CZ2001454A3 - Short oligonucleotides intended for inhibition of VEGF expression, process of their preparation and their use - Google Patents

Abstract

The present invention relates to short oligonucleotides or derivatives thereof having a sequence corresponding to a particular portion of a nucleic acid sequence that encodes VEGF (vascular endothelial growth factor) and which is at most 15 nucleotides in length. It further relates to a process for preparing such oligonucleotides and their use. A short oligonucleotide or derivative thereof having a length of 10 to 15 nucleotides and corresponding to a portion of the VEGF coding sequence has one of the following SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a portion thereof: SEQ ID NO. 1 is 5-CCCGGCCCGGTCGGGCCTCCG-3 ', SEQ ID NO. No. 2 is 5'-CGGGCCTCCGAAACC-3 ', SEQ. ID. No. 3 is 5'-GCTCTACCTCCACCATGCCAA-3 ', SEQ ID NO. No. 4 is 5'-GTGGTCCCAGGCTGCACCCATGGC-3 ', SEQ ID NO. No. 5 is 5'-CATCTTCAAGCCATCC-3 ', SEQ ID NO. No. 6 is 5'-TGCGGGGGCTGCTGC-

Description

Technical field

The present invention relates to short oligonucleotides or derivatives thereof having a sequence corresponding to a certain portion of a nucleic acid encoding a VEGF (vascular endothelial growth factor) that is at most 15 nucleotides in length, and further to a process for preparing such oligonucleotides and their use.

BACKGROUND OF THE INVENTION

VEGF, sometimes permeability), mitotic effect (Ferrara, Trends

Angiogenesis, defined as the growth of new blood capillaries, plays a fundamental role in growth and development. In an adult, the ability to initiate an angiogenic response is present in all tissues but is kept under strict control. Angiogenesis is only mobilized under specific circumstances, such as recovery of injured tissue or regulation of the uterine mucosa. The regulation of angiogenesis is the result of a delicate balance between numerous inhibitory and stimulating factors.

also called VPF (vascular factor is a key regulator of angiogenesis and is specific for

Cardiovasc. Copper.

it exists in at least four different isoforms (VEGF121, VEGF _165, VEGF ₁₈₉ and VEGF206), which exhibit similar biological activity and result from different mRNA splicing. VEGF is expressed at abnormally high levels in human tumors and diseased tissue, characterized by a high rate of vascularization or high vascular permeability, such as its cells

VEGF endothelial (1993) 3, 244). For example, diabetic retinopathy, psoriasis, macular, such as diabetic retinopathy. age-dependent degeneration, rheumatoid arthritis and other inflammatory diseases. Thus, agents that selectively reduce VEGF levels can be used to treat malignancies and other angiogenesis disorders.

Monoclonal antibodies against VEGF have been shown to suppress the growth of some tumors in nude mice (Kim et al., Nature (1993) 362, 841). Another way to lower the level

VEGF is the use of antisense oligonucleotides that are optionally modified to improve their properties (E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990); S.

Agrawal, T1BTECH 1996, 376; EP 0653439 A2). Antisense oligonucleotides are believed to bind to specific mRNA sequences, leading to mRNA degradation and / or inhibition of protein synthesis.

EP 0769 552 A1 in the claims discloses oligonucleotides of 8 nucleotides or longer, directed against VEGF, which inhibit VEGF expression to 30% or less. These oligonucleotides were tested in a cell-free system in the form of phosphodiesters, but which are not stable in vivo. Selected oligonucleotides were also tested in the form of all-phosphothioates (A085R-S, A087P-S, A227-S, A237-S, A311-S and A419-S), which showed 30-46% inhibition of VEGF expression using 20 μΜ all -phosphothioate oligonucleotide in A549 cell assay. Most effective was the all-phosphothioate oligonucleotide (A419-S), 20mer having the sequence of SEQ ID NO: 2. No. 100: 5'-TGGTGAGGTTTGATCCGCAT-3 '.

SUMMARY OF THE INVENTION

The present invention provides a short oligonucleotide or derivative thereof that corresponds to a portion of a VEGF coding sequence, wherein a portion of a VEGF coding sequence that corresponds to a VEGF coding sequence.

oligonucleotide, has id. one of SEQ ID NOs. 1, id. 2, id. No. 5 and id. 6, wherein No. 3, id. C. 4, Sequence id. C. 1: 5'- CCCGGCCCCGGTCGGGCCTCCG - 3 ' Sequence id. C. 2: 5'-CGGGCCTCCGAAACC-3 ' Sequence id. C. 3: 5'- GCTCTACCTCCACCATGCCAA -3 ' Sequence id. C. 4: 5'- GTGGTCCCAGGCTGCACCCATGGC -3 ' Sequence id. C. 5: 5'-CATCTTCAAGCCATCC-3 ' Sequence id. C. 6: 5'- TGCGGGGGCTGCTGC -3 '. Sequence id. 1, 2, 3, 4, 5 and 6 are core sections,

which have been identified as being particularly suitable for the design of oligonucleotides. SEQ ID NO. Nos. 1, 2, 3, 4, 5 and 6 are equivalent to nucleotides 30 to 51 (SEQ ID NO: 1, which is termed core region 1), nucleotides 42 to 56 (SEQ ID NO: 2, which the nucleotide sequence 2), the nucleotide sequence 101 to 121 (SEQ ID NO 3, which is termed the nucleus 3), the nucleotide sequence 122 to 145 (SEQ ID NO 4, which is termed the nucleus 4), the sequence nucleotides 268 to 284 (SEQ ID NO: 5, which is termed core region 5) and nucleotides 303 'to 317 (SEQ ID NO: 6, which is termed core region 6). This numbering refers to the nucleotide sequence encoding human VEGF, i.e., sequence id. No. 93 (Table 1). The localization of these core sequences in sequence id. The nucleotide sequence of the human VEGF cDNA is set forth in Figure 1B of Leung et al (1989), Science 8, 1307. No. 93 corresponds to the 5'-end (nucleotides 1-480) of the sequence of Figure 1B in Leung et al.

The oligonucleotide has a sequence that corresponds to a portion of the nucleic acid that encodes VEGF. The term corresponding means that the base sequence of the oligonucleotide is complementary to the portion of the nucleic acid sequence that encodes VEGF (e.g.

gene sequence, cDNA, mRNA), thus allowing the oligonucleotide to hybridize (ie, bind) to the sense portion of the nucleic acid encoding VEGF (which is preferably VEGF mRNA). Therefore, the name antisense oligonucleotide is also used. In a preferred embodiment of the invention, the oligonucleotide is just an antisense oligonucleotide. In another preferred embodiment of the invention, the oligonucleotide is a ribozyme. Ribozyme is a catalytic nucleic acid that cleaves mRNA. Preferred ribozymes are selected from the group comprising hammer ribozymes (see Uhlmann and Peyman, 1990).

The oligonucleotide of the invention is identical to one of the following SEQ ID NOs. Nos 7 to 12 or parts thereof:

SEQ ID NO. 7: 3'- GGGCCGGGGCCAGCCCGGAGGC-5 ’

5 '- CGGAGGCCCGACCGGGGCCGGG-3' (corresponds to SEQ ID NO: 1)

SEQ ID NO. No. 8

3’- GCCCGGAGGCTTTGG -5 ’

5 ^T - GGTTTCGGAGGCCCG-3 '(corresponds to SEQ ID NO: 2)

SEQ ID NO. No. 9: 3'- CGAGATGGAGGTGGTACGGTT -5 '

5'- TTGGCATGGTGGAGGTAGAGC-3 '(corresponds to SEQ ID NO 3)

SEQ ID NO. No. 10: 3'- CACCAGGGTCCGACGTGGGTACCG -5 '5'- GCCATGGGTGCAGCCTGGGCAACA-3' (corresponds to SEQ ID NO 4)

SEQ ID NO. No 11: 3'- GTAGAAGTTCGGTAGG -5 '

5'- GGATGGCTTGAAGATG -3 '(corresponds to SEQ ID NO 5)

9 9 99 99 9 9 9 9

9.9 9 · 9 9 9 · ·· · 9 in · <i titt 9 9 9 9 9

SEQ ID NO. No. 12: 3'- ACGCCCCCGACGACG -5 '

5'- GCAGCAGCCCCCGCA-3 '(corresponds to SEQ ID NO: 6)

The portion of the VEGF-encoding nucleic acid sequence corresponding to the oligonucleotides is 10, 11, 12, 13, 14 or nucleotides in length, preferably the oligonucleotide of the invention is 12 nucleotides in length from the VEGF-encoding sequence. Thus, the oligonucleotide of the present invention has a size of 10 (10mer), 11 (11mer), 12 (12mer), 13 (13mer), 14 (14mer) or 15 (15mer) nucleotides.

In a preferred embodiment of the invention, the oligonucleotide is 12 nucleotides in size, e.g., one of SEQ ID NOs. No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 and 56, wherein:

SEQUENCE ID C. 14: 3 ^T - CCAGCCCGGAGG -5 ', 5'- GGAGGCCCGACC-3' (equivalent to SEQ ID NO: 7),. SEQUENCE ID C. 16: 3'- CGGAGGCTTTGG -5 ', 5'- GGTTTCGGAGGC-3' (equivalent to SEQ ID NO: 8), SEQUENCE ID C. 27: 3'- GATGGAGGTGGT -5 ', 5'-TGGTGGAGGTAG-3' (equivalent to SEQ ID NO: 9), SEQUENCE ID C. 28: 3'- GGAGGTGGTACG -5 ', 5'-GCATGGTGGAGG-3' (equivalent to SEQ ID NO: 9), SEQUENCE ID C. 29: 3 '- GGTGGTACGGTT -5', 5'-TTGGCATGGTGG-3 ' (equivalent to SEQ ID NO: 9), SEQUENCE ID C. 33: 3'- CACCAGGGTCCG -5 ', 5'-GCCTGGACCAC-3' (equivalent to SEQ ID NO: 10), SEQUENCE ID C. 34: 3'- CCAGGGTCCGAC -5 ', 5'-CAGCCTGGGACC-3' (equivalent to SEQ ID NO: 10), SEQUENCE ID C. 35: 3 '- AGGGTCCGACGT -5', 5'-TGCAGCCTGGGA-3 ' (equivalent to SEQ ID NO: 10), SEQUENCE ID C. 36: 3 '- GGGTCCGACGTG -5', 5'-GTGCAGCCTGGG-3 '

(equivalent to SEQ ID NO: 10), &quot; &quot;

SEQUENCE ID C. 37: 3'- GGTCCGACGTGG -5 ', 5' -GGTGCAGCCTGG-3 ' (equivalent parts sequence id. No. 10), SEQUENCE ID C. 38: 3'- CCGACGTGGGTA -5 ' ', ATGGGTGCAGCC-3' (equivalent parts) sequence id. No. 10), SEQUENCE ID C. 52: 3'- GTAGAAGTTCGG -5 ' 5'-GCCTTGAAGATA-3 ' (equivalent parts sequence id. No. 11) SEQUENCE ID C. 55: 3'- ACGCCCCCGACG -5 ’ ', GCAGCCCCCGCA-3' (equivalent parts sequence id. No. 12), SEQUENCE ID C. 56: 3'- CCCCCGACGACG -5 ' ', GCAGCAGCCCCC-3' (equivalent parts sequence id. No. 12).

In another embodiment of the invention, the oligonucleotide is 13 nucleotides in length, such an oligonucleotide having, for example, one of the following sequences of SEQ ID NO. No 73, 74 or 75:

SEQUENCE ID C. 73: 3 '- GGAGGTGGTACGG -5', 5'-GGCATGGTGGAGG (equivalent to SEQ ID NO: 9) ·, SEQUENCE ID C. 74: 3'- GGGTCCGACGTGG -5 ', 5'-GGTGCAGCCTGGG (equivalent to SEQ ID NO: 10), SEQUENCE ID C. 75: 3'-GCCCCCGACGACG - 5 ', 5'-GCAGCAGCCCCCG (equivalent to SEQ ID NO: 12).

In another oligonucleotide of the following embodiments, the size 14 sequences of SEQ ID NO: 2.

of the present invention has nucleotides and has eg one

No 76, 77, 78 or 79:

SEQ ID NO. No. 76:

3'-CCCGGAGGCTTTGG-5 ', 5'-GGTTTCGGAGGCCC-3' (equivalent to SEQ ID NO: 8),

SEQ ID NO. No. 77:

3'- CGAGATGGAGGTGG -5 ', 5'-GGTGGAGGTAGAGC-3' (equivalent to SEQ ID NO 9),

SEQ ID NO. No. 78:

3'- GGGTCCGACGTGGG -5 ', 5'-GGGTGCAGCCTGGG-3' · (equivalent parts) · · · · · · · · · · · · · · · · · · SEQ ID NO: 10)

SEQ ID NO. No. 79:

3'-CGCCCCCGACGACG - 5 ', 5'-GCAGAGAGCCCCCGC-3' (equivalent to SEQ ID NO 12).

In yet another embodiment of the present invention, the oligonucleotide is 15 nucleotides in length, such an oligonucleotide having, for example, one of the following sequences of Id. Nos 80 to 88:

SEQ ID NO. No. 80:

3'- GGGCCGGGGCCAGCC -5 ', 5'-CCGACCGGGGCCGGG-3' (equivalent to SEQ ID NO: 10),

SEQ ID NO. No. 81:

3'- CCGGGGCCAGCCCGG -5 ', 5´-GGCCCGACCGGGGCC-3' (equivalent to SEQ ID NO 10),

SEQ ID NO. No. 82:

3'- GGCCGGGGCCA.GCCC -5 ', 5 ^! -CCCGACCGGGGCCGG-3 '(equivalent to SEQ ID NO: 10),

SEQ ID NO. No 83:

3’- CCCCGGAGGCTTTGG -5 ', 5'-GGTTTCGGAGGCCCC-3' (equivalent to SEQ ID NO 10),

SEQ ID NO. No. 84:

3’- A.TGGAGGTGGTACGG -5 ', 5' -GGCATGGTGGAGGTA-3 '(equivalent to SEQ ID NO 10),

SEQ ID NO. No 85:

3'- GGAGGTGGTACGGTT -5 ', 5’-TTGGCATGGTGGAGG-3' (equivalent to SEQ ID NO 10),

SEQ ID NO. No. 86:

3'- CCAGGGTCCGACGTG - 5 ', 5'-GTGCAGCCTGGACC-3' (equivalent to SEQ ID NO 10),

SEQ ID NO. No. 87:

3'- GTAGAAGTTCGGTAG -5 ', 5'-GATGGCTTGAAGATG-3' (equivalent to SEQ ID NO: 10), · · · · · · · · · · · · · · · · ·

Β · · · · · · · · ·

SEQ ID NO. No. 88:

3'- TAGAAGTTCGGTAGG -5 ', 5' -GGATGGCTTGAAGAT-3 '(equivalent to SEQ ID NO: 10).

