Modified antisense oligonucleotide for inhibition of FoxP3 expression
20220372482 · 2022-11-24
Inventors
- Frank Jaschinski (Puchheim, DE)
- Richard KLAR (München, DE)
- Sven Michel (Bernried, DE)
- Julia FESTAG (Eggenfelden, DE)
Cpc classification
C12N2310/3231
CHEMISTRY; METALLURGY
C12N2320/35
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention refers to an oligonucleotide comprising 12 to 25 nucleotides, wherein at least one of the nucleotides comprises a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, a 2′Fluoro modified nucleotide, a 2 O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA and a combination thereof. The oligonucleotide hybridizes with a nucleic acid sequence of Foxp3 of SEQ ID NO.1 and/or of SEQ ID NO.2 resulting in a reduction of the expression of FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof. The invention is further directed to a pharmaceutical composition comprising an oligonucleotide of the present invention and to the oligonucleotide and pharmaceutical composition, respectively for use in a method of preventing and/or treating a disorder, where FoxP3 imbalance is involved.
Claims
1. Oligonucleotide comprising 12 to 25 nucleotides, wherein at least one of the nucleotides comprises a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, a 2′Fluoro modified nucleotide, a 2 O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA and a combination thereof, and hybridizing with a nucleic acid sequence of Foxp3 of SEQ ID NO.1 and/or of SEQ ID NO.2 resulting in a reduction of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof of 40% to 99% within 6 to 240 h or within 12 to 120 h from first administration of the oligonucleotide compared to an untreated control.
2. Oligonucleotide according to claim 1 resulting in a reduction of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof of 40% to 99% within 24 to 72 h from first administration of the oligonucleotide.
3. Oligonucleotide according to claim 1 or 2 hybridizing with Foxp3 of SEQ ID NO.1 and/or SEQ ID NO.2, wherein the oligonucleotide hybridizes within a region of position 1510 to 2109 or of position 1810-2109 of SEQ ID NO.2.
4. Oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide comprises SEQ ID NO.58, SEQ ID NO.81, SEQ ID NO.103, SEQ ID NO.56, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.59, SEQ ID NO.83, SEQ ID NO.84, SEQ ID NO.85, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, SEQ ID NO.89, SEQ ID NO.90, SEQ ID NO.102, SEQ ID NO.104, SEQ ID NO.105, SEQ ID NO.106, SEQ ID NO.107, SEQ ID NO.108, SEQ ID NO.167, SEQ ID NO.194, SEQ ID NO.195, SEQ ID NO.196, SEQ ID NO.197, SEQ ID NO.198, SEQ ID NO.199, SEQ ID NO.200, SEQ ID NO.201, SEQ ID NO.202, SEQ ID NO.203, SEQ ID NO.204, SEQ ID NO.205, SEQ ID NO.82, SEQ ID NO.57, SEQ ID NO.23, SEQ ID NO.101, SEQ ID NO.166, SEQ ID NO.187, SEQ ID NO.188, SEQ ID NO.189, SEQ ID NO.190, SEQ ID NO.191, SEQ ID NO.192, SEQ ID NO.193 or a combination thereof.
5. Oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide is selected from the group consisting of TABLE-US-00021 (A25073H; SEQ ID NO. 58) +C*+G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G, (A25126H; SEQ ID NO. 81) +G*+A*+A*G*T*A*A*T*C*T*G*T*G*C*G*+A*+G*+C, (A25150H; SEQ ID NO. 103) +A*+T*+G*C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T, (A25097H, SEQ ID NO. 56) +G*A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G, (A25098H, SEQ ID NO. 56) +G*+A*G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G, (A25099H; SEQ ID NO. 58) +C*G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*+T*+G, (A25100H, SEQ ID NO. 58) +C*G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G, (A25102H; SEQ ID NO. 24) +T*G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G, (A25103H; SEQ ID NO. 24) +T*G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G, (A25106H, SEQ ID NO. 25) +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A, (A25107H, SEQ ID NO. 26) +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T, (A25108H, SEQ ID NO. 26) +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*G*+T, (A25109H, SEQ ID NO. 27) +A*G*+C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A, (A25110H; SEQ ID NO. 26) +G*C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T, (A25111H; SEQ ID NO. 59) +A*T*+G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25128H; SEQ ID NO. 83) +G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G, (A25129H; SEQ ID NO. 84) +T*G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T, (A25130H, SEQ ID NO. 85) +G*+G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*G*+A, (A25131H; SEQ ID NO. 86) +A*+A*+T*T*C*T*A*A*C*A*G*G*C*C*G*+T*+G*+T, (A25132H; SEQ ID NO. 87) -G*+T*+G*A*A*T*T*C*T*A*A*C*A*G*G*+C*+C*+G, (A25133H; SEQ ID NO. 88) +T*+A*+T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25134H; SEQ ID NO. 89) +C*+A*+T*A*T*G*C*G*T*G*A*G*A*T*A*+C*+A*+C, (A25135H; SEQ ID NO. 90) +C*+T*+C*G*G*C*T*G*C*A*G*T*T*T*A*+T*+T*+G, (A25149H, SEQ ID NO. 102) +G*+A*+A*T*T*C*T*A*A*C*A*G*G*C*C*G*+T*+G*+T, (A25151H, SEQ ID NO. 104) +T*+A*+T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G*+G, (A25152H; SEQ ID NO. 105) +A*+T*+A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25153H; SEQ ID NO. 105) +A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*A*+C*+A*+G, (A25154H; SEQ ID NO. 105) +A*T*+A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25155H; SEQ ID NO. 105) +A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25156H, SEQ ID NO. 106) +G*+T*G*C*A*T*A*T*G*C*G*T*G*A*G*A*+T*A*+C, (A25157H; SEQ ID NO. 106) +G*+T*G*C*A*T*A*T*G*C*G*T*G*A*G*A*T*+A*+C, (A25158H, SEQ ID NO. 107) +G*+C*+T*C*G*G*C*T*G*C*A*G*T*T*T*A*+T*+T*+G, (A25159H; SEQ ID NO. 108) +G*+G*+A*G*C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A, (A25219H; SEQ ID NO. 167) +T*+C*+G*G*C*T*G*C*A*G*T*T*T*A*T*+T*+G*+G, (A25249H; SEQ ID NO. 194) +T*+G*T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G, (A25250H; SEQ ID NO. 195) +G*+T*G*T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T, (A25251H; SEQ ID NO. 196) +G*+G*C*C*G*T*G*T*G*T*G*T*G*A*G*C*G*+A*+G, (A25252H; SEQ ID NO. 197) +T*C*+T*A*A*C*A*G*G*C*C*G*T*G*T*G*+T*+G*+T, (A25253H; SEQ ID NO. 198) +A*+A*+T*T*C*T*A*A*C*A*G*G*C*C*G*T*+G*+T*+G, (A25254H, SEQ ID NO. 199) +T*+G*+A*A*T*T*C*T*A*A*C*A*G*G*C*C*+G*+T*+G, (A25255H; SEQ ID NO. 200) +G*+T*+G*A*A*T*T*C*T*A*A*C*A*G*G*C*+C*+G*+T, (A25256H; SEQ ID NO. 201) +G*G*+T*G*A*A*T*T*C*T*A*A*C*A*G*G*C*++C*+G, (A25257H; SEQ ID NO. 201) +G*G*+T*G*A*A*T*T*C*T*A*A*C*A*G*G*+C*C*+G, (A25258H; SEQ ID NO. 202) +C*+A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*+A*+C*+A, (A25259H; SEQ ID NO. 203) +G*+T*+T*C*C*T*C*T*G*C*A*G*T*C*T*A*+A*+G*+C, (A25260H; SEQ ID NO. 204) +G*+T*+A*G*T*T*C*C*T*C*T*G*C*A*G*T*+C*+T*+A, (A25261H, SEQ ID NO. 205) +C*+T*+C*G*G*C*T*G*C*A*G*T*T*T*A*T*+T*+G*+G, (A25028H; SEQ ID NO. 24) +T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G, (A25029H, SEQ ID NO. 25) +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A, (A25030H; SEQ ID NO. 26) +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T, (A25069H; SEQ ID NO. 56) +G*+A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G, (A25096H; SEQ ID NO. 56) +G*+A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G, (A25101H; SEQ ID NO. 58) +C*+G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G, (A25127H; SEQ ID NO. 82) +G*+T*+T*G*T*T*T*G*A*G*T*G*T*A*C*+T*+G*+A, (A25104H; SEQ ID NO. 24) #T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G, (A25070H, SEQ ID NO. 24) #T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G, (A25071H; SEQ ID NO. 57) +G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T, (A25072; SEQ ID NO. 25) +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A, (A25074H; SEQ ID NO. 26) +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*G*+G*+T, (A25075H, SEQ ID NO. 26) +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*G*+T, (A25076H, SEQ ID NO. 26) +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T, (A25077H; SEQ ID NO. 59) +A*+T*+G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G, (A25078H; SEQ ID NO. 27) +A*G*+C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A, (A25105H; SEQ ID NO. 23) +C*G*+C*T*G*C*T*T*C*T*G*T*G*T*+A*+G*+G, (A25147H; SEQ ID NO. 101) +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*G*+G*C*+C, (A25148H; SEQ ID NO. 101) +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*G*G*+C*+C, (A25218H, SEQ ID NO. 166) +T*+G*+A*A*G*T*A*A*T*C*T*G*T*G*C*+G*+A*+G, (A25242H; SEQ ID NO. 187) +C*+T*+G*A*A*G*T*A*A*T*C*T*G*T*G*C*+G*+A*+G, (A25243H; SEQ ID NO. 188) +C*+C*+T*G*A*A*G*T*A*A*T*C*T*G*T*G*+C*+G*+A, (A25244H; SEQ ID NO. 189) +G*+T*+T*G*T*T*T*G*A*G*T*G*T*A*C*T*+G*+A*+G, (A25245H, SEQ ID NO. 190) +G*+G*+T*T*G*T*T*T*G*A*G*T*G*T*A*C*+T*+G*+A, (A25246H; SEQ ID NO. 191) +A*+C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*+G*G*+C, (A25247H; SEQ ID NO. 192) +G*+A*+C*G*C*T*G*C*T*T*C*T*G*T*G*T*A*+G*+G, (A25248H; SEQ ID NO. 193) +G*+G*+T*A*C*T*G*A*C*G*C*T*G*C*T*T*C*+T*+G, (A25027H, SEQ ID NO. 23) +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*+A*+G*+G , (A25068H; SEQ ID NO. 23) +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*+A*G*+G and a combination thereof, wherein + indicates an LNA modified nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.
6. Oligonucleotide according to any one of claims 1 to 5, wherein the oligonucleotide inhibits the expression of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof at a nanomolar or micromolar concentration.
7. Pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof.
8. Pharmaceutical composition of claim 7, further comprising an antitumor active agent such as a chemotherapeutic (e.g., platinum, gemcitabine), an immune stimulating agent, disease specific agent or an agent that reverses tumor- or infection-mediated immunosuppression, another oligonucleotide, an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin, a small molecule or a combination thereof.
9. Pharmaceutical composition of claim 8, wherein the antitumor active agent, the disease specific agent, the other oligonucleotide, the antibody, the carbohydrate-modified antibody, the peptide-based therapeutic, the protein-based therapeutic, the therapeutic vaccine, the HERA fusion protein, the ligand trap, the Fab fragment, the nanobody, the BiTe, the DARPin and/or the small molecule inhibits expression or activity of an immune suppressive factor selected from the group consisting of IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, MTDH, Xbp1, Chop and a combination thereof, or stimulates expression or activity of an immune stimulatory factor selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof.
10. Pharmaceutical composition of claim 8 or 9, wherein the antitumor active agent, the disease specific agent, the other oligonucleotide, the antibody, the carbohydrate-modified antibody, the peptide-based therapeutic, the protein-based therapeutic, the therapeutic vaccine, the HERA fusion protein, the ligand trap, the Fab fragment, the nanobody, the BiTe, the DARPin and/or the small molecule inhibits expression or activity of a factor involved in cancer progression and/or metastasis selected from the group consisting of SND1, MTDH, HER-2, BRAF, KRAS, VEGF, EGFR1, EGFR2, BCR/ABL, ABL, MET, ALK, JAK2, BTK, miR-223, CCL18, CCL20, Lcn2, CCL5/CCR9, DDR2, PHD2, IL6, SDF-1/CXCL12 and a combination thereof.
11. Oligonucleotide according to any one of claims 1 to 6 or pharmaceutical composition according to any one of claims 7 to 10 for use in a method of preventing and/or treating a disorder, where an imbalance of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof is involved.
12. Oligonucleotide or pharmaceutical composition for use according to claim 11, wherein the disorder is a malignant and/or benign tumor, a chronic infectious disease, a chronic inflammatory disease caused by infection or a combination thereof.
13. Oligonucleotide or pharmaceutical composition for use according to any one of claims 10 to 12, wherein the malignant tumor is 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.
14. Oligonucleotide or pharmaceutical composition for use according to any one of claims 10 to 12, wherein the chronic infectious disease is for example selected from the group consisting of hepatitis B and/or C virus, human immune deficiency virus, cytomegalovirus, Herpes Simplex virus, Measles virus, respiratory syncytial virus, Helicobacter pylori infection or a combination thereof, or wherein the chronic inflammatory disease caused by infection is selected from the group consisting of chronic inflammatory diseases of the liver such as liver fibrosis, liver cirrhosis or a combination thereof.
15. Oligonucleotide or the pharmaceutical composition for use according to any one of claims 10 to 14, wherein the oligonucleotide and/or the composition is suitable to be administered locally or systemically.
Description
DESCRIPTION OF FIGURES
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
DETAILED DESCRIPTION
[0033] The present invention provides human and mouse-specific oligonucleotides such as antisense oligonucleotides which hybridize with mRNA and pre-mRNA sequences of FoxP3 and inhibit the expression, functionality and downstream effects, respectively, of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof. Thus, the oligonucleotides such as antisense oligonucleotides of the present invention represent promising and highly efficient tools for use in a method of preventing and/or treating disorders, where the FoxP3 expression, functionality, and downstream effects, respectively, deviates from the expression, functionality and downstream effects in a healthy subject. The FoxP3 expression for example is involved in the induction and/or maintenance of the disease and/or mediates resistance to another therapy. The oligonucleotide such as the antisense oligonucleotide of the present invention hybridizes for example with a nucleic acid sequence of FoxP3 of SEQ ID NO.1 (human mRNA), of SEQ ID NO. 2 (human pre-mRNA), of SEQ ID NO.324 (mouse mRNA) and/or of SEQ ID NO.325 (mouse pre-mRNA), wherein the antisense oligonucleotide inhibits at least 40% of the FoxP3 expression within 6 to 240 h, 12 to 216 h, 18 to 120 h or 24 to 72 h, or 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h, 132 h, 144 h, 156 h, 168 h, 180 h, 192 h, 204 h, 216 h, 228 h or 240 h from administration of the antisense oligonucleotide.
[0034] An oligonucleotide of the present invention is an aptamer, a siRNA, preferably an antisense oligonucleotide.
[0035] The present invention provides for example oligonucleotides for reducing the levels of the transcription factor FoxP3. In particular, the present invention relates to compounds, particularly oligonucleotides, which in preferred embodiments, hybridize with mRNA and/or pre-mRNA encoding FoxP3 thereby subsequently recruiting RNaseH. Such compounds reduce FoxP3 mRNA and/or FoxP3 pre-mRNA levels and decrease the amount of functional FoxP3 transcription factor, such that the effect and/or expression of further downstream effectors are impaired.
[0036] Inhibiting according to the present invention includes reducing an effect such as expression in different percentages and amounts, respectively.
[0037] The concept of the present invention is the provision of an oligonucleotide such as an antisense oligonucleotide mediating the limitation of available FoxP3 mRNA for protein expression. In order to limit protein expression, the oligonucleotide requires the presence of a complementary mRNA and/or pre-mRNA representing a hybridization target which allows the formation of heteroduplexes. The oligonucleotides of the present invention hybridize with RNAs of SEQ ID NO.1 and/or SEQ ID NO.2. The formation of a heteroduplex between the oligonucleotide and the target RNA leads to RNaseH-mediated degradation or inactivation of the target RNA and thus, reduces the amount of available FoxP3 mRNA for protein expression.
[0038] In the following, the elements of the present invention will be described in more detail. These elements 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 elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
[0039] 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.
[0040] The oligonucleotide such as an antisense oligonucleotide of the present invention consists of or comprises for example 12 to 25 nucleotides, 12 to 15 nucleotides, 15 to 20 nucleotides, 12 to 16 nucleotides, or 15 to 19 nucleotides. The oligonucleotide such as an antisense oligonucleotides for example consist of or comprise 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The oligonucleotide such as an antisense oligonucleotide of the present invention comprises 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), ENA, a 2′Fluoro modified nucleotide, a 2′O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA or a combination thereof. The oligonucleotide such as an antisense oligonucleotide of the present invention comprises nucleotides that have for example the same or different modifications. The oligonucleotide such as an antisense oligonucleotide of the present invention comprises for example a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate.
