U.S. patent application number 11/073230 was filed with the patent office on 2005-07-07 for antisense oligonucleotides against tenascin for the treating of vitiligo.
This patent application is currently assigned to Aventis Pharma Deutschland GmbH. Invention is credited to Peyman, Anuschirwan, Uhlmann, Eugen, Weiser, Caroline.
Application Number | 20050148017 11/073230 |
Document ID | / |
Family ID | 7848863 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050148017 |
Kind Code |
A1 |
Peyman, Anuschirwan ; et
al. |
July 7, 2005 |
Antisense oligonucleotides against tenascin for the treating of
vitiligo
Abstract
Antisense oligonucleotides against tenascin for the treatment of
vitiligo The invention relates to specific, optionally modified
oligonucleotides having a length of up to 18 nucleotides, which
correspond to sections of tenascin-coding sequences or can bind to
these sequences, to their preparation and to the use thereof, for
example for the specific inhibition of the expression of tenascin
and for the production of pharmaceuticals which can be used for the
treatment of vitiligo.
Inventors: |
Peyman, Anuschirwan;
(Kelkheim, DE) ; Uhlmann, Eugen; (Glashutten,
DE) ; Weiser, Caroline; (Hattersheim, DE) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Assignee: |
Aventis Pharma Deutschland
GmbH
Frankfurt am Main
DE
|
Family ID: |
7848863 |
Appl. No.: |
11/073230 |
Filed: |
March 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073230 |
Mar 4, 2005 |
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09554267 |
Jul 24, 2000 |
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6878547 |
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09554267 |
Jul 24, 2000 |
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PCT/EP98/06868 |
Oct 29, 1998 |
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Current U.S.
Class: |
435/456 ;
514/44R; 536/23.1 |
Current CPC
Class: |
C12N 2310/318 20130101;
C12N 2310/351 20130101; A61P 17/00 20180101; C12N 2310/346
20130101; C12N 2310/321 20130101; C12N 2310/321 20130101; C12N
2310/33 20130101; C12N 2310/321 20130101; C12N 2310/345 20130101;
C12N 2310/32 20130101; C12N 15/113 20130101; C12N 2310/315
20130101; A61K 38/00 20130101; C12N 2310/3525 20130101; C12N
2310/317 20130101; C12N 2310/3181 20130101; C12N 2310/3521
20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/02; A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 1997 |
DE |
19750702.6-44 |
Claims
1-23. (canceled)
24. An oligonucleotide or physiologically tolerable salt thereof,
comprising a sequence selected from the group consisting of SEQ ID
NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17,
SEQ ID NO. 19, and SEQ ID NO. 20, wherein the oligonucleotide has a
maximum length of 7 to 17 nucleotide units:
25. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO: 13.
26. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO: 14.
27. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO:15.
28. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO: 16.
29. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO: 17.
30. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO:19.
31. The oligonucleotide of claim 24, wherein the oligonucleotide
consists of SEQ ID NO:20.
Description
[0001] The invention relates to specific, optionally modified
oligonucleotides having a length of up to 18 nucleotides,
preferably a length of 7-15 nucleotides, which correspond to
sections of tenascin-coding sequences and can bind to these
sequences, to their preparation and to the use thereof, for example
for the specific inhibition of the expression of tenascin and for
the production of medicaments which can be used for the treatment
of vitiligo.
[0002] Vitiligo is understood as meaning an acquired lack of
melanocytes, by means of which hypopigmented areas of skin result,
which as a rule are sharply demarcated and often symmetrically
arranged, form one or two spots or cover almost the entire skin.
The hair in hypopigmented regions is normally white and appears
white even in the Wood light. The affected skin sites are
susceptible to sunburn. The cause of the disorder is unknown.
Although vitiligo is considered as a disease which is acquired in
the course of life, a familial cluster is occasionally found
(autosomally dominant, with incomplete penetrance and variable
pronouncement). It can also follow an unusual physical trauma, in
particular a skull injury. The association of vitiligo with
Addison's disease, diabetes mellitus, pernicious anemia or thyroid
gland dysfunction and the increased occurrence of antibodies
against thyroglobulin, cells of the adrenal gland and border cells
of the stomach in the serum have led to an immunological or
neurochemical cause being suspected. Antibodies against melanin
were found in some patients.
[0003] All available therapeutic methods lead to satisfactory
therapeutic results in only some of the patients (F. Wach et al.,
H+G 71 (1996) 206). The present therapies (S. P. W. Kumarasinghe,
Ceylon Medical Journal 40 (1995) 94) include photochemotherapies
(PUVA) for example with methoxypsoralen, phenylalanin or khellin,
the transplantation of cultured melanocytes, epidermal grafting,
and treatment with steroids or placenta extracts. Recently,
treatment with pseudocatalase was reported (Schallreuter et al.,
Dermatology 190 (1995) 223). Small foci can also be covered with
cosmetic make-up or tannic acid solutions.
[0004] Poole et al. (British Journal of Dermatol. 137 (1997) 171)
were able to show that the vitiligo-affected skin has a high
content of tenascin in comparison with normal skin. The high
tenascin content can contribute to the loss of pigmentation and
prevent repigmentation. Tenascin (Crossin, J. Cell. Biol. 61 (1996)
592) is an extracellular matrix glycoprotein, which consists of six
identical subunits which are linked to the amino terminus via
disulfide bridges. The tenascin subunits have a characteristic
domain structure: a cysteine-rich sequence at the amino-terminal
end is followed by three sequence sections, in each case
constructed of repeating units, made of units homologous to EGF, of
units homologous to fibronectin (type II) and of units homologous
to fibrinogen.
[0005] A number of isoforms of the tenascin subunits exists
(designated below as tenascin isoforms), which differ in the number
of repeating units which are homologous to fibronectin type III.
These isoforms are formed by alternative splicing of the tenascin
pre-mRNA and subsequent translation of the various splice variants
(A. Leprini et al., Perspectives in Developmental Neurobiology 2
(1994) 117-123). A cDNA from human tenascin was described (sequence
in Table 1) by A. Siri et al. (Nucl. Acids Res. 19 (1991) 525-531).
This cDNA is stored under the accession number X56160 in gene
databases and can be obtained under this number, for example under
EMBUGenbank/DDBJ/NBRF-PIR. This cDNA contains a sequence section
which codes for 12 repeating units which are homologous to
fibrinogen type II. The cDNAs of the other isoforms of human
tenascin are truncated in this sequence section and code for less
than 12 of these repeating units.
[0006] The expression of tenascin is limited spatially and
temporally and a significance is ascribed to it during the
development of an organism and in pathological changes (Crossin,
vide supra). Such pathological changes are, for example, vitiligo,
tumors and inflammation.
[0007] Antisense oligonucleotides offer one possibility for the
regulation of gene expression (E. Uhlmann and A. Peyman, Chemical
Reviews 90, 543 (1990); S. Agrawal. TIBTECH 1996, 376). WO 94/21664
(L. Denner et al.) describes antisense oligonucleotides against
tenascin, which are employed for the inhibition of the
proliferation of the smooth cell musculature. The oligonucleotides
described there have a length of at least 18 nucleotides.
[0008] It was an object of the present invention to make available
novel oligonucleotides which have advantageous properties and which
can be used for the complete and/or partial inhibition of the gene
expression of tenascin.
[0009] It has surprisingly been found that oligonucleotides which
have a length of up to 18 nucleotides can effectively influence the
expression of tenascin. The present invention relates to
oligonucleotides having 7-17 nucleotide units which are optionally
modified. In particular embodiments of the invention, the
oligonucleotide has a length of 17, 16, 15, 14, 13, 12, 1 1, 10, 9,
8 or 7 nucleotides. The oligonucleotide corresponds to sections of
tenascin-coding sequences (i.e. the oligonucleotide has a sequence
which is complementary to the corresponding section of a
tenascin-coding sequence) and the oligonucleotide binds
specifically to this tenascin-coding sequence (nucleic acid), for
example to the tenascin gene and/or tenascin mRNA and/or tenascin
cDNA, the tenascin-coding sequence preferably being of human origin
(e.g. human tenascin gene, human tenascin mRNA, human tenascin
cDNA). The section of the tenascin-coding sequence which
corresponds to the oligonucleotide or is complementary to the
oligonucleotide preferably has a length of 17, 16, 15, 14, 13, 12,
11, 10, 9, 8 or 7 nucleotide units (this applies in particular to
the determination of the length of a modified and/or chimeric
oligonucleotide or of oligonucleotide analogs).
[0010] A particular embodiment of the invention relates to an
oligonucleotide which binds to a nucleic acid which codes for one
of the isoforms of human tenascin or parts thereof and inhibits its
expression, where the oligonucleotide has a length of 7 to 15
nucleotides and can optionally be modified, and the physiologically
tolerable salts of the oligonucleotide.
[0011] A particular embodiment of the invention relates to an
oligonucleotide which is directed against one or more specific
regions of a tenascin-coding sequence, for example the translation
start, the 5'-nontranslated region, the coding region and/or the
3'-noncoding region. In a particular embodiment of the invention,
the oligonucleotide can also be directed against one or more
regions of a tenascin-coding sequence which codes, for example, for
certain domains of the tenascin, for example against the
cysteine-rich domain, against a domain homologous to EGF, against a
domain homologous to fibronectin type III and/or against a domain
homologous to fibrinogen.
[0012] One embodiment of the invention relates to an
oligonucleotide which binds to a nucleic acid which codes for one
of the isoforms of human tenascin or parts thereof and inhibits its
expression, where the oligonucleotide can bind to a region of the
nucleic acid which comprises
[0013] a) a part of the 5'-noncoding region and/or the translation
start or
[0014] b) the translation start and/or a part of the coding region
or
[0015] c) a part of the coding region and/or a part of the
3'-noncoding region.
[0016] The invention relates in particular to an oligonucleotide
which corresponds to a sequence section of the human cDNA according
to SEQ ID NO. 1 (Table 1). The invention furthermore relates to an
oligonucleotide which corresponds to a sequence section of the cDNA
which is stored in gene databases under the accession number
X56160.
