are among the most recent additions to the wide variety of nucleic acid-based molecules trialed to selectively and potently silence gene expression.8 This review focuses primarily on the limitations of conventional T2DM therapies, leading to critical appraisal of the status of traditional and modified siRNA-based therapeutics, with emphasis placed on potential new targets for more effective and selective treatment strategies to combat T2DM.

2. TYPE 2 DIABETES MELLITUS 2.1. The Root Cause

The T2DM-affected population mainly comprises individuals where insulin resistance (IR) 4b and pancreatic β-cell B


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Figure 3. Downstream insulin signaling and the underlying molecular pathways implicated in IR.

[chemokine (C−C motif) ligand 4].17a,21,22 The release of TNF-α and activation of its receptor (TNFR) stimulates JunN-terminal kinase-1 (JNK-1), a negative regulator of insulin receptor substrate and protein kinase B (Akt).23 Separately, IL6 interacts with its receptor, initiating PKC-δ-mediated stimulation of suppressor of cytokine signaling-3 (SOCS-3), this being a negative regulator of Akt in the insulin-signaling pathway.24 Another crucial piece of the IR puzzle takes place in the various insulin-sensitive organs and involves the unfolded protein response (UPR) responsible for JNK1 activation.25 Here, free fatty acid enters mitochondria and is incorporated into the tricarboxylic acid (TCA) cycle. This in turn generates reactive oxygen species (ROS), resulting in mitochondrial dysfunction and stress within endoplasmic reticulum (ER). This ER stress is the underlying mechanism for the UPR, which triggers activation of JNK-1.26 Along similar lines, malate, esters of malic acid and an intermediate byproduct of the TCA cycle, takes part in glucose synthesis (gluconeogenesis) and this increases glucose output from the liver, exacerbating underlying hyperglycemia.27 During the later stages of IR when blood glucose levels become chronically elevated, glucose is party to interactions with various other proteins, which lead to the production of Schiff’s bases referred to collectively as advanced glycation end products (AGEs). AGEs, upon interaction with their receptors, stimulate further ROS production and propagate NF-κB signaling, thus exacerbating IR.28 2.1.2. Pancreatic β-Cell Dysfunction. Pancreatic β-cell dysfunction is characterized primarily by reduced β-cell mass, and the underlying molecular mechanism proposed for this reduction is an increase in apoptosis not compensated by adequate β-cell regeneration.29 Furthermore, ultrastructural analysis on T2DM-affected islets has shown a reduction in insulin granules and morphological changes in several β-cell organelles, such as mitochondria and endoplasmic reticulum.30 Subsequently, several qualitative and quantitative studies on

Figure 2. Pathophysiology of T2DM.

2.1.1. Insulin Resistance. One of the key functions of insulin is to regulate glucose delivery to the key insulin-sensitive organs/tissue: liver, adipocytes, skeletal muscle, and brain.16 IR is a complex metabolic disorder in which normal or elevated levels of endogenous (or exogenous) insulin produces an attenuated biological response, i.e., impaired sensitivity to insulin-mediated glucose clearance.17 Thus, insulin-resistant organs fail to consume adequate levels of glucose, which contributes to hyperglycemia.18 In spite of extensive research aimed at deciphering the molecular mechanism of IR, there remain many missing links and threads to identifying the primary or root causes.17a What is clear from documented evidence is that, in the insulin-resistant state, defects stem from downstream insulin-signaling processes rather than from the insulin molecule or its receptor.19 Researchers have identified a handful of mechanisms and pathways proposed to participate in the phenomenon of IR. These include increased plasma free fatty acid concentrations, chronic inflammation, oxidative and nitrative stress, altered gene expression, and mitochondrial dysfunction (Figure 3).20 In patients where obesity and T2DM coexist, circulating free fatty acid levels are permanently elevated, resulting in excess uptake by hepatocytes and skeletal muscle. These free fatty acids in turn activate the serine/threonine cascade via protein kinase C-ε (in liver) or protein kinase C-θ (in muscle), which are negative regulators of the insulin receptor and insulin receptor substrate, respectively, through the serine phosphorylation mechanism.21 Overexpression of protein kinase C (ε/θ) leads to activation of various intracellular inflammatory pathways, such as IkB-kinase (IkK), which in turn stimulates NF-κB-mediated transcription and release of various proinflammatory chemokines, namely, TNF-α, IL-6, and CCL4 C


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Figure 4. Intracellular fate of glucose: 1, GLUT; 2, hexokinase; 3a, gluconeogenesis; 3b, glycolysis; 4a, glycogenesis; 4b, glycogenolysis; 5, triacylglycerol synthesis; 6, pentose phosphate pathway; 7, hexosamine biosynthesis; 8, aldose reductase; 9, AGE formation.

isolated diseased islets demonstrated defects in β-cell function that encompassed alterations in early-phase glucose-stimulated insulin release.31 Separately, genome-wide association studies have identified >40 susceptible genes closely associated with T2DM,32 with the majority of these risk variants in the healthy population acting by impairing insulin secretion (i.e., through pancreatic β-cell dysfunction) rather than insulin action (i.e., through IR). Among the acquired factors, of particular relevance is the role that glucotoxicity,33 lipotoxicity,34 and chronic inflammation35 play in disease progression, with elevated levels of glucose/free fatty acids having been implicated in the natural history of β-cell damage in T2DM.

pathway 2). Hexokinase II levels were noted to drop in the skeletal muscle of subjects with T2DM, and thus, glucose phosphorylation is also hampered.39 Glucokinase (hexokinase IV) expression in the liver is also compromised in T2DM patients, leading to its increased popularity as a target candidate for novel antidiabetic medication development.40 Separately, intracellular glucose may also participate in oxidative catabolism (glycolysis) (Figure 4, pathway 3), resulting in its conversion to pyruvate;41 conversely, it may follow the anabolic pathway and be stored as the polysaccharide glycogen (Figure 4, pathway 4).41 Separately, an insulin-resistant liver also increases glucose output by increasing both gluconeogenesis and glycogenolysis (Figure 4, pathways 3a and 4b).27 Glucose can, albeit to a lesser extent, also be converted to other sugars and metabolites essential in anabolic and catabolic metabolism. Metabolites such as glycerol-3-phosphate (Figure 4, pathway 5), for example, are utilized in triglyceride synthesis via mono- and diacylglycerol formation,42 the latter of which activates PKC, a key driver of insulin resistance in the liver and skeletal muscle.21 The pentose phosphate shunt (Figure 4, pathway 6) is another important pathway that glucose follows for the generation of various intermediates, such as reducing cofactor equivalents (e.g., NADH) and ribose-5-phosphate (nucleic acid precursor).43 The hexosamine biosynthetic pathway (Figure 4, pathway 7) is primarily associated with post-translational protein modification through glycosylation and synthesis of glycolipids, proteoglycans, and the glycosyl phosphatidyl

2.2. Fate of Glucose in T2DM

Glucose is a central molecule in carbohydrate metabolism as well as the primary source of energy for living cells.36 In the insulin resistant state, however, liver glucose output (through glycogenolysis and gluconeogenesis) increases, and uptake by insulin-sensitive organs decreases, leading to the hyperglycemic condition common to T2DM patients.27 Glucose uptake into cells occurs through a range of glucose transporters (GLUTs), which are either insulin-dependent or -independent (Figure 4, pathway 1).37 Of the GLUT family, GLUT-4 in particular has been widely studied due to its being down-regulated by IR, this being prevalent in skeletal muscle and adipocytes.38 Upon cellular entry, glucose rapidly undergoes phosphorylation by various hexokinases into glucose-6-phosphate (Figure 4, D


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Figure 5. Mechanism of AGE formation.

Figure 6. A selection of AGEs arising from T2DM: pyrraline,60 pentosidine,61 glyoxal−lysine dimer,62 1-alkyl-2-formyl-3,4-diglycosyl pyrrole,63 methyl-glyoxal−lysine dimer,64 N-ε-carboxy-methyl-lysine,62 N-ε-carboxy-ethyl-lysine,65 imidazolone,66 and 2-(2-furoyl)-4(5)-1H-imidazole.67

inositol anchor.44 The hexosamine pathway is a mediator of adverse effects arising from glucotoxicity and it contributes to the development of insulin resistance, insulin secretory dysfunction, and various other complications.45 In the insulinresistant state, as hyperglycemia intensifies, glucose participates beyond its conventional metabolism, acting along aldose reductase pathways (Figure 4, pathway 8), which increases oxidative stress, resulting in the formation of AGEs (Figure 4, pathway 9), which facilitate the incidence of micro- and macrovascular complications.46

number of clinical symptoms, with NAFLD being a very common condition seen in obese patients with T2DM.48 Here, if obesity is left unchallenged, it leads to excessive triglyceride accumulation within the liver, otherwise known as steatosis or “fatty liver”, which can ultimately accelerate into fibrosis as nonalcoholic steatohepatitis (NASH),49 then cirrhosis, and eventually end-stage liver failure.50 In the latter stages of T2DM, pancreatic β-cells also become affected due to deposition of misfolded β-amyloid protein,15 which ultimately leads to depleted insulin production (discussed below). Hyperglycemia (glucotoxicity) and hyperlipidemia (lipotoxicity) intensify insulin resistance, which impair insulin secretion;14 this also contributes to the formation of AGEs.28 These are just some of the serious clinical

2.3. Clinical Consequences

DM is a risk factor for fibrosis in several different liver diseases, including hemochromatosis and nonalcoholic fatty liver disease (NAFLD).47 Obesity and insulin resistance are key drivers of a E