[0041] The oligonucleotide such as an antisense oligonucleotide of the present invention comprises the one or more modified nucleotides at the 3′- and/or 5′-end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow for example 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. The following Table 1 presents embodiments of oligonucleotides such as antisense oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides such as antisense oligonucleotides consisting of or comprising the sequences of Table 1 may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. Antisense oligonucleotides of Table 1 hybridize with the mRNA of human FoxP3 (SEQ ID NO.1; NM_014009.3) or with intronic regions of the pre-mRNA of human FoxP3 (SEQ ID NO.2; GRCh38.p13 (GCF_000001405.39, Chr X (NC_000023.11): 49,249,986K—49,226,382—pre-mRNA positions), indicated by “I” in the following Table 1:
TABLE-US-00001 TABLE 1 List of human FoxP3-specific antisense oligonucleotides and a control oligonucleotide. An “H” after the antisense oligonucleotide ID indicates a human FoxP3- specific sequence that binds to the FoxP3 mRNA and/or an exonic region of the pre- mRNA, a “HM” after the antisense oligonucleotide ID indicates a human/mouse cross- reactive FoxP3 sequence that binds to an exonic region of the pre-mRNA and a “HI” after the antisense oligonucleotide ID indicates a human FoxP3-specific sequence that binds to an intronic region of the pre-mRNA. *refers to exon spanning oligonucleotides such as antisense oligonucleotides, position depicted in Table 1 indicates position on mRNA SEQ ID NO. 1 for exon spanning oligonucleotides. position on pre-mRNA (GRCh38.pl3 Seq Antisense Sequence Antisense (GCF_000001405.39, ID Name 5′-3′ Sequence 5′-3′ with PTO (*) and LNA (+) Chr X (NC_000023.11) 3 A25004H TTCGAAGACCTTCTCAC +T*+T*+C*G*A*A*G*A*C*C*T*T*C*T*+C*+A*+C 7838 4 A25005H GAAGATGGTCCGCCTGG +G*+A*+A*G*A*T*G*G*T*C*C*G*C*C*+T*+G*+G 6847 5 A25006H CAGAAGATGGTCCGCCT +C*+A*+G*A*A*G*A*T*G*G*T*C*C*G*+C*+C*+T 6845 6 A25008H TCCAGAAGATGGTCCGC +T*+C*+C*A*G*A*A*G*A*T*G*G*T*C*+C*+G*+C 6843 7 A25009H ATCCAGAAGATGGTCCG +A*+T*+C*C*A*G*A*A*G*A*T*G*G*T*+C*+C*+G 6842 8 A25011H CTTGTCGGATGATGCC +C*+T*+T*G*T*C*G*G*A*T*G*A*T*+G*+C*+C 5119 9 A25012H CTACGATGCAGCAGGAG +C*+T*+A*C*G*A*T*G*C*A*G*C*A*G*+G*+A*+G 5101 10 A25013H CGTGGCGTAGGTGAAAG +C*+G*+T*G*G*C*GMT*A*G*G*T*G*A*+A*+A*+G 4205 11 A25014H ATGAGCGTGGCGTAGGT +A*+T*+G*A*G*C*G*T*G*G*C*G*T*A*+G*+G*+T 4200 12 A25015H ATGAGCGTGGCGTAGG +A*+T*+G*A*G*C*G*T*G*G*C*G*T*+A*+G*+G 4199 13 A25016H GATGAGCGTGGCGTAGG +G*+A*+T*G*A*G*C*G*T*G*G*C*G*T*+A*+G*+G 4199 14 A25017H ATGAGCGTGGCGTAG +A*+T*+G*A*G*C*G*T*G*G*C*G*+T*+A*+G 4198 15 A25018H GATGAGCGTGGCGTAG +G*+A*+T*G*A*G*C*G*T*G*G*C*G*+T*+A*+G 4198 16 A25019H GGATGAGCGTGGCGTAG +G*+G*+A*T*G*A*G*C*G*T*G*G*C*G*+T*+A*+G 4198 17 A25020H GGATGAGCGTGGCGTA +G*+G*+A*T*G*A*G*C*G*T*G*G*C*+G*+T*+A 4197 18 A25021H CGGATGAGCGTGGCGTA +C*+G*+G*A*T*G*A*G*C*G*T*G*G*C*+G*+T*+A 4197 19 A25022H CCAGCGGATGAGCGTG +C*+C*+A*G*C*G*G*A*T*G*A*G*C*+G*+T*+G 4192 20 A25023H CAGTGGTAGATCTCATT +C*+A*+G*T*G*G*T*A*G*A*T*C*T*C*A*+T*+T 2780 21 A25025H GACTCAGGTTGTGGCGG +G*+A*C*T*C*A*G*G*T*T*G*T*G*G*C*+G*+G 2526 22 A25026H GCGGAACTCCAGCTCAT +G*+C*+G*G*A*A*C*T*C*C*A*G*C*T*+C*+A*+T 2455 23 A25027H CGCTGCTTCTGTGTAGG +C*+G*C*T*G*C*T″T*C*T*G*T*G*T*+A*+G*+G 1820 24 A25028H TGAGCGAGCACGTGTTG +T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G 1778 25 A25029H GCCGTGTGTGTGAGCGA +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A 1768 26 A25030H GCGTGAGATACACAGGT +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T 1739 27 A25031H AGCTCGGCTGCAGTTTA +A*+G*C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A 1510 28 A25032HI GATCGATGGAGTGTGGT +G*+A*+T*C*G*A*T*G*G*A*G*T*G*T*+G*+G*+T 15174 29 A25033HI TCGGCGACATTACTATT +T*+C*G*G*C*G*A*C*A*T*T*A*C*T*+A*+T*+T 15058 30 A25034HI CCTCGGCGACATTACT +C*+C*+T*C*G*G*C*G*A*C*A*T*T*+A*+C*+T 15055 31 A25035HI GTCCAACAATCGGCACT +G*+T*C*C*A*A*C*A*A*T*C*G*G*C*+A*+C*+T 14551 32 A25036HI CGTGGATCGTCCAACCT +C*G*+T*G*G*A*T*C*G*T*C*C*A*A*+C*+C*+T 12736 33 A25037HI TCGTGGATCGTCCAAC +T*+C*+G*T*G*G*A*T*C*G*T*C*C*+A*+A*+C 12734 34 A25038HMI CACAGGTTTCGTTCCGA +C*+A*+C*A*G*G*T*T*T*C*G*TT*C*+C*+G*+A 11933 35 A25039HI GCTTCATCGACACCACG +G*+C*+T*T*C*A*T*C*G*A*C*A*C*C*+A*+C*+G 11886 36 A25040HI TTTCCGCCATTGACGTC +T*+T**C*C*G*C*C*A*T*T*G*A*C*G*+T*+C 11849 37 A25041HI TTTCGTTCCGAGAACT +T*+T*+T*C*G*T*T*C*C*G*A*G*A*+A*+C*+T 11938 38 A25044HI TCAGATGCCGAGTTCCG +T*+C*+A*G*A*T*G*C*C*G*A*G*T*T*+C*+C*+G 10832 39 A25045HI CCGAGTTCCGTAGTCC +C*+C*+G*A*G*T*T*C*C*G*T*A*G*T*+C*+C 10838 40 A25046HI GATCATGCACGGATCCA +G*+A*+T*C*A*T*G*C*A*C*G*G*A*T*C*+C*+A 10450 41 A25047HI CGGACTTTCTCCTCGGA +C*+G*+G*A*C*T*T*T*C*T*C*C*T*C*G*+G*+A 9995 42 A25048HI GATACTCGACCACCTGA +G*+A*+T*A*C*T*C*G*A*C*C*A*C*C*+T*+G*+A 9645 43 A25049HI GTATGAGATACTCGACC +G*+T*+A*T*G*A*G*A*T*A*C*T*C*G*+A*+C*+C 9639 44 A25050HI ACGGCCATTCGCAGGTG +A*+C*+G*G*C*C*A*T*T*C*G*C*A*G*+G*+T*+G 8247 45 A25051HI AAGACGGCCATTCGCAG +A*+A*+G*A*C*G*G*C*C*A*T*T*C*G*+C*+A*+G 8244 46 A25052HI AAGACGGCCATTCGCA +A*+A*+G*A*C*G*G*C*C*A*T*T*C*+G*+C*+A 8243 47 A25053HI GTGCGGATGTCGTATGT +G*+T*+G*C*G*G*A*T*G*T*C*G*T*A*+T*+G*+T 5608 48 A25054HI CAGGTGCGGATGTCGTA +C*+A*+G*G*T*G*C*G*G*A*T*G*T*C*+G*+T*+A 5605 49 A25055HI CAGGTGCGGATGTCGT +C*+A*+G*G*T*G*C*G*G*A*T*G*T*+C*+G*+T 5604 50 A25057HI TTAGGTGTGGCGCTAGG +T*+T*+A*G*G*T*G*T*G*G*C*G*C*T*+A*+G*+G 3617 51 A25060HI GTTCAGAGACAGTCGG +G*+T*+T*C*A*G*A*G*A*C*A*G*T*+C*+G*+G 3558 52 A25061HI GTTCGGTGTGGAGTGA +G*+T*T*C*G*G*T*G*T*G*G*A*G*+T*+G*+A 3431 53 A25062HI TCGAGTATCTTACGTG +T*+C*G*A*G*T*A*T*C*T*T*A*C*+G*+T*+G 3361 54 A25063HI CGAGTATCTTACGTG +C*+G*+A*G*T*A*T*C*T*T*A*C*+G*+T*+G 3361 55 A25065H GTCGCATGTTGTGGAAC +G*T*+C*G*C*A*T*G*T*T*G*T*G*G*+A*+A*+C 4225 23 A25068H CGCTGCTTCTGTGTAGG +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*+A*G*+G 1820 56 A25069H GAGCGAGCACGTGTTGG +G*+A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G 1779 24 A25070H TGAGCGAGCACGTGTTG +T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G 1778 57 A25071H GTGAGCGAGCACGTGTT +G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T 1777 25 A25072H GCCGTGTGTGTGAGCGA +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A 1768 58 A25073H CGTGAGATACACAGGTG +C*+G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G 1740 26 A25074H GCGTGAGATACACAGGT +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*G*+G*+T 1739 26 A25075H GCGTGAGATACACAGGT +G*+C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*G*+T 1739 26 A25076H GCGTGAGATACACAGGT +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T 1739 59 A25077H ATGCGTGAGATACACAG +A*+T*+G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 27 A25078H AGCTCGGCTGCAGTTTA +A*G*+C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A 1510 60 A25079HI TCGATGGAGTGTGGTCA +T*+C*+G*A*T*G*G*A*G*T*G*T*G*G*+T*+C*+A 15176 61 A25080HI AGATCGATGGAGTGTGG +A*+G*+A*T*C*G*A*T*G*G*A*G*T*G*+T*+G*+G 15173 62 A25081HI CCTCGGCGACATTACTA +C*+C*+T*C*G*G*C*G*A*C*A*T*T*A*+C*+T*+A 15056 63 A25082HI CTCGGCGACATTACTA +C*+T*+C*G*G*C*G*A*C*A*T*T*A*+C*+T*+A 15056 64 A25083HI GCTAAACTACGGTTGAC +G*+C*+T*A*A*A*C*T*A*C*G*G*TT*+G*+A*+C 14882 65 A25084HI GTTTCGTTCCGAGAACT +G*+T*+T*T*C*G*T*T*C*C*G*A*G*A*A*+C*+T 11938 66 A25085HMI AGGTTTCGTTCCGAGAA +A*+G*+G*T*T*T*C*G*T*T*C*C*G*A*+G*+A*+A 11936 67 A25086HI GATGCCGAGTTCCGTAG +G*+A*T*G*C*C*G*A*G*T*T*C*C*G*+T*+A*+G 10835 68 A25087HI AGATGCCGAGTTCCGTA +A*+G*+A*T*G*C*C*G*A*G*TT*C*C*G*+T*+A 10834 69 A25088HI GTGATCATGCACGGATC +G*+T*G*A*T*C*A*T*G*C*A*C*G*G*+A*+T*+C 10448 70 A25089HI TTAAAGACGGCCATTCG +T*+T*+A*A*A*G*A*C*G*G*C*C*A*T*+T*+C*+G 8241 71 A25090HI AGGTGCGGATGTCGTAT +A*+G*+G*T*G*C*G*G*A*T*G*T*C*G*+T*+A*+T 5606 72 A25091HI GTGCGGATGTCGTATG +G*+T*+G*C*G*G*A*T*G*T*C*G*T*A*+T*+G 5607 73 A25092HI AGGTGCGGATGTCGTA +A*+G*+G*T*G*C*G*G*A*T*G*T*C*+G*+T*+A 5605 74 A25093HI ACAGGTGCGGATGTCG +A*C*+A*G*G*T*G*C*G*G*A*T*G*+T*+C*+G 5603 75 A25095HI GTTAGGTGTGGCGCTAG +G*+T*+T*A*G*G*T*G*T*G*G*C*G*C*+T*+A*+G 3616 56 A25096H GAGCGAGCACGTGTTGG +G*+A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G 1779 56 A25097H GAGCGAGCACGTGTTGG +G*A*+G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G 1779 56 A25098H GAGCGAGCACGTGTTGG +G*+A*G*C*G*A*G*C*A*C*G*T*G*T*T*+G*+G 1779 58 A25099H CGTGAGATACACAGGTG +C*G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*+T*+G 1740 58 A25100H CGTGAGATACACAGGTG +C*G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G 1740 58 A25101H CGTGAGATACACAGGTG +C*+G*+T*G*A*G*A*T*A*C*A*C*A*G*+G*T*+G 1740 24 A25102H TGAGCGAGCACGTGTTG +T*G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G 1778 24 A25103H TGAGCGAGCACGTGTTG +T*G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G 1778 24 A25104H TGAGCGAGCACGTGTTG +T*+G*+A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G 1778 23 A25105H CGCTGCTTCTGTGTAGG +C*G*+C*T*G*C*T*T*C*T*G*T*G*T*+A*+G*+G 1820 25 A25106H GCCGTGTGTGTGAGCGA +G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*+G*+A 1768 26 A25107H GCGTGAGATACACAGGT +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T 1739 26 A25108H GCGTGAGATACACAGGT +G*+C*G*T*G*A*G*A*T*A*C*A*C*A*+G*G*+T 1739 27 A25109H AGCTCGGCTGCAGTTTA +A*G*+C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A 1510 26 A25110H GCGTGAGATACACAGGT +G*C*+G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T 1739 59 A25111H ATGCGTGAGATACACAG +A*T*+G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 34 A25112H CACAGGTTTCGTTCCGA +C*+A*+C*A*G*G*T*T*T*C*G*T*T*C*C*+G*+A 11933 34 A25113H CACAGGTTTCGTTCCGA +C*A*+C*A*G*G*T*T*T*C*G*T*T*C*+C*G*+A 11933 34 A25114H CACAGGTTTCGTTCCGA +C*+A*+C*A*G*G*T*T*T*C*G*T*T*C*+C*G*+A 11933 49 A25115H CAGGTGCGGATGTCGT +C*+A*+G*G*T*G*C*G*G*A*T*G*T*C*+G*+T 5604 66 A25116H AGGTTTCGTTCCGAGAA +A*+G*G*T*TT*C*G*T*T*C*C*G*A*+G*A*+A 11936 73 A25117H AGGTGCGGATGTCGTA +A*G*+G*T*G*C*G*G*A*T*G*T*C*+G*+T*+A 5605 76 A25118H GAAAAACCACGCTGTACG +G*+A*+A*A*A*A*C*C*A*C*G*C*T*G*T*+A*+C*+G 15817 77 A25120H ATCCAGAAGATGGTCCGC +A*+T*+C*C*A*G*A*A*G*A*T*G*G*T*C*+C*+G*+C 