NLRP3 oligonucleotide for use in preventing and/or treating an inflammatory disease

Abstract

The present invention refers to oligonucleotide consisting of 12 to 20 nucleotides comprising at least one modified nucleotide hybridizing with a nucleic acid sequence of NLR family pyrin domain containing 3 (NLRP3) and a pharmaceutical composition 5 comprising such oligonucleotide together with a pharmaceutically acceptable carrier, excipient and/or dilutant to inhibit the expression of NLRP3 for example for preventing and/or treating an inflammatory disease.

Claims

1. Oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO. 59, SEQ ID NO. 335, SEQ ID NO. 297, and SEQ ID NO. 133 comprising at least one modified nucleotide, hybridizing with a nucleic acid sequence of a NLR family pyrin domain containing 3 (NLRP3) of human SEQ ID NO. 1 and/or human SEQ ID NO. 2 resulting in a reduction of the level of NLRP3, NLRP3 mRNA, NLRP3 pre-mRNA or a combination thereof.

2. The oligonucleotide of claim 1, wherein the modified nucleotide is selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2Fluoro modified nucleotide, 2O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA and a combination thereof.

3. The oligonucleotide of claim 1 or 2, wherein the reduction of the level of NLRP3, NLRP3 mRNA, NLRP3 pre-mRNA or a combination thereof is 30 to 99% compared to an untreated control.

4. The oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide comprises a modification selected from the group consisting of TABLE-US-00014 (A31109Hi;SEQIDNO.59) +G*+T*+A*A*T*G*T*C*A*A*C*G*G*A*+T*+C, (A31055Hi,SEQIDNO.59) +G*+T*+A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C, (A31110Hi,SEQIDNO.59) +G*+T*A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C, (A31111Hi,SEQIDNO.59) +G*+T*+A*+A*T*G*T*C*A*A*C*G*G*+A*+T*+C, (A31112Hi,SEQIDNO.59) +G*T*+A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C, (A31368Hi,SEQIDNO.59) +G*+T*+A*A*+T*G*T*C*A*A*C*G*G*+A*+T*+C, (A31369Hi,SEQIDNO.59) +G*+T*+A*A*T*+G*T*C*A*A*C*G*G*+A*+T*+C, (A31370Hi,SEQIDNO.59) +G*+T*+A*A*T*G*+T*C*A*A*C*G*G*+A*+T*+C, (A31371Hi,SEQIDNO.59) +G*+T*+A*A*T*G*T*C*A*A*C*+G*G*+A*+T*+C, (A31372Hi,SEQIDNO.59) +G*+T*+A*A*T*G*T*C*A*A*+C*G*G*+A*+T*+C, (A31373Hi,SEQIDNO.59) +G*+T*+A*+A*T*G*T*C*A*A*C*G*+G*+A*+T*+C, (A31149H,SEQIDNO.133) +G*+A*C*T*G*T*C*A*C*G*T*C*T*C*+G*+G*+C, (A31314Hi,SEQIDNO.297) +C*+A*+T*A*G*T*T*C*T*C*T*G*C*A*A*C*+A*+G*+G, (A31352Hi,SEQIDNO.335) +T*+A*+G*T*A*T*C*A*C*T*G*T*A*T*G*T*+C*+C*+A, and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

5. The oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide comprises the modification selected from the group consisting of TABLE-US-00015 (A31109Hi;SEQIDNO.59) +G*+T*+A*A*T*G*T*C*A*A*C*G*G*A*+T*+C, (A31149H,SEQIDNO.133) +G*+A*C*T*G*T*C*A*C*G*T*C*T*C*+G*+G*+C, (A31314Hi,SEQIDNO.297) +C*+A*+T*A*G*T*T*C*T*C*T*G*C*A*A*C*+A*+G*+G, (A31352Hi,SEQIDNO.335) +T*+A*+G*T*A*T*C*A*C*T*G*T*A*T*G*T*+C*+C*+A, and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

6. The oligonucleotide of any one of claims 1 to 5, wherein the oligonucleotide hybridizes with at least one exon or intron of SEQ ID NO. 1 and/or with the mRNA of SEQ ID NO. 2.

7. The oligonucleotide of any one of claims 1 to 6, wherein the oligonucleotide inhibits the expression of NLRP3, NLRP3 mRNA, NLRP3 pre-mRNA or a combination thereof at a nanomolar or micromolar concentration.

8. Pharmaceutical composition comprising an oligonucleotide of any one of claims 1 to 7 and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof.

9. The pharmaceutical composition of claim 8, further comprising another active agent selected from the group consisting of an oligonucleotide, an antibody, a small molecule, a polypeptide, a lipid, a sugar and a combination thereof.

10. The pharmaceutical composition of claim 9, wherein the oligonucleotide and the other active agent inhibit the same target or a different target.

11. The pharmaceutical composition of claim 10, wherein the other active agent modulates the target selected from the group consisting of NLRP3, CD39, CD73, IL-1, IL-1 receptor, IL-1R accessory protein, IL-18, IL-18 receptor, ASC, NLRC4, AIM2, Caspase-1, RIPK3, Gasdermin D, MLKL, TLR4, Caspase-8, P2X7, NFB, RORt, TGF-, IL-21, IL-17, IL-22, IL-23, IL-6, TNF-, CCR6, CCL20, STAT3, MMP-1, MMP-8, ADAMTS-5, HMG-CoA, Myd-88, HMGB-1, ROS, TAK-1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

12. The oligonucleotide of any one of claims 1 to 7 or the pharmaceutical composition of any one of claims 8 to 11 for use in a method of preventing and/or treating a disorder, where an NLRP3 imbalance is involved.

13. The oligonucleotide or the pharmaceutical composition for use according to claim 12, wherein the disorder is selected from the group consisting of a hyperproliferative disorder such as cancer, an inflammatory or autoimmune disorder, neurodegenerative disease, a neurological disorder, cardiovascular, metabolic disorder, renal disorder, liver disorder, lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ transplantation, fibrotic disorder and a combination thereof.

14. The oligonucleotide or the pharmaceutical composition for use according to claim 12 or 13, wherein the disorder is selected from the group consisting of Alzheimer's disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, transplantation of kidney, lung, liver and/or heart, fibrotic disorder of kidney, lung, liver and/or heart, hyperinflammation following influenza infection, graft-versus-host disease, interstitial cystitis, uveitis, sinusitis, periodontal disease, optic neuritis, myelodysplastic syndrome, cryopyrin-associated periodic syndromes (CAPS) including familial cold autoinflammatory syndrome (FCAS), the Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disease (NOMID), gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity and a combination thereof, and/or a cancer selected from the group consisting of breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, epidermoid carcinoma and a combination thereof.

15. The oligonucleotide or the pharmaceutical composition for use according to any one of claims 12 to 13, wherein the oligonucleotide or the composition is administered locally or systemically.

Description

DESCRIPTION OF FIGURES

[0024] FIG. 1 is a schematic presentation of the signal cascade of the NLRP3 activation (see McAllister et al., Osteoarthritis and Cartilage, 2018). NLRP3 is part of the NLRP3 inflammasome comprising NLRP3, adaptor protein ASC and (pro-) caspase-1. Two signals are necessary for the activation of NLRP3: signal 1 induces transcription and translation of pro-IL-1, pro-IL-18 and NLRP3 via NFB. Signal 2 leads to the activation of the proteolytic activity of the inflammasome complex. Activated caspase-1 induces cleaving of pro-IL-1 and pro-IL-18 to mature IL-1 and IL-18 which are secreted into the extracellular space. NLRP3 induced Caspase-1 activity results additionally in a specific form of cell death which is pyroptosis.

[0025] FIG. 2 shows a single concentration efficacy screen in human THP-1 cells in a first screening round for human NLRP3-specific antisense oligonucleotides (ASOs) after three days treatment.

[0026] FIG. 3 depicts a single concentration efficacy screen in human U87MG cells in a first screening round for human NLRP3-specific ASOs after three days treatment.

[0027] FIG. 4 depicts a single concentration efficacy screen in human PBMC-derived macrophages of selected human NLRP3-specific ASOs after six days treatment.

[0028] FIG. 5 depicts a single concentration efficacy screen of human NLRP3-specific ASOs in human U87MG cells in a second screening round for human NLRP3-specific ASOs after three days treatment.

[0029] FIG. 6 shows a single concentration efficacy screen of human NLRP3-specific ASOs in human PBMC-derived macrophages in a second screening round for human NLRP3-specific ASOs after six days treatment.

[0030] FIG. 7 depicts concentration-dependent NLRP3 mRNA knockdown by selected human NLRP3-specific ASOs from the first and second screening round in human PBMC-derived macrophages after six days treatment.

[0031] FIG. 8 shows a single concentration efficacy screen in human THP-1-derived macrophages in a third screening round for human NLRP3-specific ASOs after three days treatment.

[0032] FIG. 9 shows a single concentration efficacy screen in human PBMC-derived macrophages in a third screening round for human NLRP3-specific ASOs after six days treatment FIG. 10 shows a single concentration efficacy screen in human THP-1-derived macrophages in a fourth screening round for human NLRP3-specific ASOs after three days treatment.

[0033] FIG. 11 shows a single concentration efficacy screen in human PBMC-derived macrophages in a fourth screening round for human NLRP3-specific ASOs after six days treatment.

[0034] FIG. 12 depicts concentration-dependent NLRP3 mRNA knockdown by selected human NLRP3-specific ASOs from the first, second, third and fourth screening round in human THP-1-derived macrophages after three days treatment.

[0035] FIG. 13 shows the efficacy of selected human NLRP3-specific ASOs in human microglial HMC3 cells after three days treatment.

[0036] FIG. 14 shows a single concentration screen in human TPH-1-derived macrophages in a fifth screening round after three days treatment with human NLRP3-specific ASOs.

[0037] FIG. 15 shows a single concentration screen in human PBMC-derived macrophages in a fifth screening round after six days treatment with human NLRP3-specific ASOs.

[0038] FIG. 16 depicts functional assays investigating selected human NLRP3-specific ASOs in differentiated human THP-1-derived macrophages after six days treatment. FIG. 16A shows residual NLRP3 mRNA expression in ASO-treated THP-1-derived macrophages compared to mock-treated cells. FIG. 16B shows the inhibitory effects of the ASOs on caspase-1 activity. FIG. 16C shows IL-1 protein expression of pro-IL-1 and mature-IL-1 in cell lysates and cell culture supernatants of ASO-treated THP-1-derived macrophages analyzed by Western Blot.

[0039] FIGS. 17A and 17B show single concentration efficacy screens in mouse 4T1 cells in a first screening round for mouse NLRP3-specific ASOs after three days treatment. FIG. 17A depicts residual NLRP3 mRNA expression in ASO-treated 4T1 cells compared to mock-treated cells after three days treatment. FIG. 17B depicts cell viability of 4T1 cells after three days ASO treatment assessed by cell titer blue assay.

[0040] FIG. 18 depicts a single concentration efficacy screen in mouse Raw246.7 cells in a first screening round for mouse NLRP3-specific ASOs after three days treatment

[0041] FIGS. 19A and 19 B show single concentration screens in mouse Raw246.7 cells in a second screening round for mouse NLRP3-specific ASOs after three days treatment.

[0042] FIG. 20 shows a single concentration screen in mouse 4T1 cells for selected mouse NLRP3-specific from the second screening round after three days treatment.

[0043] FIG. 21 depicts concentration-dependent NLRP3 mRNA knockdown by selected NLRP3-specific ASOs from the first and second screening round in mouse Raw246.7 cells after three days treatment.

[0044] FIG. 22 depicts functional assays investigating the mouse NLRP3-specific ASOs A31087Mi (SEQ ID NO. 419) in mouse bone marrow derived macrophages (BMDM) after six days treatment. FIG. 22 A depicts residual NLRP3 mRNA expression in ASO-treated BMDM compared to mock-treated cells. FIG. 22 B shows NLRP3 protein expression and IL-1 protein expression of pro-IL-1 and mature-IL-1 in cell lysates and cell culture supernatants of ASO-treated BMDM cells analyzed by Western Blot.

[0045] FIG. 23 shows the efficacy of selected mouse NLRP3-specific ASOs in mouse Raw246.7 cells after three days treatment.

[0046] FIG. 24 shows the efficacy of selected mouse NLRP3-specific antisense oligonucleotides in mouse microglial IMG cells after three days treatment.

DETAILED DESCRIPTION

[0047] The present invention provides for the first time human and murine oligonucleotides which hybridize with mRNA sequences of NLR family pyrin domain containing 3 (NLRP3) and inhibit the expression and activity, respectively, of NLRP3 of human SEQ ID NO. 1 (GRCh38_Chr1: 247412861-247452403, in particular GRCh38.p12_Chr1: 247412861-247452403, comprising 39543 nucleotides) and/or human SEQ ID NO. 2 (RefSeq ID NM_004895.4), murine SEQ ID NO. 3 (GRmCh38_Chr11: 59539030-59569495, in particular GRmCh38.p4_Chr11: 59539030-59569495, comprising 30466 nucleotides), and/or SEQ ID NO. 4 (RefSeq ID NM_145827). Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating a disorder, where the NLRP3 expression and activity, respectively, is imbalanced, e.g., increased or decreased for example in an inflammatory disease.

[0048] In the following, the features of the present invention will be described in more detail. These features are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described features in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0049] Throughout this specification and the claims, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms a and an and the and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as, for example), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0050] Oligonucleotides of the present invention are for example antisense oligonucleotides (ASO) consisting of or comprising 10 to 25 nucleotides, 12 to 20 nucleotides, 11 to 15 nucleotides, 13 to 18 nucleotides, or 14 to 17 nucleotides. The oligonucleotides for example consist of or comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 nucleotides. The oligonucleotides of the present invention comprise at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2,4-LNA), cET, ENA, a 2Fluoro modified nucleotide, a 2O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA or a combination thereof. The oligonucleotide of the present invention comprises nucleotides having for example one or more, two or more, three or more, four or more, five or more or six or more, for example six of the same or different modifications. Further, the oligonucleotide of the present invention comprises a phosphate backbone, optionally a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate or methylphosphonate or a combination thereof, or where sulfur, amines or hydrocarbons are substituted for the bridging of non-bridging atoms in the phosphodiester bond. Modified nucleotide backbones include for example further chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3-alkylene phosphonate and/or chiral phosphonate, phosphinate, phosphoramidate including 3-amino phosphoramidate and/or aminoalkylphosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and borano phosphate having 3-5-linkage and/or 2-5-linkages.

[0051] The oligonucleotide of the present invention comprises one or more modified nucleotide at the 3- and/or 5-end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides.

[0052] The following Tables 1 and 2 present embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides consisting of or comprising the sequences of Tables 1 and 2 (mRNA (Antisense) sequence 5-3), respectively, may comprise any other modified nucleotide and any other combination of modified and unmodified nucleotides. Some oligonucleotides are exon spanning, i.e., the oligonucleotide hybridizes with one or more different exons. Oligonucleotides of Table 1 hybridize with human NLRP3 mRNA:

TABLE-US-00002 TABLE1 ListofhumanNLRP3-specificASOshybridizingwithhumanNLRP3for exampleofSEQIDNO.1;Neg1,R01002andR01011areantisenseoligonucleotides representinganegativecontrolwhichisnothybridizingwithNLRP3ofSEQIDNO.1or SEQIDNO.2.AnHaftertheASOIDindicatesahumanNLRP3-specificsequencethat bindstoanexonicregionofthepre-mRNAandaHiaftertheASOIDindicatesahuman NLRP3-specificsequencethatbindstoanintronicregionofthepre-mRNA.**=exon spanningoligo,positiondepictedinTable1indicatespositionsonmRNAofSEQIDNO. 2(RefSeqIDNM_004895.4)forexonspanningoligonucleotides. Positionon pre-mRNA (GRCh38, Chr1: Nameof 247412861- SEQ theLNA- 247452403; ID AntisenseSequence modified AntisenseSequence SEQID NO. 5-3 ASO 5-3withPTO(*)andLNA(+) NO.1) 5 ACGATGCCATCTTGACC A31001H +A*+C*+G*A*T*G*C*C*A*T*C*T*T*G*A*+C*+C 5280 6 ACGGTGAACAACCACTT A31002H +A*+C*+G*G*T*G*A*A*C*A*A*C*C*A*+C*+T*+T 5298 7 TACGGTGAACAACCAC A31003H +T*+A*+C*G*G*T*G*A*A*C*A*A*C*+C*+A*+C 5300 8 TCGATCCAGGAGTGTGT A31004H +T*+C*+G*A*T*C*C*A*G*G*A*G*T*G*+T*+G*+T 5394 9 ACACGGCACACGGATGA A31005H +A*+C*+A*C*G*G*C*A*C*A*C*G*G*A*+T*+G*+A 5861 10 TCGGTCCACACTAAGAT A31006H +T*+C*+G*G*T*C*C*A*C*A*C*T*A*A*+G*+A*+T 5890 11 CTTAGGCTTCGGTCCAC A31007H +C*+T*+T*A*G*G*C*T*T*C*G*G*T*C*+C*+A*+C 5898 12 ATCATTAGCGTGGCTAG A31008H +A*+T*+C*A*T*T*A*G*C*G*T*G*G*C*+T*+A*+G 6094 13 GCAGCGAAGATCCACAC A31009H +G*+C*+A*G*C*G*A*A*G*A*T*C*C*A*+C*+A*+C 6148 14 TTGATCGCAGCGAAGA A31010H +T*+T*+G*A*T*C*G*C*A*G*C*G*A*+A*+G*+A 6155 15 TGTTGATCGCAGCGAAG A31011H +T*+G*+T*T*G*A*T*C*G*C*A*G*C*G*+A*+A*+G 6156 16 ACGTGCATTATCTGAAC A31012H +A*+C*+G*T*G*C*A*T*T*A*T*C*T*G*+A*+A*+C 10370 17 TTCGAAACACGTGCAT A31013H +T*+T*+C*G*A*A*A*C*A*C*G*T*G*+C*+A*+T 10379 18 ATTCTCGAAAGGTACTC A31014H +A*+T*+T*C*T*C*G*A*A*A*G*G*T*A*+C*+T*+C 10450 19 CGTGTGTAGCGTTTGTT A31015H +C*+G*+T*G*T*G*T*A*G*C*G*T*T*T*+G*+T*+T 11076 20 CGTGTGTAGCGTTTGT A31016H +C*+G*+T*G*T*G*T*A*G*C*G*T*T*+T*+G*+T 11077 21 GCAGTCGTGTGTAGCGT A31017H +G*+C*+A*G*T*C*G*T*G*T*G*T*A*G*+C*+G*+T 11081 22 CTCTCACACGTCTTGGT A31018H +C*+T*+C*T*C*A*C*A*C*G*T*C*T*T*G*+G*+T 11160 23 AGAGCGGTCCTATGTGC A31019H +A*+G*+A*G*C*G*G*T*C*C*T*A*T*G*+T*+G*+C 11522 24 AGAGCGGTCCTATGTG A31020H +A*+G*+A*G*C*G*G*T*C*C*T*A*T*+G*+T*+G 11523 25 AGAAGACGTACACCGCG A31021H +A*+G*+A*A*G*A*C*G*T*A*C*A*C*C*+G*C*+G 11903 26 AACGTTCGTCCTTCCTT A31022H +A*+A*+C*G*T*T*C*G*T*C*C*T*T*C*+C*+T*+T 12205 27 GGAAGCTTCAAACGACT A31023H +G*+G*A*A*G*C*T*T*C*A*A*A*C*G*+A*+C*+T 12228 28 GGACTGTCACGTCTCG A31024H +G*+G*+A*C*T*G*T*C*A*C*G*T*C*+T*+C*+G 12249 29 TCGAATTTGCCATAGTT A31025H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*A*+G*+T*+T 12273 30 TCGAATTTGCCATAGT A31026H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 31 CTCGTACAAACAGTAGA A31027H +C*+T*+C*G*T*A*C*A*A*A*C*A*G*T*+A*+G*+A 12475 32 AGGTCCAATTCAGTTAG A31028H +A*+G*+G*+T*C*C*A*A*T*T*C*A*G*T*+T*+A*+G 16795 33 AGCGTTTCACACAACAC A31029H +A*+G*+C*G*T*T*T*C*A*C*A*C*A*A*+C*+A*+C 16846 34 ATCTCCGAATGTTACAG A31030H +A*+T*+C*T*C*C*G*A*A*T*G*T*T*A*+C*+A*+G 16875 35 GTCGAAGCAGCACTCAT A31031H +G*+T*C*G*A*A*G*C*A*G*C*A*C*T*+C*+A*+T 21266 36 GGACCAAGGAGATGTCG A31032H +G*+G*+A*C*C*A*A*G*G*A*G*A*T*G*+T*+C*+G 21279 37 ATTCCGAAGTCACCGAG A31033H +A*+T*+T*C*C*G*A*A*G*T*C*A*C*C*+G*+A*+G 21343 38 TCACATAAAATTGCGAC A31034H +T*+C*+A*C*A*T*A*A*A*A*T*T*G*C*+G*+A*+C 23225 39 AGACTGACGTAAGGCCA A31035H +A*+G*+A*C*T*G*A*C*G*T*A*A*G*G*C*+C*+A 31124 40 GTAAAGGTGCGTGAGAT A31036H +G*+T*+A*A*A*G*G*T*G*C*G*T*G*A*+G*+A*+T 31180 41 GTGTGACGTGAGGTTG A31037H +G*+T*+G*T*G*A*C*G*T*G*A*G*G*+T*+T*+G 31803 42 CAGTGTGACGTGAGGTT A31038H +C*+A*+G*T*G*T*G*A*C*G*T*G*A*G*+G*+T*+T 31804 43 CTAACGCACTTTTTGTC A31039H +C*+T*+A*A*C*G*C*A*C*T*T*T*T*T*+G*+T*+C 35572 44 CTCAAAGACGACGGTCA A31040H +C*+T*+C*A*A*A*G*A*C*G*A*C*G*G*+T*+C*+A 35616 45 CGGAGAACACTGGCGTC A31041H +C*+G*+G*A*G*A*A*C*A*C*T*G*G*C*G*+T*+C 35665 46 ATGGATCGCAGCTCTCT A31042H +A*+T*+G*G*A*T*C*G*C*A*G*C*T*C*T*+C*+T 35710 47 CGACACTCCACCGGAAG A31043H +C*+G*+A*C*A*C*T*C*C*A*C*C*G*G*+A*+A*+G 35754 48 TCTCCGACACTCCACCG A31044H +T*+C*+T*C*C*G*A*C*A*C*T*C*C*A*+C*+C*+G 35758 49 CGTCGGCAAGCTCTCTT A31045H +C*+G*+T*C*G*G*C*A*A*G*C*T*C*T*+C*+T*+T 35774 50 CGTCGGCAAGCTCTCT A31046H +C*+G*+T*C*G*G*C*A*A*G*C*T*C*+T*+C*+T 35775 51 AAGGCATCGTCGGCAAG A31047H +A*+A*+G*G*C*A*T*C*G*T*C*G*G*C*+A*+A*+G 35781 52 CTAACTGAGGCGCTGTG A31048H +C*+T*+A*A*C*T*G*A*G*G*C*G*C*T*+G*+T*+G 35879 53 TAGCACTACCGTGAGAG A31049Hi +T*+A*+G*C*A*C*T*A*C*C*G*T*G*A*+G*+A*+G 7906 54 CGTGCCTAATCTAGTG A31050Hi +C*+G*+T*G*C*C*T*A*A*T*C*T*+A*+G*+T*+G 9849 55 ACGACCAGTCTATTTCG A31051Hi +A*+C*G*A*C*C*A*G*T*C*T*A*T*T*+T*+C*+G 9863 56 GCTTAATCACCGCCGCT A31052Hi +G*+C*+T*T*A*A*T*C*A*C*C*G*C*C*+G*+C*+T 10682 57 TCGGAAGCGAGTGGTAA A31053Hi +T*+C*+G*G*A*A*G*C*G*A*G*T*G*G*+T*+A*+A 10894 58 CATCGGAAGCGAGTGGT A31054Hi +C*+A*+T*C*G*G*A*A*G*C*G*A*G*T*+G*+G*+T 10896 59 GTAATGTCAACGGATC A31055Hi +G*+T*+A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 60 CCGTAGAAACCGTACC A31056Hi +C*+C*+G*T*A*G*A*A*A*C*C*G*T*A*+C*+C 14070 61 CCGTAAGGTGAACCTTC A31057Hi +C*+C*+G*T*A*A*G*G*T*G*A*A*C*C*T*+T*+C 14184 62 GACGTATCAGGCGATGG A31058Hi +G*+A*+C*G*T*A*T*C*A*G*G*C*G*A*+T*+G*+G 14401 63 TGACGTATCAGGCGAT A31059Hi +T*+G*+A*C*G*T*A*T*C*A*G*G*C*+G*+A*+T 14403 64 TCGGACGGAGAGCATG A31060Hi +T*+C*+G*G*A*C*G*G*A*G*A*G*C*+A*+T*+G 16512 65 CTAGAGTGGCGGACTTT A31061Hi +C*+T*+A*G*A*G*T*G*G*C*G*G*A*C*+T*+T*+T 18501 66 GAGTTACACTAAGGCCG A31062Hi +G*+A*+G*T*T*A*C*A*C*T*A*A*G*G*+C*+C*+G 19318 67 CCGGCCGTAAACAGTG A31063Hi +C*+C*G*G*C*C*G*T*A*A*A*C*A*+G*+T*+G 20629 68 TCCGTTGAAGGACGT A31064Hi +T*+C*C*G*T*T*G*A*A*G*G*A*+C*+G*+T 22478 69 TCGAGTCTAGCTTATC A31065Hi +T*+C*+G*A*G*T*C*T*A*G*C*T*T*+A*+T*+C 22594 70 TCGCCGTCACTCCAGTT A31066Hi +T*+C*+G*C*C*G*T*C*A*C*T*C*C*A*+G*T*+T 24910 71 GAGGCACTCGCCAAACG A31067Hi +G*+A*+G*G*C*A*C*T*C*G*C*C*A*A*A*+C*+G 24945 72 GGACCTGTACGACCTAT A31068Hi +G*+G*+A*C*C*T*G*T*A*C*G*A*C*C*+T*+A*+T 25310 73 TAGCGCAGGTGATTGC A31069Hi +T*+A*+G*C*G*C*A*G*G*T*G*A*T*T*+G*+C 29495 74 GGTGGCGTAATTCCTAG A31070Hi +G*+G*+T*G*G*C*G*T*A*A*T*T*C*C*+T*+A*+G 29545 75 TCTACGAAGTTCGGTT A31071Hi +T*+C*+T*A*C*G*A*A*G*T*T*C*G*+G*+T*+T 32254 76 TAGACGAGTTCTACGAA A31072Hi +T*+A*+G*A*C*G*A*G*T*T*C*T*A*C*+G*+A*+A 32262 77 ATGATGCGAGTTACGTT A31073Hi +A*+T*+G*A*T*G*C*G*A*G*T*T*A*C*+G*+T*+T 33476 78 TGATGCGAGTTACGT A31074Hi +T*+G*+A*T*G*C*G*A*G*T*T*A*+C*+G*+T 33477 79 CCTAATGTACGGTACTT A31075Hi +C*+C*+T*A*A*T*G*T*A*C*G*G*T*A*+C*+T*+T 34555 80 GCTCGTCGTGTCCTTCG A31076Hi +G*+C*+T*C*G*T*C*G*T*G*T*C*C*T*+T*+C*+G 35277 81 GCTCGTCGTGTCCTT A31077Hi +G*+C*+T*C*G*T*C*G*T*G*T*C*+C*+T*+T 35279 82 AGCCGGTGCTCGTCGTG A31078Hi +A*+G*+C*C*G*G*T*G*C*T*C*G*T*C*+G*+T*+G 35284 83 ATGGCCGCGAGACTCCG A31079Hi +A*+T*+G*G*C*C*G*C*G*A*G*A*C*T*C*+C*+G 35330 84 CGCCGCCGAGACCAAGA A31080Hi +C*+G*+C*C*G*C*C*G*A*G*A*C*C*A*+A*+G*+A 35346 29 TCGAATTTGCCATAGTT A31081H +T*+C*G*A*A*T*T*T*G*C*C*A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31082H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*A*G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31083H +T*C*+G*A*A*T*T*T*G*C*C*A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31084H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*A*+G*T*+T 12273 30 TCGAATTTGCCATAGT A31085H +T*+C*G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31086H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*A*+G*+T 12274 30 TCGAATTTGCCATAGT A31087H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*+A*G*+T 12274 30 TCGAATTTGCCATAGT A31088H +T*C*+G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 85 TTCGAATTTGCCATAGT A31089H +T*+T*+C*G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 85 TTCGAATTTGCCATAGT A31090H +T*+T*C*G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 85 TTCGAATTTGCCATAGT A31091H +T*+T*+C*G*A*A*T*T*T*G*C*C*A*T*A*+G*+T 12274 86 TTTCGAATTTGCCATAG A31092H +T*+T*+T*C*G*A*A*T*T*T*G*C*C*A*+T*+A*+G 12275 86 TTTCGAATTTGCCATAG A31093H +T*+T*+T*+C*G*A*A*T*T*T*G*C*C*A*+T*+A*+G 12275 86 TTTCGAATTTGCCATAG A31094H +T*+T*+T*C*G*A*A*T*T*T*G*C*C*+A*+T*+A*+G 12275 87 TTGATCGCAGCGAAGAT A31095H +T*+T*+G*A*T*C*G*C*A*G*C*G*A*A*+G*+A*+T 6154 88 TCGAAAGGTACTCCAGT A31096H +T*+C*+G*A*A*A*G*G*T*A*C*T*C*C*+A*+G*+T 10446 89 GCTTCTCACGTACTTTC A31097H +G*+C*+T*T*C*T*C*A*C*G*T*A*C*T*+T*+T*+C 11005 90 TGTAGCGTTTGTTGAGG A31098H +T*+G*+T*A*G*C*G*T*T*T*G*T*T*G*+A*+G*+G 11072 91 CAGTCGTGTGTAGCGTT A31099H +C*+A*+G*T*C*G*T*G*T*G*T*A*G*C*+G*+T*+T 11080 92 ACGCAGTCGTGTGTAGC A31100H +A*+C*+G*C*A*G*T*C*G*T*G*T*G*T*+A*+G*+C 11083 93 TGAGACGCAGTCGTGTG A31101H +T*+G*+A*G*A*C*G*C*A*G*T*C*G*T*+G*+T*+G 11087 94 TGATGAGACGCAGTCGT A31102H +T*+G*+A*T*G*A*G*A*C*G*C*A*G*T*+C*+G*+T 11090 95 GTGCAGAGCGGTCCTAT A31103H +G*+T*+G*C*A*G*A*G*C*G*G*T*C*C*+T*+A*+T 11526 96 ATCTCGTACAAACAGTA A31104H +A*+T*+C*T*C*G*T*A*C*A*A*A*C*A*+G*+T*+A 12477 97 CTCCGAATGTTACAGCC A31105H +C*+T*+C*C*G*A*A*T*G*T*T*A*C*A*+G*+C*+C 16873 98 CATAAAATTGCGACTCC A31106H +C*+A*+T*A*A*A*A*T*T*G*C*G*A*C*+T*+C*+C 23222 99 AAGGCTCAAAGACGACG A31107H +A*+A*+G*G*C*T*C*A*A*A*G*A*C*G*+A*+C*+G 35620 100 GCATCGTCGGCAAGCTC A31108H +G*+C*+A*T*C*G*T*C*G*G*C*A*A*G*+C*+T*+C 35778 59 GTAATGTCAACGGATC A31109Hi +G*+T*+A*A*T*G*T*C*A*A*C*G*G*A*+T*+C 13706 59 GTAATGTCAACGGATC A31110Hi +G*+T*A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31111Hi +G*+T*+A*+A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31112Hi +G*T*+A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 101 GGTAATGTCAACGGATC A31113Hi +G*+G*+T*A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 101 GGTAATGTCAACGGATC A31114Hi +G*+G*+T*A*A*T*G*T*C*A*A*C*G*G*A*+T*+C 13706 101 GGTAATGTCAACGGATC A31115Hi +G*+G*T*A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 101 GGTAATGTCAACGGATC A31116Hi +G*+G*+T*+A*A*T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 102 GGTAATGTCAACGGAT A31117Hi +G*+G*+T*A*A*T*G*T*C*A*A*C*G*+G*+A*+T 13707 102 GGTAATGTCAACGGAT A31118Hi +G*+G*+T*+A*A*T*G*T*C*A*A*C*G*+G*+A*+T 13707 103 ACGCTTAATCACCGCCG A31119Hi +A*+C*+G*C*T*T*A*A*T*C*A*C*C*G*+C*+C*+G 10684 104 CGAGTGGTAACCGGAA A31120Hi +C*+G*+A*G*T*G*G*T*A*A*C*C*G*+G*+A*+A 10888 105 TGTCATCGGAAGCGAGT A31121Hi +T*+G*+T*C*A*T*C*G*G*A*A*G*C*G*+A*+G*+T 10899 106 ACGTATCAGGCGATGG A31122Hi +A*+C*+G*T*A*T*C*A*G*G*C*G*A*+T*+G*+G 14401 107 GACGTATCAGGCGATG A31123Hi +G*+A*+C*G*T*A*T*C*A*G*G*C*G*+A*+T*+G 14402 108 CCTAGAGTGGCGGACTT A31124Hi +C*+C*+T*A*G*A*G*T*G*G*C*G*G*A*+C*+T*+T 18502 109 AGACGAGTTCTACGA A31125Hi +A*+G*+A*C*G*A*G*T*T*C*T*A*+C*+G*+A 32263 110 GATGCGAGTTACGTTT A31126Hi +G*+A*+T*G*C*G*A*G*T*T*A*C*G*+T*+T*+T 33475 111 GTGATATGATGCGAGT A31127Hi +G*+T*+G*A*T*A*T*G*A*T*G*C*G*+A*+G*+T 33482 112 TTCTCCTGTTGGCTCGA A31128H +T*+T*C*T*C*C*T*G*T*T*G*G*C*T*+C*+G*+A 5407 113 ACCAGGCAGTGAACACG A31129H +A*+C*C*A*G*G*C*A*G*T*G*A*A*C*+A*+C*+G 5873 114 TTAGGCTTCGGTCCACA A31130H +T*+T*+A*G*G*C*T*T*C*G*G*T*C*C*+A*+C*+A 5897 115 GGTCCTTAGGCTTCGGT A31131H +G*+G*T*C*C*T*T*A*G*G*C*T*T*C*+G*+G*+T 5902 116 TCGATCATTAGCGTGGC A31132H +T*+C*G*A*T*C*A*T*T*A*G*C*G*T*+G*+G*+C 6097 117 CGAAGATCCACACGGCC A31133H +C*+G*+A*A*G*A*T*C*C*A*C*A*C*G*G*+C*+C 6144 118 TCCTTGATGAGACGCAG A31134H +T*+C*C*T*T*G*A*T*G*A*G*A*C*G*+C*+A*+G 11094 119 TTGCCGATGGCCAGAAG A31135H +T*+T*+G*C*C*G*A*T*G*G*C*C*A*G*+A*+A*+G 11142 120 TCTTGGTCTTGCCGATG A31136H +T*+C*+T*T*G*G*T*C*T*T*G*C*C*G*+A*+T*+G 11150 121 CACACGTCTTGGTCTTG A31137H +C*+A*+C*A*C*G*T*C*T*T*G*G*T*C*+T*T*+G 11156 122 GCTCTCACACGTCTTGG A31138H +G*C*+T*C*T*C*A*C*A*C*G*T*C*T*+T*+G*+G 11161 123 GCAGCTCATCGAAGCCG A31139H +G*C*+A*G*C*T*C*A*T*C*G*A*A*G*+C*+C*+G 11489 124 GCTCGTCAAAGGCACCT A31140H +G*+C*+T*C*G*T*C*A*A*A*G*G*C*A*+C*C*+T 11507 125 CAGAGCGGTCCTATGTG A31141H +C*A*+G*A*G*C*G*G*T*C*C*T*A*T*+G*+T*+G 11523 126 CGGCCTTCTGCCAGTCA A31142H +C*+G*+G*C*C*T*T*C*T*G*C*C*A*G*+T*+C*+A 11543 127 CACATGCCGAGGATGGT A31143H +C*+A*C*A*T*G*C*C*G*A*G*G*A*T*+G*+G*+T 11671 128 TTGGCCTCGGAGAAACC A31144H +T*+T*+G*G*C*C*T*C*G*G*A*G*A*A*+A*C*+C 11697 129 GTGAAGAGGACCTCGTT A31145H +G*+T*+G*A*A*G*A*G*G*A*C*C*T*C*+G*T*+T 11787 130 GAAGACGTACACCGCG A31146H +G*+A*+A*G*A*C*G*T*A*C*A*C*C*+G*C*+G 11903 131 AGGAAGAAGACGTACAC A31147H +A*+G*+G*A*A*G*A*A*G*A*C*G*T*A*+C*+A*+C 11907 132 GCTGTAGAACTTCTCGC A31148H +G*C*+T*G*T*A*G*A*A*C*T*T*C*T*+C*+G*+C 12124 133 GACTGTCACGTCTCGGC A31149H +G*+A*C*T*G*T*C*A*C*G*T*C*T*C*+G*+G*+C 12247 134 TGCACGAAGTCCTCCTC A31150H +T*+G*+C*A*C*G*A*A*G*T*C*C*T*C*+C*+T*+C 12498 135 TCTCCGAATGTTACAGC A31151H +T*+C*+T*C*C*G*A*A*T*G*T*T*A*C*+A*+G*+C 16874 136 TCATGCGAGAGGCCACA A31152H +T*+C*+A*T*G*C*G*A*G*A*G*G*C*C*A*+C*+A 21253 137 CACTCATGCGAGAGGCC A31153H +C*+A*+C*T*C*A*T*G*C*G*A*G*A*G*G*+C*+C 21256 138 AGAAGTCTGATTCCGAA A31154H +A*+G*+A*A*G*T*C*T*G*A*T*T*C*C*+G*+A*+A 21352 139 GGTGCGTGAGATTCTGA A31155H +G*+G*+T*G*C*G*T*G*A*G*A*T*T*C*+T*+G*+A 31175 140 CTCGCAGGTAAAGGTGC A31156H +C*+T*+C*G*C*A*G*G*T*A*A*A*G*G*+T*+G*+C 31187 141 CCTGAAGCTTGCAGTCG A31157H +C*+C*+T*G*A*A*G*C*T*T*G*C*A*G*+T*+C*+G 31259 142 CGTGAGGTTGCAGTTGT A31158H +C*+G*+T*G*A*G*G*T*T*G*C*A*G*T*+T*+G*+T 31796 143 GTGTGACGTGAGGTTGC A31159H +G*+T*G*T*G*A*C*G*T*G*A*G*G*T*+T*+G*+C 31802 144 CAGGCTCAGCTTTCGCA A31160H +C*+A*+G*G*C*T*C*A*G*C*T*T*T*C*+G*+C*+A 31862 145 ATCGCAGCTCTCTCCAC A31161H +A*+T*C*G*C*A*G*C*T*C*T*C*T*C*+C*+A*+C 35706 146 TGGCCTGGATGGATCGC A31162H +T*+G*+G*C*C*T*G*G*A*T*G*G*A*T*C*+G*+C 35718 147 GCAAGCTCTCTTCTCCG A31163H +G*+C*+A*A*G*C*T*C*T*C*T*T*C*T*+C*+C*+G 35769 148 GAAGGCATCGTCGGCAA A31164H +G*+A*+A*G*G*C*A*T*C*G*T*C*G*G*+C*+A*+A 35782 149 AGGAAGGCATCGTCGGC A31165H +A*+G*+G*A*A*G*G*C*A*T*C*G*T*C*+G*+G*+C 35784 150 TGAGGCGCTGTGATGAC A31166H +T*+G*A*G*G*C*G*C*T*G*T*G*A*T*+G*+A*+C 35874 151 ACGTGTTCTTAGGATA A31167Hi +A*+C*+G*T*G*T*T*C*T*T*A*G*G*+A*+T*+A 7195 152 GCACTACCGTGAGAGG A31168Hi +G*C*+A*C*T*A*C*C*G*T*G*A*G*+A*+G*+G 7905 153 CACGGTTCTTCAAGGAG A31169Hi +C*+A*+C*G*G*T*T*C*T*T*C*A*A*G*+G*+A*+G 8158 154 AGACGGTGTAGTGGTT A31170Hi +A*G*+A*C*G*G*T*G*T*A*G*T*G*+G*+T*+T 8871 155 GTCACTAGATAGCATA A31171Hi +G*+T*+C*A*C*T*A*G*A*T*A*G*C*+A*+T*+A 9654 156 TATTTCGTGCCTAATC A31172Hi +T*+A*+T*T*T*C*G*T*G*C*C*T*A*+A*+T*+C 9854 157 AGAACGACCAGTCTAT A31173Hi +A*+G*+A*A*C*G*A*C*C*A*G*T*C*+T*+A*+T 9867 158 GAACCTATAGTATGGC A31174Hi +G*+A*+A*C*C*T*A*T*A*G*T*A*T*+G*G*+C 10573 159 TAATCACCGCCGCTGG A31175Hi +T*+A*+A*T*C*A*C*C*G*C*C*G*C*+T*G*+G 10680 160 GAAACGCTTAATCACC A31176Hi +G*+A*+A*A*C*G*C*T*T*A*A*T*C*+A*+C*+C 10688 161 TGTGTAGCTACCAGCC A31177Hi +T*+G*+T*G*T*A*G*C*T*A*C*C*A*+G*+C*+C 10767 162 CCGAGAATCTCTGGTG A31178Hi +C*+C*G*A*G*A*A*T*C*T*C*T*G*G*+T*+G 10826 163 CGAGTGGTAACCGGAAT A31179Hi +C*+G*+A*G*T*G*G*T*A*A*C*C*G*G*+A*+A*+T 10887 164 TGTCATCGGAAGCGAG A31180Hi +T*+G*+T*C*A*T*C*G*G*A*A*G*C*+G*+A*+G 10900 165 CCACCTGAGATGCGCTC A31181Hi +C*+C*+A*C*C*T*G*A*G*A*T*G*C*G*+C*T*+C 10928 166 GTCAACGGATCAAGAA A31182Hi +G*+T*+C*A*A*C*G*G*A*T*C*A*A*+G*+A*+A 13701 167 CCTTCATGGCCAAACCA A31183Hi +C*+C*+T*T*C*A*T*G*G*C*C*A*A*A*+C*+C*+A 13748 168 GCACTTGTTTCCGGAC A31184Hi +G*+C*+A*C*T*T*G*T*T*T*C*C*G*+G*+A*+C 13843 169 CGCTGGTGAACAGGTTC A31185Hi +C*+G*C*T*G*G*T*G*A*A*C*A*G*G*T*+T*+C 13926 170 GCCGTAGAAACCGTAC A31186Hi +G*+C*+C*G*T*A*G*A*A*A*C*C*G*+T*+A*+C 14071 171 ACTGTTTGCTGCCGTA A31187Hi +A*+C*+T*G*T*T*T*G*C*T*G*C*C*G*+T*+A 14081 172 AGGCACCATCGACCAGG A31188Hi +A*+G*+G*C*A*C*C*A*T*C*G*A*C*C*+A*+G*+G 14266 173 TATGAACTTGCACTCAC A31189Hi +T*+A*+T*G*A*A*C*T*T*G*C*A*C*T*+C*+A*+C 16921 174 CTAGAGTGGCGGACTT A31190Hi +C*+T*+A*G*A*G*T*G*G*C*G*G*A*+C*+T*+T 