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Figure 7. Pathways by which commonly prescribed oral hypoglycemic agents achieve glucose homeostasis.

hyperglycemia, and the extent of oxidative stress in the intraand extracellular environment.58 The formation of AGEs (Maillard reaction) (Figure 5) starts with Schiff base formation between the aldose sugar (glucose) and amino groups in proteins, lipids, or nucleic acids, which are subsequently converted to Amadori products. It is well-known that open-chain Amadori products are free to rearrange and produce α-oxoaldehydes, glyoxal, methylglyoxal, and 3deoxyglucosone. These highly reactive intermediates can readily react with amino, sulfhydryl, and guanidine functional groups in proteins, resulting in denaturation of and cross-linking with the target protein.55 In addition, the α-dicarbonyl products react readily with lysine and arginine residues, leading to the formation of AGEs such as Nε-(carboxymethyl)lysine, Nε-(carboxyethyl)lysine, imidazolone, glyoxal−lysine dimer, and many others, as depicted in Figure 6.56 AGEs have been shown to accumulate in the wall of vessels, where they perturb cell structure and function, which is a key factor responsible for micro- and macrovascular complications of DM.59

consequences following chronic mismanagement of T2DM, which are elaborated below. 2.3.1. Misfolded β-Amyloid Protein Accumulation. Deposition of amyloid derived from the neuropancreatic hormone islet amyloid polypeptide is the most typical islet alteration in T2DM.51 It is accepted that β-cell dysfunction and loss of β-cell mass are key features of T2DM that are correlated to several factors, including islet inflammation, glucotoxicity, lipotoxicity, and islet amyloid formation.52 In early-stage T2DM, islet amyloid polypeptide overproduction and misprocessing, along with other yet unknown factors, lead to islet amyloidosis, β-cell dysfunction, and cell death.53 Much progress has been made in better understanding the pathological effects of islet amyloid polypeptide formation, although many questions still remain unanswered. These include the mechanism by which islet amyloid forms, its cytotoxicity, the initial site of amyloid deposition, and the mechanism by which it is cleared.54 2.3.2. Advanced Glycation End Products. AGEs are nonenzymatic glycation and subsequent oxidation products of proteins or lipids after contact with aldose sugars.55 Generally, glycation and oxidation processes result in the formation of Schiff’s bases and Amadori products, but further glycation of proteins and lipids generate AGEs.56 These products are responsible for inflicting oxidative stress and bind to specific cell surface receptors.57 AGE formation in T2DM depends on the availability of proteins for glycoxidation, the degree of

3. ORAL HYPOGLYCEMIC AGENTS Currently prescribed antidiabetic drug therapies universally aim to reduce blood glucose levels by targeting one or more of the six key organs/tissues, i.e., the pancreas, liver, skeletal muscle, small intestine, kidneys, and adipose tissue (Figure 7).68 F


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Table 1. Antidiabetic Medication Classes with Named Examples and Their Adverse Effect Profile antidiabetic medication class sulfonylureas

drug examples

biguanides

acetohexamide, chlorpropamide, tolazamide, tolbutamide, glibenclamide, gliclazide, glipizide, gliquidone, glimepiride phenformin, metformin, buformin, proguanil

meglitinides α-glucosidase inhibitors 2,4-thiazolidinedione

repaglinide, nateglinide acarbose, miglitol, voglibose ciglitazone, troglitazone, pioglitazone, rosiglitazone

glucagon-like peptide-1 (GLP-1) receptor agonists DPP-IV inhibitors

exenatide, liraglutide

SGLT2 inhibitors

dapagliflozin, canagliflozin

associated adverse effects hypoglycemia, weight gain, possible increased mortality from cardiovascular disease, possible increased incidence of cancer91 hepatotoxicity, gastrointestinal disturbances, including diarrhea, nausea, abdominal pain; lactic acidosis; vitamin B12 deficiency; metallic taste92 hypoglycemia and weight gain91a GI disturbance, including diarrhea, abdominal cramping, and flatulence93 hepatotoxicity, edema, congestive heart failure, weight gain, bone fractures, possible increased risk of ischemic heart disease, bladder cancer94 significant but transient GI disturbance, including nausea and vomiting; possible association with medullary thyroid cancer; possible association with acute pancreatitis95 nasopharyngitis and upper respiratory tract related infection, possible association with acute pancreatitis, possible association with exfoliative dermatitis95 volume reduction,96 genitourinary tract infection,97

vildagliptin, saxagliptin, sitagliptin

3.1. Mechanism of Action

agents has also been shown to be elevated in the small intestine of diabetic animal models.80 The α-glucosidase inhibitors are saccharides that act as competitive inhibitors of enzymes whose primary function is to digest carbohydrates, specifically, the αglucosidase enzymes present in the brush border region of the small intestine.81 Adipose tissue is primarily responsible for glucose utilization, while adipocytes also secrete a diverse range of proinflammatory cytokines, such IL-6 and TNF-α, as well as anti-inflammatory cytokines, such as adiponectin.82 The 2,4thiazolidinediones (TZDs) show antidiabetic properties by activating PPAR-γ (peroxisome proliferator-activated receptorγ) through binding to DNA in conjunction with the retinoid Xreceptor, increasing transcription of a number of genes.83 TZDs induce a coordinated up-regulation of transcripts of proteins involved in fatty acid uptake/binding, β-oxidation, electron transport, and oxidative phosphorylation in adipose tissue. These changes are associated with an increase in total body fatty acid oxidation, a decrease in plasma free fatty acid levels, and an increase in insulin-stimulated glucose uptake (i.e., improved insulin sensitivity).83b,84 The kidneys actively participate in maintaining glucose homeostasis through a number of pathways, including gluconeogenesis and glucose uptake from the systemic circulation, as well as its reabsorption following glomerular filtration.85 Typically, each day ∼180 g of glucose is filtered by the kidneys, which is then reabsorbed in the proximal tubules predominantly via the sodium-glucose cotransporter 2 (SGLT2, 90%; SGLT1, 10%).86 In T2DM, when a patient’s plasma glucose levels exceed natural threshold levels (160−180 mg/ dL), SGLT2 receptor saturation results, and this triggers the renal elimination of glucose.87 Similarly, the action of SGLT2 inhibitors prevents reabsorption of filtered glucose by the kidneys, and this results in a glucose-lowering effect.88 In addition to the chemotherapeutic options outlined above, the American Diabetes Association and European Association for the Study of Diabetes recommend dietary modification in conjunction with regular exercise as first-line therapy.89 To summarize, there are different classes of antidiabetic medications available for clinical use today, each of them displaying unique pharmacologic actions (Figure 7).

The physiological regulation of insulin secretion from pancreatic β-cells is well-reported and well-understood.69 The sulfonylurea-based hypoglycemic agents have been shown to bind specifically to ATP-dependent K+ (KATP) channels on the membrane of pancreatic β-cells, resulting in K+ efflux, which triggers cell depolarization, leading to an increase in intracellular Ca2+. This phenomenon ultimately drives fusion of cytosolic insulin granulae with the cell membrane, thereby facilitating extracellular secretion of (pro)insulin.70 Important modulators of glucose are the incretins,71 which encompass the gut-derived glucagon-like peptide-1 (GLP-1), secreted by intestinal L cells post cibum.72 Once in the systemic circulation, its major function involves controlling hyperglycemia via interaction with its receptor on pancreatic βcells, which in turn stimulates insulin release while inhibiting glucagon secretion.73 The half-life of GLP-1 in the circulation is a mere 2 min, with its rapid metabolism being driven by the enzyme dipeptidyl peptidase-4 (DPP-4).74 Thus, therapeutic intervention through development of long-acting GLP-1 analogues75 and DPP-4 inhibition (i.e., via gliptins) has led to each being recognized as a new and highly potent class of hypoglycemic agent.76 The liver is the primary organ manipulating glucose (through glycogenesis, gluconeogenesis, and glycogenolysis) and lipid (through β-oxidation of fatty acids and de novo synthesis of fatty acids) processing. Hepatic insulin stimulates glycogen and triglyceride synthesis while inhibiting glycogenolysis, ketogenesis, and gluconeogenesis. Hence, increased circulating insulin concentrations suppress overall hepatic glucose output.77 Thus, the liver is considered a potential target for regulating hyperglycemia and hyperlipidemia. The biguanides are a class of agent that counteract hyperglycemia primarily through suppression of glucose production in the liver via AMP-activated protein kinase.78 In skeletal muscle, specifically, insulin stimulates glucose uptake, protein synthesis, and glycogen synthesis and inhibits protein degradation and glycogenolysis.79 Skeletal muscle is responsible for approximately 75% of insulin-stimulated glucose uptake by the entire body, so in addition to suppressing hepatic glucose production, biguanides also increase insulin sensitivity and enhance glucose uptake in skeletal muscle through AMPmediated protein kinase activation.78 The primary absorption site of glucose and lipids is via the small intestine, and interestingly, the absorption of therapeutic

3.2. Conventional Therapies and Their Limitations

The number of regulatory approvals for oral hypoglycemic agents has grown significantly over the past 2 decades, yet the glucose-lowering interventions used today remain far from G


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Figure 8. siRNA duplex composition.

ideal.5 According to the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, the frequency of hypoglycemic episodes increased with overly stringent glycemic control, although this did not correlate to increased rates of mortality.90 Thus, findings from the ACCORD study certainly point toward a need to design pharmacological agents and strategies that achieve optimal glycemic control.90 Further, and more crucially, none of the currently available pharmacological agents used to manage T2DM has demonstrated any capacity to halt the progressive decline of pancreatic β-cell function that reduces insulin secretion,6 with treatment of T2DM patients limited not only by the efficacy of the agents but also by their adverse effect profile (Table 1).