6843 78 A25122H CGTGGCGTAGGTGAAAGG +C*+G*+T*G*G*C*G*T*A*G*G*T*G*A*A*+A*+G*+G 4206 79 A25123H GGATGAGCGTGGCGTAGG +G*+G*+A*T*G*A*G*C*G*T*G*G*C*G*T*+A*+G*+G 4199 80 A25125H TGCGGAACTCCAGCTCAT +T*+G*+C*G*G*A*A*C*T*C*C*A*G*C*T*+C*+A*+T 2455 81 A25126H GAAGTAATCTGTGCGAGC +G*+A*+A*G*T*A*A*T*C*T*G*T*G*C*G*+A*+G*+C 2069 82 A25127H GTTGTTTGAGTGTACTGA +G*+T*+T*G*T*T*T*G*A*G*T*G*T*A*C*+T*+G*+A 1966 83 A25128H GTGAGCGAGCACGTGTTG +G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*T*+G 1778 84 A25129H TGTGAGCGAGCACGTGTT +T*G*+T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T 1777 85 A25130H GGCCGTGTGTGTGAGCGA +G*+G*+C*C*G*T*G*T*G*T*G*T*G*A*G*+C*G*+A 1768 86 A25131H AATTCTAACAGGCCGTGT +A*+A*+T*T*C*T*A*A*C*A*G*G*C*C*G*+T*+G*+T 1758 87 A25132H GTGAATTCTAACAGGCCG +G*+T*+G*A*A*T*T*C*T*A*A*C*A*G*G*+C*+C*+G 1755 88 A25133H TATGCGTGAGATACACAG +T*+A*+T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 89 A25134H CATATGCGTGAGATACAC +C*+A*+T*A*T*G*C*G*T*G*A*G*A*T*A*+C*+A*+C 1735 90 A25135H CTCGGCTGCAGTTTATTG +C*+T*+C*G*G*C*T*G*C*A*G*T*T*T*A*+T*+T*+G 1513 91 A25136H AGAAAAACCACGCTGTACG +A*+G*+A*A*A*A*A*C*C*A*C*G*C*T*G*T*+A*+C*+G 15817 92 A25138H TCGCATGTTGTGGAACTTG +T*+C*+G*C*A*T*G*T*T*G*T*G*G*A*A*C*+T*+T*+G 4228 93 A25139H GCGTGGCGTAGGTGAAAGG +G*+C*+G*T*G*G*C*G*T*A*G*G*T*G*A*A*+A*+G*+G 4206 94 A25140H AGCGTGGCGTAGGTGAAAG +A*+G*+C*G*T*G*G*C*G*T*A*G*G*T*G*A*+A*+A*+G 4205 95 A25141H GAGCGTGGCGTAGGTGAAA +G*+A*+G*C*G*T*G*G*C*G*T*A*G*G*T*G*+A*+A*+A 4204 96 A25142H TGAGCGTGGCGTAGGTGAA +T*+G*+A*G*C*G*T*G*G*C*G*T*A*G*G*T*+G*+A*+A 4203 97 A25143H ATGAGCGTGGCGTAGGTGA +A*+T*+G*A*G*C*G*T*G*G*C*G*T*A*G*G*+T*+G*+A 4202 98 A25144H ATCTCATTGAGTGTCCGCT +A*+T*+C*T*C*A*T*T*G*A*G*T*G*T*C*C*+G*+C*+T 2791 99 A25145H GATCTCATTGAGTGTCCGC +G*+A*+T*C*T*C*A*T*T*G*A*G*T*G*T*C*+C*+G*+C 2790 100 A25146H GGCTCCGTTTCTTGCGGAA +G*+G*+C*T*C*C*G*T*TT*C*T*T*G*C*G*+G*+A*+A 2444 101 A25147H CGCTGCTTCTGTGTAGGCC +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*G*+G*C*+C 1822 101 A25148H CGCTGCTTCTGTGTAGGCC +C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*G*G*+C*+C 1822 102 A25149H GAATTCTAACAGGCCGTGT +G*+A*+A*T*T*C*T*A*A*C*A*G*G*C*C*G*+T*+G*+T 1758 103 A25150H ATGCGTGAGATACACAGGT +A*+T*+G*C*G*T*G*A*G*A*T*A*C*A*C*A*+G*+G*+T 1739 104 A25151H TATGCGTGAGATACACAGG +T*+A*+T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G*+G 1738 105 A25152H ATATGCGTGAGATACACAG +A*+T*+A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 105 A25153H ATATGCGTGAGATACACAG +A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*A*+C*+A*+G 1737 105 A25154H ATATGCGTGAGATACACAG +A*T*+A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 105 A25155H ATATGCGTGAGATACACAG +A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*A*C*+A*+G 1737 106 A25156H GTGCATATGCGTGAGATAC +G*+T*G*C*A*T*A*T*G*C*G*T*G*A*G*A*+T*A*+C 1733 106 A25157H GTGCATATGCGTGAGATAC +G*+T*G*C*A*T*A*T*G*C*G*T*G*A*G*A*T*+A*+C 1733 107 A25158H GCTCGGCTGCAGTTTATTG +G*+C*+T*C*G*G*C*T*G*C*A*G*T*T*T*A*+T*+T*+G 1513 108 A25159H GGAGCTCGGCTGCAGTTTA +G*+G*+A*G*C*T*C*G*G*C*T*G*C*A*G*T*+T*+T*+A 1510 109 A25160HI CCTCGGCGACATTACTAT +C*+C*+T*C*G*G*C*G*A*C*A*T*T*A*C*+T*+A*+T 15040 110 A25161HI CGTGGATCGTCCAACCTG +C*+G*+T*G*G*A*T*C*G*T*C*C*A*A*C*+C*+T*+G 12720 111 A25162HI AGATGCCGAGTTCCGTAG +A*+G*+A*T*G*C*C*G*A*G*TT*C*C*G*+T*+A*+G 10818 112 A25163HI CTTAAAGACGGCCATTCG +C*+T*+T*A*A*A*G*A*C*G*G*C*C*A*T*+T*+C*+G 8224 113 A25164HI AGGTGCGGATGTCGTATG +A*+G*+G*T*G*C*G*G*A*T*G*T*C*G*T*+A*+T*+G 5590 114 A25165HI CAGGTGCGGATGTCGTAT +C*+A*+G*G*T*G*C*G*G*A*T*G*T*C*G*+T*+A*+T 5589 115 A25166HI GGTTAGGTGTGGCGCTAG +G*+G*+T*T*A*G*G*T*G*T*G*G*C*G*C*+T*+A*+G 3599 116 A25167HI ATTATCGAGTATCTTACG +A*+T*+T*A*T*C*G*A*G*T*A*T*C*T*T*+A*+C*+G 3342 117 A25168HI AGGAGATCGATGGAGTGTG +A*+G*+G*A*G*A*T*C*G*A*T*G*G*A*G*T*+G*+T*+G 15154 118 A25169HI CCTCGGCGACATTACTATT +C*+C*+T*C*G*G*C*G*A*C*A*T*T*A*C*T*+A*+T*+T 15040 119 A25170HI GGTCTCCTCTAAAGCGATA +G*+G*+T*C*T*C*C*T*C*T*A*A*A*G*C*G*+A*+T*+A 14919 120 A25171HI GGTAGGTCCACACAGCTAA +G*+G*+T*A*G*G*T*C*C*A*C*A*C*A*G*C*+T*+A*+A 14852 121 A25172HI AACAATCGGCACTTGGTCA +A*+A*+C*A*A*T*C*G*G*C*A*C*T*T*G*G*+T*+C*+A 14539 122 A25173HI TGTGCGAGAGGAGGATTGC +T*+G*+T*G*C*G*A*G*A*G*G*A*G*G*A*T*+T*+G*+C 13198 123 A25174HI CACGCTCTGGCCAACTAGG +C*+A*+C*G*C*T*C*T*G*G*C*C*A*A*C*T*+A*+G*+G 12632 124 A25175HI GCCTTCGCCAATACAGAGC +G*+C*+C*T*T*C*G*C*C*A*A*T*A*C*A*G*+A*+G*+C 12509 125 A25176HI CTCAGTATGTGTAGGCCAG +C*+T*+C*A*G*T*A*T*G*T*G*T*A*G*G*C*+C*+A*+G 12245 126 A25177HI CGTTCCGAGAACTGGCTGC +C*+G*+TT*C*C*G*A*G*A*A*C*T*G*G*C*+T*+G*+C 11926 127 A25178HI TCGTTCCGAGAACTGGCTG +T*+C*+G*T*T*C*C*G*A*G*A*A*C*T*G*G*+C*+T*+G 11925 128 A25179HI TTTCGTTCCGAGAACTGGC +T*+T*+T*C*G*T*T*C*C*G*A*G*A*A*C*T*+G*+G*+C 11923 129 A25180HI GTTTCGTTCCGAGAACTGG +G*+T*+T*T*C*G*T*T*C*C*G*A*G*A*A*C*+T*+G*+G 11922 130 A25181HI ACAGGTTTCGTTCCGAGAA +A*+C*+A*G*G*T*T*T*C*G*T*T*C*C*G*A*+G*+A*+A 11918 131 A25182HI CACAGGTTTCGTTCCGAGA +C*+A*+C*A*G*G*T*T*T*C*G*TT*C*C*G*+A*+G*+A 11917 132 A25183HI CCACAGGTTTCGTTCCGAG +C*+C*+A*C*A*G*G*T*TT*C*G*T*T*C*C*+G*+A*+G 11916 133 A25184HI TTTCGGTGCAAATGGATGT +T*+T*+T*C*G*G*T*G*C*A*A*A*T*G*G*A*+T*+G*+T 11469 134 A25185HI AGGACCGAGCTGACATTAC +A*+G*+G*A*C*C*G*A*G*C*T*G*A*C*A*T*+T*+A*+C 10257 135 A25186HI ATACTCGACCACCTGAGCC +A*+T*+A*C*T*C*G*A*C*C*A*C*C*T*G*A*+G*+C*+C 9630 136 A25187HI ATGAGATACTCGACCACCT +A*+T*+G*A*G*A*T*A*C*T*C*G*A*C*C*A*+C*+C*+T 9625 137 A25188HI CATTCGCAGGTGCTGACAT +C*+A*+T*T*C*G*C*A*G*G*T*G*C*T*G*A*+C*+A*+T 8236 138 A25189HI AAAGACGGCCATTCGCAGG +A*+A*+A*G*A*C*G*G*C*C*A*T*T*C*G*C*+A*+G*+G 8227 139 A25190HI GTACATTCGCATCATGAGA +G*+T*+A*C*A*T*T*C*G*C*A*T*C*A*T*G*+A*+G*+A 5717 140 A25191HI GTGCGGATGTCGTATGTGG +G*+T*+G*C*G*G*A*T*G*T*C*G*T*A*T*G*+T*+G*+G 5592 141 A25192HI AGGTGCGGATGTCGTATGT +A*+G*+G*T*G*C*G*G*A*T*G*T*C*G*T*A*+T*+G*+T 5590 142 A25193HI CAGGTGCGGATGTCGTATG +C*+A*+G*G*T*G*C*G*G*A*T*G*T*C*G*T*+A*+T*+G 5589 143 A25194HI ACAGGTGCGGATGTCGTAT +A*+C*+A*G*G*T*G*C*G*G*A*T*G*T*C*G*+T*+A*+T 5588 144 A25195HI AGCATGAGCCGTATTTATT +A*+G*+C*A*T*G*A*G*C*C*G*T*A*T*T*T*+A*+T*+T 5564 145 A25196HI GATGGCCGAATATAGTAGC +G*+A*+T*G*G*C*C*G*A*A*T*A*T*A*G*T*+A*+G*+C 4677 146 A25197HI TGTGGCGCTAGGATGAAGG +T*+G*+T*G*G*C*G*C*T*A*G*G*A*T*G*A*+A*+G*+G 3606 147 A25198HI GGTTCGGTGTGGAGTGAGG +G*+G*+T*T*C*G*G*T*G*T*G*G*A*G*T*G*+A*+G*+G 3415 148 A25199HI TTATCGAGTATCTTACGTG +T*+T*+A*T*C*G*A*G*T*A*T*C*T*T*A*C*+G*+T*+G 3343 149 A25200H CTTCGAAGACCTTCTCAC +C*+T*+T*C*G*A*A*G*A*C*C*T*T*C*T*+C*+A*+C 7838 150 A25201H AGAAGATGGTCCGCCTGG +A*+G*+A*A*G*A*T*G*G*T*C*C*G*C*C*+T*+G*+G 6847 151 A25202H CATCCAGAAGATGGTCCG +C*+A*+T*C*C*A*G*A*A*G*A*T*G*G*T*+C*+C*+G 6842 152 A25204H CTACGATGCAGCAGGAGC +C*+T*+A*C*G*A*T*G*C*A*G*C*A*G*G*+A*+G*+C 5102 153 A25205H GCCAGCAGCTACGATGCA +G*+C*+C*A*G*C*A*G*C*T*A*C *G*A*T*+G*+C*+A 5094 154 A25206H GTGCCTCCGGACAGCAAA +G*+T*+G*C*C*T*C*C*G*G*A*C*A*G*C*+A*+A*+A 5019 155 A25207H TCGCATGTTGTGGAACTT +T*+C*+G*C*A*T*G*T*T*G*T*G*G*A*A*+C*+T*+T 4227 156 A25208H GCGTGGCGTAGGTGAAAG +G*+C*+G*T*G*G*C*G*T*A*G*G*T*G*A*+A*+A*+G 4205 157 A25209H AGCGTGGCGTAGGTGAAA +A*+G*+C*G*T*G*G*C*G*T*A*G*G*T*G*+A*+A*+A 4204 158 A25210H TGAGCGTGGCGTAGGTGA +T*+G*+A*G*C*G*T*G*G*C*G*T*A*G*G*+T*+G*+A 4202 159 A25211H ATGAGCGTGGCGTAGGTG +A*+T*+G*A*G*C*G*T*G*G*C*G*T*A*G*+G*+T*+G 4201 160 A25212H CGGATGAGCGTGGCGTAG +C*+G*+G*A*T*G*A*G*C*G*T*G*G*C*G*+T*+A*+G 4198 161 A25213H GCGGATGAGCGTGGCGTA +G*+C*+G*G*A*T*G*A*G*C*G*T*G*G*C*+G*+T*+A 4197 162 A25214H AGCGGATGAGCGTGGCGT +A*+G*+C*G*G*A*T*G*A*G*C*G*T*G*G*+C*+G*+T 4196 163 A25215H CAGCGGATGAGCGTGGCG +C*+A*+G*C*G*G*A*T*G*A*G*C*G*T*G*+G*+C*+G 4195 164 A25216H ATCTCATTGAGTGTCCGC +A*+T*+C*T*C*A*T*T*G*A*G*T*G*T*C*+C*+G*+C 2790 165 A25217H AGACTCAGGTTGTGGCGG +A*+G*+A*C*T*C*A*G*G*T*T*G*T*G*G*+C*+G*+G 2526 166 A25218H TGAAGTAATCTGTGCGAG +T*+G*+A*A*G*T*A*A*T*C*T*G*T*G*C*+G*+A*+G 2068 167 A25219H TCGGCTGCAGTTTATTGG +T*+C*+G*G*C*T*G*C*A*G*T*T*T*A*T*+T*+G*+G 1514 168 A25220H GAAGAAAAACCACGCTGTA +G*+A*+A*G*A*A*A*A*A*C*C*A*C*G*C*T*+G*+T*+A 15815 169 A25221H TTGGTGAAGTGGACTGACA +T*+T*+G*G*T*G*A*A*G*T*G*G*A*C*T*G*+A*+C*+A 15731 170 A25223H TCGAAGACCTTCTCACATC +T*+C*+G*A*A*G*A*C*C*T*T*C*T*C*A*C*+A*+T*+C 7841 171 A25224H TTCGAAGACCTTCTCACAT +T*+T*+C*G*A*A*G*A*C*C*T*T*C*T*C*A*+C*+A*+T 7840 172 A25225H TCATCCAGAAGATGGTCCG +T*+C*+A*T*C*C*A*G*A*A*G*A*T*G*G*T*+C*+C*+G 6842 173 A25227H CTACGATGCAGCAGGAGCC +C*+T*+A*C*G*A*T*G*C*A*G*C*A*G*G*A*+G*+C*+C 5103 174 A25228H GGTGCCTCCGGACAGCAAA +G*+G*+T*G*C*C*T*C*C*G*G*A*C*A*G*C*+A*+A*+A 5019 175 A25229H CATGTTGTGGAGGAACTCT +C*+A*+T*G*T*T*G*T*G*G*A*G*G*A*A*C*+T*+C*+T 4255 176 A25230H TAGTCCATGTTGTGGAGGA +T*+A*+G*T*C*C*A*T*G*T*T*G*T*G*G*A*+G*+G*+A 4250 177 A25231H GATGAGCGTGGCGTAGGTG +G*+A*+T*G*A*G*C*G*T*G*G*C*G*T*A*G*+G*+T*+G 4201 178 A25232H CGGATGAGCGTGGCGTAGG +C*+G*+G*A*T*G*A*G*C*G*T*G*G*C*G*T*+A*+G*+G 4199 179 A25233H GCGGATGAGCGTGGCGTAG +G*+C*+G*G*A*T*G*A*G*C*G*T*G*G*C*G*+T*+A*+G 4198 180 A25234H CAGCGGATGAGCGTGGCGT +C*+A*+G*C*G*G*A*T*G*A*G*C*G*T*G*G*+C*+G*+T 4196 181 A25235H GCGTGTGAACCAGTGGTAG +G*+C*+G*T*G*T*G*A*A*C*C*A*G*T*G*G*+T*+A*+G 2772 182 A25237H ACTCAGGTTGTGGCGGATG +A*+C*+T*C*A*G*G*T*T*G*T*G*G*C*G*G*+A*+T*+G 2529 183 A25238H CTTGTGCAGACTCAGGTTG +C*+T*+T*G*T*G*C*A*G*A*C*T*C*A*G*G*+T*+T*+G 2520 184 A25239H TGCGGAACTCCAGCTCATC +T*+G*+C*G*G*A*A*C*T*C*C*A*G*C*T*C*+A*+T*+C 2456 185 A25240H TTGCGGAACTCCAGCTCAT +T*+T*+G*C*G*G*A*A*C*T*C*C*A*G*C*T*+C*+A*+T 2455 186 A25241H TCTGGCTCCGTTTCTTGCG +T*+C*+T*G*G*C*T*C*C*G*T*T*T*C*T*T*+G*+C*+G 2441 187 A25242H CTGAAGTAATCTGTGCGAG +C*+T*+G*A*A*G*T*A*A*T*C*T*G*T*G*C*+G*+A*+G 2068 188 A25243H CCTGAAGTAATCTGTGCGA +C*+C*+T*G*A*A*G*T*A*A*T*C*T*G*T*G*+C*+G*+A 2067 189 A25244H GTTGTTTGAGTGTACTGAG +G*+T*+T*G*T*T*T*G*A*G*T*G*T*A*C*T*+G*+A*+G 1967 190 A25245H GGTTGTTTGAGTGTACTGA +G*+G*+T*T*G*T*T*T*G*A*G*T*G*T*A*C*+T*+G*+A 1966 191 A25246H ACGCTGCTTCTGTGTAGGC +A*+C*+G*C*T*G*C*T*T*C*T*G*T*G*T*A*+G*G*+C 1821 192 A25247H GACGCTGCTTCTGTGTAGG +G*+A*+C*G*C*T*G*C*T*T*C*T*G*T*G*T*A*+G*+G 1820 193 A25248H GGTACTGACGCTGCTTCTG +G*+G*+T*A*C*T*G*A*C*G*C*T*G*C*T*T*C*+T*+G 1814 194 A25249H TGTGAGCGAGCACGTGTTG +T*+G*T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T*+G 1778 195 A25250H GTGTGAGCGAGCACGTGTT +G*+T*G*T*G*A*G*C*G*A*G*C*A*C*G*T*G*+T*+T 1777 196 A25251H GGCCGTGTGTGTGAGCGAG +G*+G*C*C*G*T*G*T*G*T*G*T*G*A*G*C*G*+A*+G 1769 197 A25252H TCTAACAGGCCGTGTGTGT +T*C*+T*A*A*C*A*G*G*C*C*G*T*G*T*G*+T*+G*+T 1762 198 A25253H AATTCTAACAGGCCGTGTG +A*+A*+T*T*C*T*A*A*C*A*G*G*C*C*G*T*+G*+T*+G 1759 199 A25254H TGAATTCTAACAGGCCGTG +T*+G*+A*A*T*T*C*T*A*A*C*A*G*G*C*C*+G*+T*+G 1757 200 A25255H GTGAATTCTAACAGGCCGT +G*+T*+G*A*A*T*T*C*T*A*A*C*A*G*G*C*+C*+G*+T 1756 201 A25256H GGTGAATTCTAACAGGCCG +G*G*+T*G*A*A*T*T*C*T*A*A*C*A*G*G*C*+C*+G 1755 201 A25257H GGTGAATTCTAACAGGCCG +G*G*+T*G*A*A*T*T*C*T*A*A*C*A*G*G*+C*C*+G 1755 202 A25258H CATATGCGTGAGATACACA +C*+A*+T*A*T*G*C*G*T*G*A*G*A*T*A*C*+A*+C*+A 1736 203 A25259H GTTCCTCTGCAGTCTAAGC +G*+T*+T*C*C*T*C*T*G*C*A*G*T*C*T*A*+A*+G*+C 1579 204 A25260H GTAGTTCCTCTGCAGTCTA +G*+T*+A*G*T*T*C*C*T*C*T*G*C*A*G*T*+C*+T*+A 1576 205 A25261H CTCGGCTGCAGTTTATTGG +C*+T*+C*G*G*C*T*G*C*A*G*T*T*T*A*T*+T*+G*+G 1514 206 A25262HI GTGTAGCGACAGACAGAT +G*+T*+G*T*A*G*C*G*A*C*A*G*A*C*A*+G*+A*+T 15244 207 A25263HI TCGATGGAGTGTGGTCAA +T*+C*+G*A*T*G*G*A*G*T*G*T*G*G*T*+C*+A*+A 15160 208 A25264HI AGATCGATGGAGTGTGGT +A*+G*+A*T*C*G*A*T*G*G*A*G*T*G*T*+G*+G*+T 15157 209 A25265HI CTCGGCGACATTACTATT +C*+T*+C*G*G*C*G*A*C*A*T*T*A*C*T*+A*+T*+T 15041 109 A25266HI CCTCGGCGACATTACTAT +C*+C*+T*C*G*G*C*G*A*C*A*T*T*A*C*+T*+A*+T 15040 210 A25267HI TCCTCGGCGACATTACTA +T*+C*+C*T*C*G*G*C*G*A*C*A*T*T*A*+C*+T*+A 15039 211 A25268HI CCTCTAAAGCGATACAAG +C*+C*+T*C*T*A*A*A*G*C*G*A*T*A*C*+A*+A*+G 14924 212 A25269HI GCTAAACTACGGTTGACA +G*+C*+T*A*A*A*C*T*A*C*G*G*T*T*G*+A*+C*+A 14866 213 A25270HI AGCTAAACTACGGTTGAC +A*+G*+C*T*A*A*A*C*T*A*C*G*G*T*T*+G*+A*+C 14865 214 A25271HI ACAATCGGCACTTGGTCA +A*+C*+A*A*T*C*G*G*C*A*C*T*T*G*G*+T*+C*+A 14540 110 A25272HI CGTGGATCGTCCAACCTG +C*+G*+T*G*G*A*T*C*G*T*C*C*A*A*C*+C*+T*+G 12720 215 A25273HI TCGTGGATCGTCCAACCT +T*+C*+G*T*G*G*A*T*C*G*T*C*C*A*A*+C*+C*+T 12719 216 A25274HI ACGCTCTGGCCAACTAGG +A*+C*+G*C*T*C*T*G*G*C*C*A*A*C*T*+A*+G*+G 12633 217 A25275HI CTTCGCCAATACAGAGCC +C*+T*+T*C*G*C*C*A*A*T*A*C*A*G*A*+G*+C*+C 12511 218 A25276HI AATACATGGCCACTCCGC +A*+A*+T*A*C*A*T*G*G*C*C*A*C*T*C*+C*+G*+C 12408 219 A25277HI TTTCGTTCCGAGAACTGG +T*+T*+T*C*G*T*T*C*C*G*A*G*A*A*C*+T*+G*+G 11923 220 A25278HI GTTTCGTTCCGAGAACTG +G*+T*+T*T*C*G*T*T*C*C*G*A*G*A*A*+C*+T*+G 11922 221 A25279HI ACAGGTTTCGTTCCGAGA +A*+C*+A*G*G*T*T*T*C*G*T*T*C*C*G*+A*+G*+A 11918 222 A25280HI CCACAGGTTTCGTTCCGA +C*+C*+A*C*A*G*G*T*T*T*C*G*T*T*C*+C*+G*+A 11916 ill A25281HI AGATGCCGAGTTCCGTAG +A*+G*+A*T*G*C*C*G*A*G*TT*C*C*G*+T*+A*+G 10818 223 A25282HI GATCATGCACGGATCCAG +G*+A*+T*C*A*T*G*C*A*C*G*G*A*T*C*+C*+A*+G 10434 224 A25283HI CCGAGCTGACATTACCTG +C*+C*+G*A*G*C*T*G*A*C*A*T*T*A*C*+C*+T*+G 10261 225 A25284HI GTATGAGATACTCGACCA +G*+T*+A*T*G*A*G*A*T*A*C*T*C*G*A*+C*+C*+A 9623 226 A25285HI AAGACGGCCATTCGCAGG +A*+A*+G*A*C*G*G*C*C*A*T*T*C*G*C*+A*+G*+G 8228 112 A25286HI CTTAAAGACGGCCATTCG +C*+T*+T*A*A*A*G*A*C*G*G*C*C*A*T*+T*+C*+G 8224 227 A25287HI GCTTAAAGACGGCCATTC +G*+C*+TT*A*A*A*G*A*C*G*G*C*C*A*+T*+T*+C 8223 228 A25288HI GCGTGTGACCTATGTGGT +G*+C*+G*T*G*T*G*A*C*C*T*A*T*G*T*+G*+G*+T 5798 229 A25289HI GTACATTCGCATCATGAG +G*+T*+A*C*A*T*T*C*G*C*A*T*C*A*T*+G*+A*+G 5717 230 A25290HI GGTGCGGATGTCGTATGT +G*+G*+T*G*C*G*G*A*T*G*T*C*G*T*A*+T*+G*+T 5591 113 A25291HI AGGTGCGGATGTCGTATG +A*+G*+G*T*G*C*G*G*A*T*G*T*C*G*T*+A*+T*+G 5590 114 A25292HI CAGGTGCGGATGTCGTAT +C*+A*+G*G*T*G*C*G*G*A*T*G*T*C*G*+T*+A*+T 5589 231 A25293HI GAGCCGTATTTATTAGAG +G*+A*+G*C*C*G*T*A*T*T*T*A*T*T*A*+G*+A*+G 5569 232 A25294HI CAGCATGAGCCGTATTTA +C*+A*+G*C*A*T*G*A*G*C*C*G*T*A*T*+T*+T*+A 5563 233 A25295HI CGTGTAGTGCAAGGACCA +C*+G*+T*G*T*A*G*T*G*C*A*A*G*G*A*+C*+C*+A 4943 234 A25296HI CGACACTCGAGACCATAT +C*+G*+A*C*A*C*T*C*G*A*G*A*C*C*A*+T*+A*+T 4755 235 A25297HI GATGGCCGAATATAGTAG +G*+A*+T*G*G*C*C*G*A*A*T*A*T*A*G*+T*+A*+G 4677 236 A25298HI GCGGAGTAACTTGCACAC +G*+C*+G*G*A*G*T*A*A*C*T*T*G*C*A*+C*+A*+C 4470 237 A25299HI CACATTTGAGGCACGGCT +C*+A*+C*A*T*T*T*G*A*G*G*C*A*C*G*+G*+C*+T 4022 238 A25300HI GTGTGGCGCTAGGATGAA +G*+T*+G*T*G*G*C*G*C*T*A*G*G*A*T*+G*+A*+A 3605 239 A25301HI TTAGGTGTGGCGCTAGGA +T*+T*+A*G*G*T*G*T*G*G*C*G*C*T*A*+G*+G*+A 3601 115 A25302HI GGTTAGGTGTGGCGCTAG +G*+G*+T*T*A*G*G*T*G*T*G*G*C*G*C*+T*+A*+G 3599 240 A25303HI GGTTCGGTGTGGAGTGAG +G*+G*+T*T*C*G*G*T*G*T*G*G*A*G*T*+G*+A*+G 3415 241 A25304HI CGAGTATCTTACGTGTCA +C*+G*+ A*G*T*A*T*C*T*T*A*C*G*T*G*+T*+C*+A 3347 242 A25305HI TATCGAGTATCTTACGTG +T*+A*+T*C*G*A*G*T*A*T*C*T*T*A*C*+G*+T*+G 3344 116 A25306HI ATTATCGAGTATCTTACG +A*+T*+T*A*T*C*G*A*G*T*A*T*C*T*T*+A*+C*+G 3342 243 A25307HI TACCTGGCTGGAATCACGG +T*+A*+C*C*T*G*G*C*T*G*G*A*A*T*C*A*+C*+G*+G 15579 244 A25308HI CGTATCAATTGATGAATTC +C*+G*+T*A*T*C*A*A*T*T*G*A*T*G*A*A*+T*+T*+C 15478 245 A25309HI TAGCGACAGACAGATGGCG +T*+A*+G*C*G*A*C*A*G*A*C*A*G*A*T*G*+G*+C*+G 15247 246 A25310HI TAAACGCCAGCTGTGTACA +T*+A*+A*A*C*G*C*C*A*G*C*T*G*T*G*T*+A*+C*+A 15061 247 A25311HI ATTAAACGCCAGCTGTGTA +A*+T*+T*A*A*A*C*G*C*C*A*G*C*T*G*T*+G*+T*+A 15059 248 A25312HI CTCGGCGACATTACTATTA +C*+T*+C*G*G*C*G*A*C*A*T*T*A*C*T*A*+T*+T*+A 15041 249 A25313HI TAAAGGTCCTCGGCGACAT +T*+A*+A*A*G*G*T*C*C*T*C*G*G*C*G*A*+C*+A*+T 15033 250 A25314HI TCCTCTAAAGCGATACAAG +T*+C*+C*T*C*T*A*A*A*G*C*G*A*T*A*C*+A*+A*+G 14923 251 A25315HI CGGTTGACAATGGTGTGAA +C*+G*+G*T*T*G*A*C*A*A*T*G*G*T*G*T*+G*+A*+A 14875 252 A25316HI AGCTAAACTACGGTTGACA +A*+G*+C*T*A*A*A*C*T*A*C*G*G*T*T*G*+A*+C*+A 14865 253 A25317HI AATCGGCACTTGGTCAAAT +A*+A*+T*C*G*G*C*A*C*TT*G*G*T*C*A*+A*+A*+T 14542 254 A25318HI ACAATCGGCACTTGGTCAA +A*+C*+A*A*T*C*G*G*C*A*C*T*T*G*G*T*+C*+A*+A 14540 255 A25319HI CAACAATCGGCACTTGGTC +C*+A*+A*C*A*A*T*C*G*G*C*A*C*T*T*G*+G*+T*+C 14538 256 A25320HI AATAGTCAGTCCATTATCC +A*+A*+T*A*G*T*C*A*G*T*C*C*A*T*T*A*+T*+C*+C 13537 257 A25321HI GTGCGAGAGGAGGATTGCC +G*+T*+G*C*G*A*G*A*G*G*A*G*G*A*T*T*+G*+C*+C 13199 258 A25322HI GGTTAAGTCATTAGGTGTC +G*+G*+T*T*A*A*G*T*C*A*T*T*A*G*G*T*+G*+T*+C 13015 259 A25323HI CTTCTACGCTGTCTGGTTA +C*+T*+T*C*T*A*C*G*C*T*G*T*C*T*G*G*+T*+T*+A 13001 260 A25324HI CGTGGATCGTCCAACCTGT +C*+G*+T*G*G*A*T*C*G*T*C*C*A*A*C*C*+T*+G*+T 12720 261 A25325HI TCGTGGATCGTCCAACCTG +T*+C*+G*T*G*G*A*T*C*G*T*C*C*A*A*C*+C*+T*+G 12719 262 A25326HI ATCATCACCACGCTCTGGC +A*+T*+C*A*T*C*A*C*C*A*C*G*C*T*C*T*+G*+G*+C 12624 263 A25327HI CCTTCGCCAATACAGAGCC +C*+C*+T*T*C*G*C*C*A*A*T*A*C*A*G*A*+G*+C*+C 12510 264 A25328HI AGCCTTCGCCAATACAGAG +A*+G*+C*C*T*T*C*G*C*C*A*A*T*A*C*A*+G*+A*+G 12508 265 A25329HI CAGCCTTCGCCAATACAGA +C*+A*+G*C*C*T*T*C*G*C*C*A*A*T*A*C*+A*+G*+A 12507 266 A25330HI TCAGCCTTCGCCAATACAG +T*+C*+A*G*C*C*T*T*C*G*C*C*A*A*T*A*+C*+A*+G 12506 267 A25331HI ATAGTATAACACCAGGACC +A*+T*+A*G*T*A*T*A*A*C*A*C*C*A*G*G*+A*+C*+C 12142 268 A25332HI TTCATCGACACCACGGAGG +T*+T*+C*A*T*C*G*A*C*A*C*C*A*C*G*G*+A*+G*+G 11872 269 A25333HI GCTTCATCGACACCACGGA +G*+C*+T*T*C*A*T*C*G*A*C*A*C*C*A*C*+G*+G*+A 11870 270 A25334HI TTCCGCCATTGACGTCATG +T*+T*+C*C*G*C*C*A*T*T*G*A*C*G*T*C*+A*+T*+G 11834 271 A25335HI CAGATGCCGAGTTCCGTAG +C*+A*+G*A*T*G*C*C*G*A*G*T*T*C*C*G*+T*+A*+G 10817 272 A25336HI GCTCAGATGCCGAGTTCCG +G*+C*+T*C*A*G*A*T*G*C*C*G*A*G*T*T*+c*+C*+G 10814 273 A25337HI GATCATGCACGGATCCAGC +G*+A*+T*C*A*T*G*C*A*C*G*G*A*T*C*C*+A*+G*+C 10434 274 A25338HI TGATCATGCACGGATCCAG +T*+G*+A*T*C*A*T*G*C*A*C*G*G*A*T*C*+C*+A*+G 10433 275 A25339HI GTGTTTGCTCATCTTGCCG +G*+T*+G*T*T*T*G*C*T*C*A*T*C*T*T*G*+C*+C*+G 9950 276 A25340HI GATACTCGACCACCTGAGC +G*+A*+T*A*C*T*C*G*A*C*C*A*C*C*T*G*+A*+G*+C 9629 277 A25341HI TGAGATACTCGACCACCTG +T*+G*+A*G*A*T*A*C*T*C*G*A*C*C*A*C*+C*+T*+G 9626 278 A25342HI TATGAGATACTCGACCACC +T*+A*+T*G*A*G*A*T*A*C*T*C*G*A*C*C*+A*+C*+C 9624 279 A25343HI GTATGAGATACTCGACCAC +G*+T*+A*T*G*A*G*A*T*A*C*T*C*G*A*C*+C*+A*+C 9623 280 A25344HI GCGGTATGAGATACTCGAC +G*+C*+G*G*T*A*T*G*A*G*A*T*A*C*T*C*+G*+A*+C 9620 281 A25345HI AGTGCCACAGTAAAGGTCG +A*+G*+T*G*C*C*A*C*A*G*T*A*A*A*G*G*+T*+C*+G 9270 282 A25346HI TCATGGAGATCGAGTAACT +T*+C*+A*T*G*G*A*G*A*T*C*G*A*G*T*A*+A*+C*+T 8954 283 A25347HI ACGGCCATTCGCAGGTGCT +A*+C*+G*G*C*C*A*T*T*C*G*C*A*G*G*T*+G*+C*+T 8231 284 A25348HI AAGACGGCCATTCGCAGGT +A*+A*+G*A*C*G*G*C*C*A*T*T*C*G*C*A*+G*+G*+T 8228 285 A25349HI TTAAAGACGGCCATTCGCA +T*+T*+A*A*A*G*A*C*G*G*C*C*A*T*T*C*+G*+C*+A 8225 286 A25350HI AGCTTAAAGACGGCCATTC +A*+G*+C*T*T*A*A*A*G*A*C*G*G*C*C*A*+T*+T*+C 8222 287 A25351HI GAAGCTTAAAGACGGCCAT +G*+A*+A*G*C*T*T*A*A*A*G*A*C*G*G*C*+C*+A*+T 8220 288 A25352HI CGTGTGACCTATGTGGTTA +C*+G*+T*G*T*G*A*C*C*T*A*T*G*T*G*G*+T*+T*+A 5799 289 A25353HI TGTACATTCGCATCATGAG +T*+G*+T*A*C*A*T*T*C*G*C*A*T*C*A*T*+G*+A*+G 5716 290 A25354HI TCTGTACATTCGCATCATG +T*+C*+T*G*T*A*C*A*T*T*C*G*C*A*T*C*+A*+T*+G 5714 291 A25355HI TGAGCCGTATTTATTAGAG +T*+G*+A*G*C*C*G*T*A*T*T*T*A*T*T*A*+G*+A*+G 5568 292 A25356HI CAGCATGAGCCGTATTTAT +C*+A*+G*C*A*T*G*A*G*C*C*G*T*A*T*T*+T*+A*+T 5563 293 A25357HI ACAGCATGAGCCGTATTTA +A*+C*+A*G*C*A*T*G*A*G*C*C*G*T*A*T*+T*+T*+A 5562 294 A25358HI CCGACACTCGAGACCATAT +C*+C*+G*A*C*A*C*T*C*G*A*G*A*C*C*A*+T*+A*+T 4754 295 A25359HI CGAATATAGTAGCTGGAGT +C*+G*+A*A*T*A*T*A*G*T*A*G*C*T*G*G*+A*+G*+T 4683 296 A25360HI TCTGATGGCCGAATATAGT +T*+C*+T*G*A*T*G*G*C*C*G*A*A*T*A*T*+A*+G*+T 4674 297 A25361HI AGTGGATAGGTGAGCTCGG +A*+G*+T*G*G*A*T*A*G*G*T*G*A*G*C*T*+C*+G*+G 4579 298 A25362HI GCGGAGTAACTTGCACACC +G*+C*+G*G*A*G*T*A*A*C*T*T*G*C*A*C*+A*+C*+C 4470 299 A25363HI CATTTGAGGCACGGCTTGG +C*+A*+T*T*T*G*A*G*G*C*A*C*G*G*C*T*+T*+G*+G 4024 300 A25364HI GTTTGGATTTGCGGACAGG +G*+T*+T*T*G*G*A*T*T*T*G*C*G*G*A*C*+A*+G*+G 3977 301 A25365HI TAGGTTTGGATTTGCGGAC +T*+A*+G*G*T*TT*G*G*A*T*TT*G*C*G*+G*+A*+C 3974 302 A25366HI GGCGCTAGGATGAAGGTTC +G*+G*+C*G*C*T*A*G*G*A*T*G*A*A*G*G*+T*+T*+C 3609 303 A25367HI GTGGCGCTAGGATGAAGGT +G*+T*+G*G*C*G*C*T*A*G*G*A*T*G*A*A*+G*+G*+T 3607 304 A25368HI GTGTGGCGCTAGGATGAAG +G*+T*+G*T*G*G*C*G*C*T*A*G*G*A*T*G*+A*+A*+G 3605 305 A25369HI TAGGTGTGGCGCTAGGATG +T*+A*+G*G*T*G*T*G*G*C*G*C*T*A*G*G*+A*+T*+G 3602 306 A25370HI GGTTAGGTGTGGCGCTAGG +G*+G*+T*T*A*G*G*T*G*T*G*G*C*G*C*T*+A*+G*+G 3599 307 A25371HI TTAGGTGGTTAGGCTCAGG +T*+T*+A*G*G*T*G*G*T*T*A*G*G*C*T*C*+A*+G*+G 3374 308 A25372HI GTTAGGTGGTTAGGCTCAG +G*+T*+T*A*G*G*T*G*G*TT*A*G*G*C*T*+C*+A*+G 3373 309 A25373HI CGAGTATCTTACGTGTCAG +C*+G*+A*G*T*A*T*C*T*T*A*C*G*T*G*T*+C*+A*+G 3347 322 A25374HI TCGAGTATCTTACGTGTCA +T*+C*+G*A*G*T*A*T*C*T*T*A*C*G*T*G*+T*+C*+A 3346 310 A25375HI ATTATCGAGTATCTTACGT +A*+T*+T*A*T*C*G*A*G*T*A*T*C*T*T*A*+C*+G*+T 3342 311 A25376HI ATGGTTTGAATTATCGAGT +A*+T*+G*G*T*T**G*A*A*T*T*A*T*C*G*+A*+G*+T 3333 312 A25010H* CTTGTCGGATGATGCCA +C*+T*+T*G*T*C*G*G*A*T*G*A*T*G*+C*+C*+A 1003 313 A25024H* GATGGCGTTCTTCCAGG +G*+A*+T*G*G*C*G*T*T*C*T*T*C*C*+A*+G*+G 1327 314 A25119H* CCGTTGAGAGCTGGTGCA +C*+C*+G*T*T*G*A*G*A*G*C*T*G*G*T*+G*+C*+A 496 315 A25121H* CCTTGTCGGATGATGCCA +C*+C*+T*T*G*T*C*G*G*A*T*G*A*T*G*+C*+C*+A 1003 316 A25124H* GATGGCGTTCTTCCAGGT +G*+A*+T*G*G*C*G*T*T*C*T*T*C*C*A*+G*+G*+T 1326 317 A25137H* CCGTTGAGAGCTGGTGCAT +C*+C*+G*T*T*G*A*G*A*G*C*T*G*G*T*G*+C*+A*+T 495 318 A25203H* CTTGTCGGATGATGCCAC +C*+T*+T*G*T*C*G*G*A*T*G*A*T*G*C*+C*+A*+C 1002 319 A25222H* CGTTGAGAGCTGGTGCATG +C*+G*+TT*G*A*G*A*G*C*T*G*G*T*G*C*+A*+T*+G 494 320 A25226H* TCGGATGATGC CACAGATG +T*+C*+G*G*A*T*G*A*T*G*C*C*A*C*A*G*+A*+T*+G 997 321 A25236H* GATGGCGTTCTTCCAGGTG +G*+A*+T*G*G*C*G*T*T*C*T*T*C*C*A*G*+G*+T*+G 1325 323 Control +C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T oligo
