Biodistribution and metabolism studies of lipid nanoparticle-formulated internally [3H]-labeled siRNA in mice.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice.

Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular weight metabolites.

A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs.

Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2'-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity.

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Control of the SOST bone enhancer by PTH using MEF2 transcription factors.

UNLABELLED: Expression of the osteocyte-derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH. INTRODUCTION: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52-kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness. MATERIALS AND METHODS: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast-like UMR-106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody-induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT-PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA-mediated RNA knockdown. RESULTS: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co-localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co-expression of a dominant negative MEF2C mutant. Finally, siRNA-mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR-106 osteoblast-like cells. CONCLUSIONS: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.

siRNAs in drug discovery: target validation and beyond.

'Omics' technologies, combined with knowledge management, have changed the way pharmaceutical companies approach drug discovery activities. The rapid generation of more reliable putative drug targets from gain- or loss-of-function genome scale screens means that there is a strong need to filter and prioritize targets that will be considered in the drug discovery process. Short interfering RNAs (siRNAs) can be used to validate genomic drug targets. The outlook for this approach is promising, provided that special attention is given to the use of optimal reagents and to careful development of animal models.

Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators.

Sphingosine-1-phosphate, a lipid mediator produced by sphingosine kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of sphingosine kinase type 1 (SPHK1) enzyme activity upon stimulation with both TNF-alpha or IL-1beta. This transient activation of SPHK1 was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK1 enzyme activity), but also a dominant-negative SPHK1 mutant (suppressing induced SPHK1 activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-1beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-1, and VCAM-1) was found to require SPHK1 activation. Suppression of SPHK1 activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK1 activation. Inhibition of basal SPHK1 activity by N,N-dimethylsphingosine or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK1 activation in proinflammatory signalling and of SPHK1 basal activity in survival of A549 lung carcinoma cells.

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.

Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.

mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity.

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3'-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs.

siRNA relieves chronic neuropathic pain.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

ADME studies of [5-(3)H]-2'-O-methyluridine nucleoside in mice: a building block in siRNA therapeutics.

The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine.

Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain.

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.

Analysis of ribosyl-modified, mixed backbone analogs of a bcl-2/bcl-xL antisense oligonucleotide.

Progress in oligonucleotide chemistry has provided second-generation antisense oligonucleotides with increased efficacy and reduced non-antisense-related toxicity. The ability of the 2'-O-(2-methoxyethylribose) (2'-MOE)-modified phosphorothioate gapmer oligonucleotide 4625, which matches the bcl-2 mRNA and has three base-mismatches to bcl-xL, to inhibit bcl-2 and bcl-xL expression and induce tumor cell apoptosis has been described. Here we investigated the consequences of adding of 2'-MOE or 2'-Me modifications to ribonucleotides at either the two ends of the sequence, or the center region together with different combinations of phosphodiester/phosphorothioate backbones on the activity of oligonucleotide 4625. The ability of the various 4625 analogs, including the parental first-generation oligonucleotide 3005, to inhibit bcl-2 and bcl-xL expression, and diminish cell growth or induce tumor cell death was assessed in SW2 lung cancer cells using real-time PCR, Western blotting and cell viability assays. Only oligonucleotide 4625 exhibited a potent bispecific antisense activity against bcl-2 and bcl-xL, which effectively reduced tumor cell viability. The other antisense oligonucleotides were either uniquely active against bcl-2 or completely inactive. Our data suggest that the 2'-MOE modification in combination with the phophorothioate gapmer chemistry is the optimal format of the 4625 sequence in terms of antisense activity and biological efficacy.

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides.

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.

A role for oligonucleotide-based RNA-knock down technologies in functional genomics.

Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process.

Dual Recognition of Double-Stranded DNA by 2'-Aminoethoxy-Modified Oligonucleotides.

Simultaneous interaction of the 2'-aminoethoxy-modified oligonucleotides with the phosphodiester backbone (shown on the right, A) and with the bases through Hoogsteen base contacts (B) is seen at each base-pair step of the duplex DNA target. The electrostatic interaction between the protonated amino group and the negatively charged phosphate group provides for a dramatic increase in the binding affinity and the association rate constant.

Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction.

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by rpL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5'-transcriptional start site (5'-TOP mRNAs), including that encoding rpL11, on impairment of 40S ribosome biogenesis. Increased 5'-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins, specific targeting of rpL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction.

Increased exploratory activity of APP23 mice in a novel environment is reversed by siRNA.

Genetic abnormalities in amyloid precursor protein (APP) are associated with Down's syndrome and familial Alzheimer's disease where hallmark plaques contain A beta peptides derived from APP. Both APP and its derivatives are implicated in neurodegenerative processes and may play important physiological and pathophysiological roles in synaptic function. Here, we show that young APP23 transgenic mice overexpressing human APP with the Swedish double mutation display altered novelty seeking behavior before the age of plaque onset. Using short interfering RNA (siRNA) targeted against APP, we investigate the direct contribution of APP and its derivatives to this behavioral deficit. After validating siRNAs targeting human APP in vitro, siRNAs were infused directly into the brain of APP23 mice for 2 weeks. Behavioral analysis shows that infusion of siRNA targeted against APP completely reverses increased exploratory activity in APP23 mice. Collectively, these data suggest that excessive APP and/or its derivatives, causes a hyperactive phenotype in APP23 mice when placed in a novel environment, which is fully reversible and not linked to plaque deposits.

Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34.

Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones. Here we show that AAs induce a rise in intracellular Ca(2+) ([Ca(2+)](i)), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca(2+)](i) increases the direct binding of Ca(2+)/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression.

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells.

BACKGROUND: The mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase. RESULTS: Here we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions. CONCLUSION: This approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.