[0017] In specific embodiments of the invention, an oligonucleotide
can have, for example, one of the following sequences or parts
thereof:
1 SEQ ID NO. 2: 3'-GGTTTGGGTGGAGGTGG-5' SEQ ID NO. 3:
3'-GGAGGTGGTACCCCCGG-5' SEQ ID NO. 4: 3'-GGTGGTACCCCCGG-5' SEQ ID
NO. 5: 3'-GGAGGTGGTACCCC-5' SEQ ID NO. 6: 3'-AGAAAGAACGAAAGGAA-5'
SEQ ID NO. 7: 3'-GGAGGTGGTACC-5' SEQ ID NO. 8:
3'-GGAGCGATGGCTTCCA-5' SEQ ID NO. 9: 3'-AAAGGAACGGGAGCG-5' SEQ ID
NO. 10: 3'-GGTCGGTTTGGGTGG-5' SEQ ID NO. 11: 3'-CTTACAGGTCCGTTGA-5'
SEQ ID NO. 12: 3'-GGCCGTGTTCGCTGT-5' SEQ ID NO. 13:
3'-TCACCCCTCTTTCTGG-5' SEQ ID NO. 14: 3'-GGACACCGACACGG-5' SEQ ID
NO. 15: 3'-AACGGGAGCGATGG-5' SEQ ID NO. 16: 3'-ATCTCGGGGTCGTC-5'
SEQ ID NO. 17: 3'-AAAGAACGAAAGGAA-5' SEQ ID NO. 18:
3'-GGTGGTACCCC-5' SEQ ID NO. 19: 3'-CCCGGTACTGA-5' and SEQ ID NO.
20: 3'-CGACAGAAAGAAC-5'.
[0018] The sequences SEQ ID NO. 2 to SEQ ID NO. 20 correspond to
sections of the tenascin-coding cDNA, as is shown in Table 1. An
oligonucleotide which has one of the sequences SEQ ID NO. 2 to SEQ
ID NO. 20 is complementary to a corresponding section of a
tenascin-coding nucleic acid, e.g. a human tenascin cDNA, and can
bind to this nucleic acid. Sequences SEQ ID NO. 3, SEQ ID NO. 4,
SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 18 are examples of
oligonucleotides which have a sequence which is directed against
the translation start of the tenascin-coding sequences.
[0019] The invention also relates to derivatives of an
oligonucleotide, for example its salts, in particular its
physiologically tolerable salts. Physiologically tolerable salts
are understood as meaning compounds which are readily soluble,
soluble or poorly soluble in water, for example according to the
definition in the "Deutsches Arzneibuch" [German Pharmacopeia] (9th
Edition 1986, official edition, Deutscher Apotheker Verlag
Stuttgart), page 19. A specific embodiment of the invention relates
to the sodium salt of the oligonucleotide according to the
invention. Derivatives are also modified oligonucleotides.
[0020] An oligonucleotide can be synthesized completely or
partially from the natural nucleotides adenosine phosphate,
guanosine phosphate, inosine phosphate, cytidine phosphate, uridine
phosphate and thymidine phosphate. One embodiment of the invention
relates to an oligonucleotide which is synthesized from the natural
nucleotides adenosine, guanosine, inosine, cytidine, uridine and
thymidine and in which the nucleosides are linked to one another
via phosphoric acid diester internucleoside bridges ("phosphoric
acid diester bridges").
[0021] In other embodiments of the invention, an oligonucleotide
can optionally contain one or more modifications, for example
chemical modifications. An oligonucleotide can have a number of
identical and/or different modifications. Modifications can be
localized on certain nucleotide positions (nucleobase and/or
.beta.-D-2'-deoxyribose unit) and/or certain internucleoside
bridges.
[0022] Examples of chemical modifications are known to the person
skilled in the art and are described, for example, in E. Uhlmann
and A. Peyman, Chemical Reviews 90 (1990) 543 and "Protocols for
Oligonucleotides and Analogs" Synthesis and Properties &
Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press,
Totowa, USA 1993, S. T. Crooke, F. Bennet, Ann. Rev. Pharmacol.
Toxicol. 36 (1996) 107-129 and J. Hunziber and C. Leumann (1995)
Mod. Synt. Methods, 7, 331-417.
[0023] The chemical modification of an oligonucleotide can mean,
for example, a) the complete or partial replacement of the
phosphoric acid diester bridges (internucleoside bridges) by
modified phospho bridges, phosphorothioate, phosphorodithioate,
NR.sup.1R.sup.1-phosphoramidate, boranophosphate,
phosphate-(C.sub.1-C.sub.21)-O-alkyl ester,
phosphate-[(C.sub.6-C.sub.12)-aryl-(C.sub.1-C.sub.21)-O-alkyl]
ester, (C.sub.1-C.sub.8)alkylphosphonate and/or
(C.sub.6-C.sub.12)-arylphosphona- te bridges being examples of
modified phospho bridges, where
[0024] R.sup.1 and R.sup.1 independently of one another are
hydrogen, (C.sub.1-C18)-alkyl, (C.sub.6-C.sub.20)-aryl,
(C.sub.6-C.sub.14)-aryl-(C.- sub.1-C.sub.8)-alkyl, preferably
hydrogen, (C.sub.1-C.sub.8)-alkyl and/or methoxyethyl, particularly
preferably hydrogen, (C.sub.1-C.sub.4)-alkyl and/or methoxyethyl
or
[0025] R.sup.1 and R.sup.1', together with the nitrogen atom
carrying them, form a 5- to 6-membered heterocyclic ring, which can
additionally contain a further heteroatom from the group consisting
of O, S and N; and/or
[0026] b) the complete or partial replacement of the 3' and/or 5'
phosphoric acid diester internucleoside bridges ("phosphoric acid
diester bridges") by "dephospho" bridges (described, for example,
in Uhlmann, E. and Peyman, A. in "Methods in Molecular Biology",
Vol. 20, "Protocols for Oligonukleotides and Analogs", S. Agrawal,
Ed., Humana Press, Totowa 1993, Chapter 16, 355ff), formacetal,
3'-thioformacetal, methylhydroxylamine, oxime,
methylenedimethylhydrazo, dimethylenesulfone and/or silyl groups
being examples of dephospho bridges; and/or
[0027] c) the complete or partial replacement of the sugar
phosphate backbone (replacement of sugar phosphate units) by other
units, the other unit being suitable, for example, to synthesize a
"morpholine derivative" oligomer (described, for example, in E. P.
Stirchak et al., Nucleic Acids Res. 17 (1989) 6129) (i.e.
replacement by a morpholino derivative unit) and/or being suitable
to synthesize a polyamide nucleic acid ("PNA") described, for
example, in P. E. Nielsen et al., Bioconj. Chem. 5 (1994) 3 (EP 0
672 677) (i.e. replacement by a PNA unit, for example
2-aminoethyl-glycine) and/or being suitable to synthesize a
phosphomonoacid ester nucleic acid ("PHONA", "PMENA") (described,
for example, in Peyman et al., Angew. Chem. Int. Ed. Engl. 35
(1996) 2632-2638, EP 0 739 898) (i.e. replacement by a PHONA unit);
and/or
[0028] d) the complete or partial replacement of the
.beta.-D-2'-deoxyribose (.beta.-D-2'-deoxyribose unit) by modified
sugar units, .alpha.-D-2'-deoxyribose, L-2'-deoxy-ribose,
2'-F-2'-deoxyribose, 2'-O--(C.sub.1-C.sub.6)alkylribose, preferably
2'-O-methylribose, 2'-O--(C.sub.2-C.sub.6alkenylribose,
2'-[O--(C.sub.1-C.sub.6) alkyl-O--(C.sub.1-C.sub.6alkyl)]ribose,
2'--NH.sub.2-2'-deoxyribose, .beta.-D-xylofuranose,
.alpha.-arabinofuranose, 2,4-dideoxy-.beta.-D-eryt-
hrohexopyranose, carbocyclic sugar analogs (described, for example,
in Froehler, J.Am.Chem.Soc. 114 (1992) 8320), open-chain sugar
analogs (described, for example, in Vandendriessche et al.,
Tetrahedron 49 (1993) 7223) and bicyclo sugar analogs (described,
for example, in M. Tarkov et al., Helv. Chim. Acta 76 (1993) 481)
being examples of modified sugar units; and/or
[0029] e) the modification or the complete or partial replacement
of the natural nucleoside bases by modified (nucleoside) bases
("nucleobases"), 5-(hydroxymethyl)uracil, 5-aminouracil,
pseudouracil, dihydrouracil, 5-(C.sub.1-C.sub.6-alkyluracil,
5-(C.sub.2-C.sub.6)-alkenyluracil,
5-(C.sub.2-C.sub.6)alkynyluracil, 5-(C.sub.1-C.sub.6)alkylcytosine,
5-(C.sub.2-C.sub.6)alkenylcytosine,
5-(C.sub.2-C.sub.6)alkynylcytosine, 5-fluorouracil,
5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil,
5-bromocytosine, 7-deaza-7-substituted purines,
7-deaza-8-substituted purines, 8-azapurines, 2,4-diaminopurines,
5-bromocytosine, 5-bromouracil, 5-chlorocytosine, 5-chlorouracil,
5-fluorocytosine, 5-fluorouracil, hypoxanthine and uracil being
examples of modified bases; and/or
[0030] f) the conjugation to one or more molecules (oligonucleotide
conjugates) which adapt the property(ies) of the oligonucleotide to
specific requirements or favorably influence the properties (e.g.
cell penetration, nuclease stability, affinity for the
tenascin-coding target sequence, pharmacokinetics) of the
oligonucleotide (e.g. antisense oligonucleotide, triple
helix-forming oligonucleotide) and/or in the hybridization of the
oligonucleotide on the target sequence can attack this with binding
and/or crosslinking, polylysine, intercalators such as pyrene,
acridine, phenazine, phenanthridine, fluorescent compounds such as
fluorescein, crosslinkers such as psoralen, azidoproflavine,
lipophilic molecules such as (C.sub.12-C.sub.20)alkyl, lipids such
as 1,2-dihexadecyl-rac-glycerol, steroids such as cholesterol,
testosterone, vitamins such as vitamin E, poly- or oligoethylene
glycol, (C.sub.12-C.sub.18)alkyl phosphate diesters and
--O--CH.sub.2--CH(OH)--O-- -(C.sub.12-C.sub.18)-alkyl being
examples of molecules which can be conjugated to an
oligonucleotide, where such molecules can be conjugated to the
oligonucleotide at the 5' and/or at the 3' end and/or within the
sequence, e.g. via a nucleobase; and/or
[0031] g) the conjugation to a 2'5'-linked oligoadenylate or a
derivative thereof, a 2'5'-linked triadenylate, a 2'5'-linked
tetraadenylate, a 2'5'-linked pentaadenylate etc. being examples of
2'5'-linked oligoadenylates and cordycepin (2'5'-linked
3'-deoxyadenylate) being an example of a derivative of a
2'5'-linked oligoadenylate, the conjugation preferably taking place
via a linker, where the 5'-end of the 2'5'-linked oligoadenylate
can preferably be a phosphate, diphosphate or triphosphate group,
where the linker, for example, can be an oligoethylene glycol,
triethylene glycol, tetraethylene glycol and hexaethylene glycol
being examples of oligoethylene glycol linkers; and/or
[0032] h) the introduction of a 3'-3' and/or 5'-5' inversion at the
3' and/or at the 5' end of the oligonucleotide, this type of
chemical modification being known to the person skilled in the art
and being described, for example, in M. Koga et al., J. Org. Chem.