[0042] The oligonucleotides such as antisense oligonucleotides of the present invention hybridize for example with mRNA of human FoxP3 of SEQ ID NO.1 and/or introns of the pre-mRNA of human FoxP3 of SEQ ID NO.2. Such antisense oligonucleotides are called FoxP3 antisense oligonucleotides. The antisense oligonucleotides hybridize for example within a hybridizing active area which is one or more region(s) on the FoxP3 mRNA, e.g., of SEQ ID NO.1 and/or the FoxP3 pre-mRNA, e.g., of SEQ ID NO.2, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the FoxP3 expression. In the present invention surprisingly several hybridizing active regions were identified for example selected from position 1510 to 2109, position 1510 to 1809, position 1810 to 2109, position 2410 to 2709, position 2710 to 3009, position 3310 to 3609, position 3610 to 3909, position 3910 to 4209, position 4210 to 4509, position 4510 to 4809, position 4810 to 5109, position 5110 to 5409, position 5410 to 5709, position 5710 to 6009, position 6610 to 6909, position 7810 to 8109, position 8110 to 8409, position 8710 to 9009, position 9010 to 9309, position 9610 to 9909, position 9910 to 10209, position 10210 to 10509, position 10810 to 11109, position 11410 to 11709, position 11710 to 12009, position 12010 to 12309, position 12310 to 12609, position 12610 to 12909, position 12910 to 13209, position 13510 to 13809, position 14410 to 14709, position 14710 to 15009, position 15010 to 15309, position 15310 to 15609, position 15610 to 15909 or a combination thereof (including the terminal figures of the ranges) of FoxP3 pre-mRNA for example of SEQ ID NO.2. Antisense oligonucleotides hybridizing with these regions are indicated in the following Table 2:
TABLE-US-00002 First Region of SEQ position SEQ ID NO.2/ on SEQ ID ID ASO name NO.2 NO Region 1510-1809 A25028H 1778 24 A25029H 1768 25 A25030H 1739 26 A25069H 1779 56 A25070H 1778 24 A25071H 1777 57 A25072H 1768 25 A25073H 1740 58 A25074H 1739 26 A25075H 1739 26 A25076H 1739 26 A25077H 1737 59 A25078H 1510 27 A25096H 1779 56 A25097H 1779 56 A25098H 1779 56 A25099H 1740 58 A25100H 1740 58 A25101H 1740 58 A25102H 1778 24 A25103H 1778 24 A25104H 1778 24 A25106H 1768 25 A25107H 1739 26 A25108H 1739 26 A25109H 1510 27 A25110H 1739 26 A25111H 1737 59 A25128H 1778 83 A25129H 1777 84 A25130H 1768 85 A25131H 1758 86 A25132H 1755 87 A25133H 1737 88 A25134H 1735 89 A25135H 1513 90 A25149H 1758 102 A25150H 1739 103 A25151H 1738 104 A25152H 1737 105 A25153H 1737 105 A25154H 1737 105 A25155H 1737 105 A25156H 1733 106 A25157H 1733 106 A25158H 1513 107 A25159H 1510 108 A25219H 1514 167 A25249H 1778 194 A25250H 1777 195 A25251H 1769 196 A25252H 1762 197 A25253H 1759 198 A25254H 1757 199 A25255H 1756 200 A25256H 1755 201 A25257H 1755 201 A25258H 1736 202 A25259H 1579 203 A25260H 1576 204 A25261H 1514 205 Region 1810-2109 A25027H 1820 23 A25068H 1820 23 A25105H 1820 23 A25126H 2069 81 A25127H 1966 82 A25147H 1822 101 A25148H 1822 101 A25218H 2068 166 A25242H 2068 187 A25243H 2067 188 A25244H 1967 189 A25245H 1966 190 A25246H 1821 191 A25247H 1820 192 A25248H 1814 193 Region 2410-2709 A25025H 2526 21 A25026H 2455 22 A25125H 2455 80 A25146H 2444 100 A25217H 2526 165 A25237H 2529 182 A25238H 2520 183 A25239H 2456 184 A25240H 2455 185 A25241H 2441 186 Region 2710-3009 A25023H 2780 20 A25144H 2791 98 A25145H 2790 99 A25216H 2790 164 A25235H 2772 181 Region 3310-3609 A25060HI 3558 51 A25061HI 3431 52 A25062HI 3361 53 A25063HI 3361 54 A25166HI 3599 115 A25167HI 3342 116 A25197HI 3606 146 A25198HI 3415 147 A25199HI 3343 148 A25300HI 3605 238 A25301HI 3601 239 A25302HI 3599 115 A25303HI 3415 240 A25304HI 3347 241 A25305HI 3344 242 A25306HI 3342 116 A25366HI 3609 302 A25367HI 3607 303 A25368HI 3605 304 A25369HI 3602 305 A25370HI 3599 306 A25371HI 3374 307 A25372HI 3373 308 A25373HI 3347 309 A25374HI 3346 322 A25375HI 3342 310 A25376HI 3333 311 Region 3610-3909 A25057HI 3617 50 A25095HI 3616 75 Region 3910-4209 A25013H 4205 10 A25014H 4200 11 A25015H 4199 12 A25016H 4199 13 A25017H 4198 14 A25018H 4198 15 A25019H 4198 16 A25020H 4197 17 A25021H 4197 18 A25022H 4192 19 A25122H 4206 78 A25123H 4199 79 A25139H 4206 93 A25140H 4205 94 A25141H 4204 95 A25142H 4203 96 A25143H 4202 97 A25208H 4205 156 A25209H 4204 157 A25210H 4202 158 A25211H 4201 159 A25212H 4198 160 A25213H 4197 161 A25214H 4196 162 A25215H 4195 163 A25231H 4201 177 A25232H 4199 178 A25233H 4198 179 A25234H 4196 180 A25299HI 4022 237 A25363HI 4024 299 A25364HI 3977 300 A25365HI 3974 301 Region 4210-4509 A25065H 4225 55 A25138H 4228 92 A25207H 4227 155 A25229H 4255 175 A25230H 4250 176 A25298HI 4470 236 A25362HI 4470 298 Region 4510-4809 A25196HI 4677 145 A25296HI 4755 234 A25297HI 4677 235 A25358HI 4754 294 A25359HI 4683 295 A25360HI 4674 296 A25361HI 4579 297 Region 4810-5109 A25012H 5101 9 A25204H 5102 152 A25205H 5094 153 A25206H 5019 154 A25227H 5103 173 A25228H 5019 174 A25295HI 4943 233 Region 5110-5409 A25011H 5119 8 Region 5410-5709 A25053HI 5608 47 A25054HI 5605 48 A25055HI 5604 49 A25090HI 5606 71 A25091HI 5607 72 A25092HI 5605 73 A25093HI 5603 74 A25115H 5604 49 A25117H 5605 73 A25164HI 5590 113 A25165HI 5589 114 A25191HI 5592 140 A25192HI 5590 140 A25193HI 5589 142 A25194HI 5588 143 A25195HI 5564 144 A25290HI 5591 230 A25291HI 5590 113 A25292HI 5589 114 A25293HI 5569 231 A25294HI 5563 232 A25355HI 5568 291 A25356HI 5563 292 A25357HI 5562 293 Region 5710-6009 A25190HI 5717 139 A25288HI 5798 228 A25289HI 5717 229 A25352HI 5799 288 A25353HI 5716 289 A25354HI 5714 290 Region 6610-6909 A25005H 6847 4 A25006H 6845 5 A25008H 6843 6 A25009H 6842 7 A25120H 6843 77 A25201H 6847 150 A25202H 6842 151 A25225H 6842 172 Region 7810-8109 A25004H 7838 3 A25200H 7838 149 A25223H 7841 170 A25224H 7840 171 Region 8110-8409 A25050HI 8247 44 A25051HI 8244 45 A25052HI 8243 46 A25089HI 8241 70 A25163HI 8224 112 A25188HI 8236 137 A25189HI 8227 138 A25285HI 8228 226 A25286HI 8224 112 A25287HI 8223 227 A25347HI 8231 283 A25348HI 8228 284 A25349HI 8225 285 A25350HI 8222 286 A25351HI 8220 287 Region 8710-9009 A25346HI 8954 282 Region 9010-9309 A25345HI 9270 281 Region 9610-9909 A25048HI 9645 42 A25049HI 9639 43 A25186HI 9630 135 A25187HI 9625 136 A25284HI 9623 225 A25340HI 9629 276 A25341HI 9626 277 A25342HI 9624 278 A25343HI 9623 279 A25344HI 9620 280 Region 9910-10209 A25047HI 9995 41 A25339HI 9950 275 Region 10210-10509 A25046HI 10450 40 A25088HI 10448 69 A25185HI 10257 134 A25282HI 10434 223 A25283HI 10001 224 A25337HI 10434 273 A25338HI 10433 274 Region 10810-11109 A25044HI 10832 38 A25045HI 10838 39 A25086HI 10835 67 A25087HI 10834 68 A25162HI 10818 111 A25281HI 10818 111 A25335HI 10817 271 A25336HI 10814 272 Region 11410-11709 A251841HI 11469 133 Region 11710-12009 A25038HMI 11933 34 A25039HI 11886 35 A25040HI 11849 36 A25041HI 11938 37 A25084HI 11938 65 A25085HMI 11936 66 A25112H 11933 34 A25113H 11933 34 A25114H 11933 34 A25116H 11936 66 A25177HI 11926 126 A25178HI 11925 127 A25179HI 11923 128 A25180HI 11922 129 A25181HI 11918 130 A25182HI 11917 131 A25183HI 11916 132 A25277HI 11923 219 A25278HI 11922 220 A25279HI 11918 221 A25280HI 11916 222 A25332HI 11872 268 A25333HI 11870 269 A25334HI 11834 270 Region 12010-12309 A25176H1 12245 125 A25331HI 12142 267 Region 12310-12609 A25175HI 12509 124 A25275HI 12511 217 A25276HI 12408 218 A25327HI 12510 263 A25328HI 12508 264 A25329HI 12507 265 A25330HI 12506 266 Region 12610-12909 A25036HI 12736 32 A25037HI 12734 33 A25161HI 12720 110 A25174HI 12632 123 A25272HI 12720 110 A25273HI 12719 215 A25274HI 12633 216 A25324HI 12720 260 A25325HI 12719 261 A25326HI 12624 262 Region 12910-1309 A25173HI 13198 122 A25321HI 13199 257 A25322HI 13015 258 A25323HI 13001 259 Region 13510-13809 A25320HI 13537 256 Region 14410-14709 A25035HI 14551 31 A25172HI 14539 121 A25271HI 14540 214 A25317HI 14542 253 A25318HI 14540 254 A25319HI 14538 255 Region 14710-15009 A25083HI 14882 64 A25170HI 14919 119 A25171HI 14852 120 A25268HI 14924 211 A25269HI 14866 212 A25270HI 14865 213 A25314HI 14923 250 A25315HI 14875 251 A25316HI 14865 252 Region 15010-15309 A25032HI 15174 28 A25033HI 15058 29 A25034HI 15055 30 A25079HI 15176 60 A25080HI 15173 61 A25081HI 15056 62 A25082HI 15056 63 A25160HI 15040 109 A25168HI 15154 117 A25169HI 15040 118 A25262HI 15244 206 A25263HI 15160 207 A25264HI 15157 208 A25265HI 15041 209 A25266HI 15040 109 A25267HI 15039 210 A25309HI 15247 245 A25310HI 15061 246 A25311HI 15059 247 A25312HI 15041 248 A25313HI 15033 249 Region 15310-15609 A25307HI 15579 243 A25308HI 15478 244 Region 15610-15909 A25118H 15817 76 A25136H 15817 91 A25220H 15815 168 A25221H 15731 169
[0043] Table 2 shows some hybridizing active regions and antisense oligonucleotides hybridizing in this region.