SEQ ID NO. No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55, and 56, as well as SEQ ID NOs. Nos. 73 to 88 correspond to one of the core sequences or portions thereof (are equivalent to one of SEQ ID NOs 7 to 12 or portions thereof). SEQ ID NO. Nos. 13, 15, 17 to 26, 30 to 32, 39 to 51, 53, 54 and 57 to 72 do not correspond to the core sequence (oligonucleotides in Example 1, Fig. 1). All of these sequences are derived from the human VEGF cDNA sequence shown herein as SEQ ID NO. No. 93 or the VEGF sequence published by Leung et al. The present invention (Science 1989, 246, 1306). further relates to derivatives of oligonucleotides, eg salts thereof, in particular physiologically tolerable salts. Salts and physiologically tolerable salts are described, for example, in Remingtons Pharmaceuticals Science (1985) Mack Publishing Company, Easton, PA (p. 1418). Derivatives also include modified oligonucleotides having one or more modifications (e.g., at a particular nucleoside position and / or at a particular internucleoside linkage), oligonucleotide analogues (e.g., polyamide-nucleic acids, PNA, phosphonic acid monoesters nucleic acids, PHONA = PMENA, oligonucleotide chimeras (eg, composed of DNA and PNA portions or DNA and PHONA portions)).

A preferred aspect of the invention relates to an oligonucleotide having a sequence that corresponds to sequence id. 1 to 6 or a portion thereof (a sequence that is equivalent to one of SEQ ID NOs 7 to 12 or a portion thereof), preferably one of SEQ ID NOs: 1 to 6 or a portion thereof. No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55, and 56, as well as SEQ ID NOs. No. 73 to 88, which is modified to some extent. Most preferably, the oligonucleotide is modified to improve its properties, eg, to increase its nuclease resistance or to become nuclease resistant, or to improve its binding affinity. to a complementary nucleic acid encoding a VEGF, e.g., mRNA, or to increase oligonucleotide uptake by a cell.

Thus, the present invention preferably relates to oligonucleotides having the sequence as above and which additionally have one or more chemical modifications as compared to natural DNA which is composed of natural nucleosides deoxyadenosine (adenine + β-Ό-2'-deoxyribose) ), deoxyguanosine (guanine + β-Ώ-2'-deoxyribose), deoxycytidine (cytosine + β-ϋ-2'-deoxyribose) and thymidine (thymine + β-ϋ-2'deoxyribose) linked via phosphodiester internucleoside bridges. Oligonucleotides comprise one or more modifications of the same type and / or modifications of different types, each modification independently selected from all types of modifications known to modify the oligonucleotides.

Examples of chemical modifications are known to those skilled in the art and have been described, for example, in E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543, Protocols for Oligonucleotides and Analogs Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, New Jersey, United States 1993, ST Crooke, F., Bennet, Ann. Roar. Pharmacol. Toxicol. 36 (1996) 107-129, by J. Hunziker and C. Leuman (1995) Mod. Synt. Methods, 7, 331-417.

For example, when compared to natural DNA, the phosphophodiester internucleoside bridge, β-ϋ-2´-deoxyribose and / or the natural nucleoside base (adenine, guanine, cytosine, thymine) may be modified or replaced. The oligonucleotide of the present invention has one or more modifications, each modification being located in a particular phosphodiester internucleoside bridge and / or in a particular β-ϋ-2'deoxyribose unit and / or at a certain natural position. · Nucleoside base, as compared to an oligonucleotide with the same natural DNA sequence .

For example, the present invention relates to an oligonucleotide that comprises one or more modifications, each of which is independently selected from the following:

a) replacement of the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside with a modified internucleoside bridge,

b) replacing the phosphodiester bridge located at the 3 'and / or 5' end of the nucleoside with a defospho bridge,

(c) replacement of a sugar-phosphate backbone unit with another unit;

(d) replacement of the β-ϋ-2´-deoxyribose unit with a modified sugar unit;

e) replacement of the natural nucleoside base with a modified nucleoside base,

f) conjugation to a molecule that affects the properties of the oligonucleotide,

g) conjugation to the 2'5'-linked oligoadenylate or derivative thereof, optionally via a suitable linker,

h) inserting a 3'-3 'and / or 5'-5' inversion at the 3'- and / or 5'-end of the oligonucleotide.

More detailed examples of chemical modifications of oligonucleotides are provided below:

a) replacing the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside with a modified internucleoside bridge, wherein the modified internucleoside bridge is selected from, for example, phosphothioate, phosph odithioate, N-R ¹ 'phosphoramidite, boranphosphate, esters O -alkyl phosphate having 1 to 21 carbon atoms, aryl-O-alkyl phosphate having 6 to 12 aryl groups

9 uhlíku uhlíku 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 a carbon atoms and an alkyl group containing 1 to 21 carbon atoms, alkylphosphonate having an alkyl group having 1 to 8 carbon atoms and / or α-hydroxymethylaryl having an aryl group having 7 to 15 carbon atoms (see WO 95/01363), wherein the aryl group having 6 to 12 carbon atoms, the aryl group containing The C 6 -C 20 aryl group and the C 6 -C 14 aryl group are optionally replaced by halogen, alkyl, alkoxy, nitro or cyano, and R ¹ and R ¹ are, independently of each other, hydrogen, an alkyl group containing containing up to 14 carbon atoms up to 8 carbon atoms, up to 18 carbon atoms, aryl group up to 20 carbon atoms, arylalkyl group having an aryl group containing 6 and an alkyl group containing 1 preferably is hydrogen, an alkyl group having bsahující 1-8 carbon atoms and / or- methoxyethyl, particularly preferably hydrogen, alkyl having 1 to 4 carbon aroma or methoxyethyl, or R ¹ and R ¹ forming together with the nitrogen atom which carries them, a heterocyclic ring of 5 up to 6 member, which may additionally contain another heteroatom selected from the group consisting of O, S and N.

replacement of the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside by a defrost bridge (defrost bridge have been described, for example, in Uhlmann, E. and Peyman, A. in Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, 355ff), wherein the defosfo bridge is selected from, for example, the group consisting of formacetal, 3'-thioformacetal, methylhydroxylamine and oxime, methylene-dimethylhydrazo, dimethylenesulfone, and the like. and / or silyl groups.

c) replacing the sugar phosphate unit (β-Ώ-2'-deoxyribose and phosphodiester internucleoside bridge together to form a sugar-phosphate unit) in the sugar-phosphate backbone (sugar-phosphate co-constituent of sugar phosphate units) with another unit unit is able to create eg.

a morpholine derivative of an oligomer (as described e.g.

in E.P. Stirchak et al., Nucieic Acids Res. 17 (1989) 6129), or a morpholine derivative unit,

polyamide-nucleic acid (PNA) (as described, for example, in PE Nielsen et al., Bioconj. Chem. 5 (1994) 3 and in EP 0672677 A2), thus replacing the PNA unit in the backbone, e.g. 2-aminoethylglycine, phosphonic acid nucleic acid monoester (PHONA) (as described in Peyman et al., Angew. Chem. Int. Ed. Engl. 35 (1996) 26322638 and in EP 0739898 A2). replacing a unit in the skeleton with a PHONA unit

d) replacing the β-ϋ-2'-deoxyribose unit with a modified sugar unit, wherein the modified sugar unit is selected for example from the group consisting of β-D-ribose, α-D2'-deoxyribose, L-2'-deoxyribose, 2'- F-2'-deoxyribose,

2'-O-alkylribose having an alkyl group having 1 to 6 carbon atoms, wherein 2'-O-alkylribose having an alkyl group having 1 to 6 carbon atoms is preferably 2'-Omethylribose, 2'-O-alkenylribose having an alkenyl group containing 2 up to 6 carbon atoms, 2 '- [O- (alkyl-O-alkyl) ribose with alkyl groups containing 1 to 6 carbon atoms, 2' -NH ₂ -2'-deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-dideoxy-4-D-erythrohexopyranose,

and carbocyclic (described, for example, in Froehler, J. Am. Chem. Soc. 114 (1992) 8320), carbohydrate analogues and / or open-chain carbohydrate analogues (described, for example, in Vandendriessche et al., Tetrahedron 49 (1993)) 7223) and / or bicycloacacidide analogs (described, for example, in M. Tarkov et al., Helv. Chim. Acta 76 (1993) 481), replacing the natural nucleoside base with a modified nucleoside base, wherein the modified nucleoside base is selected from the group consisting of uracil. , hypoxanthine, 5- (hydroxymethyl) uracil, N ² -dimethylguanosine 5- (hydroxymethyl) uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5-fluorouracil, 5-fluorocytosine,

5-chlorouracil, 5-chlorocytosine, 5-bromouracil,

5-bromocytosine, 2,4-diaminopurine, 8-azapurine,

7-deazapurine, preferably at the 7-position and / or substituted at the 8-position or other modifications of the natural nucleoside base, e.g., the modified nucleoside base described in EP 0 710 667 A2 and EP 0 680 969 A2,

7-deazapurine

7-deazapurine substituted

f) conjugation to a molecule that affects the properties of the oligonucleotide, wherein conjugating the oligonucleotide to one or more molecules that (preferably) affect the properties of the oligonucleotide (eg, the ability of the oligonucleotide to cross the cell membrane or enter the cell, oligonucleotide stability against nucleases; sequence, oligonucleotide pharmacokinetics, ability of the antisense oligonucleotide / ribozymes to attack the VEGF encoded target sequence, i.e., the ability to bind and / or crosslink when the oligonucleotide or oligonucleotide conjugated molecule hybridizes to the VEGF encoded target · · · · · · · · · · · · · · · · · · · · examples of molecules that can be conjugated to an oligonucleotide include polylysine, intercalating agents such as pyrene, acridine, phenazine or phe nantridine, fluorescent agents such as fluorescein, crosslinking agents such as psoralen or azidoproflavin, lipophilic molecules such as C 12 -C 20 alkyls, lipids such as 1,2-dihexadecyl-rac-glycerol, steroids such as cholesterol or testosterone, vitamins such as vitamin E, poly- or oligoethylene glycol, preferably bound to the oligonucleotide via a phosphate group (e.g. triethylenglykolfosfát) alkyl phosphate diesters having an alkyl group containing 12 to 18 carbon atoms and / or O-CH ₂ -CH (OH} -O-alkyl having an alkyl group containing 12 to 18 carbon atoms. These molecules are conjugated to the 3'- and / Methods for preparing oligonucleotide conjugates are known to those skilled in the art and have been described, for example, in Uhlmann, E. & Peyman, A., Chem. Rev. 90 (1990) 543 and M. Manoharan, Antisense Research and Applications, Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chapter 17, p. 303ff, and EP-A 0 552 766,

g) conjugation to a 2'5'-linked oligoadenylate, preferably via a suitable linker molecule, wherein

The 2'5'-linked oligoadenylate is selected from the group consisting of 2'5'-linked triadenylate molecules, 2'5'-linked tetraadenylate molecules, 2'5'-linked pentaadenylate molecules, 2'5'-linked hexaadenylate molecules and 2 ' 5'-linked heptaadenylate molecules or derivatives thereof, wherein the 2'5'-linked oligoadenylate derivative is eg Cordycepin (2'5'-linked 3'-deoxyadenylate), and an example of a suitable linker / linker) is triethylglycol, and wherein 5 ' the end of the 2'5'-linked oligoadenylate must carry a phosphate, diphosphate or triphosphate moiety where one or more oxygen atoms can be replaced by, for example, a sulfur atom, with phosphate or thiophosphate substitution being preferred,

h) insertion of a 3'-3 'and / or 5'-5' inversion at the 3'- and / or 5'-end of the oligonucleotide, which kind of modification is known to those skilled in the art and has been described, e.g. J. Org. Chem. 56 (1991) 3757 and in EP 0 464 638 EP 0 593 901.

The replacement of a sugar phosphate unit from a sugar-phosphate backbone with another unit, eg a PNA or PHONA, is a skeleton-forming unit, preferably a replacement of a nucleotide eg PNA or PHONA with a unit that already contains natural nucleoside bases and / or modified nucleoside bases eg nucleoside bases from the group comprising uracil, hvpoxanthin, 5- (hydroxymethyl) uracil. · N-dimethylguanosine, 5- (hydroxymethyl) uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil,

5-bromocytosine, 2,4-diaminopurine, 8-azapurine, substituted 7-deazapurine, preferably 7-deazapurine substituted at position 7 and 7-deazapurine substituted at position 8, or other modifications of natural nucleoside bases (modified nucleoside bases have been described e.g. EP 0 710 667 A2 and EP 0 680 969 A2). Oligonucleotide modifications described in the patent. EP 0 710 667 A2, EP 0 680 969 A2, EP 0 464 638, EP 0 593 901, WO 55/01363, EP 0 672 677 A2, EP 0 739 898 A2 and EP 0 552 766 are incorporated by reference in the present application .

In a particular embodiment of the present invention, one or more phosphodiester bridges in the oligonucleotide are modified, preferably one or more internucleoside phosphodiester bridges are replaced with a phosphothioate internucleoside bridge and / or an arylphosphonate bridge with an aryl group containing 6 to 12 carbon atoms, preferably an α-hydroxybenzylphosphonate bridge, wherein the benzyl group is preferably substituted, for example, by a nitro group, an internucleoside group or oligonucleotides with a methyl halogen. In all-phosphothioate, all phosphodiester bridges are modified with phosphothioate.

The present invention preferably relates to oligonucleotides wherein not all phosphodiester internucleoside bridges have been uniformly modified by internucleoside bridges, either one or a nucleoside phosphothioate (phosphothioate)

Preferably, the at least one internucleoside bridge has another type of modification or is not modified. In particular, the invention relates to oligonucleotides which additionally comprise at least one other type of modification.

In another particular embodiment of the present invention, a plurality of nucleosides (β-0-2'-deoxyribose and / or base) in sequence; oligonucleotides are modified, preferably β-Ό-2'-deoxyribose is replaced by 2'-O-alkylribose having an alkyl group containing 1 to 6 carbon atoms, preferably 2'-O-methylribose, and / or the nucleoside base is replaced by 8-azapurine, 7- deazapurine substituted at position 7 and / or 7-deazapurine substituted at position 8 (purine: adenine, guanine). Preferably, the invention relates to oligonucleotides wherein not all nucleosides are modified uniformly. Preferably, the invention relates to oligonucleotides which additionally comprise at least one other type of modification.