18502 175 ACTGCGTGTCCAGGCAA A31191Hi +A*+C*T*G*C*G*T*G*T*C*C*A*G*G*C*+A*+A 18654 176 CATTCAGTGCGTGCTTT A31192Hi +C*+A*+T*T*C*A*G*T*G*C*G*T*G*C*+T*+T*+T 18775 177 TAAGCCACCGAACAGC A31193Hi +T*+A*+A*G*C*C*A*C*C*G*A*A*C*+A*+G*+C 18893 178 TGTTTGACGATTTCATC A31194Hi +T*+G*+T*T*T*G*A*C*G*A*T*T*T*C*+A*+T*+C 20418 179 AGAGAGCTCCGGAATA A31195Hi +A*+G*+A*G*A*G*C*T*C*C*G*G*A*+A*+T*+A 20719 20845 20887 21055 21097 180 GGACACACCTGACAAGA A31196Hi +G*+G*+A*C*A*C*A*C*C*T*G*A*C*A*+A*+G*+A 20984 181 CGCCTCGCTGGCAGAA A31197Hi +C*+G*+C*C*T*C*G*C*T*G*G*C*A*+G*+A*+A 21442 182 CCGTTGAAGGACGTTT A31198Hi +C*+C*+G*T*T*G*A*A*G*G*A*C*G*+T*+T*+T 22476 183 ATCCGTTGAAGGACGT A31199Hi +A*+T*+C*C*G*T*T*G*A*A*G*G*A*+C*+G*+T 22478 184 TTGTAGTGTGTACTAG A31200Hi +T*+T*+G*T*A*G*T*G*T*G*T*A*C*+T*+A*+G 22544 185 TGTGAGTGTCTTGGAAG A31201Hi +T*+G*+T*G*A*G*T*G*T*C*T*T*G*G*+A*+A*+G 23557 186 AATATTCTAAGGCGAT A31202Hi +A*+A*+T*A*T*T*C*T*A*A*G*G*C*+G*+A*+T 24672 187 CGCCGTCACTCCAGTT A31203Hi +C*+G*+C*C*G*T*C*A*C*T*C*C*A*+G*+T*+T 24910 188 CCTGTACGACCTATAA A31204Hi +C*+C*+T*G*T*A*C*G*A*C*C*T*A*+T*+A*+A 25308 189 ACATGGATGACGACTT A31205Hi +A*+C*+A*T*G*G*A*T*G*A*C*G*A*+C*+T*+T 28147 190 ACGGATATCTTGCCAT A31206Hi +A*+C*+G*G*A*T*A*T*C*T*T*G*C*+C*+A*+T 28750 191 CAACAGACGGTGCAGT A31207Hi +C*+A*+A*C*A*G*A*C*G*G*T*G*C*+A*+G*+T 28958 192 GGTGTCTACGAGGCAGG A31208Hi +G*+G*+T*G*T*C*T*A*C*G*A*G*G*C*+A*+G*+G 29430 193 ATGCATCAATCTCGAT A31209Hi +A*+T*+G*C*A*T*C*A*A*T*C*T*C*+G*+A*+T 29648 194 GAACGTCCATTGTGAC A31210Hi +G*+A*+A*C*G*T*C*C*A*T*T*G*T*+G*+A*+C 30615 195 AGCCTTGTATGCTGGTA A31211Hi +A*+G*+C*C*T*T*G*T*A*T*G*C*T*G*+G*+T*+A 31637 196 GGACTAACTAGCTTCA A31212Hi +G*+G*+A*C*T*A*A*C*T*A*G*C*T*+T*+C*+A 31657 197 CCTTGGCCATGATCGCA A31213Hi +C*+C*+T*T*G*G*C*C*A*T*G*A*T*C*+G*+C*+A 32216 198 TCGGTTCCTTGTGTAG A31214Hi +T*+C*+G*G*T*T*C*C*T*T*G*T*G*+T*+A*+G 32244 199 AGACGAGTTCTACGAA A31215Hi +A*+G*+A*C*G*A*G*T*T*C*T*A*C*+G*+A*+A 32262 200 GTGACCATCGACTGAA A31216Hi +G*+T*+G*A*C*C*A*T*C*G*A*C*T*+G*+A*+A 33199 201 AGTGATATGATGCGAG A31217Hi +A*+G*+T*G*A*T*A*T*G*A*T*G*C*+G*+A*+G 33483 202 GATAAGAAGACGATGT A31218Hi +G*+A*+T*A*A*G*A*A*G*A*C*G*A*+T*+G*+T 33506 203 AGCGGAATCTTCGGAA A31219Hi +A*+G*+C*G*G*A*A*T*C*T*T*C*G*+G*+A*+A 33588 204 TGCCTCTCACATCTTCG A31220Hi +T*+G*+C*C*T*C*T*C*A*C*A*T*C*T*+T*+C*+G 34727 205 GCTCGTCGTGTCCTTC A31221Hi +G*+C*+T*C*G*T*C*G*T*G*T*C*C*+T*+T*+C 35278 206 TGCTCGTCGTGTCCTT A31222Hi +T*+G*+C*T*C*G*T*C*G*T*G*T*C*+C*+T*+T 35279 207 CGCCGCCGAGACCAAG A31223Hi +C*+G*+C*C*G*C*C*G*A*G*A*C*C*+A*+A*+G 35347 208 GCTCCGCGTCCTCGCCG A31224Hi +G*+C*+T*C*C*G*C*G*T*C*C*T*C*G*+C*+C*+G 35370 209 ACCAGGCAGTGAACACGG A31225H +A*+C*+C*A*G*G*C*A*G*T*G*A*A*C*A*+C*+G*+G 5872 210 GGTGCTTGCCATCTTCAT A31226H +G*+G*+T*G*C*T*T*G*C*C*A*T*C*T*T*+C*+A*+T 5935 211 GATAGTCCTCTAAGTGCAT A31227H +G*+A*+T*A*G*T*C*C*T*C*T*A*A*G*T*G*+C*+A*+T 6013 212 TAGTCCTCTAAGTGCATC A31228H +T*+A*+G*T*C*C*T*C*T*A*A*G*T*G*C*+A*+T*+C 6012 213 AGCGTGGCTAGATCCACAT A31229H +A*+G*+C*G*T*G*G*C*T*A*G*A*T*C*C*A*+C*+A*+T 6086 214 GCTAGATCCACATGGTCT A31230H +G*+C*+T*A*G*A*T*C*C*A*C*A*T*G*G*+T*+C*+T 6081 215 CATTAGCGTGGCTAGATC A31231H +C*+A*+T*T*A*G*C*G*T*G*G*C*T*A*G*+A*+T*+C 6091 216 ACACGTGCATTATCTGAAC A31232H +A*+C*+A*C*G*T*G*C*A*T*T*A*T*C*T*G*+A*+A*+C 10370 217 TCTCGAAAGGTACTCCAG A31233H +T*+C*+T*C*G*A*A*A*G*G*T*A*C*T*C*+C*+A*+G 10447 218 AATCTGCTTCTCACGTAC A31234H +A*+A*+T*C*T*G*C*T*T*C*T*C*A*C*G*+T*+A*+C 11009 219 GCGTTTGTTGAGGCTCACA A31235H +G*+C*+G*T*T*T*G*T*T*G*A*G*G*C*T*C*+A*+C*+A 11066 220 GTGTAGCGTTTGTTGAGGC A31236H +G*+T*+G*T*A*G*C*G*T*T*T*G*T*T*G*A*+G*+G*+C 11071 221 CGTGTGTAGCGTTTGTTGA A31237H +C*+G*+T*G*T*G*T*A*G*C*G*T*T*T*G*T*+T*+G*+A 11074 222 GCAGTCGTGTGTAGCGTTT A31238H +G*+C*+A*G*T*C*G*T*G*T*G*T*A*G*C*G*+T*+T*+T 11079 223 TGTAGCGTTTGTTGAGGC A31239H +T*+G*+T*A*G*C*G*T*T*T*G*T*T*G*A*+G*+G*+C 11071 224 AGTCGTGTGTAGCGTTTG A31240H +A*+G*+T*C*G*T*G*T*G*T*A*G*C*G*T*+T*+T*+G 11078 225 GCAGTCGTGTGTAGCGTT A31241H +G*+C*+A*G*T*C*G*T*G*T*G*T*A*G*C*+G*+T*+T 11080 226 CTCCGGTGCTCCTTGATG A31242H +C*+T*+C*C*G*G*T*G*C*T*C*C*T*T*G*+A*+T*+G 11102 227 GTCTTGGTCTTGCCGATGG A31243H +G*+T*+C*T*T*G*G*T*C*T*T*G*C*C*G*A*+T*+G*+G 11149 228 TAGTCAAACCTGTCTTGGT A31244H +T*+A*+G*T*C*A*A*A*C*C*T*G*T*C*T*T*+G*+G*+T 11329 229 TAGAACAGATAGTCAAACC A31245H +T*+A*+G*A*A*C*A*G*A*T*A*G*T*C*A*A*+A*+C*+C 11338 230 CGACAGTGGATATAGAACA A31246H +C*+G*+A*C*A*G*T*G*G*A*T*A*T*A*G*A*+A*+C*+A 11350 231 AGAACAGATAGTCAAACC A31247H +A*+G*+A*A*C*A*G*A*T*A*G*T*C*A*A*+A*+C*+C 11338 232 CGACAGTGGATATAGAAC A31248H +C*+G*+A*C*A*G*T*G*G*A*T*A*T*A*G*+A*+A*+C 11351 233 TCGTCAAAGGCACCTTGCA A31249H +T*+C*+G*T*C*A*A*A*G*G*C*A*C*C*T*T*+G*+C*+A 11503 234 CAGTGCAGAGCGGTCCTAT A31250H +C*+A*+G*T*G*C*A*G*A*G*C*G*G*T*C*C*+T*+A*+T 11526 235 TCGTCAAAGGCACCTTGC A31251H +T*+C*+G*T*C*A*A*A*G*G*C*A*C*C*T*+T*+G*+C 11504 236 CGGCCTTCTGCCAGTCAGT A31252H +C*+G*+G*C*C*T*T*C*T*G*C*C*A*G*T*C*+A*+G*+T 11541 237 AGAGCGGTCCTATGTGCT A31253H +A*+G*+A*G*C*G*G*T*C*C*T*A*T*G*T*+G*+C*+T 11521 238 GGTCTCGTGGTGATGAGCA A31254H +G*+G*+T*C*T*C*G*T*G*G*T*G*A*T*G*A*+G*+C*+A 11620 239 CGAGGATGGTCCAGCAAGT A31255H +C*+G*+A*G*G*A*T*G*G*T*C*C*A*G*C*A*+A*+G*+T 11662 240 AGGATCTCCACATGCCGA A31256H +A*+G*+G*A*T*C*T*C*C*A*C*A*T*G*C*+C*+G*+A 11678 241 CACGATCCAGCAGACCAGG A31257H +C*+A*+C*G*A*T*C*C*A*G*C*A*G*A*C*C*+A*+G*+G 11819 242 GACACATCCGCCTTCTGCA A31258H +G*+A*+C*A*C*A*T*C*C*G*C*C*T*T*C*T*+G*+C*+A 12064 243 GCAGACACATCCGCCTTC A31259H +G*+C*+A*G*A*C*A*C*A*T*C*C*G*C*C*+T*+T*+C 12068 244 TACATGGCGGCAAAGAACT A31260H +T*+A*+C*A*T*G*G*C*G*G*C*A*A*A*G*A*+A*+C*+T 12163 245 GTACATGGCGGCAAAGAA A31261H +G*+T*+A*C*A*T*G*G*C*G*G*C*A*A*A*+G*+A*+A 12165 246 GGTAGTACATGGCGGCAA A31262H +G*+G*+T*A*G*T*A*C*A*T*G*G*C*G*G*+C*+A*+A 12169 247 TCGAATTTGCCATAGTTT A31263H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*A*G*+T*+T*+T 12272 248 CTCGTACAAACAGTAGAAC A31264H +C*+T*+C*G*T*A*C*A*A*A*C*A*G*T*A*G*+A*+A*+C 12473 249 TCGTACAAACAGTAGAAC A31265H +T*+C*+G*T*A*C*A*A*A*C*A*G*T*A*G*+A*+A*+C 12473 250 TTGCACGAAGTCCTCCTC A31266H +T*+T*+G*C*A*C*G*A*A*G*T*C*C*T*C*+C*+T*+C 12498 251 GCATGTTATGGAGAAACC A31267H +G*+C*+A*T*G*T*T*A*T*G*G*A*G*A*A*+A*+C*+C 12628 252 GCGTTTCACACAACACTCT A31268H +G*+C*+G*T*T*T*C*A*C*A*C*A*A*C*A*C*+T*+C*+T 16843 253 CACAATCTCCGAATGTTAC A31269H +C*+A*+C*A*A*T*C*T*C*C*G*A*A*T*G*T*+T*+A*+C 16877 254 GTCGAAGCAGCACTCATGC A31271H +G*+T*+C*G*A*A*G*C*A*G*C*A*C*T*C*A*+T*+G*+C 21264 255 GACCAAGGAGATGTCGAAG A31272H +G*+A*+C*C*A*A*G*G*A*G*A*T*G*T*C*G*+A*+A*+G 21276 256 TGTCGAAGCAGCACTCAT A31273H +T*+G*+T*C*G*A*A*G*C*A*G*C*A*C*T*+C*+A*+T 21266 257 GGACCAAGGAGATGTCGA A31274H +G*+G*+A*C*C*A*A*G*G*A*G*A*T*G*T*+C*+G*+A 21278 258 TTCTTCAGATTGCACAAC A31275H +T*+T*+C*T*T*C*A*G*A*T*T*G*C*A*C*+A*+A*+C 21390 259 GCGTGAGATTCTGATTAGT A31276H +G*+C*+G*T*G*A*G*A*T*T*C*T*G*A*T*T*+A*+G*+T 31170 260 AGGTGCGTGAGATTCTGAT A31277H +A*+G*+G*T*G*C*G*T*G*A*G*A*T*T*C*T*+G*+A*+T 31174 261 GTAAAGGTGCGTGAGATTC A31278H +G*+T*+A*A*A*G*G*T*G*C*G*T*G*A*G*A*+T*+T*+C 31178 262 TTGTCTCCGAGAGTGTTGC A31279H +T*+T*+G*T*C*T*C*C*G*A*G*A*G*T*G*T*+T*+G*+C 31204 263 CGTGAGATTCTGATTAGT A31280H +C*+G*+T*G*A*G*A*T*T*C*T*G*A*T*T*+A*+G*+T 31170 264 GTGAGGTTGCAGTTGTCTA A31281H +G*+T*+G*A*G*G*T*T*G*C*A*G*T*T*G*T*+C*+T*+A 31793 265 TGACGTGAGGTTGCAGTTG A31282H +T*+G*+A*C*G*T*G*A*G*G*T*T*G*C*A*G*+T*+T*+G 31797 266 CCTCGCAGGTAAAGGTGC A31283H +C*+C*+T*C*G*C*A*G*G*T*A*A*A*G*G*+T*+G*+C 31187 267 TTGTCTCCGAGAGTGTTG A31284H +T*+T*+G*T*C*T*C*C*G*A*G*A*G*T*G*+T*+T*+G 31205 268 TTTCGCAGGCTCTGGCTGG A31285H +T*+T*+T*C*G*C*A*G*G*C*T*C*T*G*G*C*+T*+G*+G 31850 269 ACGTGAGGTTGCAGTTGT A31286H +A*+C*+G*T*G*A*G*G*T*T*G*C*A*G*T*+T*+G*+T 31796 270 TCTTGAAGTGTTTCTAACG A31287H +T*+C*+T*T*G*A*A*G*T*G*T*T*T*C*T*A*+A*+C*+G 35583 271 TCTTGAAGTGTTTCTAAC A31288H +T*+C*+T*T*G*A*A*G*T*G*T*T*T*C*T*+A*+A*+C 35584 272 TACATGAGGTCACCAAGA A31289H +T*+A*+C*A*T*G*A*G*G*T*C*A*C*C*A*+A*+G*+A 35906 273 TGCACAATGTGAGTGTACC A31290Hi +T*+G*+C*A*C*A*A*T*G*T*G*A*G*T*G*T*+A*+C*+C 6630 274 CACCTTCTCATAGCACTAC A31291Hi +C*+A*+C*C*T*T*C*T*C*A*T*A*G*C*A*C*+T*+A*+C 7914 275 TGAGTATATAAGTACTGAC A31292Hi +T*+G*+A*G*T*A*T*A*T*A*A*G*T*A*C*T*+G*+A*+C 8621 276 AGTACTTCTACAGTTGTGA A31293Hi +A*+G*+T*A*C*T*T*C*T*A*C*A*G*T*T*G*+T*+G*+A 8664 277 AGACAGATAACTTCAGGTG A31294Hi +A*+G*+A*C*A*G*A*T*A*A*C*T*T*C*A*G*+G*+T*+G 9563 278 TATAGGAGAAATTCAGGTT A31295Hi +T*+A*+T*A*G*G*A*G*A*A*A*T*T*C*A*G*+G*+T*+T 9619 279 AAGGCATTCTATAGGAGA A31296Hi +A*+A*+G*G*C*A*T*T*C*T*A*T*A*G*G*+A*+G*+A 9629 280 AACAACTTCTGCTGTCCAC A31297Hi +A*+A*+C*A*A*C*T*T*C*T*G*C*T*G*T*C*+C*+A*+C 10160 281 ACATGGCTCTGAGTTGCAG A31298Hi +A*+C*+A*T*G*G*C*T*C*T*G*A*G*T*T*G*+C*+A*+G 10236 282 ATTGAAACCTGGCCTAATA A31299Hi +A*+T*+T*G*A*A*A*C*C*T*G*G*C*C*T*A*+A*+T*+A 10297 283 AATCTCTGGTGTCAAGTTT A31300Hi +A*+A*+T*C*T*C*T*G*G*T*G*T*C*A*A*G*+T*+T*+T 10818 284 AGAAGAAGCTGGCGAGGA A31301Hi +A*+G*+A*A*G*A*A*G*C*T*G*G*C*G*A*+G*+G*+A 12776 285 GACTGACTCTTAGGCACTA A31302Hi +G*+A*+C*T*G*A*C*T*C*T*T*A*G*G*C*A*+C*+T*+A 13059 286 CAGACTTGGCCACTAGCTT A31303Hi +C*+A*+G*A*C*T*T*G*G*C*C*A*C*T*A*G*+C*+T*+T 13473 287 TCATTGCACAGACTTGGC A31304Hi +T*+C*+A*T*T*G*C*A*C*A*G*A*C*T*T*+G*+G*+C 13482 288 GACTCAACCACTTCATTG A31305Hi +G*+A*+C*T*C*A*A*C*C*A*C*T*T*C*A*+T*+T*+G 13494 289 CAGTCCTTCATGGCCAAAC A31306Hi +C*+A*+G*T*C*C*T*T*C*A*T*G*G*C*C*A*+A*+A*+C 13750 290 GTGCTTTGGATACTCTGC A31307Hi +G*+T*+G*C*T*T*T*G*G*A*T*A*C*T*C*+T*+G*+C 13900 291 TTCAAATGCAAAGTCTAGC A31308Hi +T*+T*+C*A*A*A*T*G*C*A*A*A*G*T*C*T*+A*+G*+C 15571 292 TCACCTGTAGCTCTCATT A31309Hi +T*+C*+A*C*C*T*G*T*A*G*C*T*C*T*C*+A*+T*+T 15588 293 GGTCCTGAAGATCTTTCTC A31310Hi +G*+G*+T*C*C*T*G*A*A*G*A*T*C*T*T*T*+C*+T*+C 16964 294 ACAAAGCATTCTGACCAGG A31311Hi +A*+C*+A*A*A*G*C*A*T*T*C*T*G*A*C*C*+A*+G*+G 18120 295 TTGATAAGACTGGTGACA A31312Hi +T*+T*+G*A*T*A*A*G*A*C*T*G*G*T*G*+A*+C*+A 18559 296 GGTGACTATTCATGTGAG A31313Hi +G*+G*+T*G*A*C*T*A*T*T*C*A*T*G*T*+G*+A*+G 23568 297 CATAGTTCTCTGCAACAGG A31314Hi +C*+A*+T*A*G*T*T*C*T*C*T*G*C*A*A*C*+A*+G*+G 23845 298 AGTGCATGATTATTAGTGG A31315Hi +A*+G*+T*G*C*A*T*G*A*T*T*A*T*T*A*G*+T*+G*+G 23964 299 CTGAGTTCCACTACCTTCT A31316Hi +C*+T*+G*A*G*T*T*C*C*A*C*T*A*C*C*T*+T*+C*+T 24162 300 CGTTTGTCTACACTGAGT A31317Hi +C*+G*+T*T*T*G*T*C*T*A*C*A*C*T*G*+A*+G*+T 24175 301 GAATGCTGATCTGGCTGGT A31318Hi +G*+A*+A*T*G*C*T*G*A*T*C*T*G*G*C*T*+G*+G*+T 24450 302 TCTTCCGGCTCCTTCACAG A31319Hi +T*+C*+T*T*C*C*G*G*C*T*C*C*T*T*C*A*+C*+A*+G 25010 303 GTTCCATCTGCTTTGCCTG A31320Hi +G*+T*+T*C*C*A*T*C*T*G*C*T*T*T*G*C*+C*+T*+G 25035 304 TACACCAAGCATGTGTTAC A31321Hi +T*+A*+C*A*C*C*A*A*G*C*A*T*G*T*G*T*+T*+A*+C 25951 305 ACTATTGTAAATGGCTCTC A31322Hi +A*+C*+T*A*T*T*G*T*A*A*A*T*G*G*C*T*+C*+T*+C 26028 306 CACAAAGCTCTGATACTC A31323Hi +C*+A*+C*A*A*A*G*C*T*C*T*G*A*T*A*+C*+T*+C 26214 307 GACTTGTGTTCTTTCACAG A31324Hi +G*+A*+C*T*T*G*T*G*T*T*C*T*T*T*C*A*+C*+A*+G 26674 308 ATTAATGCCACTTATAGAT A31325Hi +A*+T*+T*A*A*T*G*C*C*A*C*T*T*A*T*A*+G*+A*+T 27090 309 ATAGGCTATACCTAACAC A31326Hi +A*+T*+A*G*G*C*T*A*T*A*C*C*T*A*A*+C*+A*+C 27163 27199 27235 27253 310 ACATAGGCTATACCTAAC A31327Hi +A*+C*+A*T*A*G*G*C*T*A*T*A*C*C*T*+A*+A*+C 27237 27255 311 GCCAAGCTAAGATGTCAC A31328Hi +G*+C*+C*A*A*G*C*T*A*A*G*A*T*G*T*+C*+A*+C 27640 312 CAGTGATGTCCACCTATA A31329Hi +C*+A*+G*T*G*A*T*G*T*C*C*A*C*C*T*+A*+T*+A 27890 313 GTAGGAGCTTCTTAGATG A31330Hi +G*+T*+A*G*G*A*G*C*T*T*C*T*T*A*G*+A*+T*+G 28024 314 GATATCTTGCCATGTACT A31331Hi +G*+A*+T*A*T*C*T*T*G*C*C*A*T*G*T*+A*+C*+T 28745 315 CAGGCTTTACCACTATTTC A31332Hi +C*+A*+G*G*C*T*T*T*A*C*C*A*C*T*A*T*+T*+T*+C 29041 316 TGAGTGGCTTCTACTAAG A31333Hi +T*+G*+A*G*T*G*G*C*T*T*C*T*A*C*T*+A*+A*+G 29223 317 TTTAAGCTCTTTGGTGTC A31334Hi +T*+T*+T*A*A*G*C*T*C*T*T*T*G*G*T*+G*+T*+C 29441 318 TTTGATGTACTATTCTTGA A31335Hi +T*+T*+T*G*A*T*G*T*A*C*T*A*T*T*C*T*+T*+G*+A 29617 319 GTTTCTCTCAAGGCCACAG A31336Hi +G*+T*+T*T*C*T*C*T*C*A*A*G*G*C*C*A*+C*+A*+G 29800 320 TGACCTTCTAAGCTGTAG A31337Hi +T*+G*+A*C*C*T*T*C*T*A*A*G*C*T*G*+T*+A*+G 30601 321 CCGTAACACTGCAGACAC A31338Hi +C*+C*+G*T*A*A*C*A*C*T*G*C*A*G*A*+C*+A*+C 30732 322 ACACCTCACTTCTAAGATA A31339Hi +A*+C*+A*C*C*T*C*A*C*T*T*C*T*A*A*G*+A*+T*+A 31371 323 GAAGTGGAATGTGTAGTTT A31340Hi +G*+A*+A*G*T*G*G*A*A*T*G*T*G*T*A*G*+T*+T*+T 32126 324 CCTTAAAGCCAAGCAGTGA A31341Hi +C*+C*+T*T*A*A*A*G*C*C*A*A*G*C*A*G*+T*+G*+A 32843 325 ACGTTTGAGAGGTTTATT A31342Hi +A*+C*+G*T*T*T*G*A*G*A*G*G*T*T*T*+A*+T*+T 33123 326 AAAGAGACAGTGACGACA A31343Hi +A*+A*+A*G*A*G*A*C*A*G*T*G*A*C*G*+A*+C*+A 33252 327 GAGTTTGTTGCTCTGATT A31344Hi +G*+A*+G*T*T*T*G*T*T*G*C*T*C*T*G*+A*+T*+T 33530 328 GGAGAGGCTGGATTATGTG A31345Hi +G*+G*+A*G*A*G*G*C*T*G*G*A*T*T*A*T*+G*+T*+G 33566 329 GATGACAGTGTTCTTGCAA A31346Hi +G*+A*+T*G*A*C*A*G*T*G*T*T*C*T*T*G*+C*+A*+A 33654 330 GATCATTAATATGGACAGA A31347Hi +G*+A*+T*C*A*T*T*A*A*T*A*T*G*G*A*C*+A*+G*+A 33941 331 GCTTGTTATCGCAGCCTGA A31348Hi +G*+C*+T*T*G*T*T*A*T*C*G*C*A*G*C*C*+T*+G*+A 34241 332 CAGCTTGTTATCGCAGCC A31349Hi +C*+A*+G*C*T*T*G*T*T*A*T*C*G*C*A*+G*+C*+C 34244 333 ACCAAATTCATCAGAGACT A31350Hi +A*+C*+C*A*A*A*T*T*C*A*T*C*A*G*A*G*+A*+C*+T 34331 334 CTCCTCTTTAATAAAGTTT A31351Hi +C*+T*+C*C*T*C*T*T*T*A*A*T*A*A*A*G*+T*+T*+T 34695 335 TAGTATCACTGTATGTCCA A31352Hi +T*+A*+G*T*A*T*C*A*C*T*G*T*A*T*G*T*+C*+C*+A 34843 336 GGTCATGTCAACTTGTTTC A31353Hi +G*+G*+T*C*A*T*G*T*C*A*A*C*T*T*G*T*+T*+T*+C 35001 337 TCAGAGTCGTTAACCTTA A31354Hi +T*+C*+A*G*A*G*T*C*G*T*T*A*A*C*C*+T*+T*+A 35479 29 TCGAATTTGCCATAGTT A31355H +T*+C*+G*A*+A*T*T*T*G*C*C*A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31356H +T*+C*+G*A*A*+T*T*T*G*C*C*A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31357H +T*+C*+G*A*A*T*+T*T*G*C*C*A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31358H +T*+C*+G*A*A*T*T*T*G*C*C*A*T*+A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31359H +T*+C*+G*A*A*T*T*T*G*C*C*A*+T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31360H +T*+C*+G*A*A*T*T*T*G*C*C*+A*T*A*+G*+T*+T 12273 29 TCGAATTTGCCATAGTT A31361H +T*+C*+G*A*A*T*T*T*G*+C*C*A*T*A*+G*+T*+T 12273 30 TCGAATTTGCCATAGT A31362H +T*+C*+G*A*+A*T*T*T*G*C*C*A*T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31363H +T*+C*+G*A*A*+T*T*T*G*C*C*A*T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31364H +T*+C*+G*A*A*T*+T*T*G*C*C*A*T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31365H +T*+C*+G*A*A*T*T*T*G*C*C*A*+T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31366H +T*+C*+G*A*A*T*T*T*G*C*C*+A*T*+A*+G*+T 12274 30 TCGAATTTGCCATAGT A31367H +T*+C*+G*+A*A*T*T*T*G*C*C*A*+T*+A*+G*+T 12274 59 GTAATGTCAACGGATC A31368Hi +G*+T*+A*A*+T*G*T*C*A*A*C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31369Hi +G*+T*+A*A*T*+G*T*C*A*A*C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31370Hi +G*+T*+A*A*T*G*+T*C*A*A*C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31371Hi +G*+T*+A*A*T*G*T*C*A*A*C*+G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31372Hi +G*+T*+A*A*T*G*T*C*A*A*+C*G*G*+A*+T*+C 13706 59 GTAATGTCAACGGATC A31373Hi +G*+T*+A*+A*T*G*T*C*A*A*C*G*+G*+A*+T*+C 13706 Position onmRNA Nameof (NM_ SEQ theLNA- 004895.4; ID AntisenseSequence modified AntisenseSequence SEQID NO. 5-3 ASO 5-3withPTO(*)andLNA(+) NO.2) 338 CCACAATCTCCGAATGTTA A31270H** +C*+C*+A*C*A*A*T*C*T*C*C*G*A*A*T*G*+T*+T*+A 16878 339 CGTTTAGGCTATGTACTT Neg1 +C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T Control 340 TACGCGCGGTTGTTTA R01002 +T*+A*+C*G*C*G*C*G*G*T*T*G*T*+T*+T*+A Control 341 GATCATTCGCGGACAAC R01011 +G*+A*+T*C*A*T*T*C*G*C*G*G*A*C*+A*+A*+C Control

