STABILIZED POLYRIBONUCLEOTIDE CODING FOR AN ELASTIC FIBROUS PROTEIN

Abstract

The invention relates to a polyribonucleotide, a cosmetic and pharmaceutical compound that has the polyribonucleotide and a medicinal product that has the polyribonucleotide and the compound.

Claims

1-18. (canceled)

19. A method for the induction of the synthesis of elastic fibrous protein in an organism, comprising the following steps: (1) providing a polyribonucleotide, and (2) administering said polyribonucleotide in or to an organism, wherein said polyribonucleotide encodes elastin/tropoelastin and comprises at least one polyribonucleotide stabilizing chemical modification, wherein at least approximately 25% of the uridines of the polyribonucleotide are replaced by pseudouridines (Ψ) and at least approximately 25% of the cytidines of the polyribonucleotide are replaced by 5-methylcytidines (m5C) to stabilize the polyribonucleotide.

20. The method of claim 19, wherein said polyribonucleotide is an mRNA.

21. The method of claim 19, wherein said polyribonucleotide further comprises a chemically modified uridine selected from the group consisting of: 2-thiouridine, 5-methyluridine, 5-idouridine, 4-thiouridine, 5-bromouridine, 2′-methyl-2′-deoxyuridine, 2′-amino-2′-deoxyuridine, 2′-azido-2′-deoxyuridine, 2′-fluoro-2′-deoxyuridine, and combinations thereof.

22. The method of claim 19, wherein said polyribonucleotide further comprises a chemically modified cytidine selected from the group consisting of 5-methylcytidine, 3-methylcytidine, 2-thiocytidine, 2′-methyl-2′-deoxcytidin, 2′-amino-2′-deoxycytidine, 2′-fluoro-2′-deoxycytidine, 5-iodcytidine, 5-bromocytidine, and 2′-azido-2′-deoxycytidine, and combinations thereof.

23. The method of claim 22, wherein said polyribonucleotide further comprises a chemical modification selected from the group consisting of: a 5′ cap structure, a poly (A) tail, a cap structure analog, and a strengthening of a translation-initiation sequence at the start codon AUG by the sequence (CCCCGC)aucGagAUG.

24. The method of claim 19, wherein said polyribonucleotide comprises the sequence of SEQ ID NO:1, wherein at least about 25% of the uridines of the polyribonucleotide are replaced by pseudouridines and at least about 25% of the cytidines of the polyribonucleotide are replaced by 5-methylcytidines.

25. The method of claim 19, wherein said polyribonucleotide encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

26. The method of claim 19, wherein said polyribonucleotide is configured for any of the following indications: the induction of the synthesis of elastin/tropoelastin, in age-related loss of elasticity of the skin (wrinkle formation), for promoting wound healing, and for the recovery of the elasticity of vaginal tissue, soft and hard tissue of the periodontium.

27. The method of claim 19, wherein said polyribonucleotide is configured for the treatment of a deficient synthesis of elastin/tropoelastin.

28. The method of claim 19, wherein said polyribonucleotide is configured for the treatment of a disease selected from the group consisting of: arteriosclerosis, aortic stenosis, aortic aneurysm, pulmonary emphysema, dermatochalasis, Williams-Beuren syndrome, and sub valvular innate aortic stenosis (SVAS).

29. The method of claim 19, wherein said polyribonucleotide is provided in a pharmaceutically acceptable formulation.

30. The method of claim 19, wherein said polyribonucleotide is provided in a cosmetically acceptable formulation.

31. The method of claim 19, wherein the modified polyribonucleotide is configured to increase elastin/tropoelastin synthesis by at least approximately threefold.

32. The method of claim 19, wherein in said polyribonucleotide approximately 100% of the uridines of the polyribonucleotide are replaced by pseudouridines (Ψ) and approximately 100% of the cytidines of the polyribonucleotide are replaced by 5-methylcytidines (m5C) to stabilize the polyribonucleotide, and the Ψ/m5C modifications are configured to increase elastin/tropoelastin synthesis from the polyribonucleotide.