In another particular embodiment of the invention, one or more sugar phosphate backbone units are replaced by PNA units, preferably 2-amonoethylglycine units. Preferably, the sugar-phosphate units that are replaced are at least to some extent linked together. Preferably, the invention relates to oligonucleotides wherein not all sugar-phosphate units have been uniformly replaced. In particular, the invention relates to chimeric oligonucleotides, eg, those that are composed of one or more portions of PNA and one or more portions of DNA. Such oligonucleotides have, for example, the following modifications, but are not limited to: DNA-PNA, PNA-DNA, DNAPNA-DNA, PNA- DNA-PNA, DNA-PNA-DNA-PNA, PNA-DNA-PNA-DNA. Similar modification patterns are also possible for chimeric molecules composed of DNA and PHONA portions, e.g., DNA-PHONA, PHONADNA, DNA-PHONA-DNA, PHONA-DNA-PHONA, DNA-PHONA-DNA-PHONA, PHONA-DNA-PHONA- DNA. In addition, chimeric molecules that comprise portions of three different species, that is, a portion (or portions) of DNA, a portion (or portions) of PNA, and a portion (or portions) of PHONA, are of course also possible. Preferably, the invention relates to oligonucleotides which additionally comprise at least one further modification.

In another embodiment of the invention, the oligonucleotide is at its 3'-end and / or 5'-end with an alkyl moiety of 12 to 18 carbon atoms, preferably an alkyl moiety of 16 carbon atoms, a triethylene glycol or hexaethlene glycol moiety - these moieties preferably being attached to an oligonucleotide via a phosphate group. Preferably, the invention relates to oligonucleotides that do not have both ends (5'-end and 3'-end) uniformly modified. Preferably, the invention relates to oligonucleotides which additionally comprise at least one further modification.

In a preferred embodiment of the invention, only some particular positions in the oligonucleotide sequence are modified (it is a partially modified oligonucleotide). A partially modified oligonucleotide is also referred to in some publications as a minimal modified oligonucleotide. Within the sequence of the oligonucleotides, the modification is located at some specific positions (on a particular nucleotide, on a certain nucleoside, on a certain nucleoside base, on a particular internucleoside bridge).

In a particular embodiment of the present invention, a partially modified oligonucleotide is prepared such that only some of the phosphodiester bridges have been replaced.

3'- and / or replacement with modified internucleoside bridges, e.g.

phosphothioate bridges and / or α-hydroxybenzylphosphonate bridges. Thus, the invention particularly relates to oligonucleotides that are only modified to some extent.

Particular embodiments of the present invention relates to an oligonucleotide wherein the 1 to 5 terminal nucleotide units at the 3 ^r -, and / or the 5'-end of the oligonucleotide are protected by modified internucleoside bridges located 5'-end of the corresponding nucleoside, preferably a phosphodiester internucleoside bridge phosphorothioate bridges and / or? -hydroxybenzylphosphonate bridges. Most preferably, 1 to 5 terminal nucleotide units at the 3'-end of the oligonucleotide are protected by modification of internucleoside bridges located at the 3'- and / or 5'-end of the corresponding nucleosides. Optionally, 1 to 5 terminal nucleotide units at the 5'-end of the oligonucleotide are additionally protected by modification of internucleoside bridges located at the 5'- and / or 3'-end of the corresponding nucleosides. Optionally, the oligonucleotides comprise further modifications at other positions.

The present invention further relates to an oligonucleotide wherein at least one internal pyrimidine nucleoside and / or an internucleoside bridge located at the 5'-end and / or the 3'-end of this pyrimidine nucleoside with a pyrimidine base such as cytosine has been modified, preferably replaced by one phosphodiester internucleoside bridges. phosphothioate bridges and / or α-hydroxybenzylphosphonate bridges.

In a preferred embodiment of the invention, 1 to 5 terminal at the 3 'modification (nucleoside thymine) is or more and / or the 5'-end of the internucleoside nucleotide units of the oligonucleotide are protected by bridges located 3' and / or 5'-end of the corresponding nucleosides. an inner pyrimidine nucleoside and / or an internucleoside bridge located at the ·

The 5'-end of the pyrimidine nucleoside and / or located at the 3'-end of the pyrimidine nucleoside is modified.

The principle of partially modified oligonucleotides has been described in A. Peyman, E. Uhlmann, Biol. Chem. HoppeSeyler, 377 (1996) 67-70 and EP 0 653 439, incorporated herein by reference). In this case, 1 to 5 terminal nucleotide units located at the 3'- and / or 5'-end of the oligonucleotide are protected, for example, by replacing the phosphodiester internucleoside bridges located at the 3'- and / or 5'-end of the corresponding nucleosides, e.g. phosphothioate internucleoside bridges. In addition, preferably at least one internal pyrimidine nucleoside (or nucleotide) position is modified, preferably the 5'-internucleoside bridge of the pyrimidine is modified, e.g., by a phosphothioate. Partially modified oligonucleotides exhibit particularly advantageous properties, e.g. have a particularly high degree of nuclease stability in conjunction with minimal modification. They also have significantly lower propensity for non-antisense effects, which are often associated with the use of all-phosphothioate oligonucleotides

Antisense Res. Dev. 4, 67).

oligonucleotides also exhibit higher binding affinity than all-phosphothioate oligonucleotides.

In particular, the present invention relates to partially / minimally modified oligonucleotides. Examples of oligonucleotides having one of SEQ ID NOs.

27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 and 56 certain internucleoside

3'- and / or nucleoside, (Stein and Krieg, 1994, Partially modified modified c

and bridges, such 14, 16, where are the following ON 1, ON4, ON15, 0N16, ON 17, ON 21, ON 22, ON 23, ON 24, ON 25, ON 26, ON 22, ON 39, ON 40, ON 58 and ON 100 to ON 113:

· · · · · * * * * * * * * * * * * * * * * * * * * *

ON 2 ON 3

ON 15

ON 16

ON100

ON101

ON102

ON103

ON 17 ON 21

ON24

ON1Q4

ON105

ON 106

ON107

ON108

ON 109

ON110

ON111

ON112

ON113

ON 25 ON 26 ON 58 ON 39 ON 40

3'-C * C * AGC * C * C * GGAG * G -5 '3'-C * G * G AGG C * T * T * TG * G T * GG * T -5 '

3'-G * AGG * TGG * TAC'G -5 '3'-AG * GG * T * GG * TAC'G -5' 3'-AGG * T * GG * TA * C * G -5 '3'-G * G * AGG * T * G * TA * C * G -5' 3'-G * G * AGG * TG * T * AC * G -5 '

3'-G * G * T * GGT * A C'G GT * T -5 '3'-C * A * C * CAGGGT * C * C * G * C * CGA * C -5'3'-A'G * GGT * C * CGA C'G'T -5 '

3'-G * GG * T'C * CGA C'G G * G -5 '3'-G * G * G * GT * C * CGAC * GT * G -5' 3'-G * G'G T * C * CGAC * G 'T * G -5' 3'-G * G * G T'C * CGAC * G * TG -5 '3'-G * GG * T * C * CGAC * G * T * G -5 '3'-G * G'G * T * C * CGAC * GT * G -5' 3'-G * G'G * T * C * CGAC * G * T'G -5 '3'- G * G * GT * C * GAC * G'T * G -5 '3'-G * G * G T * C * GAC * GT * G -5' 3'-G * G * G * T * C * CGAC * GT * G -5 '3'-G * G * T * C * CGAC * G * T * G -5'

3'-G * G * TC * C * GAC * GT * GG -5 '3'-C * C * GA * C G'T GGG * T * A * C * G * G -5 '3' -A * C * GC * C * CC * CGAC'G -5 '3'-C * C * C * CC * GA * CGAC'G -5'

(Ex sec. id. . No. 14) (Ex sec. id. . No. 16) (Ex sec. id. No. 27) (Ex sec. id. No. 23) (Ex sec. id. No. 28) (Ex sec. id. No. 23) (Ex sec. id. No. 28) (Ex sec. id. No. 23) (Ex sec. id. No. 29) (Ex sec. id. No. 33) (Ex sec. id. No. 34) (Ex sec. id. No. 35) (Ex sec. id. C.' 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 36) (Ex sec. id. No. 37) (Ex sec. id. No. 38) (Ex sec. id. No. 52) (Ex sec. id. No. 55)

444 where it means localization of the internucleoside modification

bridge bridge. Yippee phosphothioate internucleoside Another example special design submitted ' of the invention therefore concerns partly modified

an oligonucleotide having a nucleoside modification, eg, a nucleoside base modification and / or a modification of a β-Ο-2 deoxyribose unit. Preferably, β-ϋ-2'-deoxyribose is replaced by 2'-O-alkylribose having an alkyl group of 1 to 6 carbon atoms, most preferably it is replaced by 2'-O-methylribose (ie β-ϋ-2'-deoxyribonucleoside is replaced by 2'O (methylribonucleoside). Examples of such oligonucleotides having e.g. . Nos. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 and 56 show the nucleoside modification patterns in the ON 114 to ON 138 oligonucleotides (only N "modifications, not internucleoside modifications) .

The oligonucleotide of the invention may contain other types of modifications in addition to modification of one type.

In another preferred embodiment of the invention, the oligonucleotide comprises modified internucleoside bridges at certain positions, and additionally a modification of the nucleoside at a certain position, preferably replacing β-Ώ-2'-deoxyribose. In a preferred embodiment of the invention, the internucleoside modification of the phosphodiester bridge replacement is a phosphothioate bridge and the modification of β-ϋ-2'-deoxyribose is the replacement of 2'-O-methylribose, in which case the oligonucleotide is a chimeric oligonucleotide composed of modified and unmodified DNA and RNA portions , and which comprises the nucleosides 2'-O-methyl-ribonucleoside and β-ϋ-2'-deoxyribonucleoside and phosphodiester and phosphothioate internucleoside bridges.

• · ····

Examples of such oligonucleotides having one of SEQ ID NOs. . No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 and 56 and modifications of certain internucleoside bridges and additionally certain nucleoside positions. modification formulas):

0N114 ON115 ON116 ON117 ON118 ON119 ON120 ON121 ON122 ON123 ON124 ON125 ON126 ON 127 ON128 ON130 ON131 ON132 ON133 ON134 ON135 ON136 ON137 ON138

3'-C * C * A G * C * C * C * G * G * G-5 (eg for SEQ ID NO.

3'-C * G * G A G G C * T * T * T G * G-5 (eg for SEQ ID NO.

3'-G * A * TGGAGG * T * GG ^w T-5 (pf for SEQ ID NO.

3'-G * G * A * G * T * G * T * C * G-5 (eg for SEQ ID NO.

3'-G * G * T * G G * T * A C * G * T * T-5 (eg for SEQ ID NO.

3'-C * A * C * C * G * G * T * C * C * G-5 (eg for SEQ ID NO.

3'-C * C * A G G G * C * C G A * C-5 (Ex. For SEQ ID NO.

3'-A * G * G G * T * C * G G * C * G * T-5 (Ex. For SEQ ID NO.

3'-G * G G * T * C * C G A C * G T * G-5 (Ex. For SEQ ID NO.

3'-G * G * T * C * G * C * G * G * 5 (Ex.

3'-C * C * G A * C G * T G G * T * A-5 (Ex. For SEQ ID NO.

3'-C’C * C * C CPG A * C G A C * G-'5 (Ex.

3'-G * G * G T * C * C G A C * G T * G-5 (Ex. For SEQ ID NO.

3'-G * G * G T * C * C G A C * G T * G-5 (Ex. For SEQ ID NO.

3'-G * G * G T * C * C G A C * G T * G-5 (Ex. For SEQ ID NO.

3'-G * G * G T * C * C G A C * G T * G-5 (Ex. For SEQ ID NO.

3'-G * G * G T * C * C G A C * G T »G-5 (Ex. For SEQ ID NO.

3'-G * G * A G G * T G G * T A C * G-5 (Ex. For SEQ ID NO.

3'-G ^w G * AGG * TGG * TAC * G-5 (Ex. For SEQ ID NO.

3'-G * G * A G G * T G G ~ T A C * G-5 (Ex. For SEQ ID NO.

3'-G * G * A G G * T G G * T A C * G-5 (Ex. For SEQ ID NO.

3'-G * G * A G G * T G G * T A C * G-5 (Ex. For SEQ ID NO.

3'-G * G * A G G * T G G * T A C * G-5 (Ex. For SEQ ID NO.

3'-G * G * AGG ^W TGG * TAC * G-'5 (Ex. For SEQ ID NO.

14)

16)

27)

28) 29)

33)

34)

35)

36)

37)

38) 56) 36) 36) 36) 36) 36) 28) 28) 28) 28) 28) 28) 28) where denotes the position of the internucleoside bridge modification and N denotes the position of the modified nucleoside (ie nucleoside base modification and / or modification) β-ϋ-2'deoxyribose). Preferably, the phosphothioate bridge is and N is the F-position of the 2'-O-alkylribonucleoside containing 1 to 6 methylribonucleoside atoms.

Other examples are oligonucleotides wherein each nucleoside is replaced by a 2'-O-alkylribonucleoside (totally composed of 2'-O-alkylribonucleosides, 2'-O-alkyl-RNA). Such oligonucleotides can be stabilized against nuclease action by partial replacement of phosphodiester

bridges: . (Ex. For sec. id. C. 14) (Ex. For sec. id. C. 16) , (Ex. For sec. id. C. 27) (Ex. For sec. id. C. 23) (Ex. For sec. id. C. 29) (Ex. For sec. id. C.· 33) (Ex. For sec. id. C. 34) (Ex. For sec. id. C. 35) '(Ex. For sec. id. C. 36) AND (Ex. For sec. id. C. 37) (Ex. For sec. id. C. 33) (Ex. For sec. id. C. 55) (Ex. For sec. id. C. 56) 1 (Ex. For sec. id. C. 52)

ON139 3'-C * C * G * C * C * C * G * G * G-5

ON140 3'-C * G * G A G G * T «T * T G * 5N141 3'-G * A * T * G G * T * G * T-5 '

ON142 3'-G * G * A GTT G G * T AND C * G-5 '

ON143 3´-G »G * -G * G * G G * T-5 '

0N144 3'-C * A * C * CAGGGT * C * C * G-5 '0N145 3'-C * C * AGGGT »C * CG A'C-5' 0N146 3'-A * G * GGT» C * CGAC * G * T-5 '

0N147 3'-G * G G * T * C * C G A C »G T ^ G-5 '

ONI48 3'-G * G * T * C * G * C * G T * G G-5 '

ON149 3'-C * C * G A * C G * T G G * T »A-5 '

0N150 3'-A * C * C * C * C * C * C G * 5-ON151 3'-C * C * C * C * G * C * C G * 5

ON152 3'-G * T * AGAAG * TT * C * G * G-5 'where denotes internucleoside bridge modification position and N denotes modified nucleoside position (ie nucleoside base modification and / or β-0-2'deoxyribose modification) . Preferably, it is a phosphothioate bridge and N is the position of the 2'-O-alkylribonucleoside, preferably 2'-O-methylribonucleoside (in this case T is 2'-O-methyluridine).