[0053] The following Table 2 shows oligonucleotides hybridizing with murine NLRP3 mRNA:

TABLE-US-00003 TABLE2 ListofmouseNLRP3-specificASOshybridizingwithmouseNLRP3forexampleofSEQIDNO.3; S5isanantisenseoligonucleotiderepresentinganegativecontrolwhichisnothybrid- izingwithNLRP3ofSEQIDNO.3orSEQIDNO.4.AnMaftertheASOIDindicatesamouse NLRP3-specificsequencethatbindstoanexonicregionofthepre-mRNAandaMiafterthe ASOIDindicatesamouseNLRP3-specificsequencethatbindstoanintronicregionofthe pre-mRNA.**=exonspanningoligo,positiondepictedinTable2indicatespositions onmRNAofSEQIDNO.4(RefSeqIDNM_145827)forexonspanningoligonucleotides.AnR aftertheASOIDindicatesamouseNLRP3-specificwithcross-reactivitytorat. Positionon pre-mRNA (GRmCh38, Chr11: Nameof 59539030- SEQ theLNA- 59569495; ID AntisenseSequence modified AntisenseSequence SEQID NO. 5-3 ASO 5-3withPTO(*)andLNA(+) NO.3) 342 CTTGATCCAGACGTAT A31001M +C*+T*+T*G*A*T*C*C*A*G*A*C*G*+T*+A*+T 3807 343 AACGGACACTCGTCATC A31002M +A*+A*C*G*G*A*C*A*C*T*C*G*T*C*+A*+T*+C 4083 344 TTGCAACGGACACTCGT A31003M +T*+T*+G*C*A*A*C*G*G*A*C*A*C*T*+C*+G*+T 4087 345 CTCGCCTGTTGATCGCA A31004M +C*+T*+C*G*C*C*T*G*T*T*G*A*T*C*+G*+C*+A 4305 346 CTCGCCTGTTGATCGC A31005M +C*+T*+C*G*C*C*T*G*T*T*G*A*T*+C*+G*+C 4306 347 CTCGCCTGTTGATCG A31006M +C*+T*+C*G*C*C*T*G*T*T*G*A*+T*+C*+G 4307 348 TCTCGCCTGTTGATCG A31007M +T*+C*+T*C*G*C*C*T*G*T*T*G*A*+T*+C*+G 4307 349 AGACGCGCGTTCCTGT A31008M +A*+G*A*C*G*C*G*C*G*T*T*C*C*T*+G*+T 9015 350 CACCTAGACGCGCGTTC A31009M +C*+A*C*C*T*A*G*A*C*G*C*G*C*G*T*+T*+C 9019 351 CGTGTAGCGACTGTTGA A31010M +C*+G*+T*G*T*A*G*C*G*A*C*T*G*T*+T*+G*+A 9048 352 CGTGTAGCGACTGTTG A31011M +C*+G*+T*G*T*A*G*C*G*A*C*T*G*+T*+T*+G 9049 353 GCGTGTAGCGACTGTTG A31012M +G*+C*+G*T*G*T*A*G*C*G*A*C*T*G*+T*+T*+G 9049 354 GCGTGTAGCGACTGTT A31013M +G*+C*+G*T*G*T*A*G*C*G*A*C*T*+G*+T*+T 9050 355 TGCGTGTAGCGACTGTT A31014M +T*+G*+C*G*T*G*T*A*G*C*G*A*C*T*+G*+T*+T 9050 356 GGAGCTGCGTGTAGCGA A31015M +G*+G*A*G*C*T*G*C*G*T*G*T*A*G*C*+G*+A 9055 357 TAGTCCGGCCGATGGT A31016M +T*+A*+G*T*C*C*G*G*C*C*G*A*T*+G*+G*+T 9122 358 TTAGTCCGGCCGATGGT A31017M +T*+T*+A*G*T*C*C*G*G*C*C*G*A*T*+G*+G*+T 9122 359 ACCGGCCTCGTCGTTAT A31018M +A*+C*C*G*G*C*C*T*C*G*T*C*G*T*T*+A*+T 9599 360 CTACCGGCCTCGTCGTT A31019M +C*+T*+A*C*C*G*G*C*C*T*C*G*T*C*G*+T*+T 9601 361 CTACCGGCCTCGTCGT A31020M +C*+T*A*C*C*G*G*C*C*T*C*G*T*C*+G*+T 9602 362 AAGGCTACCGGCCTCGT A31021M +A*+A*G*G*C*T*A*C*C*G*G*C*C*T*+C*+G*+T 9605 363 TAGACGGCCGTAGTGGT A31022M +T*+A*+G*A*C*G*G*C*C*G*T*A*G*T*G*+G*+T 9869 364 TAGACGGCCGTAGTGG A31023M +T*+A*+G*A*C*G*G*C*C*G*T*A*G*+T*+G*+G 9870 365 GTAGACGGCCGTAGTGG A31024M +G*+T*+A*G*A*C*G*G*C*C*G*T*A*G*+T*+G*+G 9870 366 GTAGACGGCCGTAGTG A31025M +G*+T*+A*G*A*C*G*G*C*C*G*T*A*+G*+T*+G 9871 367 ACGTAGACGGCCGTAG A31026M +A*+C*+G*T*A*G*A*C*G*G*C*C*G*+T*+A*+G 9873 368 AGAAGACGTAGACGGC A31027M +A*+G*+A*A*G*A*C*G*T*A*G*A*C*+G*+G*+C 9878 369 AGCGGAGACGTCAGTCT A31028M +A*+G*+C*G*G*A*G*A*C*G*T*C*A*G*T*+C*+T 10044 370 GGACCTTCACGTCTCGG A31029M +G*+G*+A*C*C*T*T*C*A*C*G*T*C*T*+C*+G*+G 10228 371 TAGTAGGACCTTCACGT A31030M +T*+A*+G*T*A*G*G*A*C*C*T*T*C*A*+C*+G*+T 10233 372 TAGTAGGACCTTCACG A31031M +T*+A*+G*T*A*G*G*A*C*C*T*T*C*+A*+C*+G 10234 373 CTAGTAGGACCTTCACG A31032M +C*+T*+A*G*T*A*G*G*A*C*C*T*T*C*+A*+C*+G 10234 374 ACAACACGCGGATGTGA A31033M +A*+C*+A*A*C*A*C*G*C*G*G*A*T*G*+T*+G*+A 19371 375 GCACGTTTTGTTTCACG A31034M +G*+C*+A*C*G*T*T*T*T*G*T*T*T*C*+A*C*+G 27157 376 ACGTTATCGATGTGAGG A31035M +A*+C*+G*T*T*A*T*C*G*A*T*G*T*G*+A*+G*+G 27405 377 ACGTTATCGATGTGAG A31036M +A*+C*+G*T*T*A*T*C*G*A*T*G*T*+G*+A*+G 27406 378 TTCACGTTATCGATGT A31037M +T*+T*+C*A*C*G*T*T*A*T*C*G*A*+T*+G*+T 27409 379 TTTCACGTTATCGATGT A31038M +T*+T*+T*C*A*C*G*T*T*A*T*C*G*A*+T*+G*+T 27409 380 GTTTCACGTTATCGAT A31039M +G*+T*+T*T*C*A*C*G*T*T*A*T*C*+G*+A*+T 27411 381 AGTTTCACGTTATCGAT A31040M +A*+G*+T*T*T*C*A*C*G*T*T*A*T*C*+G*+A*+T 27411 351 CGTGTAGCGACTGTTGA A31041M +C*+G*+T*G*T*A*G*C*G*A*C*T*G*+T*+T*+G*+A 9048 351 CGTGTAGCGACTGTTGA A31042M +C*+G*+T*+G*T*A*G*C*G*A*C*T*G*T*+T*+G*+A 9048 351 CGTGTAGCGACTGTTGA A31043M +C*+G*T*G*T*A*G*C*G*A*C*T*G*T*+T*+G*+A 9048 351 CGTGTAGCGACTGTTGA A31044M +C*+G*+T*G*T*A*G*C*G*A*C*T*G*T*T*+G*+A 9048 375 GCACGTTTTGTTTCACG A31045M +G*+C*+A*+C*G*T*T*T*T*G*T*T*T*C*+A*C*+G 27157 375 GCACGTTTTGTTTCACG A31046M +G*+C*+A*C*G*T*T*T*T*G*T*T*T*+C*+A*C*+G 27157 375 GCACGTTTTGTTTCACG A31047M +G*C*+A*+C*G*T*T*T*T*G*T*T*T*C*A*+C*+G 27157 376 ACGTTATCGATGTGAGG A31048M +A*+C*+G*T*T*A*T*C*G*A*T*G*T*+G*+A*+G*+G 27405 376 ACGTTATCGATGTGAGG A31049M +A*+C*G*T*T*A*T*C*G*A*T*G*T*+G*+A*+G*+G 27405 382 ACGTGTACCTTACTGTA A31050Mi +A*+C*+G*T*G*T*A*C*C*T*T*A*C*T*+G*+T*+A 4411 383 CTACCTGCACGAGGAAT A31051Mi +C*+T*+A*C*C*T*G*C*A*C*G*A*G*G*+A*+A*+T 4618 384 GAACAATTACGGCTGC A31052Mi +G*+A*+A*+C*A*A*T*T*A*C*G*G*C*+T*+G*+C 5367 385 CCACATAGGTGCCGTAG A31053Mi +C*+C*+A*C*A*T*A*G*G*T*G*C*C*G*+T*+A*+G 5517 386 GCACGGATAGTAATAGG A31054Mi +G*+C*+A*+C*G*G*A*T*A*G*T*A*A*T*+A*+G*+G 6279 387 CTTACTATGACCGGCC A31055Mi +C*+T*+T*A*C*T*A*T*G*A*C*C*G*G*+C*+C 6355 388 TACTCTTACTATGACCG A31056Mi +T*+A*+C*T*C*T*T*A*C*T*A*T*G*A*+C*+C*+G 6358 389 GTGCAACCGCGAACCT A31057Mi +G*+T*+G*C*A*A*C*C*G*C*G*A*A*C*+C*+T 6509 390 CGTGCAACCGCGAACCT A31058Mi +C*+G*T*G*C*A*A*C*C*G*C*G*A*A*+C*+C*+T 6509 391 ACATTCGTGCAACCGC A31059Mi +A*+C*+A*T*T*C*G*T*G*C*A*A*C*+C*+G*+C 6515 392 AACATTCGTGCAACCGC A31060Mi +A*+A*+C*A*T*T*C*G*T*G*C*A*A*C*+C*+G*+C 6515 393 CTCAGTTGGATGTACGT A31061Mi +C*+T*+C*A*G*T*T*G*G*A*T*G*T*A*+C*+G*+T 7132 394 CTCAGTTGGATGTACG A31062Mi +C*+T*+C*A*G*T*T*G*G*A*T*G*T*+A*+C*+G 7133 395 CACTGCTCGAGTGTATC A31063Mi +C*+A*C*T*G*C*T*C*G*A*G*T*G*T*+A*+T*+C 8170 396 TGGTATGACCGGACAGA A31064Mi +T*+G*+G*T*A*T*G*A*C*C*G*G*A*C*+A*+G*+A 8490 397 ATAGAGCGAGCTGTCG A31065Mi +A*+T*+A*G*A*G*C*G*A*G*C*T*G*+T*+C*+G 8756 398 TATAGAGCGAGCTGTCG A31066Mi +T*+A*+T*A*G*A*G*C*G*A*G*C*T*G*+T*+C*+G 8756 399 AGGATTTACGAACAAC A31067Mi +A*+G*+G*A*T*T*T*A*C*G*A*A*C*+A*+A*+C 8836 400 TAAGCTGCTAGCGGACC A31068Mi +T*+A*+A*G*C*T*G*C*T*A*G*C*G*G*A*+C*+C 8885 401 GCATGGAAGCGATATTC A31069Mi +G*+C*+A*T*G*G*A*A*G*C*G*A*T*A*+T*+T*+C 11021 13044 402 TCTCAGAACGACGGAAT A31070Mi +T*+C*+T*C*A*G*A*A*C*G*A*C*G*G*+A*+A*+T 11341 403 GAGCATGTTTGCCGATA A31071Mi +G*+A*+G*C*A*T*G*T*T*T*G*C*C*G*+A*+T*+A 12045 404 CCGTTGGACTGGTATCA A31072Mi +C*+C*+G*T*T*G*G*A*C*T*G*G*T*A*+T*+C*+A 13244 405 TTCACGTATCAGCACA A31073Mi +T*+T*+C*A*C*G*T*A*T*C*A*G*C*+A*+C*+A 13672 406 GTGAACGTAATGGCAGG A31074Mi +G*+T*+G*A*A*C*G*T*A*A*T*G*G*C*+A*+G*+G 13906 407 TTAAAGACCGGTGTGCC A31075Mi +T*+T*+A*A*A*G*A*C*C*G*G*T*G*T*G*+C*+C 14147 408 ACCTCGAGTTTGGTTCA A31076Mi +A*+C*+C*T*C*G*A*G*T*T*T*G*G*T*T*+C*+A 14378 409 GACCGGTGATCTCATAA A31077Mi +G*+A*+C*C*G*G*T*G*A*T*C*T*C*A*+T*+A*+A 15826 410 GAACTGACCGGTGATC A31078Mi +G*+A*+A*C*T*G*A*C*C*G*G*T*G*A*+T*+C 15832 411 CCGTGCGTTAGGAGAAT A31079Mi +C*+C*+G*T*G*C*G*T*T*A*G*G*A*G*+A*+A*+T 15893 412 TCTTTCCGTGCGTTAG A31080Mi +T*+C*+T*T*T*C*C*G*T*G*C*G*T*+T*+A*+G 15899 413 TGAATGCACCAAGCTCG A31081Mi +T*+G*+A*A*T*G*C*A*C*C*A*A*G*C*+T*+C*+G 16071 414 CCGACCATATAATGCTA A31082Mi +C*+C*+G*+A*C*C*A*T*A*T*A*A*T*G*+C*T*+A 16351 415 AGCGTTCAGTAAGGACC A31083Mi +A*+G*+C*G*T*T*C*A*G*T*A*A*G*G*A*+C*+C 17288 416 TACGTTCCTACTAGAGC A31084Mi +T*+A*+C*G*T*T*C*C*T*A*C*T*A*G*+A*+G*+C 17943 417 ACGGTTGCATAGCTGGC A31085Mi +A*+C*+G*G*T*T*G*C*A*T*A*G*C*T*G*+G*+C 18058 418 TGTTACTGAGTACGACC A31086Mi +T*+G*+T*T*A*C*T*G*A*G*T*A*C*G*A*+C*+C 20304 419 AGCAATTTGGTAACGTC A31087Mi +A*+G*+C*A*A*T*T*T*G*G*T*A*A*C*+G*+T*+C 20517 420 CCGTGGCAGCACCTTAA A31088Mi +C*+C*+G*T*G*G*C*A*G*C*A*C*C*T*+T*+A*+A 20545 421 AACTGAGCGTAGTGAAG A31089Mi +A*+A*+C*T*G*A*G*C*G*T*A*G*T*G*+A*+A*+G 22910 422 TAACTTGCGGTATTATC A31090Mi +T*+A*+A*C*T*T*G*C*G*G*T*A*T*T*+A*+T*+C 23548 423 GTCTAACTTGCGGTAT A31091Mi +G*+T*C*T*A*A*C*T*T*G*C*G*G*+T*+A*+T 23552 424 CAATCCTCGTTGTTCC A31092Mi +C*+A*+A*T*C*C*T*C*G*T*T*G*T*+T*+C*+C 23744 425 TACGTCAGCAATCCTCG A31093Mi +T*+A*+C*G*T*C*A*G*C*A*A*T*C*C*+T*+C*+G 23751 426 AAGTTTGCTGTCCGGTA A31094Mi +A*+A*+G*T*T*T*G*C*T*G*T*C*C*G*+G*+T*+A 23854 427 CGAGAAGCGATGCTTAG A31095Mi +C*+G*+A*+G*A*A*G*C*G*A*T*G*C*T*+T*+A*+G 24285 428 GCTTGGATCCACGAGAG A31096Mi +G*+C*+T*T*G*G*A*T*C*C*A*C*G*+A*G*+A*+G 25786 429 CACTGCGAGCTGCATC A31097Mi +C*+A*C*T*G*C*G*A*G*C*T*G*C*+A*+T*+C 25939 430 CGCATATACAAAGTCAC A31098M +C*+G*+C*A*T*A*T*A*C*A*A*A*G*T*+C*+A*+C 3705 431 CTTGATCCAGACGTATG A31099M +C*+T*+T*G*A*T*C*C*A*G*A*C*G*T*+A*+T*+G 3806 432 TTGATCCAGACGTATG A31100M +T*+T*+G*A*T*C*C*A*G*A*C*G*T*+A*+T*+G 3806 433 GCTTGATCCAGACGTAT A31101M +G*+C*+T*T*G*A*T*C*C*A*G*A*C*G*+T*+A*+T 3807 434 CGGACACTCGTCATCTT A31102M +C*+G*+G*A*C*A*C*T*C*G*T*C*A*T*+C*+T*+T 4081 435 AGCTCGTTGGAGAAATA A31103M +A*+G*+C*T*C*G*T*T*G*G*A*G*A*A*+A*+T*+A 9710 436 CTGTAGAATCTCTCGCA A31104M +C*+T*+G*T*A*G*A*A*T*C*T*C*T*C*+G*+C*+A 10097 437 ATAGTACATAGCAGCGA A31105M +A*+T*+A*G*T*A*C*A*T*A*G*C*A*G*+C*+G*+A 10143 438 TAGTACATAGCAGCGA A31106M +T*+A*+G*T*A*C*A*T*A*G*C*A*G*+C*+G*+A 10143 439 CAAACTTGCCGTAATTT A31107M +C*+A*+A*A*C*T*T*G*C*C*G*T*A*A*+T*+T*+T 10252 440 TAGAACAGTTCCAGTTG A31108M +T*+A*+G*A*A*C*A*G*T*T*C*C*A*G*+T*+T*+G 10442 441 TGCTTCGTAGATAGAGG A31109M +T*+G*+C*T*T*C*G*T*A*G*A*T*A*G*+A*+G*+G 26089 442 CATTGCTTCGTAGATAG A31110M +C*+A*+T*T*G*C*T*T*C*G*T*A*G*A*+T*+A*+G 26092 443 CGCCAAGATCATTGTTG A31111M +C*+G*+C*C*A*A*G*A*T*C*A*T*T*G*+T*+T*+G 26906 444 GCACAGATCGCCAAGAT A31112M +G*+C*+A*C*A*G*A*T*C*G*C*C*A*A*+G*+A*+T 26914 445 CGTTTTGTTTCACGATT A31113M +C*+G*+T*T*T*T*G*T*T*T*C*A*C*G*+A*+T*+T 27154 375 GCACGTTTTGTTTCACG A31114M +G*+C*+A*C*G*T*T*T*T*G*T*T*T*C*+A*+C*+G 27157 446 GAAATCTCGAAGACTAT A31115M +G*+A*+A*A*T*C*T*C*G*A*A*G*A*C*+T*+A*+T 27211 447 CGTTATCGATGTGAGGA A31116M +C*+G*+T*T*A*T*C*G*A*T*G*T*G*A*+G*+G*+A 27404 448 GCAGTTTCACGTTATCG A31117M +G*+C*+A*G*T*T*T*C*A*C*G*T*T*A*+T*+C*+G 27413 343 AACGGACACTCGTCATC A31118M +A*+A*+C*G*G*A*C*A*C*T*C*G*T*C*+A*+T*+C 4083 449 GAGCCAGCTTGCAACG A31119M +G*+A*+G*C*C*A*G*C*T*T*G*C*A*+A*+C*+G 4096 450 GTCTCGCCTGTTGATCG A31120MR +G*+T*+C*T*C*G*C*C*T*G*T*T*G*A*+T*+C*+G 4307 451 GTCTCGCCTGTTGATC A31121MR +G*+T*+C*T*C*G*C*C*T*G*T*T*G*+A*+T*+C 4308 452 GTCGTCTGTACATCTTA A31122M +G*+T*+C*G*T*C*T*G*T*A*C*A*T*C*+T*+T*+A 8968 453 AGCGACTGTTGAGGTCC A31123M +A*+G*+C*G*A*C*T*G*T*T*G*A*G*G*+T*+C*+C 9043 454 AGTTGGAGCTGCGTGTA A31124M +A*+G*+T*T*G*G*A*G*C*T*G*C*G*T*+G*+T*+A 9059 455 GTCCAACATAATCTTCC A31125MR +G*+T*+C*C*A*A*C*A*T*A*A*T*C*T*+T*+C*+C 9267 456 GTCGTTATGAGCAGAGA A31126M +G*+T*+C*G*T*T*A*T*G*A*G*C*A*G*+A*+G*+A 9590 359 ACCGGCCTCGTCGTTAT A31127M +A*+C*+C*G*G*C*C*T*C*G*T*C*G*T*+T*+A*+T 9599 457 TACCGGCCTCGTCGTT A31128M +T*+A*+C*C*G*G*C*C*T*C*G*T*C*+G*+T*+T 9601 458 ACGTAGACGGCCGTAGT A31129M +A*+C*+G*T*A*G*A*C*G*G*C*C*G*T*+A*+G*+T 9872 459 CCGTGCTTCCGCAGATC A31130M +C*+C*+G*T*G*C*T*T*C*C*G*C*A*G*+A*+T*+C 10019 460 GCCGTGCTTCCGCAGAT A31131M +G*+C*+C*G*T*G*C*T*T*C*C*G*C*A*+G*+A*+T 10020 369 AGCGGAGACGTCAGTCT A31132M +A*+G*+C*G*G*A*G*A*C*G*T*C*A*G*+T*+C*+T 10044 461 CTCTCGCAGTCCACTTC A31133M +C*+T*+C*T*C*G*C*A*G*T*C*C*A*C*+T*+T*+C 10088 462 ATCTCTCGCAGTCCACT A31134M +A*+T*+C*T*C*T*C*G*C*A*G*T*C*C*+A*+C*+T 10090 463 GAACCAATGCGAGATCC A31135MR +G*+A*+A*C*C*A*A*T*G*C*G*A*G*A*+T*+C*+C 19390 464 AGTAAGGCCGGAATTC A31137MR +A*+G*+T*A*A*G*G*C*C*G*G*A*A*+T*+T*+C 26017 465 GGACTCGTGTCTGACCA A31138M +G*+G*+A*C*T*C*G*T*G*T*C*T*G*A*+C*+C*+A 3686 466 CGCGTTCCTGTCCTTGA A31139M +C*+G*+C*G*T*T*C*C*T*G*T*C*C*T*+T*+G*+A 9009 467 TAGACGCGCGTTCCTGT A31140M +T*+A*+G*A*C*G*C*G*C*G*T*T*C*C*+T*+G*+T 9015 468 TAGACGCGCGTTCCTG A31141M +T*+A*+G*A*C*G*C*G*C*G*T*T*C*+C*+T*+G 9016 469 CTCACCTAGACGCGCG A31142M +C*+T*+C*A*C*C*T*A*G*A*C*G*C*+G*+C*+G 9022 470 CTCTCACCTAGACGCG A31143M +C*+T*+C*T*C*A*C*C*T*A*G*A*C*+G*+C*+G 9024 471 GCTGCGTGTAGCGACTG A31144M +G*+C*+T*G*C*G*T*G*T*A*G*C*G*A*+C*+T*+G 9052 472 CGGCCGATGGTCAGGAG A31145M +C*+G*+G*C*C*G*A*T*G*G*T*C*A*G*+G*+A*+G 9116 473 CATTTTAGTCCGGCCGA A31146M +C*+A*+T*T*T*T*A*G*T*C*C*G*G*C*+C*+G*+A 9126 474 GCCTCGTCGTTATGAG A31147M +G*+C*+C*T*C*G*T*C*G*T*T*A*T*+G*+A*+G 9596 475 CAAGGCTACCGGCCTCG A31148M +C*+A*+A*G*G*C*T*A*C*C*G*G*C*C*+T*+C*+G 9606 476 CGGCCGTAGTGGTCTT A31149M +C*+G*+G*C*C*G*T*A*G*T*G*G*T*+C*+T*+T 9866 477 AGACGTAGACGGCCG A31150M +A*+G*+A*C*G*T*A*G*A*C*G*G*+C*+C*+G 9876 478 AGAAGACGTAGACGGCC A31151M +A*+G*+A*A*G*A*C*G*T*A*G*A*C*G*+G*+C*+C 9877 479 GCAGGCCGTGCTTCCGC A31152M +G*+C*+A*G*G*C*C*G*T*G*C*T*T*C*+C*+G*+C 10024 480 TCGGTTCAGAAGATCTG A31153M +T*+C*+G*G*T*T*C*A*G*A*A*G*A*T*+C*+T*+G 10215 481 GGTTGGTGCTTAGACTT A31154M +G*+G*+T*T*G*G*T*G*C*T*T*A*G*A*+C*+T*+T 12790 482 TGTCGAAGCAGCATTGA A31155M +T*+G*+T*C*G*A*A*G*C*A*G*C*A*T*+T*+G*+A 16743 483 CGCTTCTAAGGCACGTT A31156M +C*+G*+C*T*T*C*T*A*A*G*G*C*A*C*+G*+T*+T 27167 484 TCACGTTATCGATGTGA A31157M +T*+C*+A*C*G*T*T*A*T*C*G*A*T*G*+T*+G*+A 27407 Positionon Nameof mRNA SEQ theLNA- (NM_145827; ID AntisenseSequence modified AntisenseSequence SEQID NO. 5-3 ASO 5-3withPTO(*)andLNA(+) NO.4) 485 GCCGGAATTCACCAACC A31136MR** +G*+C*+C*G*G*A*A*T*T*C*A*C*C*A*+A*+C*+C 26010 486 TTATGTCCGGTTATTTC S5 +T*+T*+A*T*G*T*C*C*G*G*T*T*A*T*+T*+T*+C Control

