Oligonucleotide sequences targeting transcription factor TSC22D4 for the treatment of insulin resistance

Abstract

The present invention relates to oligonucleotide inhibitors of the TSC22D4 activity or expression and their uses for the prevention, treatment, and/or regulation of insulin resistance, metabolic syndrome and/or diabetes and/or for improving insulin sensitivity in a mammal.

Claims

1. An inhibitor of expression and/or biological activity of transforming growth factor beta1 stimulated clone 22 D4 (TSC22D4) wherein said inhibitor is an oligonucleotide that is an interfering ribonucleic acid, protein nucleic acid (PNA) or locked nucleic acid (LNA), and wherein said oligonucleotide comprises SEQ ID NO: 1, or the complementary sequence thereof.

2. The inhibitor according to claim 1, wherein the interfering ribonucleic acid is a small interfering ribonucleic acid (siRNA) or small hairpin ribonucleic acid (shRNA) or micro ribonucleic acid (miRNA) or a combination thereof.

3. The inhibitor according to claim 2, wherein the siRNA has a length of 19 to 30 nucleotides.

4. The inhibitor according to claim 2, wherein the siRNA consists of SEQ ID NO: 1.

5. The inhibitor according to claim 1, wherein the functional variants thereof comprise at least one modified or substituted nucleotide.

6. A recombinant vector, comprising an oligonucleotide according to claim 1.

7. A recombinant cell, comprising an oligonucleotide according to claim 1.

8. A pharmaceutical composition, comprising at least one of the inhibitor according to claim 1, together with a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein said pharmaceutical composition is formulated for administration orally, rectally, transmucosally, transdermally, intestinally, parenterally, intramuscularly, intrathecally, direct intraventricularly, intravenously, intraperitoneally, intranasally, intraocularly, or subcutaneously.

10. A method for prevention, regulation, and/or treatment of a disease, and/or for improving insulin sensitivity, wherein said method comprises administering, to a subject in need of such prevention, regulation, treatment, and/or improvement, the inhibitor according to claim 1.

11. The method, according to claim 10, wherein said disease is selected from insulin resistance, hypertension, dyslipidemia, coronary artery disease, metabolic syndrome and diabetes type 1.

12. The method, according to claim 10, wherein the insulin resistance is diet-induced insulin resistance and/or obesity-induced insulin resistance.

13. A therapeutic kit, comprising the inhibitor according to claim 1, optionally together with suitable buffers and excipients, and instructions for use.

14. The therapeutic kit according to claim 13 with instructions for use in the prevention, regulation, and/or treatment of a disease, wherein said disease is selected from insulin resistance, hypertension, dyslipidemia, coronary artery disease, metabolic syndrome and/or diabetes type 1 or 2, and/or for improving insulin sensitivity.

15. The method, according to claim 10, for improving insulin sensitivity in the context of a tumorous disease.

16. The inhibitor according to claim 1, wherein the interfering ribonucleic acid is a siRNA.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the knockdown efficiency of color-coded, selected TSC22D4-directed siRNAs in murine hepatoma cells. Relative mRNA levels are shown. All other tested siRNA sequences did not show any significant TSC22D4 knockdown in these experiments (not shown).

(2) FIG. 2 shows the knockdown efficiency of mhD4-siRNA1 upon transfection into murine Hepa1-6 hepatoma cells towards murine TSC22D4. Relative mRNA levels are shown.

(3) FIG. 3 shows the knockdown efficiency of mhD4-siRNA1 upon transfection into human Huh7 hepatoma cells towards human TSC22D4. Relative mRNA levels are shown.

BRIEF DESCRIPTION OF THE SEQUENCES

(4) SEQ ID NOs: 1 to 6 show oligonucleotide sequences according to the present invention.

DETAILED DESCRIPTION

Examples

(5) Recombinant Viruses

(6) Adenoviruses expressing a TSC22D4 or a non-specific shRNA under the control of the U6 promoter, or the TSC22D4 cDNA under the control of the CMV promoter were cloned using the BLOCK-iT Adenoviral RNAi expression system (Invitrogen, Karlsruhe, Germany). Viruses were purified by the cesium chloride method and dialyzed against phosphate-buffered-saline buffer containing 10% glycerol prior to animal injection, as described previously (Herzig S, Hedrick S, Morantte I, Koo S H, Galimi F, Montminy M. CREB controls hepatic lipid metabolism through nuclear hormone receptor PPAR-gamma. Nature. 2003; 426: 190-193. Herzig S, Long F, Jhala U S, Hedrick S, Quinn R, Bauer A, Rudolph D, Yoon C, Puigserver P, Spiegelman B, et al. CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature. 2001; 413: 179-183). AAVs encoding control or TSC22D4-specific miRNAs under the control of a hepatocyte-specific promoter were established as described previously (Rose A J, Frosig C, Kiens B, Wojtaszewski J F, Richter E A. Effect of endurance exercise training on Ca2+ calmodulin-dependent protein kinase II expression and signaling in skeletal muscle of humans. J Physiol. 2007; 583: 785-795).