A further preferred embodiment of the invention provides oligonucleotides having at their 3 'and / or 5' end one or more C 12 -C 18 alkyl radicals, preferably an C 16 -C 16 alkyl radical. be bound eg as a phosphodiester as described in patent, document EP 0 552 766 A2 (which is incorporated by reference) or as a 3'-phosphodiester of O-CH ₂ -CH (OH) -O-alkyl containing in the alkyl group 12 up to 18 carbon atoms. Preferred is an oligonucleotide having an alkyl residue of 16 carbon atoms attached at its 3'- and / or 5'-end.

Examples of such oligonucleotides are ON 153 to ON 164 (oligonucleotides having one of SEQ ID NOs: 14, 16,

27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 and 56 and modifications in certain internucleoside bridges, similar to ON 1, CN 4, ON 15, ON 16, ON 17, CN 21 , ON 22, ON 23, ON 24, ON 25, ON 26, ON 22, ON 39, ON 40 and ON 58 and in addition an alkyl radical containing 16 carbon atoms bound at the 3'- and / or 5'-end) oligonucleotides may have other sequences and other patterns of modifications):

ON153 3'-C * C * A G * C * C * G * G * G-C16-5 ',

ON154 3'-C * G * G AGG C * T * T * T * G-C16-5 ',

ON155 3'-G * A * T * G * G * T * G * T-C 16-5 '

ON156 3'-G * G * A G * G * T * C * G-C16-5 ',

ON157 3'-G * G * T * G * T * A * C * G * PT-C16-5 ',

ON 158 3‘-C * AC * C * G * G * C * C'G-C16-5 ',

ON159 3'-C * C * A GGG T * C * C G * C-C16-5 ',

ON160 3'-A * G'G G T * C * C G * C * G * T-C16-5 ‘

ON161 3'-G * G * T * C * C * G * C * G PG-C16-5 ',

ON162 3'-G * G 'C * C' G & C * G 'G-C16-5'

ON163 3'-O * C * G A * C * G * G * T * A-C16-5 '

ON164 3'-C * C * C * CC * GA * CGAC * G-C16-5 ', * where it shows the position of the modification of the internucleoside bridge, preferably the location of the phosphothioate bridge, and Cig shows the position of the modification at the 5'-end of hexadecyl phosphate.

The present invention further relates to an oligonucleotide wherein the 3'-end and / or the 5'-end is attached to an oligoethylene residue, preferably triethylene glycol or hexaethylene glycol, most preferably via a phosphodiester (tri- or hexaethylene glycol phosphoric acid ester). Such an oligonucleotide of the invention may contain other modifications. An example of such an oligonucleotide having the sequence id. No. 36 are ON 165, ON 166 and ON 167:

ON165 3'-teq-G * G * G T * C * C G * C * G T * G -5 \

ON16S 3'-teq-G 'G * G T * C * C G A C * G T * G -5'.

ON167 3'-teg-GV ^ GT'C * CGAC * GT * G -5 ', wherein teg is an oligoethylene glycol moiety bound as a phosphate ester to an oligonucleotide, preferably teg is a triethyleneeglycol- or hexaethylene glycol ester of phosphoric acid, is a modified internucleoside bridge, N is modified nucleoside (eg, nucleoside base modification and / or β-ϋ-2'-deoxyribose modification). Preferably, it is a phosphothioate bridge, N is 2'-O-alkylribonucleoside, preferably 2'-O-methylribonucleoside (in which case T is 2'-O-alkyluridine)

In another specific embodiment of the invention, the oligonucleotide is linked via a linker to a 2'5'-linked oligoadenylate-5 (thio) phosphate. The linker can be, for example, an oligoethylene glycol phosphate, preferably a triethylene glycol phosphate, a tetraethylene glycol phosphate or a hexaethylene glycol phosphate residue. The 2'5'-linked oligoadenylate is preferably attached via its 2'-end as a tetra- or pentaadenylate whose 5'-hydropxyl. the functional group is substituted with phosphate or thiophosphate 9 9 99 99 9999 99 9999 999 9999

9 · <· «· 9 9 9 9 99 9 rest. 2'5'-oligoadenylate is known to induce RNase L cleavage of the target mRNA (Torrence et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90, 1300). 2'5'-oligoadenylate serves to activate ribonuclease L (RNase L), which then degrades VEGF mRNA. Also, a 2'5'-linked 3'-deoxyadenylate derived from the nucleoside analogue of Cordycepin may be used in place of the 2'5'-linked adenylate. In such a case, the oligonucleotide portion that is complementary to the target nucleic acid is preferably modified at certain positions by a 2'-O-alkylribonucleoside having an alkyl group containing 1 to 6 carbon atoms (preferably 2'-O-methylribonucleoside) or PNA. An example of such an oligonucleotide having the sequence id. No. 36 is ON 168 (these oligonucleotides may also have any other oligonucleotide sequence of the invention):

ON 168 5'-p * - (2'5 ^, -rA * rA * rA * rA) * (teg) G * TG * CAGC * C * T * GG * G-3 'wherein teg is an oligoethylene glycol residue, preferably triethylene glycol residue, N is β-D-2'-deoxyribonucleoside substituted with 2'-O-alkyl residue, preferably 2'-O-CH ₃ (T is 2'-O-methyluridine), rA is ribo-A, Co is 3 ' -deoxy-A (Cordycepin), p * is 5'-thiophosphate and is a modified internucleoside bridge

Another preferred embodiment of the invention comprises replacing one or more natural nucleoside bases with a non-natural or modified nucleoside base, preferably 8azapurines and / or substituted 7-deazapurines, preferably 7-deazapurine substituted at the 7-position and 7-deazapurine substituted at the 8-position, e.g. in EP 0 171 066 and EOP 0 680 969. Examples of such oligonucleotides are ON 169 and ON 170 (both having SEQ ID NO: 36 and, in addition to the nucleoside base modification, other types of modifications):

0N169 3'-G * G * G T »C» C G A C * G T * G -5 ',

ON 170 3'-teq-G * G * GT * C * CGAC * GT * G -5 ', where G is 8-azadeoxyguanosine, A is 8-azadeoxyadenosine, teg is oligoethylene glycol residue, preferably triethylene glycol residue, N' is 2 ' -O-alkylribonucleoside, preferably 2'-O-methylribonucleoside, wherein (T is 2'-Omethyluridine).

In another preferred embodiment of the invention, the oligonucleotide has a 3'3'and / or 5'5'end at the 5'-end and / or 3'-end as described in the patent, EP 0 464 638 and EP 0 593 901. such an oligonucleotide is ON 171 having the sequence id. No. 36 and other types of modifications besides the 3'3'inversion at the 3'-end

ON171

3'-G (3'3 ') G * G T * C * C G A C * G T * G-5', a phosphodiester internucleoside wherein (3'3 ') is a 3'3' phosphodiester bond and. is a modified internucleoside bridge, preferably a phosphothioate internucleoside bridge.

Another preferred embodiment of the present invention relates to the replacement of one or more phosphodiester bridges with an α-hydroxybenzylphosphonate bridge as described in WO 95/01363. An example of such an oligonucleotide is ON 172 having the sequence id. C.

bridge of phosphodiester bridge with phosphothioate internucleoside bridge:

ON172 3'-G (hbp) G'G T * C * CGAC * GT * G-5 ', and in addition to the α-hydroxybenzylphosphonate replacement, it also contains a replacement where (hbp) is an α-hydroxybenzylphosphonate bridge, preferably α-hydroxy (o- nitrophenyl) methylphosphonate bridge and is a phosphothioate bridge.

· · Φ «·« ♦ «« «« «« «« «« «« 9 9 ·

In another preferred embodiment, the oligonucleotides comprise sugar-phosphate backbone modifications, preferably PNA units. Examples of such DNA-PNA chimeras having one of SEQ ID NOs. 52, 55 and 56, DNA) (synthesis

ON173

ON174

ON175

ON176

ON177

ON178

ON179

ON180

0N181

ON182

ON183

ON184. No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, have the following modification formulas (for the PNA-a pattern of PNA-DNA chimeras see EP 0 672 677):

(3j-ca. g ccc GG AG G-5 '(3j-cggaggc Τ Τ TG G-5' (3j-g atggaGGTGG T-5 ') (3 jg gaggt GGTAC G-5' (3 jg gtggtaCGGT T-5 '( 3j-caccaggGTC C G-5 '(3') - c cagggtCCGA C-5 '(3 and gggtccGACG T-5' (3j-gggtcc GACGT G-5 ') (3 jg gtccgaCGTG G-5' (3 jc cgacgtGGGT A- 5 '(3j-c cccc CG AC G-5'

(Ex sec. id. C. 14) (Ex sec. id. C. 16) (Ex sec. id. C. 27) (Ex sec. id. C. 28) (Ex sec. id. C. 29) (Ex sec. id. C. 33) (Ex sec. id. C. 34) (Ex sec. id. C. 35) (Ex sec. id. C. 36) (Ex sec. id. C.· 37) (Ex sec. id. C. 38) (Ex sec. id. C. 56)

where lowercase letters denote PNA units, underlined letters denote hydoxyethyglycine-PNA units, uppercase denotes DNA.

Other modification patterns, such as DNA-PNADNA or PNA-DNA, are also possible. Comparable formulas of modifications are also possible with PHONA / DNA chimeras. These modification patterns can be combined with any other type of modification and similar modification patterns are possible for any oligonucleotide of the present invention. Examples of oligonucleotides that are derived from ON 173 to ON 184 oligonucleotides but which, in addition to replacing the sugar-phosphate backbone units with PNA backbone units, additionally have modifications of the phosphodiester internucleoside linkage at certain positions in the DNA portion are:

ON185 ON186 ON187 ON188 ON189 ON190 0N191 ON 192 ON193 ON194 ON195 ON196 (3 jc cagccc GG * AG * G-5 '< ^ex · P ^r ° ^sec (3j-cggaggc T * T * TG'G-5' (Ex. sec (3j-ggggt GG * T * GG * T-5 '(Ex for sec (3j-ggggt GG * TAG * G-5') (Ex for sec (3j-ggtggta C * GG T'T-5 ' (eg for sec (3j-caccagg GT * C * C * G-5 ') (eg for sec (3 jc cagggt C * C * GA * C-5') (eg for sec (3) -a gggtccGAC G T-5 (pf, _{P for} sec (3 jg ggtcc GAC * G T'G-5 ') _{(for> sec} (3j-ggtccga C'G T * G G-5'_{(for> sec} (3 jc cgacgt) GG G'T'A-5 ' _{{ex for sec} (3 jc ccccga C * GAC * G-5' ₍ ex _for SEQ ID NO ID NO ID NO ID NO ID NO ID No. ID No. ID No. ID No.

14)

16)

27)

28) 29)

33)

34)

35)

36)

37)

38) 56) where lowercase letters denote PNA units, underlined letters denote hydoxyethyglycine-PNA units, uppercase denotes DNA and is a modified internucleoside bridge, preferably a phosphothioate bridge.

The above specific oligonucleotides - certain sequences with a particular type of modification (s) at a particular position (specific pattern of modifications) serve only as examples of various embodiments of the invention. The present invention is not limited to said oligonucleotides. Other combinations of sequences and modification patterns are possible according to the invention.

The oligonucleotide of the present invention specifically inhibits expression of a target protein (which is VEGF) or a target sequence (a nucleic acid that encodes VEGF, preferably VEGF mRNA). Preferably, the oligonucleotide of the invention specifically inhibits VEGF expression. This leads to a decrease in the level of VEGF protein as compared to unaffected expression. For example, specificity can be demonstrated by determining the effect of the oligonucleotides of the invention on VEGF expression as compared to the effect of the same oligonucleotides in beta-actin expression on mRNA and / or protein levels: when treated with oligonucleotide. according to the invention, only mRNA and / or VEGF protein levels have been reduced, while, for example, mRNA and / or beta-actin protein levels (structural protein of the basic metabolic pathway, i.e. house-keeping protein) remained unchanged. In particular, the effect of the oligonucleotide can be demonstrated by determining the amount of VEGF mRNA and / or VEGF protein (e.g., when compared in a similar experiment without an oligonucleotide). For example, the inhibitory action of an oligonucleotide can be determined in vitro by treating tissue cultures with an oligonucleotide. Then, for example, the level of mRNA in cell lysates can be determined, for example as described in Example 4. The level of VEGF protein (e.g., absolute amount of VEGF protein in grams or, e.g., relative to percent compared to untreated cells) can be determined from supernatant ( (e.g., culture medium) (amount of secreted VEGF) and / or membrane preparations (amount of membrane bound VEGF) and / or cell lysates. For example, the amount of secreted VEGF protein can be determined by ELISA, eg as described in Example 3.

In a particular embodiment, the oligonucleotide inhibits VEGF mRNA expression and / or reduces the level of VEGF protein, eg, in a cell culture with an IC _{50 of} about 1 µ přibližně or less, eg, 500 nM, 200 nM, 100 nM or less.

In another particular embodiment of the invention, the inhibition specific to the oligonucleotide of the invention, in these cases only the oligonucleotide having the sequence of the invention, reduces the level of VEGF protein and / or VEGF mRNA. Compared to these specific oligonucleotides, these levels do not change to the same extent, if demonstrably altered, when an oligonucleotide with a sequence containing a mismatch or a mixed sequence (scrambled) is used. These oligonucleotides are used as control oligonucleotides, such as ON 200, ON 201, ON 203, and ON 204 oligonucleotides. ON 200 and ON 201 have 2 and 4 mismatch positions. (Mismatch) relative to the ON 24 sequence (SEQ ID NO: 36), but all three oligonucleotides have the same phosphorothioate modification pattern (position * *). These four oligonucleotides were used in comparative experiments with ON 16 and ON 24. Control oligonucleotides did not inhibit VEGF mRNA expression in cell culture at concentrations of 1 μ 1 and below (Table 3).