56 (1991) 3757.
[0033] In preferred embodiments of the invention, the
oligonucleotide has one or more chemical modifications which
independently of one another are selected from
[0034] a) the complete or partial replacement of the phosphoric
acid diester bridges by phosphorothioate and/or
(C.sub.1-C.sub.8)alkylphosphon- ate bridges,
[0035] b) the complete or partial replacement of the sugar
phosphate backbone by PNA units and/or PHONA units,
[0036] c) the complete or partial replacement of the
.beta.-D-2'-deoxyribose units by 2'-F-2'-deoxyribose,
2'-O--(C.sub.1-C.sub.6)alkylribose and/or
2'-[O--(C.sub.1-C.sub.6)alkyl-O-
--(C.sub.1-C.sub.6)alkyl]ribose,
[0037] d) the complete or partial replacement of the natural
nucleoside bases by 5-(C.sub.2-C.sub.6)-alkynyluracil and/or
5-(C.sub.2-C.sub.6)alky- nylcytosine,
[0038] e) the conjugation of the oligonucleotide to one or more
molecules which independently of one another can be selected from
the group comprising lipophilic molecules, e.g.
(C.sub.12-C.sub.20)alkyl, lipids, e.g.
1,2-dihexadecyl-rac-glycerol, steroids, e.g. cholesterol and/or
testosterone, vitamins, e.g. vitamin E, poly- or oligoethylene
glycol, (C.sub.12-C.sub.18)-alkyl phosphate diesters and
--O--CH.sub.2--CH(OH)--O- --(C.sub.12-C.sub.18)-alkyl and
[0039] f) one or more 3'-3' inversions at the 3' end of the
oligonucleotide, In another preferred embodiment of the invention,
the oligonucleotide has one or more chemical modifications which
independently of one another can be selected from the group
comprising
[0040] a) the complete or partial replacement of the phosphoric
acid diester bridges (phosphodiester bridges) by phosphorothioate
bridges,
[0041] b) the complete or partial replacement of the
.beta.-D-2'-deoxyribose units by 2'-F-2'-deoxyribose,
2'-O--(C.sub.1-C.sub.6)alkylribose and/or
2'-[O--(C.sub.1-C.sub.6)alkyl-O-
--(C.sub.1-C.sub.6)alkyl]ribose,
[0042] c) conjugation to lipophilic molecules, e.g.
(C.sub.12-C.sub.20)-alkyl, to lipids, e.g.
1,2-dihexadecyl-rac-glycerol, to (C.sub.12-C.sub.18)alkyl phosphate
diesters and/or to
--O--CH.sub.2--CH(OH)--O--(C.sub.12-C.sub.18)-alkyl.
[0043] Processes for the preparation of an oligonucleotide
conjugate are known to the person skilled in the art and are
described, for example, in Uhlmann, E. & Peyman, A., Chem. Rev.
90 (1990) 543 and/or M. Manoharan in "Antisense Research and
Applications", Crooke and Lebleu, Eds., CRC Press, Boca Raton,
1993, Chapter 17, p.303ff. and/or EP-A 0 552 766.
[0044] In a particular embodiment of the invention, an
oligonucleotide is made available which can have one or more
modifications and which has one of the sequences SEQ ID NO. 2-SEQ
ID NO. 20 or which corresponds to one of the sequences SEQ ID NO. 2
to SEQ ID NO. 20 or which corresponds to the appropriate sequence
sections of a tenascin-coding sequence and can bind to this section
of the tenascin-coding sequence.
[0045] In a particular embodiment of the invention, oligonucleotide
is made available in whose sequence each nucleotide (base and/or
sugar and/or internucleoside bridge) is modified. In a particular
embodiment of the invention, for example, the oligonucleotide is
completely synthesized from phosphorothioates (universally modified
phosphorothioate, all internucleoside bridges modified). In a
further specific embodiment of the invention, an oligonucleotide is
made available which corresponds to one of the sequences SEQ ID NO.
2-SEQ ID NO. 20, but where the phosphodiester bridges between the
individual nucleosides (i.e. the internucleoside bridges between
the individual nucleosides) are completely replaced by
phosphorothioate bridges (i.e. phosphorothioate groups between the
nucleosides).
[0046] In a further particular embodiment of the invention, an
oligonucleotide is made available by only replacing some of the
phosphodiester bridges by phosphorothioate bridges. In particular,
the invention comprises oligonucleotides which are only minimally
(or partially) modified. The principle of the minimally modified
oligonucleotides is described in A. Peyman, E. Uhlmann, Biol. Chem.
Hoppe-Seyler, 377 (1996) 67-70. In this case, 1-5, preferably 1-3
terminal nucleotide units (preferably the corresponding
internucleoside bridges) at the 5' and/or at the 3' end and, if
appropriate, additionally selected internal pyrimidine positions or
preferably the corresponding internucleoside bridges which are
located at the 3' and/or 5' end of the corresponding pyrimidine
nucleoside, are modified or replaced, internucleoside bridges
preferably being replaced by phosphorothioate bridges.
Oligonucleotides minimally modified in this way have particularly
advantageous properties, for example they exhibit particular
nuclease stability on minimal modification.
[0047] A particular embodiment of the invention relates to an
oligonucleotide in which selected internucleoside bridges are
replaced by modified internucleoside bridges, preferably by
phosphorothioate bridges.
[0048] The invention relates to an oligonucleotide in which
either
[0049] a) only certain phosphodiester internucleoside bridges
or
[0050] b) all phosphodiester internucleoside bridges are
modified.
[0051] The invention furthermore relates to an oligonucleotide in
which 1-5 terminal internucleoside bridges are modified at the 5'
and/or at the 3' end of the oligonucleotide. The invention also
relates to an oligonucleotide in which the internucleoside bridges
located at the 3' and/or 5' end of nonterminal nucleosides which
contain a pyrimidine base (internal pyrimidine nucleosides) are
modified.
[0052] Specific embodiments of the invention comprise a minimally
modified oligonucleotide which has one of the sequences selected
from the group consisting of the sequences SEQ ID NO. 21 to SEQ ID
NO. 39, where
2 SEQ ID NO. 21: is 3'-GsGsTsTsTGGGTsGGAGGsTsGsG-5', SEQ ID NO. 22:
is 3'-GsGsAsGGTsGGTsACsCCsCCsGsG- 5', SEQ ID NO. 23: is
3'-GsGsTGGTsACsCsCCsCsGsG-5', SEQ ID NO. 24: is
3'-GsGsAGGTsGGTsACsCsCsC-5', SEQ ID NO. 25: is
3'-AsGsAAAGAAsCsGAAAGGsAsA-5', SEQ ID NO. 26: is
3'-GsGsAGGTsGGTsAsCsC-5', SEQ ID NO. 27: is
3'-GsGsAGCsGATsGGCsTsTsCsCsA-5', SEQ ID NO. 28: is
3'-AsAsAGGAACsGGGAGsCsG-5', SEQ ID NO. 29: is
3'-GsGsTCGGTsTsTGGGTsGsG-5', SEQ ID NO. 30: is
3'-CsTsTACAGGTsCsCGTsTsGsA-5', SEQ ID NO. 31: is
3'-GsGsCsCGsTGTsTCGCsTsGsT-5', SEQ ID NO. 32: is
3'-TsCsACsCCsCTsCsTTsTsCsTsGsG- 5', SEQ ID NO. 33: is
3'-GsGsAsCACsCGACsACsGsG-5', SEQ ID NO. 34: is
3'-AsAsCsGGGAGCGATsGsG-5', SEQ ID NO. 35: is
3'-AsTsCsTCGGGGTsCsGsTsC-5', SEQ ID NO. 36: is
3'-AsAsAGAACsGAAAGGsAsA-5', SEQ ID NO. 37: is
3'-GsGsTGGTsACsCsCsC-5', SEQ ID NO. 38: is
3'-CsCsCsGGTsACsTsGsA-5', SEQ ID NO. 39: is
3'-CsCsAsCAGAAAGsAsAsC-5' and
[0053] "s" indicating the position of a modified internucleoside
bridge or dephospho bridge, "s" preferably indicating the position
of a phosphorothioate bridge.
[0054] The sequences SEQ ID NO. 21 to SEQ ID NO. 39 correspond to
the sequences SEQ ID NO. 2-SEQ ID NO. 20, i.e. they can bind to the
same regions of a tenascin-coding sequence, where, however, in
contrast to the SEQ ID NO. 2-20, some of the phosphodiester bridges
are replaced by modified phosphodiester bridges or dephospho
bridges, preferably by phosphorothioate bridges (in the sequence
marked by an "s").
[0055] A further embodiment of the invention relates to chimeric
oligonucleotides. A chimeric oligonucleotide is synthesized from at
least two different sequence sections, for example from a DNA
section and a modified section, e.g. a PNA section and/or a PHONA
section. These different sections impart particular properties to
the entire oligonucleotide.
[0056] A particular form of chimeric oligonucleotides is described,
for example, in Matteucci and Wagner, Nature 384 SUPP (1996) 20-22.