4. RNAI-BASED THERAPEUTICS Advances in the fields of genetics and functional genomics have yielded great insight into both the underlying molecular mechanisms that regulate gene expression and the counter mechanisms, which ultimately lead to their dysfunction, in T2DM.32,98 The Nobel prize winning discovery of RNAi by Fire and Mello revealed that small RNA duplexes were capable of degrading specific mRNAs complementary to their antisense sequence.8 Ultimately, this led drug discovery toward the identification of therapeutic agents that may turn off genes by targeting the mRNA that encodes for target protein synthesis rather than the proteins themselves.99 Thus, mRNA an intermediate between genes and proteins has become an ideal target for drug discovery and development processes.100 Chemically, small interfering RNAs (siRNAs) are doublestranded oligoribonucleotides comprising 21−23 base pairs that exhibit affinity for sequence-specific gene silencing (Figure 8).101 The underlying mechanism of RNAi in silencing genes is the sequence-specific degradation of cytoplasmic mRNA containing the same sequence as the dsRNA trigger (Figure 9).102 RNAi has displayed tremendous potential not least due to its high target specificity, which is absent of unwanted interferon responses.103 From a drug development perspective and following the first report of RNAi in 1998, within a mere 2 decades over 15 RNAi-based therapeutic candidates have reached various stages of clinical trials.104 Despite several unresolved, predominantly safety issues surrounding RNAi-based therapeutics, numerous companies are firmly invested toward developing RNAi-based treatments for diseases such as DM and its related complications.7 Sirna Therapeutics, Inc. is a notable example, having systemically delivered chemically modified siRNA against the hepatitis C

Figure 9. Transcription: (1) mRNA synthesis, (2) migration of mRNA to cytoplasm. Translation: (3 and 4) synthesis of protein from mRNA, tRNA, and ribosomal subunits. siRNA-mediated gene silencing: (5) RNA-induced gene-silencing complex (RISC) formation, (6) sequence-specific RNase H-mediated degradation of cytoplasmic mRNA.

virus. The development of a nanoparticulate delivery technology that efficiently and specifically delivers siRNA to hepatocytes has assisted Sirna Therapeutics in also addressing other liver-associated diseases, such as DM.105 Separately, CytRx Corp. initiated a collaborative program with the University of Massachusetts Medical School in 2003 to develop drug compounds based on RNAi technology for the treatment of obesity and T2DM.106 Other companies, such as Alnylam Pharmaceuticals, Inc., have developed a chemically modified siRNA for direct, systematic delivery, opening the possibility of RNAi therapeutics for a wider range of metabolic and cardiovascular disorders.107 4.1. Targeting the Root Cause of T2DM

Obesity is a key driver that chronically elevates levels of circulating free fatty acids in T2DM patients. These free fatty acids in turn activate the serine/threonine cascade, which is a well-known negative regulator of the insulin receptor and insulin receptor substrate (Figure 10, pathway 1).21 Additional consequences of obesity include inflammation of adipose tissue H


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Figure 10. Targeting IR with siRNA-mediated gene silencing.

activated protein kinase isoform 4 (MAP4K4) (Figure 10, pathway 4)115 or osteopontin,116 may serve as a useful strategy for enhancing insulin signaling. To verify such claims, siRNAmediated gene silencing of MAP4K4 in human skeletal muscle has indeed been able to prevent TNF-α-induced insulin resistance, as well as increase glucose utilization.117 Another mechanism of TNF-α-mediated IR is via activation of inhibitor of nuclear factor κB kinase β (IKKβ) (Figure 10, pathway 5) with siRNA-mediated gene silencing of IKKβ preventing TNFα-mediated impairment of insulin action by way of Akt phosphorylation, glucose uptake, and metabolism in human skeletal muscle.118 The intraperitoneal administration of siRNA encapsulated in glucan shells selectively silenced the inflammatory cytokines TNF-α or osteopontin in epididymal adipose tissue macrophages, resulting in significant improvements in glucose tolerance among obese mice.119 Other pro-inflammatory cytokines, such as IL-6, also play a crucial role in the development of IR, with the proposed mechanism for such events modulated through activation of STAT3 (signal transducer and activator of transcription 3) and consequently SOCS3 (suppressor of cytokine signaling 3) (Figure 10, pathway 6).24 Activated SOCS3 interferes with insulin signaling by deactivating the insulin receptor and insulin receptor substrate. siRNA-mediated silencing of STAT3 and SOCS3 yielded improved insulin signaling and glucose metabolism in skeletal muscle obtained from T2DM human subjects.120 Other than the aforementioned roles of some key proinflammatory cytokines in the development of IR, accumulated evidence also indicates that TNF-α can induce the expression of protein tyrosine phosphatase 1B (PTP-1B) (Figure 10,

and subsequent infiltration by macrophages, which are also associated with IR.108 Inflamed adipocytes are engulfed by Tlymphocytes, mast cells, and macrophages, which go on to initiate release of cytokines (TNF-α, IL, and CC chemokine ligand 2) (Figure 10, pathway 2) responsible for promoting IR.109 In some studies, gene ablation of chemokines was shown to improve insulin sensitivity, at least in obese rodent models. 110 In attempts to counter this phenomenon, administration of anti-inflammatory agents such as salsalate (a prodrug of salicylic acid) has shown reductions in hyperglycemia while glucose utilization in rodents is improved.111 As a further demonstration, C57BL/6J mice have been used to study the correlation between obesity, chronic (low-grade) inflammation, and ultimately IR. In obesity, osteopontin, an extracellular matrix protein and pro-inflammatory cytokine in adipose and liver tissue, is significantly up-regulated. C57BL/6J mice fed with a high-fat diet to induce obesity were then treated with an osteopontin-neutralizing antibody, and the resulting interference in osteopontin activity was found to significantly improve insulin sensitivity in mice.112 Importantly, such interventions have also been successfully translated to humans, where cytokine blocking using antibodies or antagonists has been shown to improve overall insulin signaling.113 From this data it is clear that an elevated level of inflammatory cytokines can be directly correlated to IR and that inhibition of these cytokines results in marked improvements in glycaemic control. It is well-documented that TNF-α drives IR via JNK activation (Figure 10, pathway 3), which in turn impairs insulin signaling and GLUT translocation.23 Therefore, suppression of JNK signaling,114 which requires activation by mitogenI


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pathway 7).121 Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that catalyze the hydrolytic removal of phosphate from tyrosine residues within proteins.122 Documented evidence firmly supports the theory of PTP-1B involvement in T2DM123 and obesity124 through negative regulation of insulin as well as leptin signaling. In vitro studies on liver- and skeletal-muscle-derived cell lines demonstrate involvement of PTP-1B in insulin signal transduction by inhibiting insulin-stimulated glycogen synthesis.125 Overexpression of PTP-1B in rat primary adipose tissue also markedly down-regulates insulin sensitivity, as well as GLUT4 translocation.126 At the genetic, molecular, biochemical, and physiological levels, PTP-1B has proven beyond reasonable doubt to be a valid target for correcting the key underlying cause of T2DM, i.e., insulin resistance.122 To date, the pursuit of traditional drug-based therapies against PTP-1B has been plagued with issues of “drugability”;127 this has meant that siRNA-based approaches (through PTP-1B silencing) are a highly attractive therapeutic proposition toward combating insulin resistance in T2DM patients.128 Using siRNA-based therapeutic strategies to partially down-regulate the causative inflammatory cytokines as well as byproduct proteins (Figure 10, Table 2) may well serve to vastly improve insulin signaling without inducing the significant adverse effects caused by the complete loss of target protein function.129

cells).132 In the insulin-resistant state, liver-mediated glucose production increases while glucose uptake and utilization fall in skeletal muscle, leading to the elevated glycaemic state observed in T2DM.27 Chronically elevated plasma glucose levels are in turn responsible for various macro- and microvascular complications common to poorly managed and late-stage diabetes.133 To counter this, various first-line antidiabetic agents are prescribed, which aim to normalize glucose back to safe levels, and this is achieved through various well-known mechanisms (Figure 7). Various genetic drivers regulating glucose efflux from hepatocytes have been identified, and some of the genes responsible are either overexpressed or undergo mutations in the insulin-resistant state.134 Where causative genes are known to be overexpressed, RNAi has a vital role to play in the future therapeutic intervention of T2DM. A notable example is peroxisome proliferator-activated receptor (PPAR)-γ-coactivator 1 (PGC-1), a nuclear hormone receptor coactivator that regulates genes involved in gluconeogenesis and fatty acid oxidation (Figure 11, A).135 Hepatic levels of PGC-1 are elevated in T2DM mouse models,136 while its RNAi-mediated gene silencing significantly reduced the key enzymes implicated in gluconeogenesis, i.e., phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase.137 Furthermore, fasting hypoglycemia and improved insulin sensitivity were also observed in the same mouse model.136 Hepatic post-transcriptional gene silencing of PEPCK, the rate-limiting enzyme in gluconeogenesis, was achieved in mice with a 50% decrease in protein level, and this was sufficient to lower blood glucose back to “safe” levels.138 Another important transcriptional regulator of hepatic gluconeogenesis is transducer of regulated cAMP-responsive element-binding protein activity 2 (TORC2) (Figure 11, A), which is reported to increase in hyperglycemic rodent models. Liver-specific knockdown of TORC2 in mice fed a high-fat diet and Zucker diabetic fatty rats has been achieved once again with siRNA, this time delivered via a novel lipidic nanoparticulate system.139 PGC-1-deficient mice upon RNAi delivery to the liver undergo fasting hypoglycemia and thus enhanced insulin sensitivity with reduced expression of the mammalian Tribbles homologue-3 (TRIB3).140 TRIB3 functions as a negative regulator of Akt, and with its expression being induced in the liver under fasting conditions, it too has been found to interrupt insulin signaling. The mRNA and protein levels of TRIB3 were reported to be elevated in db/db diabetic mice, while its knockdown was found to improve glucose tolerance in mouse models.141 Glucagon is another key regulator of glucose homeostasis, with its actions opposing those of insulin.131 Upon binding to its receptor, it stimulates hepatic output of glucose by mediating various signaling pathways, which activates glycogen phosphorylase (Figure 11, B).142 This enzyme is responsible for the release of glucose-1-phosphate from glycogenated polymer, so inhibition of the glucagon receptor may serve as another plausible therapeutic intervention strategy for managing T2DM.143 To test this hypothesis, the effects of siRNAmediated hepatic glucagon receptor inhibition in db/db mice were assessed, with significant reductions in plasma glucose levels observed.144 An unexpected finding, however, was a sharp rise in total plasma cholesterol, most likely due to overexpression of the hepatic lipogenic gene.144 The unexpected rise in cholesterol during this study further highlights the