ON16 3'-G * G * AGG * TGG * TA C'G-5 ' _antisense oligonucleotide

ON200 3'-G * G * A G PG G G * T A C * G-5 '2 mismatched positions

ON201 3'-G * G * C G PG G G * T A A * G-5 ‘4 mismatched positions

0N24 3'-G * G * PC * C * G * C * G PG-5 'antisense oligonucleotide

ON203 3‘-G * G * PC * C AG C‘G PG-5 ’2 mismatched positions

ON204 3'-G * GG * C * C * CAG PG PG-5 ' ⁴ mismatched positions where mismatched positions are underlined in ON 200 and ON 201 relative to ON 16 and in ON 203 and ON 204 relative to ON 24,

HE 200 has sequence id. C. _8g . 3'-GGAGTGGGTACG-5 ' HE 201 has sequence id. C. 90: 3'-GGCGTGGGTA AG — 5 HE 203 has sequence id. C. 91: 3'-GGGTCCAGCGTG-5 \ HE 204 has sequence id. C. 92: 3'-GGGCCCAGTGTG-5 '. Oligonucleotide according to of the invention effectively inhibits

VEGF protein in cell cultures as compared to control oligonucleotides. Giant. 2 shows inhibition of VEGF secretion from U-87 cells treated with one of 52 different 12-nucleotide antisense oligonucleotides at a concentration of 3 μΜ. The corresponding oligonucleotide sequences are shown in Table 2, where for some oligonucleotides also IC ₅₀ values are given.

The oligonucleotide of the invention inhibits VEGF protein expression by 55%, preferably 65%, most preferably 75%, relative to control and / or VEGF compared to control. cells, e.g. the secreted VEGF is reduced by 55, 65 or 75% when the cells are treated with the oligonucleotide of the invention at a concentration of 3 μΜ, preferably even at a lower concentration such as 1 μΜ or less, preferably 0.5 μΜ or even lower (see Fig. 2).

The oligonucleotide of the invention effectively inhibits the expression of VEGF (isoforms) in human cells and / or has the ability to inhibit tumor growth in vertebrates. Preferably, the oligonucleotide of the invention reduces the level of VEGF mRNA protein in tumors of untreated individuals. Preferably, the oligonucleotide of the invention reduces tumor volume in vertebrates, e.g., mice, as compared to untreated mice or compared to tumor volume in the same animal prior to treatment.

The invention also relates to a process for preparing the oligonucleotide of the invention. The method of preparation involves chemical synthesis of the oligonucleotide. Preferably, the chemical synthesis is carried out by a known standard method used for oligonucleotide synthesis, for example by the Caruthers phosphoramidite method (Tetrahedron Letters, 24, 245, 1983), by the H-phosphonate method (Todd et al., J. Chem. Soc., 3291, 1957) ) or by the phosphotriester method (Sonveaux, Bioorg. Chem., 14, 274, 1986, Gait, MJ, Oligonucleotide Synthesis, A Practical Approach, IRL Press, Oxford, 1984) or by improved or modified methods derived from these standard methods. For example, an oligonucleotide of the invention may be prepared as described in Example 1. Preferably, an oligonucleotide of the invention is synthesized on a solid support by condensation of suitably protected monomers (e.g. nucleosides) to form internucleoside bridges between these monomers. The invention relates to a process for the preparation of an oligonucleotide or a derivative thereof, wherein a nucleotide unit having a 3'- or 2'-terminal phosphorus group (V) and a free 5'-hydroxyl or mercapto group reacts with another nucleotide unit having a phosphorus group (III). Or the phosphorus (V) at the 3D position, or its activated derivatives, and optionally using protective groups that can be temporarily introduced by the dd oligonucleotide to protect other functional groups and which are removed after synthesis before the oligonucleotide can be which has been cleaved from the carrier, optionally converted to a physiologically tolerated salt. For the synthesis of modified oligonucleotides, the standard methods have been altered to some extent. However, these variations are known to the person skilled in the art and have been described, for example, by Agrawal S. ("Protocols for oligonucleotides and analogs, Human Press, Inc., Totowa, New Jersey, USA, 1993). The preparation of modified oligonucleotides has also been described in EP 0 710 667, EP 0 630 969, EP 0 464 638, EP 0 593 901, WO 95/01363, EP 0 672 677, EP 0 739 898 and EP 0 552 766. Methods for preparing the modified oligonucleotides described in the above documents are incorporated by reference.

The invention further relates to a method of inhibiting VEGF expression and / or modulating the expression of a VEGF-encoding nucleic acid, wherein the oligonucleotide of the invention is contacted with an mRNA, cDNA), wherein the oligonucleotide is contacted with a VEGF-encoding nucleic acid. (i.e., binds a nucleic acid encoding VEGF.

Thus, the invention also relates to an oligonucleotide contacted with nucleic acids encoding VEGF (eg, mRNA, cDNA), for example by introducing the oligonucleotide into a cell by known methods, for example by incubating cells with said oligonucleotide or a formulation thereof. such as lipofectin, lipofectamine, Cellfectin or polycations (e.g. polylysine).

For example, to introduce an oligonucleotide into a cell, an oligonucleotide that has been pre-incubated with Cellfectin for 30 minutes at room temperature is incubated for about 5 hours or less with the cells.

The invention further relates to the use of an oligonucleotide, preferably as an antisense oligonucleotide (binding of the oligonucleotide to mRNA encoding VEGF) or as a ribozyme (binding to mRNA encoding VEGF and cleavage of this mRNA). In another particular embodiment of the invention, the oligonucleotide can be used to induce cleavage of mRNA encoding VEGF by RNase H, resulting in a decrease in VEGF expression.

The invention relates to the use of an oligonucleotide for modulation as well as total or partial inhibition of VEGF expression (eg, VEGF121, VEGFiss, VEGFig, VEGF ₂₀₆ ) and / or splice variants thereof and / or mutants thereof, for example for total or partial inhibition of VEGF encoding mRNA.

The invention relates to the use of oligonucleotides for inhibiting, preventing or modulating angiogenesis, neovascularization, tumor growth and metastasis, particularly in vertebrates. The invention generally relates to the use of the oligonucleotide of the invention for the treatment or prevention of diseases in which VEGF is overexpressed. Such diseases in which VEGF is overexpressed are, for example, cancer, age-dependent macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis, and other inflammatory diseases.

The invention further relates to the use of an oligonucleotide as a medicament and to the use of an oligonucleotide for the preparation of a pharmaceutical composition. In particular, the oligonucleotide may be used in a pharmaceutical composition which is used to prevent and / or treat diseases that are associated with the expression or overexpression (overexpression) of VEGF, and for the treatment of diseases in which VEGF is involved or is a causal factor thereof. overexpression.

The invention further relates to a pharmaceutical composition comprising an oligonucleotide and / or its physiologically tolerated salts in addition to pharmaceutically acceptable excipients or excipients.

The invention relates to a pharmaceutical composition comprising at least one oligonucleotide according to the invention, which can be used for the treatment of diseases which are associated with abnormal vascular permeability, cell proliferation, cell permeation, angiogenesis, neovascularization, tumor cell growth and neoplastic cell metastasis.

The invention further relates to a process for the preparation of a pharmaceutical composition comprising admixing one or more oligonucleotides of the invention with a physiologically acceptable excipient and optionally other substances, e.g., where appropriate, with suitable additives and / or excipients.

In particular, the invention relates to the use of an oligonucleotide or a pharmaceutical composition prepared therefrom for the treatment of cancer, eg for inhibiting tumor growth and tumor metastasis, and for the treatment of diabetic retinopathy, age-dependent macular degeneration, psoriasis, rheumatoid arthritis and other inflammatory diseases. For example, an oligonucleotide or a pharmaceutical composition prepared therefrom can be used to treat solid tumors such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colorectal cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, liver carcinoma, tongue carcinoma, neuroblastoma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, retinoblastoma, Wilms' tumor, multiple myeloma, and for the treatment of skin cancer such as meianoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of an oligonucleotide of the invention or a pharmaceutical composition thereof for inhibiting VEGF expression and / or inhibiting accumulation of ascites fluid and pleural effusion in various types of cancers, such as breast cancer, lung cancer, neck cancer, brain cancer, abdominal cancer, colorectal cancer, colorectal cancer, oesophageal cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, bsteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilms tumor, multiple myeloma , lymphomas and blood cancer. Due to the inhibitory effect on VEGF expression and / or formation of ascites fluid and pleural effusion, the oligonucleotide of the invention or the pharmaceutical composition prepared therefrom can increase the quality of life. In a preferred embodiment of the invention, the oligonucleotide of the invention or a pharmaceutical composition prepared therefrom may inhibit the accumulation of ascites fluid in ovarian cancer.

The invention further relates to the use of an oligonucleotide of the invention or a pharmaceutical composition prepared therefrom, for example for the treatment of cancer or for preventing tumor metastasis, or for treating age-dependent macular degeneration, rheumatoid arthritis, psoriasis, and diabetic retinopathy in combination with other drugs and / or other treatment methods, eg known drugs and / or known treatment methods, such as those currently used to treat cancer and / or to prevent tumor metastases. A combination of radiation therapy and chemotherapeutic agents such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen is preferred.

The oligonucleotide and / or its physiologically tolerated salt may be administered to the animal, preferably to a mammal, and in particular to a human, alone, in admixture with another oligonucleotide (or physiologically tolerated salt), or in the form of a pharmaceutical formulation that allows local, percutaneous, parenteral or enteral use; which comprises, as an active ingredient, an effective dose of at least one oligonucleotide, in addition to the usual pharmaceutically acceptable excipients and excipients. This pharmaceutical composition normally contains about 0.1 to 90% (by weight) of the therapeutically active oligonucleotide (s). The dose may vary within wide limits and is adapted to the individual situation in each individual case. In the treatment of psoriasis, topical use is preferred. In the case of cancer, infusions, oral and rectal administration, or nasal aerosol administration are preferred, while in the case of diabetic retinopathy, local, vitreous and oral administration is preferred.

The pharmaceutical composition may be manufactured in a manner known per se (eg, see Remingtons Pharmaceutical Siences, Mack Publ. Co., Easton, PA, 1985) using pharmaceutically inert inorganic and / or organic excipients. For example, lactose, corn starch and / or derivatives thereof, talc, stearic acid and / or salts thereof, and the like can be used to prepare pills, tablets, coated tablets and hard gelatin capsules. Examples of excipients for soft gelatin capsules and / or suppositories are fats , waxes, semi-solid and liquid polyols, natural and / or hardened oils, and the like. Examples of suitable excipients for preparing solutions and / or syrups are water, sucrose, invert carbohydrates, glucose, polyols, and the like. Suitable excipients for injectable solutions are water, alcohols, glycerol, polyols, vegetable oils and the like. Suitable excipients for microcapsules, implants and / or sticks are mixed polymers of glycolic acid and lactic acid. In addition, liposome formulations such as those described by N. Weiner (Drug Develop. Ind. Pharm., 15, 1523, 1989, &quot; Liposome Dermatics, Springer Verlag, 1992) and Hayashi (Gene Therapy, 3, 878, 1996). The pharmaceutical composition may also include a formulation that enhances the oral availability of the oligonucleotide, such as intestinal permeabilization enhancers, e.g., mannitol, urea, bile acids, such as CDCA (chenodeoxocholate) (2%). Cutaneous administration can also be performed, for example, using iontophoretic methods and / or by electroporation. In addition, lipofectins and other carrier systems, for example those used in gene therapy, may be used. Particularly suitable are systems that can be used to introduce oligonucleotides in a highly efficient manner into eukaryotic cells or into nuclei of eukaryotic cells. The pharmaceutical composition may also comprise two or more different oligonucleotides and / or their physiologically tolerated salts and, in addition to at least one oligonucleotide, one or more different therapeutically active ingredients.

In addition to the active ingredients and excipients, the pharmaceutical composition may also contain additives such as fillers, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavoring or flavoring agents, thickeners, diluents or buffering agents, and in addition, diluents and / or solvents and / or agents for achieving a slow release effect, as well as salts for varying the osmotic pressure, coating agents and / or antioxidants.

Description of the picture

Figure 1 (parts A to E) shows the location of the antisense VEGF oligonucleotides (SEQ ID NOs 13 to 72) relative to the cDNA sequence of the VEGF clone (both strands), whose nucleotide sequence is shown in Table 1. Also, the localization of the core regions 1 to 6 and the sequences of id. Nos. 1 to 12 are shown (underlined portions of the sequence).

Figure 2 summarizes the inhibitory effect of different oligonucleotides (each used at a concentration of 3 μΜ) on VEGF protein secretion in cell culture (secretion from human U87-MG cells).

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The inhibitory effect is related to control cells not treated with oligonucleotide (the amount of VEGF secreted from the oligonucleotide treated cells to the amount of VEGF secreted from the untreated cells). The results show that ON 2, ON 4, ON 15, ON 16, ON 17, ON 21, ON 22, ON 23, ON 24, ON 25, ON 26, ON 39 and ON 40 showed the best inhibitory effect relative to control cells . Denomination: 1 is ON 300, 2 is ON 2, 3 is ON 301, 4 is ON 4, 5 is ON 302, 6 is ON 303, 7 is ON

304 , 8 is ON 305, 9 Yippee HE 306, 10 Yippee ON 307, 11 Yippee ON 308, 12 Yippee HE 309, 13 is ON 31 0, 14 is ON 311, 15 'is ON 15 Dec , 16 is HE 16, 17 is ON 17, 18 Yippee HE 312, 19 Yippee ON 313, 20 Yippee ON 314, 21 Yippee HE 33, 22 j e ON 22, 23 is ON 23 t 24 is ON 24, 25 is ON 25, 26 Yippee ON 2 6, 27 is HE 315 , 28 j e ( HE 316, 29 is HE 317, 30 Yippee

ON 318, 31 is ON 319, 32 is ON 320, 33 is ON 321, 34 is ON

322, 35 is ON 323, 36 is ON 324, 37 is ON 325, 38 is ON 326, is ON 39, 40 is ON 40, 41 is ON 327, 42 is ON 328, 43 is

ON 329, 44 is ON 330, 45 is ON 331, 46 is ON 332, 47 is ON

333, 48 is ON 334, 49 is ON 335, 50 is ON 336, 51 is ON 337 and 60 is ON 345.

Figure 3: Inhibition of tumor growth by ON 24. Nude mice bearing U87-MG xenografts were treated with ON 24 at various concentrations (1 mg / kg, 4 mg / kg, V 12 mg / kg (ie mg oligonucleotide per kg body weight) Tumor size was analyzed on day 27. Tumor size was also determined in untreated mice for comparison (*).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Synthesis of oligonucleotides

Oligonucleotides (ON) were synthesized using an automated DNA synthesizer Applied Biosystems 394 DNA Syntehetizer (Perkin ELmer Applied Biosystems, Inc., Foster City, USA) using standard phosphoramidite chemical synthesis techniques (see, eg, F. Eckstein (Ed.) "Oligonucleotides and Analogues. A Practical Approach, IR Press, Oxford, 1991). After condensation, the phosphothioate linkages were introduced by sulfurization with Beaucage reagent followed by blocking with acetic anhydride and N-methylimidazole. After cleavage from the solid support and final deprotection with concentrated ammonia, they were purified by gel electrophoresis. 2'-O-methyl-modified by replacing the standard 2'-O-methylribooligonucleotides with polyacrylamide oligonucleotides were prepared by phosphoramidites in the corresponding nucleoside phosphoramidites. All oligonucleotides were analyzed by negative ion electrospray mass spectroscopy (Fisions BioQ), which in all cases confirmed the calculated mass.