A chimeric oligonucleotide can contain, for example,
[0057] 1. a so-called core sequence, which consists of
approximately seven nucleotides and which can activate the RNase H,
and
[0058] 2. one or more flanking sequences which increase the
affinity, specificity and/or nuclease stability of the
oligonucleotide.
[0059] For example, the core sequence can have internucleoside
bridges modified in certain positions, for example the core
sequence can contain phosphorothioate and/or phosphodiester
bridges. Suitable flanking sequences are, for example, sequences in
which the sugar phosphate backbone (replacement of one or more
sugar phosphate units) and/or .beta.-D-2'-deoxyribose units are
replaced. Suitable flanking sequences are, for example, PNAs and/or
2'-O-alkyl derivatives such as, for example, 2'-O-methyl and/or
2'-O-propyl and/or 2'-methoxyethoxy derivatives.
[0060] A particular embodiment of the invention relates to a
chimeric oligonucleotide which has one of the sequences SEQ ID NO.
40-SEQ ID NO. 58, where
[0061] x independently of one another represents an unmodified or a
modified phosphodiester internucleoside bridge or a dephospho
bridge, preferably phosphorothioate and/or phosphorus diester
and
[0062] y independently of one another represents the replacement of
a sugar phosphate unit or a .beta.-D-2'-deoxyribose unit,
preferably 2'-O-methyl-, 2'-O-propyl- and/or 2'-methoxyethoxyribose
or a PNA unit, where
3 SEQ ID NO. 40: is 3'-GyGyTyTyTyGxGxGxTxGxGxAxGyGy TyGyG-5', SEQ
ID NO. 41: is 3'-GyGyAyGyGyTxGxGxTxAxCxCxCy- Cy CyGyG-5', SEQ ID
NO. 42: is 3'-GyGyTxGxGxTxAxCxCxCxCyCyGyG- 5', SEQ ID NO. 43: is
3'-GyGyAyGyGxTxGxGxTxAxCyCyCyC- 5', SEQ ID NO. 44: is
3'-AyGyAyAxAxGxAxAxCxGxAxAxAyGy GyAyA-5, SEQ ID NO. 45: is
3'-GyGyAxGxGxTxGxGxTxAyCyC-5', SEQ ID NO. 46: is
3'-GyGyAxGxCxGxAxTxGyGyCyTyTyCy CyA-5', SEQ ID NO. 47: is
3'-AyAyAyGxGxAxAxCxGxGyGyAyGyCy G-5', SEQ ID NO. 48: is
3'-GyGyTyCxGxGxTxTxTxGxGyGyTyGy G-5', SEQ ID NO. 49: is
3'-CyTyTyAxCxAxGxGxTxCxCxGyTyTy GyA-5', SEQ ID NO. 50: is
3'-GyGyCyCxGxTxGxTxTxCxGyCyTyGy T-5', SEQ ID NO. 51: is
3'-TyCyAyCxCxCxCxTxCxTxTy- TyCyTy GyG-5', SEQ ID NO. 52: is
3'-GyGyAyCxAxCxCxGxAxCxAyCyGyG- 5', SEQ ID NO. 53: is
3'-AyAyCyGxGxGxAxGxCxGxAyTyGyG- 5, SEQ ID NO. 54: is
3'-AyTyCyTxCxGxGxGxGxTxCxGyTyC- 5', SEQ ID NO. 55: is
3'-AyAyAyGxAxAxCxGxAxAxAxGyGyAy A-5', SEQ ID NO. 56: is
3'-GyGyTxGxGxTxAxCxCyCyC-5', SEQ ID NO. 57: is
3'-CyCxCxGxGxTxAxCyTyGyA-5', SEQ ID NO. 58: is
3'-CyCyAxCxAxGxAxAxAxGyAyAyC-5'
[0063] The sequences SEQ ID NO. 40-SEQ ID NO. 58 correspond to the
abovementioned sequences SEQ ID NO. 2 to SEQ ID NO. 20, i.e. they
bind to the corresponding sequence sections of a tenascin-coding
sequence, where, however, the modifications mentioned are
present.
[0064] The invention relates to processes for the preparation of
the oligonucleotides. The oligonucleotides described can be
prepared with the 10 aid of various known, chemical processes, e.g.
applying the standard phosphoramidite chemistry using iodine or TED
(tetraethylthiuram disulfide) as oxidant. This process is
described, for example, in Eckstein, F. (1991) "Oligonucleotides
and Analogues, A Practical Approach", IRL Press, Oxford. The
oligonucleotides can also be prepared by processes which optionally
contain one or more enzymatic steps.
[0065] The invention relates to the use of the oligonucleotides.
The oligonucleotides can be used for hybridization or binding to
tenascin-coding (single-stranded and/or double-stranded) nucleic
acids, for example DNA (e.g. genes, cDNA) and/or RNA (e.g.
pre-mRNA, mRNA). In particular, this relates to the use of the
oligonucleotides for hybridization with or binding to nucleic acids
which have the sequence SEQ ID NO. 1 according to Table 1 or with
nucleic acids which have parts of this sequence (for example
sequences which code for tenascin isoforms) or with nucleic acids
whose sequence differs slightly from these sequences (which have,
for example, one or more point mutations).
[0066] The invention furthermore relates to the use of the
oligonucleotides for the modulation and for the complete or partial
inhibition of the expression of tenascin or various tenascin
isoforms or of mutants thereof, for example for the complete or
partial inhibition of transcription and/or of translation.
[0067] The invention relates, for example, to the use of the
oligonucleotides as antisense oligonucleotides. Moreover, the
oligonucleotides can be used as aids in molecular biology.
[0068] The invention furthermore relates to the use of the
oligonucleotides as pharmaceutical and/or diagnostic or the use of
the oligonucleotides for the production of pharmaceuticals and/or
diagnostics. In particular, the oligonucleotides can be employed in
pharmaceuticals which are suitable for the prevention and/or
treatment of diseases which accompany the expression of an
overexpression of tenascin. Since the expression of tenascin is
normally, i.e., for example, in the healthy person, limited
spatially and temporally, a deviation from this normal spatial and
temporal expression can be regarded as overexpression. Furthermore,
the oligonucleotides can be employed in diagnostic processes. Such
diagnostic processes can be employed, for example, for the
diagnosis or early recognition of diseases which accompany abnormal
expression (e.g. overexpression) of tenascin.
[0069] The invention also relates to a test kit which contains one
or more oligonucleotides according to the invention and, if
appropriate, further components. Such a test kit can be employed,
for example, in diagnosis and as a precaution, for example against
skin cancer disorders.
[0070] The invention further relates to the use of the
oligonucleotides or of pharmaceuticals which contain these
oligonucleotides for the treatment of diseases in which tenascin or
an overexpression of tenascin is the cause or is involved.
[0071] The invention relates in particular to the use of the
oligonucleotides or of pharmaceuticals which contain these
oligonucleotides for the treatment and/or prevention of diseases in
which a dysregulation or disorder of the immigration or of the
presence or of the inclusion of melanocytes in epithelial cell
layers, for example in the epithelial cell layer of the epidermis,
of the choroid membrane of the eye or of the substantia nigra as
the basis serves or is involved and of Addison's disease, diabetes
mellitus, pernicious anemia and/or thyroid gland dysfunctions.
[0072] The invention relates in particular to the use of the
oligonucleotides or of pharmaceuticals which contain these
oligonucleotides for the treatment and/or prevention of vitiligo
and other depigmentation diseases or depigmentation disorders (e.g.
of the skin, hair, eyes) for example albinism and/or for the
treatment of psoriasis and/or for the treatment of cancer, e.g. for
the inhibition of tumor growth and tumor metastasis, for example in
melanomas and/or for the treatment of inflammations, in particular
as antiinflammatories and/or for the treatment and/or prophylaxis
of cardiovascular disorders, for example of restenosis.
[0073] In particular, the invention relates to the use of the
oligonucleotides for the treatment of vitiligo or for the
production of pharmaceuticals which can be used for the treatment
of vitiligo. The invention moreover relates quite generally (i.e.
also oligonucleotides having a length of greater than or equal to
18 nucleotides) to the use of oligonucleotides for the treatment of
vitiligo or the production of pharmaceuticals which can be used for
the treatment of vitiligo.
[0074] The invention furthermore relates to the use for the
treatment of vitiligo in combination with known therapeutic
processes, for example in combination a) with photochemotherapy
(PUVA), e.g. using methoxypsoralen, phenylalanine and/or khellin
and/or b) with the transplantation of cultured melanocytes
(epidermal grafting) and/or c) with a steroid treatment and/or d)
with a treatment with placenta extracts and/or e) with a treatment
with pseudocatalase.
[0075] The invention furthermore relates to processes for the
production of pharmaceuticals (pharmaceutical preparations). For
the production of pharmaceuticals, one or more different
oligonucleotides or their physiologically tolerable salts are
mixed, it optionally being possible to add further pharmaceutical
vehicles and/or additives.
[0076] The invention furthermore relates to pharmaceutical
preparations (pharmaceuticals), which contain one or more different
oligonucleotides and/or their physiologically tolerable salts, and,
if appropriate, pharmaceutical vehicles and/or additives.
[0077] The oligonucleotide(s) and/or its/their physiologically
tolerable salts can be administered to animals, preferably to
mammals, in particular to humans as pharmaceuticals on its/their
own, in mixtures with one another or in the form of pharmaceutical
preparations. The pharmaceuticals can make possible topical,
percutaneous, parenteral and/or enteral administration. The
administration form preferred in each case depends on the specific
conditions in each case. For the treatment of vitiligo, for
example, a topical application, e.g. in the form of ointments,
lotions or tinctures, emulsions or suspensions, is preferred.
Likewise, the frequency of the administration depends on the
individual conditions. For the treatment of vitiligo, for example,
a topical composition can be applied to the depigmented skin site
one to two times during the day.
[0078] As active constituent, pharmaceuticals or pharmaceutical
preparations can contain an efficacious dose of at least one
oligonucleotide and/or a mixture of a number of oligonucleotides
and, if appropriate, additional, pharmaceutically innocuous
vehicles and/or additives. Pharmaceutical preparations can contain
approximately 0.1% (percent by weight) or less up to approximately
90% (percent by weight) or more of the therapeutically active
oligonucleotide or the pharmaceutically active oligonucleotide.
