Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides.

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3-5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

Integration of microRNA miR-122 in hepatic circadian gene expression.

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.

microRNA-33 Regulates ApoE Lipidation and Amyloid-ß Metabolism in the Brain.

Dysregulation of amyloid-ß (Aß) metabolism is critical for Alzheimer's disease (AD) pathogenesis. Mounting evidence suggests that apolipoprotein E (ApoE) is involved in Aß metabolism. ATP-binding cassette transporter A1 (ABCA1) is a key regulator of ApoE lipidation, which affects Aß levels. Therefore, identifying regulatory mechanisms of ABCA1 expression in the brain may provide new therapeutic targets for AD. Here, we demonstrate that microRNA-33 (miR-33) regulates ABCA1 and Aß levels in the brain. Overexpression of miR-33 impaired cellular cholesterol efflux and dramatically increased extracellular Aß levels by promoting Aß secretion and impairing Aß clearance in neural cells. In contrast, genetic deletion of mir-33 in mice dramatically increased ABCA1 levels and ApoE lipidation, but it decreased endogenous Aß levels in cortex. Most importantly, pharmacological inhibition of miR-33 via antisense oligonucleotide specifically in the brain markedly decreased Aß levels in cortex of APP/PS1 mice, representing a potential therapeutic strategy for AD. SIGNIFICANCE STATEMENT: Brain lipid metabolism, in particular Apolipoprotein E (ApoE) lipidation, is critical to Aß metabolism and Alzheimer's disease (AD). Brain lipid metabolism is largely separated from the periphery due to blood-brain barrier and different repertoire of lipoproteins. Therefore, identifying the novel regulatory mechanism of brain lipid metabolism may provide a new therapeutic strategy for AD. Although there have been studies on brain lipid metabolism, its regulation, in particular by microRNAs, is relatively unknown. Here, we demonstrate that inhibition of microRNA-33 increases lipidation of brain ApoE and reduces Aß levels by inducing ABCA1. We provide a unique approach for AD therapeutics to increase ApoE lipidation and reduce Aß levels via pharmacological inhibition of microRNA in vivo.

A screen in mice uncovers repression of lipoprotein lipase by microRNA-29a as a mechanism for lipid distribution away from the liver.

UNLABELLED: Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy-dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR-29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR-29a levels causes lipids to accumulate in mouse livers. CONCLUSION: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR-29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR-29a's known antifibrotic effect, suggest miR-29a is a therapeutic target in fatty liver disease.

De-repression of FOXO3a death axis by microRNA-132 and -212 causes neuronal apoptosis in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder for which the mechanisms leading to profound neuronal loss are incompletely recognized. MicroRNAs (miRNAs) are recently discovered small regulatory RNA molecules that repress gene expression and are increasingly acknowledged as prime regulators involved in human brain pathologies. Here we identified two homologous miRNAs, miR-132 and miR-212, downregulated in temporal cortical areas and CA1 hippocampal neurons of human AD brains. Sequence-specific inhibition of miR-132 and miR-212 induces apoptosis in cultured primary neurons, whereas their overexpression is neuroprotective against oxidative stress. Using primary neurons and PC12 cells, we demonstrate that miR-132/212 controls cell survival by direct regulation of PTEN, FOXO3a and P300, which are all key elements of AKT signaling pathway. Silencing of these three target genes by RNAi abrogates apoptosis caused by the miR-132/212 inhibition. We further demonstrate that mRNA and protein levels of PTEN, FOXO3a, P300 and most of the direct pro-apoptotic transcriptional targets of FOXO3a are significantly elevated in human AD brains. These results indicate that the miR-132/miR-212/PTEN/FOXO3a signaling pathway contributes to AD neurodegeneration.

MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.