[0054] The oligonucleotides of the present invention hybridize for example with mRNA and/or pre-mRNA of human NLRP3 of SEQ ID NO. 2 and SEQ ID NO 1, respectively, and/or of mouse NLRP3 of SEQ ID NO. 4 and SEQ ID NO 3, respectively. Such oligonucleotides are called NLRP3 antisense oligonucleotides. Oligonucleotides of the present invention, which are for example antisense oligonucleotides, are shown in Tables 1 and 2. The present invention further refers to oligonucleotides such as antisense oligonucleotides having 80 to 99%, 85 to 98%, 90 to 95 or 93% sequence homology to an oligonucleotide of Table 1 and/or Table 2. Such oligonucleotides still show an inhibitory activity of 70 to 100%, 80 to 90%, 70%, 75%80%, 85%, 90%, 95% or 100% compared to the oligonucleotide having the nucleic acid sequence shown in Table 1 or 2.

[0055] Each nucleotide of the sequence can be modified, wherein ASOs of the present invention preferably comprise a core of 6 to 8 unmodified nucleotides. ASOs of the present invention comprise for example one or more modified nucleotides, e.g., 1, 2, 3, 4 or 5 nucleotides at the 5- and/or 3-end of the oligonucleotide, i.e., on the 5- and/or 3-side of the core. The 5- and 3-end are modified identically or differently. If the 5- and 3-ends are modified identically the nucleotides are modified at the same positions counted from the 5- and 3-end (in each case starting the counting with 1 from the end), respectively, having the same modification for example LNA-modification. If the 5- and 3-ends are modified differently the position of the modified nucleotide and/or the type of modification at the 5- and 3-ends differ; the type of nucleotide modification is the same (e.g., LNA) or different. Modified nucleotides such as LNA-modified nucleotides need not to follow in a row, but may be separated by one or more unmodified nucleotides. In the following some modification patterns at the 5- and 3-end of the ASOs of the present invention are described, wherein an unmodified nucleotide is indicated by _ and the figure refers to the number of modified nucleotides such as LNA-modified nucleotides in a row. The modified nucleotide(s) is/are at any position of the 5- and/or 3-end of the ASO as shown in the following Table 3:

TABLE-US-00004 TABLE 3 LNA modification at the 5- LNA modification at the 3- side of the core side of the core Abbreviation 3 3 3 + 3 3 2 3 + 2 2 3 2 + 3 1_1 3 1_1 + 3 1_1 2 1_1 + 2 1_1 1_1 1_1 + 1_1 3 1_1 3 + 1_1 2 1_1 2 + 1_1 2 2 2 + 2 4 3 4 + 3 4 2 4 + 2 4 1_1 4 + 1_1 2_1 3 2_1 + 3 2_1 1_1 2_1 + 1_1 2_1 2 2_1 + 2 3 4 3 + 4 2 4 2 + 4 1_1 4 1_1 + 4 3 1_2 3 + 1_2 1_1 1_2 1_1 + 1_2 2 1_2 2 + 1_2

[0056] Typical modification patterns of each ASO of the present invention, comprising for example LNA-modified nucleotides, are shown for example in the following

TABLE-US-00005 TABLE 4 Position of the modification Position of the modification at the 5-end (counted from at the 3-end (counted from the 5-end starting with 1) the 3-end starting with 1) Abbreviation nucleotides 1 to 5 nucleotides 1 to 5 5 + 5 nucleotides 1 to 4 nucleotides 1 to 4 4 + 4 nucleotides 1 to 3 nucleotides 1 to 3 3 + 3 nucleotides 1 and 2 nucleotides 1 and 2 2 + 2 nucleotide 1 nucleotide 1 1 + 1 nucleotides 1 to 5 nucleotides 1 to 4 5 + 4 nucleotides 1 to 4 nucleotides 1 to 3 4 + 3 nucleotides 1 to 3 nucleotides 1 and 2 3 + 2 nucleotides 1 and 2 nucleotide 1 2 + 1 nucleotide 1 nucleotides 1 to 5 1 + 5 nucleotides 1 to 5 nucleotides 1 to 3 5 + 3 nucleotides 1 to 4 nucleotides 1 and 2 4 + 2 nucleotides 1 to 3 nucleotide 1 3 + 1 nucleotides 1 and 2 nucleotides 1 to 5 2 + 5 nucleotide 1 nucleotides 1 to 4 1 + 4 nucleotides 1 to 5 nucleotides 1 and 2 5 + 2 nucleotides 1 to 4 nucleotide 1 4 + 1 nucleotides 1 to 3 nucleotides 1 to 5 3 + 5 nucleotides 1 and 2 nucleotides 1 to 4 2 + 4 nucleotide 1 nucleotides 1 to 3 1 + 3 nucleotides 1 to 5 nucleotide 1 5 + 1 nucleotides 1 to 4 nucleotides 1 to 5 4 + 5 nucleotides 1 to 3 nucleotides 1 to 4 3 + 4 nucleotides 1 and 2 nucleotides 1 to 3 2 + 3 nucleotide 1 nucleotides 1 and 2 1 + 2 nucleotides 1 and 2 nucleotides 1 and 3 2 + 1_1 nucleotides 1 and 3 nucleotides 1 to 3 1_1 + 3 nucleotides 1 and 3 nucleotides 1 and 3 1_1 + 1_1 nucleotides 1 to 3 nucleotides 1 and 3 3 + 1_1 nucleotides 1 to 4 nucleotides 1 and 3 4 + 1_1 nucleotides 1, 2 and 4 nucleotides 1 to 3 2_1 + 3 nucleotides 1, 2 and 4 nucleotides 1 and 3 2_1 + 1_1 nucleotides 1, 2 and 4 nucleotides 1 and 2 2_1 + 2 nucleotides 1 and 3 nucleotides 1 to 4 1_1 + 4 nucleotides 1 to 3 nucleotides 1, 2 and 4 3 + 1_2 nucleotides 1 and 3 nucleotides 1, 2 and 4 1_1 + 1_2 nucleotides 1 and 2 nucleotides 1, 2 and 4 2 + 1_2
which indicates specific positions of the LNA modifications at the 5- and 3-end of each ASO:

[0057] An oligonucleotide of the present invention further or alternatively hybridizes for example with the NLRP3 nucleic acid sequence of SEQ ID NO. 1 and SEQ ID NO. 2 (human) or SEQ ID NO. 3 and SEQ ID NO. 4 (murine) in a hybridizing active area. A hybridizing active area is an area on the NLRP3 pre-mRNA of SEQ ID NO. 1 (human) or SEQ ID NO. 3 (mouse), wherein binding of an oligonucleotide most likely leads to potent knockdown of the NLRP3 expression. An oligonucleotide of the present invention hybridizes for example within these positions or overlaps with a terminal position.