[0079] The pharmaceutically efficacious dose of the respective
oligonucleotide or of an oligonucleotide which is a constituent of
a mixture of various oligonucleotides can vary within wide limits
and is to be adapted to the individual conditions in each
individual case.
[0080] The production of the pharmaceutical preparations can be
carried out in a manner known per se, e.g. described in Remingtons
Pharmaceutical Sciences (1985), Mack Publ. Co., Easton, Pa., it
optionally being possible to use pharmaceutically inert inorganic
and/or organic vehicles. For the production of pills, tablets,
coated tablets and/or hard gelatin capsules, it is possible to use,
for example, lactose, cornstarch and/or derivatives thereof, talc,
stearic acid and/or its salts. Vehicles which can be used for soft
gelatin capsules and/or suppositories are, for example, fats,
waxes, semisolid and/ or liquid polyols, natural and/or hardened
oils. Vehicles which can be used for the production of solutions
and/or syrups are, for example, water, sucrose, invert sugar,
glucose and/or polyols. Vehicles which can be used for the
production of injection solutions are, for example, water,
alcohols, glycerol, polyols and/or vegetable oils. Vehicles which
can be used for microcapsules, implants and/or rods are, for
example, copolymers, e.g. of glycolic acid and lactic acid.
Moreover, liposome formulations which are known to the person
skilled in the art (N. Weiner, Drug Develop Ind Pharm 15 (1989)
1523; "Liposome Dermatics, Springer Verlag 1992), for example HVJ
liposomes (Hayashi, Gene Therapy 3 (1996) 878) are suitable. Dermal
administration can be carried out, for example, also with the aid
of ionophoretic methods and/or with the aid of electroporation.
Moreover, lipofectins and/or other (nucleic acid or DNA) carrier
systems, for example those which are used in gene therapy, can be
used. In particular, suitable systems are those with whose aid
oligonucleotides can be introduced into eukaryotic cells or the
nuclei of eukaryotic cells with great efficiency. In addition to
the active compounds and vehicles, a pharmaceutical preparation can
additionally contain additives, such as, for example, fillers,
extenders, disintegrants, binding agents, lubricants, wetting
agents, stabilizers, emulsifiers, preservatives, sweeteners,
colorants, flavorings or aromatizers, thickening agents, diluents,
buffer substances, furthermore solvents and/or solubilizers and/or
agents for achieving a depot effect, and salts for changing the
osmotic pressure, coating agents and/or antioxidants. They can also
contain two or more different oligonucleotides and/or their
physiologically tolerable salts and furthermore, in addition to at
least one oligonucleotide, one or more other therapeutically active
substances.
EXAMPLES
Example 1
Oligonucleotide Synthesis
[0081] The oligonucleotide was synthesized on an automatic DNA
synthesizer (Applied Biosystems Model 380B or 394) using the
standard phosphoramidite chemistry and oxidation with iodine (F.
Eckstein, Ed "Oligonucleotides and Analogs, A Practical Approach",
IRL Press, Oxford, 1991). For the introduction of phosphorothioate
bridges in mixed phosphorothioates and phosphodiester
oligonucleotide, oxidation was carried out with TETD
(tetraethylthiuram disulfide) instead of iodine (Applied Biosystems
User Bulletin 65). After removal of solid carrier (CPG or Tentagel)
and removal of the protective groups with conc. NH.sub.3 at
55.degree. C. (18 h), the oligonucleotide was first purified by
butanol precipitation (Sawadogo, Van Dyke, Nucl. Acids Res. 19
(1991) 674). The sodium salt was then obtained by precipitation
from a 0.5 M NaCl solution using 2.5 parts by volume of
ethanol.
[0082] The oligonucleotide was analyzed with the aid of
[0083] a) analytical gel electrophoresis (gel: 20% acrylamide, 8M
urea; running buffer: 454M tris borate buffer, pH 7.0) and/or
[0084] b) HPLC analysis (column material: Waters GenPak FAX;
gradient: CH.sub.3CN (400 ml), H.sub.2O (1.6 l), NaH.sub.2PO.sub.4
(3.1 g), NaCl (11.7 g) pH 6.8 (0.1 M in NaCl) after CH.sub.3CN (400
ml), H.sub.2O (1.6 l), NaH.sub.2PO.sub.4 (3.1 g), NaCl (175.3 g),
pH 6.8 (1.5 M in NaCl)) and/or
[0085] c) capillary gel electrophoresis (Beckmann capillary
eCAP.TM., U100P gel column, 65 cm length, 100 mm I.D., window 15 cm
from one end; buffer: 140 .mu.M tris, 360 mM boric acid, 7M urea)
and/or
[0086] d) electrospray mass spectroscopy.
[0087] The analysis of the oligonucleotide showed that this was in
each case present in a purity of greater than 90%. The methods for
the analysis of oligonucleotides are described, for example, in
Schweiber and Engler "Analysis of oligonucleotides" (in
"Antisense--from technology to therapy", a laboratory manual and
textbook, Schlingensiepen et al. eds., Biol. Science, Vol. 6 (1997)
p. 78-103).
[0088] Synthesized oligonucleotide:
4 ODN1 (sequence SEQ ID NO. 24): 3'-GsGsAGGTsGGTsACsCsCs- C-5'
Example 2
Production of a Pharmaceutical Preparation
[0089] 50 mg of ODN 1 from Example 1 can be closely mixed with 1 g
of Dermatop.RTM. (Hoechst Aktiengesellschaft, Frankfurt am Main,
Germany) base cream and the mixture stored at temperatures of
<10.degree. C.
Example 3
[0090] The cream from Example 2 can then be applied twice daily (in
the morning and afternoon or evening) to a depigmented skin site of
a vitiligo patient.
5TABLE 1 Sequence SEQ ID NO. 1: Sequence of the human tenascin cDNA
according to A. Siri et al. Nucl. Acids Res. 19 (1991) 525-531.
GAATTCGCTA GAGCCCTAGA GCCCCAGCAG CACCCAGCCA AACCCACCTC CACCATGGGG
60 GCCATGACTC AGCTGTTGGC AGGTGTCTTT CTTGCTTTCC TTGCCCTCGC
TACCGAAGCT 120 GGGGTCCTCA AGAAAGTCAT CCGGCACAAG CGACAGAGTG
GGGTGAACGC CACCCTGCCA 180 GAAGAGAACC AGCCAGTGGT GTTTAACCAC
GTTTACAACA TCAAGCTGCC AGTGGGATCC 240 CAGTGTTCGG TGGATCTGGA
GTCAGCCAGT GGGGAGAAAG ACCTGGCACC GCCTTCAGAG 300 CCCAGCCAAA
GCTTTCAGGA GCACACAGTA GATGGGGAAA ACCAGATTGT CTTCACACAT 360
CGCATCAACA TCCCCCGCCG GGCCTGTGGC TGTGCCGCAG CCCCTGATGT TAAGGAGCTG
420 CTGAGCAGAC TGGAGGAGCT GGAGAACCTG GTGTCTTCCC TGAGGGAGCA
ATGTACTGCA 480 GGAGCAGGCT GCTGTCTCCA GCCTGCCACA GGCCGCTTGG
ACACCAGGCC CTTCTGTAGC 540 GGTCGGGGCA ACTTCAGCAC TGAAGGATGT
GGCTGTGTCT GCGAACCTGG CTGGAAAGGC 600 CCCAACTGCT CTGAGCCCGA
ATGTCCAGGC AACTGTCACC TTCGAGGCCG GTGCATTGAT 660 GGGCAGTGCA
TCTGTGACGA CGGCTTCACG GGCGAGGACT GCAGCCAGCT GGCTTGCCCC 720
AGCGACTGCA ATGACCAGGG CAAGTGCGTG AATGGAGTCT GCATCTGTTT CGAAGGCTAC
780 GCGGCTGACT GCAGCCGTGA AATCTGCCCA GTGCCCTGCA GTGAGGAGCA
CGGCACATGT 840 GTAGATGGCT TGTGTGTGTG CCACGATGGC TTTGCAGGCG
ATGACTGCAA CAAGCCTCTG 900 TGTCTCAACA ATTGCTACAA CCGTGGACGA
TGCGTGGAGA ATGAGTGCGT GTGTGATGAG 960 GGTTTCACGG GCGAAGACTG
CAGTGAGCTC ATCTGCCCCA ATGACTGCTT CGACCGGGGC 1020 CGCTGCATCA
ATGGCACCTG CTACTGCGAA GAAGGCTTCA CAGGTGAAGA CTGCGGGAAA 1080
CCCACCTGCC CACATGCCTG CCACACCCAG GGCCGGTGTG AGGAGGGGCA GTGTGTATGT
1140 GATGAGGGCT TTGCCGGTGT GGACTGCAGC GACAAGAGGT GTCCTGCTGA