[0044] Table 3 specifies exon spanning oligonucleotides such as antisense oligonucleotides hybridizing for example with FoxP3 mRNA of SEQ ID NO.1:
TABLE-US-00003 Region of SEQ First position SEQ ID NO. 1/ on SEQ ID ID ASO name NO.1 NO. Exon spanning ASO A25010H 1003 312 A25024H 1327 313 A25119H 496 314 A25121H 1003 315 A25124H 1326 316 A25137H 495 317 A25203H 1002 318 A25222H 494 319 A25226H 997 320 A25236H 1325 321
[0045] The following Table 4 presents examples of oligonucleotides such as antisense oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The antisense oligonucleotides consisting of or comprising the sequences of Table 4 may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 4 hybridize with the mRNA of mouse FoxP3 (SEQ ID NO.324; NM_001199347.1) or with intronic regions of the pre-mRNA of mouse FoxP3 (SEQ ID NO.325; GRCm38.p6 (GCF_000001635.26, Chr X (NC_000086.7): 7,578,119-7,596,800), indicated by “I” in the following Table 4:
TABLE-US-00004 TABLE 4 List of mouse FoxP3-specific antisense oligonucleotides and a control oligonucleotide. An “M” after the antisense oligonucleotide ID indicates a mouse FoxP3-specific sequence that binds to an exonic region of the pre-mRNA, a “MR” after the antisense oligonucleotide ID indicates a mouse/rat cross-reactive FoxP3 sequence that binds to an exonic region of the pre-mRNA and a “MI” after the antisense oligonucleotide ID indicates a mouse FoxP3-specific sequence that binds to an intronic region of the pre-mRNA. Seq Antisense Antisense ID Name Sequence 5′-3′ Sequence 5′-3′ with PTO (*) and LNA (+) 526 A25001M GTCTCGTCTGAAGGCAG +G*+T*C*T*C*G*T*C*T*G*A*A*G*G*+C*+A*+G 527 A25002MR GGATAACGGCAGAGGAG +G*+G*+A*T*A*A*C*G*G*C*A*G*A*G*+G*+A*+G 528 A25003M TACTGGTGGCTACGATG +T*+A*+C*T*G*G*T*G*G*C*T*A*C*G*+A*+T*+G 329 A25004M TACTGGTGGCTACGAT +T*+A*C*T*G*G*T*G*G*C*T*A*C*+G*+A*+T 330 A25005M AAACAGGCCGCCGTCT +A*+A*+A*C*A*G*G*C*C*G*C*C*G*T*+C*+T 331 A25006M TGCAAACAGGCCGCCGT +T*+G*+C*A*A*A*C*A*G*G*C*C*G*C*+C*+G*+T 332 A25007M CACTGCAAACAGGCCGC +C*+A*+C*T*G*C*A*A*A*C*A*G*G*C*+C*+G*+C 333 A25008M TCGCATATTGTGGTACT +T*+C*+G*C*A*T*A*T*T*G*T*G*G*T*+A*+C*+T 334 A25009M GGTCGCATATTGTGGTA +G*+G*+T*C*G*C*A*T*A*T*T*G*T*G*+G*+T*+A 335 A25010MR GATTTCATTGAGTGTC C +G*+A*T*T*T*C*A*T*T*G*A*G*T*G*T*+C*+ C 336 A25011M GAACATGCGAGTAAAC C +G*+A*+A*C*A*T*G*C*G*A*G*T*A*A*+A*+C*+C 337 A25012M AGGCGAACATGCGAGTA +A*+G*+G*C*G*A*A*C*A*T*G*C*G*A*+G*+T*+A 338 A25013M TAGGCGAACATGCGAGT +T*+A*+G*G*C*G*A*A*C*A*T*G*C*G*+A*+G*+T 339 A25014M GTAGGCGAACATGCGAG +G*+T*+A*G*G*C*G*A*A*C*A*T*G*C*+G*+A*+G 340 A25015M GTAGGCGAACATGCGA +G*+T*+A*G*G*C*G*A*A*C*A*T*G*+C*+G*+A 341 A25016M AGTAGGCGAACATGCGA +A*+G*+T*A*G*G*C*G*A*A*C*A*T*G*+C*+G*+A 342 A25017M TCGCTCTCCACTCGCAC +T*+C*+G*C*T*C*T*C*C*A*C*T*C*G*+C*+A*+C 343 A25018MR TCATCTACGGTCCACAC +T*+C*+A*T*C*T*A*C*G*G*T*C*C*A*+C*+A*+C 344 A25019MR ATTCATCTACGGTCCAC +A*+T*+T*C*A*T*C*T*A*C*G*G*T*C*+C*+A*+C 345 A25020M CGTAGGACTTGCCTCCT +C*+G*T*A*G*G*A*C*T*T*G*C*C*T*C*+C*+T 346 A25021M TACACGTAGGACTTGCC +T*+A*+C*A*C*G*T*A*G*G*A*C*T*T*+G*+C*+C 347 A25022M ATAGGTACACGTAGGAC +A*+T*+A*G*G*T*A*C*A*C*G*T*A*G*+G*+A*+C 348 A25023M TAGCAGGCACATCATCG +T*+A*+G*C*A*G*G*C*A*C*A*T*C*A*+T*+C*+G 349 A25024M TTCACGAATGTACCAAG +T*+T*+C*A*C*G*A*A*T*G*T*A*C*C*+A*+A*+G 350 A25025MR GATCAGTTATGCCTGTG +G*+A*+T*C*A*G*T*T*A*T*G*C*C*T*+G*+T*+G 351 A25026M CTTGAGGCTGCGTATGA +C*+T*+T*G*A*G*G*C*T*G*C*G*T*A*+T*+G*+A 352 A25027M TTGCTTGAGGCTGCGTA +T*+T*+G*C*TT*G*A*G*G*C*T*G*C*+G*+T*+A 353 A25028M ATTGCTTGAGGCTGCGT +A*+T*+T*G*C*TT*G*A*G*G*C*T*G*+C*+G*+T 354 A25029M TTGGAGAGTCGGTGTGT +T*+T*+G*G*A*G*A*G*T*C*G*G*T*G*+T*+G*+T 355 A25030M TACATCTTGGAGAGTCG +T*+A*+C*A*T*C*T*T*G*G*A*G*A*G*+T*+C*+G 356 A25031MR ACGCTTAGGCATGGATT +A*+C*+G*C*T*T*A*G*G*C*A*T*G*G*+A*+T*+T 357 A25032M TTCATTTGGTATCCGCT +T*+T*+C*A*T*T*T*G*G*T*A*T*C*C*+G*+C+T 358 A25033MR GTGAGGACTACCGAGCC +G*+T*+G*A*G*G*A*C*T*A*C*C*G*A*+G*+C*+C 359 A25034MR ATCTGTGAGGACTACCG +A*+T*+C*T*G*T*G*A*G*G*A*C*T*A*+C*+C*+G 360 A25035M TGTTTTGCGCTGAGAGT +T*+G*+T*T*T*T*G*C*G*C*T*G*A*G*+A*+G*+T 361 A25036M TTCGGAAAGCCTACAAG +T*+T*+C*G*G*A*A*A*G*C*C*T*A*C*+A*+A*+G 362 A25037MR CTGTTCGGAAAGCCTAC +C*+T*+G*T*T*C*G*G*A*A*A*G*C*C*+T*+A*+C 363 A25038MR AAGGATGATGCTGTTCG +A*+A*+G*G*A*T*G*A*T*G*C*T*G*T*+T*+C*+G 364 A25039M CTCGACCGGACATTTGC +C*+T*+C*G*A*C*C*G*G*A*C*A*T*T*+T*+G*+C 365 A25040M CTCGACCGGACATTTG +C*+T*+C*G*A*C*C*G*G*A*C*A*T*+T*+T*+G 366 A25041M GCTCGACCGGACATTT +G*+C*+T*C*G*A*C*C*G*G*A*C*A*+T*+T*+T 367 A25042M AGCTCGACCGGACATTT +A*+G*+C*T*C*G*A*C*C*G*G*A*C*A*+T*+T*+T 368 A25043M GGAAGCTCGACCGGACA +G*+G*+A*A*G*C*T*C*G*A*C*C*G*G*+A*+C*+A 369 A25044M ATCTTGTCGGACACAAA +A*+T*+C*T*T*G*T*C*G*G*A*C*A*C*+A*+A*+A 370 A25045M AGATCTTGTCGGACACA +A*+G*+A*T*C*T*T*G*T*C*G*G*A*C*+A*+C*+A 371 A25046MI CCGTGATGCGATGAGC +C*+C*+G*T*G*A*T*G*C*G*A*T*G*+A*+G*+C 372 A25047MI GGAGCTATATAGCCGTA +G*+G*+A*G*C*T*A*T*A*T*A*G*C*C*+G*+T*+A 373 A25048MI TGGTCCGCTAGGACTTC +T*+G*G*T*C*C*G*C*T*A*G*G*A*C*T*+T*+C 374 A25049MI CATCGTTACTAGTGTTC +C*+A*+T*C*G*T*T*A*C*T*A*G*T*G*+T*+T*+C 375 A25050MI TCTTGCAAAGTTCGTAC +T*+C*+T*T*G*C*A*A*A*G*T*T*C*G*+T*+A*+C 376 A25051MI CCAAGTTCTATCGATTC +C*+C*+A*A*G*T*T*C*T*A*T*C*G*A*+T*+T*+C 377 A25052MI AGTCTATCCTGTAGCCG +A*+G*+T*C*T*A*T*C*C*T*G*T*A*G*+C*+C*+G 378 A25053MHI CCACAGGTTTCGTTCCG +C*+C*+*A*C*A*G*G*T*T*T*C*G*T*T*+C*+C*+G 379 125054MRHI GTCATGGCGGCCGGATG +G*+T*C*A*T*G*G*C*G*G*C*C*G*G*+A*+T*+G 380 A25055MI ACTATATTGGCTTAACC +A*+C*+T*A*T*A*T*T*G*G*C*T*T*A*+A*+C*+C 381 A25056MI CCGTGATGCGATGAGCT +C*+C*+G*T*G*A*T*G*C*G*A*T*G*A*+G*+C*+T 382 A25057MI AAGACTAGTGTGTCACG +A*+A*+G*A*C*T*A*G*T*G*T*G*T*C*+A*+C*+G 383 A25058MI CCGTTCTACTATATACT +C*+C*+G*T*T*C*T*A*C*T*A*T*A*T*+A*+C*+T 384 A25059MI ATAGTGAGGCGAGTGGT +A*+T*+A*G*T*G*A*G*G*C*G*A*G*T*+G*+G*+T 385 A25060MI TACCACTCTGTCGTGAA +T*+A*+C*C*A*C*T*C*T*G*T*C*G*T*+G*+A*+A 386 A25061MI CACACGGTAGCAACAAT +C*+A*+C*A*C*G*G*T*A*G*C*A*A*C*+A*+A*+T 387 A25062MI TGCTCCGATTCCATACC +T*+G*+C*T*C*C*G*A*T*T*C*C*A*T*+A*+C*+C 388 A25063MI GGTTGGAGTTTCCGTGA +G*+G*+T*T*G*G*A*G*TT*T*C*C*G*+T*+G*+A 389 A25064MI GACTGATAATAGCGATT +G*+A*+C*T*G*A*T*A*A*T*A*G*C*G*+A*+T*+T 390 A25065MI TACATGCGAGGTAAACT +T*+A*+C*A*T*G*C*G*A*G*G*T*A*A*+A*+C*+T 391 A25066MI TTAGATCCTTCTGCGTG +T*+T*+A*G*A*T*C*C*T*T*C*T*G*C*+G*+T*+G 392 A25067MI CTGGCCACGCAAACACG +C*+T*+G*G*C*C*A*C*G*C*A*A*A*C*+A*+C*+G 393 A25068MI ACGTTAGACAGGAGGTA +A*+C*+G*TT*A*G*A*C*A*G*G*A*G*+G*+T*+A 394 A25069MI GTAAGCAGAGTAGGCGT +G*+T*+A*A*G*C*A*G*A*G*T*A*G*G*+C*+G*+T 395 A25070MI GGTAATCGAGACACTTA +G*+G*+T*A*A*T*C*G*A*G*A*C*A*C*+T*+T*+A 396 control C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T oligo
[0046] The oligonucleotides of the present invention hybridize for example with mRNA and/or pre-mRNA of FoxP3 of SEQ ID NO. 1 and SEQ ID NO.2, respectively. Such oligonucleotides are called FoxP3 antisense oligonucleotides. Oligonucleotides of the present invention, which are for example antisense oligonucleotides, are shown in Tables 1 and 4. 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 4.
[0047] The oligonucleotide such as an antisense oligonucleotide of the present invention inhibits for example 40% to 99%, 50% to 98%, 60% to 95%, 70% to 90% or at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of FoxP3 expression (mRNA and/or pre-mRNA) such as the, e.g., human, rat or mouse, FoxP3 expression, e.g., within 6 to 240 h, 12 to 216 h, 18 to 120 h or 24 to 72 h, or 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h, 132 h, 144 h, 156 h, 168 h, 180 h, 192 h, 204 h, 216 h, 228 h or 240 h, preferably 24 to 72 h, e.g., compared to an untreated control. The untreated control is for example FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA expression or a combination thereof in a subject before an oligonucleotide of the present invention is administered or an untreated sample such as a cell, blood, urine, saliva etc.
[0048] The oligonucleotides of the present invention are for example active and inhibit expression for example in a cell, tissue, organ, or a subject. The oligonucleotide such as an antisense oligonucleotide of the present invention inhibits the expression of FoxP3 for example at a nanomolar or micromolar concentration for example in a concentration of 0.1, 1, 1.5, 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 μM.
[0049] The oligonucleotide of the present invention is for example used in a concentration of 1 to 100 nM, 5 to 90 nM, 10 to 80 nM, 15 to 70 nM, 20 to 60 nM, 25 to 50 nM, 30 to 45 nM or 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1 to 50 μM, 3 to 40 μM, 5 to 30 μM, 8 to 25 μM, 10 to 15 μM, or 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, or 1.5 nM.
[0050] The oligonucleotide such as an antisense oligonucleotide of the present invention is administered to a cell, tissue, organ or subject one or more times a day, one or more times a week, one or more times a month or one or more times a year.
[0051] In some embodiments 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 a chemotherapeutic, another disease specific active agent, another oligonucleotide, an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin and/or a small molecule which is for example effective in tumor treatment, or chronic inflammation, e.g., associated with chronic infections.
[0052] In some embodiments, the oligonucleotide such as an antisense oligonucleotide or the pharmaceutical composition of the present invention is for use in a method of preventing and/or treating a disorder. The use of the oligonucleotide or the pharmaceutical composition of the present invention for example in a method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine). The disorder is for example characterized by a FoxP3 imbalance, i.e., the FoxP3 level for example is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. Alternatively or in addition, FoxP3 expression for example is involved in the induction and/or maintenance of the disease and/or mediates resistance to another therapy. The FoxP3 level is for example increased by an increased FoxP3 expression and functionality, respectively. The FoxP3 level can be measured by any standard method such as immunohistochemistry, flow cytometry, western blot, quantitative real time PCR, HPLC, UHPLC, FPLC or QuantiGene assay known to a person skilled in the art.
