CA2390674A1 - Antisense oligonucleotides for the inhibition of expression of type i procollagen - Google Patents
Antisense oligonucleotides for the inhibition of expression of type i procollagen Download PDFInfo
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- CA2390674A1 CA2390674A1 CA002390674A CA2390674A CA2390674A1 CA 2390674 A1 CA2390674 A1 CA 2390674A1 CA 002390674 A CA002390674 A CA 002390674A CA 2390674 A CA2390674 A CA 2390674A CA 2390674 A1 CA2390674 A1 CA 2390674A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Abstract
The invention provides antisense DNA oligonucleotides which are effective in inhibiting the expression of a wild type COL1A1 gene.
Description
ANTISENSE OLIGONUCLEOTIDES
The present invention relates to antisense oligonucleotides and their use in inhibiting expression of type I procollagen.
The collagens are a family of closely related proteins, with a triple helix protein structure.
Numerous collagen types have been identified (>10) of which type I procollagen (consisting of two alphal chains and one alpha2 chain) is the principal component of bone, skin, and tendon.
io It has been recognised for many years that many pathological conditions are caused by overproduction of collagen fibres in the form of scars and excess fibrous tissues. For example, liver cirrhosis is a two-step process in which normal liver tissue is first destroyed by a virus or by alcohol and other toxins, and then excessive amounts of collagen fibres ~s replace the damaged cells before normal liver cell regeneration. Idiopathic pulmonary fibrosis is a lethal condition in which normal lung tissue is gradually replaced by excessive amounts of collagen fibres. Progressive systemic sclerosis (scleroderma) is a frequently lethal disease where skin and many internal organs become leather-like because of excessive depositions of collagen fibres. In many individuals, wounds or surgical incisions 2o in the skin are followed by excessive depositions of collagen in the form of hypertrophic scars and keloids that present cosmetic problems and sometimes more serious consequences. Also, excessive scarring frequently occurs in normal individuals following trauma and surgical procedures. In these and related conditions, a means of specifically inhibiting collagen synthesis and deposition would be of tremendous benefit.
PCT Patent Application Publication No. WO 94/11494 discloses a DNA or RNA
oligonucleotide comprising from 5 to 200 nucleotides substantially complementary to a mutant collagen nucleotide sequence or a normal wild type collagen nucleotide sequence which is capable of inhibiting collagen gene expression. Preferred oligonucleotides are 3o said to be antisense oligonucleotides. The Examples of WO 94/11494 describe a series of DNA oligonucleotides, some of which are antisense, that were synthesised primarily with regard to the region at the 3' end of exon 1 (from nucleotides 198 to 222) and the first two nucleotides of intron 1 of the human gene for the proal chains of type I
procollagen (COL1A1). The synthesised oligonucleotides were found to vary considerably in their s ability to inhibit expression of an internally deleted mutant COLlAl gene of human origin.
The effectiveness of the oligonucleotides in inhibiting the expression of the human wild type COLlAI gene was not however demonstrated. Since the structure and conformation of the RNA transcripts of the human, mutant and wild type COL1A1 genes would most likely differ, it would not necessarily follow that oligonucleotides which are effective io inhibitors of the expression of the mutant COLIAl gene would also be effective inhibitors of the expression of the wild type COL1A1 gene.
It would be desirable to identify antisense DNA oligonucleotides that are capable of inhibiting the expression of a wild type COL1A1 gene.
is In accordance with the present invention, there is therefore provided an antisense DNA
oligonucleotide comprising from 18 to 25 nucleotides which is complementary to a nucleotide sequence from position 750 to position 3900 inclusive of SEQ ID
NO:1, wherein SEQ ID NO:l comprises a nucleotide sequence encoding a polypeptide zo comprising an amino acid sequence according to SEQ ID N0:2, the oligonucleotide being capable of inhibiting expression of the polypeptide in a cell that expresses it.
SEQ ID NO:1 is identical to the nucleotide sequence registered under EMBL
accession no.
274615. SEQ ID N0:2 is the amino acid sequence of the polypeptide encoded by the is nucleotide sequence of SEQ ID NO:I. The polypeptide encoded by SEQ ID NO:1 is a precursor of the wild type, proal chain of type I procollagen ("prepro-alphal (I) collagen").
The antisense DNA oligonucleotide according to the invention comprises 18, 19, 20, 21, 30 22, 23, 24 or 25 nucleotides and is preferably 20 nucleotides in length.
The present invention relates to antisense oligonucleotides and their use in inhibiting expression of type I procollagen.
The collagens are a family of closely related proteins, with a triple helix protein structure.
Numerous collagen types have been identified (>10) of which type I procollagen (consisting of two alphal chains and one alpha2 chain) is the principal component of bone, skin, and tendon.
io It has been recognised for many years that many pathological conditions are caused by overproduction of collagen fibres in the form of scars and excess fibrous tissues. For example, liver cirrhosis is a two-step process in which normal liver tissue is first destroyed by a virus or by alcohol and other toxins, and then excessive amounts of collagen fibres ~s replace the damaged cells before normal liver cell regeneration. Idiopathic pulmonary fibrosis is a lethal condition in which normal lung tissue is gradually replaced by excessive amounts of collagen fibres. Progressive systemic sclerosis (scleroderma) is a frequently lethal disease where skin and many internal organs become leather-like because of excessive depositions of collagen fibres. In many individuals, wounds or surgical incisions 2o in the skin are followed by excessive depositions of collagen in the form of hypertrophic scars and keloids that present cosmetic problems and sometimes more serious consequences. Also, excessive scarring frequently occurs in normal individuals following trauma and surgical procedures. In these and related conditions, a means of specifically inhibiting collagen synthesis and deposition would be of tremendous benefit.
PCT Patent Application Publication No. WO 94/11494 discloses a DNA or RNA
oligonucleotide comprising from 5 to 200 nucleotides substantially complementary to a mutant collagen nucleotide sequence or a normal wild type collagen nucleotide sequence which is capable of inhibiting collagen gene expression. Preferred oligonucleotides are 3o said to be antisense oligonucleotides. The Examples of WO 94/11494 describe a series of DNA oligonucleotides, some of which are antisense, that were synthesised primarily with regard to the region at the 3' end of exon 1 (from nucleotides 198 to 222) and the first two nucleotides of intron 1 of the human gene for the proal chains of type I
procollagen (COL1A1). The synthesised oligonucleotides were found to vary considerably in their s ability to inhibit expression of an internally deleted mutant COLlAl gene of human origin.
The effectiveness of the oligonucleotides in inhibiting the expression of the human wild type COLlAI gene was not however demonstrated. Since the structure and conformation of the RNA transcripts of the human, mutant and wild type COL1A1 genes would most likely differ, it would not necessarily follow that oligonucleotides which are effective io inhibitors of the expression of the mutant COLIAl gene would also be effective inhibitors of the expression of the wild type COL1A1 gene.
It would be desirable to identify antisense DNA oligonucleotides that are capable of inhibiting the expression of a wild type COL1A1 gene.
is In accordance with the present invention, there is therefore provided an antisense DNA
oligonucleotide comprising from 18 to 25 nucleotides which is complementary to a nucleotide sequence from position 750 to position 3900 inclusive of SEQ ID
NO:1, wherein SEQ ID NO:l comprises a nucleotide sequence encoding a polypeptide zo comprising an amino acid sequence according to SEQ ID N0:2, the oligonucleotide being capable of inhibiting expression of the polypeptide in a cell that expresses it.
SEQ ID NO:1 is identical to the nucleotide sequence registered under EMBL
accession no.
274615. SEQ ID N0:2 is the amino acid sequence of the polypeptide encoded by the is nucleotide sequence of SEQ ID NO:I. The polypeptide encoded by SEQ ID NO:1 is a precursor of the wild type, proal chain of type I procollagen ("prepro-alphal (I) collagen").
The antisense DNA oligonucleotide according to the invention comprises 18, 19, 20, 21, 30 22, 23, 24 or 25 nucleotides and is preferably 20 nucleotides in length.