Table 2. siRNA-Mediated Silencing of Various Key Genes Implicated in Insulin Resistance target gene MAP4K4117

IKBKB118

STAT3120

SOSC3120

PTPN1128

protein and its functiona (a) MAP4K4 (b) serine/threonine kinases (c) JNK1 activation (a) IKKβ (b) serine/threonine kinases (c) TNF-α-mediated insulin resistance (a) STAT3 (b) STAT (c) IL-6-mediated insulin resistance (a) SOSC3 (b) SOCS (c) IL-6 mediated insulin resistance (a) PTP-1B

siRNA delivery method

pharmacological/ molecular tools

Lipofectamine

primary human skeletal muscle

Lipofectamine

satellite cells isolated from muscle biopsies

calcium phosphate transfection

rat L6 muscle cells

calcium phosphate transfection

rat L6 muscle cells

Lipofectamine

Hepa1-6 mouse hepatoma cells db/db diabetic mice ob/ob obese mice

(b) tyrosine phosphatase (c) negative regulator of insulin and leptin signaling proteins a

Labels: (a) protein, (b) protein family, and (c) pathway involved in insulin resistance.

4.2. Normalizing Hyperglycemia

Glucose supplies the central nervous system with a continuous source of energy,36 which needs to be maintained at a concentration of ∼100 mg/dL. The liver, on the other hand, plays a central role in maintaining glucose homeostasis,130 this being achieved through the actions of glucagon (secreted by pancreatic α cells)131 and insulin (secreted by pancreatic β J


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Figure 11. Liver as the target for siRNA-mediated gene silencing for normalizing hyperglycemia at (A) the glucagon receptor and (B) gluconeogenic genes.

Table 3. siRNA-Mediated Silencing of Various Key Genes Implicated in Hyperglycemia target gene PPARGC1A

PEPCK138

CRTC2139

TRIB3141

GCGR144

a

137

protein and its functiona

mode of siRNA delivery

(a) PGC-1 (b) transcriptional coactivator (c) regulates genes involved in gluconeogenesis (a) PEPCK (b) lyase (c) rate-limiting enzyme in gluconeogenesis (a) TORC2 (b) transcriptional coactivator (c) regulates genes involved in gluconeogenesis (a) TRIB3 (b) protein kinase (c) negative regulator of Akt in insulin signaling (a) glucagon receptor (b) G-protein coupled receptor (c) release of glucose-1-phosphate from glycogen in liver

FuGENE

polyethylenimine

pharmacological/molecular tools db/db diabetic mice rat primary hepatocytes HEPG2 cells human hepatoma cell line Huh-7 streptozotocin-induced diabetic mice

lipid nanoparticles

C57BL/6 mice mouse hepatocytes

FuGENE

db/db diabetic mice rat primary hepatocytes HEPG2 cells db/db diabetic mice

lipid nanoparticles

Labels: (a) protein, (b) protein family, and (c) pathway leading to hyperglycemia in T2DM.

immensely complex interplay between various signaling pathways that are at play, and much effort is still needed to better understand and comprehensively unraveled their associations. In summarizing, the aforementioned studies utilized RNAi technology to target key genes involved in the regulation of glucose metabolism, thus demonstrating proof-of-concept for

the future development of RNAi-based therapies for T2DM (Table 3). 4.3. Targeting Vascular Complications of T2DM

The direct and indirect consequences of chronic hyperglycemia and their presentation on the human vascular tree are a major cause for concern in DM. Generally, the deleterious effects resulting from hyperglycemia are collectively referred to as K


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micro- and macrovascular complications.133 The tissue damage arising from microvascular complications in particular encompasses specific cell types, namely, capillary endothelial cells within the retina (retinopathy),145 mesangial cells in the renal glomerulus (nephropathy),146 and Schwann cells that line peripheral nerves (neuropathy).147 The primary underlying mechanisms/processes driving such complications have been identified as the polyol pathway (Figure 4, pathway 8),46,148 generation of AGEs (Figure 4, pathway 9 and Figure 5),149 PKC activation (Figure 10, pathway 1),150 increased activity along the hexosamine pathway (Figure 4, pathway 7),45 and increased oxidative stress due to elevated ROS production.151 Here, RNAi-based intervention through post-transcriptional gene silencing has emerged as a viable therapeutic approach in targeting the underlying causes of microvascular complications, examples of which are elaborated on below. Many questions remain unanswered as to the precise etiology of diabetic retinopathy,145 a slow progressing disease and a leading cause of vision loss in many developed nations. What is abundantly clear is that hyperglycemia remains primarily responsible for driving the accumulation of sorbitol36,113 and AGEs,149 increased oxidative stress,151 activation of the renin angiotensin system,152 and up-regulation of vascular endothelial growth factor (VEGF) production.153 To date, laser-mediated retinal photocoagulation is the only supportive therapy available for diabetic retinopathy;154 however, the wider effects of laser use are associated with a range of side effects that render this approach far from optimal.155 Improved avenues of treatment focusing on direct intraocular administration of antiVEGF agents, such as ranibizumab and bevacizumab, have undergone trial with some success.156 Site-specific delivery of therapeutic antibodies by intravitreous injection circumvents the peripheral side effects, such as hypertension, stroke, myocardial infarction, and nonocular hemorrhaging, which may otherwise be triggered through broader down-regulation of VEGF following systemic administration.157 That said, the protective role VEGF has to play on retinal ganglion cells against peroxide-mediated oxidative stress is another important, and often overlooked, consideration that calls for upstream VEGF-based interventions, over direct inhibition.158 To this end, phase 2 clinical trials where patients were administered a range of doses (0.4, 1, and 3 mg) of siRNA designed to block a specific hypoxia-induced stress protein pathway (RTP801) were compared with laser photocoagulation therapy. The overall outcome pointed to a vast improvement in visual acuity for subjects treated with siRNA at 1 and 3 mg, when compared to laser treatment.159 Separately, hyperglycemia has also been reported to induce extracellular matrix gene expression, resulting in excessive synthesis of retinal capillary basement membrane components, such as fibronectin, collagen type IV, and laminin.160 Collectively, an elevation in these components leads to retinal capillary basement membrane thickening, which promotes characteristic lesion formation, vascular leakage, and an overall disturbance in vascular homeostasis. Keeping this in mind, siRNA-mediated gene silencing of fibronectin in streptozotocin-induced diabetic rats has resulted in significant reductions of basement membrane thickening.161 Diabetic nephropathy,146 another poorly understood disease process, is a primary cause of chronic renal failure, characterized by deposition of extracellular matrix in glomerular and tubulointerstitial compartments. Other specific features of diabetic nephropathy include thickening and hyalinization of intrarenal vasculature.146 Other than the common cellular

mechanisms45,36,113,149,150 involved in microvascular complications, overexpression of transforming growth factor-β (TGFβ),162 GTP-binding protein,163 and ROS151 are also seen in patients with diabetic nephropathy. Of these, TGF-β1 in particular is reported to play a key role in the accumulation of extracellular matrix within the kidneys.164 The inhibitory effect of siRNA on the high glucose-induced overexpression of TGFβ1 in rat mesangial cells was accompanied by a down-regulation of plasminogen activator inhibitor 1 and collagen type I.165 However, because of its pleiotropic actions, TGF-β may not be an ideal target in treating or circumventing diabetic nephropathy.166 Other than TGF-β, increased gene expression of connective tissue growth factor,167 VEGF, 168 bone morphogenetic proteins (BMPs),169 and gremlin (a BMP antagonist)170 have also been identified as possible targets. Under normal physiological conditions, gremlin levels are relatively low within adult kidneys, being highly expressed in biopsy specimens taken from patients with diabetic nephropathy, where it colocalizes with TGF-β1.170 siRNA-induced silencing of gremlin has been examined as a viable therapeutic target in vivo with progression of diabetic nephropathy monitored in a mouse model. In vivo inhibition of gremlin significantly decreases proteinuria and collagen IV accumulation as well as inhibition of renal cell proliferation and apoptosis in streptozotocin-induced mouse models.171 Diabetic neuropathy,147 which presents primarily as chronic pain172 with associated numbness/tingling sensations of the extremities, is a very common complication of DM, and although improved glycaemic control reduces its risk, the overall incidence is still high (∼50%).173 The underlying pathophysiology is once again poorly understood, although hyperglycemia induced by sorbitol,36,113 AGEs,149 oxidative,151 and inflammatory stress correlate closely with diabetic neuropathy. In the peripheral nervous system, sensory neurons, Schwann cells, and the microvascular endothelium are susceptible to oxidative and inflammatory stress in the presence of altered metabolic states, as is observed in DM.174 To date there are no approved interventions that effectively prevent or halt painful diabetic neuropathy, and only symptomatic pain therapies,172 with variable levels of efficacy, modest at best, are available. Given the great potential and promise that siRNA holds, it is hoped that various selective therapies can be developed that can effectively target the underlying pathophysiological mechanisms associated with diabetic nephropathy. 4.4. Engineering siRNA’s for Enhanced Target Specificity and Bio/Thermostability