33. The method of claim 19, wherein said polyribonucleotide comprises at least two polyribonucleotide modifications.

34. The method of claim 19, wherein in addition an immune-suppressive agent is administered in or to said organism.

35. The method of claim 19, wherein the polyribonucleotide encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

36. The method of claim 19, wherein the polyribonucleotide encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and which composition is configured to cause a lower level of IL-6 activation as compared to a level of IL-6 activation caused by a comparable amount of the immunostimulant polyinosinic/polycytidylic acid.

37. The method of claim 19, wherein said polyribonucleotide is provided as comprised by a medical patch or a vascular implant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1: Plasmid preparations of four individually selected bacteria colonies were tested for correct insert by means of ELN-specific PCR. Detection of PCR products by means of agarose gel electrophoresis, 1.2% (120 V, 30 min). PCR cycles: denaturating 3 min, 94° C., 1. 40 sec, 94° C.-2. 1 min, 58° C.-3. 2.5 min, 72° C.; final amplification: 7 min, 72° C.;

[0057] FIG. 2: Distinctively cutting restriction enzymes were determined by means of test digests of the plasmid and analysis in the following on a 1% agarose gel (120 V, 40 min). Bands: 1-marker/DNA ladder, 2-5: test digest of the plasmid with different downstream (3′) cutting restriction enzymes: 2: Not I, 3: Xba I, 4: Bfa I, 5: Xho I, 6: digest with downstream (5′) cutting enzyme, EcoRI, 7-10: downstream cutting enzymes were combined with Ecor I (double digest), order as 2-5. kb =kilobases;

[0058] FIG. 3: In vitro synthesized ELN-mRNA in denaturating agarose gel electrophoresis 1% (16% formaldehyde, 100 V, 45 min). 1: IVT reaction after 3 h incubation; 2: IVT reaction with polyadenylating mix and 25 min incubation; 3: as in 2, after 45 min; 4: marker; 5-6: purified, polyadenylated ELN-mRNAs; 5, 6: with Ambion MEGAclear Kit (catalog number AM1908); 7: with Qiagen RNeasy Midi Kit, double RNA amount (catalog number: 75142);

[0059] FIG. 4: Detection of reporter gene expression: detection of the Luciferase expression after transfection of IVT-Luciferase-mRNA into EA.hy926. hpt=hours post transfection; dpt=days post transfection;

[0060] FIG. 5: In vitro synthesized ELN protein on the basis of four different in vitro synthesized ELN-mRNAs and controls: 1, 2: marker; 3-6: protein synthesis with ELN-mRNAs from different in vitro transcription reactions (5: mRNA with modified nucleotides); 7: in vitro translation reaction without mRNA (negative control); 8: as in 7, but with addition of human, soluble elastin protein from Calbiochem (catalog number: 324751) after incubation time; 9: only water solved elastin protein from Calbiochem—according to data sheet the protein should run as a smear band between 5 and 60 kDa. Native elastin protein is detected by means of the used antibody at a level of 50 kDa.

[0061] FIG. 6: Detection of the elastin expression by measuring the amount of tropoelastin in EA.hy926 cells after transfection at three successive days with IVT-ELN-mRNA; they contained different fractions of modified nucleic acids (mCTP/ΨUTP) and Lipofectamin2000. RNA or Lipofectamin2000 alone were used as negative controls.

[0062] FIG. 7: Detection of the elastin expression in the supernatant of the EA.hy926 cells after transfection with 100% modified (mCTP/ΨUTP) IVT-ELN-mRNA by means of dot-blot assays; M: only cell culture medium, M+L: cell culture medium plus transfection reagent.

[0063] FIG. 8: Immune activation of EA.hy926 cells after the transfection of IVT-ELN-mRNA; the expressions were normalized to the housekeeping gene GAPDH. The negative controls were set to 1.