The antisense DNA oligonucleotide is preferably complementary to a nucleotide sequence in one of the following regions of SEQ ID NO:1, s Region 1 - from position 750 to position 900 inclusive, Region from position 1200 to position 1300 2 - inclusive, Region from position 1400 to position 1500 3 - inclusive, Region from position 1450 to position 1550 4 - inclusive, Region 5 - from position 1850 to position 2000 inclusive, io Region 6 - from position 2500 to position 2600 inclusive, Region 7 - from position 2850 to position 2950 inclusive, Region 8 - from position 3800 to position 3900 inclusive.
Particularly preferred antisense DNA oligonucleotides are those which are complementary is to a nucleotide sequence in Region 2, 4, 6 or 8 of SEQ ID NO:1.
The oligonucleotides of the invention may be prepared by any suitable method known in the art. The oligonucleotides are very conveniently prepared by synthetic chemical methods, for example, phosphoramidite chemistry by sulfurization with tetraethylthiuram zo disulfide in acetonitrile as described in Tetrahedron Lett., 1991, 32, 30005-30008.
The oligonucleotides of the present invention are advantageous in that they inhibit expression of the wild type COLlAl gene. They are therefore useful in the treatment or prevention of conditions/disorders caused by overproduction of collagen fibres, for zs example, liver cirrhosis, kidney, liver and heart fibrosis, scleroderma, hypertrophic scars and keloids.
Accordingly, the present invention provides an antisense DNA oligonucleotide according to the invention for use in therapy.
Particularly preferred antisense DNA oligonucleotides are those which are complementary is to a nucleotide sequence in Region 2, 4, 6 or 8 of SEQ ID NO:1.
The oligonucleotides of the invention may be prepared by any suitable method known in the art. The oligonucleotides are very conveniently prepared by synthetic chemical methods, for example, phosphoramidite chemistry by sulfurization with tetraethylthiuram zo disulfide in acetonitrile as described in Tetrahedron Lett., 1991, 32, 30005-30008.
The oligonucleotides of the present invention are advantageous in that they inhibit expression of the wild type COLlAl gene. They are therefore useful in the treatment or prevention of conditions/disorders caused by overproduction of collagen fibres, for zs example, liver cirrhosis, kidney, liver and heart fibrosis, scleroderma, hypertrophic scars and keloids.
Accordingly, the present invention provides an antisense DNA oligonucleotide according to the invention for use in therapy.
In another aspect, the invention provides the use of an antisense DNA
oligonucleotide according to the invention in the manufacture of a medicament for use in therapy.
In the context of the present specification, the term "therapy" also includes "prophylaxis"
unless there are specific indications to the contrary. The terms "therapeutic"
and "therapeutically" should be construed accordingly.
The invention further provides a method of treating, or reducing the risk of, a collagen disorder in a patient suffering from, or at risk of, the disorder, which comprises io administering to the patient a therapeutically effective amount of an antisense DNA
oligonucleotide according to the invention.
For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the oligonucleotide employed, the mode of administration, the treatment desired and is the disorder indicated. Effective dosages are those which are able to inhibit collagen protein production in cells at a level which eliminates or reduces the symptoms or conditions associated with the collagen protein production.
The oligonucleotides according to the invention will generally be administered in the form Zo of a pharmaceutical composition in which the oligonucleotide is formulated with a pharmaceutically acceptable adjuvant, diluent or carrier.
Thus, the present invention also provides a pharmaceutical composition comprising an antisense DNA oligonucleotide according ao the invention in association with a 2s pharmaceutically acceptable adjuvant, diluent or Garner.
The invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing the antisense DNA
oligonucleotide with a pharmaceutically acceptable adjuvant, diluent or carrier.
S
The pharmaceutical composition of the invention may be administered topically in the form of, for example, a creme, lotir.~n or ointment; or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules, or by parenteral administration in the form of sterile solutions or suspensions.
The present invention will now be further explained by reference to the following illustrative Examples.
EXAMPLES
io Example 1 Oligonucleotide synthesis Phosphorothioate oligodeoxynucleotides synthesis was carried out at a 1 p.m scale on PE
~s Biosystems 394 DNA synthesizer using phosphoramidite chemistry with TETD/
acetonitrile sulphurizing reagent. Oligonucleotides were purified on Poly-Pak TM II
cartridges (Glen Research), desalted on NAPTM 10 columns (Amersham Pharmacia Biotech AB) and ion-exchanged using Dowex SOWXB-100 ion exchange resin (Aldrich).
Twelve antisense DNA oligonucleotides (ASOs) were prepared having the following zo sequences (5' -~ 3 ~:
1. GGACGACCAGGTT"ITCCAGC (SEQ ID N0:3) 2. GCAGCACCAGCAGGGCCAGG (SEQ ID N0:4) 3. GCCAGGAGCACCAGGTTCAC (SEQ ID NO:S) zs CTTCCTCTCCAGCAGGGCCA (SEQ ID N0:6) 4.
5. GCCTTGCCGGGCTCTCCAGC (SEQ ID N0:7) 6. CGGGAACACCTCGCTCTCCA (SEQ ID N0:8) 7. GCAGGACCGACAGCGCCAGG (SEQ ID N0:9) 8. TCCATCTTTGCCAGCAGGAC (SEQ ID NO:10) 30 GGTCCCTGAGCTCCAGCCTC (SEQ ID NO:11 9. ) 10. TTGGCCGTCAGCACCAGGG (SEQ ID N0:12) 11. TTTCTCGCCAGCAGGGCCAG (SEQ ID N0:13) 12. CTCGATCTGCTGGCTCAGGC (SEQ ID N0:14) s Example 2 Treatment of cells The human cell line WI-26 was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. The cells were plated in 48-well plates or 6-well plates io (Costar, Corning Inc.) to obtain 70-80% confluence. After 24 hours, the cells were washed two times with pre-warmed DMEM and 0.35m1 (for 48-well experiments) or lml (6-well experiments) DMEM containing Spg/ml lipofectin (Gibco BRL) or 2.Sp,g/ml cytofectin GSV (Glen Research Ltd) and oligonucleotides at 200nM were added to each well.
After 4-5 hours at 37°C the cells were washed two times with pre-warmed DMEM
and 0.35m1 is DMEM (48-well plates) or lml DMEM (6-well plates) was added together with ascorbic acid at 20ug/ml. The cells were incubated for 20 hours prior to analysis of collagen levels.
Example 3 Protein analysis Zo At the end of the experiment, 150p1 of medium was removed and the amount of secreted type I procollagen determined using an ELISA kit (AmershamPharmacia Ltd) and the results expressed as nanograms of procollagen in the medium/10,000 cells. To correct for cell numbers, plates were washed with pre-warmed PBS, cells treated with trypsin and cell is numbers determined using an automated Coulter counter. For 6-well experiments, the cells were counted, treated with lml TRI reagent (SIGMA Ltd) and proteins and RNA
extracted according to the manufacturers guidelines. The protein pellet was re-suspended in 1%
SDS containing protease inhibitors. 30-100ugs cellular proteins were heated at 100°C for 5 mins and then lectrophoresed in a 4-12% SDS polyacrylamide gel. Proteins were 3o electrophoretically transferred to nitrocellulose filters and hybridised with an antibody against a synthetic peptide corresponding to human proal (I) chain of type 1 collagen (obtained from Dr Larry Fisher, NIH, USA). The proal ( 1 ) band was detected using an anti-rabbit secondary antibody coupled to HRP (Biorad Ltd) and developed using ECL
(Pierce Ltd). Protein loading was determined by treating the membrane with an antibody to GAPDH (Advanced Immunochemicals). Protein loading was normalised to GAPDH
levels using desitometry.
Example 4 RNA analysis io RNA was extracted using TRI reagent and the final pellet was re-suspended in 0.5% SDS.
One to three micrograms of total RNA were electrophoresed in a formaldehyde denaturing gel according to standard procedures (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ~s Cold Spring Harbor, New York). RNA was transferred to Hybond-N membranes (Amersham) and hybridised for 24 hours to an alphal(1) cDNA probe radiolabeled using a T7 polymerase kit (AmershamPharmacia). Following washing, the filter was exposed to X-ray film and the film developed 4-24 hours later. The autoradiographic images of the alphal(1) transcripts (4.8kb & 5.8kb) were analysed by densitometric analysis and RNA
Zo loading was corrected using the intensity of the GAPDH transcript or the intensity of the 28S rRNA as internal controls.