(7) Animal Experiments

(8) Male 8-12 week old C57Bl/6 and 10 week old db/db mice were obtained from Charles River Laboratories (Brussels, Belgium) and maintained on a 12 h light-dark cycle with regular unrestricted diet. Prior to insulin and glucose tolerance tests, animals were fasted for 4 h. Otherwise, animals were fed ad libitum and had free access to water. For adenovirus injections, 1-2×10.sup.9 plaque-forming units (pfu) per recombinant virus were administered via tail vein injection. For AAV experiments, 5×10.sup.11 viruses were injected via the tail vein. In each experiment, 6-12 animals received identical treatments and were analyzed under fasted (18 hrs fasting), random fed or fed (18 hrs fasting followed by 6 hrs re-feeding) conditions as indicated. Organs including liver, epididymal and abdominal fat pads, and gastrocnemius muscles were collected after specific time periods, weighed, snap-frozen and used for further analysis. Total body fat content was determined by an Echo MRI body composition analyzer (Echo Medical Systems, Houston, USA). Animal handling and experimentation was done in accordance with NIH guidelines and approved by local authorities.

(9) For the insulin tolerance tests a stock solution of 1 U Insulin/mL was prepared using 0.9% sodium chloride. Mice were fasted for 4 hours prior to the experiment. The body weight of all animals was determined and the blood glucose levels were measured by cutting the tail with a razor blade. The blood drop was put onto a glucometer strip and measured. 1 U insulin/kg body weight was injected to C57Bl/6 and 1.5 U insulin/kg body weight was injected to db/db mice intraperitoneally. The blood glucose levels were monitored after 20, 40, 60, 80 and 120 min.

(10) For the glucose tolerance tests a stock solution of 20% glucose was prepared using 0.9% sodium chloride. Mice were fasted for 4 hours prior to the experiment. The body weight of all animals was determined and the blood glucose levels were measured by cutting the tail with a razor blade. The blood drop was put onto a glucometer strip and measured. 5 μL per gram of 20% glucose solution was injected to C57Bl/6 and db/db mice intraperitoneally. The blood glucose levels were monitored after 20, 40, 60, 80 and 120 min.

(11) Quantitative Taqman RT-PCR

(12) Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

(13) Human TSC22D4 mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using the TaqMan assay, and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany). Total RNA was isolated using TRIzol (Life technologies, Grand Island, N.Y.), and 1 μg RNA was reverse transcribed with standard reagents (Life Technologies, Grand Island, N.Y.). From each RT-PCR, 2 μl were amplified in a 26 l PCR reaction using the Brilliant SYBR green QPCR Core reagent kit from stratagene (La Jolla, Calif.) according to the manufacturer's instructions. Samples were incubated in the ABI PRISM 7000 sequence detector for an initial denaturation at 95° C. for 10 min, followed by 40 PCR cycles, each cycle consisting of 95° C. for 15 s, 60° C. for 1 min and 72° C. for 1 min. Human TSC22D4 and Obp2a (LCN13) (determined by Hs00229526_ml and Hs01062934_g1, respectively) (Applied Biosystems, Darmstadt, Germany) mRNA expression was calculated relative to the mRNA expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1), determined by a premixed assay on demand for HPRT1 (Hs01003267_ml) (Applied Biosystems, Darmstadt, Germany). Amplification of specific transcripts was confirmed by melting curve profiles (cooling the sample to 68° C. and heating slowly to 95° C. with measurement of fluorescence) at the end of each PCR. The specificity of the PCR was further verified by subjecting the amplification products to agarose gel electrophoresis.

(14) Protein Analysis

(15) Protein was extracted from frozen organ samples or cultured hepatocytes in cell lysis buffer (Rose A J, Frosig C, Kiens B, Wojtaszewski J F, Richter E A. Effect of endurance exercise training on Ca2+ calmodulin-dependent protein kinase II expression and signaling in skeletal muscle of humans. J Physiol. 2007; 583: 785-795) and 20 μg of protein were loaded onto 4-12% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Western blot assays were performed as described (Herzig et al, 2001) using antibodies specific for TSC22D4 (Abcam, Cambridge, UK or Sigma, Munich, Germany), AKT, p-AKT, GSK, p-GSK (Cell signaling, Danvers, USA) or VCP (Abcam).