[0064] FIG. 9: Detection of the reporter gene expression: microscopic images of “stented” vessels, focus on the border between stent and tissue. A, B: negative controls (uncoated stents). C-E: eGPF-mRNA-coated stents. The size of the images corresponds to an area of 30×30 μm. Upper picture: original image. Lower picture: images with FIDSAM and contrast correction.

[0065] FIG. 10: Detection of the Luciferase expression in pig skin after transfection with Luciferase mRNA

EXAMPLES

[0066] 1. Plasmids Constructs for the RNA Synthesis

[0067] An elastin, transcript variant 1 encoding (SEQ ID No. 2) Sp6-promoter containing plasmid, pCMV-Sp6_ELN, cloned into E. coli, was purchased from Thermo Scientific.

[0068] The Luciferase encoding, T7-promoter containing plasmid, pCMV-GLuc-1 used as a reporter gene, was purchased from Nanolight Technology, Inc. and cloned into Qiagen EZ competent E. coli from the Qiagen PCT cloning kit.

[0069] The plasmids contain sites for sequencing with primers such as the M13 forward and reverse primers, and promoter regions for polymerases, such as Sp6 and T7. These sequences can be found 5′ or 3′ to the inserted sequence of interest, respectively. Additionally, the insert region is flanked by short recognition sequences for specific restriction enzymes.

[0070] 2. Verification of Plasmid Inserts

[0071] The plasmids were isolated with the Qiagen Plasmid Maxi kit (Qiagen).

[0072] Insert-specific primers were designed with a free primer designing tool from NCBI and produced by Eurofins MWG Operon. The plasmid inserts were verified by PCR with the insert-specific primer pairs (FIG. 1), and the following cycling parameters were used: denature 3 min at 94° C.; 1. 40 sec at 94° C.; 2. 1 min at 58° C.; 3. 2.5 min at 72° C.; repeat steps 1-3, 28×; final amplification 7 min, 72° C.

[0073] Additionally, plasmid inserts were sequenced completely through the company GATC Biotech with self-designed insert-specific primers and with M13-forward and reverse primers. The sequence assembly was done with the DNA-baser program.

[0074] 3. In Vitro Synthesis of ELN-mRNA with Different Amounts of Modified Nucleic Acids

[0075] For the in vitro transcription (IVT) plasmids were linearized downstream of the gene of interest with the Fast Digest Enzyme System (Fermentas/Thermo Scientific) and purified with the MiniElute PCR clean up kit (Qiagen). Test digests were performed prior to the experiment (FIG. 2).

[0076] The in vitro transcription was performed according to the manufacturer's instructions with MEGAscript Sp6 kit. For each 40 μL IVT reaction 1 μg of linearized template was used. To optimize the stability and cytocompatibility of IVT-mRNAs in the reactions different ratios of the nucleic acid triphosphates 5-methylcytidine and pseudouridine (TriLink Biotechnologies) were combined with standard nucleotides from the kit.

TABLE-US-00001 TABLE 1 nucleotide mixtures in the IVT reactions with different amounts of 5-methylcytidine and pseudouridine CTP 5-methylcitidine UTP Pseudouridine  25% modification 5.25 mM 1.75 mM 5.25 mM 1.75 mM  50% modification  3.5 mM  3.5 mM  3.5 mM  3.5 mM 100% modification —   7 mM —   7 mM

[0077] In each IVT reaction 6 mM ATP, 3 mM GTP (from the kit) and 2 mM 3′-O-Me-m.sup.7G(5′)ppp(5′)G (anti-reverse cap-analog from New England Biolabs) were used. Further components were added as indicated in the manual of the kit.

[0078] The reactions were incubated for 3.5 hours at 37° C. and treated with DNase I from the kit at the end of the incubation period in compliance with the instructions of the manufacturer, in order to eliminate the template DNA.

[0079] The polyadenylation was made with the PolyA Tailing kit (Ambion) according to the instructions of the manufacturer.

[0080] The mRNA was purified with the RNeasy mini kit (Qiagen). The detection of the elastin mRNA was made by denaturating agarose gel electrophoresis (FIG. 3).

[0081] The concentrations of mRNAs were measured by means of BioPhotometer6131 (Eppendorf).