Results zs Table I below shows the average percentage (%) collagen inhibition which relates to either collagen levels in the medium or collagen mRNA levels. In the treated cell assay used, there was a very good correlation between percentage collagen inhibition as measured in the medium and percentage inhibition of intracellular collagen mRNA levels.
Table I
ASO AVERAGE % COLLAGEN
INHIBITION
(SEQ ID N0:3) (SEQ ID N0:4) (SE ID NO:S) (SEQ ID N0:6) (SE ID N0:7) (SE ID N0:8) (SEQ ID N0:9) (SEQ ID NO:10) (SEQ ID NO:11 ) (SEQ ID N0:12) (SEQ ID N0:13) (SEQ ID N0:14) SEQUENCE LISTING
<110> AstraZeneca AB
<120> ANTISENSE OLIGONUCLEOTIDES
<130> 270611 <140>
<141>
<160> 14 <170> PatentIn Ver. 2.1 <210> 1 <211> 6728 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (120)..(4511) <220>
<221> sig~eptide <222> (120)..(185) <300>
<308> EMBL/Z74615 <309> 1996-07-O1 <400> 1 agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc 60 atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagac 119 atg ttc agc ttt gtg gac ctc cgg ctc ctg ctc ctc tta gcg gcc acc 167 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr gcc ctc ctg acg cac ggc caa gag gaa ggc caa gtc gag ggc caa gac 215 Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp gaa gac atc cca cca atc acc tgc gta cag aac ggc ctc agg tac cat 263 Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His gac cga gac gtg tgg aaa ccc gag ccc tuc cgg atc tgc gtc tgc gac 311 Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp aac ggc aag gtg ttg tgc gat gac gtg atc tgt gac gag acc aag aac 359 Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn tgc ccc ggc gcc gaa gtc ccc gag ggc gag tgc tgt ccc gtc tgc ccc 407 Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro gac ggc tca gag tca ccc acc gac caa gaa acc acc ggc gtc gag gga 455 Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly ccc aag gga gac act ggc ccc cga ggc cca agg gga ccc gca ggc ccc 503 Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro cct ggc cga gat ggc atc cct gga cag cct gga ctt ccc gga ccc ccc 551 Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro gga ccc ccc gga cct ccc gga ccc cct ggc ctc gga gga aac ttt get 599 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala ccc cag ctg tct tat ggc tat gat gag aaa tca acc gga gga att tcc 647 Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser gtg cct ggc ccc atg ggt ccc tct ggt cct cgt ggt ctc cct ggc ccc 695 Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro cct ggt gca cct ggt ccc caa ggc ttc caa ggt ccc cct ggt gag cct 743 Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro ggc gag cct gga get tca ggt ccc atg ggt ccc cga ggt ccc cca ggt 791 Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly ccc cct gga aag aat gga gat gat ggg gaa get gga aaa cct ggt cgt 839 Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg cct ggt gag cgt ggg cct cct ggg cct cag ggt get cga gga ttg ccc 887 Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro gga aca get ggc ctc cct gga atg aag gga cac aga ggt ttc agt ggt 935 Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly ttg gat ggt gcc aag gga gat get ggt cct get ggt cct aag ggt gag 983 Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu cct ggc agc cct ggt gaa aat gga get cct ggt cag atg ggc ccc cgt 1031 Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg ggc ctg cct ggt gag aga ggt cgc cct gga gcc cct ggc cct get ggt 1079 Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly get cgt gga aat gat ggt get act ggt get gcc ggg ccc cct ggt ccc 1127 Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro acc ggc ccc get ggt cct cct ggc ttc cct ggt get gtt ggt get aag 1175 Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys ggt gaa get ggt ccc caa ggg ccc cga ggc tct gaa ggt ccc cag ggt 1223 Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly gtg cgt ggt gag cct ggc ccc cct ggc cct get ggt get get ggc cct 1271 Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro get gga aac cct ggt get gat gga cag cct ggt get aaa ggt gcc aat 1319 Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn ggt get cct ggt att get ggt get cct ggc ttc cct ggt gcc cga ggc 1367 Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly ccc tct gga ccc cag ggc ccc ggc ggc cct cct ggt ccc aag ggt aac 1415 Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn agc ggt gaa cct ggt get cct ggc agc aaa gga gac act ggt get aag 1463 Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys gga gag cct ggc cct gtt ggt gtt caa gga ccc cct ggc cct get gga 1511 Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly gag gaa gga aag cga gga get cga ggt gaa ccc gga ccc act ggc ctg 1559 Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu ccc gga ccc cct ggc gag cgt ggt gga cct ggt agc cgt ggt ttc cct 1607 Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro ggc gca gat ggt gtt get ggt ccc aag ggt ccc get ggt gaa cgt ggt 1655 Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly tct cct ggc ccc get ggc ccc aaa gga tct cct ggt gaa get ggt cgt 1703 Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg ccc ggt gaa get ggt ctg cct ggt gcc aag ggt ctg act gga agc cct 1751 Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro ggc agc cct ggt cct gat ggc aaa act ggc ccc cct ggt ccc gcc ggt 1799 Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly caa gat ggt cgc ccc gga ccc cca ggc cca cct ggt gcc cgt ggt cag 1847 Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln get ggt gtg atg gga ttc cct gga cct aaa ggt get get gga gag ccc 1895 Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro ggc aag get gga gag cga ggt gtt ccc gga ccc cct ggc get gtc ggt 1943 Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly cct get ggc aaa gat gga gag get gga get cag gga ccc cct ggc cct 1991 Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro get ggt ccc get ggc gag aga ggt gaa caa ggc cct get ggc tcc ccc 2039 Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro gga ttc cag ggt ctc cct ggt cct get ggt cct cca ggt gaa gca ggc 2087 Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly aaa cct ggt gaa cag ggt gtt cct gga gac ctt ggc gcc cct ggc ccc 2135 Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro tct gga gca aga ggc gag aga ggt ttc cct ggc gag cgt ggt gtg caa 2183 Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln ggt ccc cct ggt cct get gga ccc cga ggg gcc aac ggt get ccc ggc 2231 Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly aac gat ggt get aag ggt gat get ggt gcc cct gga get ccc ggt agc 2279 Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser cag ggc gcc cct ggc ctt cag gga atg cct ggt gaa cgt ggt gca get 2327 Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala ggt ctt cca ggg cct aag ggt gac aga ggt gat get ggt ccc aaa ggt 2375 Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly get gat ggc tct cct ggc aaa gat ggc gtc cgt ggt ctg acc ggc ccc 2423 Ala Asp G1y Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro att ggt cct cct ggc cct get ggt gcc cct ggt gac aag ggt gaa agt 2471 Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser ggt ccc agc ggc cct get ggt ccc act gga get cgt ggt gcc ccc gga 2519 Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly gac cgt ggt gag cct ggt ccc ccc ggc cct get ggc ttt get ggc ccc 2567 Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro cct ggt get gac ggc caa cct ~:~gt get aaa ggc gaa cct ggt gat get 2615 Pro Gly Ala Asp Gly Gln Pro ~ 1y Ala Lys Gly Glu Pro Gly Asp Ala ggt gcc aaa ggc gat get ggt ccc cct ggg cct gcc gga ccc get gga 2663 Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly ccc cct ggc ccc att ggt aat gtt ggt get cct gga gcc aaa ggt get 2711 Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala cgc ggc agc get ggt ccc cct ggt get act ggt ttc cct ggt get get 2759 Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala ggc cga gtc ggt cct cct ggc ccc tct gga aat get gga ccc cct ggc 2807 Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly cct cct ggt cct get ggc aaa gaa ggc ggc aaa ggt ccc cgt ggt gag 2855 Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu act ggc cct get gga cgt cct ggt gaa gtt ggt ccc cct ggt ccc cct 2903 Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro ggc cct get ggc gag aaa gga tcc cct ggt get gat ggt cct get ggt 2951 Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly get cct ggt act ccc ggg cct caa ggt att get gga cag cgt ggt gtg 2999 Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val gtc ggc ctg cct ggt cag aga gga gag aga ggc ttc cct ggt ctt cct 3047 Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro ggc ccc tct ggt gaa cct ggc aaa caa ggt ccc tct gga gca agt ggt 3095 Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly gaa cgt ggt ccc ccc ggt ccc atg ggc ccc cct gga ttg get gga ccc 3143 Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro cct ggt gaa tct gga cgt gag ggg get cct get gcc gaa ggt tcc cct 3191 Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro gga cga gac ggt tct cct ggc gcc aag ggt gac cgt ggt gag acc ggc 3239 Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly ccc get gga ccc cct ggt get cct ggt get cct ggt gcc cct ggc ccc 3287 Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro gtt ggc cct get ggc aag agt ggt gat cgt ggt gag act ggt cct get 3335 Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala ggt ccc gcc ggt ccc gtc ggc ccc gtc ggc gcc cgt ggc ccc gcc gga 3383 Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly ccc caa ggc ccc cgt ggt gac aag ggt gag aca ggc gaa cag ggc gac 3431 Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp aga ggc ata aag ggt cac cgt ggc ttc tct ggc ctc cag ggt ccc cct 3479 Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro ggc cct cct ggc tct cct ggt gaa caa ggt ccc tct gga gcc tct ggt 3527 Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly cct get ggt ccc cga ggt ccc cct ggc tct get ggt get cct ggc aaa 3575 Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys gat gga ctc aac ggt ctc cct ggc ccc att ggg ccc cct ggt cct cgc 3623 Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg ggt cgc act ggt gat get ggt cct gtt ggt ccc ccc ggc cct cct gga 3671 Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly cct cct ggt ccc cct ggt cct ccc agc get ggt ttc gac ttc agc ttc 3719 Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe ctg ccc cag cca cct caa gag aag get cac gat ggt ggc cgc tac tac 3767 Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr cgg get gat gat gcc aat gtg gtt cgt gac cgt gac ctc gag gtg gac 3815 Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp acc acc ctc aag agc ctg agc cag cag atc gag aac atc cgg agc cca 3863 Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro gag gga agc cgc aag aac ccc gcc cgc acc tgc cgt gac ctc aag atg 3911 Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met tgc cac tct gac tgg aag agt gga gag tac tgg att gac ccc aac caa 3959 Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln ggc tgc aac ctg gat gcc atc aaa gtc ttc tgc aac atg gag act ggt 4007 Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly gag acc tgc gtg tac ccc act cag ccc agt gtg gcc cag aag aac tgg 4055 Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp tac atc agc aag aac ccc aag gac aag agg cat gtc tgg ttc ggc gag 4103 Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu agc atg acc gat gga ttc cag ttc gag tat ggc ggc cag ggc tcc gac 4151 Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp cct gcc gat gtg gcc atc cag ctg acc ttc ctg cgc ctg atg tcc acc 4199 Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr gag gcc tcc cag aac atc acc tac cac tgc aag aac agc gtg gcc tac 4247 Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr atg gac cag cag act ggc aac ctc aag aag gcc ctg ctc ctc aag ggc 4295 Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly tcc aac gag atc gag atc cgc gcc gag ggc aac agc cgc ttc acc tac 4343 Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr agc gtc act gtc gat ggc tgc acg agt cac acc gga gcc tgg ggc aag 4391 Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys aca gtg att gaa tac aaa acc acc aag tcc tcc cgc ctg ccc atc atc 4439 Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile gat gtg gcc ccc ttg gac gtt ggt gcc cca gac cag gaa ttc ggc ttc 4487 Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe gac gtt ggc cct gtc tgc ttc ctg taaactccct ccatcccaac ctggctccct 4541 Asp Val Gly Pro Val Cys Phe Leu cccacccaac caactttccc cccaacccgg aaacagacaa gcaacccaaa ctgaaccccc 4601 ccaaaagcca aaaaatggga gacaatttca catggacttt ggaaaatatt tttttccttt 4661 gcattcatct ctcaaactta gtttttatct ttgaccaacc gaacatgacc aaaaaccaaa 4721 agtgcattca accttaccaa aaaaaaaaaa aaaaaaaaaa gaataaataa ataagttttt 4781 aaaaaaggaa gcttggtcca cttgcttgaa gacccatgcg ggggtaagtc cctttctgcc 4841 cgttgggtta tgaaacccca atgctgccct ttctgctcct ttctccacac cccccttggc 4901 ctcccctcca ctccttccca aatctgtctc cccagaagac acaggaaaca atgtattgtc 4961 tgcccagcaa tcaaaggcaa tgctcaaaca cccaagtggc ccccaccctc agcccgctcc 5021 tgcccgccca gcacccccag gccctgggga cctggggttc tcagactgcc aaagaagcct 5081 tgccatctgg cgctcccatg gctcttgcaa catctcccct tcgtttttga gggggtcatg 5141 ccgggggagc caccagcccc tcactgggtt cggaggagag tcaggaaggg ccacgacaaa 5201 gcagaaacat cggatttggg gaacgcgtgt catcccttgt gccgcaggct gggcgggaga 5261 gactgttctg ttctgttcct tgtgtaactg tgttgctgaa agactacctc gttcttgtct 5321 tgatgtgtca ccggggcaac tgcctggggg cggggatggg ggcagggtgg aagcggctcc 5381 ccatttttat accaaaggtg ctacatctat gtgG.tgggtg gggtggggag ggaatcactg 5441 gtgctataga aattgagatg cccccccagg ccagcaaatg ttcctttttg ttcaaagtct 5501 atttttattc cttgatattt tttctttctt tttttttttt tttgtggatg gggacttgtg 5561 aatttttcta aaggtgctat ttaacatggg aggagagcgt gtgcgctcca gcccagcccg 5621 ctgctcactt tccaccctct ctccacctgc ctctggcttc tcaggcctct gctctccgac 5681 ctctctcctc tgaaaccctc ctccacagct gcagcccatc ctcccggctc cctcctagtc 5741 tgtcctgcgt cctctgtccc cgggtttcag agacaacttc ccaaagcaca aagcagtttt 5801 tccctagggg tgggaggaag caaaagactc tgtacctatt ttgtatgtgt ataataattt 5861 gagatgtttt taattatttt gattgctgga ataaagcatg tggaaatgac ccaaacataa 5921 tccgcagtgg cctcctaatt tccttctttg gagttggggg aggggtagac atggggaagg 5981 ggccttgggg tgatgggctt gccttccatt cctgcccttt ccctccccac tattctcttc 6041 tagatccctc cataacccca ctcccctttc tctcaccctt cttataccgc aaacctttct 6101 acttcctctt tcattttcta ttcttgcaat ttccttgcac cttttccaaa tcctcttctc 6161 ccctgcaata ccatacaggc aatccacgtg cacaacacac acacacactc ttcacatctg 6221 gggttgtcca aacctcatac ccactcccct tcaagcccat ccactctcca ccccctggat 6281 gccctgcact tggtggcggt gggatgctca tggatactgg gagggtgagg ggagtggaac 6341 ccgtgaggag gacctggggg cctctccttg aactgacatg aagggtcate tggcctctgc 6401 tcccttctca cccacgctga cctcctgccg aaggagcaac gcaacaggag aggggtctgc 6461 tgagcctggc gagggtctgg gagggaccag gaggaaggcg tgctccctgc tcgctgtcct 6521 ggccctgggg gagtgaggga gacagacacc tgggagagct gtggggaagg cactcgcacc 6581 gtgctcttgg gaaggaagga gacctggccc tgctcaccac ggactgggtg cctcgacctc 6641 ctgaatcccc agaacacaac ccccctgggc tggggtggtc tggggaacca tcgtgccccc 6701 gcctcccgcc tactcctttt taagctt 6728 <210> 2 <211> 1464 <212> PRT
<213> Homo Sapiens <400> 2 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly G1u Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu <210> 3 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 3 ggacgaccag gttttccagc 20 <210> 4 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 4 gcagcaccag cagggccagg 20 <210> 5 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 5 gccaggagca ccaggttcac 20 <210> 6 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 6 cttcctctcc agcagggcca 20 <210> 7 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 7 gccttgccgg gctctccagc 20 <210> 8 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 8 cgggaacacc tcgctctcca 20 <210>9 <211>20 <212>DNA
<213>Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 9 gcaggaccga cagcgccagg 20 <210> 10 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 10 tccatctttg ccagcaggac <210> 11 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 11 ggtccctgag ctccagcctc 20 <210> 12 <211> 19 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 12 ttggccgtca gcaccaggg <210> 13 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 13 tttctcgcca gcagggccag 20 <210>14 <211>20 <212>DNA
<213>Homo sapiens <220>
<223> Antisense oligonucleotide <400> 14 ctcgatctgc tggctcaggc 20
oligonucleotide according to the invention in the manufacture of a medicament for use in therapy.