Clβ-modified oligonucleotides were synthesized using hexadecyloxy (cyanoethoxy) N, N-diisopropyaminophosphane as the phosphitylating agent in the last step of the oligonucleotide instead of the standard amidite, or the synthesis began on a solid support correspondingly derivatized. The tetraethyl glycol linker is available from Glenn Research Corporation. Adenosine or cordycepin 2'-phosphoamidite were obtained from Chem Genes Corp. and Cemogen Corp. The introduction of the 5'-phosphate or thiophosphate moiety was carried out as described in the synthesis, such that in a commercial manner, 9999 99, by a commercial method. in Uhlman and Engels, 1986, Tetrahedron Lett. 27, 1023. PNA-DNA chimeras were prepared as described in the patent document EP 0 672 677.

Oligonucleotide analysis was performed as follows:

(a) analytical gel electrophoresis on 20% acrylamide with 8M urea, 45 μΜ Tris-borate buffer, pH 7,0 and / or

b) HPLC analysis on a Waters GenPak FAX column, gradient: CH ₃ CH (400 mL), H ₂ O (1.6 L), NaHClPCL (3.1 g), NaCl (11.7 g), pH 6.8 (0.1 M NaCl) followed by CK ₃ CH (400 mL), H ₂ O (1.6 L), NaH ₂ PO ₄ (3.1 g), NaCl (175.3 g), pH 6.8 ( 1.5M NaCl) and / or

(c) capillary electrophoresis using a Beckmann eCAP ™ U100P capillary gel column, 65 cm long, 100 mm internal diameter, 15 cm window from one end, buffer: 140 μΜ Tris, 360 mM borate, 7M urea) and / or

(d) Negative electron spray mass spectroscopy, which in all cases confirmed the expected molecular mass.

The methods of analysis of oligonucleotides a), b), c) and d) used are known to those skilled in the art. These methods have already been described (eg, in the chapter Schweitzrt and Engei, Analisis of oligonucleotides, from Antisense-Technology Technology to Therapy, A Laboratory Manual and Textbook, Schlingensiepen et al., Eds., Biol. Science, Vol. 6 (1997) ), pp. 78-103).

The following oligonucleotides were prepared (as described above) and tested:

ON 300 3'-G * G * C * C A G C‘C G G 'A-5'

ON 2 3'-C * C * C * C * C * C * G * G 'G-5'

ON 301 3'-G * C * C * C * G * G * C'T * T-5 '

ON 4 3'-C’G 'G A G G' G-5 '

ON 302 3'-G'G * C Τ'Τ'Τ GGT'AC * T-5 '(Ex. For SEQ ID NO. 13) (Ex. For SEQ ID NO. 14) (Ex. for SEQ ID No. 15) (Ex. for SEQ ID No. 16) (Ex. for SEQ ID No. 17) • 9 · * ·· »· ♦ · · · · · · · · 9 9 9 9 9 9 9 9 9 9 9

999 99 99 99 99

ON 303 3'-T * T * G G * T * C * T * T G * A-d

ON 304 3'-G'G * T * C * T * G * A A * G-5 '

ON 305 3'-C'T * T * G A A A G A C * G * A-5 '

ON 306 3'-G'A * AAGA * C * GA * CA * G-5 '(Ex for SEQ ID NO: 18) (Ex for SEQ ID NO: 19) (Ex for SEQ ID NO: 18) (SEQ ID NO: 20) (Ex. for SEQ ID NO: 21)

ON 307 3'-G * A * C * C AC * AG A * C'C-5 '

ON 308 3'-G * A * C * A GAA * C * C * C-5 '

ON 309 3'-G * A * A * C * C * A * A-5 '

ON 310 3'-C * G * A * C * G * A * T * G * A-5 '

ON 311 3'-C * G * A * A * T * G * G G * T-5 ‘

ON 15 3'-G * A * T * G * G * T * G * T-5 '

ON 16 3‘-G * G * A G * G * T * C * G-5 '

ON 17 3'-G * G * G * G G * T

ON 312 3'-G * G'T A * C G G * T * C A * C-5 '

ON 313 3'-A * C * G G 'T * C A * C A * G-5'

ON 314 3'-G * G * T * C * A * C * C * G * G-5 '

ON 21 3'-C * A * C * C * G * G * T * C * C * G-5 '

ON 22 3‘-C * C * A GGG T * C'C G A'C-5 ’

ON 23 3'-A * G * G G T * C'C G A C'G'T-5 '

ON 24 3'-G * G G * T * C * C G * C T 'G-5'

ON 25 3'-G'G * T C * C * G AND C * G T'G G-5 '

ON 26 3'-C * C * G A 'C G * T G G' T 'A-5'

ON 315 3'-C * C * A C'T * T'C AAG T'A-5 '

ON 316 3'-C * T'C A A G * T'C * C'T-5 '

ON 317 3'-C'A * A G * T * C * C * T * C-5 '

ON 318 3'-G * T * C * C * C * A G 'T-5'

ON 319 3'-A'C * C'T A * C A G A * T A'G-5 '

ON 320 3'-C * T * A * C AG * A * T * G * T * C-5 '

ON 321 3'-C * A * G A * T A G'T * C G C'G-5 '

ON 322 3'-G'A * T & G T * C G * C'G T * C-5 ’

ON 323 3'-G * T'C G'C G * T'C G A T'G-5 '

ON 324 3'-C'G * C * G T'C G A'T G A'C-5 '

ON 325 3'-C * G * T * C * A G * 5 *

ON 326 3'-C * G * A * T G A 'C G G * T A' G-5 '

ON 39 3'-A'C * GC * C'C C * CGAC * G-5 '(Ex for sec. (Ex for sec. (Ex for sec. (Ex for sec.) (Ex for Sec. (Ex for Sec. (Ex for Sec.) (Ex for Sec. (Ex for Sec.) (Ex for Sec (Ex for Sec (Ex for Sec) (Ex for Sec (Ex for Sec (Ex for Sec (Ex for Sec (Ex for Sec) (Ex for Sec (Ex for Sec (Ex for Sec) (Ex for Sec (Ex for Sec) (Ex for Sec (Ex for Sec. sec.

id. 22) id. No. 23) id. 24) id. No. 25) id. 26) id. 27) id. No. 28) id. 29) id. No. 30) id. No. 31) id. No. 32) id. 33) id. 34) id. 35) id. 36) id. 37) id. 38) id. 39) id. 40) id. 41) id. 42) id. 43) id. 44) id. No. 45) id. No. 46) id. No. 47) id. 43) id. No. 49) id. No. 50) id. No. 55)

ON 40 ON 327 ON 328 ON 329 ON 330 ON 331

ON 332 ON 333 ON 334 ON 335 ON 336 ON 337 ON 338 ON 339 ON 340 ON 341 ON 342 ON 343 ON 58 ON 344 ON 345

3'-C'C * C * C'GA * CGA C'G-5 '3'-C * G * ACGT * TA * C * TG'C-5'3'-C'T'C C * CGGAC * C * T * C-5'3'-C'G * GA C'C'T'CACA * C-5'3'-G * A * C * CT * C A'C AC A'C-5 ¹ 3'-G * A * TGT * CGT'GT * T * G-5 '

3'-C * G * TGT * TGT * TT * A'C-5 '3'-C * TTA * CGT * CT'G-5 ¹ 3-CTT A'CGT'C'T G GT -5 '

3'-G * T * GG'TA * CGT'C'T'-5 ¹ 3'-G * G * TA * CGT * C * TAA'T-5 '3'-C'G * T'C T'AA T'A C'G'C-5'3'-C * T * AA'TA * CGC'C'T'A-5'3'-C * G * CC'TAGT * T'TG 'G-5'3'-A'G'T * T'TGGAG * T G'G-5'3'-G * rrc a rG τά ga'a-5 ¹ 3'-G'T'A GAAG'TT 'C'G'G-5 ¹ 3'-G * A'G GT * rC'G G'TA'G-5'3'-C'G'G * TAGGA'CA'C'A-5 ¹

• · · · · · · · · · • · «9 • · · · · «« · · · • • · · • · · · · • · · · · · (Ex . pro sek . id . No. 56) (Ex • for sec. . id. . No. 57) (Ex . pro sek, . id. . No. 58) (Ex. - for sec. . id. . No. 59) (Ex. . pro sek. id. No. 60) - (Ex.  pro sek. id. No. 67) (Ex. pro sek. id. No. 68) (Ex. pro sek. id. No. 69) (Ex. pro sek. id. No. 70) (Ex. pro sek. id. No. 71) (Ex. pro sek. id. No. 72) (Ex. pro sek. id. No. 61) (Ex. pro sek. id. No. 62) (Ex. pro sek. id. No. 63) (Ex. pro sek. id. No. 64) (Ex. pro sek. id. No. 65) (Ex. pro sek. id. No. 66) (Ex. pro sek. id. No. 51) (Ex. pro sek. id. No. 52) (Ex. pro sek. id. No. 53) (Ex. pro sek. id. No. 54)

ON104 3'-G'G * GT * C * CGA C'G ^Tg-5-one ON105 3’-G * G‘G T * C * C G AND C‘G‘T‘G-5 ', ON106 3'-G * G * G GT'C'CGA C'G'T ^5-one, ON114 3'-C'C'AGC'C * C'GGA G'G-51 ¹ ON115 3'-C * G * G AND G G C * rrT G * G-5 ' ON116 3'-G * T * * A'TGGAGG GG'T 5 ^1st ON117 3'-G * G * A * G * T * G * T * C * G-5 '. ON118 3’-G * G * T * G G T‘A C * G G T‘T-5 ’. ON119 3'-C * A * C * CAGGGT'C'C'G-5 ¹ . ON120 3'-C'C * A G G G‘C * C G A‘C-5 ' ON121 3'-A * * G'GGT C'CGAC'G'T ^5-one ON122 3’-G * G G‘T‘C’C G A C‘G T‘G-5 '

4 4 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

ON123

ON124

ON125

ON139

ON140

0N141

0N142

0N143

ON144

ON145

ON146

ON147

0N148

0N149

GN150

0N151

ON152

ON153

ON154

ON155

ON156

ON157

ON158

ON159

ON160

0N161

ON162

ON163

ON164

3'-G * G * TC * C * GAC * GT * G G-5 ' CC * GA * CGAC * G-5 '

3'-C * C * AGC * C * C * GGAG * G-5 ', 3'-C * G * G * GGGC * T * T * TG * G-5', 3'-G * A * TGGAGG * T * GG * T-5 ', 3'-G * GGG * TGG * TAC * G-5', 3'-GG * T * GGT * AC * GGT * T-5 ', 3'-C * A * C * CAGGGT * C * C * G-5 ', 3'-C ^w * C * C * AGGGT CGA C-5', 3'-A * G * GGT * C * G * CGAC * T -5 ', 3'-G * GG * T * C * CGAC * GT * G-5', 3'-G * G * TC * C * GAC * GT * G G-5 ', 3'-C * C * GA * CG * TGGG * T * A-5 ', 3'-A * C * GC * C * CC * CGAC * G-5', 3'-C ^w C »C * CC * GA * CGAC * G-5 ', 3'-G ^w * AGA AGT T * C »G * G-5',

3'-C'C * AGC * C * C * GGAG * G-C16-5 ', 3' ~ C * G * GAGGC * T * T * TG * G-C16-5 ', 3'-G * A * TGGAG G'T * GG * T-C16-5 ', 3'-G * G' GG * TGG * TAC * G-C16-5 ', 3'-G * G * T * GGT * AC * GGT * T-C16-5 '. 3'-C * A * C * CGGGT * C'C * G-C16-5 ', 3'-C * C * AGGGT * C * CGA * C-C16-5', 3'-A * G * GGT * C'CGAC * G * T-C16-5 ', 3'-G * GG * T * C * CGAC * GT * G-C16-5', 3'-G'G * TC * C'GAC * GT G * G-C16-5 ', 3'-C * C * ^w GA * TGGG CG * T * A-C16-5', 3'-C * C * C ^w CC * GA * CGAC * G-C16 5 ', ·· · · · · · · · · · · · · 9 · 9 9 9 9 · 9 9 9 «· 9 9 9 9 9 4 9 9 9 9 9 9 9 4 '

ON165 3'-teq-G * G * G * C * C * G * G * G * G *

ON 166 3'-teq-G * G * G * T * C * C * G * G * G *

ON167 3'-teq-G * G * G T * C * C G * C * G T * G. -5 ',

ON346 3'-C * C * A G * C * C * G * G * G-vitE -5 ',

ON347 3‘-G * A * T G G A G G * T'G G * T-vitE -5 ‘,

ON348 3'-G * G * A G * G * T * C * G-vitE -5 \

ON349 3'-G * G * T * G G * T * A C * G * T-vitE -5 ',

ON350 3'-C * A * C * C * G * G * T * C * C * G *

ON351 3'-C * C * A G G T * C * C G A * C-vii -5 ', where is a phosphothioate internucleoside bridge,

N is 2'-O-methylribonucleoside (in which case T is 2'O-methyluridine), teg is a triethyleneglycol phosphate linker,

016 is hexadecyl phosphate and vitE is vitamin E-glycerol phosphate

Example 2

Treatment of cells with antisense oligonucleotides

Cells were plated in 96-well plates at 30000 cells / well in 150 μΐ well per well (type of cell-dependent media). The following day Cellfectin (GibcoBRL) was diluted to 400 μg / ml in water (solution A). the oligonucleotides were diluted to a concentration 40 times higher than the desired (4OX) in water (solution B). Thereafter, equal amounts of solution A and solution B of the desired volume were then mixed together. such that the Cellfectin concentration was 200 gg / ml and the oligonucleotide concentration was 20X, and the mixture was left at room temperature for 30 minutes. Then, 19 volumes of Optimem medium (Gibco BRL) were added to obtain a solution with a Cellfectin concentration of 10 μς / πιΐ and oligonucleotide IX (solution C). The media was removed from the cells, the wells were rinsed twice with Optimem medium, and 150 μΐ of solution C was added to each well. The plates were then incubated again. After 5 hours the Cellfectin / oligonucleotide solution was removed and replaced with 150 μΐ of normal growth medium. VEGF protein and VEGF mRNA analyzes were performed 19 hours later.

Example 5

Inhibition of VEGF Expression by Antisense Oligonucleotide in Cell Culture (VEGF Protein Assay)

Conditioned media samples were taken from the desired wells and assayed for human VEGF using the human VEGF ELISA kit (from R & D Systems). The test protocol was included in the kit.