The tremendous potential that siRNA-mediated gene silencing presents in treating metabolic diseases like T2DM has been convincingly showcased across various preclinical models of the disease.7 Yet, despite the immense therapeutic attractiveness of gene knockdown technologies, and siRNA in particular, such biomolecules are considered far from optimal “druglike” molecules.175 Serum instability, due to the abundance of exo/ endonucleases, and a tendency for the duplexes to dissociate in the systemic circulation are major primary obstacles to their use.176 siRNAs, with their large molecular volume and anionic surface charge, are rendered ideal candidates for extensive binding with albumin and rapid renal clearance.177 Secondary to the systemic challenges giving rise to inefficient/unreliable delivery (by vectors) is the propensity for off-target effects to occur.178 In efforts to address these shortfalls, a range of structural modifications have been proposed, on the L


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Figure 12. Various nucleobase modifications: 2,4-difluorotoluene,184 5-bromouracil,185 5-methyluracil,186 5-iodouracil,185 N-methyladenine derivatives,187 2-thiouracil, pseudouracil, dihydrouracil,188 3-methyluracil,185 5-propynyluracil,186 4-thiouracil,185 diaminopurine,186 dichlorobenzene,189 4,6-difluorobenzimidazole,190 and 7-deazagaunine.191

sugar-modified analogues have been reported since the late 1990s, with bridged or locked nucleic acids (BNA/LNA) being among the most prominent and potent analogues.196 These nucleotide analogues are now more widely recognized as LNA, a term first coined by preeminent nucleic acid biologist Jesper Wengel.196b Unique to LNA is the ribose unit, which is locked through an oxymethylene bridge connecting the C(2′)- and C(4′)-positions; this imposes spatial restrictions that force them to adopt an N-type (C3′-endo) sugar puckering conformation.196,197 It was further demonstrated that these constrained analogues can be successfully utilized to form stable oligonucleotide duplexes in synthetic DNA, as well as RNA.196,197 Thermal denaturation studies prove that LNAbased oligonucleotides display high binding affinity to complementary DNA and RNA oligonucleotides,196b,198 while NMR spectroscopy reveals that LNA-containing oligonucleotides possess an A-type duplex geometry.199 LNA-modified oligonucleotides have proven to be uniquely stable,198d being extensively used across the field of chemical biology, therapeutics, and biotechnology198c (siRNA,200 antisense oligonucleotide,201 DNAzyme,202 miRNA targeting probes,203 aptamers204) due to their favorable properties compared to conventional DNA or RNA, including high bio/thermostability and improved binding affinity.205 A broad range of sugar modifications206 with enhanced stability against serum endo/ exonucleases have also been proposed, many of which are depicted in Figure 13. 4.4.3. Internucleotidic-Linkage (Phosphate) Modifications. Structural modification of the bridge within the internucleotide linkage has involved replacement of nonbridging oxygen atoms in the phosphate backbone (Figure 14). Here, the most notable modifications reported included phosphorothioates (PS),224 methylphosphonates (Me− P),225 borane phosphonates (P−BH3), and cationic phosphoramidites.226 In contrast, researchers have also been successful in removing the nucleotide backbone altogether, this being achieved through the use of peptide nucleic acids (PNAs) instead.227 The improvement in potency and biostability that phosphate backbone modification brings to nucleotides has attracted considerable interest in the field of oligonucleotidebased therapeutics.228

nucleobase, sugar moiety, and phosphate linkage of a nucleotide monomer, leading to a library of semisynthetic siRNA’s with superior stability, safety, and efficacy (Figure 12).179 Modified nucleic acids also bring with them a greater spectrum of applications not only in therapeutics180 but extending into diagnostics, e.g., as chemical probes,98b,181 while facilitating an expansion of the current knowledge of interaction/dynamic phenomena, such as protein−nucleic acid interactions.182 4.4.1. Nucleobase Modifications. The primary purposes of nucleobase (purine and pyrimidine) modifications are to enhance the binding strength and kinetics of duplexed oligonucleotides.183 Classic examples of base-modified nucleosides include 2,4-difluorotoluene;184 5-bromo-,185 5-methyl-,186 5-iodouracil;185 and N-methyladenine derivatives (Figure 12).187 Subtle conformational changes within the minor/ major grooves can also impact the physicochemical properties of the duplexes, leading to melting temperature (Tm) fluctuations, which improve their thermostability.186 Comparing the properties of 2-thiouracil, pseudouracil, and dihydrouracil derivatives, researchers saw no effect on Tm for the former two, while introducing dihydrouracil presented a 3−5 °C rise in Tm. From a potency perspective, 2-thiouracil and pseudouracil modifications on the 3′-end and dihydrouracil modifications on the 5′-end improve gene-silencing potency by 25−30%, when compared to native siRNA, which are devoid of any modifications.188 4.4.2. Sugar Modifications. Modification to the sugar unit has attracted considerable interest, with antisense oligonucleotides demonstrating superior antisense properties. For example, 2′-O-modifications on sugar bearing electronegative substitutions considerably improved the binding affinity to target mRNA.192 Other factors that greatly influence affinity toward the target and nuclease resistance are length, net electronegativity, and the conformation of 2′-substitution.193 Methyl substituted or 2′-halo moieties possess remarkably high affinity and conformational stability toward complementary RNA, although their resistance to nucleases is somewhat diminished.194 Increasing overall steric bulk at the 2′-position certainly improves nuclease stability, but this does not necessarily lead to enhanced potency.195 A number of 2′M


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Figure 15. Architecturally modified siRNA.

silencing reported, while also displaying lower levels of immunogenicity compared to their unmodified counterparts.235 To date, canonical synthetic 21-mer RNA duplexes remain the most widely studied construct, with annealing affording a 19-bp duplex with 2-nt overhangs at both 3′-ends.236 Alternatively, lengthier (27-mer) design types such as dicersubstrate siRNAs (DsiRNAs) are also employed, as they can mimic various dicer substrates and so improve their union into the RNAi pathway, while also substantially enhancing gene silencing.236,237 Other lengthier versions include short hairpin siRNA (shRNA)238 and dumbbell-shaped nanocircular RNA (23−27-mer),239 which can be cleaved by dicer-forming dsRNA’s that are reported to be more potent and display prolonged interference. Efforts to mitigate off-target effects caused by the sense strand240 have also been addressed by eliminating the sense strand altogether (i.e., single-stranded siRNAs),241 although sense-strand shortening has also proven to be another effective strategy.242 Examples of such modifications include small internally segmented interfering RNAs (sisiRNA)243 and asymmetric siRNAs (asiRNA).242 The former three-stranded “sisiRNA” construct consists of one intact antisense strand complemented by two shorter (10−12 nt) sense strands;243 this considerably reduces off-target effects caused by the sense strand and ensures that only the antisense strand is available for incorporation into the RISC. Conversely, asiRNA’s are primarily characterized by a long antisense and short sense strand, with the latter strand far less likely to be incorporated into RISC, once again resulting in less off-target silencing compared to native siRNA.242 Interestingly, the off-target behavior of the sense strand has been successfully exploited to target alternative genes, this being achieved through the concept of “dual-targeting siRNA”, whereby the sense strand is replaced by an antisense strand directed toward another target gene.244 Other promising modifications include forked-siRNA, where the sense strand possesses a few nucleotide mismatches at the 3′-end against the antisense strand in the duplex. This modification is not only responsible for enhanced RNAi activity but has gone on to display superior stability against nucleases compared to traditional siRNA.245 4.4.5. siRNA Overhangs and Termini Modifications. The key quality determinants of an siRNA’s capacity to silence a target gene depends on its stability against exo- and endonucleases, thermodynamics, unwinding capability, loading into the RISC, and the rate at which it cleaves target mRNA.232,234 The plasma half-life of traditional naked siRNA typically ranges from several minutes to 1 h. 246 The susceptibility of naked siRNA toward degradation by exo- or endonucleases present in serum has profoundly limited their application in vivo.247 Such limitations can be overcome

Figure 13. Various sugar modifications: 2′-OMe,207 2′-fluoro,208 2′amino,209 locked nucleic acid (LNA),196b 2′-O-Me-4′-thioRNA (MeSRNA),210 unlocked nucleic acid (UNA),211 oxetane-LNA (OXE),212 2′-methoxyethyl (2′-MOE),213 cyclohexenyl,214 hexitol nucleic acid (HNA),215 α-L-LNA,216 4-thio,217 2′-fluoro-4′-thioRNA (F-SRNA),210 2′-aminoethyl (EA),218 2′-cyanoethyl (CE),218 4′-C-hydroxymethyl (HM),219 ethylene bridge nucleic acids (ENA),220 2′-fluoro-β-Darabinonucleotide (FANA),221 4′-thio-FANA (4′S-FANA),222 5′OMe.223

