Patent application title: MODULATION OF ENDOTHELIAL LIPASE EXPRESSION
Inventors:
Kenneth W. Dobie (Del Mar, CA, US)
Assignees:
Isis Pharmaceuticals, Inc.
IPC8 Class: AA61K317088FI
USPC Class:
514 44
Class name: N-glycoside nitrogen containing hetero ring polynucleotide (e.g., rna, dna, etc.)
Publication date: 2009-06-18
Patent application number: 20090156538
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Patent application title: MODULATION OF ENDOTHELIAL LIPASE EXPRESSION
Inventors:
Kenneth W. Dobie
Agents:
JONES DAY for;Isis Pharmaceuticals, Inc.
Assignees:
Isis Pharmaceuticals, Inc.
Origin: NEW YORK, NY US
IPC8 Class: AA61K317088FI
USPC Class:
514 44
Abstract:
Compounds, compositions and methods are provided for modulating the
expression of endothelial lipase. The compositions comprise
oligonucleotides, targeted to nucleic acid encoding endothelial lipase.
Methods of using these compounds for modulation of endothelial lipase
expression and for diagnosis and treatment of disease associated with
expression of endothelial lipase are provided.Claims:
1. A composition comprising an antisense compound or a salt form thereof,
wherein the antisense compound is 15 to 30 nucleobases in length and is
at least 90% complementary within nucleobases 1099 to 1143 of SEQ ID
NO:4, wherein the antisense compound is double-stranded and a
pharmaceutically acceptable carrier or diluent
2. The composition of claim 1, wherein the antisense compound comprises a DNA oligonucleotide.
3. The composition of claim 1, wherein the antisense compound comprises an RNA oligonucleotide.
4. The composition of claim 1, wherein the antisense compound comprises a chimeric antisense oligonucleotide.
5. The composition of claim 1, wherein the antisense compound comprises at least one modified internucleoside linkage, sugar moiety, or nucleobase.
6. The composition of claim 1, wherein the antisense compound comprises at least one 2'-O-methoxyethyl sugar moiety.
7. The composition of claim 1, wherein the antisense compound comprises at least one phosphorothioate internucleoside linkage.
8. The composition of claim 1, wherein the antisense compound comprises at least one 5-methylcytosine.
9. The composition of claim 4, wherein the chimeric antisense oligonucleotide comprises a plurality of 2'-deoxynucleotides flanked on each side by at least one 2'-O-methoxyethyl nucleotide.
10. A composition comprising an antisense compound or a salt form thereof, wherein the antisense compound is 15 to 30 nucleobases in length and is at least 90% complementary to at least 8 consecutive nucleobases within nucleobases 1104-1123 of SEQ ID NO:4, wherein the antisense compound is double-stranded, wherein the antisense compound is a chimeric antisense oligonucleotide, and a pharmaceutically acceptable carrier or diluent.
11. The composition of claim 10, wherein the antisense compound comprises at least one modified internucleoside linkage, sugar moiety, or nucleobase.
12. The composition of claim 11 wherein the modified internucleoside linkage is a phosphorothioate linkage.
13. The composition of claim 11 wherein the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety.
14. The composition of claim 11 wherein the modified nucleobase is a 5-methylcytidine.
15. The composition of claim 10, wherein the antisense compound comprises a plurality of 2'-deoxynucleotides flanked on each side by at least one 2'-O-methoxyethyl nucleotide.
16. The composition of claim 1, wherein the antisense compound is at least 95% complementary within nucleobases 1099 to 1143 of SEQ ID NO:4.
17. The composition of claim 1, wherein the antisense compound is fully complementary within nucleobases 1099 to 1143 of SEQ ID NO:4.
18. The composition of claim 10, wherein the antisense compound is at least 95% complementary to at least 8 consecutive nucleobases within nucleobases 1104-1123 of SEQ ID NO:4.
19. The composition of claim 10, wherein the antisense compound is fully complementary to at least 8 consecutive nucleobases within nucleobases 1104-1123 of SEQ ID NO:4.
20. The composition of claim 15, wherein the antisense compound comprises ten 2'-deoxynucleotides flanked on each side by five 2'-O-methoxyethyl nucleotides.
21. The composition of claim 20, wherein the antisense compound is 20 nucleobases in length.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of pending U.S. patent application Ser. No. 10/319,915, filed Dec. 12, 2002, herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled RTS0447USC 1 SEQ.txt created on Oct. 30, 2008 which is 164 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003]The present invention provides compositions and methods for modulating the expression of endothelial lipase. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding endothelial lipase. Such compounds are shown herein to modulate the expression of endothelial lipase.
BACKGROUND OF THE INVENTION
[0004]Atherosclerosis is the major causative factor of heart disease and stroke, and the leading cause of death in Western countries is cardiovascular disease. Dyslipidaemia is a primary contributor to atherosclerosis. Because triglycerides are insoluble in the bloodstream, they are packaged for plasma transport into micelle-like lipoprotein particles composed of protein and phospholipid shells surrounding a non-polar core of acylglycerols, free cholesterol, and cholesterol esters. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons (which transport dietary lipids from intestine to tissues); very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL), (all of which transport triacylglycerols and cholesterol from the liver to tissues); and high density lipoproteins (HDL) (which transport endogenous cholesterol from tissues to the liver, as well as mediating selective cholesteryl ester delivery to steroidogenic tissues). All of these particles undergo continuous metabolic processing and have somewhat variable properties and compositions. Plasma concentrations of LDL and HDL are directly and inversely related, respectively, to the risk of atherosclerotic cardiovascular disease (Krieger, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 4077-4080).
[0005]HDL protect the arterial wall from the development of atherosclerosis by promoting efflux of excess cholesterol from cells in the arterial wall and returning it to the liver for excretion into the bile, as well as by protecting LDL from oxidation, thereby reducing the inflammatory response of epithelial cells, inhibiting the coagulation pathway, and promoting the availability of nitric oxide. The metabolism of HDL is influenced by several members of the triacylglycerol (TG) lipase family of proteins, which hydrolyze triglycerides, phospholipids and cholesteryl esters, generating fatty acids to facilitate intestinal absorption, energy production or storage. Of the TG lipases, lipoprotein lipase (LPL) influences the metabolism of HDL cholesterol by hydrolyzing triglycerides in triglyceride-rich lipoproteins, resulting in the transfer of lipids and apolipoproteins to HDL and is responsible for hydrolyzing chylomicron and VLDL in muscle and adipose tissues. Hepatic lipase (HL) hydrolyzes HDL triglyceride and phospholipids, generating smaller, lipid-depleted HDL particles, and plays a role in the uptake of HDL cholesterol (Jin et al., Trends Endocrinol. Metab., 2002, 13, 174-178; Wong and Schotz, J. Lipid Res., 2002, 43, 993-999). Endothelial lipase (also known as EDL, EL, LIPG, endothelial-derived lipase and endothelial cell-derived lipase) was identified using differential display to isolate mRNAs which were differentially regulated in response to oxidized-LDL (Jaye et al., Nat. Genet., 1999, 21, 424-428). Independently, the human endothelial lipase gene was identified in human umbilical vein endothelial cells (HUVECs) undergoing tube formation in a model of vascular formation (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175).
[0006]In humans, the endothelial lipase gene has been assigned to chromosome 18, and the rat endothelial lipase gene was identified and mapped to rat chromosome 18 in the vicinity of a quantitative trait locus that affects serum HDL levels after a high fat diet (Bonne et al., DNA Seq., 2001, 12, 285-287).
[0007]At least 50% of the variation in HDL cholesterol levels is genetically determined. The phenotype of elevated HDL cholesterol is often dominantly inherited, but homozygous deficiency of HL or of the cholesteryl ester transfer protein (CETP), which result in elevated HDL cholesterol, are recessive conditions. Recently, several genetic variations in the human endothelial lipase gene have been identified, 6 of which potentially produce functional variants of the protein, and the frequencies of these variants were found to be associated with elevated levels of HDL cholesterol in human subjects (deLemos et al., Circulation, 2002, 106, 1321-1326).
[0008]Notably, the endothelial lipase-mediated binding and uptake of HDL particles and the selective uptake of HDL-derived cholesterol esters have been reported to be independent of its enzymatic lipolytic activity (Strauss et al., Biochem. J., 2002).
[0009]Recombinant endothelial lipase protein has substantial phospholipase activity but has been reported to have less hydrolytic activity toward triglyceride lipids (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428). However, endothelial lipase does exhibit triglyceride lipase activity ex vivo in addition to its HDL phospholipase activity, and endothelial lipase was found to hydrolyze HDL more efficiently than other lipoproteins (McCoy et al., J. Lipid Res., 2002, 43, 921-929). Overexpression of the human endothelial lipase gene in the livers of mice markedly reduces plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I (apoA-I) (Jaye et al., Nat. Genet., 1999, 21, 424-428).
[0010]On the basis of its amino acid sequence homology to other members of the TG lipase family, including the presence of a characteristic 19-amino acid "lid" domain predicted to form an amphipathic helix covering the catalytic pocket of the enzyme and confers substrate specificity to the enzymes of the TG lipase family, and its demonstrated phospholipase activity, the endothelial lipase protein is believed to be involved in lipoprotein metabolism and vascular biology (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428).
[0011]Endothelial lipase was cloned from epithelial cells but has been demonstrated to be abundantly expressed in a variety of tissues including ovary, testis, thyroid gland, liver, lung, kidney and placenta, the latter suggesting the potential for a role in development (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428). Interestingly, endothelial lipase mRNA levels were upregulated in HUVEC and coronary artery endothelial cells upon treatment with inflammatory cytokines implicated in vascular disease etiology and vascular remodeling, including TNF-alpha and IL-1 beta. Thus, endothelial lipase is predicted to be intricately involved in modulating vessel wall lipid metabolism and to play a role in vascular diseases such as atherosclerosis (Hirata et al., Biochem. Biophys. Res. Commun., 2000, 272, 90-93).
[0012]Disclosed and claimed in U.S. Pat. No. 6,395,530 is an isolated nucleic acid which hybridizes at high stringency to a nucleic acid having a sequence selected from a group of which endothelial lipase is a member or to a target consisting of nucleotides from 44-79 of the endothelial lipase gene wherein the complement of said isolated nucleic acid encodes a polypeptide having triacylglycerol lipase or phospholipase A activity. Further claimed are a vector, a composition, a recombinant cell and method for preparing a polypeptide. Antisense nucleic acids are generally disclosed (Jaye et al., 2002).
[0013]Disclosed and claimed in PCT Publications WO 01/40466 and WO 00/73452 is an isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes the endothelial lipase protein or to a nucleotide sequence selected from a group of which the endothelial lipase gene is a member, a vector, a host cell, a process for producing a polypeptide, an isolated polypeptide, a chimeric molecule, and antibody, a method of detecting said polypeptide in a sample, a method of linking a bioactive molecule to a cell expressing said polypeptide, a method of modulating at least one biological activity of a cell expressing said polypeptide, methods for stimulating the release of TNF-alpha from human blood, for modulating the uptake of glucose or FFA by skeletal muscle or adipocyte cells, for stimulating the proliferation or differentiation of chondrocyte cells, for stimulating the proliferation of inner ear utricular supporting cells, endothelial cells or T-lymphocyte cells, for stimulating the proliferation of or gene expression in pericyte cells, for stimulating the release of proteoglycans from cartilage, for stimulating the release of a cytokine from PBMC cells, for inhibiting the binding of A-peptide to factor VIIA, for inhibiting the differentiation of adipocyte cells, for detecting the presence of tumor in an mammal, an oligonucleotide probe derived from any of several nucleotide sequences cited, a composition useful for the treatment of immune related diseases, use of a polypeptide to prepare said composition, a method of diagnosing an immune related disease in a mammal, an immune related disease diagnostic kit, a method for identifying an agonist or a compound capable of inhibiting the expression and/or activity of a polypeptide, a vector, and an ex vivo producer cell. Antisense oligonucleotide agonists or antagonists are generally disclosed (Ashkenazi et al., 2000; Baker et al., 2001).
[0014]Disclosed and claimed in PCT Publication WO 01/96388 is an isolated polynucleotide comprising a sequence selected from a group of nucleotide sequences, complements of said sequences, sequences consisting of at least 20 contiguous residues of one of said sequences, sequences that hybridize to said sequences, sequences having at least 75% identity to said sequence, and degenerate variants of said sequence, an isolated polypeptide, an expression vector, a host cell, an isolated antibody, a method for detecting the presence of a cancer in a patient, a fusion protein, an oligonucleotide that hybridizes to said sequence, a method for stimulating and/or expanding T cells specific for a tumor protein, an isolated T cell population, a composition comprising a first component selected from the group consisting of physiologically acceptable carriers and immunostimulants, and a second component selected from the group consisting of said polypeptides, polynucleotides, antibodies, fusion proteins, T cell populations and antigen presenting cells that express a polypeptide, a method for stimulating an immune response in a patient, a method for the treatment of a cancer in a patient, a method for determining the presence of a cancer in a patient, a diagnostic kit comprising at least one oligonucleotide or antibody, and a method for inhibiting the development of a cancer in a patient. Antisense oligonucleotides are generally disclosed (Jiang et al., 2001).
[0015]Currently, there are no known therapeutic agents which effectively inhibit the synthesis of endothelial lipase.
[0016]Consequently, there remains a long felt need for agents capable of effectively inhibiting endothelial lipase function.
[0017]Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of endothelial lipase expression.
[0018]The present invention provides compositions and methods for modulating endothelial lipase expression.
SUMMARY OF THE INVENTION
[0019]The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding endothelial lipase, and which modulate the expression of endothelial lipase. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of endothelial lipase and methods of modulating the expression of endothelial lipase in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of endothelial lipase are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0020]The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding endothelial lipase. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding endothelial lipase. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding endothelial lipase" have been used for convenience to encompass DNA encoding endothelial lipase, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
[0021]The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of endothelial lipase. In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.
[0022]In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
[0023]An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
[0024]In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
[0025]"Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
[0026]It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
B. Compounds of the Invention
[0027]According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
[0028]While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.
[0029]The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).
[0030]In the context of this invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
[0031]While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.
[0032]The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.
[0033]In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.
[0034]In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.
[0035]Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.
[0036]Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
[0037]Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.
C. Targets of the Invention
[0038]"Targeting" an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes endothelial lipase.
[0039]The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.
[0040]Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding endothelial lipase, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0041]The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions which may be targeted effectively with the antisense compounds of the present invention.
[0042]The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
[0043]Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.
[0044]Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
[0045]It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.
[0046]Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
[0047]It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.
[0048]The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as "preferred target segments." As used herein the term "preferred target segment" is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.
[0049]While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.
[0050]Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
[0051]Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
[0052]Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
D. Screening and Target Validation
[0053]In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of endothelial lipase. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding endothelial lipase and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding endothelial lipase with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding endothelial lipase. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding endothelial lipase, the modulator may then be employed in further investigative studies of the function of endothelial lipase, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
[0054]The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.
[0055]Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).
[0056]The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between endothelial lipase and a disease state, phenotype, or condition. These methods include detecting or modulating endothelial lipase comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of endothelial lipase and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.
E. Kits, Research Reagents, Diagnostics, and Therapeutics
[0057]The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
[0058]For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
[0059]As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
[0060]Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
[0061]The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding endothelial lipase. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective endothelial lipase inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding endothelial lipase and in the amplification of said nucleic acid molecules for detection or for use in further studies of endothelial lipase. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding endothelial lipase can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of endothelial lipase in a sample may also be prepared.
[0062]The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
[0063]For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of endothelial lipase is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a endothelial lipase inhibitor. The endothelial lipase inhibitors of the present invention effectively inhibit the activity of the endothelial lipase protein or inhibit the expression of the endothelial lipase protein. In one embodiment, the activity or expression of endothelial lipase in an animal is inhibited by about 10%. Preferably, the activity or expression of endothelial lipase in an animal is inhibited by about 30%. More preferably, the activity or expression of endothelial lipase in an animal is inhibited by 50% or more.
[0064]For example, the reduction of the expression of endothelial lipase may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding endothelial lipase protein and/or the endothelial lipase protein itself.
[0065]The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
F. Modifications
[0066]As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
Modified Internucleoside Linkages (Backbones)
[0067]Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
[0068]Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
[0069]Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
[0070]Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
[0071]Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
Modified Sugar and Internucleoside Linkages-Mimetics
[0072]In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
[0073]Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH2--NH--O--CH2--, --CH2--N(CH3)--O--CH2-- [known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --O--N(CH3)--CH2--CH2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH2--] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified Sugars
[0074]Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2, also described in examples hereinbelow.
[0075]Other preferred modifications include 2'-methoxy (2'-O--CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2--CH═CH2), 2'-O-allyl (2'-O--CH2--CH═CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
[0076]A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH2--), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
Natural and Modified Nucleobases
[0077]Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C≡C--CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
[0078]Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.
Conjugates
[0079]Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.
[0080]Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
Chimeric Compounds
[0081]It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
[0082]The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
[0083]Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
G. Formulations
[0084]The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
[0085]The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.
[0086]The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0087]The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
[0088]The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0089]The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[0090]Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
[0091]Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0092]Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
[0093]Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0094]The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0095]In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0096]One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
[0097]Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
[0098]For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.
[0099]Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.
[0100]Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
[0101]Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
[0102]In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.
H. Dosing
[0103]The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
[0104]While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0105]The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N-benzoyl-5-methylcytidin-- 3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2'-Fluorodeoxyadenosine, 2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine, 2'-O-(2-Methoxyethyl) modified amidites, 2'-O-(2-methoxyethyl)-5-methyluridine intermediate, 5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi- n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytid- ine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N4-benzo- yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N6-benzo- yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su- p.4 isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit- e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites, 2'-(Dimethylaminooxyethoxy) nucleoside amidites, 5'-O-tert-Butyldiphenylsilyl-O2-2'-anhydro-5-methyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine, 2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine, 5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri- dine, 5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-meth- yluridine, 2'-O-(dimethylaminooxyethyl)-5-methyl uridine, 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe- thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(- 4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora- midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethyl-aminoethoxy)-ethyl)]-5-methyl uridine and 5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0106]The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
[0107]Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.
[0108]Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0109]3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.
[0110]Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.
[0111]Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.
[0112]3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.
[0113]Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0114]Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.
[0115]Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligo-nucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.
[0116]Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.
[0117]Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.
Example 3
RNA Synthesis
[0118]In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5'-hydroxyl in combination with an acid-labile orthoester protecting group on the 2'-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2' hydroxyl.
[0119]Following this procedure for the sequential protection of the 5'-hydroxyl in combination with protection of the 2'-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.
[0120]RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3'- to 5'-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3'-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5'-end of the first nucleoside. The support is washed and any unreacted 5'-hydroxyl groups are capped with acetic anhydride to yield 5'-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5'-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.
[0121]Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2'-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.
[0122]The 2'-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.
[0123]Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand, 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).
[0124]RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.
Example 4
Synthesis of Chimeric Oligonucleotides
[0125]Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate Oligonucleotides
[0126]Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides
[0127][2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy Phosphorothioate]--[2'-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides
[0128][2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy phosphorothioate]-[2'-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
[0129]Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.
Example 5
Design and Screening of Duplexed Antisense Compounds Targeting Endothelial Lipase
[0130]In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target endothelial lipase. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.
[0131]For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 280) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:
##STR00001##
[0132]RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (-20° C.) and freeze-thawed up to 5 times.
[0133]Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate endothelial lipase expression.
[0134]When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.
Example 6
Oligonucleotide Isolation
[0135]After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32+/-48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.
Example 7
Oligonucleotide Synthesis
96 Well Plate Format
[0136]Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
[0137]Oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis
96-Well Plate Format
[0138]The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE® MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE® 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0139]The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.
T-24 Cells:
[0140]The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.
[0141]For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
A549 Cells:
[0142]The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.
NHDF Cells:
[0143]Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.
HEK Cells:
[0144]Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier.
[0145]Cells were routinely maintained for up to 10 passages as recommended by the supplier.
HuVEC Cells:
[0146]The human umbilical vein endothilial cell line HuVEC was obtained from the American Type Culture Collection (Manassas, Va.). HuVEC cells were routinely cultured in EBM (Clonetics Corporation Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence were maintained for up to 15 passages. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/well for use in RT-PCR analysis.
[0147]For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
Primary Mouse Hepatocytes
[0148]Primary mouse hepatocytes were prepared from CD-1 mice purchased from Charles River Labs. Primary mouse hepatocytes were routinely cultured in Hepatoyte Attachment Media (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone (Sigma), 10 nM bovine insulin (Sigma). Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/well for use in RT-PCR analysis.
[0149]For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
Treatment with Antisense Compounds:
[0150]When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM®-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM®-1 containing 3.75 μg/mL LIPOFECTIN® (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
[0151]The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS13920), JNK2 (for ISIS18078) or c-raf (for ISIS15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.
Example 10
Analysis of Oligonucleotide Inhibition of Endothelial Lipase Expression
[0152]Antisense modulation of endothelial lipase expression can be assayed in a variety of ways known in the art. For example, endothelial lipase mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
[0153]Protein levels of endothelial lipase can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to endothelial lipase can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
Example 11
Design of Phenotypic Assays and In Vivo Studies for the Use of Endothelial Lipase Inhibitors
Phenotypic Assays
[0154]Once endothelial lipase inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.
Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of endothelial lipase in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
[0155]In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with endothelial lipase inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
[0156]Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
[0157]Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the endothelial lipase inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
In Vivo Studies
[0158]The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
[0159]The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study.
[0160]To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or endothelial lipase inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a endothelial lipase inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.
[0161]Volunteers receive either the endothelial lipase inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding endothelial lipase or endothelial lipase protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.
[0162]Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.
[0163]Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and endothelial lipase inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the endothelial lipase inhibitor show positive trends in their disease state or condition index at the conclusion of the study.
Example 12
RNA Isolation
[0164]Poly(A)+ mRNA Isolation
[0165]Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
[0166]Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
[0167]Total RNA was isolated using an RNEASY 96® kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96® well plate attached to a QIAVAC® manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96® plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96® plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96® plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC® manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC® manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
[0168]The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of Endothelial Lipase mRNA Levels
[0169]Quantitation of endothelial lipase mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.
[0170]A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM® Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
[0171]Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing).
[0172]Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
[0173]PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
[0174]Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen® (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen® are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
[0175]In this assay, 170 μL of RiboGreen® working reagent (RiboGreen® reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
[0176]Probes and primers to human endothelial lipase were designed to hybridize to a human endothelial lipase sequence, using published sequence information (GenBank accession number NM--006033.1, incorporated herein as SEQ ID NO:4). For human endothelial lipase the PCR primers were:
forward primer: CCGGACGGGAGCTGAATAT (SEQ ID NO: 5)reverse primer: CAGTTTCCGCTGGGTTTCC (SEQ ID NO: 6) and the PCR probe was: FAM-AGGCGCATCCGGGTGAAGTC-TAMRA(SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)reverse primer: GAAGATGGTGATGGGATTTC GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:9) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.
[0177]Probes and primers to mouse endothelial lipase were designed to hybridize to a mouse endothelial lipase sequence, using published sequence information (GenBank accession number BC020991.1, incorporated herein as SEQ ID NO:11). For mouse endothelial lipase the PCR primers were:
forward primer: GCTGAATGCCACAAACACCTT (SEQ ID NO:12)reverse primer: CAGGTAAGTCGCATCTTCAAGAGA (SEQ ID NO: 13) and the PCR probe was: FAM-CTTGTCTACACTGAGGAGGACTTGGGCG-TAMRA(SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15)reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and the PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.
Example 14
Northern Blot Analysis of Endothelial Lipase mRNA Levels
[0178]Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL® (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND®-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER® UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB® hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
[0179]To detect human endothelial lipase, a human endothelial lipase specific probe was prepared by PCR using the forward primer CCGGACGGGAGCTGAATAT (SEQ ID NO: 5) and the reverse primer CAGTTTCCGCTGGGTTTCC (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0180]To detect mouse endothelial lipase, a mouse endothelial lipase specific probe was prepared by PCR using the forward primer GCTGAATGCCACAAACACCTT (SEQ ID NO: 12) and the reverse primer CAGGTAAGTCGCATCTTCAAGAGA (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0181]Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER® and IMAGEQUANT® Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.
Example 15
Antisense Inhibition of Human Endothelial Lipase Expression by Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and a Deoxy Gap
[0182]In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human endothelial lipase RNA, using published sequences (GenBank accession number NM--006033.1, incorporated herein as SEQ ID NO: 4, a genomic sequence of endothelial lipase represented by the complement of residues 3262-78294 of GenBank accession number NT--025012.8, incorporated herein as SEQ ID NO: 18, GenBank accession number AW450414.1, the complement of which is incorporated herein as SEQ ID NO: 19, and GenBank accession number A1676039.1, the complement of which is incorporated herein as SEQ ID NO: 20). The compounds are shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human endothelial lipase mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which HuVEC cells were treated with oligonucleotides 259869-259946 (SEQ ID NOs: 21-98) of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, "N.D." indicates "no data".
TABLE-US-00001 TABLE 1 Inhibition of human endothelial lipase mRNA levels by chimeric phosphorothioate oligonucleotides having 2'- MOE wings and a deoxy gap TARGET SEQ CONTROL ISIS SEQ ID TARGET % ID SEQ ID # REGION NO SITE SEQUENCE INHIB NO NO 259869 5'UTR 4 48 tcaacggcttgccccagaac 61 21 1 259870 5'UTR 4 103 ggtggaaaatgaaaacttgg 11 22 1 259871 5'UTR 4 218 cccctcccaagaaacagaag 46 23 1 259872 Start 4 245 ggagttgctcatcctgcccc 29 24 1 Codon 259873 Coding 4 317 ctctggaccaaaaggtacgg 19 25 1 259874 Coding 4 342 tgtggagcttatcttccagc 70 26 1 259875 Coding 4 382 ctcacagatggtttgacctc 68 27 1 259876 Coding 4 437 gacggagaggtagcatcctt 53 28 1 259877 Coding 4 560 cagggctgacacgagtttgt 56 29 1 259878 Coding 4 641 attgaccgcatccgtgtaaa 63 30 1 259879 Coding 4 700 tccttctcctgcagccagtc 62 31 1 259880 Coding 4 702 cgtccttctcctgcagccag 66 32 1 259881 Coding 4 725 gtggacattcccgagagaaa 66 33 1 259882 Coding 4 747 ctccgaggctgtagccgatc 82 34 1 259883 Coding 4 830 aaacatgggcccggcaggat 53 35 1 259884 Coding 4 988 cagcctggctggaagtcacc 60 36 1 259885 Coding 4 1099 tcctgattcaccagagagtc 79 37 1 259886 Coding 4 1104 gcttgtcctgattcaccaga 84 38 1 259887 Coding 4 1112 aaaactcggcttgtcctgat 75 39 1 259888 Coding 4 1124 gcactggaaggcaaaactcg 84 40 1 259889 Coding 4 1142 gaagcgattggagtcagtgc 67 41 1 259890 Coding 4 1266 tgaaaggcatgcctgcccgg 82 42 1 259891 Coding 4 1283 ctgataatggtaaactctga 67 43 1 259892 Coding 4 1312 ttcttgtaactgaagacatg 82 44 1 259893 Coding 4 1343 gacgtaaaaggtgggctcaa 73 45 1 259894 Coding 4 1398 gctccactatttccagtggc 60 46 1 259895 Coding 4 1427 gaaggtgttggtggcattct 53 47 1 259896 Coding 4 1459 aggtctcccaagtcctcctc 56 48 1 259897 Coding 4 1491 aggccccctcccaggtgagc 21 49 1 259898 Coding 4 1532 gtagctgcgaaactccttcc 48 50 1 259899 Coding 4 1571 gatattcagctcccgtccgg 70 51 1 259900 Coding 4 1619 aaatgtcagtttccgctggg 79 52 1 259901 Coding 4 1641 tgttctcagggtcttctgta 55 53 1 259902 Coding 4 1724 cacagtgggactggtttcgt 66 54 1 259903 Stop 4 1743 ggcaccctcagggaagctcc 83 55 1 Codon 259904 3'UTR 4 1771 ttgctgccttgctggcaaga 51 56 1 259905 3'UTR 4 1817 gtcctcagcagtaactttcc 58 57 1 259906 3'UTR 4 1966 acagaggtttggagttagag 68 58 1 259907 3'UTR 4 2044 ccaatccagtgtgcacgaga 83 59 1 259908 3'UTR 4 2107 gggcctcttcggagccagcg 61 60 1 259909 3'UTR 4 2241 caacccatgagaaccccaac 36 61 1 259910 3'UTR 4 2279 aggacggaatggctaagacg 57 62 1 259911 3'UTR 4 2361 caatagacatttgctcaatt 82 63 1 259912 3'UTR 4 2438 atcccaactccactgggttc 12 64 1 259913 3'UTR 4 2496 aggtgcctttccccatgcat 63 65 1 259914 3'UTR 4 2527 tgcttatatcctatagcctc 70 66 1 259915 3'UTR 4 2552 ccacttaaagcctcagggtc 55 67 1 259916 3'UTR 4 2635 tagtgatcaaacacgtcact 85 68 1 259917 3'UTR 4 2752 acatatgtcataacttctat 68 69 1 259918 3'UTR 4 2873 caacaagaaaaggcactggt 78 70 1 259919 3'UTR 4 2918 ttaattcaaatcaagatcta 34 71 1 259920 3'UTR 4 2980 ctaattaggcaatgcataca 69 72 1 259921 3'UTR 4 3039 ctcactaagcctcagttttg 69 73 1 259922 3'UTR 4 3085 tggctgcaccactaactagt 62 74 1 259923 3'UTR 4 3118 gactttgcctccaggaaatc 40 75 1 259924 3'UTR 4 3151 ggctgcaaaagtcttcatgg 57 76 1 259925 3'UTR 4 3218 atggatggctctatataaaa 38 77 1 259926 3'UTR 4 3237 taaaagggcttaggatttta 68 78 1 259927 3'UTR 4 3264 acagatgttctcctggttat 76 79 1 259928 3'UTR 4 3285 ataaaaagtccaaccgttgg 64 80 1 259929 3'UTR 4 3301 ctcccgaatctcagccataa 86 81 1 259930 3'UTR 4 3325 ctctcctgcttggtgtcaca 61 82 1 259931 3'UTR 4 3335 atcattcttcctctcctgct 80 83 1 259932 3'UTR 4 3386 ggcacgatacagattaaaac 85 84 1 259933 3'UTR 4 3410 gaagtttaacagtgatacaa 76 85 1 259934 3'UTR 4 3441 ttgaaataagactcaactgg 74 86 1 259935 3'UTR 4 3796 ccaaaactttttgagcacca 71 87 1 259936 Intron 18 12206 agaccttttactttttgcaa 22 88 1 259937 Intron 18 28424 cctagcctgggaacccaaac 19 89 1 259938 Intron: 18 37297 ggatccaaacctgcagcaga 8 90 1 exon junction 259939 Intron: 18 47525 tgaaggttacctctgaaagg 5 91 1 exon junction 259940 Intron: 18 48229 taatggtaaactgcagtgac 53 92 1 exon junction 259941 Intron: 18 49641 aggcacttacttccgctggg 61 93 1 exon junction 259942 Intron 18 50496 gtcatagatgacgaatgtaa 72 94 1 259943 Intron 18 55242 gaattctgccagcagactgc 12 95 1 259944 Intron 18 29605 ttcttaagaagattgggttt 44 96 1 259945 3'UTR 20 105 atatacaattaaggcttcaa 19 97 1 259946 3'UTR 20 114 agtatcattatatacaatta 0 98 1
[0183]As shown in Table 1, SEQ ID NOs 21, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 65, 66, 67, 68, 69, 70, 72, 73, 74, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 92, 93 and 94 demonstrated at least 50% inhibition of human endothelial lipase expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 38, 68 and 84. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found.