Inhibition of VEGF expression in U87-MG cells by various 12 nucleotide (12mer) antisense oligonucleotides is shown in Table 2 and Figure 2. ON 2, ON 4, ON 15, ON 16, ON 17, ON 24 and ON 40), while other oligonucleotides were virtually ineffective under the same conditions (eg ON 315 and ON 316. Phosphothioate modification patterns used in the 12mer used varied within limits, as described above So ON 24, ON 104, ON 105 · ·> · · * * ON ON ON ON ON ON ON ON ON ON ON ON ON ON ON 9 9 9 9 9 9 9

999 9 9 99 99 9 9 9 9 9 and ON 106 had the same inhibitory effect, while ON 104 was somewhat more effective than the other three with the same sequence. Partial derivatization to 2'-O-methyl-KNA, e.g. ON 117, further enhanced the inhibitory effect compared to ON 16 DNA of the same sequence.

Example 4

VEGF mRNA assay

The media in the 96 well plates of the previous example was removed and cell lysates were prepared for quantification of VEGF mRNA using an Applied Biosystems 7700 Analyzer. The resulting VEGF data was normalized to β-actin data.

Example 5

Determination of _IC50 values

IC ₅₀ values were calculated based on 100% of the amount of VEGF protein or VEGF mRNA in cells treated with Cellfectin but without oligonucleotides. For the ELISA assay, the amount of VEGF in the conditioned medium was normalized to the number of cells in each sample. Cell counts were determined using the CYQuant assay (Molecular Probes, Inc.).

• · ···· ·· · ·. · A · A A A A A A A A A A A A A A A A A A A A A A A A A A A

Example 6

In vivo studies

Experiments were performed with 4-6 week old female nude (nu / nu) mice. Tumors were induced by subcutaneous implantation of cells (eg 2,000,000 U87-MG cells at 200 μΐ). Oligonucleotides were dissolved in phosphate buffered saline and injected subcutaneously or intravenously (into the tail) at a volume of 100 μΐ. When tumor cells were implanted s.c. at day 0, treatment was then initiated on day 1-4 of daily i.v. injection (into the vessel in the tail).

• · · · · · · ·

Table 1

Nucleotide sequence of human VEGF (SEQ ID NO: 93)

CAGTGTGCTGGCGGCCCGGCGCGAGCCGGCCCGGCCCCGGTCGGGCCTCCGAAACC ATGAACTTTCTGCTGTCTTGGGTGCATTGGAGCCTCGCCTTGCTGCTCTACCTCCA CCATGCCAAGTGGTCCCAGGCTGCACCCATGGCAGAAGGAGGAGGGCAGAATCATC ACGAAGTGGTGAAGTTCATGGATGTCTATCAGCGCAGCTACTGCCATCCAATCGAG ACCCTGGTGGACATCTTCCAGGAGTACCCTGATGAGATCGAGTACATCTTCAAGCC ATCCTGTGTGCCCCTGATGCGATGCGGGGGCTGCTGCAATGACGAGGGCCTGGAGT GTGTGCCCACTGAGGAGTCCAACATCACCATGCA.GATTATGCGGATCAAACCTCAC CAAGGCCAGCACATAGGAGAGATGAGCTTCCTACAGCACAACAAATGTGAATGCAG CAAAGAAAG AC AG AT A.G. C AAG AAG AC AAAATA C

Table 3

Inhibitory effect of ON 18 and ON 24 oligonucleotides compared to oligonucleotides having 2 and 4 mismatch positions relative to oligonucleotides 18 and 24

oligonucleotide IC ₅₀ (%) note ON 18 0, 65 antisense ON 200 ineffective 2 mismatched positions ON 201 ineffective 4 mismatched positions ON 24 0, 55 antisense ON 203 ineffective 2 mismatched positions ON 204 ineffective 4 mismatched positions

Φ Φ · Φ •

Φ ΦΦΦΦ

ΦΦ ΦΦΦ ΦΦ φφ Φ

Table 2

Inhibitory effect of antisense VEGF oligonucleotides on VEGF protein secretion

Oligonucleotide ICsofpM] ON 300 3'-G * G * C * C & G C * C * C GG * A-5 ' ON 2 3'-C * C * G * C * C * G * G G * G-5 ' ON 301 3'-G * C * C * CGGAGG * C * T * T-5 ^y ON 4 0.4 3'-C * G * G AGG * T * T * T * G-5 ' ON 302 3'-G * G * CT ⁺ T * TGGT * AC * T-5 ' ON 303 3> -T * T * G G * T A * A-5 ' ON 304 3> -G * G * T * C * T * G * A * G-5 ON 305 3'-C * T * T * G * A * A * C * G * A-5 ' ON 306 3'-G * A * A A G * C * G * C * G-5 ' ON 307 3'-G * A * C * G * C * A * G * A * C-5 ' ON 308 3'-G * A * C * A * G * A * C * C * 5 * ON 309 3'-G * A A * C * C * A * C * A * A-5 ' ON 310 3'-C * G * AC * GAGA * TG G-5 ^r ON 311 3'-C * G * A G * T * G GAG * T-5 ' ON 15 3> -G * A * T * G * G * T * G * T-5 ' ON 16 0.65 3'-G * G * A G G * T G * T * C-G ' ON 17 2 3'-G'G * T * G G T'A C * G G T * T-5 ' ON 312 3'-G * G * T * C G G * T * C * C-5 ' ON 313 3'-A * C * G G * T * C * C * C * G-5 ' ON 314 3'-G * G * T * C * A * C * C * G * G-5 ' ON 21 0.85 3'-C * A * C * C AGG G * C * C * G-5 ' ON 22 3'-C + C * A GGG T * C'C G A * C-5 ' ON 23 3'-A * G * G G 'C * C G * C * G * T-5' ON 24 0.55 3'-G * G G * T * C 'C G & C * G T * G-5' ON 25 1 3'-G * G * T * C * G * C * G * G * 5 ' ON 26 2.2 3'-C * C * G A * C * G * G * G * T-5 ' ON 315 > 3 3'-C * C * A * T * T * C * AAG T * A-5 ’ ON 316 > 3 3'-C * T * T * C * A * G * T * C * C * T-5 ' ON 317 > 3 3'-C * A * A * G * T * C * T * C * A-S '

• · ···· · · · · · · · · · · · · · ·

(Oligonucleotide IC _S0 [pM] ON 318 3'-G ⁺ T ' ^to AC * C * TAC * AGA * T-5' ON 319 3'-A * C * C * T A * C A G A * T A * G-5 ' ON 320 3'-C * T * A * C * G A * T * G * T * C-5 ’ ON 321 3 3'-C * A * G * T A G * T * C G * G-5 ' ON 322 3'-G'A * T & G T * C G * C * G T * C-5 ' ON 323 3'-G * T ⁺ CG * CG * T * CGAT * G-5 ' ON 324 3’-C * G * C * G * G * A * G * C-5 ' ON 325 3'-C * G * T'C G A * T G * C G * G-5 ' ON 326 3’-C * G * A * T G * C G G * T A * G-5 ' ON 39 1 3'-A'C + G C * C C * C G C * G-5 ' ON 40 1.2 3'-ε * 6 * 0 * σ * C * G * G * C * G-5 ’ ON 327 3'-C'G * A C G T * T * C * T G * C-5 ' ON 328 3'-C'T * C C'C GGA C * C * T * C-5 ' ON 329 3'-C'G * G A C'C * T * C A C * C-5 ' ON 330 3'-G * A * C * CT ⁺ CA * CACA * C-5 '. ON 331 3'-G * A * T G T * C G T'G T * T * G-5 ' ON 332 3'-C * G * T G * T G * T * A * C-5 ' ON 333 3'-C * A * C * T T * C G * T * C * G ~ 5 ' ON 334 1.9 3'-C * T * T A * C G * T * C * T G * T-5 ' ON 335 3'-C * G'T C * T G G * T T * C * T-5 ' ON 336 2 · - c * ψ * G g TTT * C τ * TT * C ^- 5 ' ON 337 3'-G * T * G * T * A * C * G * C * T * A-5 ' ON 338 3> G * G * T A * C * G * T * A * T-5 ' ON 339 > 3 3> -C * G * T * C * T * A * T * A * G * C-5 ' ON 340 > 3 3'-C * T'A A * T A * C G * C * T * A-5 ' ON 341 3 3'-C * G * C * T * G * T * T * G-5 ' ON 342 0.9 3'-A * G ⁺ T * T 'T GGA G * TG * G-5' ON 343 3 3'-G * C * T * C * T * G * T * A * A-5 ' ON 58 0.25 3'-G * T * A GAA G * T * C * G * 5 · ON 344 3'-G * A * A * G * T * C * G * T A 'G-5' ON 345 3'-C * G ⁺ G * TAGGA * CA * C * A-5 '

• · ·

ON 104 2.3 3'-G * G * G * C * C * G * C *, G * G-5 ' ON 105 2.0 3'-G * G * G T * C * 'C G & C * G * T * G-5' ON 106 > 3.0 3'-G * G'G T’C'C G A C * G * T G-5 ' ON 114 1.0 3'-C * C * G * C * C * G * G * G * G-5 ON 115 3.0 3'-C * G * G AND G G C * T * T * T G * G-5 ON 116 1.7 3> -G * A * T * G G * G * T * G * T-5 ON 117 0.75 3'-G * G * A * G * T * G * T * C * G-5 ON 118 > 3.0 3'-G * G * T * G * G * T * A * G * G * T-5 ON 119 2.3 3'-C * A * C * C * G * G * T * C * C * G-5 ON 120 1.6 3'-C * C * A G G * T * C * C G * C-'5 ON 121 1.7 3'-A * G * G * T * C * C * G * C * G * T-5 ON 122 0.6 3'-G * G * T * C * C * G A C * G * T * G-5 ON 123 1.0 3'-G * G * T * G * C * G * C * G * G * -5 ON 124 > 3.0 3'-C * C * G * C * G * T * G * T * A-'5 ON 125 3.0 3'-C * C * C ⁺ CC * GA * CGAC * G-5 ON 126 0.6 3'-G * G * G * C * C * C * G * C * G * G-5 ON 139 > 3.0 3'-C * C * G * C * C * G * G G * G-5 ' ON 140 > 3.0 3'-C * G * G * G * G * T * T * T * G-5 ' ON 142 3.0 3'-G ⁺ G'GG * 7GG * TAC * G-5 ' ON 143 > 3.0 3'-G * G * T * G * G * A * G * G * T-5 ' ON 146 3.0 3'-A * G ⁺ GGT * C * CGA * C'G'T-5 ON 165 0.4 3'-teg-G * G ⁺ G 7'C'C GA C'G T'G-5 ' ON 166 2.5 3'-teg-G * G * G T * C * C G * C * G T * G-5 ' ON 167 3.0 3'-tea-G ⁺ G'G T * C * CGAC * GT * G-5 '

• · · · ··············

LIST OF SEQUENCES (2) INFORMATION FOR SEQUENCE WITH IDENTIFICATION NUMBER 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear

(ii) MOLECULA TYPE: DNA (genomic) (iv) OTHER ORIENTATION: Yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 1:

CCCGGCCCCG GTCGGGCCTC CG 22 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of bass (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 2:

CGGGCCTCCG AAACC 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 pairs of bass (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) • · (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 3:

GCTCTACCTCCACCATGCCA A 21 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 4:

GTGGTCCCAG GCTGCACCCA TGGC 24 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..16 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 5:

CATCTTCAAG CCATCC

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of bass (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 6:

TGCGGGGGCT GCTGC 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear

(ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (i.x) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..22 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 7: CGGAGGCCCG ACCGGGGCCG GG 22nd

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) OTHER ORIENTATION: yes 9 9 9 9 9 9 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 99 99 (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 8:

GGTTTCGGAG GCCCG 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 pairs (b) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 9: ·

TTGGCATGGT GGAGGTAGAG C 21 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 10:

GCCATGGGTG CAGCCTGGGC AACA (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 pairs of bait (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iv) ) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..16 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 11:

GGATGGCTTG AAGATG 16 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 12:

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 12:

GCAGCAGCCC CCGCA 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic

0000 0 * 00 • 0

00000 · * · ·

0 0 0 0 0 0 (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: '1.. 12 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER 13 SEQUENCE:

AGGCCCGACC GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 14:

GGAGGCCCGA CC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 15:

TTCGGAGGCC CG 9 9 9 · «« TC TC TC TC TC TC TC TC TC TC TC TC TC TC (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 16:

GGTTTCGGAG GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv). ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TCATGGTTTC GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic

0000 • 0 • 009 0 0 0 0 0

0 0000 00 00 090 00 0009 99 (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 18: AAGTTCATGG TT 12 (2) INFORMATION FOR SEQUENCE WITH IDENTIFICATION NUMBER 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes.

(ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 19:

GAAAGTTCAT GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 20:

AGCAGAAAGT TC (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 21:: φ φ · · · · φ φ φ φ

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 21:

GACAGCAGAA AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 22:

CCAAGACAGC AG .12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) TYPE. FIBERS: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 23:

CACCCAAGAC AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 24:

AATGCACCCA AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs (3) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 25:

AGGTAGAGCA CG (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 26:

TGGAGGTAGA GC 12

LENGTH: 12 base pair TYPE: nucleic acid FIBER TYPE: simple TOPOLOGY: linear

MOLECULES: other nucleic acid DESCRIPTION: / desc = "synthetic (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 27:

(i) SEQUENCE CHARACTERISTICS:

(A) (3) (C) (D) (ii) TYPE (A) (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 27:

TGGTGGAGGT AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 28:

GCATGGRGGA GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 29:

TTGGCATGGT GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 30:

CACTTGGCAT GG (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 31:

GACCACTTGG CA 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear - (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic • (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 32 GGGACCACTT GG 12

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic • · ( (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 33:

GCCTGGGACC AC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 34:

CAGCCTGGGA CC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear

(ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 35 TGCAGCCTGG GA 12

0 · 0 Φ · · 0 · 0 · «· (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 36:

GTGCAGCCTG GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) DESCRIPTION OF SEQUENCE: SEQUENCE WITH IDENTIFICATION NUMBER 37:

GGTGCAGCCT GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 38:

ATGGGTGCAG CC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 39:

ATGAACTTCA CC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bass (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc ~ 'synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 40:

TCCATGAACT TC • · (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 41:

ACATCCATGA AC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 42:

(and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 42 TAGACATCCA TG 12

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (s) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 43: GATAGACATC CA 12 (2) INFORMATION FOR SEQUENCE WITH IDENTIFICATION NUMBER 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: excn (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 44:

CTGATAGACA TC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) DESCRIPTION OF SEQUENCE: SEQUENCE WITH IDENTIFICATION NUMBER 45:

GCGCTGATAG AC ·

«·· · * ···· · · · · (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 46:

CTGCGCTGAT AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 47:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 pairs of bases (B) TYPE: nuclic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear »» (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = 'synthetic (iv) REVERSE ORIENTATION: yes (ix) FEATURES:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 47:

GTAGCTGCGC TG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" · · « · · · · · · · · ·

(iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 48:

CAGTAGCTGC GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 49:

GGCAGTAGCT GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear

(ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 50 GATGGCAGTA GC 12

9 9 9 9

9 9 99 9 9 9 99 9 9 9 9 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 51:

AAGATGTACT CG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc - "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 52:

GGCTTGAAGA TG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic • · ' (Iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 53:

GATGGCTTGA AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 54:

ACACAGGATG GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 55:

GCAGCCCCCG CA • · (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 56:

GCAGCAGCCC CC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 57: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear - (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic - (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 57 »» CGTCATTGCA GC 12

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" · · · (Iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 58:

CTCCAGGCCC TC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 59:

CACACTCCAG GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) OPTIONAL ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (Xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 60:

CACACACTCC AG

0

0 0 · · ·

000 0000 00

00000 00 ·

0000 0000 00 0000 000 00 000 00 0000 00 ·· (2) INFORMATION FOR SEQUENCE WITH IDENTIFICATION NUMBER 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bass (B) TYPE: nucleic acid (O) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 61:

ATCTGCATGG TG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (O) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 62:

TAATCTGCAT GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 63:

CGCATAATCT GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) FOSITION: I..12 (Xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 64:

ATCCGCATAA TC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 65:

GGTTTGATCC GC * (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 66:

1111

I 9 »»

1111 (i) SEQUENCE CHARACTERISTICS :.