Figure 14. Various internucleotidic-linkage modifications: 2′,5′linked,229 phosphorothioate,224 amide-linked,230 peptide nucleic acids (PNA),227 and boranophosphate.231

4.4.4. siRNA Design Type Modifications. In recent years, nucleic acid researchers the world over have focused on structural modifications (sugar, base, or internucleotidic linkage)232 and various delivery approaches,233 in an attempt to improve the pharmacokinetic/dynamic outcomes of siRNAbased therapies. Here, architectural modification has provided a useful tool for designing efficacious and target-specific siRNA’s, and this can ultimately be achieved through chemical synthesis.234 Several chemically modified siRNA’s (Figure 15) have shown promise, with superior stability, specificity and gene N


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Figure 16. Conjugation strategies to enhance in vivo pharmacokinetic/dynamic behavior of siRNA. (Carbohydrates: lactose264 and Nacetylgalactosamine.265 Lipids: cholesterol266 and α-tocopherol.267 CPPs: HIV-TAT,268 penetratin,269 and transportan.270 Antibody: monoclonal antibody to the transferrin receptor.271 Aptamers: HNA272 and LNA.273)

through introduction of various chemical modifications in the nucleic acid structure and architecture, as discussed earlier. One of those structural modifications includes various overhangs or termini alternations, which serve to build siRNA constructs that are more resistant to nuclease degradation.232,234 Traditionally, canonical siRNA constructs possess two deoxythymidines at both (sense and antisense strand) 3′-termini.248 Studies have shown that siRNA duplexes absent of specific nucleotide overhangs (i.e., blunt siRNA’s) are more efficient at PTEN gene silencing in HeLa cells, this compared to 3′- or 5′-overhangcontaining siRNAs.249 Blunt siRNA’s with varying duplex lengths (21−45-mer) were compared to those siRNA with 3′or 5′-overhangs, the former of which (a 27-mer) showed maximum RNAi activity.250 Accumulating evidence also suggests that blunt siRNA’s are more resistant to 3′exonucleases compared to traditional siRNA duplexes.251 Furthermore, 3′-overhang modifications on both sense as well as antisense strands, through addition of aromatic compounds252 and PNA,253 can enhance siRNA duplex stability. Sticky siRNA, a relatively new class of siRNA developed by Bolcato-Bellemin et al., consists of a short complementary “A5−8/T5−8” 3′-overhang (a.k.a. sticky overhang). The rationale for developing sticky siRNA’s has been 2-fold, with it not only enhancing the stability of siRNA/vector complexes against nucleases but also significantly improving gene silencing.254 4.4.6. siRNA Modification through Chemical Conjugations. Despite the immense therapeutic attractiveness and appeal of RNAi, its clinical applicability remains questionable due to the undesirable pharmacokinetic properties of siRNA, as well as its poor cellular uptake and inefficient delivery.255 Thus, all these factors should be borne in mind when designing siRNA delivery system constructs, so as to improve tissuespecific targeting and promoting cellular uptake of siRNA.255,256 An array of delivery vectors have been investigated for these purposes, being either viral (e.g., lentiviruses, adenoviruses)257 or nonviral (e.g., liposomes, dendrimers, polymers, peptides) in origin.258

Some nonviral cationic vectors are able to condense siRNA into nanosized polyelectrolyte complexes through complementary charge−charge interactions.259 By virtue of the condensing process cationic polyelectrolyte complexes are effective at protecting their cargo from enzymatic degradation, going on to be internalized by endocytosis.260 Despite advancements in the engineering of cationic-vector systems, commercially available synthetic vectors display significant cytotoxicity, limiting their clinical application.261 To overcome some of these limitations, researchers are increasing their focus on covalent conjugation of siRNA to peptides, proteins, lipids, aptamers, small molecules (cholesterol, tocopherol, folic acid), or polymers (Figure 16).262 Importantly, the targeting ligand itself directly or indirectly influences the in vivo pharmacokinetic/dynamic behavior of siRNA.263 Given that traditional siRNA is composed of two complementary strands, theoretically there exist four possible conjugation sites at the termini (3′ and 5′) of each sense and antisense strand. That said integrity of the 5′-termini of the antisense strand is considered essential for incorporation into the RNAi machinery, which primarily leaves the 3′- and 5′termini of the sense strand and the 3′-termini of antisense strand as key sites for conjugation, while a negligible impact on gene silencing is endured.274 A significant body of work exists in the literature describing conjugation strategies for preparing targeted siRNA’s, with most employing selectively cleavable acid-labile (cleaved in the acidic endosomal compartment) 264 and redox-sensitive (cleaved in reductive cytosolic space)275 linkages that facilitate release of intact siRNA intracellularly. 4.4.6.1. siRNA−Carbohydrate Conjugates. Lactose was among the first carbohydrates to be conjugated to siRNA via the sense strand 5′-termini, while also PEG and an acid-labile βthiopropionate spacer were incorporated.264 The Lac−PEG− siRNA conjugate was prepared as a micellar formulation with luciferase expression in Huh-7 cell lines then determined. A 100-fold enhancement of luciferase gene silencing resulted; this comparison to the free siRNA value highlights the potential of O


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targeting ligand−siRNA conjugation strategies.264 Tethering of multiple (one, two or four) consecutive glucose or galactose units at the 5′-termini of sense strands on siRNA has also been investigated, with preference noted for galactose over glucose units in vitro.276 Although carbohydrates clearly exploit receptor-mediated pathways for cellular entry, individual carbohydrate−protein interactions are relatively weak, which presents opportunities for their oligomeric forms in promoting endocytosis.277 A case in point is the trident form of Nacetylgalactosamine (GalNAc)3, which uses asialoglycoprotein receptors present specifically in hepatocytes for cellular entry. When conjugated to an oligonucleotide, it was found to substantially improve cellular uptake in hepatocellular carcinoma cells (HepG2), while delivery to human fibrosarcoma (HT1080) and human promyelocytic leukemia (HL-60) cells was relatively poor.265 Similarly, in vitro and in vivo delivery of siRNA to hepatocytes through conjugation with triantennary (GalNAc)3 generated constructs with enhanced (5-fold greater) gene-silencing efficacy and systemic stability following either systemic or subcutaneous administration.278 4.4.6.2. siRNA−Lipid Conjugates. Among the various nonviral vectors that underwent trials for siRNA delivery, lipid conjugation to nucleic acids remains one of the most promising strategies, leading to improved membrane permeability, as well as cell-specific uptake and delivery.279 Two broad strategies exist for lipid-mediated siRNA delivery, the first requiring formation of lipid vesicles that entrap siRNA/coat the vesicle surface and the second involving direct covalent tethering of lipid and siRNA.279,280 The vesicular approach most typically relies upon the formation of electrostatic complexes between siRNA and cationic lipids, and this also generates highly stable lipoplexes.281 That said, lipid−siRNA chemical conjugates have also shown promise through an improved circulatory half-life in plasma and more potent gene silencing observed both in vitro and in vivo.282 Cholesterol, one of the most extensively studied lipids for siRNA delivery, has been conjugated to the 3′-sense strand end of siRNA through a highly stable pyrrolidone linkage. The resulting conjugate significantly increased RNAi activity in vitro. Moreover, in vivo administration led to significant levels of gene silencing in the liver and jejunum, with improved pharmacokinetic behavior also observed.266 Tissue distribution studies also showed that the cholesterol−siRNA construct accumulated in the liver, heart, kidneys, and adipose tissues, remaining detectable 24 h after initial iv administration of the formulation.266 In addition to siRNA conjugates with cholesterol, those with bile acids and long-chain saturated and unsaturated fatty acids have also been investigated, showing varying degrees of tissue specificity and effectiveness in gene silencing.283 Once administered, such hydrophobic conjugates interact with lipoproteins (high and low density), lipoprotein receptors, and transmembrane proteins, each of which play a vital role in facilitating their distribution through tissue and also cellular uptake.283a siRNA conjugates with relatively high affinity for lipoproteins have shown superior gene-silencing ability, which highlights the role of lipoproteins in facilitating cellular uptake and gene delivery. Interestingly, it is possible to predict tissue distribution of siRNA−lipid conjugates on the basis of the type of lipoprotein they associate with following administration. For instance, siRNA−lipid conjugates binding preferentially to lowdensity lipoproteins primarily distribute to the liver, while those bound to high-density lipoproteins more evenly distribute