[0058] The human NLRP3 oligonucleotides of the present invention hybridize for example with hybridizing active areas of human NLRP3 pre-mRNA for example of SEQ ID NO. 1. Hybridizing active areas of SEQ ID NO. 1 are listed for example in the following

TABLE-US-00006 TABLE 5 Region of First position on SEQ ID NO. 1 SEQ ID NO. 1 SEQ ID NO. 5280-5779 A31001H 5280 5 A31002H 5298 6 A31003H 5300 7 A31004H 5394 8 A31128H 5407 112 5780-6279 A31005H 5861 9 A31006H 5890 10 A31007H 5898 11 A31008H 6094 12 A31009H 6148 13 A31010H 6155 14 A31011H 6156 15 A31095H 6154 87 A31129H 5873 113 A31130H 5897 114 A31131H 5902 115 A31132H 6097 116 A31133H 6144 117 A31225H 5872 209 A31226H 5935 210 A31227H 6013 211 A31228H 6012 212 A31229H 6086 213 A31230H 6081 214 A31231H 6091 215 6280-6779 A31290Hi 6630 273 6780-7279 A31167Hi 7195 151 7780-8279 A31049Hi 7906 53 A31168Hi 7905 152 A31169Hi 8158 153 A31291Hi 7914 274 8280-8779 A31292Hi 8621 275 A31293Hi 8664 276 8780-9279 A31170Hi 8871 154 9280-9779 A31171Hi 9654 155 A31294Hi 9563 277 A31295Hi 9619 278 A31296Hi 9629 279 9780-10279 A31050Hi 9849 54 A31051Hi 9863 55 A31172Hi 9854 156 A31173Hi 9867 157 A31297Hi 10160 280 A31298Hi 10236 281 10280-10779 A31012H 10370 16 A31013H 10379 17 A31014H 10450 18 A31052Hi 10682 56 A31096H 10446 88 A31119Hi 10684 103 A31174Hi 10573 158 A31175Hi 10680 159 A31176Hi 10688 160 A31177Hi 10767 161 A31232H 10370 216 A31233H 10447 217 A31299Hi 10297 282 10780-11279 A31015H 11076 19 A31016H 11077 20 A31017H 11081 21 A31018H 11160 22 A31053Hi 10894 57 A31054Hi 10896 58 A31097H 11005 89 A31098H 11072 90 A31099H 11080 91 A31100H 11083 92 A31101H 11087 93 A31102H 11090 94 A31120Hi 10888 104 A31121Hi 10899 105 A31134H 11094 118 A31135H 11142 119 A31136H 11150 120 A31137H 11156 121 A31138H 11161 122 A31178Hi 10826 162 A31179Hi 10887 163 A31180Hi 10900 164 A31181Hi 10928 165 A31234H 11009 218 A31235H 11066 219 A31236H 11071 220 A31237H 11074 221 A31238H 11079 222 A31239H 11071 223 A31240H 11078 224 A31241H 11080 225 A31242H 11102 226 A31243H 11149 227 A31300Hi 10818 283 11280-11779 A31019H 11522 23 A31020H 11523 24 A31103H 11526 95 A31139H 11489 123 A31140H 11507 124 A31141H 11523 125 A31142H 11543 126 A31143H 11671 127 A31144H 11697 128 A31244H 11329 228 A31245H 11338 229 A31246H 11350 230 A31247H 11338 231 A31248H 11351 232 A31249H 11503 233 A31250H 11526 234 A31251H 11504 235 A31252H 11541 236 A31253H 11521 237 A31254H 11620 238 A31255H 11662 239 A31256H 11678 240 11780-12279 A31021H 11903 25 A31022H 12205 26 A31023H 12228 27 A31024H 12249 28 A31025H 12273 29 A31026H 12274 30 A31081H 12273 29 A31082H 12273 29 A31083H 12273 29 A31084H 12273 29 A31085H 12274 30 A31086H 12274 30 A31087H 12274 30 A31088H 12274 30 A31089H 12274 85 A31090H 12274 85 A31091H 12274 85 A31092H 12275 86 A31093H 12275 86 A31094H 12275 86 A31145H 11787 129 A31146H 11903 130 A31147H 11907 131 A31148H 12124 132 A31149H 12247 133 A31257H 11819 241 A31258H 12064 242 A31259H 12068 243 A31260H 12163 244 A31261H 12165 245 A31262H 12169 246 A31263H 12272 247 A31355H 12273 29 A31356H 12273 29 A31357H 12273 29 A31358H 12273 29 A31359H 12273 29 A31360H 12273 29 A31361H 12274 29 A31362H 12274 30 A31363H 12274 30 A31364H 12274 30 A31365H 12274 30 A31366H 12274 30 A31367H 12274 30 12280-12779 A31027H 12475 31 A31104H 12477 96 A31150H 12498 134 A31264H 12473 248 A31265H 12473 249 A31266H 12498 250 A31267H 12628 251 A31301Hi 12776 284 12780-13279 A31302Hi 13059 285 13280-13779 A31055Hi 13706 59 A31109Hi 13706 59 A31110Hi 13706 59 A31111Hi 13706 59 A31112Hi 13706 59 A31113Hi 13706 101 A31114Hi 13706 101 A31115Hi 13706 101 A31116Hi 13706 101 A31117Hi 13707 102 A31118Hi 13707 102 A31182Hi 13701 166 A31183Hi 13748 167 A31303Hi 13473 286 A31304Hi 13482 287 A31305Hi 13494 288 A31306Hi 13750 289 A31368Hi 13706 59 A31369Hi 13706 59 A31370Hi 13706 59 A31371Hi 13706 59 A31372Hi 13706 59 A31373Hi 13706 59 13780-14279 A31056Hi 14070 60 A31057Hi 14184 61 A31184Hi 13843 168 A31185Hi 13926 169 A31186Hi 14071 170 A31187Hi 14081 171 A31188Hi 14266 172 A31307Hi 13900 290 14280-14779 A31058Hi 14401 62 A31059Hi 14403 63 A31122Hi 14401 106 A31123Hi 14402 107 15280-15779 A31308Hi 15571 291 A31309Hi 15588 292 16280-16779 A31060Hi 16512 64 16780-17279 A31028H 16795 32 A31029H 16846 33 A31030H 16875 34 A31105H 16873 97 A31151H 16874 135 A31189Hi 16921 173 A31268H 16843 252 A31269H 16877 253 A31310Hi 16964 293 17780-18279 A31311Hi 18120 294 18280-18779 A31061Hi 18501 65 A31124Hi 18502 108 A31190Hi 18502 174 A31191Hi 18654 175 A31192Hi 18775 176 A31312Hi 18559 295 18780-19279 A31193Hi 18893 177 19280-19779 A31062Hi 19318 66 20280-20779 A31063Hi 20629 67 A31194Hi 20418 178 A31195Hi 20719 179 20780-21279 A31031H 21266 35 A31032H 21279 36 A31152H 21253 136 A31153H 21256 137 A31196Hi 20984 180 A31271H 21264 254 A31272H 21276 255 A31273H 21266 256 A31274H 21278 257 21280-21779 A31033H 21343 37 A31154H 21352 138 A31197Hi 21442 181 A31275H 21390 258 22280-22779 A31064Hi 22478 68 A31065Hi 22594 69 A31198Hi 22476 182 A31199Hi 22478 183 A31200Hi 22544 184 22780-23279 A31034H 23225 38 A31106H 23222 98 23280-23779 A31201Hi 23557 185 A31313Hi 23568 296 23780-24279 A31314Hi 23845 297 A31315Hi 23964 298 A31316Hi 24162 299 A31317Hi 24175 300 24280-24779 A31202Hi 24672 186 A31318Hi 24450 301 24780-25279 A31066Hi 24910 70 A31067Hi 24945 71 A31203Hi 24910 187 A31319Hi 25010 302 A31320Hi 25035 303 25280-25779 A31068Hi 25310 72 A31204Hi 25308 188 25780-26279 A31321Hi 25951 304 A31322Hi 26028 305 A31323Hi 26214 306 26280-26779 A31324Hi 26674 307 26780-27279 A31325Hi 27090 308 A31326Hi 27163 309 A31327Hi 27237 310 27280-27779 A31328Hi 27640 311 27780-28279 A31205Hi 28147 189 A31329Hi 27890 312 A31330Hi 28024 313 28280-28779 A31206Hi 28750 190 A31331Hi 28745 314 28780-29279 A31207Hi 28958 191 A31332Hi 29041 315 A31333Hi 29223 316 29280-29779 A31069Hi 29495 73 A31070Hi 29545 74 A31208Hi 29430 192 A31209Hi 29648 193 A31334Hi 29441 317 A31335Hi 29617 318 29780-30279 A31336Hi 29800 319 30280-30779 A31210Hi 30615 194 A31337Hi 30601 320 A31338Hi 30732 321 30780-31279 A31035H 31124 39 A31036H 31180 40 A31155H 31175 139 A31156H 31187 140 A31157H 31259 141 A31276H 31170 259 A31277H 31174 260 A31278H 31178 261 A31279H 31204 262 A31280H 31170 263 A31283H 31187 266 A31284H 31205 267 31280-31779 A31211Hi 31637 195 A31212Hi 31657 196 A31339Hi 31371 322 31780-32279 A31037H 31803 41 A31038H 31804 42 A31071Hi 32254 75 A31072Hi 32262 76 A31125Hi 32263 109 A31158H 31796 142 A31159H 31802 143 A31160H 31862 144 A31213Hi 32216 197 A31214Hi 32244 198 A31215Hi 32262 199 A31281H 31793 264 A31282H 31797 265 A31285H 31850 268 A31286H 31796 269 A31340Hi 32126 323 32780-33279 A31216Hi 33199 200 A31341Hi 32843 324 A31342Hi 33123 325 A31343Hi 33252 326 33280-33779 A31073Hi 33476 77 A31074Hi 33477 78 A31126Hi 33475 110 A31127Hi 33482 111 A31217Hi 33483 201 A31218Hi 33506 202 A31219Hi 33588 203 A31344Hi 33530 327 A31345Hi 33566 328 A31346Hi 33654 329 33780-34279 A31347Hi 33941 330 A31348Hi 34241 331 A31349Hi 34244 332 34280-34779 A31075Hi 34555 79 A31220Hi 34727 204 A31350Hi 34331 333 A31351Hi 34695 334 34780-35279 A31076Hi 35277 80 A31077Hi 35279 81 A31221Hi 35278 205 A31222Hi 35279 206 A31352Hi 34843 335 A31353Hi 35001 336 35280-35779 A31039H 35572 43 A31040H 35616 44 A31041H 35665 45 A31042H 35710 46 A31043H 35754 47 A31044H 35758 48 A31045H 35774 49 A31046H 35775 50 A31078Hi 35284 82 A31079Hi 35330 83 A31080Hi 35346 84 A31107H 35620 99 A31108H 35778 100 A31161H 35706 145 A31162H 35718 146 A31163H 35769 147 A31223Hi 35347 207 A31224Hi 35370 208 A31287H 35583 270 A31288H 35584 271 A31354Hi 35479 337 35780-36279 A31047H 35781 51 A31048H 35879 52 A31164H 35782 148 A31165H 35784 149 A31166H 35874 150 A31289H 35906 272
as well as the oligonucleotides hybridizing in these areas:

[0059] Table 5 shows some hybridizing active regions of SEQ ID NO. 1 and human NLRP3 antisense oligonucleotides hybridizing in these regions.

[0060] The mouse NLRP3 oligonucleotides of the present invention hybridize with hybridizing active regions of NLRP3 pre-mRNA for example of SEQ ID NO. 3 Hybridizing active areas of SEQ ID NO. 3 are for example listed in the following

TABLE-US-00007 TABLE 6 Region of First position on SEQ ID NO. 3 SEQ ID NO. 3 SEQ ID NO. 3686-4185 A31001M 3807 342 A31002M 4083 343 A31003M 4087 344 A31098M 3705 430 A31099M 3806 431 A31100M 3806 432 A31101M 3807 433 A31102M 4081 434 A31118M 4083 343 A31119M 4096 449 A31138M 3686 465 4186-4685 A31004M 4305 345 A31005M 4306 346 A31006M 4307 347 A31007M 4307 348 A31050Mi 4411 382 A31051Mi 4618 383 A31120MR 4307 450 A31121MR 4308 451 5186-5685 A31052Mi 5367 384 A31053Mi 5517 385 6186-6685 A31054Mi 6279 386 A31055Mi 6355 387 A31056Mi 6358 388 A31057Mi 6509 389 A31058Mi 6509 390 A31059Mi 6515 391 A31060Mi 6515 392 6686-7185 A31061Mi 7132 393 A31062Mi 7133 394 7686-8185 A31063Mi 8170 395 8186-8685 A31064Mi 8490 396 8686-9185 A31008M 9015 349 A31009M 9019 350 A31010M 9048 351 A31011M 9049 352 A31012M 9049 353 A31013M 9050 354 A31014M 9050 355 A31015M 9055 356 A31016M 9122 357 A31017M 9122 358 A31041M 9048 351 A31042M 9048 351 A31043M 9048 351 A31044M 9048 351 A31065Mi 8756 397 A31066Mi 8756 398 A31067Mi 8836 399 A31068Mi 8885 400 A31122M 8968 452 A31123M 9043 453 A31124M 9059 454 A31139M 9009 466 A31140M 9015 467 A31141M 9016 468 A31142M 9022 469 A31143M 9024 470 A31144M 9052 471 A31145M 9116 472 A31146M 9126 473 9186-9685 A31018M 9599 359 A31019M 9601 360 A31020M 9602 361 A31021M 9605 362 A31125MR 9267 455 A31126M 9590 456 A31127M 9599 359 A31128M 9601 457 A31147M 9596 474 A31148M 9606 475 9686-10185 A31022M 9869 363 A31023M 9870 364 A31024M 9870 365 A31025M 9871 366 A31026M 9873 367 A31027M 9878 368 A31028M 10044 369 A31103M 9710 435 A31104M 10097 436 A31105M 10143 437 A31106M 10143 438 A31129M 9872 458 A31130M 10019 459 A31131M 10020 460 A31132M 10044 369 A31133M 10088 461 A31134M 10090 462 A31149M 9866 476 A31150M 9876 477 A31151M 9877 478 A31152M 10024 479 10186-10685 A31029M 10228 370 A31030M 10233 371 A31031M 10234 372 A31032M 10234 373 A31107M 10252 439 A31108M 10442 440 A31153M 10215 480 10686-11185 A31069Mi 11021 401 11186-11685 A31070Mi 11341 402 11686-12185 A31071Mi 12045 403 12686-13185 A31154M 12790 481 13186-13685 A31072Mi 13244 404 A31073Mi 13672 405 13686-14185 A31074Mi 13906 406 A31075Mi 14147 407 14186-14685 A31076Mi 14378 408 15686-16185 A31077Mi 15826 409 A31078Mi 15832 410 A31079Mi 15893 411 A31080Mi 15899 412 A31081Mi 16071 413 16186-16685 A31082Mi 16351 414 16686-17185 A31155M 16743 482 17186-17685 A31083Mi 17288 415 17686-18185 A31084Mi 17943 416 A31085Mi 18058 417 19186-19685 A31033M 19371 374 A31135MR 19390 463 20186-20685 A31086Mi 20304 418 A31087Mi 20517 419 A31088Mi 20545 420 22686-23185 A31089Mi 22910 421 23186-23685 A31090Mi 23548 422 A31091Mi 23552 423 23686-24185 A31092Mi 23744 424 A31093Mi 23751 425 A31094Mi 23854 426 24186-24685 A31095Mi 24285 427 25686-26185 A31096Mi 25786 428 A31097Mi 25939 429 A31109M 26089 441 A31110M 26092 442 A31137MR 26017 464 26686-27185 A31034M 27157 375 A31045M 27157 375 A31046M 27157 375 A31047M 27157 375 A31111M 26906 443 A31112M 26914 444 A31113M 27154 445 A31114M 27157 375 A31156M 27167 483 27186-27685 A31035M 27405 376 A31036M 27406 377 A31037M 27409 378 A31038M 27409 379 A31039M 27411 380 A31040M 27411 381 A31048M 27405 376 A31049M 27405 376 A31115M 27211 446 A31116M 27404 447 A31117M 27413 448 A31157M 27407 484
as well as the oligonucleotides hybridizing in these areas:

[0061] Table 6 shows some hybridizing active regions of SEQ ID NO. 3 and mouse NLRP3 antisense oligonucleotides hybridizing in these regions.

[0062] The oligonucleotide of the present invention inhibits for example at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of NLRP3 such as the, e.g., human or murine, NLRP3 expression compared to an untreated control. The oligonucleotide is for example administered via gymnotic delivery i.e., without a transfection reagent. The oligonucleotides of the present invention inhibit NLRP3 expression for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of NLRP3 at a nanomolar or micromolar concentration for example in a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1, 10 or 100 M.

[0063] The oligonucleotide of the present invention is used for example in a concentration of 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6 or 10 M.

[0064] An oligonucleotide of the present invention hybridizes for example with at least one exon and/or intron of SEQ ID NO. 1 and optionally with the mRNA of SEQ ID NO. 2. The oligonucleotide hybridizes for example with 2, 3, 4 or 5 exons and/or with 2, 3, 4 or 5 introns.

[0065] In addition, the present invention refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient and/or dilutant. The pharmaceutical composition further comprises for example another active agent for example selected from the group consisting of an oligonucleotide, an antibody, a small molecule, a polypeptide, a lipid, sugar and a combination thereof.

[0066] The oligonucleotide or the pharmaceutical composition of the present invention is for example for use in a method of preventing and/or treating a disorder. The disorder is for example characterized by an NLRP3 imbalance, i.e., the NLRP3 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The NLRP3 level can be measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR, HHPLC, FPLC or QuantiGene assay known to a person skilled in the art.

[0067] A disorder treatable by an oligonucleotide of the present invention or a pharmaceutical composition comprising such oligonucleotide is for example selected from the group consisting of an inflammatory or autoimmune disorder, a neurological disorder, cardiovascular or metabolic disorder, renal disorder, liver disorder lung disorder, skin disorder, ocular disorder, disorder of the gastro-intestinal tract, joint inflammation, organ transplantation, fibrotic disorder and a combination thereof. Furthermore, the disorder is a hyperproliferative disorder such as a cancer. A cancer is for example selected from the group consisting of breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, epidermoid carcinoma and a combination thereof.

[0068] In more detail such disorder is for example selected from the group consisting of Alzheimer's disease, multiple sclerosis, autoimmune encephalitis, stroke, traumatic brain injury, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, hypertension, myocardial infarction, acute kidney injury, ischemia reperfusion injury, chronic kidney diseases, crystal-induced nephropathies, glomerulonephritis, silicosis, asthma, allergic airway inflammation, inflammatory bowel disease, colitis ulcerosa, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, transplantation of kidney, lung, liver and/or heart, fibrotic disorder of kidney, lung, liver and/or heart, hyperinflammation following influenza infection, graft-versus-host disease, interstitial cystitis, uveitis, sinusitis, periodontal disease, myelodysplastic syndrome, cryopyrin-associated periodic syndromes, gout, obesity-induced inflammation, insulin resistance, type 1 and type 2 diabetes, contact hypersensitivity, psoriasis, alopecia and a combination thereof.

[0069] An oligonucleotide or a pharmaceutical composition of the present invention is administered locally or systemically for example orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, intraventricular, transdermal, rectal, intraarticular, intraocular, intravitreal, subconjunctival, retro bulbar, intra nasal, intracameral, intratracheal, intrapleural, per inhalation, intraurethral and/or intra vesical. In addition, an oligonucleotide or a pharmaceutical composition of the present invention is for example used in an ex vivo treatment of a transplant.

[0070] According to the present invention, one or more oligonucleotides of the present invention can be administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. Alternatively, one or more oligonucleotides of the present invention are for example administered together with another active agent such as another oligonucleotide (i.e., not being part of the present invention), an antibody, a small molecule, a polypeptide, a statin, a vaccine, an adjuvant, a chemotherapeutic agent, a cytotoxic agent, an allergen, an antibiotic, a siRNA molecule, a TLR antagonist, an activated cell, a cell therapy product, a peptide, a polypeptide, a protein, a gene therapy vector, and/or a co-stimulatory molecule (e.g., a cytokine, a chemokine, a protein ligand, a trans-activating factor, a peptide or peptide comprising modified amino acid and/or a therapeutic mRNA), at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. The other active agent interacts or inhibits the same target and/or a different target than the oligonucleotide of the present invention, wherein interacting means that the active agent has an indirect effect on the target. Another active agent interacts or inhibits for example a target selected from the group consisting of NLRP3, CD39, CD73, IL-1, IL-1 receptor, IL-1R accessory protein, IL-18, IL-18 receptor, ASC, NLRC4, AIM2, Caspase-1, RIPK3, Gasdermin D, MLKL, TLR4, Caspase-8, P2X7, NFB, RORt, TGF-, IL-21, IL-17, IL-22, IL-23, IL-6, TNF-, CCR6, CCL20, STAT3, MMP-1, MMP-8, ADAMTS-5, HMG-CoA, Myd-88, HMGB-1, ROS, TAK-1, Chop, FPR1, LIMCH1, caspase inhibitor and a combination thereof.