[0053] An oligonucleotide such as an antisense oligonucleotide or a pharmaceutical composition of the present invention is for example administered locally or systemically for example orally, sublingually, nasally, inhaled, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, transdermally, and/or rectally. Alternatively or in combination an immune cell ex vivo treated with an oligonucleotide such as an oligonucleotide of the present invention is administered. In another alternative an oligonucleotide of the present invention is used in a cell therapy method and for example administered in combination with CAR-T cells, transgenic TCR-T cells or ex vivo expanded TILs. The oligonucleotide such as an antisense oligonucleotide of the present invention is administered alone or in combination with another oligonucleotide of the present invention and optionally in combination with another compound such as a chemotherapeutic (e.g., platinum, gemcitabine), another disease specific agent, another oligonucleotide (e.g., an oligonucleotide not being part of the present invention), an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin and/or a small molecule. The other compound such as a chemotherapeutic, another disease specific agent, another oligonucleotide (i.e., lot being part of the present invention), the antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin and/or the small molecule are for example effective in preventing and/or treating a malignant and/or benign tumor, a chronic infection, a chronic inflammatory disease or a combination thereof.
[0054] An oligonucleotide such as an antisense oligonucleotide or a pharmaceutical composition of the present invention is for example for use in a method of preventing and/or treating a chronic inflammatory disease, a chronic infection, a malignant and/or benign tumor or a combination thereof. Examples of tumors preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are 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, osteosarcoma, 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.
[0055] An oligonucleotide such as an antisense oligonucleotide or a pharmaceutical composition of the present invention is for example for use in a method of preventing and/or treating a chronic infectious disease, wherein the chronic infectious disease is for example selected from the group consisting of hepatitis B and C virus, human immune deficiency virus, cytomegalovirus, Herpes Simplex virus, Measles virus, respiratory syncytial virus, Helicobacter pylori infection or a combination thereof.
[0056] An oligonucleotide such as an antisense oligonucleotide or a pharmaceutical composition) of the present invention is for example for use in a method of preventing and/or treating a chronic inflammatory disease caused by infection, wherein the chronic inflammatory disease caused by infection is for example selected from the group consisting of chronic inflammatory diseases of the liver such as liver fibrosis, liver cirrhosis or a combination thereof.
[0057] In some embodiments two or more oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals for example as a pharmaceutical composition. Alternatively or in addition, one or more oligonucleotides of the present invention are administered together with another compound such as a chemotherapeutic, a disease specific agent, another oligonucleotide (i.e., not being part of the present invention), an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin and/or a small molecule, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals.
[0058] The oligonucleotide such as an antisense oligonucleotide of the present invention inhibits for example the expression and functionality, respectively, of FoxP3 and an antitumor active agent such as a chemotherapeutic, a disease specific agent, another oligonucleotide (i.e., not being part of the present invention), an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a therapeutic vaccine, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a DARPin and/or small molecule inhibits (antagonist) an immune suppressive factor and/or stimulates (agonist) an immune stimulatory factor or inhibits another target that is involved in cancer progression and/or metastasis directly and/or indirectly. The immune suppressive factor is for example selected from the group consisting of IDOL IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STATS, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, MTDH, Xbp1, Chop and a combination thereof. The immune stimulatory factor is for example selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof. The factor involved in cancer progression and/or metastasis is for example selected from the group consisting of SND1, MTDH, HER-2, BRAF, KRAS, VEGF, EGFR1, EGFR2, BCR/ABL, ABL, MET, ALK, JAK2, BTK, miR-223, CCL18, CCL20, Lcn2, CCL5/CCR9, DDR2, PHD2, IL6, SDF-1/CXCL12 and a combination hereof. An antisense oligonucleotide or a pharmaceutical composition of the present invention is for example combined with or comprises a therapeutic vaccine. In such combination the antisense oligonucleotide and the therapeutic vaccine are administered together or separately for example at the same time or at different times.
[0059] The immune suppressive factor is a factor whose expression and/or activity is for example decreased or increased in a cell, tissue, organ or subject. The immune stimulatory factor is a factor whose expression and/or activity is for example increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions. The factor involved in cancer progression and/or metastasis is a factor whose expression and/or activity is for example increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions in comparison to a healthy subject or is for example involved in the induction and/or maintenance of the disease and/or mediates resistance to another therapy.
[0060] The antisense oligonucleotide or pharmaceutical composition inhibiting the expression and/or functionality of FoxP3 results for example in an increase of the expression of a pro-inflammatory gene such as IL2 and/or IFNγ and/or Granzyme B and/or wherein the inhibition of FoxP3 results in a decrease of the expression of an immunosuppressive gene such as CD25, CD39, CD73, NRP1, TGF-beta, GARP, CCR4, Ctla4, and/or Tnfrsf18.
[0061] An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide such as an antisense oligonucleotide or the pharmaceutical composition of the present invention are for example Sunitinib, Alecitinib, Afatinib, Ibrutinib, Imatinib, Lenvatinib, Sorafenib, or Epacadostat. A chemotherapy in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example platinum or gemcitabine.
[0062] Moreover, one or more oligonucleotides such as antisense oligonucleotides of the present invention are used in determining the status of a cancer disease.
[0063] A subject of the present invention is for example a mammalian such as a human, dog, cat, horse, cow, pig etc., a bird or a fish.
EXAMPLES
[0064] 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 FoxP3, 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 IC50 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 IC50 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 hardly 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 Human FoxP3-Specific Antisense Oligonucleotides (ASOs)
[0065] For the design of ASOs with specificity for exonic regions within the human FoxP3 gene the FoxP3 mRNA sequence with the RefSeq ID NM_014009.3 was used. For ASOs with specificity for intronic regions within the human FoxP3 gene the FoxP3 pre-mRNA sequence (GRCh38.p13 (GCF_000001405.39, Chr X (NC_000023.11): 49,249,986K—49,226,382—pre-mRNA positions as annotated in FASTA format (visible range) downloaded from https://www.ncbi.nlm.nih.gov/genome/gdv/browser/?context=genome&acc=GCF_0000014 05.39) was used. An “H” after the ASO ID indicates a human FoxP3-specific sequence that binds to an exonic region of the pre-mRNA, a “HM” after the ASO ID indicates a human/mouse cross-reactive FoxP3 sequence that binds to an exonic region of the pre-mRNA and a “HI” after the ASO ID indicates a human FoxP3-specific sequence that binds to an intronic region of the pre-mRNA. 15, 16, 17, 18 and 19 mers for example were designed according to in house criteria, neg1 (described in WO2014154843 A1) was iced as control oligonucleotide in all experiments. Examples of oligonucleotides such as antisense oligonucleotides are shown in Table 1.
Example 2: Target Knockdown Efficacy Screens of Human FoxP3-Specific ASOs in T Cells in a 1.SUP.st .Screening Round
[0066] In order to investigate the knockdown efficacy of the in silico designed FoxP3 ASOs, two efficacy screening rounds were performed in human CD4.sup.+ T cells. Therefore, cells were treated with the respective ASO at a concentration of 5 μM for three days without the addition of a transfection reagent. In addition cells were treated with TGF-beta, ATRA, IL-2 and stimulated with CD3/CD28 beads (ThermoFisher) in order to increase expression levels of FoxP3. Cells were lyzed after the three days treatment period, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values. The results for the first screening round of ASOs are shown in
TABLE-US-00005 TABLE 5 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 1 compared to mock treated cells in first screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25030H 0.33 A25011H 0.96 A25027H 0.38 A25026H 0.97 A25055HI 0.39 A25010H 0.98 A25031H 0.44 A25005H 1 A25038HMI 0.48 A25012H 1.02 A25028H 0.49 A25063HI 1.03 A25029H 0.56 A25023H 1.03 A25054HI 0.71 A25045HI 1.05 A25020H 0.72 A25022H 1.06 A25021H 0.72 A25009H 1.1 A25017H 0.75 A25062HI 1.11 A25019H 0.77 A25036HI 1.11 A25052HI 0.78 A25032HI 1.12 A25016H 0.8 A25035HI 1.12 A25037HI 0.81 A25050HI 1.12 A25044HI 0.81 A25008H 1.13 A25047HI 0.83 A25060HI 1.18 A25033HI 0.83 A25013H 1.2 A25018H 0.85 A25048HI 1.22 A25015H 0.88 A25046HI 1.23 A25034HI 0.89 A25049HI 1.26 A25051HI 0.9 A25006H 1.29 A25039HI 0.91 A25061HI 1.29 A25014H 0.92 A25040HI 1.3 A25053HI 0.92 A25004H 1.33 A25025H 0.93 A25057HI 1.35 A25041HI 0.93 mock treated 1.03 cells A25024H 0.95 control oligo 1.42
TABLE-US-00006 TABLE 6 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 2 compared to mock treated cells in first screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25030H 0.26 A25034HI 0.85 A25055HI 0.29 A25045HI 0.87 A25031H 0.32 A25008H 0.88 A25027H 0.34 A25062HI 0.92 A25038HMI 0.35 A25023H 0.92 A25028H 0.39 A25018H 0.93 A25054HI 0.49 A25044HI 0.94 A25052HI 0.54 A25011H 0.94 A25020H 0.54 A25010H 0.98 A25029H 0.55 A25012H 0.99 A25051HI 0.65 A25013H 1 A25053HI 0.67 A25060HI 1 A25021H 0.68 A25050HI 1.01 A25037HI 0.69 A25022H 1.02 A25019H 0.69 A25009H 1.02 A25015H 0.7 A25032HI 1.02 A25024H 0.76 A25026H 1.03 A25039HI 0.76 A25046HI 1.05 A25016H 0.77 A25061HI 1.06 A25014H 0.78 A25006H 1.08 A25047HI 0.78 A25041HI 1.09 A25005H 0.8 A25040HI 1.14 A25036HI 0.8 A25004H 1.18 A25033HI 0.81 A25057HI 1.21 A25025H 0.82 A25049HI 1.23 A25017H 0.83 A25048HI 1.25 A25035HI 0.83 mock treated cells 1.01 A25063HI 0.85 control oligo 1.27
Example 3: Target Knockdown Efficacy Screens of Human FoxP3-Specific ASOs in T Cells in a 2.SUP.nd .Screening Round
[0067] The efficacy of 32 additional FoxP3-specific ASOs was tested in a second screening round. The ASOs were tested with regard to their knockdown efficacy together with 3 ASOs from the first screening round (A25027H (SEQ ID NO.23), A25030H (SEQ ID NO.26) and A25055HI (SEQ ID NO.49)) in CD4.sup.+ T cells of donor 1 and donor 2. As shown in
TABLE-US-00007 TABLE 7 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 1 compared to mock treated cells in second screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25073H 0.37 A25090HI 0.89 A25055HI 0.38 A25084HI 0.92 A25030HI 0.43 A25065H 0.94 A25027HI 0.44 A25081HI 0.99 A25069H 0.45 A25087HI 1.03 A25076H 0.47 A25091HI 1.08 A25085HMI 0.51 A25089HI 1.1 A25075H 0.56 A25088HI 1.12 A25092HI 0.58 A25071H 1.16 A25074H 0.63 A25083HI 1.27 A25068H 0.63 A25079HI 1.28 A25078H 0.63 A25086HI 1.31 A25093HI 0.67 A25080HI 1.55 A25070H 0.73 A25095HI 1.77 A25077H 0.75 mock treated cells 1 A25072H 0.82 control oligo 1.5 A25082HI 0.85
TABLE-US-00008 TABLE 8 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 2 compared to mock treated cells in second screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25055HI 0.49 A25070H 0.91 A25030HI 0.54 A25081HI 0.93 A25085HMI 0.54 A25088HI 0.94 A25092HI 0.54 A25087HI 0.95 A25027HI 0.54 A25091HI 0.97 A25076H 0.59 A25084HI 0.97 A25069H 0.63 A25072H 1.1 A25073H 0.64 A25083HI 1.1 A25093HI 0.67 A25086HI 1.13 A25077H 0.69 A25065H 1.22 A25078H 0.71 A25079HI 1.3 A25075H 0.72 A25095HI 1.37 A25074H 0.78 A25080HI 1.38 A25089HI 0.82 A25071H 1.43 A25082HI 0.82 mock treated cells 1 A25068H 0.84 control oligo 1.82 A25092HI 0.88
Example 4: Investigation of the Dose-Dependent Target Knockdown by Selected Human FoxP3-Specific ASOs in Regulatory T Cells
[0068] The dose-dependent knockdown of FoxP3 mRNA expression by FoxP3 ASOs in human regulatory T cells was investigated on mRNA and protein level and the respective IC50 values were calculated. Therefore, T.sub.regs were treated for three, seven or nine days with the respective ASO at the following concentrations: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, and 1.5 nM. After the treatment period, cells were lyzed, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values (
TABLE-US-00009 TABLE 9 Dose-dependent inhibition of FoxP3 mRNA expression in T.sub.regs by selected FoxP3 ASOs and respective IC.sub.50 values after 3, 7 and 9 days. IC50 Inhibition (%) mRNA ASO (nM) 6 μM 1.5 μM 375 nM 94 nM 24 nM 6 nM 1.5 nM Day 3 A25028H 324.7 61.07 53.96 41.66 16.65 14.32 17.58 19.14 A25069H 137.6 71.48 70.94 53.51 28.20 18.38 −3.48 3.61 A25073H 404.3 71.08 66.35 35.12 15.82 16.16 −5.03 1.08 Day 7 A25028H 141.2 78.50 80.38 64.20 43.13 19.94 15.12 22.64 A25069H 122.6 73.12 78.60 61.30 23.25 11.25 −9.29 −8.68 A25073H 87.2 78.80 77.52 54.16 30.39 31.99 −12.91 −3.36 Day 9 A25028H 65.4 75.99 75.36 71.31 46.93 4.24 −2.16 −10.67 A25069H 45.3 84.24 85.17 74.32 55.32 20.28 −2.71 −9.88 A25073H 57.6 83.60 84.08 72.65 55.99 22.44 12.42 1.40
TABLE-US-00010 TABLE 10 Dose-dependent inhibition of FoxP3 protein expression in T.sub.regs: IC.sub.50 values after 3, 7 and 9 days. Protein ASO IC.sub.50 (nM) Day 3 A25028H 284.7 A25069H 192.6 A25073H 309.8 Day 7 A25028H 60.8 A25069H 124.6 A25073H 100.0 Day 9 A25028H 85.0 A25069H 62.0 A25073H 47.6
Example 5: T.SUB.reg .Suppression Assay
[0069] Five human FoxP3-specific ASOs (A25028H (SEQ ID NO.24), A25031H (SEQ ID NO.27), 25038HMI (SEQ ID NO.34), A25069H (SEQ ID NO.56) and A25073H (SEQ ID NO.58)) were selected to investigate whether the knockdown of FoxP3 in T.sub.regs would diminish their suppressive capacity on responder T cells (T.sub.resp). Therefore, a T.sub.reg suppression assay was performed. Thus, a co-culture of ASO-treated T.sub.regs with T.sub.resp (stained with a cell proliferation dye) was started four days after start of ASO treatment. Proliferation of T.sub.resp was analyzed by flow cytometry three days after start of co-culture. Treatment with all of the five analyzed FoxP3-specific ASOs potently reduced the suppressive capacity of the T.sub.regs, as T.sub.resp could proliferate better than in co-cultures with mock- or control oligo-treated T.sub.regs (
TABLE-US-00011 TABLE 11 List of the mean of % suppression of T.sub.resp vs mock treated cells, IFN- γ and IL-2 concentration in supernatant of a T.sub.reg suppression assay. absolute % proliferation % % reduced number (vs mock treated suppression suppressive IFN-γ IL-2 ASO Tresp cells) of Tresp function (pg/ml) (pg/ml) A25028H 56175.33 51.23 5.12 95.00 5200.20 1815.95 A25031H 55946.33 50.63 13.10 87.00 6695.27 1359.24 A25038HMI 73447.33 97.80 0.00 100.00 5198.62 1680.29 A25069H 70586.33 90.07 0.00 100.00 7907.81 1127.24 A25073H 51707.67 39.27 18.46 81.67 3644.65 703.26 Mock treated 37136.33 0.00 100.00 0.33 1737.02 91.44 cells neg 1 38161.00 2.73 94.00 6.00 1743.51 110.82
Example 6: Design of Mouse FoxP3-Specific Antisense Oligonucleotides (ASOs)
[0070] For the design of ASOs with specificity for the mouse FoxP3 gene the FoxP3 mRNA sequence with the RefSeq ID NM_001199347.1 was used. For the design of ASOs with specificity for intronic regions within the mouse FoxP3 gene the FoxP3 pre-mRNA Sequence (GRCm38.p6 (GCF_000001635.26, Chr X (NC_000086.7): 7,578,119-7,596,800) was used. An “M” after the ASO ID indicates a mouse FoxP3-specific sequence that binds to an exonic region of the pre-mRNA, a “MR” after the ASO ID indicates a mouse/rat cross-reactive FoxP3 sequence that binds to an exonic region of the pre-mRNA and a “MI” after the ASO ID indicates a mouse FoxP3-specific sequence that binds to an intronic region of the pre-mRNA. 16 and 17mers were designed according to in house criteria, neg1 (described in WO2014154843 A1) was used as control oligonucleotide in all experiments (Table 4).