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 66:

GGTGAGGTTT GA 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 67:

GTTGTGCTGT AG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic

9 · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 (Iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 68:

CATTTGTTGT GC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION; 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 69:

GTCTGCATTC AC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 70:

TGGTCTGCAT TC (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of bases (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE TCTTTGGTCT GC

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 72:

CTTTCTTTGG TC 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" »« (Iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..13 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 73:

GGCATGGTGG AGG 13 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 74:

(and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 pairs of cows (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (A) NAME / LABEL: exon (B) POSITION: 1..13 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION SEQUENCE NUMBER 74 GGTGCAGCCT GGG 13

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 pairs (3) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..13 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 75:

GCAGCAGCCC CCG (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: I..14 (Xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 76:

GGTTTCGGAG GCCC 14 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..14 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 77:

GGTGGAGGTA GAGC 14 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic e · i I (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..14 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 78:

GGGTGCAGCC TGGG 14 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..14 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 79:

GCAGCAGCCC CCGC 14 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..15 (Xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 80:

GGGCCGGGGC CAGCC (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 81:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs (3) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) ) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 81:

CCGGGGCCAG CCCGG 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 82:

CCCGACCGGG GCCGG 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc - "synthetic" · · · * (Iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 83:

GGTTTCGGAG GCCCC 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 84:

GGCATGGTGG AGGTA 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 85:

TTGGCATGGT GGAGG (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 86:

GTGCAGCCTG GGACC 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 87:

GATGGCTTGA AGATG 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 pairs of baffles (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic" · (Iv) OTHER ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 88:

GGATGGCTTG AAGAT 15 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 89: (i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 12 pairs of cows (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) TYPE MOLECULES: other nucleic acid (AND) DESCRIPTION: / desc = "synthetic (iv) ANOTHER ORIENTATION: yes (ix) FEATURES: (AND) NAME / LABEL: exon (B) POSITION: 1..12 (Xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER

GCATGGGTGA GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 90:

(i) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 12 steams (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) TYPE MOLECULES: other nucleic acid (AND) DESCRIPTION: / desc = "synthetic

(iv) REVERSE ORIENTATION: yes

GAATGGGTGC GG (ix) FEATURES:

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 90:

(2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 91:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / DESIGNATION: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 91:

GTGCGACCTG GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 pairs of baits (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix)

(A) NAME / LABEL: exon (B) POSITION: 1..12 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 92:

GTGTGACCCG GG 12 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 480 bp (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "synthetic (iv) REVERSE ORIENTATION: yes (ix) CHARACTERS:

(A) NAME / LABEL: exon (B) POSITION: 1..480 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 93:

CAGTGTGCTG GCGGCCCGGC GCGAGCCGGC CCGGCCCCGG TCGGGCCTCC GAAACCATGA 60

ACTTTCTGCT GTCTTGGGTG CATTGGAGCC TCGCCTTGCT GCTCTACCTC CACCATGCCA 120

AGTGGTCCCA GGCTGCACCC ATGGCAGAAG GAGGAGGGCA GAATCATCAC GAAGTGGTGA 190

AGTTCATGGA TGTCTATCAG CGCAGCTACT GCCATCCAAT CGAGACCCTG GTGGACATCT 240

TCCAGGAGTA

CCCCTGATGC

CCCTGATGAG

300

ATCGAGTACA

TCTTCAAGCC

ATCCTGTGTG

GATGCGGGGG

GAGTCCAACA

CTGCTGCAAT

360

GACGAGGGCC

TGGAGTGTGT

GCCCACTGAG

TCACCATGCA

GAGATGAGCT

GATTATGCGG

420

ATCAAACCTC

ACCAAGGCCA

GCACATAGGA

TCCTACAGCA CAACAAATGT GAATGCAGAC CAAAGAAAGA TAGAGCAAGA CAAGAAAATC 48 0

Claims (14)

PATENT CLAIMS A short oligonucleotide or derivative thereof that is 10 to 15 nucleotides in length and which corresponds to a portion of the sequence encoding a VEGF, wherein the portion of the VEGF-encoding sequence that the oligonucleotide corresponds to has one of SEQ ID NOs. no. 1, 2, 3, 4 Z 5 or 6 or a portion thereof, wherein the sequences are following: sequence id. no. 1 Yippee 5’- CCCGGCCCCGGTCGGGCCTCCG - 3 ', sequence id. No 2 Yippee 5’- CGGGCCTCCGAAACC -3 ', sequence id. No 3 Yippee 5’- GCTCTACCTCCACCATGCCAA-3 ', sequence id. No. 4 Yippee 5'- GTGGTCCCAGGCTGCACCCATGGC -3 ', sequence id. No 5 Yippee 5'- CATCTTCAAGCCATCC -3 ', sequence id. No. 6 Yippee 5'- TGCGGGGGCTGCTGC -3 '. The oligonucleotide of claim 1 having one of the sequences id. No 7 3 Z 12 or a portion thereof, wherein sequence id. C . 7 is 3'- GGGCCGGGGCCAGCCCGGAGGC-5 ' sequence id. C . 8 j e 3'- GCCCGGAGGCTTTGG -5 ', sequence id. C . 9 j e 3'- CGAGATGGAGGTGGTACGGTT-5 ', sequence id. C . 10 j e 3'- CACCAGGGTCCGACGTGGGTACCG -5 ' sequence id. C . 11 is 3 '- GTAGAAGTTCGGTAGG -5', and sequence id. C . 12 j e 3'- ACGCCCCCGACGACG -5 ' 3. The oligonucleotide according to nucleotides. of claim 1 or 2, which has length 12 4. The oligonucleotide according to any of them of claims 1 to 3, which has one of the sequences id. No. 14, 16, 27, 28, 29, 33, 34, 35, 36, 37, 38, 52, 55 or 56, wherein sequence id. C. 14 Yippee sequence id. C. 16 Yippee sequence id. C. 27 Mar: Yippee sequence id. C. 28 Yippee sequence id. C. 29 Yippee sequence id. C. 33 Yippee sequence id. C. 34 Yippee sequence id. C. 35 Yippee sequence id. C. 36 Yippee sequence id. C. 37 Yippee sequence id. C. 38 Yippee sequence id. C. 52 Yippee sequence id. C. 55 Yippee sequence id. C. 56 Yippee 3'- CCAGCCCGGAGG -5 ', 3'- CGGAGGCTTTGG -5', 3'- GATGGAGGTGGT -5 ', 3'- GGAGGTGGTACG -5', 3'- GGTGGTACGGTT -5 ', 3'- CACCAGGGTCCG -5', 3 ' - CCAGGGTCCGAC -5 ', 3'- AGGGTCCGACGT -5', 3'- GGGTCCGACGTG -5 ', 3'- GGTCCGACGTGG-5', 3'- CCGACGTGGGTA -5 ', 3'- GTAGAAGTTCGG -5', 3'- ACGCCCCC 5 ', h 3’- CCCCCGACGACG -5 '. An oligonucleotide according to any one of claims 1 to 4 having one or more modifications, each of which is located in a certain phosphodiester internucleoside bridge and / or a certain β-ϋ-2'deoxyribose unit and / or a certain position of the natural nucleoside base at compared to an oligonucleotide with the same sequence composed of natural DNA. The oligonucleotide of any one of claims 1 to 5, wherein each modification is independently selected from the group consisting of: a) replacing the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside with a modified internucleoside. bridge, (b) replacement of the phosphodiester bridge located at the 3'- and / or 5'-end of the nucleoside with a defospho bridge, (c) replacement of a sugar-phosphate backbone unit with another unit; d) replacement of the β-ϋ-2'-deoxyribose unit with a modified sugar unit, e) replacement of the natural nucleoside base with a modified nucleoside base, f) conjugation to a molecule that affects the properties of the oligonucleotide, g) conjugation to a 2'5'-linked oligoadenylate or derivative thereof, optionally via a suitable linker, h) inserting a 3'-3 'and / or 5'-5' inversion at the 3'- and / or 5'-end of the oligonucleotide. The oligonucleotide of any one of claims 1 to 6, wherein each modification is independently selected from the group consisting of: a) replacing the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside with a modified internucleoside bridge, wherein the modified internucleoside bridge is selected from, for example, the group comprising bridges of the type: phosphothioate, phosphodithioate, NF / R ¹ -phosphoramidite, boranphosphate; O-alkyl phosphoric acid ester of 1 to 21 carbon atoms, aryl-O-alkyl phosphoric acid ester of aryl of 6 to 12 carbon atoms and alkyl group of 1 to 21 carbon atoms, α-hydroxymethylaryl with aryl group of 7 to 12 carbon atoms C 12 alkyl, a C 1 -C 8 alkylphosphonate and / or an C 6 -C 12 arylphosphonate wherein: R ¹ and R ¹ are, independently of each other, hydrogen, C 1 -C 18 alkyl, C 6 -C 20 aryl, arylalkyl 11; With an aryl group of 6 to 14 carbon atoms and an alkyl group of 1 to 8 carbon atoms, or R ¹ and R ¹ , together with the nitrogen atom carrying it, form a 5-6 membered heterocyclic ring , which may additionally contain a further heteroatom selected from the group consisting of O, S and N. b) replacing the phosphodiester internucleoside bridge located at the 3'- and / or 5'-end of the nucleoside with a defospho bridge, wherein the defrost bridge is selected from the group consisting of formacetal, 3'-thioformacetal, methylhydroxylamine and oxime, methylenedimethylhydrazo, dimethylenesulfone and / (c) replacing the sugar phosphate unit in the sugar-phosphate backbone with another unit selected from the group consisting of a morpholine derivative unit, a polyamide nucleic acid backbone unit, and a phosphonic acid monoester monoester of the nucleic acid backbone; d) replacing the β-ϋ-2'-deoxyribose unit with a modified sugar unit, wherein the modified sugar unit is selected from the group consisting of β-D-ribose, α-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F -2'-deoxyribose, 2'-Oalkylribose with an alkyl group containing 1 to 6 carbon atoms, 2'-O-alkenylribose with an alkenyl group containing 2 to 6 carbon atoms, 2 '- [O- (alkyl-O-alkyl) ribose C 1 -C 6 -alkyl, 2'-NH ₂ -2'-deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-dideoxy-β-D-erythrohexopyranose, carbocyclic sugar analogues and / or sugar analogues with open-chain and / or bicyclosaccharides; · · ···· · · · I · · · Λ e) replacing the natural nucleoside base with a nucleoside base, wherein the modified base is selected from. groups 5- (hydroxymethyl) uracil, 5-aminouracil, a modified nucleoside containing pseudouracil, 5-fluorocytosine, 5-bromouracil, 8-azapurines, dihydrouracil, 5-chlorouracil, 5-bromocytosine, substituted 5-fluorouracil, 5-chloro-cytosine, 2,4-diaminopurine, 7-deazapurine, preferably 7-deaza-7-substituted or 7-deaza-8-substituted purines, f) conjugating to a molecule that affects the properties of the oligonucleotide, wherein the molecule that affects the properties of the oligonucleotide is. selected from the group consisting of polylysine, intercalating agents, fluorescent agents such as fluorescein, crosslinking agents, lipophilic molecules, steroids, vitamins, poly- or oligoethylene glycol, alkyl esters of phosphoric acid with an alkyl group containing 12 to 18 carbon atoms and O-CH ₂ -CH (OH) -O-alkyl groups having an alkyl group containing 12 to 18 carbon atoms, g) conjugation to a 2'5'-linked oligoadenylate molecule or derivative thereof, optionally through a suitable linker molecule, wherein the 2'5'-linked oligoadenylate molecule is selected from tetraadenylate and heptaadenylate groups containing pentaadenylate molecules and their triadenylate, hexaadenylate derivatives, h) inserting a 3'-3 and / or 5'-5 inversion to 3'- and / or A process for the preparation of an oligonucleotide according to claims 1 to 7 characterized on a solid support by condensation suitably protected by any of the monomers. 5'-end of the oligonucleotide. Use of an oligonucleotide according to any one of claims 1 to 7 for inhibiting VEGF expression. A method of inhibiting VEGF expression, wherein the oligonucleotide of any one of claims 1 to 7 is contacted with a nucleic acid encoding VEGF. Use of an oligonucleotide according to any one of claims 1 to 7 for the manufacture of a pharmaceutical composition. A method of manufacturing a pharmaceutical composition comprising mixing one or more oligonucleotides according to any one of claims 1 to 7 with a physiologically acceptable excipient and optionally other substances. A pharmaceutical composition comprising at least one oligonucleotide according to any one of claims 1 to 7. Use of a pharmaceutical composition comprising at least one oligonucleotide according to any one of claims 1 to 7, for the treatment of diseases which are associated with abnormal vascular permeability, cell proliferation, cell permeation, angiogenesis, neovascularization, tumor cell growth and / or metastasis. Use of a pharmaceutical composition comprising at least one oligonucleotide according to any one of claims 1 to 7, in combination with other pharmaceutical compositions and / or other methods of treatment.