Example 16
Antisense Inhibition of Mouse Endothelial Lipase Expression by Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and a Deoxy Gap
[0184]In accordance with the present invention, a second series of antisense compounds were designed to target different regions of the mouse endothelial lipase RNA, using published sequences (GenBank accession number BC020991.1, incorporated herein as SEQ ID NO: 11, and GenBank accession number AF118768.1, incorporated herein as SEQ ID NO: 99). The compounds are shown in Table 2. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse endothelial lipase mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which mouse primary hepatocytes were treated with oligonucleotides 261160-261233 (SEQ ID NOs: 100-173) of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, "N.D." indicates "no data".
TABLE-US-00002 TABLE 2 Inhibition of mouse endothelial lipase mRNA levels by chimeric phosphorothioate oligonucleotides having 2'- MOE wings and a deoxy gap TARGET SEQ CONTROL ISIS SEQ ID TARGET % ID SEQ ID # REGION NO SITE SEQUENCE INHIB NO NO 261160 5'UTR 99 9 ttcccagagccgctgcgaag 65 100 1 261161 5'UTR 99 93 gaacctggaaggttggtagc 60 101 1 261162 5'UTR 99 158 cctccagagactagaagtgg 39 102 1 261163 5'UTR 99 165 actaaaacctccagagacta 61 103 1 261164 Start 99 267 cgtgtttcgcatccttcccc 80 104 1 Codon 261165 Coding 99 328 aggttgtgatacttcccgcc 60 105 1 261166 Coding 99 356 tcatctcgcagcgacccctg 47 106 1 261167 Coding 99 381 tggtactccagtgggtttat 65 107 1 261168 Coding 99 418 tgcggatgttaaaagccaca 50 108 1 261169 Coding 99 465 gtcaccaagggagagattac 68 109 1 261170 Coding 99 489 gccacagttttctaagagtt 57 110 1 261171 Coding 99 666 gttattgactgcatccgtgt 57 111 1 261172 Coding 99 674 accctggtgttattgactgc 64 112 1 261173 Coding 99 708 ccagtcaagcatcccagcta 54 113 1 261174 Coding 99 741 gttcccaagagagaactctt 30 114 1 261175 Coding 99 777 cacgtgtgctccaaggctgt 50 115 1 261176 Coding 99 907 catccacaaagtctgcatcg 59 116 1 261177 Coding 99 912 caggacatccacaaagtctg 46 117 1 261178 Coding 99 917 gtatgcaggacatccacaaa 34 118 1 261179 Coding 99 922 tgtaggtatgcaggacatcc 45 119 1 261180 Coding 99 927 cagcgtgtaggtatgcagga 66 120 1 261181 Coding 99 932 aaggacagcgtgtaggtatg 9 121 1 261182 Coding 99 937 agccaaaggacagcgtgtag 50 122 1 261183 Coding 99 942 gctcaagccaaaggacagcg 60 123 1 261184 Coding 99 947 ccaatgctcaagccaaagga 63 124 1 261185 Coding 99 952 gaatcccaatgctcaagcca 49 125 1 261186 Coding 99 957 catccgaatcccaatgctca 48 126 1 261187 Coding 99 980 tagatgtcaatgtgacccac 43 127 1 261188 Coding 99 985 tgggatagatgtcaatgtga 35 128 1 261189 Coding 99 991 cgccattgggatagatgtca 0 129 1 261190 Coding 99 1068 tttcaccatctctgagattg 54 130 1 261191 Coding 99 1098 aaagaggtgtacggctcgct 51 131 1 261192 Coding 99 1103 tcgacaaagaggtgtacggc 73 132 1 261193 Coding 99 1108 gagagtcgacaaagaggtgt 46 133 1 261194 Coding 99 1113 caccagagagtcgacaaaga 56 134 1 261195 Coding 99 1178 cagattccccttttgaagcg 65 135 1 261196 Coding 99 1214 ccaatgttattacaacggtt 63 136 1 261197 Coding 99 1318 tgtgaactttcagctggtaa 71 137 1 261198 Coding 99 1328 taagagaacatgtgaacttt 57 138 1 261199 Coding 99 1341 cccactgttattgtaagaga 59 139 1 261200 Coding 99 1412 atttccaagggcaggttctg 72 140 1 261201 Coding 99 1460 gtgtagacaaggaaggtgtt 79 141 1 261202 Coding 99 1482 gagatcgcccaagtcctcct 85 142 1 261203 Coding 99 1499 gtaagtcgcatcttcaagag 82 143 1 261204 Coding 99 1512 taccccctcccaggtaagtc 30 144 1 261205 Coding 99 1521 ggaatgggctaccccctccc 38 145 1 261206 Coding 99 1542 ctcattccacaggttgcacc 28 146 1 261207 Coding 99 1624 gggtttccccagatttgaca 56 147 1 261208 Coding 99 1703 cacttgtgaaaccacagctc 62 148 1 261209 Coding 99 1743 aaagggactggttttgtttt 38 149 1 261210 Stop 99 1766 ttgggccctcaggccaagtt 10 150 1 Codon 261211 3'UTR 99 1885 ccagcaagcaagctcctcgt 28 151 1 261212 3'UTR 99 1956 tcagcaagaacttcagcagt 56 152 1 261213 3'UTR 99 1978 caaaggtttacagctagaat 43 153 1 261214 3'UTR 99 1994 cttcctgcggcggcaacaaa 63 154 1 261215 3'UTR 99 2017 gctcacacacaagctggcct 33 155 1 261216 3'UTR 99 2036 gggctctggacactccagtg 33 156 1 261217 3'UTR 99 2085 gctcacctagcgacagggag 74 157 1 261218 3'UTR 99 2118 gccttgtgttccctgatgtt 60 158 1 261219 3'UTR 99 2136 acacagggccacttcagagc 64 159 1 261220 3'UTR 99 2141 cttccacacagggccacttc 7 160 1 261221 3'UTR 99 2167 ttcagtgaggccaggcagct 65 161 1 261222 3'UTR 99 2172 taaggttcagtgaggccagg 54 162 1 261223 3'UTR 99 2182 gacttgtcactaaggttcag 66 163 1 261224 3'UTR 11 2150 aatagaaccaggatccatca 37 164 1 261225 3'UTR 11 2480 tctgctagagatcaagggtg 51 165 1 261226 3'UTR 11 2583 gcgcagcaggtatgtagaac 68 166 1 261227 3'UTR 11 2760 tcaaactactaaagggtgtc 65 167 1 261228 3'UTR 11 2920 ccaggaaaccttgctgggtc 69 168 1 261229 3'UTR 11 2955 atggagttacagaaaggatt 48 169 1 261230 3'UTR 11 3184 acagatgcaaagaatgtgcg 57 170 1 261231 3'UTR 11 3201 tataaagctggtacaataca 51 171 1 261232 3'UTR 11 3530 aaaactaaccatagatttgt 43 172 1 261233 3'UTR 11 3559 aaatcttgaaatcggttaat 49 173 1
[0185]As shown in Table 2, SEQ ID NOs 100, 101, 103, 104, 105, 107, 108, 109, 110, 111, 112, 113, 115, 116, 120, 122, 123, 124, 130, 131, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 147, 148, 152, 154, 157, 158, 159, 161, 162, 163, 165, 166, 167, 168, 170 and 171 demonstrated at least 50% inhibition of mouse endothelial lipase expression in this experiment and are therefore preferred. More preferred are SEQ ID NOs: 104, 132 and 157. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found.
TABLE-US-00003 TABLE 3 Sequence and position of preferred target segments identified in endothelial lipase. TARGET REV SEQ SEQ ID TARGET COMP OF ID SITEID NO SITE SEQUENCE SEQ ID ACTIVE IN NO 176327 4 48 gttctggggcaagccgttga 21 H. sapiens 174 176332 4 342 gctggaagataagctccaca 26 H. sapiens 175 176333 4 382 gaggtcaaaccatctgtgag 27 H. sapiens 176 176334 4 437 aaggatgctacctctccgtc 28 H. sapiens 177 176335 4 560 acaaactcgtgtcagccctg 29 H. sapiens 178 176336 4 641 tttacacggatgcggtcaat 30 H. sapiens 179 176337 4 700 gactggctgcaggagaagga 31 H. sapiens 180 176338 4 702 ctggctgcaggagaaggacg 32 H. sapiens 181 176339 4 725 tttctctcgggaatgtccac 33 H. sapiens 182 176340 4 747 gatcggctacagcctcggag 34 H. sapiens 183 176341 4 830 atcctgccgggcccatgttt 35 H. sapiens 184 176342 4 988 ggtgacttccagccaggctg 36 H. sapiens 185 176343 4 1099 gactctctggtgaatcagga 37 H. sapiens 186 176344 4 1104 tctggtgaatcaggacaagc 38 H. sapiens 187 176345 4 1112 atcaggacaagccgagtttt 39 H. sapiens 188 176346 4 1124 cgagttttgccttccagtgc 40 H. sapiens 189 176347 4 1142 gcactgactccaatcgcttc 41 H. sapiens 190 176348 4 1266 ccgggcaggcatgcctttca 42 H. sapiens 191 176349 4 1283 tcagagtttaccattatcag 43 H. sapiens 192 176350 4 1312 catgtcttcagttacaagaa 44 H. sapiens 193 176351 4 1343 ttgagcccaccttttacgtc 45 H. sapiens 194 176352 4 1398 gccactggaaatagtggagc 46 H. sapiens 195 176353 4 1427 agaatgccaccaacaccttc 47 H. sapiens 196 176354 4 1459 gaggaggacttgggagacct 48 H. sapiens 197 176357 4 1571 ccggacgggagctgaatatc 51 H. sapiens 198 176358 4 1619 cccagcggaaactgacattt 52 H. sapiens 199 176359 4 1641 tacagaagaccctgagaaca 53 H. sapiens 200 176360 4 1724 acgaaaccagtcccactgtg 54 H. sapiens 201 176361 4 1743 ggagcttccctgagggtgcc 55 H. sapiens 202 176362 4 1771 tcttgccagcaaggcagcaa 56 H. sapiens 203 176363 4 1817 ggaaagttactgctgaggac 57 H. sapiens 204 176364 4 1966 ctctaactccaaacctctgt 58 H. sapiens 205 176365 4 2044 tctcgtgcacactggattgg 59 H. sapiens 206 176366 4 2107 cgctggctccgaagaggccc 60 H. sapiens 207 176368 4 2279 cgtcttagccattccgtcct 62 H. sapiens 208 176369 4 2361 aattgagcaaatgtctattg 63 H. sapiens 209 176371 4 2496 atgcatggggaaaggcacct 65 H. sapiens 210 176372 4 2527 gaggctataggatataagca 66 H. sapiens 211 176373 4 2552 gaccctgaggctttaagtgg 67 H. sapiens 212 176374 4 2635 agtgacgtgtttgatcacta 68 H. sapiens 213 176375 4 2752 atagaagttatgacatatgt 69 H. sapiens 214 176376 4 2873 accagtgccttttcttgttg 70 H. sapiens 215 176378 4 2980 tgtatgcattgcctaattag 72 H. sapiens 216 176379 4 3039 caaaactgaggcttagtgag 73 H. sapiens 217 176380 4 3085 actagttagtggtgcagcca 74 H. sapiens 218 176382 4 3151 ccatgaagacttttgcagcc 76 H. sapiens 219 176384 4 3237 taaaatcctaagccctttta 78 H. sapiens 220 176385 4 3264 ataaccaggagaacatctgt 79 H. sapiens 221 176386 4 3285 ccaacggttggactttttat 80 H. sapiens 222 176387 4 3301 ttatggctgagattcgggag 81 H. sapiens 223 176388 4 3325 tgtgacaccaagcaggagag 82 H. sapiens 224 176389 4 3335 agcaggagaggaagaatgat 83 H. sapiens 225 176390 4 3386 gttttaatctgtatcgtgcc 84 H. sapiens 226 176391 4 3410 ttgtatcactgttaaacttc 85 H. sapiens 227 176392 4 3441 ccagttgagtcttatttcaa 86 H. sapiens 228 176393 4 3796 tggtgctcaaaaagttttgg 87 H. sapiens 229 176398 18 48229 gtcactgcagtttaccatta 92 H. sapiens 230 176399 18 49641 cccagcggaagtaagtgcct 93 H. sapiens 231 176400 18 50496 ttacattcgtcatctatgac 94 H. sapiens 232 177667 99 9 cttcgcagcggctctgggaa 100 M. musculus 233 177668 99 93 gctaccaaccttccaggttc 101 M. musculus 234 177670 99 165 tagtctctggaggttttagt 103 M. musculus 235 177671 99 267 ggggaaggatgcgaaacacg 104 M. musculus 236 177672 99 328 ggcgggaagtatcacaacct 105 M. musculus 237 177674 99 381 ataaacccactggagtacca 107 M. musculus 238 177675 99 418 tgtggcttttaacatccgca 108 M. musculus 239 177676 99 465 gtaatctctcccttggtgac 109 M. musculus 240 177677 99 489 aactcttagaaaactgtggc 110 M. musculus 241 177678 99 666 acacggatgcagtcaataac 111 M. musculus 242 177679 99 674 gcagtcaataacaccagggt 112 M. musculus 243 177680 99 708 tagctgggatgcttgactgg 113 M. musculus 244 177682 99 777 acagccttggagcacacgtg 115 M. musculus 245 177683 99 907 cgatgcagactttgtggatg 116 M. musculus 246 177687 99 927 tcctgcatacctacacgctg 120 M. musculus 247 177689 99 937 ctacacgctgtcctttggct 122 M. musculus 248 177690 99 942 cgctgtcctttggcttgagc 123 M. musculus 249 177691 99 947 tcctttggcttgagcattgg 124 M. musculus 250 177696 99 1068 caatctcagagatggtgaaa 130 M. musculus 251 177697 99 1098 agcgagccgtacacctcttt 131 M. musculus 252 177698 99 1103 gccgtacacctctttgtcga 132 M. musculus 253 177700 99 1113 tctttgtcgactctctggtg 134 M. musculus 254 177701 99 1178 cgcttcaaaaggggaatctg 135 M. musculus 255 177702 99 1214 aaccgttgtaataacattgg 136 M. musculus 256 177703 99 1318 ttaccagctgaaagttcaca 137 M. musculus 257 177704 99 1328 aaagttcacatgttctctta 138 M. musculus 258 177705 99 1341 tctcttacaataacagtggg 139 M. musculus 259 177706 99 1412 cagaacctgcccttggaaat 140 M. musculus 260 177707 99 1460 aacaccttccttgtctacac 141 M. musculus 261 177708 99 1482 aggaggacttgggcgatctc 142 M. musculus 262 177709 99 1499 ctcttgaagatgcgacttac 143 M. musculus 263 177713 99 1624 tgtcaaatctggggaaaccc 147 M. musculus 264 177714 99 1703 gagctgtggtttcacaagtg 148 M. musculus 265 177718 99 1956 actgctgaagttcttgctga 152 M. musculus 266 177720 99 1994 tttgttgccgccgcaggaag 154 M. musculus 267 177723 99 2085 ctccctgtcgctaggtgagc 157 M. musculus 268 177724 99 2118 aacatcagggaacacaaggc 158 M. musculus 269 177725 99 2136 gctctgaagtggccctgtgt 159 M. musculus 270 177727 99 2167 agctgcctggcctcactgaa 161 M. musculus 271 177728 99 2172 cctggcctcactgaacctta 162 M. musculus 272 177729 99 2182 ctgaaccttagtgacaagtc 163 M. musculus 273 177731 11 2480 cacccttgatctctagcaga 165 M. musculus 274 177732 11 2583 gttctacatacctgctgcgc 166 M. musculus 275 177733 11 2760 gacaccctttagtagtttga 167 M. musculus 276 117734 11 2920 gacccagcaaggtttcctgg 168 M. musculus 277 177736 11 3184 cgcacattctttgcatctgt 170 M. musculus 278 177737 11 3201 tgtattgtaccagctttata 171 M. musculus 279
[0186]As these "preferred target segments" have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of endothelial lipase.
[0187]According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
Example 17
Western Blot Analysis of Endothelial Lipase Protein Levels
[0188]Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to endothelial lipase is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER® (Molecular Dynamics, Sunnyvale Calif.).
Sequence CWU
1
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 282
<210> SEQ ID NO 1
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 1
tccgtcatcg ctcctcaggg 20
<210> SEQ ID NO 2
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 2
gtgcgcgcga gcccgaaatc 20
<210> SEQ ID NO 3
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 3
atgcattctg cccccaagga 20
<210> SEQ ID NO 4
<211> LENGTH: 3927
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (253)...(1755)
<400> SEQUENCE: 4
agcagcgagt ccttgcctcc cggcggctca ggacgagggc agatctcgtt ctggggcaag 60
ccgttgacac tcgctccctg ccaccgcccg ggctccgtgc cgccaagttt tcattttcca 120
ccttctctgc ctccagtccc ccagcccctg gccgagagaa gggtcttacc ggccgggatt 180
gctggaaaca ccaagaggtg gtttttgttt tttaaaactt ctgtttcttg ggagggggtg 240
tggcggggca gg atg agc aac tcc gtt cct ctg ctc tgt ttc tgg agc ctc 291
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu
1 5 10
tgc tat tgc ttt gct gcg ggg agc ccc gta cct ttt ggt cca gag gga 339
Cys Tyr Cys Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly
15 20 25
cgg ctg gaa gat aag ctc cac aaa ccc aaa gct aca cag act gag gtc 387
Arg Leu Glu Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val
30 35 40 45
aaa cca tct gtg agg ttt aac ctc cgc acc tcc aag gac cca gag cat 435
Lys Pro Ser Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His
50 55 60
gaa gga tgc tac ctc tcc gtc ggc cac agc cag ccc tta gaa gac tgc 483
Glu Gly Cys Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys
65 70 75
agt ttc aac atg aca gct aaa acc ttt ttc atc att cac gga tgg acg 531
Ser Phe Asn Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr
80 85 90
atg agc ggt atc ttt gaa aac tgg ctg cac aaa ctc gtg tca gcc ctg 579
Met Ser Gly Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu
95 100 105
cac aca aga gag aaa gac gcc aat gta gtt gtg gtt gac tgg ctc ccc 627
His Thr Arg Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro
110 115 120 125
ctg gcc cac cag ctt tac acg gat gcg gtc aat aat acc agg gtg gtg 675
Leu Ala His Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val
130 135 140
gga cac agc att gcc agg atg ctc gac tgg ctg cag gag aag gac gat 723
Gly His Ser Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp
145 150 155
ttt tct ctc ggg aat gtc cac ttg atc ggc tac agc ctc gga gcg cac 771
Phe Ser Leu Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His
160 165 170
gtg gcc ggg tat gca ggc aac ttc gtg aaa gga acg gtg ggc cga atc 819
Val Ala Gly Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile
175 180 185
aca ggt ttg gat cct gcc ggg ccc atg ttt gaa ggg gcc gac atc cac 867
Thr Gly Leu Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His
190 195 200 205
aag agg ctc tct ccg gac gat gca gat ttt gtg gat gtc ctc cac acc 915
Lys Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr
210 215 220
tac acg cgt tcc ttc ggc ttg agc att ggt att cag atg cct gtg ggc 963
Tyr Thr Arg Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly
225 230 235
cac att gac atc tac ccc aat ggg ggt gac ttc cag cca ggc tgt gga 1011
His Ile Asp Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly
240 245 250
ctc aac gat gtc ttg gga tca att gca tat gga aca atc aca gag gtg 1059
Leu Asn Asp Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val
255 260 265
gta aaa tgt gag cat gag cga gcc gtc cac ctc ttt gtt gac tct ctg 1107
Val Lys Cys Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu
270 275 280 285
gtg aat cag gac aag ccg agt ttt gcc ttc cag tgc act gac tcc aat 1155
Val Asn Gln Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn
290 295 300
cgc ttc aaa aag ggg atc tgt ctg agc tgc cgc aag aac cgt tgt aat 1203
Arg Phe Lys Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn
305 310 315
agc att ggc tac aat gcc aag aaa atg agg aac aag agg aac agc aaa 1251
Ser Ile Gly Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys
320 325 330
atg tac cta aaa acc cgg gca ggc atg cct ttc aga gtt tac cat tat 1299
Met Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe Arg Val Tyr His Tyr
335 340 345
cag atg aaa atc cat gtc ttc agt tac aag aac atg gga gaa att gag 1347
Gln Met Lys Ile His Val Phe Ser Tyr Lys Asn Met Gly Glu Ile Glu
350 355 360 365
ccc acc ttt tac gtc acc ctt tat ggc act aat gca gat tcc cag act 1395
Pro Thr Phe Tyr Val Thr Leu Tyr Gly Thr Asn Ala Asp Ser Gln Thr
370 375 380
ctg cca ctg gaa ata gtg gag cgg atc gag cag aat gcc acc aac acc 1443
Leu Pro Leu Glu Ile Val Glu Arg Ile Glu Gln Asn Ala Thr Asn Thr
385 390 395
ttc ctg gtc tac acc gag gag gac ttg gga gac ctc ttg aag atc cag 1491
Phe Leu Val Tyr Thr Glu Glu Asp Leu Gly Asp Leu Leu Lys Ile Gln
400 405 410
ctc acc tgg gag ggg gcc tct cag tct tgg tac aac ctg tgg aag gag 1539
Leu Thr Trp Glu Gly Ala Ser Gln Ser Trp Tyr Asn Leu Trp Lys Glu
415 420 425
ttt cgc agc tac ctg tct caa ccc cgc aac ccc gga cgg gag ctg aat 1587
Phe Arg Ser Tyr Leu Ser Gln Pro Arg Asn Pro Gly Arg Glu Leu Asn
430 435 440 445
atc agg cgc atc cgg gtg aag tct ggg gaa acc cag cgg aaa ctg aca 1635
Ile Arg Arg Ile Arg Val Lys Ser Gly Glu Thr Gln Arg Lys Leu Thr
450 455 460
ttt tgt aca gaa gac cct gag aac acc agc ata tcc cca ggc cgg gag 1683
Phe Cys Thr Glu Asp Pro Glu Asn Thr Ser Ile Ser Pro Gly Arg Glu
465 470 475
ctc tgg ttt cgc aag tgt cgg gat ggc tgg agg atg aaa aac gaa acc 1731
Leu Trp Phe Arg Lys Cys Arg Asp Gly Trp Arg Met Lys Asn Glu Thr
480 485 490
agt ccc act gtg gag ctt ccc tga gggtgcccgg gcaagtcttg ccagcaaggc 1785
Ser Pro Thr Val Glu Leu Pro
495 500
agcaagactt cctgctatcc aagcccatgg aggaaagtta ctgctgagga cccacccaat 1845
ggaaggattc ttctcagcct tgaccctgga gcactgggaa caactggtct cctgtgatgg 1905
ctgggactcc tcgcgggagg ggactgcgct gctatagctc ttgctgcctc tcttgaatag 1965
ctctaactcc aaacctctgt ccacacctcc agagcaccaa gtccagattt gtgtgtaagc 2025
agctgggtgc ctggggcctc tcgtgcacac tggattggtt tctcagttgc tgggcgagcc 2085
tgtactctgc ctgacgagga acgctggctc cgaagaggcc ctgtgtagaa ggctgtcagc 2145
tgctcagcct gctttgagcc tcagtgagaa gtccttccga caggagctga ctcatgtcag 2205
gatggcaggc ctggtatctt gctcgggccc tagctgttgg ggttctcatg ggttgcactg 2265
accatactgc ttacgtctta gccattccgt cctgctcccc agctcactct ctgaagcaca 2325
catcattggc tttcctattt ttctgttcat tttttaattg agcaaatgtc tattgaacac 2385
ttaaaattaa ttagaatgtg gtaatggaca tattactgag cctctccatt tggaacccag 2445
tggagttggg atttctagac cctctttctg tttggatggt gtatgtgtat atgcatgggg 2505
aaaggcacct ggggcctggg ggaggctata ggatataagc attagggacc ctgaggcttt 2565
aagtggtttc tatttcttct tagttattat gtgccacctt cttagttatt atgtgccacc 2625
tcccctatga gtgacgtgtt tgatcactag cagaatagca agcagagtat cattcatgct 2685
ggggccagaa tgatggccgg ttgccagata taactgcttt ggagcaaatc tcttctgttt 2745
agagagatag aagttatgac atatgtaata cacatctgtg tacacagaaa ccggcacctg 2805
ccagacagag ctggttctaa gatttaatac agtgcttttt ttcctctttg aaatatttta 2865
ctttaatacc agtgcctttt cttgttgaac ttcttggaaa agccaccaat tctagatctt 2925
gatttgaatt aatacacaca atatctgaga cacttacact tttcaaaaga tttgtgtatg 2985
cattgcctaa ttagagtagg gggagaaggg caactattat tatccctatt ttacaaaact 3045
gaggcttagt gaggttcagc cacatgccta gacttatata ctagttagtg gtgcagccag 3105
ggagaggact cagatttcct ggaggcaaag tctatctctg aaactccatg aagacttttg 3165
cagccagttc ccaccaatat gccccagacg tgagacaaac aaggactttt ttttttatat 3225
agagccatcc ataaaatcct aagccctttt attaatgtat aaccaggaga acatctgtgc 3285
caacggttgg actttttatg gctgagattc gggaggaagt gtgacaccaa gcaggagagg 3345
aagaatgatt ttctttgtac ttaggttttc taaggacatt gttttaatct gtatcgtgcc 3405
aaagttgtat cactgttaaa cttctgaaga cataaccagt tgagtcttat ttcaagatat 3465
gttctcaagc caattgtgtg cttctcttgt ttctgtgatt gctttctagc caaagcgaag 3525
cttgtacagg ttgagtatcc cttatccaaa atgcttggaa ccagaagtgt ttcaaatttt 3585
agattatttt cagattttgg aatgtttgca tatacataat gagatatttt gggaatagga 3645
cccgagccta aacacaaaat tcattgatgt gtcagttaca ccttatccac atagcctgag 3705
ggtaatttta tacgatattt taaatagttg tgtacatgaa gcatggtttg tggtaactta 3765
tgtgaggggt tttcccattt tttgtcttgt tggtgctcaa aaagttttgg attttggagc 3825
atttcggatt ttggattttt ggattagggt tgctcaaccc atattattgg ctgtacatcc 3885
tggtcacttc tgacttctgt ttttactaat ggaagctttg ca 3927
<210> SEQ ID NO 5
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 5
ccggacggga gctgaatat 19
<210> SEQ ID NO 6
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 6
cagtttccgc tgggtttcc 19
<210> SEQ ID NO 7
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Probe
<400> SEQUENCE: 7
aggcgcatcc gggtgaagtc 20
<210> SEQ ID NO 8
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 8
gaaggtgaag gtcggagtc 19
<210> SEQ ID NO 9
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 9
gaagatggtg atgggatttc 20
<210> SEQ ID NO 10
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Probe
<400> SEQUENCE: 10
caagcttccc gttctcagcc 20
<210> SEQ ID NO 11
<211> LENGTH: 3610
<212> TYPE: DNA
<213> ORGANISM: Mus musculus
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (144)...(1646)
<400> SEQUENCE: 11
ccacgcgtcc gcgacacccg gcctgtccac ttctagtctc tggaggtttt agtggtttca 60
aaccaaacca acccaaacca acccaacaac aaaaaagccc aaaccaaaaa cctgcttgag 120
aggagggggc gtggcgggga agg atg cga aac acg gtt ttc ctg ctc ggc ttt 173
Met Arg Asn Thr Val Phe Leu Leu Gly Phe
1 5 10
tgg agc gtc tat tgt tac ttc ccg gcg gga agt atc aca acc ttg cgt 221
Trp Ser Val Tyr Cys Tyr Phe Pro Ala Gly Ser Ile Thr Thr Leu Arg
15 20 25
ccc gag ggg tcg ctg cga gat gag cat cat aaa ccc act gga gta cca 269
Pro Glu Gly Ser Leu Arg Asp Glu His His Lys Pro Thr Gly Val Pro
30 35 40
gct acc gcc aga ccc tct gtg gct ttt aac atc cgc act tct aag gac 317
Ala Thr Ala Arg Pro Ser Val Ala Phe Asn Ile Arg Thr Ser Lys Asp
45 50 55
cca gag cag gaa ggg tgt aat ctc tcc ctt ggt gac agc aaa ctc tta 365
Pro Glu Gln Glu Gly Cys Asn Leu Ser Leu Gly Asp Ser Lys Leu Leu
60 65 70
gaa aac tgt ggc ttc aac atg aca gcc aaa acc ttc ttc atc att cat 413
Glu Asn Cys Gly Phe Asn Met Thr Ala Lys Thr Phe Phe Ile Ile His
75 80 85 90
gga tgg acg atg agt ggc atg ttt gag agc tgg ctg cat aaa ctt gta 461
Gly Trp Thr Met Ser Gly Met Phe Glu Ser Trp Leu His Lys Leu Val
95 100 105