In the context of the present specification, the term "therapy" also includes "prophylaxis"
unless there are specific indications to the contrary. The terms "therapeutic"
and "therapeutically" should be construed accordingly.
The invention further provides a method of treating, or reducing the risk of, a collagen disorder in a patient suffering from, or at risk of, the disorder, which comprises io administering to the patient a therapeutically effective amount of an antisense DNA
oligonucleotide according to the invention.
For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the oligonucleotide employed, the mode of administration, the treatment desired and is the disorder indicated. Effective dosages are those which are able to inhibit collagen protein production in cells at a level which eliminates or reduces the symptoms or conditions associated with the collagen protein production.
The oligonucleotides according to the invention will generally be administered in the form Zo of a pharmaceutical composition in which the oligonucleotide is formulated with a pharmaceutically acceptable adjuvant, diluent or carrier.
Thus, the present invention also provides a pharmaceutical composition comprising an antisense DNA oligonucleotide according ao the invention in association with a 2s pharmaceutically acceptable adjuvant, diluent or Garner.
The invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing the antisense DNA
oligonucleotide with a pharmaceutically acceptable adjuvant, diluent or carrier.
S
The pharmaceutical composition of the invention may be administered topically in the form of, for example, a creme, lotir.~n or ointment; or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules, or by parenteral administration in the form of sterile solutions or suspensions.
The present invention will now be further explained by reference to the following illustrative Examples.
EXAMPLES
io Example 1 Oligonucleotide synthesis Phosphorothioate oligodeoxynucleotides synthesis was carried out at a 1 p.m scale on PE
~s Biosystems 394 DNA synthesizer using phosphoramidite chemistry with TETD/
acetonitrile sulphurizing reagent. Oligonucleotides were purified on Poly-Pak TM II
cartridges (Glen Research), desalted on NAPTM 10 columns (Amersham Pharmacia Biotech AB) and ion-exchanged using Dowex SOWXB-100 ion exchange resin (Aldrich).
Twelve antisense DNA oligonucleotides (ASOs) were prepared having the following zo sequences (5' -~ 3 ~:
1. GGACGACCAGGTT"ITCCAGC (SEQ ID N0:3) 2. GCAGCACCAGCAGGGCCAGG (SEQ ID N0:4) 3. GCCAGGAGCACCAGGTTCAC (SEQ ID NO:S) zs CTTCCTCTCCAGCAGGGCCA (SEQ ID N0:6) 4.
5. GCCTTGCCGGGCTCTCCAGC (SEQ ID N0:7) 6. CGGGAACACCTCGCTCTCCA (SEQ ID N0:8) 7. GCAGGACCGACAGCGCCAGG (SEQ ID N0:9) 8. TCCATCTTTGCCAGCAGGAC (SEQ ID NO:10) 30 GGTCCCTGAGCTCCAGCCTC (SEQ ID NO:11 9. ) 10. TTGGCCGTCAGCACCAGGG (SEQ ID N0:12) 11. TTTCTCGCCAGCAGGGCCAG (SEQ ID N0:13) 12. CTCGATCTGCTGGCTCAGGC (SEQ ID N0:14) s Example 2 Treatment of cells The human cell line WI-26 was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. The cells were plated in 48-well plates or 6-well plates io (Costar, Corning Inc.) to obtain 70-80% confluence. After 24 hours, the cells were washed two times with pre-warmed DMEM and 0.35m1 (for 48-well experiments) or lml (6-well experiments) DMEM containing Spg/ml lipofectin (Gibco BRL) or 2.Sp,g/ml cytofectin GSV (Glen Research Ltd) and oligonucleotides at 200nM were added to each well.
After 4-5 hours at 37°C the cells were washed two times with pre-warmed DMEM
and 0.35m1 is DMEM (48-well plates) or lml DMEM (6-well plates) was added together with ascorbic acid at 20ug/ml. The cells were incubated for 20 hours prior to analysis of collagen levels.
Example 3 Protein analysis Zo At the end of the experiment, 150p1 of medium was removed and the amount of secreted type I procollagen determined using an ELISA kit (AmershamPharmacia Ltd) and the results expressed as nanograms of procollagen in the medium/10,000 cells. To correct for cell numbers, plates were washed with pre-warmed PBS, cells treated with trypsin and cell is numbers determined using an automated Coulter counter. For 6-well experiments, the cells were counted, treated with lml TRI reagent (SIGMA Ltd) and proteins and RNA
extracted according to the manufacturers guidelines. The protein pellet was re-suspended in 1%
SDS containing protease inhibitors. 30-100ugs cellular proteins were heated at 100°C for 5 mins and then lectrophoresed in a 4-12% SDS polyacrylamide gel. Proteins were 3o electrophoretically transferred to nitrocellulose filters and hybridised with an antibody against a synthetic peptide corresponding to human proal (I) chain of type 1 collagen (obtained from Dr Larry Fisher, NIH, USA). The proal ( 1 ) band was detected using an anti-rabbit secondary antibody coupled to HRP (Biorad Ltd) and developed using ECL
(Pierce Ltd). Protein loading was determined by treating the membrane with an antibody to GAPDH (Advanced Immunochemicals). Protein loading was normalised to GAPDH
levels using desitometry.
Example 4 RNA analysis io RNA was extracted using TRI reagent and the final pellet was re-suspended in 0.5% SDS.
One to three micrograms of total RNA were electrophoresed in a formaldehyde denaturing gel according to standard procedures (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ~s Cold Spring Harbor, New York). RNA was transferred to Hybond-N membranes (Amersham) and hybridised for 24 hours to an alphal(1) cDNA probe radiolabeled using a T7 polymerase kit (AmershamPharmacia). Following washing, the filter was exposed to X-ray film and the film developed 4-24 hours later. The autoradiographic images of the alphal(1) transcripts (4.8kb & 5.8kb) were analysed by densitometric analysis and RNA
Zo loading was corrected using the intensity of the GAPDH transcript or the intensity of the 28S rRNA as internal controls.
Results zs Table I below shows the average percentage (%) collagen inhibition which relates to either collagen levels in the medium or collagen mRNA levels. In the treated cell assay used, there was a very good correlation between percentage collagen inhibition as measured in the medium and percentage inhibition of intracellular collagen mRNA levels.