[0071] Further, one or more of the oligonucleotides of the present invention are connected, e.g., via a chemical bond for example based on a linker. The chemical bond such as a linker is for example cleavable or non-cleavable.

[0072] The present invention further refers to a kit comprising an oligonucleotide and/or a pharmaceutical composition of the present invention and an instruction manual. The oligonucleotide and/or the pharmaceutical composition of the kit is for example for use in preventing and/or treating a disease caused by an imbalanced NLRP3 expression. Diseases based on such imbalanced NLRP3 expression are mentioned above. Alternatively, the kit is for example a diagnostic kit for the determination of the NLRP3 level in a sample. The sample is for example blood, saliva, urine, liquor or smear.

[0073] A subject of the present invention is for example a mammalian, a bird or a fish, wherein the mammalian is for example a human, horse, cow, cat, dog, or rabbit.

EXAMPLES

[0074] The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing NLRP3 either in an unstimulated state or after stimulation with for example LPS, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC.sub.50 values than endogenous systems which are closer to therapeutically relevant in vivo systems. Further, in the following experiments no transfecting agent is used, i.e., gymnotic delivery is performed. Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC.sub.50 value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). As artificial systems using a transfecting agent are hard or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent.

Example 1: Design of Mouse and Human NLRP3-Specific Antisense Oligonucleotides (ASOs)

[0075] For the design of human ASOs with specificity for exonic regions within the human NLRP3 gene the NRLP3 mRNA of SEQ ID NO. 2 (RefSeq ID NM_004895.4) was used. For ASOs with specificity for intronic regions within the human NLRP3 gene the NLRP3 pre-mRNA of SEQ ID NO. 1 (GRCh38, Chr1: 247412861-247452403) as annotated in FASTA format (visible range) downloaded from https://www.ncbi.nim.nih.gov/nuccore/NM 004895.4 was used. An H after the ASO ID indicates a human NLRP3-specific sequence that binds to an exonic region of the pre-mRNA and a Hi after the ASO ID indicates a human NLRP3-specific sequence that binds to an intronic region of the pre-mRNA. 16, 17, 18 and 19 mers were designed according to in house criteria.

[0076] For the design of mouse ASOs with specificity for exonic regions within the mouse NLRP3 gene the NRLP3 mRNA of SEQ ID NO. 4 (RefSeq ID NM_145827) was used. For ASOs with specificity for intronic regions within the mouse NLRP3 gene the NLRP3 pre-mRNA of SEQ ID NO. 3 (GRmCh38, Chr11: 59539030-59569495) as annotated in FASTA format (visible range) downloaded from https://www.ncbi.nlm.nih.gov/nuccore/NM_145827) was used. An M after the ASO ID indicates a mouse NLRP3-specific sequence that binds to an exonic region of the pre-mRNA and a Mi after the ASO ID indicates a mouse NLRP3-specific sequence that binds to an intronic region of the pre-mRNA. 15, 16 and 17 mers were designed according to in house criteria.

[0077] Neg1 (SEQ ID NO. 339; described in WO2014154843 A1), S5 (SEQ ID NO. 486), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341) were used as non-targeting control oligonucleotides in some experiments (Table 1 and Table 2).

Example 2: First ScreeningSingle Concentration Efficacy Screen for Human NLRP3 Antisense Oligonucleotides in THP-1 Cells

[0078] Knockdown efficacy of human NLRP3-specific ASOs were tested in human THP-1 cells (human monocytic cell line derived from an acute monocytic leukemia patient). The cells were treated with the respective human NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) at a concentration of 10 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 2.

[0079] Treatment of THP-1 cells with the A31058Hi (SEQ ID NO. 62), A31026H (SEQ ID NO. 30) and A31065Hi (SEQ ID NO. 69) ASOs resulted in a target inhibition of >40% (represented by a residual NLRP3 mRNA expression of <0.6 as compared to mock-treated cells) (FIG. 2).

Example 3: First ScreeningSingle Concentration Efficacy Screen for Human NLRP3 Antisense Oligonucleotides in U87MG Cells

[0080] Knockdown efficacy of human NLRP3-specific ASOs were tested in human U87MG cells (human primary glioblastoma cell line). The cells were treated with the respective human NLRP3-specific ASO or control oligonucleotide Neg1 at a concentration of 10 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 3.

[0081] Treatment of U87-MG cells with the A31059Hi (SEQ ID NO. 63), A31026H (SEQ ID NO. 30), A31001H (SEQ ID NO. 5) and A31025H (SEQ ID NO. 29) ASOs resulted in a target inhibition of >40% (represented by a residual NLRP3 mRNA expression of <0.6 as compared to mock-treated cells) (FIG. 3).

Example 4: First ScreeningSingle Concentration Efficacy Screen for Human NLRP3 Antisense Oligonucleotides in PBMC-Derived Macrophages

[0082] Knockdown efficacy of selected human NLRP3-specific ASOs from the first screening round were tested in human peripheral blood mononuclear cell (PBMC)-derived macrophages. PBMC were seeded in 96-well flat bottom plate and after two hours, non-adherent cells were washed away and the adherent cells (predominantly monocytes) were cultured in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4. Cells were treated with the respective human NLRP3-specific ASO or control oligonucleotide Neg1 at a concentration of 10 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, the supernatant and loose cells were removed and replaced by fresh medium containing 20 ng/ml hGM-CSF, 50 ng/ml hIL-4 and the respective human NLRP3-specific ASO or control oligonucleotide Neg1 at a concentration of 10 M. On day 6, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 4.

[0083] Treatment of PBMC-derived macrophages with the A31025H (SEQ ID NO. 29), A31026H (SEQ ID NO. 30), A31059Hi (SEQ ID NO. 63), A31065Hi (SEQ ID NO. 69) and A31055Hi (SEQ ID NO. 59) ASOs resulted in a target inhibition of >70% (represented by a residual NLRP3 mRNA expression of <0.3 as compared to mock-treated cells) (FIG. 4).

Example 5: Second ScreeningSingle Concentration Efficacy Screen of NLRP3-Specific ASOs in U87MG Cells

[0084] Knockdown efficacy of human NLRP3-specific ASOs was tested in human U87MG cells in a second screening round. The cells were treated with the respective human NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) at a concentration of 10 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 5.

[0085] Treatment of U87-MG cells with the A31111Hi (SEQ ID NO. 59), A31110Hi (SEQ ID NO. 59), A31025H (SEQ ID NO. 29), A31026H (SEQ ID NO. 30), A31092H (SEQ ID NO. 86), A31085H (SEQ ID NO. 30), A31091H (SEQ ID NO. 85) and A31083H (SEQ ID NO. 29) ASOs resulted in a target inhibition of >66% (represented by a residual NLRP3 mRNA expression of <0.34 as compared to mock-treated cells) (FIG. 5).

Example 6: Second ScreeningSingle Concentration Efficacy Screen of Human NLRP3-Specific ASOs in PBMC-Derived Macrophages

[0086] Knockdown efficacy of selected human NLRP3-specific ASOs from the second screening round were tested in PBMC-derived macrophages. Plastic adherent PBMC were treated with 10 M of the respective human NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 6.

[0087] Treatment of PBMC-derived macrophages with the A31111Hi (SEQ ID NO. 59), A31109Hi (SEQ ID NO. 59), A31085H (SEQ ID NO. 30), A31086H (SEQ ID NO. 30), A31084H (SEQ ID NO. 29), A31092H (SEQ ID NO. 86), A31089H (SEQ ID NO. 85) and A31026H (SEQ ID NO. 30) ASOs resulted in a target inhibition of at least >66% (represented by a residual NLRP3 mRNA expression of <0.34 as compared to mock-treated cells) (FIG. 6).

Example 7: Investigation of the Concentration-Dependent Target Knockdown by Selected Human NLRP3-Specific ASOs in Human PBMC-Derived Macrophages

[0088] The concentration-dependent knockdown of NLRP3 mRNA expression by selected human NLRP3-specific ASOs from the first and second screening round was investigated on mRNA level in human PBMC-derived macrophages and the respective IC.sub.50 values were calculated. Plastic adherent PBMC were cultured in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4 and treated for three days with the respective ASO at the following concentrations: 10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM and 14 nM. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides. After three days, the cell culture supernatant and loose cells were removed and replaced by fresh medium containing 40 ng/ml hGMCSF and 100 ng/ml hIL-4 and antisense oligonucleotides at a final concentration ranging from 14 nM to 10 M. On day six, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 7 and Table 7. The half maximal inhibitory concentration (IC.sub.50) values of the concentration response curve for selected candidates are indicated in the Table 8. All ASOs concentration-dependently inhibited the expression of NLRP3 mRNA.

TABLE-US-00008 TABLE 7 Concentration-dependent inhibition of NRLP3 mRNA expression in PBMC-derived macrophages by selected NLRP3-specific ASOs after six days treatment. Residual NLRP3 Residual NLRP3 mRNA expression mRNA expression compared to compared to mock-treated cells mock-treated cells ASO ID Concentration (set as 1) ASO ID Concentration (set as 1) A31025H 10 M 0.18 A31089H 10 M 0.17 (SEQ 3.3 M 0.22 (SEQ ID 3.3 M 0.32 IDNO. 29) 1.1 M 0.29 NO. 85) 1.1 M 0.45 370 nM 0.47 370 nM 0.50 123 nM 1.05 123 nM 0.59 41 nM 1.14 41 nM 0.67 14 nM 0.96 14 nM 0.76 A31026H 10 M 0.11 A31092H 10 M 0.29 (SEQ ID 3.3 M 0.31 (SEQ ID 3.3 M 0.43 NO. 30) 1.1 M 0.42 NO. 86) 1.1 M 0.60 370 nM 0.73 370 nM 0.59 123 nM 1.37 123 nM 0.96 41 nM 1.47 41 nM 0.93 14 nM 1.22 14 nM 0.77 A31055Hi 10 M 0.17 A31109Hi 10 M 0.45 (SEQ ID 3.3 M 0.47 (SEQ ID 3.3 M 0.52 NO. 59) 1.1 M 0.87 NO. 59 1.1 M 0.80 370 nM 1.12 370 nM 0.69 123 nM 1.08 123 nM 0.71 41 nM 1.65 41 nM 1.04 14 nM 1.17 14 nM 1.27 A31084H 10 M 0.20 A31110Hi 10 M 0.30 (SEQ ID 3.3 M 0.25 (SEQ ID 3.3 M 0.64 NO. 29) 1.1 M 0.33 NO. 59) 1.1 M 1.01 370 nM 0.54 370 nM 0.73 123 nM 0.95 123 nM 0.99 41 nM 1.06 41 nM 1.09 14 nM 1.39 14 nM 1.39 A31085H 10 M 0.26 A31111Hi 10 M 0.25 (SEQ ID 3.3 M 0.32 (SEQ ID 3.3 M 0.54 NO. 30) 1.1 M 0.57 NO. 59) 1.1 M 0.86 370 nM 0.79 370 nM 0.87 123 nM 1.04 123 nM 1.03 41 nM 1.44 41 nM 1.12 14 nM 1.53 14 nM 1.52 A31086H 10 M 0.17 (SEQ ID 3.3 M 0.22 NO. 30) 1.1 M 0.39 370 nM 0.59 123 nM 1.02 41 nM 1.14 14 nM 1.01

TABLE-US-00009 TABLE 8 Half maximal inhibitory concentration (IC.sub.50) values and R values of selected human NLRP3-specific ASOs after six days treatment. ASO ID IC.sub.50 values (nM) R squared A31025H 248.50 0.86 A31026H 281.00 0.82 A31055Hi 2190.00 0.71 A31084H 100.90 0.76 A31085H 154.40 0.85 A31086H 298.00 0.89 A31092H 949.90 0.55

Example 8: Third ScreeningSingle Concentration Efficacy Screen for Human NLRP3-Specific Antisense Oligonucleotides in THP-1-Derived Macrophages

[0089] Knockdown efficacy of human NLRP3-specific ASOs were tested in human THP-1-derived macrophages in a third screening round. THP-1 cells were differentiated into macrophages by addition of 10 nM PMA. The respective NLRP3-specific ASOs or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) were added at 5 M final concentration. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 8. Treatment of THP-1-derived macrophages with the A31025H (SEQ ID NO. 29), A31111Hi (SEQ ID NO. 59), A31109Hi (SEQ ID NO. 59), A31195Hi (SEQ ID NO. 179), A31112Hi (SEQ ID NO. 59), A31203Hi (SEQ ID NO. 187), A31194Hi (SEQ ID NO. 178), A31187Hi (SEQ ID NO. 171) and A31055Hi (SEQ ID NO. 59) ASOs resulted in a target inhibition of >66% (represented by a residual NLRP3 mRNA expression of <0.34 as compared to mock-treated cells) (FIG. 8).

Example 9: Third ScreeningSingle Concentration Efficacy Screen of Human NLRP3-Specific ASOs in Human PBMC-Derived Macrophages

[0090] Knockdown efficacy of human NLRP3-specific ASOs was tested in PBMC-derived macrophages in a third screening round. Adherent PBMC cells were cultured in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4. Cells were treated with the respective human NLRP3-specific ASO or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) at a concentration of 5 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides. After three days treatment, the supernatant and loose cells were removed and replaced by fresh medium containing 20 ng/ml hGM-CSF, 50 ng/ml hIL-4 and the respective human NLRP3-specific ASO or control oligonucleotide at a concentration of 5 M. On day six, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 9.

[0091] Treatment of PBMC-derived macrophages with the A31151H (SEQ ID NO. 135), A31026H (SEQ ID NO. 30), A31146H (SEQ ID NO. 130), A31135H (SEQ ID NO. 119), A31149H (SEQ ID NO. 133), A31213Hi (SEQ ID NO. 197), A31203Hi (SEQ ID NO. 187), A31150H (SEQ ID NO. 134) and A31025H (SEQ ID NO. 29) ASOs resulted in a target inhibition of >67% (represented by a residual NLRP3 mRNA expression of <0.33 as compared to mock-treated cells (untreated control)) (FIG. 9).

Example 10: Fourth ScreeningSingle Concentration Efficacy Screen for Human NLRP3-Specific Antisense Oligonucleotides in THP-1-Derived Macrophages

[0092] Knockdown efficacy of human NLRP3-specific ASOs was tested in human THP-1-derived macrophages in a fourth screening round. THP-1 cells were differentiated into macrophages by addition of 10 nM PMA. The respective NLRP3-specific ASOs or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) were added at 5 M final concentration. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 10.

[0093] Treatment of THP-1-derived macrophages with the A31352Hi (SEQ ID NO. 335), A31055Hi (SEQ ID NO. 59), A31314Hi (SEQ ID NO. 297), A31317Hi (SEQ ID NO. 300), A31332Hi (SEQ ID NO. 315), A31324Hi (SEQ ID NO. 307), A31304Hi (SEQ ID NO. 287) and A31302Hi (SEQ ID NO. 285) ASOs resulted in a target inhibition of >67% (represented by a residual NLRP3 mRNA expression of <0.33 as compared to mock-treated cells) (FIG. 10).

Example 11: Fourth ScreeningSingle Concentration Efficacy Screen of Human NLRP3-Specific ASOs in PBMC-Derived Macrophages

[0094] Knockdown efficacy of human NLRP3-specific ASOs was tested in PBMC-derived macrophages in a fourth screening round. Adherent PBMC cells were cultured in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4. Cells were treated with the respective human NLRP3-specific ASO or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) at a concentration of 5 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, the supernatant and loose cells were removed and replaced by fresh medium containing 20 ng/ml hGM-CSF, 50 ng/ml hIL-4 and the respective human NLRP3-specific ASO or control oligonucleotide at a concentration of 5 M. On day six, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 11.

[0095] Treatment of PBMC-derived macrophages with the A31302Hi (SEQ ID NO. 285), A31352Hi (SEQ ID NO. 335), A31026H (SEQ ID NO. 30), A31307Hi (SEQ ID NO. 290), A31055Hi (SEQ ID NO. 59), A31304Hi (SEQ ID NO. 287) and A31303Hi (SEQ ID NO. 286) ASOs resulted in a target inhibition of >80% (represented by a residual NLRP3 mRNA expression of <0.2 as compared to mock-treated cells) (FIG. 11).

Example 12: Investigation of the Concentration-Dependent Target Knockdown by Selected Human NLRP3-Specific ASOs in Human THP-1-Derived Macrophages

[0096] The concentration-dependent knockdown of NLRP3 mRNA expression by selected human NLRP3-specific ASOs from the first, second, third and fourth screening round was investigated on mRNA level in human THP-1-derived macrophages and the respective IC.sub.50 values were calculated. THP-1 cells differentiated to macrophages in the presence of 10 nM PMA were treated for three days with the respective ASO at the following concentrations: 10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM and 14 nM. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides. After three days, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 12 and Table 9. The half maximal inhibitory concentration (IC.sub.50) values of the dose response curve for selected candidates are indicated in the Table 10. All ASOs dose-dependently inhibited the expression of NLRP3 mRNA.

TABLE-US-00010 TABLE 9 Concentration-dependent inhibition of NRLP3 mRNA expression in THP-1-derived macrophages by selected NLRP3-specific ASOs after three days treatment. Residual NLRP3 Residual NLRP3 mRNA expression mRNA expression compared to compared to mock-treated cells mock-treated cells ASO ID Concentration (set as 1) ASO ID Concentration (set as 1) A31055Hi 14 nM 0.82 A31314Hi 14 nM 0.97 (SEQ ID 41 nM 0.86 (SEQ ID 41 nM 0.89 NO. 59) 123 nM 0.81 NO. 297) 123 nM 0.80 370 nM 0.62 370 nM 0.59 1.1 M 0.54 1.1 M 0.46 3.3 M 0.37 3.3 M 0.37 10 M 0.31 10 M 0.23 A31109Hi 14 nM 0.84 A31317Hi 14 nM 0.85 (SEQ ID 41 nM 0.93 (SEQ ID 41 nM 0.86 NO. 59) 123 nM 0.71 NO. 300) 123 nM 0.84 370 nM 0.58 370 nM 0.70 1.1 M 0.39 1.1 M 0.58 3.3 M 0.32 3.3 M 0.45 10 M 0.25 10 M 0.37 A31111Hi 14 nM 0.66 A31324Hi 14 nM 1.00 (SEQ ID 41 nM 0.91 (SEQ ID 41 nM 0.91 NO. 59) 123 nM 0.71 NO. 307) 123 nM 0.88 370 nM 0.59 370 nM 0.73 1.1 M 0.46 1.1 M 0.65 3.3 M 0.43 3.3 M 0.58 10 M 0.32 10 M 0.48 A31149H 14 nM A31332Hi 14 nM 0.93 (SEQ ID 41 nM 0.80 (SEQ ID 41 nM 0.86 NO. 133) 123 nM 0.76 NO. 315) 123 nM 0.82 370 nM 0.57 370 nM 0.70 1.1 M 0.51 1.1 M 0.58 3.3 M 0.40 3.3 M 0.44 10 M 0.30 10 M 0.27 A31203Hi 14 nM 1.05 A31334Hi 14 nM 1.09 (SEQ ID 41 nM 0.95 (SEQ ID 41 nM 1.03 NO. 187) 123 nM 0.82 NO. 317) 123 nM 0.93 370 nM 0.69 370 nM 0.85 1.1 M 0.54 1.1 M 0.67 3.3 M 0.38 3.3 M 0.60 10 M 0.29 10 M 0.48 A31243H 14 nM 0.98 A31352Hi 14 nM 0.95 (SEQ ID 41 nM 0.94 (SEQ ID 41 nM 0.90 NO. 227) 123 nM 0.74 NO. 335) 123 nM 0.61 370 nM 0.65 370 nM 0.44 1.1 M 0.51 1.1 M 0.28 3.3 M 0.37 3.3 M 0.21 10 M 0.30 10 M 0.19 A31292Hi 14 nM 1.01 A31353Hi 14 nM 1.03 (SEQ ID 41 nM 0.86 (SEQ ID 41 nM 0.94 NO. 275) 123 nM 0.71 NO. 336) 123 nM 0.82 370 nM 0.70 370 nM 0.70 1.1 M 0.63 1.1 M 0.57 3.3 M 0.51 3.3 M 0.41 10 M 0.40 10 M 0.35 A31302Hi 14 nM 0.89 A31331Hi 14 nM (SEQ ID 41 nM 0.87 (SEQ ID 41 nM 1.01 NO. 285) 123 nM 0.86 NO. 314) 123 nM 0.95 370 nM 0.73 370 nM 0.88 1.1 M 0.59 1.1 M 0.74 3.3 M 0.48 3.3 M 0.59 10 M 0.35 10 M 0.47 A31304Hi 14 nM 0.93 (SEQ ID 41 nM 0.90 NO. 287) 123 nM 0.76 370 nM 0.62 1.1 M 0.48 3.3 M 0.41 10 M 0.31

TABLE-US-00011 TABLE 10 Half maximal inhibitory concentration (IC.sub.50) values and R values of selected human NLRP3-specific ASOs after three days treatment. ASO IC50 (nM) R squared A31352Hi (SEQ ID NO. 335) 163.3 0.97 A31314Hi (SEQ ID NO. 297) 439.9 0.98 A31109Hi (SEQ ID NO. 59) 297.3 0.94 A31332Hi (SEQ ID NO. 315) 4925 0.98 A31203Hi (SEQ ID NO. 187) 589.7 0.96 A31149H (SEQ ID NO. 133) 604.6 0.90 A31243H (SEQ ID NO. 227) 411 0.94 A31304Hi (SEQ ID NO. 287) 342.7 0.97 A31055Hi (SEQ ID NO. 59) 1449 0.88 A31111Hi (SEQ ID NO. 59) 0.70 A31302Hi (SEQ ID NO. 285) 0.92 A31353Hi (SEQ ID NO. 336) 538 0.96 A31317Hi (SEQ ID NO. 300) 2972 0.88 A31292Hi (SEQ ID NO. 275) 599.7 0.87 A31331Hi (SEQ ID NO. 314) 1595 0.88 A31334Hi (SEQ ID NO. 317) 798.1 0.92 A31324Hi (SEQ ID NO. 307) 515.3 0.95

Example 13: Efficacy of Selected Human NLRP3 Antisense Oligonucleotides in Human Microglial HMC3 Cells

[0097] Knockdown efficacy of human NLRP3-specific ASOs selected from previous screening rounds were tested in human HMC3 cells (human microglial clone 3 cell line). The cells were treated with the respective human NLRP3-specific ASO or control oligonucleotides R01002 (SEQ ID NO. 340), R01011 (SEQ ID NO. 341) or Neg1 (SEQ ID NO. 339) at a concentration of 5 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 13.