tca gcc ctg cag atg aga gag aaa gat gct aac gtc gtg gtg gtt gac 509
Ser Ala Leu Gln Met Arg Glu Lys Asp Ala Asn Val Val Val Val Asp
110 115 120
tgg ctg ccc ctg gct cat cag ctg tac acg gat gca gtc aat aac acc 557
Trp Leu Pro Leu Ala His Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr
125 130 135
agg gtg gtg gga cag aga gta gct ggg atg ctt gac tgg ctg cag gag 605
Arg Val Val Gly Gln Arg Val Ala Gly Met Leu Asp Trp Leu Gln Glu
140 145 150
aag gaa gag ttc tct ctt ggg aac gtt cac ttg att ggc tac agc ctt 653
Lys Glu Glu Phe Ser Leu Gly Asn Val His Leu Ile Gly Tyr Ser Leu
155 160 165 170
gga gca cac gtg gct gga tac gct ggc aac ttt gtg aaa gga aca gtg 701
Gly Ala His Val Ala Gly Tyr Ala Gly Asn Phe Val Lys Gly Thr Val
175 180 185
ggc agg atc act ggt ctg gat ccc gcg ggt ccc atg ttt gaa ggg gtg 749
Gly Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Met Phe Glu Gly Val
190 195 200
gac atc aac aga agg ctg tcc ccg gac gat gca gac ttt gtg gat gtc 797
Asp Ile Asn Arg Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val
205 210 215
ctg cat acc tac acg ctg tcc ttt ggc ttg agc att ggg att cgg atg 845
Leu His Thr Tyr Thr Leu Ser Phe Gly Leu Ser Ile Gly Ile Arg Met
220 225 230
cct gtg ggt cac att gac atc tat ccc aat ggc ggt gac ttc cag cca 893
Pro Val Gly His Ile Asp Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro
235 240 245 250
ggc tgt gga ttc aat gat gtc atc gga tct ttt gca tat gga aca atc 941
Gly Cys Gly Phe Asn Asp Val Ile Gly Ser Phe Ala Tyr Gly Thr Ile
255 260 265
tca gag atg gtg aaa tgc gag cac gag cga gcc gta cac ctc ttt gtc 989
Ser Glu Met Val Lys Cys Glu His Glu Arg Ala Val His Leu Phe Val
270 275 280
gac tct ctg gtg aat cag gac aag ccc agc ttt gcc ttc cag tgc aca 1037
Asp Ser Leu Val Asn Gln Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr
285 290 295
gac tcc agc cgc ttc aaa agg gga atc tgc ctc agc tgc cgg aag aac 1085
Asp Ser Ser Arg Phe Lys Arg Gly Ile Cys Leu Ser Cys Arg Lys Asn
300 305 310
cgt tgt aat aac att ggc tac aac gcc aag aaa atg aga aag aag agg 1133
Arg Cys Asn Asn Ile Gly Tyr Asn Ala Lys Lys Met Arg Lys Lys Arg
315 320 325 330
aat agc aaa atg tat tta aaa acc cgg gct ggc atg cct ttc aaa gtt 1181
Asn Ser Lys Met Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe Lys Val
335 340 345
tac cat tac cag ctg aaa gtt cac atg ttc tct tac aat aac agt ggg 1229
Tyr His Tyr Gln Leu Lys Val His Met Phe Ser Tyr Asn Asn Ser Gly
350 355 360
gac acc cag ccc acc ctc tac att acc ctg tat ggt agc aac gca gac 1277
Asp Thr Gln Pro Thr Leu Tyr Ile Thr Leu Tyr Gly Ser Asn Ala Asp
365 370 375
tcc cag aac ctg ccc ttg gaa ata gtg gag aag att gag ctg aat gcc 1325
Ser Gln Asn Leu Pro Leu Glu Ile Val Glu Lys Ile Glu Leu Asn Ala
380 385 390
aca aac acc ttc ctt gtc tac act gag gag gac ttg ggc gat ctc ttg 1373
Thr Asn Thr Phe Leu Val Tyr Thr Glu Glu Asp Leu Gly Asp Leu Leu
395 400 405 410
aag atg cga ctt acc tgg gag ggg gta gcc cat tcc tgg tac aac ctg 1421
Lys Met Arg Leu Thr Trp Glu Gly Val Ala His Ser Trp Tyr Asn Leu
415 420 425
tgg aat gag ttt cgc aac tac ctg tct caa ccc agc aac ccc tcg agg 1469
Trp Asn Glu Phe Arg Asn Tyr Leu Ser Gln Pro Ser Asn Pro Ser Arg
430 435 440
gag ctg tac atc cgg cga att cgt gtc aaa tct ggg gaa acc cag cgc 1517
Glu Leu Tyr Ile Arg Arg Ile Arg Val Lys Ser Gly Glu Thr Gln Arg
445 450 455
aaa gtg aca ttt tgc act caa gac cca acg aag agt agc atc tcc cct 1565
Lys Val Thr Phe Cys Thr Gln Asp Pro Thr Lys Ser Ser Ile Ser Pro
460 465 470
ggc cag gag ctg tgg ttt cac aag tgt cag gat ggc tgg aaa atg aaa 1613
Gly Gln Glu Leu Trp Phe His Lys Cys Gln Asp Gly Trp Lys Met Lys
475 480 485 490
aac aaa acc agt ccc ttt gtg aac ttg gcc tga gggcccaaga agtcctggcg 1666
Asn Lys Thr Ser Pro Phe Val Asn Leu Ala *
495 500
tccacaccca caccccactg tccacgcaca tggaggaaaa gttactgctg aagacccact 1726
cgatggacga tctcagcctt gagccccacg aggagcttgc ttgctgggct catcctgtct 1786
cccctgacaa ctgtgacttc tcctggagag gcctgtgcac tgctgaagtt cttgctgatg 1846
attctagctg taaacctttg ttgccgccgc aggaagctga ggccagcttg tgtgtgagca 1906
ctggagtgtc cagagccctg cacactcggg gtggggggcg gggtactctc cctgtcgcta 1966
ggtgagcact ggctttgtcc aacatcaggg aacacaaggc tctgaagtgg ccctgtgtgg 2026
aaggttggca gctgcctggc ctcactgaac cttagtgaca agtctttgcc tcaggagctg 2086
actcatgcca tagagagggg actgtagacc aggctggggc tgctgccctc ccacacgagg 2146
tactgatgga tcctggttct attctgggta tttcatcctg ccttccagag caccttctga 2206
agttcacatc gttggcattt cctttttatg ttatgcatac tttggctgaa caaacattta 2266
ttgggcactt cagctcatta gcaccaggca tgtatagaca catgactcaa cttcagcatt 2326
tggagccagc gagaccagga tccctggaat ctctcaagtt cactggcccc tgagaatgcc 2386
tgtgttagtt aaagacagat gggatcaagg cgagggctac aggctgagag agcttgagct 2446
cagaggcttt tatgaaccgt ttcctctgag ggccaccctt gatctctagc agaacagcaa 2506
gcagaacacc gtgcatgctg gccacagcag tggagccaag tctctgctgg ttacggagct 2566
atacggtact gcaggagttc tacatacctg ctgcgcagag accagccccg tggacagagc 2626
tggcatgatg gttgctacag tgctctttct cccatgaagt ctttgctttt atatcagtgc 2686
cttatctgtt aagctcctgg aggaaagccc cctattctag atctttatgt gaatggataa 2746
atgtgacacc cgagacaccc tttagtagtt tgacagcttg cgtgtgtgcc gcctaattag 2806
agcacaggga gcagggcagc tgttattcct gttttaccaa gtcaaggctt agggaagttc 2866
agccacctgc ttagatgata tatcaaggtg gcagtggaag ggggtgagct aaggacccag 2926
caaggtttcc tggaggcaaa gtttatgaaa tcctttctgt aactccataa agatgtctgc 2986
agctatttcc tacccagatg ttcctgatgt gataagcagg ggactttttt ttttttcctt 3046
cagatgctat ccataaaatc caagcccttt cttaagatac aagcagaaga acggttgcgt 3106
ttttgtgggt gtgctgtcag cctggggggt ggggggtggg gggtgggaga gcgattactc 3166
tgtgtttagg tttgtcacgc acattctttg catctgtatt gtaccagctt tatagcactg 3226
tttcatgcca aagttctgat gacgtaacca gttgaccctt cacacagagt gccctttccc 3286
cccctccccc ctccccatgc ttctgtgatt gctctgtagc catgtgaagc ttgctagccg 3346
cggctgtaca tcctgtgact tccaccttgg gtttagtaat gggagcttga gattggagag 3406
ctgagtcctc tgtgggttct gtatttatcc atttggcttg aagctttgtt tatatatcgg 3466
ctgctttttt ttttttaaat gctcagacca ttatttattt cttgagtgta tataagtata 3526
aagacaaatc tatggttagt ttttacttta agattaaccg atttcaagat ttacaaaaaa 3586
aaacaacaaa aaaaaaaaaa aaaa 3610
<210> SEQ ID NO 12
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 12
gctgaatgcc acaaacacct t 21
<210> SEQ ID NO 13
<211> LENGTH: 24
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 13
caggtaagtc gcatcttcaa gaga 24
<210> SEQ ID NO 14
<211> LENGTH: 28
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Probe
<400> SEQUENCE: 14
cttgtctaca ctgaggagga cttgggcg 28
<210> SEQ ID NO 15
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 15
ggcaaattca acggcacagt 20
<210> SEQ ID NO 16
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Primer
<400> SEQUENCE: 16
gggtctcgct cctggaagat 20
<210> SEQ ID NO 17
<211> LENGTH: 27
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR Probe
<400> SEQUENCE: 17
aaggccgaga atgggaagct tgtcatc 27
<210> SEQ ID NO 18
<211> LENGTH: 75033
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(75033)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 18
agagagtgtg gctttgagcc ttggagagga tgctctcctt ctccagggat cgcctcccca 60
gcggacgcag agtttcaggg aaatgtccgc ctccgccact tgggatggca gtggggagag 120
gaggatctgg gtgtccggag gagggcagtg ggagaaagct ggagctgctg gagtcgcagc 180
tgcctgcgga gcgggcccgg gaggaagcgg ggccgagcgt gcggcgtcca cgcggtgagt 240
gtgcagcgct ttcattcaaa ctttctccag tctcggttcc ggcgtcagca aggtgtgacc 300
aatcagagcc cagagacggg aataaattat gcaaatcacc atctggcgat gggtcagatg 360
actcccatac ttttaaaaac tacctctata ggagcgtgac agcagcgagt ccttgcctcc 420
cggcggctca ggacgagggc agatctcgtt ctggggcaag ccgttgacac tcgctccctg 480
ccaccgcccg ggctccgtgc cgccaagttt tcattttcca ccttctctgc ctccagtccc 540
ccagcccctg gccgagagaa gggtcttacc ggccgggatt gctggaaaca ccaagaggtg 600
gtttttgttt tttaaaactt ctgtttcttg ggagggggtg tggcggggca ggatgagcaa 660
ctccgttcct ctgctctgtt tctggagcct ctgctattgc tttgctgcgg ggagccccgt 720
accttttggt ccagagggac ggctggaagg taacgtgaat ttgtttttat tccccccagc 780
cacttctctg tgctgggtcc cctctgtctt gctgattctt caaaccctcc ttgttcctat 840
gaaattcttc ctttcccact gcgctgcccn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960
nnnnnnnnng gccgcagtgc gctgtgaatg agccactgca ctccagcctg ggcaacagag 1020
agattccatc taagaaaaaa gttcatttaa gttttatagg aaaatcactg aaggttcaaa 1080
agacaaacta ttcactcaaa aggaaatgat ctcacaaggc ttaaatgtct tctaatctaa 1140
cccaatgctg gctgacagac tcaaatgtgg tgtgactgtt tttaattata ggcttgattt 1200
ctccctaaaa tcagagaaaa tatggttgga gtccataggg gatggagaaa gaagggataa 1260
aagatgagga aggcaaaggc cagatcatgg aaggctctta tatcatcccc aaatatttgg 1320
aacaggggcc acaggaaaaa aaaaacccaa aaaatgacat gatcattctg acagccattt 1380
tagaaaaatg actctgtcag ggccaggcgt ggtggctcac gtctgtaatc ccagcacttt 1440
gggaggctga ggtgggcgga tcacatgagg tcaggagttc aagaccagcc tggccaacat 1500
gacaaaatcc tatctctatt aaaaatacaa aaattagctg ggcatcgtgg tgggtgcctg 1560
taatcccagc tgcttgggag gctgaggcag gagaatcgct tgaacctgga ggcagaggtt 1620
gcagtgagct gagatcacac cactgcactc cagcctgggg ccacagagtg agaccttgtc 1680
tcaaaaaaaa aaaaaaaaag aaagaaagaa agaaagaaaa agaaaagaaa agaaaaatga 1740
ctgtcaggaa tatggaggat atattaaagt gcagaaataa ttagggcaag aagaccactt 1800
aaaagaataa tccagatttt gttaaataat tgtatgtgca aagacaagaa gaggggagtc 1860
tctcatcttt ctggtttaag atcctaggtg tatggggaag tcgccaaaca aggaagggag 1920
aaaggcaggg tcgcctaggg atgcagagct tggtcacaga aggctgcctt ggatcccagg 1980
gctgccactc ccagctgccg gaccttggat ttgccgggga gttccttttt tttcttcttc 2040
tttttaacac attgtagcag ccagatcaac ttcatcattt acaaatagtt gtcctaagga 2100
ttacttgagg aaatgcatgt aaaaacactg aaaatactgc ctagaatcta gtaagcttat 2160
agaatcattg tctgttactc ttgtcaccat gcaacataaa agaggaaccc ggaggagaag 2220
acaaagagtt ctgtttaagg gcatgttagg tttcaaataa cctcaggtct tcagggacct 2280
cccagaaagg atgtccaggg agtagtttgc tatcagggtc tagctcagga gacagatgtg 2340
gcgctggggt attaatctgg gagttagcat gcagttgaaa gctcatgtga ggggagggaa 2400
ttaccgagga aacaccatgg aatccagcat cagcaatgga agtggagtcg aggggagaga 2460
cttagggctg aggtctccta cactgaggtc aaggaagcaa gtgagcctag cctggagtct 2520
actctcctag agtttgagct gcctagaagg gtggtgccat gaaatagaag ccctaacaaa 2580
agctaagtgc atcactgact atgagggaat aaccagagag atgacctggg gaaggaaaaa 2640
agggaagaaa atgccaaaag gttttctcct ccagatgaag atctagaagc agcggggaaa 2700
acgaggatat tgaaccagac ccttcagtta gcgcattttt cacacaggtt ataatacaat 2760
tgattggatg acattcccat cctgagaatt tcctgattac cataattacg tctcatcctt 2820
tcatcttttt aattggccat ttacaaacca ctttcaggag tacagggcca cataatcaaa 2880
cctaactaat gtgacgaata aataaaatgt ttctttcata tctaataata cgtagcctgt 2940
cctagaacta caaacctgtt tttaattgac tgacatttaa aaccctgaat ttttatttct 3000
gtatacaaaa aagagaactt tgagacagtc cctaagtagg caattcgttt tcacacgttc 3060
ttaaaacata ttccacactc ccttcgcttt ccgccttcct gcctcctcag atctcgtttc 3120
ttcggctacg aatctcgcga gaagtcaagt tctcatgagt tctcccaaaa tccaccgctc 3180
ttcctctttc cctaagcagc ctgaggtgag tgtttctcct gcgttgctcc gagggcccaa 3240
tcctcctgcc atcgccgcca tcctggcttc gggggcgccg gcctccaggc ccccgggagg 3300
agaactccta gggctactaa atcctcgctg gaggcggtgg cttcttatgc gggaggacgt 3360
ggcggagggc ctgactttgg gagccggggt cagtcggcct ctgaggtccg cagagggacg 3420
tgatgggcgg gaatggggac taccgggctc ctccactggt gggggcgccg gcccgccgtg 3480
gggtgcgggc cgcctggggt ccgtgcggac tccggaggtc cggtgtctag tggtgagtgg 3540
tggccgcaac gaggaaaaag ttttggggga aagaaaagtc gggtggaggc gtaacacgtt 3600
actacaagag tgttgcgtac aggagggctc ttaaagtggg tcatagcccg aaggtgttga 3660
gagagacggc actcactacc tgcagccctg acagcaaagg ggtttctgta gagcgggagg 3720
gaggaggtgt agagggttac ggttgagttg tgccctgcgg atgcgtcgag tcattttacg 3780
cctggaagat ccagcattgg attgaaacag gctgtatttt cttccaaagg gttgactgga 3840
ttggtgaggc ccgtgtggct acttctgtgg aagcagtgct gtagttactg gaagataaaa 3900
gggaaagcaa gcccttggtg ggggaaagta tggctgcgat gatggcattt cttaggacac 3960
ctttggatta ataatgaaaa caactactct ctgagcagct gttcgaatca tctgatattt 4020
atactgaatg agttactgta agtacgtatt gacagaatta cactgtactt tcctctaggt 4080
gatctgtgaa aatggttcgc tattcacttg acccggagaa ccccacgaaa tgtaagtgga 4140
caggaggtag atacccattt cctacttggg gcttggcata agataccaaa aattaaccaa 4200
aacgttttat ttcctagcat gcaaatcaag aggttccaat cttcgtgttc actttaaggt 4260
atgcgattca tagttgtgat ccaacagttc ctcatgttcc actcaaaaaa ggtagctgca 4320
gtgatgactt tcttaggaca cctttggatt taccgtgaaa attaataaat tctgagcagc 4380
caccttatat ttaggcattg atgatcaggg tgtaaggatg tagcgtttgt gaaattgaga 4440
agtagtacca gaaaaccatt tgaaaaccta gtgtgcagat caagatgggg cattttaatt 4500
gtaattacat gatgatggaa tttaaacgtt ttatagtgaa taaatggagt tttggagttt 4560
tctctagctt caacgaagtt tacctggctt ttagtcacaa gtttttttaa ttgccttgcc 4620
cccaaagaat agaaataact cgacattttg aaaagctgaa aattgaaaat ttaacgttta 4680
ctttaaaaat taaagctttg atctagttaa ccaacacttt attcttcata cttagaacac 4740
tcgtgaaact gctcaggcca tcaagggtat gcatatacga aaagccacga agtatctgaa 4800
agatgtcact ttacagaaac agtgtgtacc attccgacgt tacaatggtg gagttggcag 4860
gtgtgcgcag gtgagaattc ttagttgcca tttgaaaaga cagattttaa tggaaaagtg 4920
atgggatagg ataagaatgg ctaagatctg tgctgatgaa cctatttctg gttgctgtaa 4980
tgacaggaca ccttggattg atggtgaaat aaaccatatc ctaagaaacc gtgatatcag 5040
ataaattcat cttggctgta atgttaattt aaatcaagga aaatgactca tcatctccat 5100
tttctttcag gcaagcaatg gggctggaca caaggtcggt ggcccaaaaa gagtgctgaa 5160
ttattgctgc acatgcttaa aaacgcagag agtaatgctg aacttaaggt acccaaacca 5220
ctaacatcct gtgcaacttg ggtggttgtt taattaatgt tggctgttca gatttatgtc 5280
cttgattttc ttgttttcag taattaactc atttaggatt ttttaaattt ctacctgcag 5340
tttccacaga atgaaactat ttccttttat tttttttgag tccctttatg gcaaaattct 5400
gtgaagatcc agaagcgtgc tctgtggtct ctttaagcct aacacttagt atttctagtt 5460
ttctgctgtc tttttacact ggaggtactt taccaggggc ttttacccct ttttgtgaca 5520
tacacctttg gtagtatgat taattatcaa aatgattgcc ttagcagtga ttaatgtggt 5580
ttgtggccta ccttcataat ggagagtcat gttgcatttc agaaagaaat tgtttaaaat 5640
ttcaaaaagt ttaaacaatg acaaacttgt gttgacaaaa cacaaatttc tccattttgt 5700
atattaccca ttagcctttt gcatttgaag gtctgggcca gaactggaat tagtttaggt 5760
ctgtatggct ccaaaccttc taagccatgc tgagaaattt tatcatcaag acttcaaatt 5820
gggttcttcc ttacccctca gctctccacc cgtctaagga tctttgggaa ggccactcat 5880
agacaaaatg tatagcacac tgagtttggt ggcaatttag acatgtaaag gtgaaggcag 5940
acaactggtt aaaaaaatgc tatttgcaga ataaaaggat tgcctttcaa aaatgatttc 6000
attgaggcag gactctggag caagcctggg aattcatatt tagcaatgat agtttgtggt 6060
gttttactaa ttaaggatgt tcctggtttc attctccttc ctttcttccc cagggtttag 6120
atgtagattc tctggtcatt gagcatatcc aagtgaacaa agcacctaag atgcgccgcc 6180
ggacctacag agctcatggt cggattaacc catacatgag ctctccctgc cacattgaga 6240
tgatccttac ggaaaaggaa cagattgttc ctaaaccaga agaggaggtt gcccagaaga 6300
aaaaggtaaa taagtagttg ctcggttttg tttgtgatag tagaaagatt tgtggttgct 6360
gtgatgacta tcttaggaca cctttggaat aactatgaaa gaaaactatt ctgagcaacc 6420
cttttcacct gtcttatttt ggcttttgta agtttgtttc ttgactaata ataaatggta 6480
agttttctgt aatcccagca ctttgggagg ctgaggtggg tggatcacaa ggtcaggaga 6540
tcgagaccat cctggctaac acagtgaaac cccgtctcta ctaaaaatgc aaaaaaaaat 6600
tagctgggcg tggtggcggg tatctgtagt cccagctact caggaggctg aggcaggaga 6660
atggcatgaa cccgggagga ggaactagga gtgagccgag atcgcgccac tgcactccag 6720
cctgggtgac agagcgagac gctgtctcaa aaaaaaaaaa aactggtagt tttatattca 6780
gtacctcagg cccgtatgat tatctgctga tggccccact tagatgtact tagccacttc 6840
aaaatgggcc ctctccccaa atctaagnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6960
nnnnnnnata atatatagtc actttactcc taaatatata ttatatagca tatataatat 7020
gctatattat atatcatata taatatgcta tattatatat catatactat attatatatc 7080
atatataata gtatatgtat atttaggagg aaagcgattc tggaatagag tgtagaattt 7140
gtggattatc agcctgagaa gggacaaagg gcattagggc atcatttaca cattatgagg 7200
gtgttagaac agaatatggt tatcatggat tttcagatcc actaatgttt actttccatt 7260
taaggcacat caaatcaaga gatagtcagt acacataaaa gaggagattg actttatgta 7320
tttatcaaat atttcacaaa tataaaatat tgagcatatc atgttttatt tatttattta 7380
tttatttatt tatttattta tttattggtg acagagtctc cctctgttgc ccaggctgaa 7440
gtacagtgac atgattgtat aatagctcac tgcaacttca aaatcctgga tttcaggtga 7500
tcctcctgcc tcagccttct gagtagctgg gactacaggc ttattagcta gtagtagcct 7560
ggctaatttg taaatttttg tagagacaga gttttgcttt gttgtcaagg ctggtcttga 7620
actcagcttc tggaaatcct tgtaccttag cctcccaaag tgctggaatt acaggcatga 7680
gccacagttc ctggcctgtt accaagtatt ttttaatgtt tactagtttg atgggtaaaa 7740
atgatatatt attgttttaa ttagcatatt gtactcagaa tttagtctga agatcttttc 7800
atatatttat tagctgtgag accttggcta tttcagcaag tattttttga gtaatttctc 7860
tttagcagta tgctggagat tgattaggga taaggaagaa gtgtaaggcc tgattcatgc 7920
ctctcaggaa taatctacct ggggagatat gttatatgta ctacagcaaa ataacatttc 7980
aaggcagctt ttacgtaatt gcagctttag gggagtagtg gagagtttta aggggtaaca 8040
gggaggaatg atagaatcag ttttgcacat gttaagattg agttcttgac tacattgcat 8100
tgaagatagg tcctttgaaa ggtttactac taaaagcatc agcaatagcc tacttctcat 8160
ttctagcata gagttacata aggatcattg ttaccaacaa acttactgat tatattatat 8220
atattaccct tgagttaaaa aaagtctgaa atattttaga gatatcaaca gaactggctt 8280
cctggccaga ggttgggtct agaaggagaa atttgaaagt agtatcatgg aagccactag 8340
aaaagccagg agatgggatc agttactgac agatttagag aaagaagagc tggaagccaa 8400
gggagtctaa tgatgcccag tgagggcaaa ggcagtagga ctctttggag attatccaat 8460
agtgttttct actttactct agttttcatt gataagggtc ttgagagttc caaataaatt 8520
gggagccgac gattgtgcgt gtgtgtttgg aatccttggc aggaatcatt gcagaagaga 8580
tgccattggg gctcagtttt ctactttgct ttttgtgggg gagcaggtgg atctttctgg 8640
caagagtgcc tcagtggcta ctggtcttac tatgtctgaa cttattcctc ctgtatactc 8700
caggtgccca gggtgttgcc ttcactctgt tcctagcagg tagcaccctg ttgttgcttc 8760
ctgtgggagt gagggcttag gcaggggtgt ctgctcaggt gcactagcca agcttctgtt 8820
agttgcttgt gggccttttt ctggcctttc tgcttttgtg ccggagttcc tgttcacact 8880
ctttgggatg gtgcccttga aggcccttga gcctgctctg ggatgctatt tcccttttta 8940
gcccatctcc agctgttttc tactttatgg ggattcttca cagcttctgg ccgcctgtta 9000
tttttttagc atggctgtgt aatcactggc tatttaaaaa aatatgattt atattatttc 9060
tagggtttgg tgagaaaaaa cggggaagga ggcactgtac tctttactca attggctgtc 9120
tcgatttttt ttgttaacat tctgttcagt gtctctcctg atatgcttgt aggctttatt 9180
catactgttt tagaaagatt tcatagtgtt cttggactga gcagttattt tgatttatta 9240
attttatttt tgatacaaag tcttgctctg ttgcacagtg cagtggtgta gacgtggctc 9300
actgcagcgt cgaactcctg ggctcaagcg atccttctga gtagctggga ccacaggctt 9360
gtgccaccac gcccagctag tttttaaatt ttttgtatag atggggtctt gcccaggctg 9420
gcctcgtact cctgggctca agcaatcctc ctgctctgtc tcccaaagtg ctgggattac 9480
aggtgtacag gtgtttgatt taagacttaa ttttgtggtc tatcctagcc tctttgaaaa 9540
taaagtttat ttcctataaa ccaatagaaa acagtcatag gatatgagca ggtaatggtc 9600
ttattcacgg caagagagat gcaaaataaa attgtattga aaaactgctt tttaaaaaaa 9660
atctaagttg gcaaggatca gaaagtttga ttaaacaatg gtggctgtgg agtagagaaa 9720
taggcgttgc tggtgagaat ccaagttggt acaacctctg tagaaggcag ccaggcagta 9780
tctatcaaaa ttacagatgc atataccctt tgacccagca attctatttc tctggattta 9840
tcatatctgt ctactcatac atgtgtgaaa tattttatat gcttatgtaa gtgatagtat 9900
aagatataag taatgtatat atcttattgg agaatgacat ttatttttta ttacttggag 9960
aatgatttgt aatagcaaaa gatgtagact acctaaagtc catcaatatg gtatttgaga 10020
aattataaaa tggaatgtta cacaattgta aaaaggaatg aagtgcttca tatatggata 10080
tataatgact tcctatatat tgatgagtgg gaatagcaga gtagagagga atatgtcata 10140
tgctatggtt tgtttaaaaa ggggttgaac atatgtttgt atatacacag aatgtctgtg 10200
gaatgatgcc caagaaactg tgaatgtttt ctctgagttg agaaggtggt tagaagatgg 10260
gatgggaagg agattttcac tgtctatcct ttgaagcttt tgtattttgt accatgaata 10320
ttactgatac aaaaagaaca taaaataaac agaaacatct gacaaatttt gttagtagac 10380
tctttgatag tattgtttct cgttgttgct tcacctggtg ggaggaggag gttgattcca 10440
aaatcaaatt ttgttctttt gaaaattcag cattcatgta cttaaactcc tagagtaaaa 10500
ttgcttgctt ttaagttacc acacaaaaaa agtaagatac tggtctattt aactgagaac 10560
aaattgttat gattttcatt tcttttgatc tggtgtcatt tttttcctgt taattttaat 10620
gaagcacaga gaaggaaaat tgagtacatg gtagagccag gaggacccat atttaaattg 10680
ctgtcagcaa accaacactc tttgaactaa cccactttca tccatacagt tagaggtact 10740
tattcagtac actaatatta gacctacaat cctaactgct aattagcatg agtcccctga 10800
taaatagcac actgtaaaca agtagtcaag gtctttttta ctcgtttgtt taaacatatg 10860
gtttattgag gcatattcag ggtcacaggg attattggac aaataaaaat aatttgaaag 10920
ctttggctaa aaaggaacta gcaatatcct taaggaggtg gtggaagaca aaatctgttg 10980
tatactcctc agagttaatg aaattaatgc agaatcccag aggacccaag aacattactt 11040
attttgcttt tattgaaaaa gttcaatttt gaataaaatc tgaaagaaat aatttattgg 11100
gtagctggga gaggtggctc atacctctaa ttccagtgct ttgggaagcc taggcaagag 11160
ggtctcttga ggccaggaga ttgagattag cctcagcaac atagcaagac gcccatctct 11220
ataaaaaatt taaaaaagtt agcagggtgt gatggcatgt atctgtaatc ccacctactc 11280
agaaggctaa ggcggaagga ttgcttgaat tcaggagttt gaagctacag tgaattatgt 11340
tcatgccact gcactctagt gtgggtgaca gagtgagacc ctatctctaa aataagtaaa 11400
taaatttatt ttggaaaact agtctaagtg aaagaacatt ctgtaatagg acaccgagta 11460
actagaatat gagatcctaa tatctcatca tgtttaatat ggacgatgaa gtaacaatat 11520
gtaaaaacaa tcctagtggt tgtgaaagaa agtacctatt agtctgattt gttggcacct 11580
ctctgggaca tgtagattct tttttgtttg tttgtttttg ttttttttga gatggagtgt 11640
tgttcttgtt gcccaggctg gagtgcaatg cacgatctcn nnnnnnnnnn nnnnnnnnnn 11700
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11760
nnnnnnnnnn nnnnnnnnnt ggattccaca tgtttaggtg tgtccttcaa ccccatcacc 11820
ttttcaccac tgcctgggct tgatgtgttc tctgtagaag attttttaaa atacagatag 11880
tataaaaatc cttcctgacc cctcatttgg atatttttta atggtgcctt ttttttaatt 11940
gttgacgttc aagatagact ttaaacaatc tgaaggaaaa taaatcaata tcaatgacag 12000
tcatgcacac tcaagcaatc tgtttcaaag cctaatcatt tgtcatctat tctcagtgtt 12060
tctattataa tttttattat aataatagta caggctgaat ccccagcacc tatagcagtg 12120
cctggttcag agtcgatact taaatattta tggaatgaac actaaatgtt ctctcaaaaa 12180
atgataattc agaaatatct gtatattgca aaaagtaaaa ggtcttcctt accctcccct 12240
gctccattct gatgattatg tgaaaggaaa agcctctatc cttggtgaat ctctcattta 12300
gcttttcttt tttaagaagt tgtgtattct ggtggtcttt aacagaagag ggctatgtgt 12360
tattgggggt tcattgccac ctgtaggagg caaaataacc tgtgatgagt tggccccatt 12420
gaagtaccac acctctagga agccttccct tcctcccctt ctcttgccct tgtttctgca 12480
agctctgtac aacacttctt atccgggtga ccttgcccta caggtagctg gctacctgct 12540