Example 7: Target Knockdown Efficacy Screen of Mouse FoxP3-Specific ASOs in T Cells
[0071] In order to investigate the knockdown efficacy of the in silico designed mouse FoxP3 ASOs, an efficacy screening was performed in mouse CD4.sup.+ T cells. In addition cells were treated with TGF-beta, ATRA, IL-2 and stimulated with CD3/CD28 beads (ThermoFisher) in order to increase expression levels of FoxP3. Therefore, cells were treated with the respective ASO at a concentration of 5 μM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values. The results are shown in
TABLE-US-00012 TABLE 12 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor mouse 1 compared to mock treated cells. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25063MI 0.05 A25008M 0.41 A25028M 0.08 A25044M 0.43 A25064MI 0.10 A25011M 0.44 A25032M 0.10 A25058MI 0.44 A25027M 0.11 A25057MI 0.45 A25026M 0.13 A25034MR 0.45 A25049MI 0.14 A25040M 0.46 A25013M 0.15 A25060MI 0.46 A25021M 0.16 A25003M 0.48 A25014M 0.17 A25036M 0.48 A25012M 0.17 A25004M 0.48 A25015M 0.17 A25041M 0.48 A25022M 0.18 A25018MR 0.49 A25016M 0.20 A25042M 0.49 A25024M 0.20 A25055MI 0.49 A25051MI 0.20 A25001M 0.50 A25038MR 0.21 A25056MI 0.51 A25053MHI 0.22 A25067MI 0.52 A25047MI 0.22 A25037MR 0.53 A25043M 0.23 A25010MR 0.53 A25009M 0.23 A25033MR 0.54 A25035M 0.27 A25048MI 0.55 A25025MR 0.27 A25052MI 0.56 A25039M 0.27 A25065MI 0.56 A25017M 0.27 A25054MRHI 0.57 A25019MR 0.28 A25061M 0.58 A25050MI 0.28 A25005M 0.58 A25069MI 0.28 A25045M 0.63 A25023M 0.28 A25006M 0.65 A25030M 0.29 A25007M 0.67 A25031MR 0.32 A25070MI 0.68 A25002MR 0.32 A25059MI 0.70 A25066MI 0.33 A25068MI 0.78 A25020M 0.37 A25062MI 0.79 A25029M 0.40 mock treated cells 0.96 A25046MI 0.41 control oligo 1.00
TABLE-US-00013 TABLE 13 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor mouse 2 compared to mock treated cells. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25028M 0.04 A25004M 0.46 A25063M 0.10 A25018MR 0.46 A25032M 0.11 A25066MI 0.49 A25026M 0.11 A25058MI 0.49 A25015M 0.11 A25003M 0.49 A25027M 0.12 A25055MI 0.49 A25022M 0.14 A25046MI 0.50 A25024M 0.14 A25010MR 0.51 A25016M 0.15 A25001M 0.53 A25021M 0.15 A25034MR 0.57 A25014M 0.16 A25045M 0.58 A25064MI 0.16 A25005M 0.58 A25025MR 0.16 A25057MI 0.59 A25012M 0.17 A25033MR 0.62 A25017M 0.19 A25040M 0.63 A25013M 0.19 A25056MI 0.63 A25009M 0.20 A25070MI 0.63 A25049MI 0.20 A25052MI 0.65 A25069MI 0.23 A25006M 0.65 A25038MR 0.25 A25065MI 0.66 A25030M 0.25 A25068MI 0.66 A25051MI 0.26 A25044M 0.67 A25053MHI 0.27 A25037MR 0.71 A25023M 0.29 A25054MRHI 0.72 A25043M 0.30 A25041M 0.73 A25029M 0.34 A25042M 0.74 A25019MR 0.34 A25007M 0.78 A25035M 0.35 A25048MI 0.79 A25011M 0.35 A25061MI 0.82 A25020M 0.37 A25060MI 0.82 A25047MI 0.37 A25036M 0.82 A25002MR 0.38 A25067MI 0.90 A25039M 0.38 A25059MI 0.95 A25031MR 0.39 A25062MI 0.99 A25050MI 0.40 mock treated cells 1.01 A25008M 0.43 control oligo 0.68
Example 8: Investigation of the Dose-Dependent Target Knockdown by Selected Mouse FoxP3-Specific ASOs in T Cells
[0072] The dose-dependent knockdown of FoxP3 mRNA expression by FoxP3 ASOs in mouse CD4.sup.+ T cells was investigated and the respective IC50 values were calculated. Therefore, CD4.sup.+ T cells were treated for three days with the respective ASO at the following concentrations: 6 μM, 2 μM, 600 nM, 200 nM, 60 nM, 20 nM, 6 nM, 2 nM. After the treatment period, cells were lyzed, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values. A dose-dependent knockdown of FoxP3 mRNA after treatment with all tested FoxP3 ASOs (
TABLE-US-00014 TABLE 14 Dose-dependent inhibition of FoxP3 mRNA expression in CD4.sup.+ T cells by selected FoxP3 ASOs and respective IC.sub.50 values. IC50 Inhibition (%) ASO (nM) 6 μM 2 μM 600 nM 200 nM 60 nM 20 nM 6 nM 2 nM A25014M 754.6 81.89 62.62 53.37 41.40 35.31 19.23 12.13 −4.15 A25015M 2179.7 82.96 5.63 52.29 32.48 34.56 26.82 1.71 11.44 A25021M 2304.4 73.89 59.07 59.75 37.36 29.00 27.85 15.91 1.98 A25022M 599.1 76.84 52.39 46.92 32.56 25.58 −5.76 −18.14 −33.56 A25027M 456.1 84.74 72.98 62.25 49.16 27.84 23.37 29.57 0.19 A25028M 215.2 93.22 86.17 76.55 58.16 65.65 21.20 27.18 18.60 A25032M 218.2 88.97 79.36 69.99 57.70 42.36 42.51 15.61 29.36 A25049MI 720.7 79.18 64.49 51.99 44.68 23.87 23.96 11.35 15.24 A25063MI 307.5 92.18 86.11 74.15 57.21 55.78 45.88 34.94 27.59 A25064MI 146.9 82.87 67.47 65.48 44.62 38.84 26.45 8.67 11.34
Example 9: T.SUB.reg .Suppression Assay
[0073] Seven mouse FoxP3-specific ASOs (A25014M (SEQ ID NO.339), A25015M (SEQ ID NO.340), A25021M (SEQ ID NO.346), A25027M (SEQ ID NO.352), A25032M (SEQ ID NO.357), A25049MI (SEQ ID NO.374) and A25064MI (SEQ ID NO.389)) were selected to determine the knockdown efficacy of FoxP3-specific ASOs in natural T.sub.regs on protein level. The percentage of FoxP3.sup.+ cells (pre-gated on CD4.sup.+CD25.sup.+ cells) was reduced by more than 90% after treatment with all ASOs investigated, resulting in less than 2% CD4.sup.+CD25.sup.+FoxP3.sup.+ cells (
TABLE-US-00015 TABLE 15 List of the mean of FoxP3.sup.+ cells of ASO-treated regulatory T cells compared to mock treated cells and absolute number of responder T cells in a T.sub.reg suppression assay. % FoxP3.sup.+ cells (of CD4+ absolute ASO CD25+) number T.sub.resp A25014M 1.05 2936.33 A25015M 1.17 4146.33 A25021M 1.47 2551.67 A25027M 0.95 1778.67 A25032M 0.62 1752.67 A25049MI 0.93 1831.00 A25064MI 0.86 2683.33 Mock treated cells 25.43 1411.33 neg 1 56.03 1583.00
Example 10: Target Knockdown Efficacy Screens of Human FoxP3-Specific ASOs in T Cells in a Third Screening Round
[0074] In order to investigate the knockdown efficacy of the in silico designed FoxP3 ASOs, a third efficacy screening round was performed in human CD4.sup.+ T cells. Therefore, cells were activated, treated with the respective ASO at a concentration of 5 μM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values. The results are shown in
TABLE-US-00016 TABLE 16 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 1 compared to mock treated cells in a third screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25096H 0.20 A25177HI 0.72 A25101H 0.24 A25174HI 0.73 A25105H 0.25 A25153H 0.75 A25110H 0.25 A25196HI 0.75 A25107H 0.27 A25156H 0.76 A25069H 0.29 A25128H 0.76 A25126H 0.29 A25123H 0.79 A25127H 0.30 A25195HI 0.81 A25132H 0.30 A25103H 0.84 A25073H 0.34 A25121H 0.84 A25151H 0.34 A25193HI 0.84 A25108H 0.35 A25186HI 0.87 A25028H 0.37 A25133H 0.91 A25099H 0.38 A25157H 0.91 A25113H 0.40 A25185HI 0.91 A25150H 0.40 A25163HI 0.93 A25112H 0.42 A25169HI 0.94 A25098H 0.43 A25129H 0.97 A25104H 0.43 A25119H 0.98 A25114H 0.43 A25175HI 1.00 A25109H 0.44 A25194HI 1.03 A25158H 0.45 A25187HI 1.04 A25176HI 0.45 A25118H 1.04 A25179HI 0.45 A25161HI 1.06 A25180HI 0.45 A25134H 1.07 A25190HI 0.46 A25160HI 1.08 A25116H 0.47 A25140H 1.09 A25189HI 0.49 A25167HI 1.10 A25115H 0.51 A25145H 1.11 A25147H 0.52 A25152H 1.14 A25149H 0.52 A25139H 1.15 A25182HI 0.53 A25188HI 1.15 A25097H 0.53 A25141H 1.15 A25102H 0.53 A25154H 1.16 A25192HI 0.54 A25125H 1.16 A25100H 0.55 A25184HI 1.18 A25135H 0.55 A25142H 1.21 A25191HI 0.56 A25143H 1.22 A25178HI 0.56 A25155H 1.22 A25117H 0.57 A25144H 1.22 A25159H 0.58 A25130H 1.23 A25138H 0.58 A25137H 1.24 A25148H 0.60 neg1 1.25 A25164HI 0.61 A25146H 1.26 A25106H 0.61 A25122H 1.30 A25183HI 0.62 A25173HI 1.31 A25181HI 0.63 A25162HI 1.35 A25131H 0.65 A25170HI 1.43 A25165HI 0.66 A25136H 1.45 A25120H 0.67 A25199HI 1.49 A25111H 0.67 A25197HI 1.79 A25171HI 0.69 A25166HI 1.83 A25172HI 0.71 A25168HI 1.86 A25124H 0.71 A25198HI 2.13
TABLE-US-00017 TABLE 17 List of the mean FoxP3 mRNA expression values in ASO-treated CD4.sup.+ T cells from donor 2 compared to mock treated cells in a third screening round. Expression values are normalized to HPRT1. Residual FoxP3 Residual FoxP3 expression expression (compared to (compared to mock treated mock treated ASO cells) ASO cells) A25127H 0.20 A25149H 0.76 A25126H 0.23 A25193HI 0.77 A25069H 0.24 A25133H 0.78 A25028H 0.26 A25174HI 0.79 A25096H 0.29 A25120H 0.80 A25101H 0.29 A25156H 0.81 A25073H 0.29 A25153H 0.82 A25107H 0.32 A25185HI 0.82 A25105H 0.34 A25163HI 0.82 A25132H 0.35 A25161HI 0.89 A25108H 0.35 A25111H 0.90 A25147H 0.38 A25194HI 0.91 A25182HI 0.38 A25195HI 0.92 A25135H 0.38 A25134H 0.93 A25110H 0.39 A25196HI 0.93 A25191HI 0.40 A25160HI 0.94 A25099H 0.41 A25157H 0.95 A25150H 0.42 A25103H 0.97 A25151H 0.44 A25129H 1.00 A25190HI 0.45 A25186HI 1.02 A25176HI 0.46 A25169HI 1.04 A25181HI 0.46 A25184HI 1.06 A25189HI 0.48 A25121H 1.07 A25183HI 0.48 A25125H 1.08 A25172HI 0.48 A25167HI 1.09 A25104H 0.48 A25155H 1.13 A25171HI 0.49 A25173HI 1.13 A25158H 0.50 A25162HI 1.14 A25192HI 0.50 A25140H 1.21 A25164HI 0.54 A25145H 1.22 A25179HI 0.58 A25152H 1.25 A25109H 0.58 A25144H 1.25 A25100H 0.59 A25143H 1.25 A25138H 0.59 A25187HI 1.29 A25159H 0.60 A25119H 1.29 A25112H 0.62 A25122H 1.32 A25178HI 0.63 A25170HI 1.33 A25128H 0.63 A25137H 1.35 A25116H 0.63 A25154H 1.39 A25177HI 0.63 A25118H 1.40 A25124H 0.63 A25175HI 1.41 A25097H 0.64 neg1 1.43 A25113H 0.64 A25188HI 1.43 A25114H 0.65 A25141H 1.44 A25115H 0.65 A25199HI 1.53 A25098H 0.65 A25139H 1.59 A25102H 0.66 A25146H 1.68 A25106H 0.67 A25136H 1.71 A25180HI 0.68 A25130H 1.71 A25148H 0.69 A25166HI 1.96 A25131H 0.69 A25142H 2.03 A25117H 0.71 A25197HI 2.21 A25165HI 0.71 A25168HI 2.21 A25123H 0.75 A25198HI 2.83
Example 11: Investigation of the Dose-Dependent Target Knockdown by Selected Human FoxP3-Specific ASOs in Regulatory T Cells
[0075] The dose-dependent knockdown of FoxP3 mRNA expression by FoxP3 ASOs in human regulatory T cells was investigated on mRNA level and the respective IC50 values were calculated. Therefore, T.sub.regs were treated for three days with the respective ASO at the following concentrations: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, and 1.5 nM. After the treatment period, cells were lyzed, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values (
TABLE-US-00018 TABLE 18 Dose-dependent inhibition of FoxP3 mRNA expression in regulatory T cells by selected FoxP3 ASOs and respective IC.sub.50 values after 3 days ASO treatment. IC50 Inhibition (%) ASO (nM) 6 μM 1.5 μM 375 nM 94 nM 24 nM 6 nM 1.5 nM A25096H 177 95.69 86.34 75.96 50.04 23.25 19.92 A25099H 775 85.73 75.34 53.58 42.37 29.56 54.35 50.59 A25101H 109 89.05 78.83 58.88 38.84 7.00 37.41 3.26 A25104H 933 81.02 64.23 41.70 16.03 46.69 28.93 24.79 A25108H 932 80.17 29.73 14.45 −1.41 −21.37 9.59 −10.29 A25112H 762 81.06 74.60 22.72 7.25 −12.75 45.90 26.31 A25113H 1030 66.87 64.93 46.13 7.52 36.62 31.35 36.68 A25126H 911 82.52 65.41 32.10 −39.85 −2.44 27.56 39.19 A25127H 728 89.68 70.19 46.17 18.30 7.89 38.62 25.40 A25150H 173 80.92 56.63 26.38 6.89 −28.14 −54.91 −48.92 A25151H 1758 68.59 48.81 15.75 24.95 −18.90 −6.28 19.16 A25179HI 417 79.76 54.80 9.80 5.68 24.55 −0.35 −4.60 A25182HI 341 77.97 60.60 40.01 16.19 −30.50 9.87 7.42 A25190HI 1077 63.82 24.29 8.54 −34.72 −61.19 −32.11 9.27 A25191HI 395 81.72 58.45 51.33 2.89 −31.46 −6.32
Example 12: Investigation of the Dose-Dependent Target Knockdown by Selected Human FoxP3-Specific ASOs in Regulatory T Cells
[0076] The dose-dependent knockdown of FoxP3 mRNA expression by FoxP3 ASOs in human regulatory T cells was further investigated on mRNA and protein level and the respective IC50 values were calculated. Therefore, T.sub.regs were treated for three, six or ten days with the respective ASO at the following concentrations: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, and 1.5 nM. After the treatment period, cells were lyzed, FoxP3 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the FoxP3 expression values were normalized to HPRT1 values (
TABLE-US-00019 TABLE 19 Dose-dependent inhibition of FoxP3 mRNA expression in T.sub.regs by selected FoxP3 ASOs and respective IC.sub.50 values after 3, 6 and 10 days. IC50 Inhibition (%) mRNA ASO (nM) 6 μM 1.5 μM 375 nM 94 nM 24 nM 6 nM 1.5 nM Day 3 A25073H 181.9 84.95 77.74 52.78 30.00 6.10 −12.51 0.52 A25126H 129.5 87.33 77.45 57.84 35.10 6.38 5.54 −16.32 A25150H 603.1 78.42 62.81 40.71 31.52 14.73 −0.94 3.10 Day 6 A25073H 25.2 67.64 84.39 75.00 63.89 29.58 14.21 −6.33 A25126H 63.7 64.94 72.25 65.59 50.24 16.00 9.58 9.64 A25150H 61.4 59.72 63.17 59.36 41.42 11.60 5.92 −1.49 Day A25073H 24.5 100.00 89.50 91.08 78.14 56.82 40.45 27.46 10 A25126H 46.3 90.61 99.51 94.89 83.11 55.23 48.08 39.93 A25150H 12.5 96.51 97.62 93.23 75.34 53.99 35.02 10.54
TABLE-US-00020 TABLE 20 Dose-dependent inhibition of FoxP3 protein expression in T.sub.regs: IC.sub.50 values after 3, 6 and 10 days. Protein ASO IC.sub.50 (nM) Day 3 A25073H 87.5 A25126H 161.5 A25150H 358.2 Day 6 A25073H 41.5 A25126H 61.0 A25150H 74.9 Day 10 A25073H 43.4 A25126H 30.0 A25150H 53.7