[0082] 4. In Vitro Translation of In Vitro Transcribed mRNAs

[0083] The quality of the in vitro transcribed mRNA was determined indirectly by in vitro translation with the Retic Lysate kit (Ambion), following luminescence measurement for the Luciferase or western blot analysis for elastin. The in vitro translation reactions were set up according to the instructions of the manufacturer.

[0084] 5. Cell Lines and Chemicals Used for the Transfection of IVT-mRNAs

[0085] The lung carcinoma cell lines A549 and SK-MES were used for the first trials. The endothelial cell/A549-hybridoma cell line EA-hy926 was used to establish effective transfection methods for endothelial cells and tissues.

[0086] All m RNA transfections were performed by using the transfection reagent Lipofectamine2000 (Invitrogen/Life Technologies). The medium for transfections was OptiMEM (Gibco/Life Technologies). The negative controls throughout all experiments were OptiMEM with equal amounts of Lipofectamine2000 used for mRNA transfections or mRNA without transfection reagent in OptiMEM.

[0087] 6. Transfection of IVT-mRNAs in Cell Culture

[0088] For the transfections with elastin mRNA the cells were plated with a density of 500,000 cells per well in 6-well plates one day prior to the experiments.

[0089] The transfection was made with 5 or 10 μg of elastin mRNA and 3.3 μL of transfection reagent per well diluted in OptiMEM, based on the indications of the manufacturer.

[0090] The medium with the mRNA transfection complexes was added to the cells and the plates were incubated for 4 hours under cell culture conditions. Afterwards, ⅔ of the transfection mixes were replaced with fresh culture medium and the cells were incubated overnight. This transfection method was repeated for the following 2 days. At the third day the transfection complexes were completely replaced with fresh cell culture medium.

[0091] Medium and cells were analyzed by one day after the last transfection.

[0092] 7. Expression of the Luciferase Reporter Gene In Vitro

[0093] The first assessment of the Luciferase expression was performed 5 hours after the transfection and then following every day until the expression declined. For luminescence measurements, representing the Luciferase expression, 20 μL medium was taken from each well 6/24/48/72 hours and 5/10/25 days after transfection, with medium change after each sample taking.

[0094] 8. Measurement of the Luciferase Activity

[0095] The activity of the Luciferase enzyme directly after the in vitro translation or 5 hours to 30 days after the transfection of IVT-mRNA into the cells was assessed by adding 100 μL of 2.5 ng/μL substrate coelenterazin to 20 μL cell medium from transfected cells or in vitro translation reaction, respectively. The resulting luminescence of the probes was measured in a microplate reader (Mithras LB 940, Berthold Technologies). FIG. 4 shows the measured luminescence relating to the Luciferase expression in the medium of IVT-mRNA transfected EA.hy926 cells.

[0096] The results show that even a low amount of 200 ng of IVT Luciferase mRNA can induce a significant Luciferase expression even after a short incubation period of 5 hours. A high expression can be reached with 1 μg of IVT-mRNA up to 24 hours after the transfection, however the following expression course does not differ from the probes with lower amount of IVT-mRNA. Even another increase of the amount of transfected IVT-mRNA up to 2 μg does not result in a higher expression.

[0097] 9. Detection of (Tropo-)Elastin by Western Blot

[0098] The proteins from the in vitro translation reactions were separated on a 8% SDS-PAGE and blotted on a nitrocellulose membrane for the immunodetection. The primary antibody was a rabbit polyclonal ELN antibody (central region) from Abgent and the secondary antibody was a goat anti-rabbit IgG (whole molecule) Alkaline Phosphatase Conjugate from Sigma-Aldrich. The elastin protein was revealed by precipitation of the indigo dye resulting from NBT/BCIP reaction with alkaline phosphatase (FIG. 5).