across the liver, gut, and kidneys. Furthermore, following cholesterol−siRNA administration, they bound specifically to high-density lipoproteins, possessing a 5-fold greater apolipoprotein B (apoB) gene-silencing activity in mice compared to free cholesterol−siRNA, at equivalent concentrations.283a Separately, lipid-soluble vitamins such as α-tocopherol (vitamin E) once conjugated to the 5′-termini of the antisense strand of siRNA also display liver-specific delivery and potent gene silencing without induction of inflammatory interferons.267 4.4.6.3. siRNA−Peptide Conjugates. Covalent linkage of siRNA to peptides generates chimeric molecules that show a range of properties dictated by the nature of the conjugated peptide.280 To elaborate, neutral peptides typically infer greater resistance to nucleases compared to unmodified oligonucleotides,284 while cationic peptides tend to accelerate the annealing process and duplex formation.285 Cell-penetrating peptides (CPPs), such as HIV-derived TAT,268 penetratin,269 and transportan,270 are among the most widely reported CPPs enhancing cellular delivery of therapeutics. Direct conjugation of CPPs, such as TAT to the 3′-antisense end of siRNA via a highly stable linker, showed remarkable levels of gene silencing when compared to Lipofectamine.286 Although the 5′-terminal on the antisense strand of siRNA is and remains crucial for induction into the RNAi machinery, the use of cleavable linkages here, such as a disulfide bond (−SS−), is a plausible strategy undergoing trial. The release of intact siRNA from the conjugate in the highly reductive cytosolic environment is expected to promote integration of siRNA into the RISC, thus eliminating any actual or perceived interference from CPPs stemming from their linkage with siRNA. Disulfide bonds are typically introduced by oxidation of thiol groups, resulting in CPP−SS−siRNA constructs with highly proficient gene-silencing activity across a variety of cell lines.287 Other CPPs, such as penetratin and transportan, have been linked to the 5′-terminal of the sense strand on siRNA in a similar manner, with comparable RNAi activity to that of cationic-lipid-based formulations.288 Penetratin-1−SS−siRNA conjugates have been successful in the delivery of siRNA to primary mammalian neuronal cells against superoxide dismutase (SOD) variants and caspases, demonstrating efficient gene silencing without apparent cytotoxicity.289 4.4.6.4. siRNA−Antibody Conjugates. Antibody-targeted drug therapies against cancer are clinically available,290 and a similar strategy has been pursued using siRNA to improve brain targeting. A monoclonal antibody (mAb) specific to the transferrin receptor expressed on the blood−brain barrier was coupled to biotinylated siRNA through a streptavidin-based linkage strategy, with delivery to the brain achieved and suppression of the reporter gene detected.271 Although encouraging, further studies are warranted in this area to examine the safety of targeted siRNA delivery to the central nervous system, particular for aggressive brain tumors, as well as neurodegenerative diseases. 4.4.6.5. siRNA−Aptamer Conjugates. Aptamers are essentially oligonucleotides or peptides having selective affinity toward a target protein, having found their place as effective targeting agents across a broad range of cell/disease models.291 These specialized macromolecules, ranging from ∼1 to ∼25 kDa, are characterized by their globular structure, with nucleic acid aptamers considered superior to mAb’s given their relative ease of synthesis.292 Further, subsequent chemical modificaP


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achieve and maintain euglycaemic levels, remain far from optimal clinically. This is reflected by their limited efficacy, which translates into poor glycaemic control, the latter of which exposes patients to a broad range of micro/macrovascular complications that are ultimately responsible for premature mortality. Despite the large panel of pharmacological agents at the disposal of practitioners, few are known to ameliorate insulin sensitivity, while none have shown great potential in halting the underlying causes of the disease, namely, insulin resistance and the progressive decline of pancreatic β-cell function. Over the last few decades, researchers the world over have deciphered molecular transformations characteristic of T2DM, this being achieved with the help of various animal (e.g., transgenic) models of the disease, advancements in gene transfer techniques, and next-generation pharmacological agents. Such knowledge has unlocked new, previously unexplored avenues for the development of potentially more meaningful, truly curative treatment modalities. To fulfill the potential these multimodal approaches represent, multidisciplinary teams of scientists will be required to work collaboratively to translate these genetic discoveries from the bench to bedside. In this review, we focused on recent accomplishments in the understanding and manipulation of key causative genes in T2DM, critically appraising and presenting the rationale for developing nucleic acid-based therapies through identification of viable focused enzymatic targets that have come to light most recently, and that hold much promise as effective therapies for T2DM into the future.

tions to reduce toxicity and immunogenicity are relatively straightforward to perform.293 siRNA−aptamer constructs conjugated through biotin−streptavidin linkages have targeted prostate-specific membrane antigen (PSMA)294 and HIV-1295 with good levels of gene silencing observed, although aptamer stability in vivo remains a bottleneck to their broader clinical application.296 In an effort to address issues of stability, a range of carbohydrate-modified xeno nucleic acid (XNA) aptamers have been proposed, and these include TNA (threose), HNA (hexitol), GNA (glycol), ANA (arabino), and FANA (2′fluoroarabino).204a,272,297 Through various iterations of the systematic evolution of ligand by the exponential enrichment (SELEX) method, efforts to generate modified XNA aptmers using the traditional amplification approach, via polymerase, have been attempted.298 While this process proceeds with ease for native DNA/RNA library synthesis, it remains wholly inefficient when modified nucleic acids, such as XNA aptamers, are employed. As such, significant attention has focused on refining the SELEX protocol, and some successes have emerged for HNA aptamer synthesis against HIV-transactivating response RNA and hen egg lysozyme;272 this success has most recently been extended to LNA-containing DNA aptamers against human thrombin.273 Although research into aptamer development is gathering significant momentum, chemical modifications that infer greater in vivo stability and retain specificity will be crucial to realizing the significant potential of therapies based on nucleic acid aptamers.299 4.5. Challenges in siRNA Delivery

Successful targeted delivery and gene silencing by siRNA is a complex and challenging process, most often requiring a suitable vehicle with a high capacity for siRNA loading and one that ensures its efficient and timely release in the cytosol.300 A wide array of delivery vectors have been investigated301 for this purpose, with many presently in various stages of clinical evaluation.302 These vectors can be broadly classified as being either viral303 or nonviral/chemical258 in origin, with the former having evolved over many millennia to become highly proficient at infecting host cells unchallenged, and this strategy continues to be pursued in siRNA delivery.304 That said, viral vectors are not without their significant shortfalls, having been reported to elicit mutagenic305 and immunogenic responses,306 while also possessing a broad level of tropism,178b not to mention being plagued with batch-to-batch variability during large-scale production.307 In contrast, nonviral/chemical vectors have demonstrated tremendous potential in overcoming many of these drawbacks and are emerging as viable alternatives to viruses, especially with respect to their safety, ability to deliver large payloads in a targeted manner, and being highly amenable to bulk manufacture.308 An increasing number of these nonviral/chemical vectors are now transitioning to the clinical setting, where their safety and efficacy is being evaluated.258 With continual improvements in vector design assisted by breakthroughs in nanotechnology/chemical engineering, there is an expectation that this will yield systems that rival or even supersede the efficacy of viruses, paving the way toward safer and more effective approaches to gene-silencingbased therapies.

AUTHOR INFORMATION Corresponding Authors

*R.N.V. e-mail: [email protected] or [email protected]. au. *H.S.P. e-mail: [email protected]. Author Contributions

G.R.K. and H.S.P. contributed equally to this work. Notes

The authors declare no competing financial interest. Biographies

Ganesh R. Kokil obtained his pharmacy degree from Shivaji University, Kolhapur, India (2004) and went on to receive a prestigious fellowship from the All India Council for Technical Education (AICTE) in 2005, enabling him to pursue a master’s degree in pharmaceutical chemistry from the Bharati Vidyapeeth University, Pune, India (2007). He has had a long-standing personal interest in unraveling new therapeutic avenues for type 2 diabetes mellitus, with 12 international peer-

5. CONCLUDING REMARKS AND OUTLOOK The number and class of oral hypoglycemic agents approved for human use in T2DM have grown significantly over the past 2 decades, yet such interventions, which primarily aim to Q


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iron metabolism. As an American Liver Foundation Postdoctoral Research Fellow (Dean Theil Foundation Award), he studied the cellular basis of hepatic fibrogenesis at St. Louis University Medical Center with Prof. Bruce A. Bacon and Prof. Robert S. Britton. Returning to Australia in 1995, he established the Hepatic Fibrosis Group at the Queensland Institute of Medical Research (QIMR). He is currently Principal Research Fellow and Head of Department of Cell and Molecular Biology at QIMR Berghofer Medical Research Institute, Senior Research Fellow of the National Health and Medical Research Council of Australia (NHMRC), and Professor at The University of Queensland, Brisbane, Australia. His current research areas include the cellular and molecular basis of hepatic fibrogenesis, wound healing, and liver regeneration in chronic liver disease.

reviewed publications to his credit. On the basis of these achievements, he was awarded a University of Queensland (UQ) international research scholarship, which has enabled him to further explore his research interests in Australia. He commenced his Ph.D. studies in early 2012 and is based at UQ’s recently established and state-of-theart Pharmacy Australia Centre of Excellence. His multidisciplinary supervisory team is comprised of a medicinal/formulation chemist (H.S.P.), nucleic acid biologist (R.N.V.), and liver expert (G.A.R.). His research project is aimed at designing, developing, and delivering modified nucleic acid-based therapeutics against type 2 diabetes mellitus, to target key overexpressed genes responsible for driving insulin resistance in the liver.

Rakesh N. Veedu obtained his Ph.D. in synthetic organic chemistry in 2006 from The University of Queensland, Australia, under the supervision of Prof. Curt Wentrup. He then continued his postdoctoral career under the supervision of Prof. Jesper Wengel at the Nucleic Acid Center of the University of Southern Denmark in the field of nucleic acid chemical biology. Later in 2009, he was appointed as a Research Associate Professor at the Nucleic Acid Center. He returned to The University of Queensland in mid-2010 to start his independent research career. He is currently a McCusker Fellow and Group Leader in nucleic acids chemical biology at Centre for Comparative Genomics of Murdoch University and Western Australian Neuroscience Research Institute. His research is focused on novel functional nucleic acid-based biotechnologies for developing target-specific therapies to various diseases, including neurological diseases, inherited/genetic disorders, and solid cancers. Specifically, his research involves the development and applications of nucleic acid aptamers, antisense oligonucleotides, siRNA, antimiRs, DNAzymes, etc.