gtgtctttca caggatggtg agctccttgt agacagtgct gtctttatct tgccacctcc 12600
cacagtgcct tggagggtac agttgctaaa cgttttggaa ctaaacagaa aaaaacacag 12660
tacaataaga aattaccata atgctaaatc aacaaattac tcgaatctct tcccctgaca 12720
ctttagtagc tcttgaaagg gcaatttcag tgatcttgtc aaacagctaa gtcacatggt 12780
gacctagaag agggcttgga agtttgagtc attgccgttc aatactcttt cctctagaca 12840
tcctgacccc catcctctgc aaaaccatgg gcagtgcaga gtaggtcaca agtattgtgt 12900
ttttatttgt ttgtttgttt ttagagacgg gatcttgctg tgttgcccag gctggtcttg 12960
aactcctggg ctcaagtgat ccccccaccc tggcctccca aagtcctggg attataggtg 13020
tgagccaaca tgcctggcct gagttgtaaa tcttccaggc tcgtgccact ggcttctggg 13080
agactataag atatgaccta gtgtaaatgg aaataatttt tacctatttg ggttcaaaga 13140
caccaataaa tctgttcttg gccaccgagg tcagttctat ttcttcaaat cttgcctaga 13200
gtagtgtttg cacctcatgt tatgggttga attgtgtccc tccaaaattc atttgttcaa 13260
gtcctaaccc ccagtacctc agaatttgat ctttcaaaag acaaaattag gattgggtgt 13320
ggtagctcat gcctgtaatc ccagctactc aggaggctga ggcatgagaa ctgattgaac 13380
ctgggaggtg gaggttgcag taagcccaga tcgcaccact gcactccagc ttgggcaaca 13440
gagcgagaaa aaaaaaacaa aacaaaatta caacaaattt aaatatctta actgctttta 13500
tttacaattc tagaatcaag caacacctca tcctataaaa taaaacgggt gttcccaaga 13560
gctgagcaga ggagtttggt ccaatggaga gaaaaaggct aaggaaagcg gaaacagaaa 13620
acaaaaagtg gattggttgt ttcagagtta ctttcttgtt aaggctgaag cagaggggat 13680
ttccttatca tgccggctaa aactggcttc tttggggatt tggctattat ccctcactct 13740
cttcatttct cagatggtca gataaacaaa ttagtttcaa tttggtgaca tggaacctta 13800
gcatgaatga ctccattttg gttgggccta gtgcaggagc tcagtccaag ccaatggccg 13860
cctataaatt ttatttaaca ggctttattt ggaaataggt acattgtaga tagaattagg 13920
ttataatgaa gtcaccaggg tgggccctaa cccaatatga ctggcgtcct tatataaaga 13980
ggaaatttct atgtaaacac acacacagag gaacatgtga agatgaaggt ggatataagg 14040
gtgttccttc tacaagccaa ggaatgccaa gaattgtcag tagaccagca gaaagtaggg 14100
gagagcatga aacagtttct tcctcgcagc cctcagaagg aaccaactct gacaatgcct 14160
tgattttgga cttttggcct ccaggcctat gagacaataa atgtccactg tttgtggtac 14220
tttggtatgg caccctagta aaccaatata cccaggtatt acagtcagat attacagtcc 14280
atgttagctg gaaacacaag ctaactctga ggaagccaag tgtgagtctt ccaacactgg 14340
cagtgttggc aaaaggagct ctgggacagc cttttccagt gagtgacagt gtgggctgtg 14400
cagctagact gctggtgtgt gagcctccac tggctagctt tgcaatgttg ggaattgtat 14460
ttaacttgtt gcggctgtac aatgggggac aataaataag taccttgtct tagtcctttt 14520
gagctgctat aacaaaatac cacagactgg gtaatttaca aagagcaaaa attggacagg 14580
cacagtggct catgcctgta atcccagcat tttggggtgc cgaggcaggc ggttcacgag 14640
gacaggagtt ccagaccagc ctggccaaaa aactccgcct taaaaaaaaa aaaaaaaaaa 14700
aagcaaaatt tttttcccnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14760
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnga 14820
attgtttata tgaatcattg attatgaaat agtaaagctc attctctctt atttattttg 14880
atattttttc tacttaaagt aatacttgta tattgttaga aaaggtaaat aataataaag 14940
gcaggttata aaaaatcagc agtatcctta cccatcccca ctttttaatc tcattttcag 15000
aggcagttac ttttaagaac tctgtctctc tctctctctt tttcttttgg tagagatggg 15060
gtctcactgt gttgcccagg ccgttcttga actcctagcc tcaaatggtc ctcctgcctg 15120
ggcctcccaa agtgctggga ttacaggcct gagccactgc acctggccct aaaaattctt 15180
ttaagctttt tcttttgatt cttacctctg cgtttataaa tctgcctaaa ttgctatttc 15240
gtgatttatc aattttagat aatacatatt aacttcttgt tatggtaggt ggggtattct 15300
tccctttcct tcccctattt ctaaaagcac ttccagtata ggtatataag aattcttggt 15360
taatatagtg tttagctttt atattattat gactacataa atattgttca ctgctggtga 15420
gtacttcacc atgtttcccc ttccctcccc ctagtttgct tagttttcca tggatatatt 15480
attaattttt accaaatact ttgaacggcc tgtgggattt agtgcctaag aaattgatgc 15540
aaggtgttac tctggctact gctattgcag ttgttttgtg tccctgttcc agaggtttca 15600
tgcctttttc agctttgtca gtaaagtaaa atctcagatc cccaccatgc ctgacttgac 15660
agtgtaacat cccccatttt gtttgtgctt tctttttgga actgttacta ttaatatatt 15720
aaaatcttct aaattgttct ttaaattttc tattttttct cctcctgttt ttcacttatt 15780
tgtcctcttg ttctttatta tgggagttcc cttaactttt atcttttctc cgttcttttg 15840
ttttaaattt caggtgtcct ttcttgttct ttatttcctt ttttatgaca tcctactctt 15900
gcttcatgaa gcaatatgaa acggttaaaa gagaatggtt tctgtaccta tcctacctgg 15960
gtttaaaacc tgatgtgcca ctttctagct ctataatctt gggcaagtta cttaaccttt 16020
ctttgcttta gtttattcat gtaggttagg agttggtaga catttcatct aaaggaccaa 16080
atcataaata tttcaggctt tatcagccaa gaggcaaaat gaaggatatt ttaaattatt 16140
taattatttc attatgtaag tacttacata acaagagaga aaacatttct acaagttttt 16200
tattaataaa gctcaaaatg cgatactaat aatgattgag aacaattttt gcaataaagg 16260
tttgctaatg agaagaatga atgttttttt ggaggagagg ggataacatt tggcttaatt 16320
ggagtttaaa gttagtgttc tctatcatca aaattgatta tagatgcagc attaaccacc 16380
tattaatgct gatttgtaat gagatttcat gtatttcatc tttgaaaatg tcttcttaga 16440
tactgccaaa tactgctaat agtctgtgaa catatggttt taatagagca tattcattgc 16500
ttggacgaca tttacagaat tcttttagac tcttttcttg atacttaacg tttagcaaat 16560
cattacactg cagactaatc acttccaatt gaggattagg tagaagctcc tcattgcaca 16620
gttaaatgat ttttgaaatg tggaatgttc gtttctactt gcattgaggt atgaaaaaat 16680
actgctggaa atatttgatt ttggaaaata tgtctgctgc atatttgtgt ggcagtagag 16740
atcttcttat tttaattttt gacagcatag gaagtacatg aagcatcttg acatgacttg 16800
tgacttaggc aatgttagtt gtccttgaaa tgactgtctc aatataggtt ttgcacgtag 16860
accctgttgt ccttctaact ttaggctgaa ttcattaaca gatatatcaa gtctgcagca 16920
acaatagtag ttgagggtca ttcttttcat tcagaaaaat ttgtctcagc ctggagcttg 16980
aaacatttca gtaaaactct gccattgtta agccattgta ttgtttcgtg gcaggacgcg 17040
tcaggatatt tagcttctat ttctgacaaa aatgtcatga gaacaaatga aatttacctg 17100
tgacattacg gttcaataac acatgataga ttcagatgtt ttctgcagag tacctgctga 17160
ttgagaatat gattaatagc cataagcttt aaacacctta catttcacaa ggcttgtaaa 17220
ttttgtccag ctaagccttt ttctgcttca tacatatttt taccaccatc agttgttaca 17280
catcttagca gattctactt cagattgtgc tgaattagta tttccttgat tttttggaaa 17340
atattcatag ctgtaattgt ttcatacata ctgtacttcg agccactgtt ttcatggaaa 17400
gagtaggatg ttaaagatat ttattttctc tggacacaat tctttggctg ctgtaatcaa 17460
acacaattta attaactcac cattggcaaa tactttcctt gcttgcttaa caaatgagcc 17520
atttggaaat ttacttcggt tgcagcccta tttccatttt taatttttgg caagacattc 17580
tgctctgagg aggtatatct tgttttacat ttgcccattt tcctgctcat tgctctcatg 17640
agttgggagt gaatgctgtg tggcagggca tatcgggata ttgtgatgag tgcttagtct 17700
agtaatactt cctatattgt attttgctgg cacagcttcg tgatgctttt acataataaa 17760
gtactttgac atctaactag atttaaacaa tctacagtcc actgtgactt acaagtgtga 17820
catcagagta cttctgtttt tttgacgtga tagtcactgg taaaatagtt atagtatggc 17880
aatactcatg acacttgaaa tgctgttgag taaaccgtgt cattgagatt tgtggtgcac 17940
tgagagtata aagggatgag ggcaccaaat atgatctctg tagcaactac ccaagtctgc 18000
tgttgtatca ggaaaacagc tatagaccat gtgtaaacaa atgagcatgg ctgtattcta 18060
ataagacttt attgacactg aaattttctt ttcatataat ttacacatgt cacaaaatat 18120
tcttttgatt tttttcaacg atttaaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18240
nnnnnnntga gcttgaagac ccctgagccc agatgagaac cgcggacctg cctgcatcct 18300
gattttagcc atggtgaaac cctgagcaga gaatccaact agaccatacc aggacatctg 18360
accccttaaa ctgggaggta atagatttgg gttgctttaa gcccctacat ttgtggcaat 18420
ttgttatgca tggagaacct ttatgtctag ctaagggatt gcagatacac caatcagcac 18480
tctgtgtcta gctcaaggtt tgtgaacaga ccaatcagca ccctgtgtct aggctcaggg 18540
tttgaggatg caccaatcgg cactctgtat ctagctaatc tggtggggac ttggagaatc 18600
tttatgtcta gctaagggat tgtaaataca ccaatcagca ctctgtgtct agctcaaggt 18660
ttgtaaatgt accaatccac accctgtgtc tagctcaagg tttgtaaatg caccaatcag 18720
cactctgtat ctagctcagg gtttgtaaat ataccaatca gtactctgta tctagctaat 18780
ctagggggga cttggagaac ttttgtgtcg agctcaggga ttgtacacgc accaatcagc 18840
accctgtaaa atggaccaat cagcaggatg tgggtggggc cagataaggg aataaaagca 18900
ggctgcccca accagcagtg gcaaccggct caggtcccct tccacactgc gggagctttg 18960
ttctttcgct ctttgcaata aatcttgttg ctgctcactc tttgggtcca cactgccttt 19020
atgagctgta acactcactg cgaaggtctg cagcttcact cctgaagcca gcgagagcac 19080
gaacccacca gaaggaagaa actccgaaca catccgaaat cagaaggaca aactccacac 19140
acgccgcctt taagaactgt aacactcacc gtgagggtcc gtggcttcat tcttgaagtc 19200
aatgagacca agaacccacc aattctggac acaatagcac cttctaactg aaatactgga 19260
aagagagttt tcattgttgt agaatttgct taattattat cctgatagca gggataatta 19320
ccaacaaaaa ggaagcacga aagttttact aacaccgagt ttgctagaac ttcttattgg 19380
gtttggtaat atgtcacacc ctggctatgc aaagaaggtt ataaaggaaa gagattttct 19440
gtggaagtca tgaagggatt gataactgca ggaaagatct agccaaggtt aacactaaag 19500
ttactccagc cacccaaatc caatgccact tatcctaaaa ggaatgttac ttttatatta 19560
acatttcaac aacatctggt ggaggcaaac cagtgttaca acccatcaga atagcaacag 19620
aatcaaactc caaatgatgc tgcagacaga accacacacg gacatgcctt tcttccaagg 19680
acccttagat tgaccccagg aggagcccta gctgctgatc cccacatgat gccccttttc 19740
agcaggaagt agccagaaag agtcatcacc caacaccccc taacagcagt tagggttgcc 19800
actccagagc agggaatgat gatataggag ttaacaagga attacttagg cagatagcaa 19860
gggcatggga gtcctcagta aggctgttct ttttaatgaa aagcagcccc aaatcatttt 19920
ctaacaaaga gcagcctgca agctgggagc ttgcacacga taatgcctgc agaaactaag 19980
gactagacat tttcaaaatg gcggctccat cttcccttct ctgccagcca catgtactgt 20040
aaaggagcag acaggatggt gccaatcaac tggaaagccc atttgcataa gattaggttg 20100
aggcaaccag ccttccccac acactctgta gacgtcatgc atgatcgaac caatctgtga 20160
gccctatgta aatcagacac tgccttctcc atataaaaat ctgctgcagg ccgggtgcag 20220
tggctctcgc ctgtaatctc agcactttgg gaggccgagg cgggcagatc acgaggtcag 20280
gagatcaaga ccatcctggc caacatggtg aaaccccgtc tctactaaaa tacaaaaaaa 20340
aattagccag gtgtggtggc acacgcctgt agtcccagct actagggaga ctgaggcagg 20400
gggatcacct gaacctggga ggtggaggtt gtcagtgagc tgagattttg ccactacaat 20460
ccaacctggg caacagggcc agactccatt tctaaaaaaa aaaaaaatac aaaaattagc 20520
tgggcatggt ggcaggtacc tgtaatccca gctacttggg aggctgagac aggagaatca 20580
cttgaacctg ggaggcggag gttgcagtga gccaagactg caccattgca ttccagcctg 20640
ggcaataaga gctaaactcc atctcaaaaa aacaaaacaa aaaagaaaaa gaaagtggcc 20700
acccctgtgg aaaaggactc tggagtgggg agggtggtag agcagttttt tatattaagt 20760
ttgtcatatt agttgacttt tcaaaatcac atccatatac tactttgatt aaaaataaaa 20820
tattttctgg ccaggcacag tggatcacgc ctgtaatcac agcaactcgg gaggctgagg 20880
caggaggatc acttgaggac aggggttaga taccagcctg ggcaatatag caataccctg 20940
tctctaaaaa aaatttttta attagccagg tgtggtggtg catggagatt tcaacaacaa 21000
caacaaaaaa agcacaaccc ataaaagaaa aaaatttata aactgagctt catcaaatta 21060
aaaacttttg ggccaggtgt ggtagctcat gcctataatc ccagcacttt aggaagccaa 21120
ggcaggcgga tcacttgagg tcaggagttc aagaccaggc tggccaacgt ggtaaaacct 21180
catctctact aaaaatacaa aaattagcac agcatggtgg tgtgcgccta taatcccagc 21240
ttctagggag actaaggcac gagaatcact tgaacccagg aggcagaggt ttcagtgagc 21300
caagatcatg ccactgcact tcagtctggg gaacagacca tgactccatc tcaaaaaaaa 21360
aaaaaaaaaa aagaagtgaa atgaaggaca gcaatgtata tgtgaactga aaatgcaaca 21420
gacaggcaga aagaattagg aatgcttgtt attataaggt attgtgcaaa ctgtgaagtg 21480
gtgatgtnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21540
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnntgt gtgtgtgtgg 21600
gtattttttt ttttttttct ttttttgaga caaagtgtct ctgtgtcatc caggctggag 21660
agcagtggca tgaacacggc tcactgcagc ctcaacctcc tgggctctag tgatcctctt 21720
gtcccagcca cttatgtagc tgggaccaca ggagtatgcc accatgccca gctaattttt 21780
tgatcttgta gagacaaggt ctcactttgt tgtgcaagtt ggtttcaaac tcctgggctc 21840
aagctatcct cttgcctccc aaagtgctgg gattacaggc gtgagccacc acatctatcc 21900
cctttcagat atcttaatct tggtaaattc tttcagttac taatctgtga tgaactaatt 21960
tttttcttca catatatcaa cagttgagga gctcaagctt gggaaaatgg tgtgcattcc 22020
ttgtatcgtc attccagttc tgctctggat ctacaaaaaa ttcctggagc catatatata 22080
ccctctggtt tcccccttcg ttagtcgtat atggcctaag aaagcaatac aagaatccaa 22140
tgatacaaac aaaggcaaag taaactttaa ggtaagaaca ttcacatgcc ttgaataaga 22200
gcagtgaaag ggggtggtac ttgggtgaaa cactagattt tggaaggaca agttttagaa 22260
ccagaagctt caatagtttg tgtaacattt ggcttaaatt aaagttttaa agtaggcctc 22320
tgattaatgc atttgtcctt tggccaaaat gataaccata ttttctgact ggtaagtgca 22380
atggtaaata gctaatggat gaggagagat tatgctgata caaaccagtt tcactgattg 22440
atgactttga taagagattt aattagaagg atcatagaga tttaaaaaaa aaacaatttt 22500
tttaaaaatg agtggacaac tctgttgtct ttagaattgg tgctttttta gattcagctt 22560
tgtactcagc tgcactaatt tagccatccg gcccgtttct ggaagcaaaa tgttttcact 22620
ctggagcttg tctgttttta aagagaatgt ttcttgagct gggcgcggta gctcacgcct 22680
gtaatcccag cacttgggga ggtcaagatg ggttgatcac ctgaggtcag gagtttgaga 22740
ccagcctgac caacatggtg aaacctggta attcccatat tagcccggca ctggtggggc 22800
aagcccgtag tccccactac taggggagct gagatgagag gattgcttga acccgggggg 22860
caaaaggttg cagtgagctg aggagaaact tccctgcaat cccccctggg ggacacgagc 22920
cagaccccgt ttggaaaaaa aaaaaaaagt aggaatcatt cttcaggcag cacatagggt 22980
ttagcccttg ggctgtaatt tgccaattta tgatttaggc aatgagaaaa aaaagtacca 23040
cgccatagtt tgtggtgagt gtaaaatcag ttagtatatg tgaaacagtt aagatggtga 23100
ctggggacat agtaggtgct aaatacatgt ttggtattat tgttactgtt ttcattatta 23160
tctatctaaa gatattttag tttttaattt tttcttagaa tcatctgttt tctatgattc 23220
ctttctttag tttgtttcag tttctttttt ttatttttga gacaggatct ccctctgttg 23280
cccaggctgg agtgtggtgg cacgatcaca gctcattgca gccttgacct ctcaggctca 23340
ggcaatcctc tcacctcagc ctcccaagta gcaggaacta taggcgtgca ccaccatgtc 23400
tggctaattt gtttattttt ttgtagaggc attaaaaatg tctctgtgtt gcccaggttg 23460
atctccaagt cctgggctca agtgattctt ttgtcttggc ttcccaaagc gctgggattc 23520
taggagtcag tcaccatgcc cgggctgctt tggtttctat atttcatgct ggagacttta 23580
ctcacaagtc taatcatttt tgactgtcca ttcttattta aagttgagac tacccaaaaa 23640
ctgattggaa gctctttgtg taagtagatt ttgttgagtg gtgggcattg ctgtgaggtg 23700
acgatctatt ttttaaattg gcctggcagt tcctaaattt ctgtatcttt tccctaggtt 23760
cctattcagt ttctcttgag aggaatcctt cagtctcctg ccttggaggc agggaaactc 23820
actgtggagc tgtagcaagt tgactttatc ttcattctct tgttttcttc attttccacc 23880
ttattaccct ccccaacctt ggcattggtc tctctagttg tatttctctg ggaagcacgc 23940
ataatagtct cttcctgggg agagagaccc ccaggcttta gtcctgcacc ttgccccgtc 24000
ttccccagtg ccctgtctct cgctctttag ccttcccacg gctccatggg gtgaattgcc 24060
ttccttcttg ttggtgtccc ccttggcagg aactcctttt ggggcttcct caggtcggct 24120
ggttcaggta cactcctctg tcaccttccc atcttccaca cttcccttga tctgatcttt 24180
gtgggtttat atctttcttt ccattgcttt cgtttcattg ggcggggaat acccaactgt 24240
acttaatata gcacttggag cctagttgcc tttcagagaa tgttctatta cttgtggact 24300
catggcacct gtggcattta ctctgtcctt tggctggtga ttattgccca ttatttctct 24360
gatctcctgt ttcagctttc tccttaaagt ctctgaggta caagtggggc aatatgtgtg 24420
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24480
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ggcatcccgg caagtcatgc 24540
aaaaggcaaa ggctggcagg ttgcgctggc tcttgccagc cctcccaccc ccacctcctt 24600
cccttatacg gctccctaag ccagtgctgg tgctgtccag ccagccgagc ggcctggcag 24660
cagctctgaa accttgtcct catgcctgaa cctggaggtt tgagaggcaa gaaggctgtc 24720
agagctgtgt ctcctggagc tgccttttaa tcttggtgta ggtgtcttga aagttcaggt 24780
ggcagggtag ctggctttgg tttgattcgt gcttgcaggg agcgtctggt gggtcgcttg 24840
tgcttagaag ctgccgggcc tattccccca ggttgccagc cttttcctct tcaacactct 24900
tgtgctcccc cgactcttcc tcttcccact cctcctccct ttcctcctcc tggcttttag 24960
cttctcttcc tcttgggttc tgtatggcag cctttgccag tggaggcagc ttgccctggg 25020
ggagccagag tcagtcagct ctgggggagt cagaatcaga gtgagaacta agtcagctga 25080
taagcagtgg agggctgggg agcctctaaa ctgaacccca tcatctcatc ttctagtctg 25140
ctgaggttgt gccaaatggt ctgtgtgggc ttgtgtatgt gtgggagggt tactgtgaat 25200
gaatggaaag ccttgcttta ctcaacatgt ttccaactct ggtgctaact agaagcattg 25260
cctagggcag gtctcatctc tggctcctca cctgtaaaat gctaaagttc cttctaactt 25320
ctgtgttctg ggcctctgag tatgtgtgag tcatttcctt cattgcaaaa agtatttact 25380
gcatacctac ttagtgtcag acactattct gggagcaggg gagagagcag tggacaaaat 25440
agatggcaat ctgtgccctt gtgacactta catttcagtg gtgggaagac agacagtgca 25500
ctgattaaag tatcagtggt gctcagtatt atggagaaaa atcaagcaga ggaggaaggt 25560
ggggcatgtt ggtgggggaa ggtggtgcga ttttatgatg tggtcaggga aggcctttct 25620
ggagaagttg accattgagc aaagagctga agggggtgag agaaggagcc atgaggacat 25680
ctgggtgagg aacaatcaga aagtcccaga ggctgaagtg acccaggcca aaagtagtag 25740
gaatgtacct agagaacttt ggggtcagag caggtcagac tctaagagga agagtctttg 25800
tccatatgtg ggtttatccg cagatctgtc tcaggaatcc aggagacttc ttttctccta 25860
ggtttcccca tctggcctct gcatgagtgg ctatctgggt agaaggtggg gagttgtcga 25920
actcattatg tccgtggttc aaatttctgc tggttggggc ttttgttcat tactttcatg 25980
acggttaacc tgacttccgc tcatgtgcat tcagcagcat tttctggaac cttatccctc 26040
ttacatctgc tgcattcttc cccctctctc cctcctttcc cctccgccct ctccaccact 26100
atatctcttt ccttttggta tcttcttcct tttgcttctt aactctgaga taaattaact 26160
ccaagatggt catttggcat ctttccctgc tgggtgatag gagagtttga tactctcact 26220
ttctagccag ttatccagtg atgcatccca ggatggctga aagagtgctg ggtggggtcc 26280
atttatgact ttctgaaacg gtgtcatgat gacagcacct acttagtgtt aagccacagt 26340
ggccatcagt gacatgagct gcactttggg agcagggccg tctcctaggt ggcactgtgc 26400
atgagcagtg aagctggaag gggggatttt tgttgctttt aggagtttct ccatggcttt 26460
agtcatttac ttaacaaacc agatgaatgc ctggcacatg ccatcactag aaagggcact 26520
gaggatgcct gagtttccct agatgggaac actaggggaa gcctattttc cagcaggtca 26580
acagcctctt gcctccaatt ccaaaatcca caaactctca aaattggaaa atgtgtttgg 26640
taacttgttt ggcagcaaga cctggcctta cctgatctga acttatttgg cagcaaaaac 26700
ctggcctgag ataacaggag gctattggtt atctttatat ttgccctcaa atgtgagtgg 26760
ttatgtattc tctgcagaaa tattaggatg tttgatccag ggggaatagc ccaggcccca 26820
ctggcagcat tatataatat atggtgtatg ctccatgatg cctttctaaa atctgaaaac 26880
ttctaaattc tgaaactcac ctggtcccaa gcatttcaga aaaagaactg agaacctgta 26940
ctctaaaagt aggaaggaag tagaactcct tttgatagga aagaagaggg aaatgcaaga 27000
gagagatcta tttatttcag agtgacagta attaatacta gtgactaatt aatactcttt 27060
aagtataaat gagatgtcct tttttataaa atgttcacag tttttctggg aggtgaaaag 27120
ggaacattca gcagatgtaa aaaccgaaac ccagatggct taagcaaatt gctcacgatt 27180
gaactagaat cagaccccag gtctctcact ccgcaagtct gctagatgca cccacgctct 27240
gttctgtctc cccagataag ctccacaaac ccaaagctac acagactgag gtcaaaccat 27300
ctgtgaggtt taacctccgc acctccaagg acccagagca tgaaggatgc tacctctccg 27360
tcggccacag ccagccctta gaagactgca gtttcaacat gacagctaaa acctttttca 27420
tcattcacgg atggacggtg agcccgggga gggagctctg cggctttata taagatttga 27480
ttccctttgt tttggtcaat tagaaagaat tctggtgttt ccagattcca aaccctcttc 27540
ccccctttcc ttgtgggctg cttgtatttc agacagctgt gaagaatgta acgggcattc 27600
actaaggggc aatctggact ccagcagggt ggtattaatg atggcataca acgggctttc 27660
agaaagtggc tctcctgagg gcagcagccc agctccactg gctcttaagg acccctctaa 27720
ttacacttaa ttacgctggt caaccactgc ctggaggggg ctgtcctgcc gtgagagctg 27780
agcttaacta aagcaacttg actggctctg agaacctatg agtagccagc tggcctggat 27840
tgctgactgc ccttggcata gtgccctcct gtttgcatag agatagcccc gtttaaaggc 27900
actggggctc tttatgtgcc ctagctgagc aattcacatt gaaaagccca tgacaaactt 27960
caacaaaaaa gggctttagt gctcagcttc agaagctttt gaccagccag gaatttattc 28020
attcatataa tgacagtctg tagggaaatg gcactattaa gtgagcagat ttctgcctgg 28080
gctaattgga aggggacagt cctcctctta atgatgagaa attcatatct gttacatgct 28140
tttggggtct ttaaatctgt catccatggt tgaggtcagg ggcaggagag ggtaggtgcc 28200
tgcctaccta gttgaatgaa gagatgatta agagcctgtc taaccctgct catcacacaa 28260
cctacaatca gaatcatatg tgtgaatctt tttaaggccc cctgtcaaaa tgcaaataca 28320
agctgcctgc gacttacaag taggttgcat tccagaaagt tcttttgtaa tctgtttgga 28380
acttggaaca gcagtttccc acagaagcaa gattgtaaat ggtgtttggg ttcccaggct 28440
aggcacagaa aatctatcta tttgataatg tgcttgtgga taggtttaca actggaccta 28500
gtcaccattt ataactttgt ttctgtggaa aaatacattc tgagttgtaa tttgggattc 28560
cggggacaga ttttccctac tctggggtca agagggatgg cagagatggg ttgtgggaag 28620
gggtcatttg ttaactctgc attcacctag gagacagcat ctccctgcgt ctctaagaga 28680
gaagcaattt tccagggcaa ccctccctag ctgcctttga aggatggacc aggccacttg 28740
cctgcctgta acagggactc agaccagcct catgccagta tcacccttaa cacgtatctc 28800
atagtggccc taacagacag tggggcaaca gtataggaca ttagctagga aagttttgat 28860
gggaaatact ggggatgtaa ggagcaatgg aagacaaata cctgcgtagt gattctgcct 28920
gtgctggtcc tgctcatgtg caaatgtttg gtccctgagc agccaatgtg cagattctaa 28980
aattgtctgg gggcactggg catggtggct cacgcctata atcccagcac tctggaaggc 29040
ctaggtggga agatcgcttg agcccaggag ttcaagacca gcctgggcaa catagtgaga 29100
ccctgttgct gcaaaaagtt taaaaattac ccaggcatgg tggcatatgc ctgtaaccca 29160
gtcactctgg aggctgaggt gggaggatca cctcagctca ggagttggag gctatcatga 29220
gctgagattg ttgtcagtgc actccagcct gggcaacaga gtgagaacct gagtcaaaaa 29280
aaaaaaatta attgggaaga gggtcatata gaaaggaatt ccatattttt taggatttct 29340
gaaaccaaga ataaaaaaca acttccactt ttctctgcag atgagcggta tctttgaaaa 29400
ctggctgcac aaactcgtgt cagccctgca cacaagagag aaagacgcca atgtagttgt 29460
ggttgactgg ctccccctgg cccaccagct ttacacggat gcggtcaata ataccagggt 29520
ggtgggacac agcattgcca ggatgctcga ctggctgcag gtactggggg atgagaggga 29580
gtctcctgtc accagcagga tctcaaaccc aatcttctta agaaatgcag gtcatgcatc 29640
tgttgccatg aacttctgga gtctgataaa aatctttgag attaaaagtt ttattgaatt 29700
aagtattgtt tggaatactt gaaagctggg actttttgag agccttttaa ttattgatag 29760
atctctggat ttttcctgtt acttaatttg ctgaaaattg gccttatggg ttgtttcttc 29820
ctgatcttag aaacagatgc ccttaggttg gttggggatg gccaggaacc aggccaggct 29880
tgtgatctga aattcctaca aagactgctc ttgtctctga agcatggctg tgctgagggt 29940
ggggaggggg ttatttcaaa aatgcagcct aatatctctt ttctttgaga tggagtcttg 30000
ttttgttgcc caggctggag tacagtggtg tgatcttggt tcactgcaac ctccacctcc 30060
caggttcaag cgattctcct gcctcagcct cccgagtaac tgaaactaca ggcctgtgtc 30120
accatgccca gctaattttt ttgtattttt agtagagatg ggatttcgct gtgttagcca 30180
ggatggtctt gatctcctga cctcatgatc tgcctgccta ggcctcccaa agtactggga 30240
ttacaggcat gagccaccgt gcccagcctg cctgatacct cttttataat atgaagtgtg 30300
gaaaacagat cttgaagtct tatatttact tttttttttg agacagagtc tcgctgtcac 30360
ccaggctgga gtgcagtgcc atgatcttgg ctcactgcaa cctctacctc ctgggttcaa 30420
gcaattctcc tgccttagac tcccaagtag ctggttttac aggtgtgagc caccatgccc 30480
aattaatttt tgtattttta gtagagatgg ggtttcacca tgttggccag actagtttct 30540
aactcctgac ctcaggtgat ctgcctgcct cggcctccca aagtgctggg attagagacg 30600