RATIONALE: The bile acid receptor farnesoid X receptor (FXR) regulates many aspects of lipid metabolism by variouscomplex and incompletely understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. OBJECTIVE: To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. METHODS AND RESULTS: ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma high-density lipoprotein (HDL)-cholesterol levels. Here, we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lowers hepatic ABCA1 and plasma HDL levels. We identified 2 complementary sequences to miR-144 in the 3' untranslated region of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I protein, whereas overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we used tissue-specific FXR-deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal, FXR. Finally, we identified functional FXR response elements upstream of the miR-144 locus, consistent with direct FXR regulation. CONCLUSIONS: We have identified a novel pathway involving FXR, miR-144, and ABCA1 that together regulate plasma HDL-cholesterol.

Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1.

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.

Therapeutic silencing of microRNA-33 inhibits the progression of atherosclerosis in Ldlr-/- mice--brief report.

OBJECTIVE: To study the efficacy of anti-miRNA-33 therapy on the progression of atherosclerosis. APPROACH AND RESULTS: Ldlr(-/-) mice were injected subcutaneously with PBS, control, or anti-miR-33 oligonucleotides weekly and fed a Western diet for 12 weeks. At the end of treatment, the expression of miR-33 target genes was increased in the liver and aorta, demonstrating effective inhibition of miR-33 function. Interestingly, plasma high-density lipoprotein (HDL)-cholesterol was significantly increased in anti-miR-33-treated mice but only when they were fed a chow diet. However, HDL isolated from anti-miR-33-treated mice showed an increase cholesterol efflux capacity compared with HDL isolated from nontargeting oligonucleotide-treated mice. Analysis of atherosclerosis revealed a significant reduction of plaque size and macrophage content in mice receiving anti-miR-33. In contrast, no differences in collagen content and necrotic areas were observed among the 3 groups. CONCLUSIONS: Long-term anti-miR-33 therapy significantly reduces the progression of atherosclerosis and improves HDL functionality. The antiatherogenic effect is independent of plasma HDL-cholesterol levels.

miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.

miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor.

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G(2)/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the ß2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The ß2 adrenergic pathway may play an important role in this novel mechanism.

Potent inhibition of microRNA in vivo without degradation.

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2'-fluoro/2'-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.

Metabolic Benefits of MicroRNA-22 Inhibition.

Diabesity is a growing pandemic with substantial health and financial consequences. We are developing microRNA (miRNA)-based drug candidates that transform fat storing adipocytes into fat burning adipocytes (browning effect) to treat metabolic diseases characterized by lipotoxicity. Through phenotypic screening in primary cultures of human subcutaneous adipocytes, we discovered that inhibition of miRNA-22-3p by several complementary antagomirs resulted in increased lipid oxidation, mitochondrial activity, and energy expenditure (EE). These effects may be mediated through activation of target genes like KDM3A, KDM6B, PPARA, PPARGC1B, and SIRT1 involved in lipid catabolism, thermogenesis, and glucose homeostasis. In the model of Diet-Induced Obesity in mice of various ages, weekly subcutaneous injections of various miRNA-22-3p antagomirs produced a significant fat mass reduction, but no change of appetite or body temperature. Insulin sensitivity, as well as circulating glucose and cholesterol levels, was also improved. These original findings suggest that miRNA-22-3p inhibition could become a potent treatment of human obesity and type 2 diabetes mellitus, the so-called diabesity characterized by lipotoxicity and insulin resistance.

Metabolic and energetic benefits of microRNA-22 inhibition.