CTGTCACAAT 1200 CGTGGCCGCT GTGTAGACGG GCGGTGTGAG TGTGATGATG
GTTTCACTGG AGCTGACTGT 1260 GGGGAGCTCA AGTGTCCCAA TGGCTGCAGT
GGCCATGGCC GCTGTGTCAA TGGGCAGTGT 1320 GTGTGTGATG AGGGCTATAC
TGGGGAGGAC TGCAGCCAGC TACGGTGCCC CAATGACTGT 1380 CACAGTCGGG
GCCGCTGTGT CGAGGGCAAA TGTGTATGTG AGCAAGGCTT CAAGGGCTAT 1440
GACTGCAGTG ACATGAGCTG CCCTAATGAC TGTCACCAGC ACGGCGCCTG TGTGAATGGC
1500 ATGTGTGTTT GTGATGACGG CTACACAGGG GAAGACTGCC GGGATCGCCA
ATGCCCCAGG 1560 GACTGCAGCA ACAGGGGCCT CTGTGTGGAC GGACAGTGCG
TCTGTGAGGA CGGCTTCACC 1620 GGCCCTGACT GTGCAGAACT CTCCTGTCCA
AATGACTGCC ATGGCCAGGG TCGCTGTGTG 1680 AATGGGCAGT GCGTGTGCCA
TCAAGGATTT ATGGGCAAAG ACTGCAAGGA GCAAAGATGT 1740 CCCAGTGACT
GTCATGGCCA GGGCCGCTGC GTGGACGGCC AGTGCATCTG CCACGAGGGC 1800
TTCACAGGCC TGGACTGTGG CCAGCACTCC TGCCCCAGTG ACTGCAACAA CTTAGGACAA
1860 TGCGTCTCGG GCCGCTGCAT CTGCAACGAG GGCTACAGCG GAGAAGACTG
CTCAGAGGTG 1920 TCTCCTCCCA AAGACCTCGT TGTGACAGAA GTGACGGAAG
AGACGGTCAA CCTGGCCTGG 1980 GACAATGAGA TGCGGGTCAC AGAGTACCTT
GTCGTGTACA CGCCCACCCA CGAGGGTGGT 2040 CTGGAAATGC ACTTCCGTGT
GCCTGGGGAC CAGACGTCCA CCATCATCCG GGAGCTGGAG 2100 CCTGGTGTGG
AGTACTTTAT CCGTGTATTT GCCATCCTGG AGAACAAGAA GAGCATTCCT 2160
GTCAGCGCCA GGGTGGCCAC GTACTTACCT GCACCTGAAG GCCTGAAATT CAAGTCCATC
2220 AAGGAGACAT CTGTGGAAGT GGAGTGGGAT CCTCTAGACA TTGCTTTTGA
AACCTGGGAG 2280 ATCATCTTCC GGAATATGAA TAAAGAAGAT GAGGGAGAGA
TCACCAAAAG CCTGAGGAGG 2340 CCAGAGACCT CTTACCGGCA AACTGGTCTA
GCTCCTGGGC AAGAGTATGA GATATCTCTG 2400 CACATAGTGA AAAACAATAC
CCGGGGCCCT GGCCTGAAGA GGGTGACCAC CACACGCTTG 2460 GATGCCCCCA
GCCAGATCGA GGTGAAAGAT GTCACAGACA CCACTGCCTT GATCACCTGG 2520
TTCAAGCCCC TGGCTGACAT CGATGGCATT GAGCTGACCT ACGGCATCAA AGACGTGCCA
2580 GGAGACCGTA CCACCATCGA TCTCACAGAG GACGAGAACC AGTACTCCAT
CGGGAACCTG 2640 AAGCCTGACA CTGAGTACGA GGTGTCCCTC ATCTCCCGCA
GAGGTGACAT GTCAAGCAAC 2700 CCAGCCAAAG AGACCTTCAC AACAGGCCTC
GATGCTCCCA GGAATCTTCG ACGTGTTTCC 2760 CAGACAGATA ACAGCATCAC
CCTGGAATGG AGGAATGGCA AGGCAGCTAT TGACAGTTAC 2820 AGAATTAAGT
ATGCCCCCAT CTCTGGAGGG GACCACGCTG AGGTTGATGT TCCAAAGAGC 2880
CAACAAGCCA CAACCAAAAC CACACTCACA GGTCTGAGGC CGGGAACTGA ATATGGGATT
2940 GGAGTTTCTG CTGTGAAGGA AGACAAGGAG AGCAATCCAG CGACCATCAA
CGCAGCCACA 3000 GAGTTGGACA CGCCCAAGCA CCTTCAGGTT TCTGAAACTG
CAGAGACCAG CCTGACCCTG 3060 CTCTGGAAGA CACCGTTGGC CAAATTTGAC
CGCTACCGCC TCAATTACAG TCTCCCCACA 3120 GGCCAGTGGG TGGGAGTGCA
GCTTCCAAGA AACACCACTT CCTATGTCCT GAGAGGCCTG 3180 GAACCAGGAC
AGGAGTACAA TGTCCTCCTG ACAGCCGAGA AAGGCAGACA CAAGAGCAAG 3240
CCCGCACGTG TGAAGGCATC CACTGAACAA GCCCCTGAGC TGGAAAACCT CACCGTGACT
3300 GAGGTTGGCT GGGATGGCCT CAGACTCAAC TGGACCGCGG CTGACCAGGC
CTATGAGCAC 3360 TTTATCATTC AGGTGCAGGA GGCCAACAAG GTGGAGGCAG
CTCGGAACCT CACCGTGCCT 3420 GGCAGCCTTC GGGCTGTGGA CATACCGGGC
CTCAAGGCTG CTACGCCTTA TACAGTCTCC 3480 ATCTATGGGG TGATCCAGGG
CTATAGAACA CCAGTGCTCT CTCCTGAGGC CTCCACAGGG 3540 GAAACTCCCA
ATTTGGGAGA GGTCGTGGTG GCCGAGGTGG GCTGGGATGC CCTCAAACTC 3600
AACTGGACTG CTCCAGAAGG GGCCTATGAG TACTTTTTCA TTCAGGTGCA GGAGGCTGAC
3660 ACAGTAGAGG CAGCCCAGAA CCTCACCGTC CCAGGAGGAC TGAGGTCCAC
AGACCTGCCT 3720 GGGCTCAAAG CAGCCACTCA TTATACCATC ACCATCCGCG
GGGTCACTCA GGACTTCAGC 3780 ACAACCCCTC TCTCTGTTGA AGTCTTGACA
GAGGAGGTTC CAGATATGGG AAACCTCACA 3840 GTGACCGAGG TTAGCTGGGA
TGCTCTCAGA CTGAACTGGA CCACGCCAGA TGGAACCTAT 3900 GACCAGTTTA
CTATTCAGGT CCAGGAGGCT GACCAGCTGG AAGAGGCTCA CAATCTCACG 3960
GTTCCTGGCA GCCTGCGTTC CATGGAAATC CCAGGCCTCA GGGCTGGCAC TCCTTACACA
4020 GTCACCCTGC ACGGCGAGGT CAGGGGCCAC AGCACTCGAC CCCTTGCTGT
AGAGGTCGTC 4080 CAGTGGGACG TGCCGCTCCA GTCCCCGGTG TCGTGAGCTG
GGGAACGACA TCTCCAGCAG 4140 ACAGAGGATC TCCCACAGCT GGGAGATTTA
GCCGTGTCTG AGGTTGGCTG GGATGGCCTC 4200 AGACTCAACT GGACCGCAGC
TGACAATGCC TATGAGCACT TTGTCATTCA GGTGCAGGAG 4260 GTCAACAAAG
TGGAGGCAGC CCAGAACCTC ACGTTGCCTG GCAGCCTCAG GGCTGTGGAC 4320
ATCCCGGGCC TCGAGGCTGC CACGCCTTAT AGAGTCTCCA TCTATGGGGT GATCCGGGGC
4380 TATAGAACAC CAGTACTCTC TGCTGAGGCC TCCACAGCCA AAGAACCTGA
AATTGGAAAC 4440 TTAAATGTTT CTGACATAAC TCCCCAGAGC TTCAATCTCT
CCTGGATGGC TACCGATGGG 4500 ATCTTCGACA CCTTTACCAT TGAAATTATT
GATTCCAATA GGTTGCTGGA GACTGTGGAA 4560 TATAATATCT CTGGTGCTGA
ACGAACTGCC CATATCTCAG GGCTACCCCC TAGTACTGAT 4620 TTTATTGTCT
ACCTCTCTGG ACTTGCTCCC AGCATCCGGA CCAAAACCAT CAGTGCCACA 4680
GCCACGACAG AGGCCCTGCC CCTTCTGGAA AACCTAACCA TTTCCGACAT TAATCCCTAC
4740 GGGTTCACAG TTTCCTGGAT GGCATCGGAG AATGCCTTTG ACAGCTTTCT
AGTAACGGTG 4800 GTGGATTCTG GGAAGCTGCT GGACCCCCAG GAATTCACAC
TTTCAGGAAC CCAGAGGAAG 4860 CTGGAGCTTA GAGGCCTCAT AACTGGCATT
GGCTATGAGG TTATGGTCTC TGGCTTCACC 4920 CAAGGGCATC AAACCAAGCC
CTTGAGGGCT GAGATTGTTA CAGAAGCCGA ACCGGAAGTT 4980 GACAACCTTC
TGGTTTCAGA TGCCACCCCA GACGGTTTCC GTCTGTCCTG GACAGCTGAT 5040
GAAGGGGTCT TCGACAATTT TGTTCTCAAA ATCAGAGATA CCAAAAAGCA GTCTGAGCCA
5100 CTGGAAATAA CCCTACTTGC CCCCGAACGT ACCAGGGACA TAACAGGTCT
CAGAGAGGCT 5160 ACTGAATACG AAATTGAACT CTATGGAATA AGCAAAGGAA
GGCGATCCCA GACAGTCAGT 5220 GCTATAGCAA CAACAGCCAT GGGCTCCCCA
AAGGAAGTCA TTTTCTCAGA CATCACTGAA 5280 AATTCGGCTA CTGTCAGCTG
GAGGGCACCC ACGGCCCAAG TGGAGAGCTT CCGGATTACC 5340 TATGTGCCCA
TTACAGGAGG TACACCCTCC ATGGTAACTG TGGACGGAAC CAAGACTCAG 5400
ACCAGGCTGG TGAAACTCAT ACCTGGCGTG GAGTACCTTG TCAGCATCAT CGCCATGAAG
5460 GGCTTTGAGG AAAGTGAACC TGTCTCAGGG TCATTCACCA CAGCTCTGGA
TGGCCCATCT 5520 GGCCTGGTGA CAGCCAACAT CACTGACTCA GAAGCCTTGG
CCAGGTGGCA GCCAGCCATT 5580 GCCACTGTGG ACAGTTATGT CATCTCCTAC
ACAGGCGAGA AAGTGCCAGA AATTACACGC 5640 ACGGTGTCCG GGAACACAGT
GGAGTATGCT CTGACCGACC TCGAGCCTGC CACGGAATAC 5700 ACACTGAGAA
TCTTTGCAGA GAAAGGGCCC CAGAAGAGCT CAACCATCAC TGCCAAGTTC 5760
ACAACAGACC TCGATTCTCC AAGAGACTTG ACTGCTACTG AGGTTCAGTC GGAAACTGCC
5820 CTCCTTACCT GGCGACCCCC CCGGGCATCA GTCACCGGTT ACCTGCTGGT
CTATGAATCA 5880 GTGGATGGCA CAGTCAAGGA AGTCATTGTG GGTCCAGATA
CCACCTCCTA CAGCCTGGCA 5940 GACCTGAGCC CATCCACCCA CTACACAGCC
AAGATCCAGG CACTCAATGG GCCCCTGAGG 6000 AGCAATATGA TCCAGACCAT
CTTCACCACA ATTGGACTCC TGTACCCCTT CCCCAAGGAC 6060 TGCTCCCAAG
CAATGCTGAA TGGAGACACG ACCTCTGGCC TCTACACCAT TTATCTGAAT 6120
GGTGATAAGG CTCAGGCGCT GGAAGTCTTC TGTGACATGA CCTCTGATGG GGGTGGATGG
6180 ATTGTGTTCC TGAGACGCAA AAACGGACGC GAGAACTTCT ACCAAAACTG
GAAGGCATAT 6240 GCTGCTGGAT TTGGGGACCG CAGAGAAGAA TTCTGGCTTG
GGCTGGACAA CCTGAACAAA 6300 ATCACAGCCC AGGGGCAGTA CGAGCTCCGG
GTGGACCTGC GGGACCATGG GGAGACAGCC 6360 TTTGCTGTCT ATGACAAGTT
CAGCGTGGGA GATGCCAAGA CTCGCTACAA GCTGAAGGTG 6420 GAGGGGTACA
GTGGGACAGC AGGTGACTCC ATCCCCTACC ACAATGGCAG ATCCTTCTCC 6480
ACCTTTGACA AGGACACAGA TTCAGCCATC ACCAACTGTG CTCTGTCTAC AAGGGGCTTC
6540 TGGTACAGGA ACTGTCACCG TGTCAACCTG ATGGGGAGAT ATGGGGACAA
TAACCACAGT 6600 CAGGGCGTTA ACTGGTTCCA CTGGAAGGGC CACGAACACT
CAATCCAGTT TGCTGAGATG 6660 AAGCTGAGAC CAAGCAACTT CAGAAATCTT
GAAGGCAGGC GCAAACGGGC ATAAATTGGA 6720 GGGACCACTG GGTGAGAGAG
GAATAAGGCG GCCCAGAGCG AGGAAAGGAT TTTACCAAAG 6780 CATCAATACA
ACCAGCCCAA CCATCGGTCC ACACCTGGGC ATTTGGTGAG AATCAAAGCT 6840
GACCATGGAT CCCTGGGGCC AACGGCAACA GCATGGGCCT CACCTCCTCT GTGATTTCTT
6900 TCTTTGCACC AAAGACATCA GTCTCCAACA TGTTTCTGTT TTGTTGTTTG
ATTCAGCAAA 6960 AATCTCCCAG TGACAACATC GCAATAGTTT TTTACTTCTC
