MODULATION OF HIF1 BETA EXPRESSION

Abstract

nd methods are provided for modulating the expression of HIF1-beta. The compositions comprise oligonucleotides, targeted to nucleic acid encoding HIF1-beta. Methods of using these compounds for modulation of HIF1-beta expression and for diagnosis and treatment of diseases and conditions associated with expression of HIF1-beta are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. patent application Ser. No. 12/578,468, filed Oct. 13, 2009, which is a continuation of U.S. patent application Ser. No. 11/213,593, filed Aug. 25, 2005, now U.S. Pat. No. 7,618,947, which claims the benefit of priority to U.S. provisional patent application Ser. No. 60/604,190, filed Aug. 25, 2004, and U.S. provisional patent application Ser. No. 60/649,586, filed Feb. 2, 2005, all of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002]The present invention provides compositions and methods for modulating the expression of HIF1-beta. In particular, this invention relates to antisense compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding HIF1-beta. Such compounds are shown herein to modulate the expression of HIF1-beta.

BACKGROUND OF THE INVENTION

[0003]Oxygen homeostasis in mammals is tightly regulated, necessitated by the need to maintain sufficient levels for critical oxygen-dependent processes while minimizing the production of oxygen reactive species that are capable of causing oxidative damage to DNA, lipids, and proteins. In a state of hypoxia, where oxygen demand exceeds supply, a physiological response is mounted that increases the capacity of blood to carry oxygen to tissues and alters cellular metabolism, such as facilitating ATP production by anaerobic glycolysis. The hypoxia-inducible factors (HIFs) are key transcriptional regulators of this hypoxic response. These factors have also been implicated in the pathology of many major human diseases, including cancer, myocardial infarction, ischemia and preeclampsia (Harris, Nat. Rev. Cancer, 2002, 2, 38-47); (Lee et al., N Engl J Med, 2000, 342, 626-633); (Aplin, J Clin Invest, 2000, 105, 559-560)). Cells are typically cultured in the laboratory at an ambient oxygen concentration of 21%, but cells in the human body are exposed to much lower oxygen concentrations ranging from 16% in the lungs to less than 6% in most other organs of the body and often significantly less in tumors (Semenza, Trends Mol Med, 2001, 7, 345-350).

[0004]The HIF proteins are heterodimers consisting of HIF1-beta and one of three alpha subunits, HIF1-alpha, HIF2-alpha and HIF3-alpha (Safran and Kaelin, J. Clin. Invest., 2003, 111, 779-783). The discovery of the HIF proteins was enabled by the identification of a minimal hypoxia-responsive element (HRE) in the 3' enhancer of the erythropoietin gene (Wang and Semenza, Proc Natl Acad Sci USA, 1993, 90, 4304-4308). Subsequent analysis identified the HIF protein as a phosphorylation-dependent protein that binds DNA under hypoxic conditions (Wang and Semenza, J Biol Chem, 1993, 268, 21513-21518). Purification of this DNA-binding factor revealed HIF was a heterodimeric complex consisting of a novel protein, HIF1-alpha, and the aryl hydrocarbon nuclear translocator (ARNT, also termed HIF1-beta), previously identified as a binding partner of the dioxin/aryl hydrocarbon receptor (Wang and Semenza, J. Biol. Chem., 1995, 270, 1230-1237.); (Hoffman et al., Science, 1991, 252, 954-958). HIF proteins belong to a class of transcription factors termed basic helix-loop-helix proteins, grouped by two conserved domains. The basic region consists of approximately 15 predominantly basic amino acids responsible for direct DNA binding. This region is adjacent to two amphipathic alpha helices, separated by a loop of variable length, which forms the primary dimerization interface between family members (Moore et al., Proc Natl Acad Sci USA, 2000, 97, 10436-10441).