tgagccaccg cacccagcct atttactttt aataggttaa taatctgcct gagatcagtg 30660
gatttagctg ggagaaggca gggcagggta cactgtcaaa gcggcaccat tatctgggca 30720
attccggcct ggccttcctt gttcccatgg aatgggatga attctggcag aattctggtc 30780
aggattctcg agcagtccct ctgtcctcta cctaagctct tctatagagt ttggtttccg 30840
agccatgggg ttacctcctt accccccaga tgagtgctct gacaggggac aatgcgtcaa 30900
gtctggggtc ccacatgggc ttgtgaggag gaggttggca gctcctgggg ctgggtggag 30960
gtcaggcctt agagggcagg cagagggcat tctgctgagg gctgtggttg gcactgcagt 31020
ctccagtgag tacggcagct ttgacctcct caccatcacc ctcccctctc ttagatactg 31080
ccagaaagaa caggaaatgt gtagggaaca gccacatatg cagcctccag tcataccctc 31140
caccctgtcc ccacacttgg catttgcagt gccaccaagt ccttttggcc tcccctaagc 31200
tggctgtgac ccacacatgt agcatcacca gcaaagccag tggggccctg gggagtctct 31260
gtgcccacgg cccctgcatc tcctcctgct ccgtgactga gttgttgaac tgctggtgat 31320
tctgggggct ctggaccctg ctcttctgct cacatacttt ggtgactttc tataggagaa 31380
ggacgatttt tctctcggga atgtccactt gatcggctac agcctcggag cgcacgtggc 31440
cgggtatgca ggcaacttcg tgaaaggaac ggtgggccga atcacaggtg agctccactt 31500
ccatcactaa agggctccct cagctgcgct aagccggaat gctccccaat gaggcagcag 31560
agtgagtcat agaaagttag ctttggaagg aacctggaaa gcatctaatt caaaacttcc 31620
caagcgtttt tagtcacaag gaactttttt ttttttttcc aaattgaact cgcataggcc 31680
cgcagtatat gagacacaga aaggggggtg ctgtgggtga agtggggtgg gtatatggcg 31740
gcttgtgtta gccttgccag tgtgggcgtt ccccttccct ataggcatct cagagtctca 31800
gagcaccagg gaacacagtt tgaaaaccat aggcctggtc caataggtga ggaaacaaac 31860
acggagaggt gagacaactt gcccaggggc aaacagctaa taactcaaga gcggaagctg 31920
gatagagccc tggccccaaa gcttttgggt gttcccttca ggtctccacc caaggactca 31980
ggggaaaaga atgaatagta gattttagtt tgaattctgc ctctgatttc tatcttgtct 32040
cccaatagcc tctttcccat gttaaggtag catatttatg aatgagagct accaatttgg 32100
aaagggtggt tcaactttgt aataaaagga acctttctga tattggtccc cagggggtca 32160
acagaaaatt gaattttcca tgttgaaaac tgcatcttag tgagctttta acatgagagt 32220
ttggattaaa tcatttaatg attgtgaaaa gtgtaaaagg ttttctttct catttttggg 32280
cccctcaagt atatacacag agattcctct atgctacaat taatggtaaa tttgttgccc 32340
tgaacttcta tccttcttcc agcattattt tatttaaaat tacccctagc ttccccctta 32400
ctcccaagat tctaagggtg atcacctaga aggatagatc tgaacaattc ctttccctgc 32460
agtgttttca ttttaaaaca aaactcaggc tgggtgtggt ggctcacacc tataatccca 32520
gcactttggg aggccgaggt gggtggatca cgaggtcagg agttcaagac cagcctggcc 32580
aacatgatga aacactgtct ctactaaaaa tacaaaaatt agccagctgt ggtggcgggt 32640
gcctgtaatc ccagctactt gggaggctga ggcaggagaa tcgcttgaac caaggaggta 32700
gaggttgcag tgagccgaga ttgtgccact gcactccagc ctgggtgaca gagcaagact 32760
ccgtctcaaa aaacaaaaca aaactcaggt agaagagagt ccttagctaa ataacgattt 32820
ggtaaaggta gaatttcaga tttcagtgac aagctgatat ggtagaattt taaaaagaat 32880
atttgtgaag ttttcctggc tctcctgaat cctgtccctg ggcttgagtc tagtatgtgc 32940
gtgcacacac atgagcatgc atgcacacac atgcctgttg atccaatgac ttgattcatc 33000
ccagcttgag agctttattt ttgatttcca agactgtggg gcttgagctg cccaaagcgg 33060
gctatgattg tgtggccacc catgactgct cattaacccc ttgctggtgc aaagcagttt 33120
gactggaagc cactcgggtt ccattgtctg agcgcctctt cctagcccct gaatgagagg 33180
tgtgatgttt cagatccagg cacctgagtt caaattgtag cacacaccac agtccaggcc 33240
cagaatgtca gtaacgatga caaactgcac atcttgcctt tctctatttc taaatctgaa 33300
atgagatggg gctagggagg gaactttcca gttctttccc cctgcctgtt tgagtggcat 33360
ctgctggggt gagggctttt ctcctgaggg tggcaggaga gccaggcaga atgctaatta 33420
gtgctctggg atgtttgcat ggagcatgat gcccacatgc cctgagcatg gtcccttccc 33480
tctggccttt ctctgtagag aaggggcctg gcctctgtcc agttgccaag cagcacccct 33540
gacacacgct gttgtgtatg tctgatgggg gaggctgcgt cctctccttt ccccttggta 33600
gacttctcat ggacctgtgg acaaggtgat cgcaactggt aagaggacag attgagactt 33660
gcatggggtc ctgcgtccag tgatgccact atgctaaggc caccaggcag tccttagcct 33720
gggccttgca agggtatatc tgaaccccaa tcttgcttgg accagagggt gtggatgggg 33780
gtttgctaga gttttacttt cttaggcttc tgagagaagc ctgtgatact aggaaagagg 33840
tcatatattt atagttgaac ctgtgccttg tttgctataa tcaggtaagc aagcagatga 33900
atttattcaa gaaatgtgtc catatgtgca ggctagatgc tagctaatgt caaatacctc 33960
atgtgtggcc gggcgtggcc gggcgcagtg gctcacgcct gtaaccccag cactttggga 34020
ggccgaggtg ggtggatcac ctgaggtcag gagttcaaga ccagcctggc caacatgatg 34080
aaaccccatg tctactaaaa gtacaaaaat tagccgggca tggtggtggg tgcctgtaat 34140
cccagctact caggaggctg aagcaggaga atctcttgaa cccaggatgc ggaggttgca 34200
gtgggctgag attgcgccac tgcactccag gctgggtgac aagagtgaga ctctgtctca 34260
aaaataaaaa ataaaaaata aaaaacaaac aaacaaaaaa cacctcatat atgacagcta 34320
ctctatgcta tctctgaacc acatagcaac cctgaacatt ctacggaggg ggaaactagg 34380
actcagtgat taagtaaagg gcccaatatc ctgggcccta taagtagcag agctgggact 34440
caaacccatg ctgtcagatt ccagatccca tgcacctcac tgctgtgcca tgctgcctct 34500
cttctgtgga cagagataaa gtagccaggc atggtagtgc atgcctgtaa tcctagctac 34560
tcgggatgct gaggtgggag aatcacctga gcccaggagg tggaggttgc agagagccga 34620
gactgggcca ctgcactcca gcctgggcaa cagagtgaga ccctgtctta aaaaaaaaaa 34680
aaaaaaaaaa agtagccgtt gaactacagc aggccctgcc ctgccgggcc agaatgcaca 34740
gccctggaag ttttacatgt gtgcatgtgt gtgtgctcca tttgctggac aaagcaactt 34800
tggaggagtc tatgactgaa tggaagaagc ggcggaggtt ctctatctcc tctttattta 34860
aggaggcagt tctccctgtc attcatccct agttaaaaaa gttaaatgtg ccactgtacc 34920
cctgaatctg tgggccacag atatgtgtca gggacagtca gagcagggca ggcaggctct 34980
ggctgggtag gtgaaggccc agccttgcct gccagccagc ctgtgaaacc ccatcagctg 35040
cctgcattcc tccagcacag cctggtttcc atggcactga ccgtctggtg gggacctttg 35100
ttcacaccaa gtaaagagcc tctctcttgt attgattgca aagcagaatg ttgctctctt 35160
tctctttcct ccaggggaca tggtgagagg cagggagagg ccgctgtgtc agtactgcgg 35220
tccttgcggc ctctgaaatg ggttgtcgtg ggctgttctg ggagcacact gcttttgtgc 35280
catctttctt ttgggtcctg ggctagggat aaactctcaa gcagcctggg ctccctttct 35340
gtcttggttg cacataaagt tatctaaatt tggctgggtg cagtggctta tgcctgtaat 35400
cccagctact caggaggctg aggcaggaga atcgcttgaa cccaggaggc ggaggctgca 35460
gtgagccgag atcgtgccac tgcactccag cctgggtgac agagcaagat tccaactcaa 35520
aaaaaaaaaa gaaaaaaaag ttatctaaat ttttggaaac tatttagcgt ttcagcctct 35580
accagttctc atatgagtaa tgagttctgc aagttgaccg tccactctag acggtagctc 35640
tgcctcttgt ttatcctgaa actaccatct caagtctcct gataggctcc ctggttctaa 35700
tgtgctgtgg tctggtgaac acggtcgtta accatgtcct cctttcagga ctcttgttgg 35760
tcacatctcc tcccagcctt ggtgtttcga ggccaggttt tctcagccta ggcaatattg 35820
acatttgggg ccaggtaagg ttttggtgtg gggtgtgtgt gtgttgggat tgacttgtgc 35880
actgtagaat gtttagctgt attcctggcc tggccctgct agatgctagc aactctctcc 35940
atcagaatgg gggcaataaa aaggtctcca ggaatttcca gatgtcccct ggggggcaaa 36000
gccacttgta gtggtgaacc actgttccag cggcccttcc ttcctggcac atttcatccc 36060
ttaactttct ccaggtcctt cctgtgtgtc tgtggggcaa agagctgttc tggttgggat 36120
aaatctggtt cccataatca cttgggcttg gtaatagtag ctaatactga caggcactca 36180
gtctcttaag atctcacgtg gactttgaat taaggtataa attcaggttc cattttataa 36240
atgataatga aacacaggga agtttttcaa tatttggccc acagaatacc tacttttagg 36300
atcatccggg gaataaatat ctatttctat gtgccataat tcatatctac ccactaggag 36360
ttcaggcact tgggaatctg cattttatac tttgcaagtt ctcttttgga atggttttac 36420
attaatgcag ttttcaaatc aaaagatggt gaaagtctct cttactgctc tgctacctgt 36480
ccgtttccct tacccactct ctcccccaac gcacacgcat acaggtaact agttgcttgt 36540
gtgtccttcc agattttttt gtagctgtat ggtggaattt aaatttaaca agctcccaat 36600
ttcaaatatg agaaccagtg ctctcaacag tgctgcttgc ctgctcaaca caaaatgttt 36660
ggaggcggaa aggttttctc acggtggctg caaaatagag gaattctgga ggcctgatgt 36720
atggattctc agtgtattgg gtgtcctctt ctctctcacc attccacccc tgggcctgat 36780
taaaggaaga agaataaatt cagatacaaa tagctccgtt tggggagact ggattctcct 36840
tccagaagga tgtaggcagc ttagttattc tgtaaaaatt ctgctacaat taaggacatg 36900
aaagatgctc agtggcagaa tgaatgggac aaatggctta gtttcaggca gtctgagttc 36960
tttctagaac tgcttcagtt ttaacaggag cagttccttg gagctgtgtg gcagcgaaac 37020
ttgtggctag ttgacggggt ttggctgagg tgacgggata catgaaggct tcagaagaaa 37080
gtataaattc cataacagta ggatttaatt tctgcaatga tgtcttgggg tcaactgcat 37140
atggaaatgg gtttcttatt ttcttccatg ctttccatgt cttgattctc ccagctggtc 37200
tcagaagtca tcttcattct gcacactcag acttaacttg taatcaggac tcactgacca 37260
ggtgcacctg acaccacagc tgttttgggg cctccttctg ctgcaggttt ggatcctgcc 37320
gggcccatgt ttgaaggggc cgacatccac aagaggctct ctccggacga tgcagatttt 37380
gtggatgtcc tccacaccta cacgcgttcc ttcggcttga gcattggtat tcagatgcct 37440
gtgggccaca ttgacatcta ccccaatggg ggtgacttcc agccaggctg tggactcaac 37500
gatgtcttgg gatcaattgc atatggaagt gagttccctc ttttctgctt tgtgtttgac 37560
tcagtttatt ccatctcctt ctaaatcagc cagagccttt agcactgcag gcactattta 37620
ttcattattc tcacctccca ccacagaggc ctccaatgct gtatttaata tttctcattg 37680
ttccccaaga ctaaccaacc cagtggttcc tgctttgctt tctcacgctt taaggtcaca 37740
gttgttattg attaatttct tttcttttct ttttctttct tttttttttt ttggaaaggg 37800
agtttggcct cttgttgccc aggtttagat tgcaagggtg caatcttggc tcaccacaac 37860
ttccgtctcc caggttcaaa cgattctcct gcctcagcct cctgagtagc tgggattata 37920
ggcacatgct accataccca gctaattttt gtatttttag tagggacggg gtttcaccat 37980
gtcagccagg atggtctcga tcttctgacc tcgtgatctg cctgcctcag cctcccaaag 38040
tgctgggatt acaggcataa gccaccgcac ccggcttgtt atgataattt ctaaaatgtt 38100
ttttcatttt agggtgcaga catgaatgga ttaccaacaa aaggaccaac agaaatctgt 38160
gataaaaaga aagactaaag aaattttcct aaaggacccc atcatttaaa aaatggacct 38220
gataatatga agcatcttcc ttgtaattgt ctctgacctt tttatctgag accggaattc 38280
aggataggag tctagatatt tacctgatac taatcaggaa atatatgata tccgtattta 38340
aaatgtagtt agttatattt aatgacctca ttcctaagtt cctttttcgt taatgtagct 38400
ttcatttctg ttattgctgt ttgaataata tgattaaata gaaggtttgt gccagtagac 38460
attatgttac taaatcagca ctttaaaatc tttggttctc taattcatat gaatttgctg 38520
tttgctctaa tttctttggg ctcttctaat ttgagtggag tacaattttg ttgtgaaaca 38580
gtccagtgaa actgtgcagg gaaatgaagg tagaattttg ggaggtaata atgatgtgaa 38640
acataaagat ttaataatta ctgtccaaca cagtggagca gcttgtccac aaatatagta 38700
attactattt attgctctaa ggaagattaa aaaaagatag ggaaaagggg gaaacttctt 38760
tgaaaaatga aacatctgtt acattaatgt ctaattataa aattttaatc cttactgcat 38820
ttcttctgtt cctacaaatg tattaaacat tcagtttaac tggtagttca ttttctttta 38880
aagtctattg aaatattcaa aagggaaatt tttcaccacg tcagaaggca gaatttggat 38940
gttatggcat ctgtgtagtg gtgggacaac aattactatt gttgcgtaac aagacactcc 39000
aaaatttagt ggttttcagc agcagtgact cgactgggca gttctttgct gttctgggct 39060
cactcatatg gctggctggg cctgggtctg gacctgggcc gctatgtgat ttttatcttg 39120
ggctttgaaa attcagtgta gggacctata aatattcaaa gatgatgaga tgcatcctta 39180
tatatttttt gtattcactc tgaagctggg aaagattaaa aggaactcct gcccttgggt 39240
ccctaatgaa gtctgggtcc taactcgctc tattaagttc tgtggctggt caagtcactg 39300
atctaccttg ggctttattt gatataaatc atataaatca gcaggtctca gcttttctgt 39360
ccaaagccac ctgagggata agataaattc tcattagcaa ataaactttt ttaaggtata 39420
aatactatta taaaaataac agatatttac agtacaacaa tgcaagagga tcctaaatga 39480
aaagcgaatg ttttttctca actctcattc ccttattcca gctccacaga ggtaaccaat 39540
tgtttttttt ttgttgttgt tgtatggttt gtttttgtta gacagtagtc ttttcctgaa 39600
cagtgctgct ccttgtggag caaggctaat tcataggcag tgcatccaga gtctgcccca 39660
gttgattttg tgtgtaacct tctagcaact ttttgaaaat taatttgttc ttaaatatgc 39720
acatcatttt tatttattta tgtttttgag acagggtctc actctgtcac tcaagctgga 39780
gtgcagcagc atgatcacgg ctctctatgg cctcgacctg cagtcctgaa gtgatcctct 39840
caccttagcc tctcaagtag ctgggactac aggtgcacac caccatgccc agctaatgtt 39900
ttttgtaatt ttttttatag agacagggtt tcgtcatgtt gcctgggctg atctcgatct 39960
cctgggctca agcgatcctc cactttggct tcccaaagtg ctgggattac aggcatgagc 40020
tactactgca cctggcccac acatcatctt ttaaaaatta agtaatattg caacgtttct 40080
aattgtccct acttctttgc tcctcaaagg ttttacctac tgagctgtgg gctttttaaa 40140
tttatctcca tatttataaa taaattataa aaatataaaa atataaacat tttatttttt 40200
gagacagggt ctcactgtca cccaggtgct ggagtgcagc ggcatcatca tagctcacta 40260
cagcctccaa ctcctgggct caagtgatcc tcccacctca gcctcctgaa ttgctaggac 40320
tacaggtgtg tgccaccacg cctggctaat tttttgtaga gacggggtct tgctctgttg 40380
cccaagcgag tctcaaactc ctggcctcaa agtgatcctc ttgcctcagc ctcccaaagt 40440
gtcgggatta caggcatgag ccactgcctg gctatactgt tatattttga gccatcaact 40500
tgagacattg tttcccataa tggttgagat gccgctatct tacactgcct ctgctcctcc 40560
cacacatcct cccaagtgct tttgtaaatt ttgcttacat catcagtgtt catagcatga 40620
gtgtgccagg tgtctttcat ttgcccctcc caagtctctg ctcccctcat cctgctttca 40680
gcccaaggaa gctgtataga catcaacaga cttcccatgt ctctgggttc tgctgggttt 40740
ggccaatagc gatccatagc aggagatggc tggagggagg agaatgaagg cagagtgttt 40800
attcccttgg ccctctctct gcaaggtcac ctcaggccaa ctgttcctca gccagaagtc 40860
actgtgcctc aaggaacact ccaggcagcc aactccacct gacttctttt atttttccta 40920
acctgtcctt caccctgccc ttttgggccc aggggtggtg gtatttctgt tactggctct 40980
gggctactgc ttcctctcaa gagtgctatc tggcttgctg ggacactgcc tgggagaata 41040
ctattgctca ctgcacaggt taatgcactg tgattgcctc ccttttaata tacaggtggt 41100
gtcttcctgg agtgaccagt ttaccttctc cccatacacc tctcctgcct agttgttgcc 41160
tagacctggt gcacactgca gtcctcggga ctttctgttg ccaccctcct gtattggacg 41220
tcttgtttct ggatttccta tcctcttcct tcttggttta tccctcattt tttctgtagc 41280
acagcagttt tctaattcaa tacttttagg aggttgagta tggataattt tggaaagtgt 41340
ccccgggatg attccaattt atttaagcct gttttccaga gagctgcggg tctcctaagg 41400
cctttctcat ttctaacaag tctccctctc tagaacactg gttcccagtc gttgttttca 41460
tactagcaac attttaaaag caggataggg gtggctagta tgcaattgcc aactttggat 41520
ttttccaagc aggagtatat caaaaagcta caatttatta ccagtatttc gtaaaagaca 41580
gtttgacacc agaagagtga aaaagaacat gggaatagtg gacaaaaata gccatcagtt 41640
tgaataagcg tatgaaaagt gttaatttta tgaaaaattc actattttgt tcatattttt 41700
cttattttgg aggctaattg ttaaaagctt ttatcagtta tccatgggct ggagttggta 41760
gccactgttt gataccacac aaatgtttct ttgccctaaa atatcaacat ctgtctgtgt 41820
aaaggtcagg gctgcctttt tagtaagagc taccctttgt taaacacatt gtacctgcac 41880
tgttcatggg tgtttcacac acacaaacct acctggtctg cataaacctg aaatacagtc 41940
aagaaaacag gatccaccgg aggcggtgac tcaagcctat aatcccagca ctttgggagg 42000
ccgaggtggg aggatcactt gaggccggga gttcaagacc agcctggcca acatggtgaa 42060
accctgtctc tactaaaaat acaaaaatta gtcgggcata gtggtacaca cctgtaatcc 42120
cagctactcc agaggctgag gcatgagaat cgtttgaaac caggaggcag aggttgcagt 42180
gagctgaaat tgtaccactg cactccagcc taggtgacag agcaagactg tctcaaaaga 42240
aaagaaaaag aaaaacagga tcccagcctg ggcaacatag ggagacctcg cctccaccaa 42300
aaattaaaaa tttagctagg catatggtga tgtgcacctg tggtcccagc tactctggag 42360
gctgaggtgg gaggattgct tgaacccagg aggttaaagg ctacagtgag ctgtgatcaa 42420
gccactgcac tctagcctga gcaacagagc aagaccctgt ctcaaaaaaa aaaaaatcac 42480
tattgcccca gcaatggatt taatttaatt taattttaat tttttttttt ccggaccggg 42540
attttggctc ttgtaccgca ggtgggagtg caatgggcca atcccagctc acgggaattt 42600
ccgcccccca ggtccaagca attctccttg cctcagcctc ccaagtagct gggattacag 42660
gtgcccacca tcacacctgg ctaacttttt gtatttttag tagagacagg gtttcaccac 42720
attggccagg ctggtctcaa actcctgacc tcaggtgatc cacccacctc ggcctcccaa 42780
agtgctggga ttacaggtgt gagccaccgc acctggccct aaggagtaca ttttattagc 42840
attttagttt atttaatatt cattgttcta tttaaatttt cttattctaa ttgtgacgtt 42900
ttacattttt tcagttttta tacaggtttt gaatttatat ttcataatat ctattagcat 42960
gatgactttc acatttatac cttttattca gatggaattc atttttgttt tgggggggtt 43020
gatggtttta acttctactt tagtttcagg ggtacgtatg caggtttgtt atgtagttaa 43080
atggtgtgtc acaggagttt gctgtacaga ttatttcgtc actcaggtaa taagcatagt 43140
acccaatagg tattttggaa ttcatctttg ttgcgtgtta aaataggaat ctaattttgt 43200
ttctctttgt caaatgagcc tactttctca tcatccatcc attcctttcc caaagactaa 43260
taggcccctt ttatcataca taaaatttcc gcatatatgt ggaaaggatt ttattttatt 43320
ctattctatt ttattttatt ttgagatgga gtctcactca gttgcctagg ctggagtgca 43380
gtggcgtgat ctcagctcac tgcaaactcc atctcccagg ttcgagcgat tcttctgcct 43440
cagcctccgg cgtagctggg actacaggca cgtgccacca cgcccagcta atttttgtat 43500
ttttagcaga gacgtggttt caccatattg gccaggctgg tctcgcactc ctgacctcgt 43560
gatcctccca tctcagcctc ccaaagtgct gagattacag gggtgagcca ccgtgcccac 43620
ccaggatttt atttttgtct cacactgtcc tcttatcaat tcaaatgttt acaaaacaca 43680
aatactttct tttttaccat gtacctttaa cattcacaac aaccctgagc agtattgtct 43740
ctgtttctta aatgagcaga tagagactca gaaaggctgt atttagccta aaactcacat 43800
aatgctggat gacacaccaa gaattctgat tccaaactaa ctgctctttt cccagattac 43860
ctcctgcctg tttaagcacc tagtttctta tatctaattt catctattta tttaaatatt 43920
cattgaatac ccatgatgtt ccagacacta ttctagcttc taagataacg gcagaaaaca 43980
agccagataa aaatctgtgc catcagagag cttacatggc agagggctca gggtagctct 44040
gaacacagag gtgacacctg agaaatacca tatttgatga caaagactgt tctagtacct 44100
actttgtatc catccttcca aggggtctgg ggcttcagct tgtttggaga gtcaaaggaa 44160
gtcctgtgga agttcccctg actctaacta gtaactgaac ccccaagcca aagaaggcac 44220
ttgcagtcat ccaggctatg actcttcctt tgggaaattt caacaatttt ctttactatg 44280
ccagagacac cattttctgc tttcctctga catctgaacc aagttaagaa cttatctctt 44340
attgtctaac ccaactccct acagccatta tcaagcctgg tttcagaatg tcctggtaga 44400
gatggacagt ggagtttcca ccatcactaa agccctttat aaagagcttc cttgtcattt 44460
agaaatgttt tcacacttaa gaccagaaat aaagtcaaac tgatgtaatg cattatactt 44520
catagtcata gatcaattta agattactaa aaattgacta caggatttaa tcatttaaag 44580
ggtactctgg tctatctcgc tggctttagc agagaaaaaa ctaattcctg ataaacggat 44640
ggcaagatcc aatttccctt ttaataaata gtaattggta gggggaaagg ggaaataggg 44700
acggaattgt taaaagatac aaaattacag ctagtggctg ggcccggtgg ctcacaccgg 44760
taatcccgac agtttggaag gctgaggtag gtggatcact tgaggtcagg agttcaagac 44820
cagcctggcc aacatggtga aaccttgtct ctactaaaaa tacaaaaatt agctgggtgt 44880
ggtggtgcat gcctgtaatc ccagctactt gggaggctga ggcaggagaa tcacttgaac 44940
ccaggaggcg gaggttgcag tgggccaaga tcatgcccct gcactccagc ctgggctaca 45000
gagcaagact ccatctcaaa aaaaaacaaa aaaacaaaaa acaccaaacg aaacaaacaa 45060
gattacagct agacaggagg aataaattct agtgtcctat accactgaag ggtaaaacta 45120
gagttaataa aatatggttt caagtaacta gaaggaggat attgaatgtt cccaacacaa 45180
agaaatgata catgtttgaa aggatgggta tgctaattat tccaacatga tcactatacc 45240
tgtatgtttc gaaacatcac tatttnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45300
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45360
nnnnnttgct gctaaaactg aggtgtaggg cagtgaccaa ttttgggggg gtttcttatc 45420
cttttactgc cttggctgct gacagcatat cctgtcttga ccccaggcag ctctctccac 45480
tctgatgctg tgccctccac atgtcctgcc ctgatgctca gtctttccct ctcccggttt 45540
aatccaagta gctctgcagg gtttggaggt ccatgcaagg ggattttact ccctcctcag 45600
aatataggat gatggccttt ctttcctttc ctttcctttc ctttcttttc ttttcttttc 45660
ttttcttttc ttttcttttc ttttcttttc ttttctttac tttacttttg atggagtttc 45720
actcttgttc cccaggctgg agtgcaatgg tgtgatctct gctcaccgca acctccactt 45780
ccaggttcaa gtgattctcc tgcttcagcc tcccaagtag cagggactac aggtgcttac 45840
caccatgtac agttaatttt ttgtattttt agtagaggtg gagtttctcc attttggcca 45900
ggctggtctc aaactcctgt cctcaggtga tccacccacc ttggcctccc aaagtgctgg 45960
gattacaggt gtgagccact gcccggctgg atgatagcat ttcttaaggc tgctggtgcc 46020
aatgaggcag ggttggggtt ggttatgcca aattttttgt aagatcccag aaattatttg 46080
ggccagaaaa tccaaattgt atgcctaact ttgtaaccaa ctgtctctat gaccctgagc 46140
agattacctc acccctatgg acctttgggt cataatcttt aattgaaggg attagactag 46200
aatgaggttg tcaaacatct tcttaaagca tcagctagta aatgttttag gttttgtggg 46260
ccgtattgcc tctgttccaa ctctgctgat gtagcatgaa agtagccatg acaatatata 46320
aacaaatgag ggtgactgtt ccaataaaac tttatttaca aaaacaggtg gccagcaggc 46380
atggtgtggt gcacctgtag tcccacctat ttgggaggct gaggcaggag gactgcttgg 46440
gcccaggtga ggatcacttg agcccaggag tttgaggcca gcccaggcaa cataatgaga 46500
cactgtctat aaaacaaaaa caaaacaaag caaagaaaaa aaaaagaggt agcaaacctg 46560
caagccatat tttgctgagc cctgcgtgag atgatgttta ggatctgatc catttgcagc 46620
attctgtggc tgtatagccc tattaggagg tagtgactcc tgctgcacag ctggtctgca 46680
tcaccctact tagcacttat tatatgggcg ttcttgctct ttattgcttt gtgtgtattc 46740
gtgctgtctt cccatcaaga gagtgaacag ttaggcagca tgaatggcct taaattcctc 46800
tacatcttaa tgggaaccta gcaccttgct ggacattaat gaagcattac acaggtcggg 46860
tgcagcggct cacatctgta atcccagcac tttaggaggc gaaggtggga agattgcttg 46920
aacccaggag ttcaagacca gcctgggcaa tgtggtgaaa ccctgtatct acaaaaacta 46980
caaaaattag ccaggtgtgg tggtcatgtg cctgtcgtcc cagatactgg ggaggctgag 47040
gtgagaggat cacctgaatc tcggaagttg aggctgctgt gagcgggggg tcgtgattgt 47100
cccactgcac tctaacctgg gcaagagtga gaccttgtct caacacaaat aaacaagcaa 47160
aagaattaca tgaaaatact aacagcaaca ataaacagct atcactgttg aggagtacag 47220
tatttattat ttgagtgtct aatcatcaag aagggctcat cctgcatgct ttttatctct 47280
cccaccccca gcaatcacag aggtggtaaa atgtgagcat gagcgagccg tccacctctt 47340
tgttgactct ctggtgaatc aggacaagcc gagttttgcc ttccagtgca ctgactccaa 47400
tcgcttcaaa aaggggatct gtctgagctg ccgcaagaac cgttgtaata gcattggcta 47460
caatgccaag aaaatgagga acaagaggaa cagcaaaatg tacctaaaaa cccgggcagg 47520
catgcctttc agaggtaacc ttcagtccct ggagtgtccc tgaggaaggc ccttaatacc 47580
tccttcttaa taccatgctg cagagcaggg cacatcctag cccaggagaa gtggccagca 47640
caatccaatc aaatcgttgc aaatcagatt acactgtgca tgtcctagga aagggaatct 47700
ttacaaaata aacagtgtgg accccttttg tgatgtggct atcagtccat gatcacaata 47760
tcaatatctt aaatttctat cttgcattta actttcaaag ctgtttcata tttgttttct 47820
catttgatca tcacaagaaa cctgaggcag atgaacaggg gaccaaaatg acatgttaca 47880
tttttataat gtgctgtaaa atcacaaggt tttctgggta ctgtgggtat tattttacat 47940
atgaggaagt tgaggctcag agaggttaat accttggtcc ttaatccagg ggcacgtagt 48000
tagaaagtag ctaagtgaaa atcctcaaaa gatctaactc ccaagtgcag ggctccttcc 48060
atactgcagt ggcctcttat ggcattggac agaacaagag cagatggggg ctgcaggctg 48120
ggctcaacca aaagaacacc agccctaaaa tctctgtgct ggtgactcag tttgggtcag 48180
gggtctcctg tgatgacaag caagggttac aagcatcttt gttctgctgt cactgcagtt 48240
taccattatc agatgaaaat ccatgtcttc agttacaaga acatgggaga aattgagccc 48300
accttttacg tcacccttta tggcactaat gcagattccc agactctgcc actggaaatg 48360
taagtcatcc gtttcccttg ctgggttcgg gacagagaac aggttggttt gagaatgaga 48420
gagcacaagg gagcgtgtga acgagtacag cacgcaggag agtgcagtcc gactgctcag 48480
ggaggagcca ggtggtctag ctggcctctg cagtcctctc tcccactgca ccttctctac 48540
caagccctga ctcagcgttt cggggagaaa agttcctggg atgaatcggc atcatgagaa 48600
gctgctgggt cagcagctgg atcagctggg cagaaactgc tgtttcattt caggcaatgc 48660