TTAGGTGGCT CTGGGATGGG 7020 AGAGGGGTAG GATGTACAGG GGTAGTTTGT
TTTAGAACCA GCCGTATTTT ACATGAAGCT 7080 GTATAATTAA TTGTCATTAT
TTTTGTTAGC AAAGATTAAA TGTGTCATTG GAAGCCATCC 7140 CTTTTTTTAC
ATTTCATACA ACAGAAACCA GAAAAGCAAT ACTGTTTCCA TTTTAAGGAT 7200
ATGATTAATA TTATTAATAT AATAATGATG ATGATGATGA TGAAAACTAA GGATTTTTCA
7260 AGAGATCTTT CTTTCCAAAA CATTTCTGGA CAGTACCTGA TTGTATTTTT
TTTTTAAATA 7320 AAAGCACAAG TACTTTTGAA AAAAAA 7346
[0091]
Sequence CWU 1
1
58 1 7346 DNA Homo sapiens 1 gaattcgcta gagccctaga gccccagcag
cacccagcca aacccacctc caccatgggg 60 gccatgactc agctgttggc
aggtgtcttt cttgctttcc ttgccctcgc taccgaaggt 120 ggggtcctca
agaaagtcat ccggcacaag cgacagagtg gggtgaacgc caccctgcca 180
gaagagaacc agccagtggt gtttaaccac gtttacaaca tcaagctgcc agtgggatcc
240 cagtgttcgg tggatctgga gtcagccagt ggggagaaag acctggcacc
gccttcagag 300 cccagcgaaa gctttcagga gcacacagta gatggggaaa
accagattgt cttcacacat 360 cgcatcaaca tcccccgccg ggcctgtggc
tgtgccgcag cccctgatgt taaggagctg 420 ctgagcagac tggaggagct
ggagaacctg gtgtcttccc tgagggagca atgtactgca 480 ggagcaggct
gctgtctcca gcctgccaca ggccgcttgg acaccaggcc cttctgtagc 540
ggtcggggca acttcagcac tgaaggatgt ggctgtgtct gcgaacctgg ctggaaaggc
600 cccaactgct ctgagcccga atgtccaggc aactgtcacc ttcgaggccg
gtgcattgat 660 gggcagtgca tctgtgacga cggcttcacg ggcgaggact
gcagccagct ggcttgcccc 720 agcgactgca atgaccaggg caagtgcgtg
aatggagtct gcatctgttt cgaaggctac 780 gcggctgact gcagccgtga
aatctgccca gtgccctgca gtgaggagca cggcacatgt 840 gtagatggct
tgtgtgtgtg ccacgatggc tttgcaggcg atgactgcaa caagcctctg 900
tgtctcaaca attgctacaa ccgtggacga tgcgtggaga atgagtgcgt gtgtgatgag
960 ggtttcacgg gcgaagactg cagtgagctc atctgcccca atgactgctt
cgaccggggc 1020 cgctgcatca atggcacctg ctactgcgaa gaaggcttca
caggtgaaga ctgcgggaaa 1080 cccacctgcc cacatgcctg ccacacccag
ggccggtgtg aggaggggca gtgtgtatgt 1140 gatgagggct ttgccggtgt
ggactgcagc gagaagaggt gtcctgctga ctgtcacaat 1200 cgtggccgct
gtgtagacgg gcggtgtgag tgtgatgatg gtttcactgg agctgactgt 1260
ggggagctca agtgtcccaa tggctgcagt ggccatggcc gctgtgtcaa tgggcagtgt
1320 gtgtgtgatg agggctatac tggggaggac tgcagccagc tacggtgccc
caatgactgt 1380 cacagtcggg gccgctgtgt cgagggcaaa tgtgtatgtg
agcaaggctt caagggctat 1440 gactgcagtg acatgagctg ccctaatgac
tgtcaccagc acggccgctg tgtgaatggc 1500 atgtgtgttt gtgatgacgg
ctacacaggg gaagactgcc gggatcgcca atgccccagg 1560 gactgcagca
acaggggcct ctgtgtggac ggacagtgcg tctgtgagga cggcttcacc 1620
ggccctgact gtgcagaact ctcctgtcca aatgactgcc atggccaggg tcgctgtgtg
1680 aatgggcagt gcgtgtgcca tgaaggattt atgggcaaag actgcaagga
gcaaagatgt 1740 cccagtgact gtcatggcca gggccgctgc gtggacggcc
agtgcatctg ccacgagggc 1800 ttcacaggcc tggactgtgg ccagcactcc
tgccccagtg actgcaacaa cttaggacaa 1860 tgcgtctcgg gccgctgcat
ctgcaacgag ggctacagcg gagaagactg ctcagaggtg 1920 tctcctccca
aagacctcgt tgtgacagaa gtgacggaag agacggtcaa cctggcctgg 1980
gacaatgaga tgcgggtcac agagtacctt gtcgtgtaca cgcccaccca cgagggtggt
2040 ctggaaatgc agttccgtgt gcctggggac cagacgtcca ccatcatccg
ggagctggag 2100 cctggtgtgg agtactttat ccgtgtattt gccatcctgg
agaacaagaa gagcattcct 2160 gtcagcgcca gggtggccac gtacttacct
gcacctgaag gcctgaaatt caagtccatc 2220 aaggagacat ctgtggaagt
ggagtgggat cctctagaca ttgcttttga aacctgggag 2280 atcatcttcc
ggaatatgaa taaagaagat gagggagaga tcaccaaaag cctgaggagg 2340
ccagagacct cttaccggca aactggtcta gctcctgggc aagagtatga gatatctctg
2400 cacatagtga aaaacaatac ccggggccct ggcctgaaga gggtgaccac
cacacgcttg 2460 gatgccccca gccagatcga ggtgaaagat gtcacagaca
ccactgcctt gatcacctgg 2520 ttcaagcccc tggctgagat cgatggcatt
gagctgacct acggcatcaa agacgtgcca 2580 ggagaccgta ccaccatcga
tctcacagag gacgagaacc agtactccat cgggaacctg 2640 aagcctgaca
ctgagtacga ggtgtccctc atctcccgca gaggtgacat gtcaagcaac 2700
ccagccaaag agaccttcac aacaggcctc gatgctccca ggaatcttcg acgtgtttcc
2760 cagacagata acagcatcac cctggaatgg aggaatggca aggcagctat
tgacagttac 2820 agaattaagt atgcccccat ctctggaggg gaccacgctg
aggttgatgt tccaaagagc 2880 caacaagcca caaccaaaac cacactcaca
ggtctgaggc cgggaactga atatgggatt 2940 ggagtttctg ctgtgaagga
agacaaggag agcaatccag cgaccatcaa cgcagccaca 3000 gagttggaca
cgcccaagga ccttcaggtt tctgaaactg cagagaccag cctgaccctg 3060
ctctggaaga caccgttggc caaatttgac cgctaccgcc tcaattacag tctccccaca
3120 ggccagtggg tgggagtgca gcttccaaga aacaccactt cctatgtcct
gagaggcctg 3180 gaaccaggac aggagtacaa tgtcctcctg acagccgaga
aaggcagaca caagagcaag 3240 cccgcacgtg tgaaggcatc cactgaacaa
gcccctgagc tggaaaacct caccgtgact 3300 gaggttggct gggatggcct
cagactcaac tggaccgcgg ctgaccaggc ctatgagcac 3360 tttatcattc
aggtgcagga ggccaacaag gtggaggcag ctcggaacct caccgtgcct 3420
ggcagccttc gggctgtgga cataccgggc ctcaaggctg ctacgcctta tacagtctcc
3480 atctatgggg tgatccaggg ctatagaaca ccagtgctct ctgctgaggc
ctccacaggg 3540 gaaactccca atttgggaga ggtcgtggtg gccgaggtgg
gctgggatgc cctcaaactc 3600 aactggactg ctccagaagg ggcctatgag
tactttttca ttcaggtgca ggaggctgac 3660 acagtagagg cagcccagaa
cctcaccgtc ccaggaggac tgaggtccac agacctgcct 3720 gggctcaaag
cagccactca ttataccatc accatccgcg gggtcactca ggacttcagc 3780
acaacccctc tctctgttga agtcttgaca gaggaggttc cagatatggg aaacctcaca
3840 gtgaccgagg ttagctggga tgctctcaga ctgaactgga ccacgccaga
tggaacctat 3900 gaccagttta ctattcaggt ccaggaggct gaccaggtgg
aagaggctca caatctcacg 3960 gttcctggca gcctgcgttc catggaaatc
ccaggcctca gggctggcac tccttacaca 4020 gtcaccctgc acggcgaggt
caggggccac agcactcgac cccttgctgt agaggtcgtc 4080 cagtgggacg
tgccgctcca gtccccggtg tcgtgagctg gggaacgaca tctccagcag 4140
acagaggatc tcccacagct gggagattta gccgtgtctg aggttggctg ggatggcctc
4200 agactcaact ggaccgcagc tgacaatgcc tatgagcact ttgtcattca
ggtgcaggag 4260 gtcaacaaag tggaggcagc ccagaacctc acgttgcctg
gcagcctcag ggctgtggac 4320 atcccgggcc tcgaggctgc cacgccttat
agagtctcca tctatggggt gatccggggc 4380 tatagaacac cagtactctc
tgctgaggcc tccacagcca aagaacctga aattggaaac 4440 ttaaatgttt
ctgacataac tcccgagagc ttcaatctct cctggatggc taccgatggg 4500
atcttcgaga cctttaccat tgaaattatt gattccaata ggttgctgga gactgtggaa
4560 tataatatct ctggtgctga acgaactgcc catatctcag ggctaccccc
tagtactgat 4620 tttattgtct acctctctgg acttgctccc agcatccgga
ccaaaaccat cagtgccaca 4680 gccacgacag aggccctgcc ccttctggaa
aacctaacca tttccgacat taatccctac 4740 gggttcacag tttcctggat
ggcatcggag aatgcctttg acagctttct agtaacggtg 4800 gtggattctg
ggaagctgct ggacccccag gaattcacac tttcaggaac ccagaggaag 4860
ctggagctta gaggcctcat aactggcatt ggctatgagg ttatggtctc tggcttcacc
4920 caagggcatc aaaccaagcc cttgagggct gagattgtta cagaagccga
accggaagtt 4980 gacaaccttc tggtttcaga tgccacccca gacggtttcc
gtctgtcctg gacagctgat 5040 gaaggggtct tcgacaattt tgttctcaaa
atcagagata ccaaaaagca gtctgagcca 5100 ctggaaataa ccctacttgc
ccccgaacgt accagggaca taacaggtct cagagaggct 5160 actgaatacg
aaattgaact ctatggaata agcaaaggaa ggcgatccca gacagtcagt 5220
gctatagcaa caacagccat gggctcccca aaggaagtca ttttctcaga catcactgaa
5280 aattcggcta ctgtcagctg gagggcaccc acggcccaag tggagagctt
ccggattacc 5340 tatgtgccca ttacaggagg tacaccctcc atggtaactg
tggacggaac caagactcag 5400 accaggctgg tgaaactcat acctggcgtg
gagtaccttg tcagcatcat cgccatgaag 5460 ggctttgagg aaagtgaacc