INTRODUCTION: We previously demonstrated in primary cultures of human subcutaneous adipocytes and in a mouse model of diet-induced obesity that specific microRNA-22-3p antagomirs produce a significant reduction of fat mass and an improvement of several metabolic parameters. These effects are related to the activation of target genes such as KDM3A, KDM6B, PPARA, PPARGC1B and SIRT1 involved in lipid catabolism, thermogenesis, insulin sensitivity and glucose homeostasis. RESEARCH DESIGN AND METHODS: We now report a dedicated study exploring over the course of 3 months the metabolic and energetic effects of subcutaneous administration of our first miR-22-3p antagomir drug candidate (APT-110) in adult C57BL/6 male mice. Body composition, various blood parameters and energy expenditure were measured at several timepoints between week 12 and week 27 of age. RESULTS: Weekly subcutaneous injections of APT-110 for 12 weeks produced a sustained increase of energy expenditure as early as day 11 of treatment, a significant fat mass reduction, but no change of appetite nor physical activity. Insulin sensitivity as well as circulating glucose, cholesterol and leptin were improved. There was a dramatic reduction of liver steatosis after 3 months of active treatment. RNA sequencing revealed an activation of lipid metabolism pathways in a tissue-specific manner. CONCLUSIONS: These original findings suggest that microRNA-22-3p inhibition could lead to a potent treatment of fat accumulation, insulin resistance, and related complex metabolic disorders such as obesity, type 2 diabetes mellitus and non-alcoholic fatty liver disease.

Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1.

Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase-1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.

Improved targeting of miRNA with antisense oligonucleotides.

MicroRNAs (miRNAs) are a class of 20-24 nt noncoding RNAs that regulate target mRNAs post-transcriptionally by binding with imperfect complementarity in the 3'-untranslated region (3'-UTR) and inhibiting translation or RNA stability. Current understanding of miRNA biology is limited, and antisense oligonucleotide (ASO) inhibition is a powerful technique for miRNA functionalization in vitro and in vivo, and for therapeutic targeting of miRNAs. Identification of optimal ASO chemistries for targeting miRNAs is therefore of great interest. We evaluated a number of 2'-sugar and backbone ASO modifications for their ability to inhibit miR-21 activity on a luciferase reporter mRNA. ASO modifications that improved target affinity improved miRNA ASO activity, yet the positioning of high-affinity modifications also had dramatically different effects on miRNA activity, suggesting that more than affinity determined the effectiveness of the miRNA ASOs. We present data in which the activity of a modified miRNA ASO was inversely correlated to its tolerability as an siRNA passenger strand, suggesting that a similar mechanism could be involved in the dissociation of miRNA ASOs and siRNA passenger strands. These studies begin to define the factors important for designing improved miRNA ASOs, enabling more effective miRNA functionalization and therapeutic targeting.

Therapeutic potential for microRNAs.

MiRNAs are a conserved class of non-coding RNAs that negatively regulate gene expression post-transcriptionally. Although their biological roles are largely unknown, examples of their importance in cancer, metabolic disease, and viral infection are accumulating, suggesting that they represent a new class of drug targets in these and likely many other therapeutic areas. Antisense oligonucleotide approaches for inhibiting miRNA function and siRNA-like technologies for replacement of miRNAs are currently being explored as tools for uncovering miRNA biology and as potential therapeutic agents. The next few years should see significant progress in our understanding of miRNA biology and the advancement of the technology for therapeutic modulation of miRNA activity.

Evaluation of basic amphipathic peptides for cellular delivery of antisense peptide nucleic acids.

Cellular permeation peptides have been used successfully for the delivery of a variety of cargoes across cellular membranes, including large hydrophilic biomolecules such as proteins, oligonucleotides, or plasmid DNA. For the present work, a series of short amphipathic peptides was designed to elucidate the structural requirements for efficient and nontoxic delivery of peptide nucleic acids (PNAs). On the basis of an idealized alpha-helical structure, the helical parameters were modulated systematically to yield peptides within a certain range of hydrophobicity and amphipathicity. The corresponding PNA conjugates were synthesized and characterized in terms of secondary structure, enzymatic stability, and antisense activity. The study revealed correlations between the physicochemical and biophysical properties of the conjugates and their biological activity and led to the development of potent peptide vectors for the cellular delivery of antisense PNAs. Two representative compounds were radiolabeled and evaluated for their biodistribution in healthy mice.

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs.

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.

Identification and functional validation of PNAs that inhibit murine CD40 expression by redirection of splicing.

Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.

Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid.

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.