ctgacgatgc ttcaggcaat ctgtaacttc gtaaacgtca tcctggacct ctcctaatga 48720
tgtggctatg ggaggggtgc atagcgatct gtatatgttt gttgagtatc caccagggac 48780
aaagcatcat gccaagtaat gttgggattg tgatgaaaca tcatcaaagt agctcacttc 48840
tcagggctca cagtctagct gggagaccag gtttgtacac gcacgtgcac acttacacac 48900
aggtagggca aggcagatga ggccaagcca aatgagaggg acagacagga agaggctagg 48960
agtttggggg agacagatgc ctgtctgtgg gatggatgag gtctgtgggg ggagctggtt 49020
ttccccagca tctgattttc agagcatcat taactctttc cttcagaggt tctaaggctg 49080
agcggaacag cgcacttgag tggggatgtg acttctttgt gttttcttat tgtgttcttt 49140
cttggttctg tcttttcagg aagtagcata agtggggtgg agggggtgcc caggaatcgt 49200
tttgataaga agctggggtg ctgagggcaa agttagtttg gcccatatgg agtacacctg 49260
agcttccatc ttggtcctgt tgataggaag tagagggata gaggtgggga aagcactcac 49320
actgtctctc tcctgtctgt gtggggttgt tactgccact gtgtcacccc acacagtgtg 49380
gcagttttcc agctgttagg ggagtgagat cagcttctct cccacttgta gagtggagcg 49440
gatcgagcag aatgccacca acaccttcct ggtctacacc gaggaggact tgggagacct 49500
cttgaagatc cagctcacct gggagggggc ctctcagtct tggtacaacc tgtggaagga 49560
gtttcgcagc tacctgtctc aaccccgcaa ccccggacgg gagctgaata tcaggcgcat 49620
ccgggtgaag tctggggaaa cccagcggaa gtaagtgcct cctgctcctt cttctgcctg 49680
gtgtaggtgg ggaacagaag gctgtgcctg tgacatttcc tttcctttgc tgcatctacc 49740
ctcaatcctt ccctccagca tgcaggtaaa acttcaaaca cctttgcaag gacaagtgac 49800
ttccagatca aggcttctat caaaactgtt acgcatctca tgccctgaac atgggccctc 49860
agggagacgg gtgggccagt ggaggcagta attcagccct ggctctgttt gccaggcctt 49920
gtgtcagcct ggaggagggg gacttctcct ggactgggct ttttaggagt gctgccttgt 49980
cgatgtgttt actgtggggt ccatgcagac aaaaggcttt gcttgcttct gagcaaagca 50040
gactggagta tcacttccag agagccccca tggatgaggt ctgtgggagg aactggtttt 50100
ccccagcatc tgattttcag agcattattc tctctccttc agaggttcta agtcaaggat 50160
gtggggctgt ggtcctaact gcaagtaaca gagaatcctc tgtgccacac tagctggcag 50220
tttcccattt cttttaaccc caagcctagc ataaaagatc cagatctttc tatcagctcc 50280
taacaccgtg cctggactcc ctgagcaggc ttttttctaa acagaagcca gggaagcctg 50340
cgagaccttt gtcaagaagg gagagtttcc ttaaccaagt ccttcagctg acctctacag 50400
aggtcactgc cagccttggc atgggaggag gcagctgcct ccctctttgt ccctcctctg 50460
gcattttctc cattataatt cctgccatgt ctggtttaca ttcgtcatct atgacacagc 50520
ttggcagcag agggaagtga agtggaggct tggagccctc agctgaatga ttctagtcta 50580
gaaacctgtg aactgggggt gctggcctgt ggtctagact gtgtatgttt ttacttctct 50640
gggcccttgc aggtacaaac tatacctatg gatgtgagag cagaaagatg atcatttata 50700
aataaaatat ttcaaagaaa ataaacacct attaaatcac cattgagact cacaaatgta 50760
aatagttttc catttgggtt tagatatcag atttttaaaa agtaaaacat tatagataca 50820
gttgaagccc ctgtgtcctt ctctgctcta cccttcttct ttcaaaccaa atataactaa 50880
tatttcaatg ttaacaaatg ttagtgtgtt tccttccatc atgtttttat acactttgta 50940
cttactgttt tttttgagac tgagtctctc tctttcatcc aggctggagt gcagtggcac 51000
aatcttggct cactgcaaac tccgcctccc ggattcaagt gattctcctg actcagcctt 51060
ctgagtaact gggattacag gtgtgttgcc accatgccca gctaattttt gtatttttat 51120
tacagacggg gtttcgccat gttggccagg ctggtttcag actcctgatc tcaggtgatc 51180
tgcctgcctc agcctcccaa agtgctgaga ttacaggtgt gagccaccat gcccagccgc 51240
cttagtactt ctgtctgtgt acataaacaa tgtatagaat tttggatgct tctgaatttt 51300
acatgagtgg acataatatt ttacaatttg attttttcat tctaatattg tttgtgaaat 51360
ttatccaaaa cataaagatc taatctattc ttgtttaact cctgtatgta aatcaccatt 51420
gcacaaatag ttttgccatt gttctgctga atagttgagt tgttgctttt ttgtagttac 51480
aaatggtgct gagatgaaca tcctctgcat tttccttgtg cacatgtgtg taagggctgt 51540
ttctcagcat gtgttctaaa aagtggaatt gctgggttgt agtatatgca ttctccagct 51600
tgaacagata ttgttaaatc tgtctccaaa gtggttgaac taattttcca tcttaactat 51660
ggatgaagag ttcccttttc cctccttttc acccaggtct ggtgggacat tcttgtcatt 51720
ttgttaggtg tgaatgggcc ccctgctgtt gtttgaattt gcatttcctg attgtgcgtg 51780
agataggtca tgtttttata tgtgtataaa catataggtc ctaaggtcct tagggcttcc 51840
tgggaaaggt tcttaagctc ttctgaatgg ggcttttgtc ctgcctcagc tccagggtca 51900
ggtgccccac acccttaagg attacgtggt cacgctaatc acacgcccct gaggctgcgt 51960
ggggcagcca taagttacat ggaaatggtt attaggccct gattctttct tgtggtcttg 52020
gctgtccatt tgctgaatct ttggttttga cttgcagctg agttgcagtt gnnnnnnnnn 52080
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52140
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nccagcagca gcggaaagaa atatcctgga 52200
ggaagttgaa ctttaccctg cttgggaatc tatccaggtc tgaagcccct ctaccgaggt 52260
ccccagaatg ttccccgctt tccccactga gtctgctggg gagtaaagtc caagatgaca 52320
gctcagttgt tcccacgtgg cagtgtcctc tgagggatcc aaataaacct ggagttttat 52380
tatcctgact gtgccgataa ggagactggc cgacctaggc agagaaagtg gaggccactg 52440
tccggtatct cttggcaggt cacactcatt ccttttctgt tcacatcagc tgggcttgta 52500
acagctccca gtgatagcgt atccaggctg gtcacccgac ggtggagtct tgtccatttg 52560
gaaggcccag accaatgact gaagtaaact actcacccca caatttactg ctcctagaac 52620
aaattgcttg tcttggtgat aattctcttg tgtatggctg aaactatcag ctctgggata 52680
atgtgcataa ctttgcatgg attgataaag ctccatttat ttatttggct aaacagagtt 52740
attacactat tacttcttct atgagtttct gacataaagt cagacaaact caaatgcaag 52800
cttatttatt tatttattta tttatttatt tatttattta tttatttatt tttgagactg 52860
agtcttgctc tgttgcccag gctggagtac agtggcgcaa tctcggctca ctgcaagctc 52920
cacctcccaa gttcaagtga ttctcctgcc tcagcctccc gcatagctgg gattacagat 52980
gggcaccacc acgctcggct aatttttgta ttttttagta gagacagggc ttcgccatgt 53040
tggccaggct ggtctcgaac tcctgacctc aagcgatttg cccaatttgg tctcccaaag 53100
tgctgggatt atgggcatga gccaccatgc ccggcctcaa atgcaagctt ttctatcatc 53160
atactaatcc tgctgaatga atttggagct caattcctat gggggggcaa gagcctagag 53220
gtcaaagctg tcctcttgaa tcctggtgtc gttgatgcgt gcataaggct ttgcttgttc 53280
atactctctc aggcagaagt gtaatttgag gaaaatgtag tgtgcatgtg tgtgtgtgca 53340
catgcacatg tgtgcataca tgtcctataa gaatgagaat gggtgcccac ttttcttggc 53400
tcctctccct tgtgcctctg ggcctttctg cctgaatgca cttgcctgga aatccttgtc 53460
gatctctctc attcaatgac cagagcacca actatggagt ttggaaacct ggctccagcc 53520
ctgattctgg cattgacatg ctgcataaca ttggtgaaag caactggcct ctttgaacat 53580
cccttcctgg tgtgtggcag gaaacatttg gtttccttga actgaacccg cctgacaatg 53640
ggaagatttg cctgaaccac ctcccctgct gggtagctat agggattaaa tgatgtaaca 53700
gaaggtaaag caggattatt ttttaaactt ttatctattt ttaatgccct cctatgtatc 53760
ttcaaggcca gagccttctg agacactcca gcccacggtg actgctctcc tcttatctgt 53820
cacttcttta gccttatact gctttgctta ttatgatcta tttatgtgtt gcgctttatc 53880
cacttgtctt gtccataagc tctttttctg cccttttgaa tcttttcagt ggctagtgtg 53940
ctgccttcat cataactgat ttcagtatgt ctttccagat tgactactta attgaatttg 54000
gaatatgggg aaggagactt agtgtaaagg gaccacccaa tacaaggtct gagattattt 54060
tatgccatat ttcatggtgg aataaggaat gaataacaca atctgaatga ataatgcagt 54120
gtatcatgag ttgggaaggg agaaaaaaaa gagtatttgt acatgtcatt ttttctttct 54180
ttcttttttt gtttttttga gatggagtct tgctctgtta cccaggctgc agtgtagtgg 54240
cgtgatctcg gctcactgca acctctccct cctggggtca agcgattctt ctgcctcagc 54300
cacgcaagta gctgggatta taggagcacg ccaccgcacc tggctaaatt tttgtatttt 54360
tagtagagat ggggtttcac cgtggtggcc aggctggttt caaactcctg acctcaaatg 54420
atccgcctgc ctcagcctcc caaagtgctg ggaatacagg catgagccac tgcgcccggt 54480
atatacatgt cattttcaat cattttgacc tgaatcacct ggttttatct ggtcaatgaa 54540
agcagaggac aatggtcaaa cttactattt gggtcttttg agacaaagta accagctgat 54600
cgactgtctt ttctgctcag cagaacttgc tggccctgga agccatgagg agtaactgaa 54660
ctgaggcgaa taggctcata gctggggagg aaacagcaac atagcaacat ttttactttt 54720
gccttattgg tgtaaccttg ttcacaagga agacatggaa gggaaaggac ttggagagtg 54780
agagaggaaa cagaaaagaa agattgtgag gaagggccag gaggaagcaa atagagcttc 54840
cagttcatgc ttggggagta ctcaaagtgt ccatgggaga gctacgggtg gttgcttcat 54900
atgcagggag aggcctggcc agacccacct tcctcccagc tctgacccca gagtgaggtc 54960
agtgtccagt ttccacgtga acgatttgca gaccccaccc tttccccagc aggtagcaaa 55020
ggccacctca cttgtgtcta tgcacaagcc tgggctcaga gattctctca agccctagtg 55080
tcagtgctct ccagatcaag aggaccaaca gagtatatat ctatagagaa gtttatttta 55140
aggcattggc tcacgtgatg atggaggctg gcaagtccaa aatctgcagg gtaggcctgc 55200
agcctggaaa cccagtgaag aattgcagtt tgagtccaaa ggcagtctgc tggcagaatt 55260
ccttcttgcc cctgagcaag aaggaattat tttctatgca ggccttccac tgattggatg 55320
aggcccaccc attcaatgga gggtcatcta atttactcaa agcctattga tttaaattta 55380
atctcattcc agcactttgg gaggtggagg tgggtggatc acttgaggtc agggagtcga 55440
gacnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55500
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttcaa aaaaaaaaaa 55560
aaaaaaaaaa aagaaccatg tcttcatgaa gcttacattt tttgaaaagg agatagaaat 55620
ttaagaagaa aactaataaa tgtgataatt tcattcagtg agatatgttg tgaagacaat 55680
aaaacaagat tatgtcagag aggagctcca tagactgagt gtcagggaag gagttaaggc 55740
ttcagtgact ggaagaagcc aacacgtgaa gacctagttg taaatcattg gaaggaaggg 55800
caaaagcaaa ggcatcaaga caggaatgag ttgactgtat ccctgaagca gcaaggagcc 55860
aatgtggctg gagcaaagtc agtgcagagg aaaagtatag gaaatgaggg cctggagcta 55920
gatcagcctg cacagcacag taagcagttt ggactttatt gggtgtgaaa tgtgaagtct 55980
ttcaagggtt ttaacctggg aagtgctttg gagagaagtg gggagtgtta tattatatag 56040
tttatatttt ttaaagatta cattttctgg ttatagttat catagttcta gaaactgtat 56100
tttaggacaa ccaaatcacc ccagttaata agagaaagct ctttttacaa agaatgccaa 56160
gctattcaga ttaaagaaaa ataaagagac atggcaacca aacacaatgc cttattctga 56220
atttggctct tcgtccaaaa aaaattctat aaaagatatt gttgggaaaa ttggcggcaa 56280
ttaaatatgg tctctaggtt agataatatt attatagtaa atgttaaatt ttctgattct 56340
aaaactgatt atgaaagaga acaacctggc caggcacggt ggctcatgcc tgtaatccca 56400
acactttgga aagccgagac gggcagatca cctgaggtca ggagttcaag accaacctgg 56460
ccaacatagt gaaacccagt ctgtactaaa aatacaaaaa ttagccaggc gtgatggtgg 56520
gcgactgtaa tcccagctac tcaggaggct gaggcaggag aatcacttga accccggagg 56580
cagaggttgc agtgagccga gattcgcgcc aataccactc tagcttgggt gacagcaagc 56640
ctcctgtctc aaaaataaaa ttataagaaa caacaaccag atgtcatgtg ggacctagat 56700
tgggtcttgc tttgtttctg gtttttttgt gtgtgtgtgg ctgctttggt tggttggttt 56760
gtttgtttgt ttgtttgttt gagacagagc cttgctctgt cacccaggct ggagtgtagt 56820
gacatgatct caactcactg caacctctgt ctcccagctt caagcaattc acatgcctca 56880
gcctccagag tagctgggac tacaggtgtg tgccaccaca ccctgctaat tttttctatt 56940
tttagtagag acagggattc accatgttgg ccaagctggt ttcgaactcc tggcctcaag 57000
tgatctgcct gcctcagctt cccaaagtgc tgggattaca ggtgtgagtc agtgtgcctg 57060
gcgggtcttg ctttgaacaa accaactgga aaggacattt ggggaacaac tgggataact 57120
gaaattgagg ttaggtatta gaagagatga aaatgtatca acatggtaaa attgagatta 57180
actggaaaaa aatctgatta tgaaatagca tgtacagtat aatttcacct ggtaagtata 57240
tatgtatata catacacaca tgaagtatat atctcacact atactagaag gatatattaa 57300
agcttaatct tttggaaata taaggatttt attttcttaa tctgcttacc tgctatatct 57360
gactttctat aaaacacata taaataatct taaatttttg aataatttta aaattttact 57420
tattatttat ttatttattg agatggagtc actctgtcac ccaggctgga gtgcaatggt 57480
gcgatcttgg ctcactgcaa catctgcctc ccgggttcaa gagattctcc tgcctcagcc 57540
tcccgagtac ctgggattac aggcacgcac caccatgccc ggctaatttt ttcttttctt 57600
ttcttttttc tttttctttt ttttttttga gacagagact cgctctgtca cccagactgg 57660
agtgcagtgg tgcgatctcg gctcactgca acctccgcct cccaggttca agccatgctc 57720
ctgcctcagc ctcccaagta gctgggacta caggcgtgtg ccaccatgcc cggctaattt 57780
tttgtatttt tagtagagag agatggggtt tcaccgtgtt agccaagata atctccatct 57840
cctgacctcg tgatccacct gccttggcct cccaaagtgc tgggattaca ggcgtaaggc 57900
atcttgcctg gcaacttttt ctatttttag taaagacagg gtttcaccat gttggccggg 57960
ctgatcttaa ctcctggcct caagtgatct gccaccccgg cctcccaaag tgctggggat 58020
acaagcgtga gccaccgcac ctggccaatt ttaaattttt taataaataa attgctctgg 58080
ctgttctaga tggattacag gggcaatcaa gagtggaagt aggttgtcta gttgatcagc 58140
aatatgcttt tcagtatctg tttcccaagt ctaggtggaa gccagattct tgctggaaat 58200
gaagcttctc cttcatggga atcccacata catggccaaa gagatcttac gtctgggagc 58260
acaaactggg tatgttaaag ggcttgatat tctccagggt attttgttgt ccaacaaagc 58320
tgggtagaga tttgcacagg tccacggagc tgctcagcaa agggagggtc tggaggagtg 58380
ctatccagca gactaaaggc aaggcaccag ccctctttga agttatacac tggtccagcc 58440
agggaaatca caagcacaca cacataccca acatcagtta atgacaagat aagaaagaaa 58500
atgtactttt aaatttgatc ttaaaacaat tcacaactct taaaaattat gcagaataag 58560
ccaggcgtga tccaccaagc ctggtccatg attaatagta aatgtgaagc atccggccac 58620
aacacgtggt ataggctgaa tatccctaac ccgaatatct gaaatccgac atgttccaaa 58680
atcgaaaact ttttgagaac caacataaca ccacaagtgg aaaattccac acctgacttc 58740
acatgacagg tcacagtgaa actgcaggtg cacaagccag tttatttaac atacccannn 58800
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58860
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngcc tctgttattg cagctgaata 58920
accatcccaa agtcaagcaa tattgtgtgg gattctggag aattgagaaa tagcaaaata 58980
atttttgatg gaggcagctg gcagggctag ccctgcctgc aaagttgtcg gaaaggacag 59040
gtcccacggg aaagagtagc aatatgagat agtgccaaaa ctatggaatt tgaagtcaaa 59100
tgagctgagg tttccacact gacgtctttg agcaattact tcacctctct cagccagttt 59160
cctcacctaa aatatatgca tgatgatgat tctgccatta caggaaatgt tgacatgact 59220
gtagtaataa caattcacat tatggattat tttctatttg ccatgcacta ttatttcgca 59280
aaatgaccct atggaacaga tagcaccgtt attcccatta tatagtcaat atgtgccagt 59340
gtactctgta aagtactata caaatattag gtattctttt ctagcataga ctgccagtaa 59400
attcactgag cttgcagaat tatgacagga aaacatatta aaacccaaag tagactgggc 59460
cctgtggctt atgcctgtaa tcccaatact ttgggaggct aaggtgggag gatcacttga 59520
gcccaggagt tcggaaggac cagcctgagc aacatagcga tacactgtct caacaaaaaa 59580
tacaaaaatt agctgggagt ggttggcaca ccctcggtcc cggctactcg ggaggctgat 59640
gtgggaaaat cgcttgagcc cgggaggttg aggcttcagg gagccataat ctcaccattg 59700
cactctaccc tgggcaacag aacgagacac tgtctcaaaa aaaaaaaaaa cccccaaact 59760
acctaactaa aaagccatag acactactat gcagtcattg taaagaatgc tatggatgca 59820
tatttatttg aaaacatttt catgatacac tgagtggaaa catatatagt atcaaatttt 59880
cataagtgaa ttatattagg ttggtgcaaa actaattgtg gttttgccac ttaatataat 59940
atttatatta atatattaaa tgatatatta atataacata tgtaatatat gtttactata 60000
atatttataa tacctatgta tatgtgctgt tggaaaaaag acaaataata ttaacaatat 60060
tgcaactttg ggtaatgaga tcctgtgtgg ttaaaattag tttttcccta tgttttctaa 60120
tttttctgca aggaatatga agtacatgta caattttatt ttaaaattac agcaaaaaga 60180
gactcagggc tgggcgtggt gactcatgcc tgtgatccca acactttggg aggccgaagc 60240
aggcagatca cctgaggtca gcagtttgag accagcctgc ccatcatggc gaaaccccgt 60300
ctctacgaaa aatacaaaat tagccaggca tgcgcctgta gccccaacta ctcaggaggc 60360
tgatgcagga gaatcacttg aacccagacg gtggaggttg ccacgagccg agatcatacc 60420
cctgtactcc agcctgggtg acagagccag actctgtctc aaaaaaataa aaaataaaaa 60480
aagagactca ggcatgaaaa cctaactcaa aatatttttt aatattgcct ggcacacagt 60540
ctactttccc cagactgact cagcaacagt tgtgagcatc tcctcaaaac tggctttata 60600
gacctaagaa gcaaaatatt ttatttcaag gggatcctat tagtgaaatt ttacatcgtc 60660
tccctttggt catttttgga tgacttgaaa taaacagtct tgcctgtgtt cctaggcaaa 60720
tgttataatg ttgctaggca aatatcataa atcggaagat aagattagga atatttttta 60780
ctacgtagga agtgttgagt tnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60840
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60900
nttacctccc ccaagacctc accctttttt tttttttttt gagacagagt ttcactcttg 60960
ttgtccaatc tggagtgcaa tggcactatc tcagctcaca gcaatctctg tttcccgggt 61020
tcaagcgatt ctcctgcctc agcctcctga gtagctggga ctacaggcgc acgccaccac 61080
gcccagctaa tttttgtatt tttagtagag acggggtttc accatgttgg ccaggatggt 61140
ctcggtctct tgaccttgtg atctgcctgc ctcggcctcc caaagtgctg ggattacggg 61200
cgtgagccac cgcgcctagc cctggtttgg tttttttttt tagagacagg gtctcgctca 61260
gttgcccaga ttggagtgca gtggcatgat catggctcac tgcagctgcg atctcttggg 61320
ctcaagcaat cctcctgctc cagactccca agtagctagg actacagctg tgcaccatta 61380
cgcccagcta tttttttggt agagatgggg gtctcactat gttgcttagg ctgagctgga 61440
actcctgacc tcaagtattc ctcccttctc agcctccaaa actattggaa ttgcaggcat 61500
gagccactgc acctagcccc catgttaact attattagtt tgtgactttt cctaatttgt 61560
cccagacagg aaggagacag tgacagctgc actgtgcccc ctccctaagt tctgccatca 61620
gaactgctcc tctgacatct tttctgatgg ccagaaggcc accaggaccc atagtgatca 61680
ggctgtctca acctctttcc ccaggctagg ctaagggctc aggaaaagga agaatgaatg 61740
ggggctctgt agagtgcatc taagctctcc tgcatgcagg agggactgga gaacgaactg 61800
tgtagcttct taccttatca ggcaggcaat acccaggaat gctggagtca ctgcttttgg 61860
agcattcaaa ctctctgcag cactttctga gtcttgctgc taacagctgg ccttgcaagg 61920
aattcattac catagcacca caggaggcct tagaggacgc cggttcagga gcttgctcag 61980
tcatttgtgc agctcactgg ctgtgcatct ctgtcggaat cctctcatgt ccctagaact 62040
caaatcacta acattcccta cgctgttgta actttatttt actacctgtt cagccagcct 62100
ctgcttgaac acatgcagtg attgggatct catttctctt ccacatagac catttcacta 62160
ctagtaagct taatttcact gaaccaccta tggtgaagtg attcctgagt cattaaataa 62220
ctttctactg caccctttct catagggtta tttggagact taaataagat aacagtgcct 62280
tgctcacaga aaacagcctg taatgtgaat tgacaattat tgctgctgat attgttttca 62340
gaaagttctt atatggagct gaaatctgca ttctgtaact tcgaaaactt ggtcttaatt 62400
tggctcattg gcataacaga aaataagtct aatcatgccc gcctgaggat ggttctactt 62460
atcacaagtc tcctcttagt ctagttcttc atactcagtt ccttcagccc ttccacatgg 62520
gacgtaattt ccagatctct caccatcctg actgcccttc cctggacagg ttctgtttga 62580
ccttcatccc tccttggagg agtggccagg tgctggcaac taagacatgc gagctgtcca 62640
ccatctggat tatgcagatg aactaacatt gaaaatgaca cgctacatgc attacttaat 62700
ttcgttctca cagcaccttc atgaggagaa aactgaggct cagagatgtt aagtagcttt 62760
ccccaaatcg tttagctcag aaattgtgga accagaattc aacccagatc tgtctgactc 62820
caaagtctgc tcttctttct tggggaagtg gaagcctgag tgctgttctt ggaacagcct 62880
caagacaata atctggggac tgtcttggtc ctgcttgcag agggtccttc tgttacgtat 62940
gggtcctttc tggcccttta gcttctacaa agaaagtcag tccccctcca gctctaaaga 63000
cacatgaagt ctgtttgcag tccagacctt gcaaacttgt agttatagaa acctcagagg 63060
cctaccaggg cttgaaggat atttggctcc actttacatc tccattcttc ttttcctcct 63120
gtttttaatg taacctgtaa tcctagcaat ttgggaggct gaggcaggtg gatcacctga 63180
ggtcaggagt tcaagatcag cctggccaac atggtgaaac tccgtctcta ctaaaaatat 63240
aaaaattagc tgggctaatt tttaattagt gtggtggccc gcgcctttat tcccagctac 63300
tcaggaggct gaggcaggag aattgcttga acctggaagg cggagattgc agtgagctga 63360
gattgtgcca ttgcactcca gccttggcaa caagagtgaa actccgtctc aaaaaaaaaa 63420
agtattctgt ccaccacgtg gaataatgta atatttagta ataagaacag ctgcaaggtg 63480
tagagagtta ccgtatatcc atcacccagc tttccttaac gttaacattt tatataacca 63540
cagcatcatt ttgtcaaaac tagataatca gcattggtac aatactgtta actaaactct 63600
aaacttcatt taggtttcat caggctttcc actattgcct ttttcaattc caggatgcan 63660
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63720
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt ttataagatt ttaaaactgg 63780
caaaaaatat tatggattta ttcatagcta gtaccagcat taaaattttt tttttttttt 63840
ttttttttaa gaaacagggc ctcactctgt tgctgagtac aatgctggag tacagtggcg 63900
tgattatagc tcattgtaac ctcaaactcc tgggctcaag ggagcctcat tgcctcagcc 63960
tcctaagtag ctggaactac aggtacatgc caccaaagcc cagctaatat tctgttattt 64020
ggtaaagatg gggtctcact gtgttgccca ggctggtctc gaactctttc tgccttggct 64080
tcaaccacag tgctgacatt acaagcatga gctactgagc ctggccaaga ggaattaatt 64140
taactagaaa tctatagctt ttcaagctac caattaagtt tgaaagtaga ataaaaacat 64200
tttctgattt gccaagattc aaaagacctc ccaggaacct acaaggaagc tacaagatgt 64260
actccttcca catgagggaa caagccaaga aaaaggaaga aattcaagaa acaggggatc 64320
caacacaggg cagtggtata gagaatcaca gccatgcagc aggctgggac aagagtcaaa 64380
acagctcccg gatcaaccag gagaagaaag catagaactg acggttcact tgatgcgtct 64440
aaccttattc tgtggaatgt tacaatactg taggagaatt tggaagcaat gaataatagt 64500
tatatggaga ataaagtaaa taatttcgta tagactacaa actttgggaa aaacaagtta 64560
ttcaagaaag gtcatgtaat catagtatac gactaaattc aacagtaatc aacatttata 64620
taatcactat gatataaata ctaaatattt aaccaaaaat tgtgagttaa ctctactgag 64680
aagattccag aagcagcagc aggattggtg gtctacgagt gctaaacctt catcttccat 64740
aataggtagt gctcaataga aaatatgtaa agttagtaaa tcaaaaaaca gtaaaaattt 64800
attagttata aataaggaag tagggccagg tgtggtatta tcccaacatt ttgggaggcc 64860
gaggcaggtg gatcacttaa ggccagaagt tcgagactaa cctggtcaac atagtgaaaa 64920
cccgtctcta ctaaaaatac aaaaattagc taggcatgtt ggagcatgcc cataatccca 64980
gctcctcagg aggctaaggc acgagaatca cctgaacctg ggaagtggag attgcagtga 65040
gccgagattg tgccactgca atccagcctg ggcgacagag tgaaactctg tctcaaatta 65100
aataaataag gaggtaaata gcaaaaagca acagcttata gatttttaaa tggtggccag 65160
gcgcggtcgc tcacacctgt aattccagca ccctgagagg ccaggcggat gggtcacctg 65220
agacaggagt tgagaccagc ctgaccaann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 65280
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 65340
nnnnnnnngc ggttcacgcc tgatatccca gcattttggg aggccaaggc aggcgaatca 65400
tgaggttaag agatcaagac catcctggcc aacatggtga aacctgtctc ccgttaaaat 65460
accaaaatta gcccggcatg gtggcaggta taatcccagc ttacttggga gactgaggca 65520
ggagaatcac ttggacccac ggaggcggag gttgcactga gctgagattg cacccttgca 65580
atgcagcctg ggcaaccatt gccaaactcc atttccaaaa aaaaaaaata ttcttagggt 65640
ttggggcttt tttggggggg gttggggtgt aactatggta aatgggatta ccttttaaat 65700
tttttttctt ttttttgaga tgggagtctt gctatgttgc ccaagctggt ctgaactact 65760
gggctcaagc aatctgcctg cctcagcctc ccgagtagct gggattacag acgtacacca 65820
ttgtgccatc ttgatttctt ttcttgtttg tgtctagaaa tgctacttgt ttttgaatac 65880
tgattttgta tcctgcaact ttactaaatt cacttattag ttctgagact tattgagtag 65940
agtctttata tttttctcgt tttttgagac agagtcttgc tctgttgccc aggctggagt 66000
gtggtggtgt gatctcggct ctctgcaacc cccacctccc aggttcaagt gattttccta 66060
cctcagcctc ctgagtaact gggattacag gcatgcacca ccacgcccag ctaatttttt 66120
tgtattttta gtagagacag ggtttcgcga tgttgaccag tctagtctca aactcctgac 66180
ctcaagtgat ccaccatcct ctaccaccca aagtgctggg attacaggca tgagccactg 66240
cgtccggcct gatttttcta tatataaaat catgtcatct gtaaacaggg actattttac 66300
ttccttcttt tcaaattgaa tgtccttttt tctttctttt gcctaattgc tctggctagg 66360
acttccagta ttatgttgta attcaagaaa tatttgttga gaacttatca taaacaaaaa 66420
aattattcta ggcactagag gaaaataata cagacaagaa ctatgtcttc tgccaggcac 66480
agttgctcat gcctgtaatc ccagcacttt ggaaggtcga ggtgggcgga tcacctgagc 66540
tcaggagttc gagaccagcc tggccaaaat ggcgaaaccc cctctctact aaaaatacaa 66600
aaattagccg tggtggcatg cgccggtaat cccagctact ggggaacctg aggcaggaga 66660