[0099] The detection of elastin after the in vitro translation made on the basis of IVT-elastin-mRNA confirms the integrity of the mRNA according to the invention. It is understood that a protein detection can only occur when the synthesized protein corresponds to the structures against which the antibody has been developed. For this reason the mRNA according to the invention must have been present in its entirety and functionality for the protein synthesis. Although there is apparently a non specific detection of proteins existing in the in vitro translation mix the specificity of the elastin band is unambiguous since it does not exist in the negative control which only contains the in vitro translation mix without IVT-mRNA.

[0100] 10. Detection of the Expression of Tropoelastin in the Cell Culture

[0101] The expression of elastin was analyzed with the Fastin™ Elastin Assay (Biocolor life science assays) according to the manufacturer's instructions. FIG. 6 shows the amount of tropoelastin isolated 24 hours after the last transfection with elastin-IVT-mRNAs, which contained various parts of modified nucleic acids.

[0102] After a 3-fold transfection of IVT-elastin-mRNA a significant expression of tropoelastin, i.e. of soluble and non cross-linked elastin, could be detected in the cells. It is clear that a particular large amount of 10 μg of transfected IVT-ELN-mRNA has no increasing effect on the elastin expression over only 5 μg. Also the higher amount of modified nucleotides has no positive influence on the expression. Therefore it seems that 5 μg of the IVT-ELN-mRNA with 25% of modified CTP/UTP can cause a sufficient detectable expression of elastin.

[0103] In another experiment 3×10.sup.5 cells per well of a 6 well plate were seeded. At the following day the supernatants were aspirated and the cells were washed with 1 ml PBS. Then an incubation took place for 4 hours with Opti-MEM (M), Opti-MEM with Lipofectamine 2000 (M+L), and Opti-MEM with Lipofectamine 2000 and 2.5 μg of elastin-mRNA (100% 5mCTP/WUTP). The cell supernatants were collected after 24 and 48 hours. The supernatants were analyzed by means of dot-blot. The result is shown in FIG. 7. The detection of elastin was made by means of elastin-specific AB. The cells with elastin-mRNA-incubation (M+L+elastin mRNA) show a significantly stronger staining than the cells without mRNA-transfection (M, M+L). The EA.hy926-cells without elastin-mRNA synthesize low amounts of elastin, however by the elastin-mRNA-transfection the elastin synthesis is significantly increased.

[0104] 11. Assessing the Immunogenicity of the IVT-mRNAs

[0105] Transfections for the analysis of cytokines and other markers of the immune activation were performed according to the mRNA-transfections described above. Additionally, the immunostimulant polyinosinic/polycytidylic acid (Sigma-Aldrich) was transfected at 100 ng/well as a positive control for cytokine activation. The cells were incubated with the transfection mix under cell culture conditions and the medium was replaced after 3 hours.

[0106] The following day, cells were lysed and the RNA was extracted with Aurum Total RNA Mini Kit (Biorad). The RNA-concentration was measured and 40 ng of each sample was used for cDNA-synthesis with iScript cDNA-synthesis kit (Biorad). The generated cDNA was used diluted in (qRT)-PCR reactions with the iQ SYBR Green Supermix (Biorad) in triplicates for each sample, combined with a specific primer pair for IFN-V, IL-1 B, IL-12, IL-6, IL-8, TNF-α and a GAPDH-specific primer pair. For the quantification of the immune marker expression levels a qRT-PCR was performed in 96-well plates in the CFX Connect Real-Time PCR detection system (Biorad) (FIG. 8). The results show that the in vitro synthesized elastin-mRNA does only cause a very low activation of cytokines in this highly sensitive assay.

[0107] 12. Coating of Coronary Stents with eGFP-mRNA

[0108] In another experiment the in vivo expression of eGFP via IVT-mRNA, coated on coronary stents was examined. The in vitro synthesis of the eGFP-mRNA was effected with the plasmid construct pcDNA3.3_eGFP as described in Warren et al. (I.c.). The plasmid was provided by the authors via Addgene (Cambridge, Mass., USA). BMS coronary stents 3×20 mm of Qualimed (Winsen, Germany) were dip-coated, in an emulsion of 70 μg in vitro transcribed eGFP-mRNA complexed with 20 μL of Lipofectamin2000 in nuclease free water and 150 μg of polyactic-co-glycolic acid RESOMER® RG 502 H (Sigma Aldrich) solved in ethyl acetate.