(16) Plasmids and RNA Interference

(17) For shRNA experiments, oligonucleotides targeting mouse and TSC22D4 (SEQ ID No. 1 to 3), were cloned into the pENTR/U6 shRNA vector (Invitrogen).

(18) Cell Culture and Transient Transfection Assays

(19) Primary mouse hepatocytes were isolated and cultured as described (Klingmuller U, Bauer A, Bohl S, Nickel P J, Breitkopf K, Dooley S, Zellmer S, Kern C, Merfort I, Sparna T, et al. Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways. IEE Proc Syst Biol. 2006; 153: 433-447). Briefly, male 8-12 week old C57Bl/6 mice were anaesthetized by i.p. injection of 100 mg/kg body weight ketamine hydrochloride and 5 mg/kg body weight xylazine hydrochloride. After opening the abdominal cavity, the liver was perfused at 37° C. with HANKS I (8 g NaCl, 0.4 g KCl, 3.57 g Hepes, 0.06 g Na.sub.2HPO.sub.4×2 H.sub.2O, 0.06 g KH.sub.2PO.sub.4 in 1 L distilled H.sub.2O, 2.5 mM EGTA, 0.1% glucose, adjusted to pH 7.4) via the portal vein for 5 min and subsequently with HANKS II (8 g NaCl, 0.4 g KCl, 3.57 g Hepes, 0.06 g Na.sub.2HPO.sub.4×2 H.sub.2O, 0.06 g KH.sub.2PO.sub.4 in 1 L distilled H.sub.2O, 0.1% glucose, 3 mg/ml collagenase CLSII, 5 mM CaCl.sub.2, adjusted to pH 7.4) for 5-7 min until disintegration of the liver structure was observed. The liver capsule was removed and the cell suspension was filtered through a 100 μm mesh. The cells were washed and, subsequently, viability of cells was determined by trypan blue staining. 1 000 000 living cells/well were seeded on collagen I-coated six-well plates. After 24 h, cells were infected with recombinant adenoviruses at a multiplicity of infection of 100. For stimulation experiments, primary hepatocytes were treated with PBS (control medium) or insulin at a concentration of 100 nM/6-well for 10 minutes. Cells were harvested 48 h after infection.

(20) Cistrome Analysis of Hepatic TSC22D4

(21) KEGG-Pathway analysis of Chip-Sequencing results were sorted by significance. The Insulin signaling pathway was found to be significantly regulated (p=0.00005). Chip-Sequencing was performed in liver extracts from Flag-TSC22D4 cDNA adenovirus-injected male C57Bl/6 mice 7 days after injection.

(22) Results

(23) The sequences worked very efficiently for both mouse and human TSC as seen in 4 independent experiments (see Figures). There is a nonspecific dTdT overhang attached to each sequence. The sequences matched both the mouse and the human TSC sequence to 100%.

(24) Based on these results, the sequences according to the present invention (SEQ ID No. 1 to 3) were chosen as primary candidates to be used for therapeutic purposes as it shows a superior knockdown efficiency towards TSC22D4 and targets a variety of species, including mouse, non-human primates and humans. The sequences were identified, functionally tested and validated various siRNAs directed against the TSC22D4 mRNA sequence in in vitro knockdown studies using murine Hepa1.6 as well as human Huh7 hepatoma cells. In particular the mhD4-siRNA1 showed a superior knockdown efficiency towards TSC22D4 and targets a variety of species, including mouse, non-human primates and humans.

(25) TABLE-US-00003 mhD4-siRNA1: (NM_030935.3_siRNA_1024; ORF) (SEQ ID NO: 1) Sense: 5′- GGACGUGUGUGGAUGUUUAdTdT -3′; (SEQ ID NO: 4) Antisense: 5′- UAAACAUCCACACACGUCCdTdT -3′; GC: 47% (w/o TT-overhang) mD4-siRNA2: (NM_023910.6_siRNA_993; ORF) (SEQ ID NO: 2) Sense: GGAUGUUUACGAGAGAGAUdTdT -3′; (SEQ ID NO: 5) Antisense: AUCUCUCUCGUAAACAUCCdTdT -3′; GC: 42.1% (w/o TT-overhang) mhD4-siRNA3: (SEQ ID NO: 3) Sense: 5′- AGUCCCACCUCAUGUUUGCdTdT -3′; (SEQ ID NO: 6) Antisense: 5′- GCAAACAUGAGGUGGGACUdTdT -3′; GC: 52.6% (w/o TT-overhang)