Table I
ASO AVERAGE % COLLAGEN
INHIBITION
(SEQ ID N0:3) (SEQ ID N0:4) (SE ID NO:S) (SEQ ID N0:6) (SE ID N0:7) (SE ID N0:8) (SEQ ID N0:9) (SEQ ID NO:10) (SEQ ID NO:11 ) (SEQ ID N0:12) (SEQ ID N0:13) (SEQ ID N0:14) SEQUENCE LISTING
<110> AstraZeneca AB
<120> ANTISENSE OLIGONUCLEOTIDES
<130> 270611 <140>
<141>
<160> 14 <170> PatentIn Ver. 2.1 <210> 1 <211> 6728 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (120)..(4511) <220>
<221> sig~eptide <222> (120)..(185) <300>
<308> EMBL/Z74615 <309> 1996-07-O1 <400> 1 agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc 60 atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagac 119 atg ttc agc ttt gtg gac ctc cgg ctc ctg ctc ctc tta gcg gcc acc 167 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr gcc ctc ctg acg cac ggc caa gag gaa ggc caa gtc gag ggc caa gac 215 Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp gaa gac atc cca cca atc acc tgc gta cag aac ggc ctc agg tac cat 263 Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His gac cga gac gtg tgg aaa ccc gag ccc tuc cgg atc tgc gtc tgc gac 311 Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp aac ggc aag gtg ttg tgc gat gac gtg atc tgt gac gag acc aag aac 359 Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn tgc ccc ggc gcc gaa gtc ccc gag ggc gag tgc tgt ccc gtc tgc ccc 407 Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro gac ggc tca gag tca ccc acc gac caa gaa acc acc ggc gtc gag gga 455 Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly ccc aag gga gac act ggc ccc cga ggc cca agg gga ccc gca ggc ccc 503 Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro cct ggc cga gat ggc atc cct gga cag cct gga ctt ccc gga ccc ccc 551 Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro gga ccc ccc gga cct ccc gga ccc cct ggc ctc gga gga aac ttt get 599 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala ccc cag ctg tct tat ggc tat gat gag aaa tca acc gga gga att tcc 647 Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser gtg cct ggc ccc atg ggt ccc tct ggt cct cgt ggt ctc cct ggc ccc 695 Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro cct ggt gca cct ggt ccc caa ggc ttc caa ggt ccc cct ggt gag cct 743 Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro ggc gag cct gga get tca ggt ccc atg ggt ccc cga ggt ccc cca ggt 791 Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly ccc cct gga aag aat gga gat gat ggg gaa get gga aaa cct ggt cgt 839 Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg cct ggt gag cgt ggg cct cct ggg cct cag ggt get cga gga ttg ccc 887 Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro gga aca get ggc ctc cct gga atg aag gga cac aga ggt ttc agt ggt 935 Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly ttg gat ggt gcc aag gga gat get ggt cct get ggt cct aag ggt gag 983 Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu cct ggc agc cct ggt gaa aat gga get cct ggt cag atg ggc ccc cgt 1031 Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg ggc ctg cct ggt gag aga ggt cgc cct gga gcc cct ggc cct get ggt 1079 Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly get cgt gga aat gat ggt get act ggt get gcc ggg ccc cct ggt ccc 1127 Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro acc ggc ccc get ggt cct cct ggc ttc cct ggt get gtt ggt get aag 1175 Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys ggt gaa get ggt ccc caa ggg ccc cga ggc tct gaa ggt ccc cag ggt 1223 Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly gtg cgt ggt gag cct ggc ccc cct ggc cct get ggt get get ggc cct 1271 Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro get gga aac cct ggt get gat gga cag cct ggt get aaa ggt gcc aat 1319 Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn ggt get cct ggt att get ggt get cct ggc ttc cct ggt gcc cga ggc 1367 Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly ccc tct gga ccc cag ggc ccc ggc ggc cct cct ggt ccc aag ggt aac 1415 Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn agc ggt gaa cct ggt get cct ggc agc aaa gga gac act ggt get aag 1463 Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys gga gag cct ggc cct gtt ggt gtt caa gga ccc cct ggc cct get gga 1511 Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly gag gaa gga aag cga gga get cga ggt gaa ccc gga ccc act ggc ctg 1559 Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu ccc gga ccc cct ggc gag cgt ggt gga cct ggt agc cgt ggt ttc cct 1607 Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro ggc gca gat ggt gtt get ggt ccc aag ggt ccc get ggt gaa cgt ggt 1655 Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly tct cct ggc ccc get ggc ccc aaa gga tct cct ggt gaa get ggt cgt 1703 Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg ccc ggt gaa get ggt ctg cct ggt gcc aag ggt ctg act gga agc cct 1751 Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro ggc agc cct ggt cct gat ggc aaa act ggc ccc cct ggt ccc gcc ggt 1799 Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly caa gat ggt cgc ccc gga ccc cca ggc cca cct ggt gcc cgt ggt cag 1847 Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln get ggt gtg atg gga ttc cct gga cct aaa ggt get get gga gag ccc 1895 Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro ggc aag get gga gag cga ggt gtt ccc gga ccc cct ggc get gtc ggt 1943 Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly cct get ggc aaa gat gga gag get gga get cag gga ccc cct ggc cct 1991 Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro get ggt ccc get ggc gag aga ggt gaa caa ggc cct get ggc tcc ccc 2039 Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro gga ttc cag ggt ctc cct ggt cct get ggt cct cca ggt gaa gca ggc 2087 Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly aaa cct ggt gaa cag ggt gtt cct gga gac ctt ggc gcc cct ggc ccc 2135 Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro tct gga gca aga ggc gag aga ggt ttc cct ggc gag cgt ggt gtg caa 2183 Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln ggt ccc cct ggt cct get gga ccc cga ggg gcc aac ggt get ccc ggc 2231 Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly aac gat ggt get aag ggt gat get ggt gcc cct gga get ccc ggt agc 2279 Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser cag ggc gcc cct ggc ctt cag gga atg cct ggt gaa cgt ggt gca get 2327 Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala ggt ctt cca ggg cct aag ggt gac aga ggt gat get ggt ccc aaa ggt 2375 Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly get gat ggc tct cct ggc aaa gat ggc gtc cgt ggt ctg acc ggc ccc 2423 Ala Asp G1y Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro att ggt cct cct ggc cct get ggt gcc cct ggt gac aag ggt gaa agt 2471 Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser ggt ccc agc ggc cct get ggt ccc act gga get cgt ggt gcc ccc gga 2519 Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly gac cgt ggt gag cct ggt ccc ccc ggc cct get ggc ttt get ggc ccc 2567 Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro cct ggt get gac ggc caa cct ~:~gt get aaa ggc gaa cct ggt gat get 2615 Pro Gly Ala Asp Gly Gln Pro ~ 1y Ala Lys Gly Glu Pro Gly Asp Ala ggt gcc aaa ggc gat get ggt ccc cct ggg cct gcc gga ccc get gga 2663 Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly ccc cct ggc ccc att ggt aat gtt ggt get cct gga gcc aaa ggt get 2711 Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala cgc ggc agc get ggt ccc cct ggt get act ggt ttc cct ggt get get 2759 Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala ggc cga gtc ggt cct cct ggc ccc tct gga aat get gga ccc cct ggc 2807 Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly cct cct ggt cct get ggc aaa gaa ggc ggc aaa ggt ccc cgt ggt gag 2855 Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu act ggc cct get gga cgt cct ggt gaa gtt ggt ccc cct ggt ccc cct 2903 Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro ggc cct get ggc gag aaa gga tcc cct ggt get gat ggt cct get ggt 2951 Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly get cct ggt act ccc ggg cct caa ggt att get gga cag cgt ggt gtg 2999 Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val gtc ggc ctg cct ggt cag aga gga gag aga ggc ttc cct ggt ctt cct 3047 Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro ggc ccc tct ggt gaa cct ggc aaa caa ggt ccc tct gga gca agt ggt 3095 Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly gaa cgt ggt ccc ccc ggt ccc atg ggc ccc cct gga ttg get gga ccc 3143 Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro cct ggt gaa tct gga cgt gag ggg get cct get gcc gaa ggt tcc cct 3191 Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro gga cga gac ggt tct cct ggc gcc