[0098] Treatment of HMC3 cells with the A31352Hi (SEQ ID NO. 335), A31026H (SEQ ID NO. 30), A31055Hi (SEQ ID NO. 59) and A31025H (SEQ ID NO. 29) ASOs resulted in a target inhibition of >40% (represented by a residual NLRP3 mRNA expression of <0.6 as compared to mock-treated cells) (FIG. 13).

Example 14: Fifth ScreeningSingle Concentration Efficacy Screen for Human NLRP3-Specific Antisense Oligonucleotides in THP-1-Derived Macrophages

[0099] Knockdown efficacy of human NLRP3-specific ASOs was tested in human THP-1-derived macrophages in a fifth screening round. THP-1 cells were differentiated into macrophages by addition of 10 nM PMA. The respective NLRP3-specific ASOs or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) were added at 5 M final concentration. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides. After three days treatment, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 14.

[0100] Treatment of THP-1-derived macrophages with the A31358H (SEQ ID NO. 29), A31359H (SEQ ID NO. 29) and A31026H (SEQ ID NO. 30) ASOs resulted in a target inhibition of >60% (represented by a residual NLRP3 mRNA expression of <0.4 as compared to mock-treated cells) (FIG. 14).

Example 15: Fifth ScreeningSingle Concentration Efficacy Screen of Human NLRP3-Specific ASOs in PBMC-Derived Macrophages

[0101] Knockdown efficacy of human NLRP3-specific ASOs was tested in PBMC-derived macrophages in a fifth screening round. Adherent PBMCs were cultured in the presence of 20 ng/ml hGM-CSF and 50 ng/ml hIL-4. Cells were treated with the respective human NLRP3-specific ASO or control oligonucleotides (Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) and R01011 (SEQ ID NO. 341)) at a concentration of 5 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, the supernatant and loose cells were removed and replaced by fresh medium containing 20 ng/ml hGM-CSF, 50 ng/ml hIL-4 and the respective human NLRP3-specific ASO or control oligonucleotide at a concentration of 5 M. On day six, cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 15.

[0102] Treatment of PBMC-derived macrophages with the A31365H (SEQ ID NO. 30), A31371Hi (SEQ ID NO. 59) and A31372Hi (SEQ ID NO. 59) ASOs resulted in a target inhibition of >60% (represented by a residual NLRP3 mRNA expression of <0.4 as compared to mock-treated cells) (FIG. 15).

Example 16: NLRP3 mRNA Knockdown Prevents Cleavage of Pro-IL-1 and Secretion of Mature IL-1 in Human THP-1-Derived Macrophages

[0103] In order to further investigate the target knockdown efficacy of selected human NLRP3-specific ASOs at the mRNA level, THP-1 cells were treated with the NLRP3-specific ASOs A31109Hi (SEQ ID NO. 59), A31149H (SEQ ID NO. 133), A31314Hi (SEQ ID NO. 297) or A31352Hi (SEQ ID NO. 335) or the control oligonucleotide Neg1 (SEQ ID NO. 339), R01002 (SEQ ID NO. 340) or R01011 (SEQ ID NO. 341), respectively, at a concentration of 5 M. Mock-treated cells (no antisense oligonucleotide) were cultured without the addition of oligonucleotides (untreated control). After three days treatment, medium was replaced by fresh medium containing 5 nM Phorbol 12-myristate 13-acetate (PMA) and the respective human NLRP3-specific ASO or control oligonucleotide at a concentration of 5 M. After six days treatment, cell culture supernatant was replaced by serum free medium containing 10 g/ml LPS for 4h (signal 1) and 5 mM ATP for 30 min (signal 2) in order to induce IL-1l production and activate the inflammasome complex. Afterwards the cells were lyzed. Human HPRT1 and human NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 16A. Treatment of THP-1-derived macrophages with the selected NLRP3-specific ASOs resulted in a target inhibition of >58% (represented by a residual NLRP3 mRNA expression of <0.42 as compared to mock-treated cells) (FIG. 16A).

[0104] In order to further investigate the effect of selected human NLRP3-specific ASOs in the inflammasome activation, Caspase-1 activity was assessed by Caspase-Glo 1 Inflammasome Assay in cell lysate of THP-1-derived macrophages after six days ASO treatment. Data are shown in FIG. 16B as mean and SD. ASO treatment with the selected NLRP3-specific ASOs resulted in a caspase-1 activity inhibition of >65% (represented by a caspase activity <0.35 as compared to mock treated cells set as 1) (FIG. 16B).

[0105] As part of the inflammasome complex, NLRP3 is required for cleavage of pro-IL-1 into its mature and secreted form. As a proof-of-concept it was aimed to demonstrate that IL-1 secretion by THP-1-derived macrophages can be inhibited through treatment with NLRP3 specific antisense oligonucleotides. Pro-IL1 and mature IL-1 protein expression was analyzed in cell lysates and cell culture supernatants of THP-1-derived macrophages by western blot after six days ASO treatment (FIG. 16C). Mature IL-1 was only present in the supernatants of either mock-treated cells or cells treated with the control oligonucleotides, but not in cells treated with NLRP3-specific A31109Hi (SEQ ID NO. 59), A31149H (SEQ ID NO. 133), A31314Hi (SEQ ID NO. 297) or A31352Hi (SEQ ID NO. 335) (FIG. 16C). These results demonstrated that inhibition of NLRP3 on mRNA level with antisense oligonucleotides effectively prevented the cleavage of pro-Il-1 in THP-1-derived macrophages.

Example 17: First ScreeningSingle Concentration Efficacy Screen for Mouse NLRP3-Specific Antisense Oligonucleotides in 4T1 Cells

[0106] Knockdown efficacy of mouse NLRP3-specific ASOs was tested in mouse 4T1 cells (breast cancer cell line derived from the mammary gland tissue of a mouse). The cells were treated with the respective mouse NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) at a concentration of 10 M. As unstimulated 4T1 cells did not express NLRP3 to a sufficient extent, LPS was added to the cell culture at a final concentration of 1 g/ml. After three days treatment, cells were lyzed. Mouse HPRT1 and mouse NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 17A.

[0107] Treatment of 4T1 cells with the A31034M (SEQ ID NO. 375), A31040M (SEQ ID NO. 381) and A31035M (SEQ ID NO. 376) ASOs resulted in a target inhibition of 80% (represented by a residual NLRP3 mRNA expression of <0.2 as compared to mock-treated cells) (FIG. 17A).

[0108] Cell viability of 4T1 cells after three days ASO treatment was also determined by Cell Titer Blue assay. Absolute Fluorescence Intensity (FI) normalized to mock-treated cells (no antisense oligonucleotides set as 1) is shown in FIG. 17B. Treatment of 4T1 cells with mouse NLRP3-specific ASOs for three days has no effect on cell viability (FIG. 17B).

Example 18: First ScreeningSingle Concentration Efficacy Screen for Mouse NLRP3-Specific Antisense Oligonucleotides in Raw246.7 Cells

[0109] Knockdown efficacy of mouse NLRP3-specific ASOs was tested in mouse Raw246.7 cells (murine macrophage from blood). The cells were treated with the respective mouse NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) at a concentration of 10 M. After three days treatment, cells were lyzed. Mouse HPRT1 and mouse NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 18.

[0110] Treatment of Raw246.7 cells with the A31010M (SEQ ID NO. 351), A31007M (SEQ ID NO. 348), A31011M (SEQ ID NO. 352) and A31008 (SEQ ID NO. 349) ASOs resulted in a target inhibition of >70% (represented by a residual NLRP3 mRNA expression of <0.3 as compared to mock-treated cells) (FIG. 18).

Example 20: Second ScreeningSingle Concentration Efficacy Screen for Mouse NLRP3-Specific Antisense Oligonucleotides in Raw246.7 Cells

[0111] Knockdown efficacy of mouse NLRP3-specific ASOs was tested in mouse Raw246.7 cells in a second screening round. Raw246.7 cells were treated with the respective mouse NLRP3-specific ASO or control oligonucleotide Neg1 (SEQ ID NO. 339) at a concentration of 10 M. After three days treatment, cells were lyzed. Mouse HPRT1 and mouse NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 19A and FIG. 19B

[0112] Treatment of Raw246.7 cells with A31010M (SEQ ID NO. 351), A31042M (SEQ ID NO. 351), A31041M (SEQ ID NO. 351), A31044M (SEQ ID NO. 351), A31034M (SEQ ID NO. 375), A31035M (SEQ ID NO. 376), A31045M (SEQ ID NO. 375) and A31043M (SEQ ID NO. 351) ASOs had a knockdown efficacy of >90% (represented by a residual NLRP3 mRNA expression of <0.1 as compared to mock treated cells) in Raw246.7 cells after three days treatment (FIG. 19A). Among the antisense oligonucleotides that target the introns of the NLRP3 pre-mRNA, 18 caused a target knockdown of >80% (represented by a residual NLRP3 mRNA expression of <0.2) with two antisense oligonucleotides (A31052Mi (SEQ ID NO. 384) and A31071Mi (SEQ ID NO. 403)) inhibiting target mRNA expression by >90% (FIG. 19B)

Example 20: Second ScreeningSingle Concentration Efficacy Screen for Mouse NLRP3-Specific Antisense Oligonucleotides in 4T1 Cells

[0113] Knockdown efficacy of the most potent antisense oligonucleotides in Raw246.7 cells from the second screening round were further screened in 4T1 cells. 4T1 cells were treated with the respective antisense oligonucleotides or control oligonucleotide (Neg1, SEQ ID NO. 339) at a final concentration of 10 M. To induce NLRP3 mRNA expression, cells were simultaneously treated with 1 g/ml LPS. After three days, cells were lysed and mouse HPRT1 as well as mouse NLRP3 mRNA expression was measured using the QuantiGene RNA Singleplex assay. NLRP3 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual NLRP3-mRNA expression relative to mock-treated cells (no antisense oligonucleotides set as 1) is shown as mean and SD as shown in FIG. 20.

[0114] Treatment of 4T1 cells with the A31094Mi (SEQ ID NO. 426), A31087Mi (SEQ ID NO. 419), A31056Mi (SEQ ID NO. 388), A31084Mi (SEQ ID NO. 416) and A31080Mi (SEQ ID NO. 412) ASOs resulted in a target inhibition of >85% (represented by a residual NLRP3 mRNA expression of <0.15 as compared to mock-treated cells) (FIG. 20).

Example 21: Concentration Response Curve of Selected Mouse NLRP3-Specific Antisense Oligonucleotides in Raw246.7 Cells

[0115] Selected antisense oligonucleotides from the first and second screening rounds which showed no signs of cellular toxicity in vitro were selected for IC.sub.50 determination. Raw246.7 cells were treated with the respective mouse NLRP3-specific ASO at the following concentrations: 10000 nM, 5000 nM, 2500 nM, 1250 nM, 325 nM, 313 nM and 156 nM. After three days, cells were lysed and mouse HPRT1 as well as mouse NLRP3 mRNA expression was measured using the QuantiGene RNA Singleplex assay (FIG. 21). NLRP3 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual NLRP3 mRNA expression relative to mock-treated cells (no antisense oligonucleotide set as 1) is shown as mean and SD in FIG. 21 and Table 11. The half maximal inhibitory concentration (IC.sub.50) values of the concentration response curve for selected candidates are indicated in the Table 12. All ASOs concentration-dependently inhibited the expression of NLRP3 mRNA with two candidates having an IC.sub.50 value in nanomolar range.

TABLE-US-00012 TABLE 11 Concentration-dependent inhibition of NRLP3 mRNA expression in Raw246.7 cells by selected NLRP3-specific ASOs after 3 days treatment. Residual NLRP3 Residual NLRP3 mRNA expression mRNA expression compared to compared to mock-treated cells mock-treated cells ASO ID Concentration (set as 1) ASO ID Concentration (set as 1) A31010M 10 M 0.20 A31084Mi 10 M 0.21 (SEQ ID 5 M 0.32 (SEQ ID 5 M 0.22 NO. 351) 2.5 M 0.36 NO. 416) 2.5 M 0.44 1.25 M 0.51 1.25 M 0.65 625 nM 0.68 625 nM 0.92 313 nM 0.67 313 nM 1.11 156 nM 0.73 157 nM 1.12 A31034M 10 M 0.26 A31087Mi 10 M 0.15 (SEQ ID 5 M 0.40 (SEQ ID 5 M 0.20 NO. 375) 2.5 M 0.36 NO. 419) 2.5 M 0.25 1.25 M 0.56 1.25 M 0.67 625 nM 1.12 625 nM 0.94 313 nM 1.07 313 nM 0.97 157 nM 0.82 157 nM 1.33 A31041M 10 M 0.24 A31094Mi 10 M 0.22 (SEQ ID 5 M 0.39 (SEQ ID 5 M 0.33 NO. 351) 2.5 M 0.38 NO. 426) 2.5 M 0.46 1.25 M 0.99 1.25 M 0.78 625 nM 1.27 625 nM 0.94 313 nM 1.17 313 nM 0.95 157 nM 1.05 157 nM 0.95 A31045M 10 M 0.36 A31117M 10 M 0.28 (SEQ ID 5 M 0.36 (SEQ ID 5 M 0.28 NO. 375) 2.5 M 0.49 NO. 448) 2.5 M 0.40 1.25 M 0.54 1.25 M 0.65 625 nM 0.85 625 nM 0.85 313 nM 0.98 313 nM 0.78 157 nM 1.26 157 nM 0.87 A31052Mi 10 M 0.08 (SEQ ID 5 M 0.27 NO. 384) 2.5 M 0.64 1.25 M 0.73 625 nM 0.93 313 nM 0.93 157 nM 1.03

TABLE-US-00013 TABLE 12 Half maximal inhibitory concentration (IC.sub.50) values and R values of selected mouse NLRP3-specific ASOs after three days treatment. Antisense oligonucleotide IC.sub.50 [nM] R squared A31010M (SEQ ID NO. 351) 560.9 0.76 A31034M (SEQ ID NO. 375) ~1206 0.75 A31041M (SEQ ID NO. 351) 1721 0.79 A31045M (SEQ ID NO. 375) 851.7 0.83 A31052Mi (SEQ ID NO. 384) 4039 0.89 A31084Mi (SEQ ID NO. 416) 1446 0.93 A31087Mi (SEQ ID NO. 419) 1327 0.85 A31094Mi (SEQ ID NO. 426) 1824 0.94 A31117M (SEQ ID NO. 448) 1498 0.90

Example 22: Murine NLRP3-Specific Antisense Oligonucleotides Prevent Cleavage of Pro-IL-1 in Mouse Bone Marrow Derived Macrophages (BMDM)

[0116] Knockdown efficacy of the selected mouse NLRP3-specific ASOs A31087Mi (SEQ ID NO. 419) was further screened in bone marrow derived macrophages (BMDM). For that, murine bone marrow cells were differentiated into BMDM for seven days in the presence of 50 ng/ml M-CSF. During the last four days of the BMDM differentiation protocol, cells were treated with the respective mouse NLRP3-specific ASOs A31087Mi or control oligonucleotide (Neg1 (SEQ ID NO. 339) or R01002 (SEQ ID NO. 340) at a concentration of 10 M. After seven days treatment, cell culture supernatant was replaced by serum free medium containing 200 ng/ml LPS for 4h (signal 1) and 5 mM ATP for 30 min (signal 2) in order to induce IL-1 production and activate the inflammasome complex. Afterwards the cells were lyzed. Mouse HPRT1 and mouse NLRP3 mRNA expression was measured using the QuantiGene Singleplex assay (ThermoFisher) and the NLRP3 expression values were normalized to HPRT1 values. Residual NLRP3 mRNA expression relative to mock-treated cells (set as 1) is shown as mean and SD in FIG. 22A. Treatment of BMDM with the selected NLRP3-specific ASOs resulted in a target inhibition of 94% (represented by a residual NLRP3 mRNA expression of 0.06 as compared to mock-treated cells) (FIG. 22A).

[0117] As part of the inflammasome complex, NLRP3 is required for cleavage of pro-IL-1 into its mature and secreted form. As a proof-of-concept it was aimed to demonstrate that IL-1 secretion by primary immune cells can be inhibited through treatment with NLRP3 specific antisense oligonucleotides. NLRP3, pro-IL1 and mature IL-1 protein expression was analyzed by Western Blot in cell lysates and cell culture supernatants of BMDM after six days ASO treatment (FIG. 22B). NLRP3 could be detected in the cell supernatant and cell lysates of mock-treated cells and cells treated with the control oligonucleotides, but not in cells treated with NLRP3-specific ASO A31087 Mi (SEQ ID NO. 419) (FIG. 22B). As the transcription and translation of pro-IL-1 is independent of NLRP3, pro-IL-1 could be detected in the cell lysates under all treatment conditions (cells treated with NLRP3-specific ASOs, control oligonucleotides or mock-treated cells (no ASO) (FIG. 22B). In contrast, mature IL-1 was only present in the supernatants of either mock-treated cells (no ASO, untreated control) or cells that were treated with the control oligonucleotides, but not in cells treated with NLRP3-specific ASOs A31087Mi (SEQ ID NO. 419) (FIG. 22B). These results demonstrate that inhibition of NLRP3 on mRNA level with antisense oligonucleotides effectively prevents the cleavage of pro-Il-1 in BMM.

Example 23: Efficacy of Selected Mouse NLRP3 Antisense Oligonucleotides in Raw246.7 Cells

[0118] Knockdown efficacy of selected mouse NLRP3-specific ASOs was further analyzed in Raw246.7 cells. Raw246.7 cells were treated with the respective antisense oligonucleotides or control oligo (Neg1) at a final concentration of 5 M. After three days, cells were lysed and mouse HPRT1 as well as mouse NLRP3 mRNA expression was measured using the QuantiGene RNA Singleplex assay. NLRP3 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Residual NLRP3-mRNA expression relative to mock-treated cells (no antisense oligonucleotides set as 1, untreated control) is shown as mean and SD as shown in FIG. 23.

[0119] Treatment of Raw246.7 cells with the A31087Mi (SEQ ID NO. 419), A31084Mi (SEQ ID NO. 416), A31061Mi (SEQ ID NO. 393), A31035M (SEQ ID NO. 376), A31010M (SEQ ID NO. 351) and A31041M (SEQ ID NO. 351) ASOs resulted in a target inhibition of 85% (represented by a residual NLRP3 mRNA expression of 0.15 as compared to mock-treated cells) (FIG. 23).

Example 24: Efficacy of Selected Mouse NLRP3 Antisense Oligonucleotides in Mouse Microglial IMG Cells

[0120] Knockdown efficacy of selected mouse NLRP3-specific ASOs was further assessed in IMG cells (microglial cell line isolated from the brains of adult mice). IMG cells were treated with the respective antisense oligonucleotides or control oligo (Neg1) at a final concentration of 5 M. After three days, cells were lysed and mouse HPRT1 as well as mouse NLRP3 mRNA expression was measured using the QuantiGene RNA Singleplex assay. NLRP3 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual NLRP3-mRNA expression relative to mock-treated cells (no ASO set as 1, untreated control) is shown as mean and SD as shown in FIG. 2. Treatment of IMG cells with the A31087Mi (SEQ ID NO. 419), A31084Mi (SEQ ID NO. 416) and A31034M (SEQ ID NO. 375) ASOs resulted in a target inhibition of 66% (represented by a residual NLRP3 mRNA expression of <0.34 as compared to mock-treated cells) (FIG. 24).