atcatcgaaa cccgggagtc ggaggttgca gtgggcacaa gatcacgcca ctatactcca 66720
gcctgggcga nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 66780
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn cttttttttg 66840
ggggtttccc cggccccccc cttttttttt tttttttttt tttttttttt tgtgagaccg 66900
agtcttgctc tgttgcccag gatggagtac aatggtgcaa tctctgctca ctgcaacctc 66960
cgcctcctgg gctcgagcag ttctcctgct ttagcttcct gagtagctgg gattacaggt 67020
acccaccacc atgcttggct aatttttagt agagatgggg tttcaccatg ctggccaggc 67080
tggtttcaaa ctcctgaccg taaatgatct gcctgccttg gcctcccaaa gtgctgggat 67140
tacaggcatg agtcaccatg tctggcctcc ccatttaaaa tggcaacccc tgccctgcct 67200
cctagcacct ctgtctcctt cctctgcttt attttctcta gaacacttac caccacctga 67260
catattatat atcttaacta tttctttgtt tacagtctgc cttccctgct aagatgaaag 67320
gcattcttgt ctattttgat cattgctgta tttccagtgc cagtacttaa taaatacttg 67380
ttgaataaat gcatgataaa ttctgaagct atttaaactt tttcaattgt actgagcacg 67440
ggctcagtac aaggaaatct acacacatag tttaccacat ttacacatca aaggtggatc 67500
aaaaattttt tcaataggca tttgaaaaac attagaaatg aaatacttta ataacaaggg 67560
tgattatata cttaatatac aaatgtctct acttttttaa ctcagtggta agaatttact 67620
ttttcaatct cataaagaag ggctctctaa gcaagatata aacacagaaa ataaaaggga 67680
aacataataa acttggctat acacattttt ttttttgaaa cagtcttgtt ctgtcaccca 67740
ggctagagtg cagtggtgca acctcagctc actgcaacct ccacctccca ggttcaagca 67800
attctcatgc ctcagtctga gtcatgtgcc acccgcccca acccacccca ccccaccccc 67860
agctaattgt tctattttta gtagagacag ggtttcacca tgttggccaa atttgttttt 67920
aactctgatg aaaatgagtt aaacaaagtc aaagataatt taaactaagt cacaaaatat 67980
tgcaacatat aagannnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68040
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaccaca 68100
gaatcagcaa cagttcacat agggcaaatg ttttaaataa acatttttta aactccatgt 68160
tgcaagaagt agcaacacta acaaaagcaa tatttatata aggacatact ttgtaaccag 68220
tttaaaggcc aagggagggt taggttaatg agggctcagg cagtcaggaa ggcttctgag 68280
aggctatggc cttgagaggc aggtggcctt ttggatagac agaaggaagc aggtggcaga 68340
ggtctggcag aggctgcatg aggccgaatc atggaagcct caaagtgaga cacaggttag 68400
agtccctgca gtagtgatgg ggaattctga aggcttttga gtcggggcat ggcatgaaaa 68460
tttcacccct gcctcctcac ccatctgcct tgaatctaga aagcacagct ggattccccc 68520
taaagttctg cctacatcag tggtttcttt tccctcctag actgacattt tgtacagaag 68580
accctgagaa caccagcata tccccaggcc gggagctctg gtttcgcaag tgtcgggatg 68640
gctggaggat gaaaaacgaa accaggtaac caggactttc tcacacgttc cacccaggac 68700
acgttgacat gatgatctcc tagcatgtgc tggggatgga tctgggtgcc agggacatag 68760
catgaacaaa acagataaaa atctcttcct ttaagaagtt gggccgggcg tggtggctca 68820
agcctgtaat cccagcactt tgggaggcca aggcaggcag atcacctgag gtcaggagtt 68880
tgagaccagc ctggctaaca tagtgaaacc ccatctctac taaaaaaaaa aaaaaaaaga 68940
atgccgggca tggtggtgtg tgcctgtaat cccagctact tgggaggttg aggcaggaaa 69000
atcacttgaa cctgaaaggt ggaggctgca gtgagctgaa accgcaatgt tgcactccag 69060
cctaggtgac agaaatgaag ctctgtctca aaaagaaaga aagggccagg catggaggct 69120
cacgcctgta atcccagcac tttgggaggc tgaggcaggc ggatcacgag gtcaggagat 69180
cgacacaatc ctggctaaca cagtgaaacc tcatctctct aaaaatataa aaaattagac 69240
gggcatggtg acgggtgcct gtagtcccag ctactcggga ggctgaggca ggagaatggt 69300
atgaacctgg gaggaagagc ttgcagtgag ctgagattgc accactgcac tccagcctgg 69360
ggtgacagag tgagactccg tctcaaaaaa ataaaaaaaa aaaaaaaaaa nnnnnnnnnn 69420
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69480
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aaaaaaaaaa aaaaaaaaaa aaaagaagct 69540
gaccttctgc taggagataa agaagcaagt gaaatactgt agataggatg ttggaagtgt 69600
taatgtctct tctgctcttt gtcctgcctc aacctgtagg ggttcattgt acatgaccct 69660
actggcatga gatcttctct gtaactaagt ccccaggttg gtgcccattc ctgccatctt 69720
tgaagtatct tttttttttt cctttgagac atggtcttac tctgttaccc aggctggagc 69780
gcagtggcat gatcctggct cactgcagcc tcgacctccc caggcttggg tgatccttcc 69840
gcctttgcct cctgtatagc tgggaccaca ggcacttgcc accacgctga gctaatttgt 69900
gtattttttt gcagagacgg ggtttcacca tgttgcccag tctggtcttg aactccatgg 69960
ctcaagtgat ccgcctgcct tggcctccca gagtgctggg attacaggtg tgagccactg 70020
tgcctggccc ctgccatctt tgattgacat cttctctgta ataactagag ctgtgggaac 70080
gtgcctaagg gctcctagct tccttctggc caccaggtca ggtttgggaa tggggtggga 70140
gataaagaga agcacaaagc atgttctcct ttcttaggtt ttctagaatg caggtaagtg 70200
tgggcctaat tctcccacac actttttttt tttttttttt gagacaaagt ctcactctat 70260
cgctcaggct ggagtgcagt ggagtgatct tggcttacta aaacctccgc ctcccgggtc 70320
caagtgattc tccttcctca accttccgag tagctgggat tacaagtgcc tgctaccacg 70380
cctggctaat ttttgtattt ttagttgaga tgggttttcg ccatgttggc caagttacca 70440
tgttggccaa gttggtctca aactcttgat ctcaagtgat ctgcccacct tggcctccaa 70500
aagtgctggc attacaggtt tgagccacgg tgcccaaccc caatccttcc cctttttggg 70560
aaacagaaat gcccatgtat gtggagctaa gtgagacaga ggggttgtca tgcttcacta 70620
tccccttgtc ccatgctgca atccgttatt tcagacgtga ggaaggccca tcttgtggtg 70680
tgaggcagtg ggctcatcct ggggaacagc cacacgccct ctaagccaaa acacctgcta 70740
aggaggaagg agactgtctc ctcacaccat gcctgtcgcc accctttgct ctaacaaggg 70800
tactagataa taatactgct gtggtacacc tagcacttag cttgggccca gcctgcaccc 70860
agggacttac ataatgatgt ttgtcatcaa cccactaggc aggggttgtt agccctgtgt 70920
acagatgagg aaccgaggct cagggagatg aagtttctct cccaaggccc ccagcaatgg 70980
cagagggagt tgaagcaggt cggtctaccc caaagcctgt gttgttgagc accgaggctc 71040
caagtgctca gatgatcacc tcgcgtctgt ctgggtatca attatcaggc agctgtgccc 71100
gtgctccggg gtcgcctgtg attaaccatt tgccaccccc aaggctgcgc tgcatcccag 71160
ctgctctgtc tcctggttag gagctcaaca caaccacaag caccatcatc acaggactcg 71220
tgccatctaa ttaagggact cagtctagcg taggctcgta tgattttcta ctactataac 71280
gattgtaaat ctttatgtat ttaaatgtgt acatttcaaa gtgtttccac atatattaac 71340
ttcattgatc ctccagacaa ccatgtagat tggacacacc caggaaagat gactaaggaa 71400
ggctattctt tttttattga gacagggtct tgctctgtca cccaggctgg agtacagtgg 71460
catgatcaca gctcattgca gcctcgacct ccctgggctc agatgatctt cctacctcag 71520
cctcctgagt agcttggatt acaggaatgt gccactatgc ctggctaatt tttgtagaga 71580
tgaggtttca ccatgttgcc caggctggtc tctatctcct gagctcaagt gatctgcctg 71640
cctcggcctc ccagtgctgg gtttgcaggc atgagccact gtgcccagtc aggatggcta 71700
ttcttatgat aaaggctaag atatttattc ttctttcccg ctttggaatt catatacctg 71760
agaactctat gattcaccct ctcactacta attttagaaa acaagctgtc cttttccatt 71820
ccctcaaaaa caataggagt ccaagtaata aatgaacact aggaagtcat agcatcatat 71880
gtaacatgtt tagcatcctc cctcctgaca tggatgctgt tcacatgttc actgataagg 71940
agcctgagat tcagagaggt tcagtggtgt gttcacatag ctgagactag aatccaggtc 72000
tcctaactct cagtcttgcc ccctttctgc caatacagtg tctctcttgt atttctagat 72060
caaggcaaag aggacacttt gatagttctc cccacacttg tgtgtccatg attgtgtgtg 72120
tgtgtgtgtg tgtgtgtgtg tgtgtgtatg ttgtgggtgg ataatatgta aatgcaagaa 72180
ctgtgatgta ctcaactcag ggtccagagg gtgctgcagt gtggtgtttc tcaaagtgta 72240
tctatggctt gtcaggttag ggagagaagg cagcactcgg gaccttgtcc atttattctg 72300
aaaggaatac atgtaaaata gtcccatagg ggtgtcagaa agcttggcct taaggtcaaa 72360
agagcacacc ctgaatacag gtttgcgcgt ttgctggtgt gtgagctaac aaatgccact 72420
ctcacacggt ttctttcagt cccactgtgg agcttccctg agggtgcccg ggcaagtctt 72480
gccagcaagg cagcaagact tcctgctatc caagcccatg gaggaaagtt actgctgagg 72540
acccacccaa tggaaggatt cttctcagcc ttgaacctgg agcactggga acaactggtc 72600
tcctgtgatg gctgggactc ctcgcgggag gggactgcgc tgctatagct cttgctgcct 72660
ctcttgaata gctctaactc caaacctctg tccacacctc cagagcacca agtccagatt 72720
tgtgtgtaag cagctgggtg cctggggcct ctcgtgcaca ctggattggt ttctcagttg 72780
ctgggcgagc ctgtactctg cctgacgagg aacgctggct ccgaagaggc cctgtgtaga 72840
aggctgtcag ctgctcagcc tgctttgagc ctcagtgaga agtccttccg acaggagctg 72900
actcatgtca ggatggcagg cctggtatct tgctcgggcc ctagctgttg gggttctcat 72960
gggttgcact gaccatactg cttacgtctt agccattccg tcctgctccc cagctcactc 73020
tctgaagcac acatcattgg ctttcctatt tttctgttca ttttttaatt gagcaaatgt 73080
ctattgaaca cttaaaatta attagaatgt ggtaatggac atattactga gcctctccat 73140
ttggaaccca gtggagttgg gatttctaga ccctctttct gtttggatgg tgtatgtgta 73200
tatgcatggg gaaaggcacc tggggcctgg gggaggctat aggatataag cattagggac 73260
cctgaggctt taagtggttt ctatttcttc ttagttatta tgtgccacct tcttagttat 73320
tatgtgccac ctcccctatg agtgacgtgt ttgatcacta gcagaatagc aagcagagta 73380
tcattcatgc tggggccaga atgatggccg gttgccagat ataactgctt tggagcaaat 73440
ctcttctgtt tagagagata gaagttatga catatgtaat acacatctgt gtacacagaa 73500
accggcacct gccagacaga gctggttcta agatttaata cagtgctttt tttcctcttt 73560
gaaatatttt actttaatac cagtgccttt tcttgttgaa cttcttggaa aagccaccaa 73620
ttctagatct tgatttgaat taatacacac aatatctgag acacttacac ttttcaaaag 73680
atttgtgtat gcattgccta attagagtag ggggagaagg gcaactatta ttatccctat 73740
tttacaaaac tgaggcttag tgaggttcag ccacatgcct agacttatat actagttagt 73800
ggtgcagcca gggagaggac tcagatttcc tggaggcaaa gtctatctct gaaactccat 73860
gaagactttt gcagccagtt cccaccaata tgccccagac gtgagacaaa caaggacttt 73920
tttttttata tagagccatc cataaaatcc taagcccttt tattaatgta taaccaggag 73980
aacatctgtg ccaacggttg gactttttat ggctgagatt cgggaggaag tgtgacacca 74040
agcaggagag gaagaatgat tttctttgta cttaggtttt ctaaggacat tgttttaatc 74100
tgtatcgtgc caaagttgta tcactgttaa acttctgaag acataaccag ttgagtctta 74160
tttcaagata tgttctcaag ccaattgtgt gcttctcttg tttctgtgat tgctttctag 74220
ccaaagcgaa gcttgtacag gttgagtatc ccttatccaa aatgcttgga accagaagtg 74280
tttcaaattt tagattattt tcagattttg gaatgtttgc atatacataa tgagatattt 74340
tgggaatagg acccgagcct aaacacaaaa ttcattgatg tgtcagttac accttatcca 74400
catagcctga gggtaatttt atacgatatt ttaaatagtt gtgtacatga agcatggttt 74460
gtggtaactt atgtgagggg ttttcccatt ttttgtcttg ttggtgctca aaaagttttg 74520
gattttggag catttcggat tttggatttt tggattaggg ttgctcaacc catattattg 74580
gctgtacatc ctggtcactt ctgacttctg tttttactaa tggaagcttt gcaaattgaa 74640
ttctcagtga gttgtatatt tatacacctg gcttgaagcc ttaattgtat ataatgatgc 74700
tttttaaaaa atgctatttg gaagactatt tatttctcgt gtatataatg tatataaaaa 74760
aatatggtta gtgtttacct aaggttaacc aatttcaaga ttaaaatttt taaatagtaa 74820
aataataaaa aattataaag ttcttaatgg tctgacaact caattttttt tttttttttt 74880
tttttttttg agacagagcc tctctctgtc acccaggctg gagtgcagtg gcaatcttgg 74940
ctcactacaa cctctgcctc ctgggctcag gtggtcctcc cacctcagcc ttctgagtag 75000
ctgggattac agagatgcgt caccatgtct ggc 75033
<210> SEQ ID NO 19
<400> SEQUENCE: 19
000
<210> SEQ ID NO 20
<211> LENGTH: 289
<212> TYPE: DNA
<213> ORGANISM: homo sapiens
<220> FEATURE:
<400> SEQUENCE: 20
cccatattat tggctgtaca tcctggtcac ttctgacttc tgtttttact aatggaagct 60
ttgcaaattg aattctcagt gagttgtata tttatacacc tggcttgaag ccttaattgt 120
atataatgat actttttaaa aaatgctatt tggaagacta tttatttctc gtgtatataa 180
tgtatataaa aaaatatggt tagtgtttac ctaaggttaa ccaatttcaa gattaaaatt 240
tttaaatagt aaaataataa aaaattataa aaaaaaaaaa aaaaaaaaa 289
<210> SEQ ID NO 21
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 21
tcaacggctt gccccagaac 20
<210> SEQ ID NO 22
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 22
ggtggaaaat gaaaacttgg 20
<210> SEQ ID NO 23
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 23
cccctcccaa gaaacagaag 20
<210> SEQ ID NO 24
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 24
ggagttgctc atcctgcccc 20
<210> SEQ ID NO 25
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 25
ctctggacca aaaggtacgg 20
<210> SEQ ID NO 26
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 26
tgtggagctt atcttccagc 20
<210> SEQ ID NO 27
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 27
ctcacagatg gtttgacctc 20
<210> SEQ ID NO 28
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 28
gacggagagg tagcatcctt 20
<210> SEQ ID NO 29
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 29
cagggctgac acgagtttgt 20
<210> SEQ ID NO 30
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 30
attgaccgca tccgtgtaaa 20
<210> SEQ ID NO 31
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 31
tccttctcct gcagccagtc 20
<210> SEQ ID NO 32
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 32
cgtccttctc ctgcagccag 20
<210> SEQ ID NO 33
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 33
gtggacattc ccgagagaaa 20
<210> SEQ ID NO 34
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 34
ctccgaggct gtagccgatc 20
<210> SEQ ID NO 35
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 35
aaacatgggc ccggcaggat 20
<210> SEQ ID NO 36
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 36
cagcctggct ggaagtcacc 20
<210> SEQ ID NO 37
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 37
tcctgattca ccagagagtc 20
<210> SEQ ID NO 38
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 38
gcttgtcctg attcaccaga 20
<210> SEQ ID NO 39
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 39
aaaactcggc ttgtcctgat 20
<210> SEQ ID NO 40
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 40
gcactggaag gcaaaactcg 20
<210> SEQ ID NO 41
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 41
gaagcgattg gagtcagtgc 20
<210> SEQ ID NO 42
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 42
tgaaaggcat gcctgcccgg 20
<210> SEQ ID NO 43
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 43
ctgataatgg taaactctga 20
<210> SEQ ID NO 44
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 44
ttcttgtaac tgaagacatg 20
<210> SEQ ID NO 45
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 45
gacgtaaaag gtgggctcaa 20
<210> SEQ ID NO 46
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 46
gctccactat ttccagtggc 20
<210> SEQ ID NO 47
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 47
gaaggtgttg gtggcattct 20
<210> SEQ ID NO 48
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 48
aggtctccca agtcctcctc 20
<210> SEQ ID NO 49
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 49
aggccccctc ccaggtgagc 20
<210> SEQ ID NO 50
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 50
gtagctgcga aactccttcc 20
<210> SEQ ID NO 51
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 51
gatattcagc tcccgtccgg 20
<210> SEQ ID NO 52
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 52
aaatgtcagt ttccgctggg 20
<210> SEQ ID NO 53
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 53
tgttctcagg gtcttctgta 20
<210> SEQ ID NO 54
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 54
cacagtggga ctggtttcgt 20
<210> SEQ ID NO 55
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 55
ggcaccctca gggaagctcc 20
<210> SEQ ID NO 56
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 56
ttgctgcctt gctggcaaga 20
<210> SEQ ID NO 57
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 57
gtcctcagca gtaactttcc 20
<210> SEQ ID NO 58
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 58
acagaggttt ggagttagag 20
<210> SEQ ID NO 59
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 59
ccaatccagt gtgcacgaga 20
<210> SEQ ID NO 60
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 60
gggcctcttc ggagccagcg 20
<210> SEQ ID NO 61
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 61
caacccatga gaaccccaac 20
<210> SEQ ID NO 62
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 62
aggacggaat ggctaagacg 20
<210> SEQ ID NO 63
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 63
caatagacat ttgctcaatt 20
<210> SEQ ID NO 64
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 64
atcccaactc cactgggttc 20
<210> SEQ ID NO 65
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 65
aggtgccttt ccccatgcat 20
<210> SEQ ID NO 66
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 66
tgcttatatc ctatagcctc 20
<210> SEQ ID NO 67
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 67
ccacttaaag cctcagggtc 20
<210> SEQ ID NO 68
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 68
tagtgatcaa acacgtcact 20
<210> SEQ ID NO 69
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 69
acatatgtca taacttctat 20
<210> SEQ ID NO 70
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 70
caacaagaaa aggcactggt 20
<210> SEQ ID NO 71
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 71
ttaattcaaa tcaagatcta 20
<210> SEQ ID NO 72
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 72
ctaattaggc aatgcataca 20
<210> SEQ ID NO 73
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 73
ctcactaagc ctcagttttg 20
<210> SEQ ID NO 74
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 74
tggctgcacc actaactagt 20
<210> SEQ ID NO 75
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 75
gactttgcct ccaggaaatc 20
<210> SEQ ID NO 76
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 76
ggctgcaaaa gtcttcatgg 20
<210> SEQ ID NO 77
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 77
atggatggct ctatataaaa 20
<210> SEQ ID NO 78
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 78
taaaagggct taggatttta 20
<210> SEQ ID NO 79
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 79
acagatgttc tcctggttat 20
<210> SEQ ID NO 80
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 80
ataaaaagtc caaccgttgg 20
<210> SEQ ID NO 81
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 81
ctcccgaatc tcagccataa 20
<210> SEQ ID NO 82
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 82
ctctcctgct tggtgtcaca 20
<210> SEQ ID NO 83
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 83
atcattcttc ctctcctgct 20
<210> SEQ ID NO 84
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 84
ggcacgatac agattaaaac 20
<210> SEQ ID NO 85
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 85
gaagtttaac agtgatacaa 20
<210> SEQ ID NO 86
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 86
ttgaaataag actcaactgg 20
<210> SEQ ID NO 87
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 87
ccaaaacttt ttgagcacca 20
<210> SEQ ID NO 88
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 88
agacctttta ctttttgcaa 20
<210> SEQ ID NO 89
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 89
cctagcctgg gaacccaaac 20
<210> SEQ ID NO 90
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 90
ggatccaaac ctgcagcaga 20
<210> SEQ ID NO 91
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 91
tgaaggttac ctctgaaagg 20
<210> SEQ ID NO 92
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 92
taatggtaaa ctgcagtgac 20
<210> SEQ ID NO 93
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 93
aggcacttac ttccgctggg 20
<210> SEQ ID NO 94
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 94
gtcatagatg acgaatgtaa 20
<210> SEQ ID NO 95
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 95
gaattctgcc agcagactgc 20
<210> SEQ ID NO 96
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 96
ttcttaagaa gattgggttt 20
<210> SEQ ID NO 97
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 97
atatacaatt aaggcttcaa 20
<210> SEQ ID NO 98
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 98
agtatcatta tatacaatta 20
<210> SEQ ID NO 99
<211> LENGTH: 2227
<212> TYPE: DNA
<213> ORGANISM: Mus musculus
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (275)...(1777)
<400> SEQUENCE: 99
cggcccgact tcgcagcggc tctgggaaag gacaggtcgg gctgggcagc gtgtccctcc 60
acacgctcgc tttcccggtc accgctcgca gcgctaccaa ccttccaggt tctgccagct 120
gtccacccca tcaccttctg gccgacaccc ggcctgtcca cttctagtct ctggaggttt 180
tagtggtttc aaaccaaacc aacccaaacc aacccaacaa caaaaaagcc caaaccaaaa 240
acctgcttga gaggaggggg cgtggcgggg aagg atg cga aac acg gtt ttc ctg 295
Met Arg Asn Thr Val Phe Leu
1 5
ctc ggc ttt tgg agc gtc tat tgt tac ttc ccg gcg gga agt atc aca 343
Leu Gly Phe Trp Ser Val Tyr Cys Tyr Phe Pro Ala Gly Ser Ile Thr
10 15 20
acc ttg cgt ccc cag ggg tcg ctg cga gat gag cat cat aaa ccc act 391
Thr Leu Arg Pro Gln Gly Ser Leu Arg Asp Glu His His Lys Pro Thr
25 30 35
gga gta cca gct acc gcc aga ccc tct gtg gct ttt aac atc cgc act 439
Gly Val Pro Ala Thr Ala Arg Pro Ser Val Ala Phe Asn Ile Arg Thr
40 45 50 55
tct aag gac cca gag cag gaa ggg tgt aat ctc tcc ctt ggt gac agc 487
Ser Lys Asp Pro Glu Gln Glu Gly Cys Asn Leu Ser Leu Gly Asp Ser
60 65 70
aaa ctc tta gaa aac tgt ggc ttc aac atg aca gcc aaa acc ttc ttc 535
Lys Leu Leu Glu Asn Cys Gly Phe Asn Met Thr Ala Lys Thr Phe Phe
75 80 85
atc att cat gga tgg acg atg agt ggc atg ttt gag agc tgg ctg cat 583
Ile Ile His Gly Trp Thr Met Ser Gly Met Phe Glu Ser Trp Leu His
90 95 100
aaa ctt gta tca gcc ctg cag atg aga gag aaa gat gct aac gtc gtg 631
Lys Leu Val Ser Ala Leu Gln Met Arg Glu Lys Asp Ala Asn Val Val
105 110 115
gtg gtt gac tgg ctg ccc ctg gct cat cag ctg tac acg gat gca gtc 679
Val Val Asp Trp Leu Pro Leu Ala His Gln Leu Tyr Thr Asp Ala Val
120 125 130 135
aat aac acc agg gtg gtg gga cag aga gta gct ggg atg ctt gac tgg 727
Asn Asn Thr Arg Val Val Gly Gln Arg Val Ala Gly Met Leu Asp Trp
140 145 150
ctg cag gag aag gaa gag ttc tct ctt ggg aac gtt cac ttg att ggc 775
Leu Gln Glu Lys Glu Glu Phe Ser Leu Gly Asn Val His Leu Ile Gly
155 160 165
tac agc ctt gga gca cac gtg gct gga tac gct ggc aac ttt gtg aaa 823
Tyr Ser Leu Gly Ala His Val Ala Gly Tyr Ala Gly Asn Phe Val Lys
170 175 180
gga aca gtg ggc agg atc act ggt ctg gat ccc gcg ggt ccc atg ttt 871
Gly Thr Val Gly Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Met Phe
185 190 195
gaa ggg gtg gac atc aac aga agg ctg tcc ccg gac gat gca gac ttt 919
Glu Gly Val Asp Ile Asn Arg Arg Leu Ser Pro Asp Asp Ala Asp Phe
200 205 210 215
gtg gat gtc ctg cat acc tac acg ctg tcc ttt ggc ttg agc att ggg 967
Val Asp Val Leu His Thr Tyr Thr Leu Ser Phe Gly Leu Ser Ile Gly
220 225 230
att cgg atg cct gtg ggt cac att gac atc tat ccc aat ggc ggt gac 1015
Ile Arg Met Pro Val Gly His Ile Asp Ile Tyr Pro Asn Gly Gly Asp
235 240 245
ttc cag cca ggc tgt gga ttc aat gat gtc atc gga tct ttt gca tat 1063
Phe Gln Pro Gly Cys Gly Phe Asn Asp Val Ile Gly Ser Phe Ala Tyr
250 255 260
gga aca atc tca gag atg gtg aaa tgc gag cac gag cga gcc gta cac 1111
Gly Thr Ile Ser Glu Met Val Lys Cys Glu His Glu Arg Ala Val His
265 270 275
ctc ttt gtc gac tct ctg gtg aat cag gac aag ccc agc ttt gcc ttc 1159
Leu Phe Val Asp Ser Leu Val Asn Gln Asp Lys Pro Ser Phe Ala Phe
280 285 290 295
cag tgc aca gac tcc agc cgc ttc aaa agg gga atc tgc ctc agc tgc 1207