aag ggt gac cgt ggt gag acc ggc 3239 Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly ccc get gga ccc cct ggt get cct ggt get cct ggt gcc cct ggc ccc 3287 Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro gtt ggc cct get ggc aag agt ggt gat cgt ggt gag act ggt cct get 3335 Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala ggt ccc gcc ggt ccc gtc ggc ccc gtc ggc gcc cgt ggc ccc gcc gga 3383 Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly ccc caa ggc ccc cgt ggt gac aag ggt gag aca ggc gaa cag ggc gac 3431 Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp aga ggc ata aag ggt cac cgt ggc ttc tct ggc ctc cag ggt ccc cct 3479 Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro ggc cct cct ggc tct cct ggt gaa caa ggt ccc tct gga gcc tct ggt 3527 Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly cct get ggt ccc cga ggt ccc cct ggc tct get ggt get cct ggc aaa 3575 Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys gat gga ctc aac ggt ctc cct ggc ccc att ggg ccc cct ggt cct cgc 3623 Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg ggt cgc act ggt gat get ggt cct gtt ggt ccc ccc ggc cct cct gga 3671 Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly cct cct ggt ccc cct ggt cct ccc agc get ggt ttc gac ttc agc ttc 3719 Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe ctg ccc cag cca cct caa gag aag get cac gat ggt ggc cgc tac tac 3767 Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr cgg get gat gat gcc aat gtg gtt cgt gac cgt gac ctc gag gtg gac 3815 Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp acc acc ctc aag agc ctg agc cag cag atc gag aac atc cgg agc cca 3863 Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro gag gga agc cgc aag aac ccc gcc cgc acc tgc cgt gac ctc aag atg 3911 Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met tgc cac tct gac tgg aag agt gga gag tac tgg att gac ccc aac caa 3959 Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln ggc tgc aac ctg gat gcc atc aaa gtc ttc tgc aac atg gag act ggt 4007 Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly gag acc tgc gtg tac ccc act cag ccc agt gtg gcc cag aag aac tgg 4055 Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp tac atc agc aag aac ccc aag gac aag agg cat gtc tgg ttc ggc gag 4103 Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu agc atg acc gat gga ttc cag ttc gag tat ggc ggc cag ggc tcc gac 4151 Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp cct gcc gat gtg gcc atc cag ctg acc ttc ctg cgc ctg atg tcc acc 4199 Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr gag gcc tcc cag aac atc acc tac cac tgc aag aac agc gtg gcc tac 4247 Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr atg gac cag cag act ggc aac ctc aag aag gcc ctg ctc ctc aag ggc 4295 Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly tcc aac gag atc gag atc cgc gcc gag ggc aac agc cgc ttc acc tac 4343 Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr agc gtc act gtc gat ggc tgc acg agt cac acc gga gcc tgg ggc aag 4391 Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys aca gtg att gaa tac aaa acc acc aag tcc tcc cgc ctg ccc atc atc 4439 Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile gat gtg gcc ccc ttg gac gtt ggt gcc cca gac cag gaa ttc ggc ttc 4487 Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe gac gtt ggc cct gtc tgc ttc ctg taaactccct ccatcccaac ctggctccct 4541 Asp Val Gly Pro Val Cys Phe Leu cccacccaac caactttccc cccaacccgg aaacagacaa gcaacccaaa ctgaaccccc 4601 ccaaaagcca aaaaatggga gacaatttca catggacttt ggaaaatatt tttttccttt 4661 gcattcatct ctcaaactta gtttttatct ttgaccaacc gaacatgacc aaaaaccaaa 4721 agtgcattca accttaccaa aaaaaaaaaa aaaaaaaaaa gaataaataa ataagttttt 4781 aaaaaaggaa gcttggtcca cttgcttgaa gacccatgcg ggggtaagtc cctttctgcc 4841 cgttgggtta tgaaacccca atgctgccct ttctgctcct ttctccacac cccccttggc 4901 ctcccctcca ctccttccca aatctgtctc cccagaagac acaggaaaca atgtattgtc 4961 tgcccagcaa tcaaaggcaa tgctcaaaca cccaagtggc ccccaccctc agcccgctcc 5021 tgcccgccca gcacccccag gccctgggga cctggggttc tcagactgcc aaagaagcct 5081 tgccatctgg cgctcccatg gctcttgcaa catctcccct tcgtttttga gggggtcatg 5141 ccgggggagc caccagcccc tcactgggtt cggaggagag tcaggaaggg ccacgacaaa 5201 gcagaaacat cggatttggg gaacgcgtgt catcccttgt gccgcaggct gggcgggaga 5261 gactgttctg ttctgttcct tgtgtaactg tgttgctgaa agactacctc gttcttgtct 5321 tgatgtgtca ccggggcaac tgcctggggg cggggatggg ggcagggtgg aagcggctcc 5381 ccatttttat accaaaggtg ctacatctat gtgG.tgggtg gggtggggag ggaatcactg 5441 gtgctataga aattgagatg cccccccagg ccagcaaatg ttcctttttg ttcaaagtct 5501 atttttattc cttgatattt tttctttctt tttttttttt tttgtggatg gggacttgtg 5561 aatttttcta aaggtgctat ttaacatggg aggagagcgt gtgcgctcca gcccagcccg 5621 ctgctcactt tccaccctct ctccacctgc ctctggcttc tcaggcctct gctctccgac 5681 ctctctcctc tgaaaccctc ctccacagct gcagcccatc ctcccggctc cctcctagtc 5741 tgtcctgcgt cctctgtccc cgggtttcag agacaacttc ccaaagcaca aagcagtttt 5801 tccctagggg tgggaggaag caaaagactc tgtacctatt ttgtatgtgt ataataattt 5861 gagatgtttt taattatttt gattgctgga ataaagcatg tggaaatgac ccaaacataa 5921 tccgcagtgg cctcctaatt tccttctttg gagttggggg aggggtagac atggggaagg 5981 ggccttgggg tgatgggctt gccttccatt cctgcccttt ccctccccac tattctcttc 6041 tagatccctc cataacccca ctcccctttc tctcaccctt cttataccgc aaacctttct 6101 acttcctctt tcattttcta ttcttgcaat ttccttgcac cttttccaaa tcctcttctc 6161 ccctgcaata ccatacaggc aatccacgtg cacaacacac acacacactc ttcacatctg 6221 gggttgtcca aacctcatac ccactcccct tcaagcccat ccactctcca ccccctggat 6281 gccctgcact tggtggcggt gggatgctca tggatactgg gagggtgagg ggagtggaac 6341 ccgtgaggag gacctggggg cctctccttg aactgacatg aagggtcate tggcctctgc 6401 tcccttctca cccacgctga cctcctgccg aaggagcaac gcaacaggag aggggtctgc 6461 tgagcctggc gagggtctgg gagggaccag gaggaaggcg tgctccctgc tcgctgtcct 6521 ggccctgggg gagtgaggga gacagacacc tgggagagct gtggggaagg cactcgcacc 6581 gtgctcttgg gaaggaagga gacctggccc tgctcaccac ggactgggtg cctcgacctc 6641 ctgaatcccc agaacacaac ccccctgggc tggggtggtc tggggaacca tcgtgccccc 6701 gcctcccgcc tactcctttt taagctt 6728 <210> 2 <211> 1464 <212> PRT
<213> Homo Sapiens <400> 2 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly G1u Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu <210> 3 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 3 ggacgaccag gttttccagc 20 <210> 4 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 4 gcagcaccag cagggccagg 20 <210> 5 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 5 gccaggagca ccaggttcac 20 <210> 6 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 6 cttcctctcc agcagggcca 20 <210> 7 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 7 gccttgccgg gctctccagc 20 <210> 8 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 8 cgggaacacc tcgctctcca 20 <210>9 <211>20 <212>DNA
<213>Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 9 gcaggaccga cagcgccagg 20 <210> 10 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 10 tccatctttg ccagcaggac <210> 11 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<223> Antisense oligonucleotide <400> 11 ggtccctgag ctccagcctc 20 <210> 12 <211> 19 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 12 ttggccgtca gcaccaggg <210> 13 <211> 20 <212> DNA
<213> Homo sapiens <220>
<223> Antisense oligonucleotide <400> 13 tttctcgcca gcagggccag 20 <210>14 <211>20 <212>DNA
<213>Homo sapiens <220>
<223> Antisense oligonucleotide <400> 14 ctcgatctgc tggctcaggc 20
Claims (15)
1. An antisense DNA oligonucleotide comprising from 18 to 25 nucleotides which is complementary to a nucleotide sequence from position 750 to position 3900 inclusive of SEQ ID NO:1, wherein SEQ ID NO:1 comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence according to SEQ ID NO:2, the oligonucleotide being capable of inhibiting expression of the polypeptide in a cell that expresses it.
2. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 750 to position 900 inclusive of SEQ ID NO:1.
3. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 1200 to position 1300 inclusive of SEQ ID NO:1.
4. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 1400 to position 1500 inclusive of SEQ ID NO:1.
5. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 1450 to position 1550 inclusive of SEQ ID NO:1.
6. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 1850 to position 2000 inclusive of SEQ ID NO:1.
7. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 2500 to position 2600 inclusive of SEQ ID NO:1.
8. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 2850 to position 2950 inclusive of SEQ ID NO:1.
9. An oligonucleotide according to claim 1 which is complementary to a nucleotide sequence from position 3800 to position 3900 inclusive of SEQ ID NO:1.