Johannes B. Prins is Director and CEO of the Mater Research InstituteUniversity of Queensland and Senior Consultant Endocrinologist (pre-eminent status) at the Princess Alexandra Hospital in Brisbane, Australia. His research has had significant impact in the areas of the relationship between obesity and type 2 diabetes and its complications and in the potential for lifestyle modification as a therapeutic modality. Further, he has combined this research approach with ongoing significant clinical and teaching roles (including as Departmental and Divisional Head and as Professor of Endocrinology). His leadership has had a national influence via substantive roles on the Health and Medical Research Council and the Royal Australasian College of Physicians. This has facilitated a great impact via training a large cohort of diabetes research scientists and researchactive clinician−scientists. His mentees include a number of laboratory and clinical department heads worldwide. His basic research mostly involves study of human tissues and primary cultures and thus is readily translatable into the clinical setting. His observations regarding basic adipose biology and the relationship between adipose function and diabetes have been major influences in current approaches into the study of the global problems of diabetes and obesity (“diabesity”). Prior to his observations, human adipose tissue was regarded as a static, relatively inert tissue. His demonstration that adipose is dynamic has directly led to explorations of interventions to alter adipose cell number as an approach to diabetes and obesity. He has patented some of his research findings and spun out a biotech company, and his research has directly led to a novel drug now in phase 1 clinical trials in the United States. His work has been highly cited (an average of >50 cites per paper) with over 800 citations per year. He has consistently published in the leading diabetes journals and is a recognized key opinion leader in diabetes, sitting on numerous national and international committees and advisory boards. He has attracted over $15 million in grant income plus contributed to a large number of additional research grants as coinvestigator. His leadership role extends to a substantial service commitment in diabetes via grant review activities for Australian, US, UK, Singapore, and Canadian agencies;

Grant A. Ramm received his Ph.D. in medicine in 1993 from The University of Queensland, Brisbane, Australia, under the mentorship of Prof. June W. Halliday and Prof. Lawrie W. Powell working in hepatic R


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interfering RNA; SOCS-3, suppressor of cytokine signaling-3; STAT3, signal transducer and activator of transcription 3; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; TGF-β, transforming growth factor-β; TNFR, tumor necrosis factor receptor; TNF-α, tumor necrosis factor alpha; TORC2, transducer of regulated cAMP-responsive elementbinding protein activity 2; TRIB3, Tribbles homologue-3; TZDs, 2,4-thiazolidinediones; UPR, unfolded protein response; VEGF, vascular endothelial growth factor

about 30 journal reviews per year; and more than 50 lectures/ presentations and workshops related to diabetes per year. His mentorship and research training are renowned, and he continues to attract trainees from around Australia and internationally.

REFERENCES (1) Aguirre, F.; Brown, A.; Cho, N. H.; Dahlquist, G.; The Diabetes Education Consultative Section; Dodd, S.; Dunning, T.; Hirst, M.; Hwang, C.; Magliano, D.; Patterson, C.; Scott, C.; Shaw, J.; Soltesz, G.; Usher-Smith, J.; Whiting, D. In IDF Diabetes Atlas, 6th ed.; Guariguata, L., Nolan, T., Beagley, J., Linnenkamp, U., Jacqmain, O., Eds.; International Diabetes Federation: Brussels, Belgium, 2014; http:// www.idf.org/diabetesatlas/introduction. (2) (a) World Health Organization. http://www.who.int/ mediacentre/factsheets/fs236/en/. (b) King, H.; Aubert, R. E.; Herman, W. H. Diabetes Care 1998, 21, 1414. (c) Rathmann, W.; Giani, G. Diabetes Care 2004, 27, 2568. (d) Narayan, K. M. V. Clin. Diabetes 2005, 23, 2. (e) Sicree, R.; Shaw, J.; Zimmet, P. The Global BurdenDiabetes and Impaired Glucose Tolerance. IDF Diabetes Atlas, 4th ed.; International Diabetes Federation: Brussels, Belgium, 2009. (f) Shaw, J. E.; Sicree, R. A.; Zimmet, P. Z. Diabetes Res. Clin. Pract. 2010, 87, 4. (g) Ashcroft, F. M.; Rorsman, P. Cell 2012, 148, 1160. (3) (a) Karamitsos, D. T. Diabetes Res. Clin. Pract. 2011, 93 (Suppl1), S2. (b) Baganz, H. M.; Carfagno, S. C.; Cowan, B. Y.; Dillon, E. S. Am. J. Med. Sci. 1951, 222, 1. (c) Levine, R. Diabetes Care 1984, 7 (Suppl 1), 3. (d) Bailey, C. J.; Day, C. Pract. Diabetes Int. 2004, 21, 115. (e) Ribes, G.; Trimble, E. R.; Wollheim, C. B.; Blayac, J. P.; Loubatieres-Mariani, M. M. Am. J. Physiol. 1983, 244, E380. (f) Cheatham, W. W. Am. J. Clin. Nutr. 2010, 91, 262S. (g) Bischoff, H.; Ahr, H. J.; Schmidt, D.; Stoltefuß, J. Nach. Chem. Technol. Lab. 1994, 42, 1119. (h) Drucker, D.; Easley, C.; Kirkpatrick, P. Nat. Rev. Drug Discovery 2007, 6, 109. (4) (a) Naik, S. R.; Kokil, G. R. In Studies in Natural Products Chemistry; Atta ur, R., Ed.; Elsevier: New York, 2013; Vol. 39. (b) Kokil, G. R.; Rewatkar, P. V.; Verma, A.; Thareja, S.; Naik, S. R. Curr. Med. Chem. 2010, 17, 4405. (5) Rotenstein, L. S.; Kozak, B. M.; Shivers, J. P.; Yarchoan, M.; Close, J.; Close, K. L. Clin. Diabetes 2012, 30, 10. (6) Lebovitz, H. E. Nat. Rev. Endocrinol. 2011, 7, 408. (7) Czech, M. P.; Aouadi, M.; Tesz, G. J. Nat. Rev. Endocrinol. 2011, 7, 473. (8) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806. (9) (a) Lorenzo, C.; Wagenknecht, L. E.; D’Agostino, R. B.; Rewers, M. J.; Karter, A. J.; Haffner, S. M. Diabetes Care 2010, 33, 67. (b) Marchetti, P.; Lupi, R.; Del Guerra, S.; Bugliani, M.; Marselli, L.; Boggi, U. Adv. Exp. Med. Biol. 2010, 654, 501. (10) Cerf, M. E. Front. Endocrinol. (Lausanne, Switz.) 2013, 4, 37. (11) (a) LeRoith, D. Am. J. Med. 2002, 113 (Suppl 6A), 3s. (b) Taylor, R. Diabetes 2012, 61, 778. (12) Prins, J. B. Proc. Nutr. Soc. 1997, 56, 889. (13) Bener, A.; Zirie, M.; Al-Rikabi, A. Croat. Med. J. 2005, 46, 302. (14) Del, P. S. Diabet. Med. 2009, 26, 1185. (15) Hull, R. L.; Westermark, G. T.; Westermark, P.; Kahn, S. E. J. Clin. Endocrinol. Metab. 2004, 89, 3629. (16) Rhodes, C. J.; White, M. F. Eur. J. Clin. Invest. 2002, 32 (Suppl 3), 3. (17) (a) Samuel, V. T.; Shulman, G. I. Cell 2012, 148, 852. (b) Wilcox, G. Clin. Biochem. Rev. 2005, 26, 19. (18) Robinson, L. E.; van Soeren, M. H. AACN Clin. Issues 2004, 15, 45. (19) Vollenweider, P.; Ménard, B.; Nicod, P. Diabetes 2002, 51, 1052.

Harendra S. Parekh obtained his pharmacy degree from the University of Portsmouth (1997) and Ph.D. in medicinal chemistry from the University of Nottingham (2002), in the UK. He relocated to Australia in 2003, where he spent 2 years developing nonviral gene delivery vectors at the School of Chemistry, The University of Queensland (UQ). In 2005, he was appointed Lecturer, establishing his independent research group in the School of Pharmacy at UQ, focusing on the development of chemical carriers for drug and gene delivery. He was granted tenure in 2009 and was promoted to Senior Lecturer in 2012. He is an adjunct-faculty member at Manipal University, Manipal, India, and the National University of Singapore’s (NUS) Nanoscience and Nanotechnology Institute. He has gone on to establish satellite research/commercialization laboratories in both India and Singapore. Active projects and collaborations underway with counterparts in India, China, Singapore, Germany, UK, and Brazil explore the application of dendrimers, liposomes, and bubble liposomes, as well as traditional Chinese and Indian medicine for health and cosmetic benefit.

ACKNOWLEDGMENTS H.S.P. wishes to thank The University of Queensland for a UQI Ph.D. Research Scholarship in support of G.R.K. R.N.V. acknowledges the funding from McCusker Charitable Foundation and Western Australian Neuroscience Research Institute. G.A.R. is supported by a National Health and Medical Research Council of Australia (NHMRC) Senior Research Fellowship (APP1061332). ABBREVIATIONS USED AGEs, advanced glycation end products; Akt, protein kinase B; CCL4, CC chemokine ligand 4; DM, diabetes mellitus; GLUTs, glucose transporters; IkK, IkB-kinase; IL-6, interleukin 6; IR, insulin resistance; JNK-1, Jun-N-terminal kinase-1; LNA, locked nucleic acid; MAP4K4, mitogen-activated protein kinase kinase kinase kinase isoform 4; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor (PPAR)-γ-coactivator 1; PKC-δ, protein kinase C-δ; PNAs, peptide nucleic acids; PPAR-γ, peroxisome proliferator-activated receptor-γ; PTP-1B, protein tyrosine phosphatase 1B; RISC, RNA-induced gene-silencing complex; ROS, reactive oxygen species; siRNA, small S


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