Gln Cys Thr Asp Ser Ser Arg Phe Lys Arg Gly Ile Cys Leu Ser Cys
300 305 310
cgg aag aac cgt tgt aat aac att ggc tac aac gcc aag aaa atg aga 1255
Arg Lys Asn Arg Cys Asn Asn Ile Gly Tyr Asn Ala Lys Lys Met Arg
315 320 325
aag aag agg aat agc aaa atg tat tta aaa acc cgg gct ggc atg cct 1303
Lys Lys Arg Asn Ser Lys Met Tyr Leu Lys Thr Arg Ala Gly Met Pro
330 335 340
ttc aaa gtt tac cat tac cag ctg aaa gtt cac atg ttc tct tac aat 1351
Phe Lys Val Tyr His Tyr Gln Leu Lys Val His Met Phe Ser Tyr Asn
345 350 355
aac agt ggg gac acc cag ccc acc ctc tac att acc ctg tat ggt agc 1399
Asn Ser Gly Asp Thr Gln Pro Thr Leu Tyr Ile Thr Leu Tyr Gly Ser
360 365 370 375
aac gca gac tcc cag aac ctg ccc ttg gaa ata gtg gag aag att gag 1447
Asn Ala Asp Ser Gln Asn Leu Pro Leu Glu Ile Val Glu Lys Ile Glu
380 385 390
ctg aat gcc aca aac acc ttc ctt gtc tac act gag gag gac ttg ggc 1495
Leu Asn Ala Thr Asn Thr Phe Leu Val Tyr Thr Glu Glu Asp Leu Gly
395 400 405
gat ctc ttg aag atg cga ctt acc tgg gag ggg gta gcc cat tcc tgg 1543
Asp Leu Leu Lys Met Arg Leu Thr Trp Glu Gly Val Ala His Ser Trp
410 415 420
tgc aac ctg tgg aat gag ttt cgc aac tac ctg tct caa ccc agc aac 1591
Cys Asn Leu Trp Asn Glu Phe Arg Asn Tyr Leu Ser Gln Pro Ser Asn
425 430 435
ccc tcg agg gag ctg tac atc cgg cga att cgt gtc aaa tct ggg gaa 1639
Pro Ser Arg Glu Leu Tyr Ile Arg Arg Ile Arg Val Lys Ser Gly Glu
440 445 450 455
acc cag cgc aaa gtg aca ttt tgc act caa gac cca acg aag agt agc 1687
Thr Gln Arg Lys Val Thr Phe Cys Thr Gln Asp Pro Thr Lys Ser Ser
460 465 470
atc tcc cct ggc cag gag ctg tgg ttt cac aag tgt cag gat ggc tgg 1735
Ile Ser Pro Gly Gln Glu Leu Trp Phe His Lys Cys Gln Asp Gly Trp
475 480 485
aaa atg aaa aac aaa acc agt ccc ttt gtg aac ttg gcc tga 1777
Lys Met Lys Asn Lys Thr Ser Pro Phe Val Asn Leu Ala *
490 495 500
gggcccaaga agtcctggcg tccacaccca caccccactg tccacgcaca tggaggaaaa 1837
gttactgctg aagacccact cgatggacga tctcagcctt gagccccacg aggagcttgc 1897
ttgctgggct catcctgtct cccctgacaa ctgtgacttc tcctggagag gcctgtgcac 1957
tgctgaagtt cttgctgatg attctagctg taaacctttg ttgccgccgc aggaagctga 2017
ggccagcttg tgtgtgagca ctggagtgtc cagagccctg cacactcggg gtggggggcg 2077
gggtactctc cctgtcgcta ggtgagcact ggctttgtcc aacatcaggg aacacaaggc 2137
tctgaagtgg ccctgtgtgg aaggttggca gctgcctggc ctcactgaac cttagtgaca 2197
agtctttgcc tcaggagctg actcatgccg 2227
<210> SEQ ID NO 100
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 100
ttcccagagc cgctgcgaag 20
<210> SEQ ID NO 101
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 101
gaacctggaa ggttggtagc 20
<210> SEQ ID NO 102
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 102
cctccagaga ctagaagtgg 20
<210> SEQ ID NO 103
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 103
actaaaacct ccagagacta 20
<210> SEQ ID NO 104
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 104
cgtgtttcgc atccttcccc 20
<210> SEQ ID NO 105
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 105
aggttgtgat acttcccgcc 20
<210> SEQ ID NO 106
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 106
tcatctcgca gcgacccctg 20
<210> SEQ ID NO 107
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 107
tggtactcca gtgggtttat 20
<210> SEQ ID NO 108
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 108
tgcggatgtt aaaagccaca 20
<210> SEQ ID NO 109
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 109
gtcaccaagg gagagattac 20
<210> SEQ ID NO 110
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 110
gccacagttt tctaagagtt 20
<210> SEQ ID NO 111
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 111
gttattgact gcatccgtgt 20
<210> SEQ ID NO 112
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 112
accctggtgt tattgactgc 20
<210> SEQ ID NO 113
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 113
ccagtcaagc atcccagcta 20
<210> SEQ ID NO 114
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 114
gttcccaaga gagaactctt 20
<210> SEQ ID NO 115
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 115
cacgtgtgct ccaaggctgt 20
<210> SEQ ID NO 116
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 116
catccacaaa gtctgcatcg 20
<210> SEQ ID NO 117
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 117
caggacatcc acaaagtctg 20
<210> SEQ ID NO 118
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 118
gtatgcagga catccacaaa 20
<210> SEQ ID NO 119
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 119
tgtaggtatg caggacatcc 20
<210> SEQ ID NO 120
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 120
cagcgtgtag gtatgcagga 20
<210> SEQ ID NO 121
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 121
aaggacagcg tgtaggtatg 20
<210> SEQ ID NO 122
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 122
agccaaagga cagcgtgtag 20
<210> SEQ ID NO 123
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 123
gctcaagcca aaggacagcg 20
<210> SEQ ID NO 124
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 124
ccaatgctca agccaaagga 20
<210> SEQ ID NO 125
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 125
gaatcccaat gctcaagcca 20
<210> SEQ ID NO 126
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 126
catccgaatc ccaatgctca 20
<210> SEQ ID NO 127
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 127
tagatgtcaa tgtgacccac 20
<210> SEQ ID NO 128
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 128
tgggatagat gtcaatgtga 20
<210> SEQ ID NO 129
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 129
cgccattggg atagatgtca 20
<210> SEQ ID NO 130
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 130
tttcaccatc tctgagattg 20
<210> SEQ ID NO 131
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 131
aaagaggtgt acggctcgct 20
<210> SEQ ID NO 132
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 132
tcgacaaaga ggtgtacggc 20
<210> SEQ ID NO 133
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 133
gagagtcgac aaagaggtgt 20
<210> SEQ ID NO 134
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 134
caccagagag tcgacaaaga 20
<210> SEQ ID NO 135
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 135
cagattcccc ttttgaagcg 20
<210> SEQ ID NO 136
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 136
ccaatgttat tacaacggtt 20
<210> SEQ ID NO 137
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 137
tgtgaacttt cagctggtaa 20
<210> SEQ ID NO 138
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 138
taagagaaca tgtgaacttt 20
<210> SEQ ID NO 139
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 139
cccactgtta ttgtaagaga 20
<210> SEQ ID NO 140
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 140
atttccaagg gcaggttctg 20
<210> SEQ ID NO 141
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 141
gtgtagacaa ggaaggtgtt 20
<210> SEQ ID NO 142
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 142
gagatcgccc aagtcctcct 20
<210> SEQ ID NO 143
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 143
gtaagtcgca tcttcaagag 20
<210> SEQ ID NO 144
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 144
taccccctcc caggtaagtc 20
<210> SEQ ID NO 145
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 145
ggaatgggct accccctccc 20
<210> SEQ ID NO 146
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 146
ctcattccac aggttgcacc 20
<210> SEQ ID NO 147
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 147
gggtttcccc agatttgaca 20
<210> SEQ ID NO 148
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 148
cacttgtgaa accacagctc 20
<210> SEQ ID NO 149
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 149
aaagggactg gttttgtttt 20
<210> SEQ ID NO 150
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 150
ttgggccctc aggccaagtt 20
<210> SEQ ID NO 151
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 151
ccagcaagca agctcctcgt 20
<210> SEQ ID NO 152
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 152
tcagcaagaa cttcagcagt 20
<210> SEQ ID NO 153
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 153
caaaggttta cagctagaat 20
<210> SEQ ID NO 154
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 154
cttcctgcgg cggcaacaaa 20
<210> SEQ ID NO 155
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 155
gctcacacac aagctggcct 20
<210> SEQ ID NO 156
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 156
gggctctgga cactccagtg 20
<210> SEQ ID NO 157
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 157
gctcacctag cgacagggag 20
<210> SEQ ID NO 158
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 158
gccttgtgtt ccctgatgtt 20
<210> SEQ ID NO 159
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 159
acacagggcc acttcagagc 20
<210> SEQ ID NO 160
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 160
cttccacaca gggccacttc 20
<210> SEQ ID NO 161
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 161
ttcagtgagg ccaggcagct 20
<210> SEQ ID NO 162
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 162
taaggttcag tgaggccagg 20
<210> SEQ ID NO 163
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 163
gacttgtcac taaggttcag 20
<210> SEQ ID NO 164
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 164
aatagaacca ggatccatca 20
<210> SEQ ID NO 165
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 165
tctgctagag atcaagggtg 20
<210> SEQ ID NO 166
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 166
gcgcagcagg tatgtagaac 20
<210> SEQ ID NO 167
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 167
tcaaactact aaagggtgtc 20
<210> SEQ ID NO 168
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 168
ccaggaaacc ttgctgggtc 20
<210> SEQ ID NO 169
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 169
atggagttac agaaaggatt 20
<210> SEQ ID NO 170
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 170
acagatgcaa agaatgtgcg 20
<210> SEQ ID NO 171
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 171
tataaagctg gtacaataca 20
<210> SEQ ID NO 172
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 172
aaaactaacc atagatttgt 20
<210> SEQ ID NO 173
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 173
aaatcttgaa atcggttaat 20
<210> SEQ ID NO 174
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 174
gttctggggc aagccgttga 20
<210> SEQ ID NO 175
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 175
gctggaagat aagctccaca 20
<210> SEQ ID NO 176
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 176
gaggtcaaac catctgtgag 20
<210> SEQ ID NO 177
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 177
aaggatgcta cctctccgtc 20
<210> SEQ ID NO 178
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 178
acaaactcgt gtcagccctg 20
<210> SEQ ID NO 179
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 179
tttacacgga tgcggtcaat 20
<210> SEQ ID NO 180
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 180
gactggctgc aggagaagga 20
<210> SEQ ID NO 181
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 181
ctggctgcag gagaaggacg 20
<210> SEQ ID NO 182
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 182
tttctctcgg gaatgtccac 20
<210> SEQ ID NO 183
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 183
gatcggctac agcctcggag 20
<210> SEQ ID NO 184
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 184
atcctgccgg gcccatgttt 20
<210> SEQ ID NO 185
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 185
ggtgacttcc agccaggctg 20
<210> SEQ ID NO 186
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 186
gactctctgg tgaatcagga 20
<210> SEQ ID NO 187
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 187
tctggtgaat caggacaagc 20
<210> SEQ ID NO 188
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 188
atcaggacaa gccgagtttt 20
<210> SEQ ID NO 189
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 189
cgagttttgc cttccagtgc 20
<210> SEQ ID NO 190
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 190
gcactgactc caatcgcttc 20
<210> SEQ ID NO 191
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 191
ccgggcaggc atgcctttca 20
<210> SEQ ID NO 192
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 192
tcagagttta ccattatcag 20
<210> SEQ ID NO 193
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 193
catgtcttca gttacaagaa 20
<210> SEQ ID NO 194
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 194
ttgagcccac cttttacgtc 20
<210> SEQ ID NO 195
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 195
gccactggaa atagtggagc 20
<210> SEQ ID NO 196
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 196
agaatgccac caacaccttc 20
<210> SEQ ID NO 197
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 197
gaggaggact tgggagacct 20
<210> SEQ ID NO 198
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 198
ccggacggga gctgaatatc 20
<210> SEQ ID NO 199
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 199
cccagcggaa actgacattt 20
<210> SEQ ID NO 200
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 200
tacagaagac cctgagaaca 20
<210> SEQ ID NO 201
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 201
acgaaaccag tcccactgtg 20
<210> SEQ ID NO 202
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 202
ggagcttccc tgagggtgcc 20
<210> SEQ ID NO 203
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 203
tcttgccagc aaggcagcaa 20
<210> SEQ ID NO 204
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 204
ggaaagttac tgctgaggac 20
<210> SEQ ID NO 205
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 205
ctctaactcc aaacctctgt 20
<210> SEQ ID NO 206
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 206
tctcgtgcac actggattgg 20
<210> SEQ ID NO 207
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 207
cgctggctcc gaagaggccc 20
<210> SEQ ID NO 208
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 208
cgtcttagcc attccgtcct 20
<210> SEQ ID NO 209
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 209
aattgagcaa atgtctattg 20
<210> SEQ ID NO 210
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 210
atgcatgggg aaaggcacct 20
<210> SEQ ID NO 211
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 211
gaggctatag gatataagca 20
<210> SEQ ID NO 212
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 212
gaccctgagg ctttaagtgg 20
<210> SEQ ID NO 213
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 213
agtgacgtgt ttgatcacta 20
<210> SEQ ID NO 214
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 214
atagaagtta tgacatatgt 20
<210> SEQ ID NO 215
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 215
accagtgcct tttcttgttg 20
<210> SEQ ID NO 216
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 216
tgtatgcatt gcctaattag 20
<210> SEQ ID NO 217
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 217
caaaactgag gcttagtgag 20
<210> SEQ ID NO 218
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 218
actagttagt ggtgcagcca 20
<210> SEQ ID NO 219
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 219
ccatgaagac ttttgcagcc 20
<210> SEQ ID NO 220
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 220
taaaatccta agccctttta 20
<210> SEQ ID NO 221
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 221
ataaccagga gaacatctgt 20
<210> SEQ ID NO 222
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 222
ccaacggttg gactttttat 20
<210> SEQ ID NO 223
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 223
ttatggctga gattcgggag 20
<210> SEQ ID NO 224
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 224
tgtgacacca agcaggagag 20
<210> SEQ ID NO 225
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 225
agcaggagag gaagaatgat 20
<210> SEQ ID NO 226
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 226
gttttaatct gtatcgtgcc 20
<210> SEQ ID NO 227
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 227
ttgtatcact gttaaacttc 20
<210> SEQ ID NO 228
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 228
ccagttgagt cttatttcaa 20
<210> SEQ ID NO 229
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 229
tggtgctcaa aaagttttgg 20
<210> SEQ ID NO 230
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 230
gtcactgcag tttaccatta 20
<210> SEQ ID NO 231
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 231
cccagcggaa gtaagtgcct 20
<210> SEQ ID NO 232
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: H. sapiens
<220> FEATURE:
<400> SEQUENCE: 232
ttacattcgt catctatgac 20
<210> SEQ ID NO 233
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 233
cttcgcagcg gctctgggaa 20
<210> SEQ ID NO 234
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 234
gctaccaacc ttccaggttc 20
<210> SEQ ID NO 235
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 235
tagtctctgg aggttttagt 20
<210> SEQ ID NO 236
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 236
ggggaaggat gcgaaacacg 20
<210> SEQ ID NO 237
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 237
ggcgggaagt atcacaacct 20
<210> SEQ ID NO 238
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 238
ataaacccac tggagtacca 20
<210> SEQ ID NO 239
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 239
tgtggctttt aacatccgca 20
<210> SEQ ID NO 240
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 240
gtaatctctc ccttggtgac 20
<210> SEQ ID NO 241
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 241
aactcttaga aaactgtggc 20
<210> SEQ ID NO 242
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 242
acacggatgc agtcaataac 20
<210> SEQ ID NO 243
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 243
gcagtcaata acaccagggt 20
<210> SEQ ID NO 244
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 244
tagctgggat gcttgactgg 20
<210> SEQ ID NO 245
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 245
acagccttgg agcacacgtg 20
<210> SEQ ID NO 246
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 246
cgatgcagac tttgtggatg 20
<210> SEQ ID NO 247
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 247
tcctgcatac ctacacgctg 20
<210> SEQ ID NO 248
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 248
ctacacgctg tcctttggct 20
<210> SEQ ID NO 249
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 249
cgctgtcctt tggcttgagc 20
<210> SEQ ID NO 250
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 250
tcctttggct tgagcattgg 20
<210> SEQ ID NO 251
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 251
caatctcaga gatggtgaaa 20
<210> SEQ ID NO 252
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 252
agcgagccgt acacctcttt 20
<210> SEQ ID NO 253
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 253
gccgtacacc tctttgtcga 20
<210> SEQ ID NO 254
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 254
tctttgtcga ctctctggtg 20
<210> SEQ ID NO 255
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 255
cgcttcaaaa ggggaatctg 20
<210> SEQ ID NO 256
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 256
aaccgttgta ataacattgg 20
<210> SEQ ID NO 257
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 257
ttaccagctg aaagttcaca 20
<210> SEQ ID NO 258
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 258
aaagttcaca tgttctctta 20
<210> SEQ ID NO 259
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 259
tctcttacaa taacagtggg 20
<210> SEQ ID NO 260
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 260
cagaacctgc ccttggaaat 20
<210> SEQ ID NO 261
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 261
aacaccttcc ttgtctacac 20
<210> SEQ ID NO 262
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 262
aggaggactt gggcgatctc 20
<210> SEQ ID NO 263
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 263
ctcttgaaga tgcgacttac 20
<210> SEQ ID NO 264
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 264
tgtcaaatct ggggaaaccc 20
<210> SEQ ID NO 265
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 265
gagctgtggt ttcacaagtg 20
<210> SEQ ID NO 266
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 266
actgctgaag ttcttgctga 20
<210> SEQ ID NO 267
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 267
tttgttgccg ccgcaggaag 20
<210> SEQ ID NO 268
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 268
ctccctgtcg ctaggtgagc 20
<210> SEQ ID NO 269
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 269
aacatcaggg aacacaaggc 20
<210> SEQ ID NO 270
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 270
gctctgaagt ggccctgtgt 20
<210> SEQ ID NO 271
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 271
agctgcctgg cctcactgaa 20
<210> SEQ ID NO 272
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 272
cctggcctca ctgaacctta 20
<210> SEQ ID NO 273
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 273
ctgaacctta gtgacaagtc 20
<210> SEQ ID NO 274
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 274
cacccttgat ctctagcaga 20
<210> SEQ ID NO 275
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 275
gttctacata cctgctgcgc 20
<210> SEQ ID NO 276
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 276
gacacccttt agtagtttga 20
<210> SEQ ID NO 277
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 277
gacccagcaa ggtttcctgg 20
<210> SEQ ID NO 278
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 278
cgcacattct ttgcatctgt 20
<210> SEQ ID NO 279
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: M. musculus
<220> FEATURE:
<400> SEQUENCE: 279
tgtattgtac cagctttata 20
<210> SEQ ID NO 280
<211> LENGTH: 19
<212> TYPE: RNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<400> SEQUENCE: 280
cgagaggcgg acgggaccg 19
<210> SEQ ID NO 281
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(19)
<223> OTHER INFORMATION: bases at these positions are RNA
<400> SEQUENCE: 281
cgagaggcgg acgggaccgt t 21
<210> SEQ ID NO 282
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: 3, 4, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20,
21
<223> OTHER INFORMATION: bases at these positions are RNA
<400> SEQUENCE: 282
ttgctctccg cctgccctgg c 21
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