10. An oligonucleotide according to claim 1 which is selected from SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
11. A pharmaceutical composition comprising an oligonucleotide as defined in any one of claims 1 to 10 in association with a pharmaceutically acceptable adjuvant, diluent or carrier.
12. A process for the preparation of a pharmaceutical composition as claimed in claim 11, which comprises mixing an oligonucleotide as defined in any one of claims 1 to 10 with a pharmaceutically acceptable adjuvant, diluent or carrier.
13. An oligonucleotide according to any one of claims 1 to 10 for use in therapy.
14. Use of an oligonucleotide according to any one of claims 1 to 10 in the manufacture of a medicament for use in therapy.
15. A method of treating, or reducing the risk of, a collagen disorder in a patient suffering from, or at risk of, the disorder, which comprises administering to the patient a therapeutically effective amount of an oligonucleotide as defined in any one of claims 1 to or a pharmaceutical composition as defined in claim 11.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9929487.8 | 1999-12-15 | ||
| GBGB9929487.8A GB9929487D0 (en) | 1999-12-15 | 1999-12-15 | Antisense oligonucleotides |
| PCT/GB2000/004741 WO2001044455A2 (en) | 1999-12-15 | 2000-12-12 | Antisense oligonucleotides for the inhibition of expression of type i procollagen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2390674A1 true CA2390674A1 (en) | 2001-06-21 |
Family
ID=10866254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002390674A Abandoned CA2390674A1 (en) | 1999-12-15 | 2000-12-12 | Antisense oligonucleotides for the inhibition of expression of type i procollagen |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US7173122B2 (en) |
| EP (3) | EP1244781B1 (en) |
| JP (1) | JP2003516752A (en) |
| KR (1) | KR20020064936A (en) |
| CN (1) | CN1423695A (en) |
| AT (2) | ATE410510T1 (en) |
| AU (1) | AU2529701A (en) |
| BR (1) | BR0016347A (en) |
| CA (1) | CA2390674A1 (en) |
| DE (2) | DE60040495D1 (en) |
| GB (1) | GB9929487D0 (en) |
| IL (1) | IL149640A0 (en) |
| MX (1) | MXPA02005871A (en) |
| NO (1) | NO20022799L (en) |
| NZ (1) | NZ519008A (en) |
| WO (1) | WO2001044455A2 (en) |
| ZA (1) | ZA200203878B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7396823B2 (en) * | 1999-12-15 | 2008-07-08 | Nath Rahul K | Therapy inhalation involving antisense oligonucleotides for treating idiopathic pulmonary fibrosis |
| CN111378657B (en) * | 2018-12-28 | 2024-03-15 | 苏州瑞博生物技术股份有限公司 | siRNA for inhibiting COL1A1 gene expression, pharmaceutical composition containing siRNA and application thereof |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6265157B1 (en) * | 1991-12-03 | 2001-07-24 | Allegheny University Of The Health Sciences | Compositions and methods for detecting altered COL1A1 gene sequences |
| WO1993011149A1 (en) * | 1991-12-03 | 1993-06-10 | Thomas Jefferson University | Methods of detecting a genetic predisposition for osteoporosis |
| CA2148687A1 (en) * | 1992-11-09 | 1994-05-26 | Darwin Prockop | Antisense oligonucleotides to inhibit expression of mutated and wild type genes for collagen |
| CA2159195A1 (en) * | 1993-03-26 | 1994-10-13 | Darwin J. Prockop | Use of a col 1a1 mini-gene construct to inhibit collagen synthesis |
| DE19508923A1 (en) * | 1995-03-13 | 1996-09-19 | Hoechst Ag | New oligo:nucleotide analogues contg. phospho:mono:ester bridges |
| ATE206131T1 (en) * | 1995-03-13 | 2001-10-15 | Aventis Pharma Gmbh | PHOSPHONOMONOESTERNUCLIC ACIDS, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| EP0736608A1 (en) * | 1995-04-08 | 1996-10-09 | Roche Diagnostics GmbH | Method for the specific amplification and the detection of DNA or RNA |
| EP0856579A1 (en) * | 1997-01-31 | 1998-08-05 | BIOGNOSTIK GESELLSCHAFT FÜR BIOMOLEKULARE DIAGNOSTIK mbH | An antisense oligonucleotide preparation method |
| US6238921B1 (en) * | 1998-03-26 | 2001-05-29 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of human mdm2 expression |
| US6007995A (en) * | 1998-06-26 | 1999-12-28 | Isis Pharmaceuticals Inc. | Antisense inhibition of TNFR1 expression |
| BR0015507A (en) * | 1999-11-12 | 2002-10-22 | Fibrogen Inc | Animal gelatins and collagen |
-
1999
- 1999-12-15 GB GBGB9929487.8A patent/GB9929487D0/en not_active Ceased
-
2000
- 2000-12-12 JP JP2001545532A patent/JP2003516752A/en active Pending
- 2000-12-12 NZ NZ519008A patent/NZ519008A/en unknown
- 2000-12-12 WO PCT/GB2000/004741 patent/WO2001044455A2/en active IP Right Grant
- 2000-12-12 BR BR0016347-3A patent/BR0016347A/en not_active Application Discontinuation
- 2000-12-12 EP EP00988966A patent/EP1244781B1/en not_active Expired - Lifetime
- 2000-12-12 CA CA002390674A patent/CA2390674A1/en not_active Abandoned
- 2000-12-12 AT AT05024183T patent/ATE410510T1/en not_active IP Right Cessation
- 2000-12-12 AT AT00988966T patent/ATE309338T1/en not_active IP Right Cessation
- 2000-12-12 DE DE60040495T patent/DE60040495D1/en not_active Expired - Lifetime
- 2000-12-12 IL IL14964000A patent/IL149640A0/en unknown
- 2000-12-12 EP EP08017586A patent/EP2025752A3/en not_active Withdrawn
- 2000-12-12 MX MXPA02005871A patent/MXPA02005871A/en unknown
- 2000-12-12 KR KR1020027007591A patent/KR20020064936A/en not_active Application Discontinuation
- 2000-12-12 CN CN00817282A patent/CN1423695A/en active Pending
- 2000-12-12 US US10/149,352 patent/US7173122B2/en not_active Expired - Fee Related
- 2000-12-12 DE DE60023943T patent/DE60023943T2/en not_active Expired - Lifetime
- 2000-12-12 AU AU25297/01A patent/AU2529701A/en not_active Abandoned
- 2000-12-12 EP EP05024183A patent/EP1698695B1/en not_active Expired - Lifetime
-
2002
- 2002-05-15 ZA ZA200203878A patent/ZA200203878B/en unknown
- 2002-06-12 NO NO20022799A patent/NO20022799L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP1698695A2 (en) | 2006-09-06 |
| WO2001044455A3 (en) | 2002-01-10 |
| EP2025752A2 (en) | 2009-02-18 |
| KR20020064936A (en) | 2002-08-10 |
| US7173122B2 (en) | 2007-02-06 |
| ZA200203878B (en) | 2003-08-15 |
| DE60023943D1 (en) | 2005-12-15 |
| IL149640A0 (en) | 2002-11-10 |
| CN1423695A (en) | 2003-06-11 |
| NO20022799D0 (en) | 2002-06-12 |
| BR0016347A (en) | 2002-09-10 |
| NO20022799L (en) | 2002-06-12 |
| EP1244781B1 (en) | 2005-11-09 |
| NZ519008A (en) | 2004-04-30 |
| AU2529701A (en) | 2001-06-25 |
| DE60040495D1 (en) | 2008-11-20 |
| GB9929487D0 (en) | 2000-02-09 |
| ATE309338T1 (en) | 2005-11-15 |
| MXPA02005871A (en) | 2002-10-23 |
| ATE410510T1 (en) | 2008-10-15 |
| JP2003516752A (en) | 2003-05-20 |
| EP1244781A2 (en) | 2002-10-02 |
| EP2025752A3 (en) | 2009-08-12 |
| EP1698695B1 (en) | 2008-10-08 |
| WO2001044455A2 (en) | 2001-06-21 |
| DE60023943T2 (en) | 2006-08-03 |
| EP1698695A3 (en) | 2007-02-14 |
| US20030105050A1 (en) | 2003-06-05 |
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