tgtctcaggg tcattcacca cagctctgga tggcccatct 5520 ggcctggtga
cagccaacat cactgactca gaagccttgg ccaggtggca gccagccatt 5580
gccactgtgg acagttatgt catctcctac acaggcgaga aagtgccaga aattacacgc
5640 acggtgtccg ggaacacagt ggagtatgct ctgaccgacc tcgagcctgc
cacggaatac 5700 acactgagaa tctttgcaga gaaagggccc cagaagagct
caaccatcac tgccaagttc 5760 acaacagacc tcgattctcc aagagacttg
actgctactg aggttcagtc ggaaactgcc 5820 ctccttacct ggcgaccccc
ccgggcatca gtcaccggtt acctgctggt ctatgaatca 5880 gtggatggca
cagtcaagga agtcattgtg ggtccagata ccacctccta cagcctggca 5940
gacctgagcc catccaccca ctacacagcc aagatccagg cactcaatgg gcccctgagg
6000 agcaatatga tccagaccat cttcaccaca attggactcc tgtacccctt
ccccaaggac 6060 tgctcccaag caatgctgaa tggagacacg acctctggcc
tctacaccat ttatctgaat 6120 ggtgataagg ctcaggcgct ggaagtcttc
tgtgacatga cctctgatgg gggtggatgg 6180 attgtgttcc tgagacgcaa
aaacggacgc gagaacttct accaaaactg gaaggcatat 6240 gctgctggat
ttggggaccg cagagaagaa ttctggcttg ggctggacaa cctgaacaaa 6300
atcacagccc aggggcagta cgagctccgg gtggacctgc gggaccatgg ggagacagcc
6360 tttgctgtct atgacaagtt cagcgtggga gatgccaaga ctcgctacaa
gctgaaggtg 6420 gaggggtaca gtgggacagc aggtgactcc atggcctacc
acaatggcag atccttctcc 6480 acctttgaca aggacacaga ttcagccatc
accaactgtg ctctgtctac aaggggcttc 6540 tggtacagga actgtcaccg
tgtcaacctg atggggagat atggggacaa taaccacagt 6600 cagggcgtta
actggttcca ctggaagggc cacgaacact caatccagtt tgctgagatg 6660
aagctgagac caagcaactt cagaaatctt gaaggcaggc gcaaacgggc ataaattgga
6720 gggaccactg ggtgagagag gaataaggcg gcccagagcg aggaaaggat
tttaccaaag 6780 catcaataca accagcccaa ccatcggtcc acacctgggc
atttggtgag aatcaaagct 6840 gaccatggat ccctggggcc aacggcaaca
gcatgggcct cacctcctct gtgatttctt 6900 tctttgcacc aaagacatca
gtctccaaca tgtttctgtt ttgttgtttg attcagcaaa 6960 aatctcccag
tgacaacatc gcaatagttt tttacttctc ttaggtggct ctgggatggg 7020
agaggggtag gatgtacagg ggtagtttgt tttagaacca gccgtatttt acatgaagct
7080 gtataattaa ttgtcattat ttttgttagc aaagattaaa tgtgtcattg
gaagccatcc 7140 ctttttttac atttcataca acagaaacca gaaaagcaat
actgtttcca ttttaaggat 7200 atgattaata ttattaatat aataatgatg
atgatgatga tgaaaactaa ggatttttca 7260 agagatcttt ctttccaaaa
catttctgga cagtacctga ttgtattttt tttttaaata 7320 aaagcacaag
tacttttgaa aaaaaa 7346 2 17 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 2 ggtggaggtg ggtttgg
17 3 17 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 3 ggcccccatg gtggagg 17 4 14 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 4 ggcccccatg gtgg 14 5 14 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 5
ccccatggtg gagg 14 6 17 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 6 aaggaaagca agaaaga
17 7 12 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 7 ccatggtgga gg 12 8 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 8 accttcggta gcgagg 16 9 15 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 9
gcgagggcaa ggaaa 15 10 15 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 10 ggtgggtttg gctgg
15 11 16 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 11 agttgcctgg acattc 16 12 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 12 tgtcgcttgt gccgg 15 13 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 13 ggtctttctc cccact 16 14 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 14 ggcacagcca cagg 14 15 14 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 15
ggtagcgagg gcaa 14 16 14 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 16 ctgctggggc tcta 14
17 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 17 aaggaaagca agaaa 15 18 11 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 18 ccccatggtg g 11 19 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 19
agtcatggcc c 11 20 13 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 20 caagaaagac acc 13
21 17 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 21 ggtggaggtg ggtttgg 17 22 17 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 22 ggcccccatg gtggagg 17 23 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 23 ggcccccatg gtgg 14 24 14 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 24
ccccatggtg gagg 14 25 17 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 25 aaggaaagca agaaaga
17 26 12 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 26 ccatggtgga gg 12 27 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 27 accttcggta gcgagg 16 28 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 28 gcgagggcaa ggaaa 15 29 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 29 ggtgggtttg gctgg 15 30 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 30 agttgcctgg acattc 16 31 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 31 tgtcgcttgt gccgg 15 32 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 32 ggtctttctc cccact 16 33 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 33 ggcacagcca cagg 14 34 14 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 34
ggtagcgagg gcaa 14 35 14 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 35 ctgctggggc tcta 14
36 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 36 aaggaaagca agaaa 15 37 11 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 37 ccccatggtg g 11 38 11 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 38
agtcatggcc c 11 39 13 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 39 caagaaagac acc 13
40 17 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 40 ggtggaggtg ggtttgg 17 41 17 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 41 ggcccccatg gtggagg 17 42 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 42 ggcccccatg gtgg 14 43 14 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 43
ccccatggtg gagg 14 44 17 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 44 aaggaaagca agaaaga
17 45 12 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 45 ccatggtgga gg 12 46 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 46 accttcggta gcgagg 16 47 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 47 gcgagggcaa ggaaa 15 48 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 48 ggtgggtttg gctgg 15 49 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 49 agttgcctgg acattc 16 50 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 50 tgtcgcttgt gccgg 15 51 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 51 ggtctttctc cccact 16 52 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 52 ggcacagcca cagg 14 53 14 DNA Artificial Sequence
Description of Artificial Sequence
Synthetic oligonucleotide 53 ggtagcgagg gcaa 14 54 14 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 54 ctgctggggc tcta 14 55 15 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 55
aaggaaagca agaaa 15 56 11 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 56 ccccatggtg g 11 57
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 57 agtcatggcc c 11 58 13 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 58 caagaaagac acc 13
* * * * *