[0109] The study was performed in accordance with the German animal welfare law and the recommendations on the care and use of laboratory animals postulated by the FELASA (Federation of European Laboratoy Animal Science Associations). All protocols and procedures were approved by the Animal Care and Welfare Commission of the University of Tubingen.

[0110] For this study two female pigs of approx. 65 kg (German land race) supplied by a local “specific pathogen-free” (SPF) breeding facility were used for this study and included in the analysis. After arrival at the animal facility of the University of Tubingen, all animals were allowed one week of adaptation prior to the intervention. During this period clinical examinations were carried out to ensure the health status, especially in consideration of the cardiovascular system.

[0111] The stents were implanted via a balloon catheter (3 mm) into the left and right coronary arteries of each pig and expanded. The location of the stents was displayed with help of an X-ray apparatus (C-Bogen) and radiopaque material. An overstretching of the arteries was provoqued intentionally. After the implantation, the animals received heparin and clopidogrel to prevent postoperative thromboses.

[0112] 44 hours after the implantation of the coated stents, the pigs were euthanized. The “stented” vessels were isolated and fixed overnight in 4% formaldehyde.

[0113] For the fluorescence analysis, the stents were embedded in methylmetacrylate based embedding system Technovit® 9100 from HeraeusKulzer (Wehrheim, Germany), and analyzed by fluorescence intensity decay shape analysis microscopy FIDSAM (fluorescence intensity decay shape analysis microscopy) at the Institute of Analytical Chemistry of the University of Tubingen.

[0114] The result is shown in FIG. 9. There the part of fluorescent tissue visible after substraction of the autofluorescence is shown. This fluorescence is only due to the induced eGFP expression and therefore the evidence for efficient uptake and translation of the IVT-mRNA encoding eGFP by cells surrounding the stent material.

[0115] 13. Pig Skin Model

[0116] A pig skin model was established to detect the synthesis of the mRNA-induced elastin in the skin. In the first experiments 2.5 μg of Luciferase mRNA/Lipofectamin 2000 complexes were injected into the skin. The skin was chopped after 24 h and for isolating the Luciferase the cells were lysed. The result is shown in FIG. 10. By means of the Luciferase Assay the successful transfection of the cells with Luciferase mRNA was demonstrated.

Sequences

[0117] SEQ ID No. 1: Nucleotide sequence of the IVT-elastin-mRNA (as used in the embodiments)

[0118] SEQ ID No. 2: Nucleotide sequence of the cDNA, derived from the mRNA of the human elastin, transcript variant 1 (NM_000501.2)

[0119] SEQ ID No. 3: Nucleotide sequence of the cDNA, derived from the mRNA of the human elastin, transcript variant 2 (NM_001081752.1)

[0120] SEQ ID No. 4: Nucleotide sequence of the cDNA, derived from the mRNA of the human elastin, transcript variant 3 (NM_001081753.1)

[0121] SEQ ID No. 5: Nucleotide sequence of the cDNA, derived from the mRNA of the human elastin, transcript variant 4 (NM_001081754.1)

[0122] SEQ ID No. 6: Nucleotide sequence of the cDNA, derived from the mRNA of the human elastin, transcript variant 5 (NM_001081755.1)

[0123] SEQ ID No. 7: Amino acid sequence of the elastin isoform a [homo sapiens] (NP_00049.2)

[0124] SEQ ID No. 8: Amino acid sequence of the elastin isoform b [homo sapiens] (NP_001075221.1)

[0125] SEQ ID No. 9: Amino acid sequence of the elastin isoform c [homo sapiens] (NP_001075222.1)

[0126] SEQ ID No. 10: Amino acid sequence of the elastin isoform d [homo sapiens] (NP_001075223.1)

[0127] SEQ ID No. 11: Amino acid sequence of the elastin isoform e [homo sapiens] (NP_001075224.1)