[0005]HIF1-beta is a key player in two major signaling pathways, the hypoxic-response pathway and the aryl hydrocarbon receptor (AHR) pathway. Since the discovery of HIF1-alpha/HIF1-beta involvement in erythropoietin transcription, HIF activity has been detected in various non-erythropoietin-producing cell lines cultured under hypoxic conditions (Wang and Semenza, Proc Natl Acad Sci USA, 1993, 90, 4304-4308); (Maxwell et al., Proc Natl Acad Sci USA, 1993, 90, 2423-2427), providing the first evidence that the HIF1 dimer not only activates the erythropoietin gene, but is part of a widespread oxygen-sensing and signal transduction mechanism. Under normoxic conditions, HIF1-alpha is rapidly degraded due to the oxygen-dependent hydroxylation of specific proline residues that mark the protein for proteasomal degradation (Jewell et al., Faseb J, 2001, 15, 1312-1314); (Gorlach et al., Biochim Biophys Acta, 2000, 1493, 125-134). Under hypoxic conditions, this hydroxylation is reversed, and the protein is further stabilized by phosphorylation (Wang et al., Biochem Biophys Res Commun, 1995, 216, 669-675). Subsequently, the protein is translocated to the nucleus, where it interacts with HIF1-beta to form a heterodimeric transcription factor (Kallio et al., Embo J, 1998, 17, 6573-6586). Studies in HIF1-beta deficient cells revealed an absolute requirement for this dimerization step for the transcriptional activation of hypoxia response element genes (Wood et al., J Biol Chem, 1996, 271, 15117-15123). Categories of genes that are activated by the HIF1 dimer include oxygen transport genes, such as erythropoietin (Semenza et al., J Biol Chem, 1994, 269, 23757-23763) and transferrin (Rolfs et al., J Biol Chem, 1997, 272, 20055-20062); genes involved in angiogenesis, such as VEGF (Levy et al., J Biol Chem, 1995, 270, 13333-13340); and genes involved in anaerobic metabolism, such as glucose transporter 1 (Ebert et al., J Biol Chem, 1995, 270, 29083-29089). Hypoxia-induced genes such as VEGF are thought to play a role in promoting angiogenesis and subsequent tumor growth (Harris, Nat. Rev. Cancer, 2002, 2, 38-47).

[0006]HIF transcriptional activity is precisely regulated by cellular oxygen concentration. Whereas changes in oxygen levels do no affect HIF1-beta protein levels, the abundance of the HIF-alpha subunits is markedly increased upon exposure of cells to hypoxia, primarily due to stabilization of the alpha subunits (Safran and Kaelin, J. Clin. Invest., 2003, 111, 779-783). HIF2-alpha mRNA and protein is expressed at low levels in tissue culture cells, but protein expression is markedly induced by exposure to 1% oxygen, a hypoxic state (Wiesener et al., Blood, 1998, 92, 2260-2268). The HIF2-alpha/HIF1-beta heterodimer protein binds to the hypoxic response element, which contains the core recognition sequence 5'-TACGTG-3' and is found in the cis-regulatory regions of hypoxia-regulated genes (Ema et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4273-4278); (Hogenesch et al., J. Biol. Chem., 1997, 272, 8581-8593). Binding of the heterodimer to the HRE induces gene expression (Wiesener et al., Blood, 1998, 92, 2260-2268).

[0007]In contrast to the HIF-alpha subunits, HIF1-beta is stable under both hypoxic and normoxic conditions, and also participates in the aryl hydrocarbon receptor (AHR) signaling pathway. AHR is a cytoplasmic receptor protein that translocates to the nucleus after ligand binding. Ligands of AHR include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxin that is a by-product of industrial processes (Poland et al., J Biol Chem, 1976, 251, 4936-4946); polycylic aromatic hydrocarbons, found in cigarette smoke and smog (Reisz-Porszasz et al., Mol Cell Biol, 1994, 14, 6075-6086); and heterocyclic amines, found in some cooked meats (Reisz-Porszasz et al., Mol Cell Biol, 1994, 14, 6075-6086). After ligand-binding and nuclear translocation, AHR forms a dimer with HIF1-beta, resulting in the activation of a number of genes involved in drug metabolism, such as the cytochromes P450, CYP1A1, CYP1A2, and CYP1B1. AHR/HIF1-beta dimers are capable of activating a range of other genes regulated by the dioxin response element (DRE), resulting in some of the toxic and carcinogenic effects associated with many of the AHR ligands, such as immunotoxicity, developmental and reproductive toxicity, disruption of endocrine pathways, a wasting syndrome, and tumor promotion (Safe, Toxicol Lett, 2001, 120, 1-7). Ohtake and colleagues (Ohtake et al., Nature, 2003, 423, 545-550) demonstrated that the AHR/HIF1-beta heterodimer directly associates with the estrogen receptors ER-alpha and ER-beta. They showed that this association results in the recruitment of unliganded estrogen receptor and coactivator p300 to estrogen-responsive gene promoters, leading to activation of transcription and estrogenic effects and giving rise to the adverse estrogen-related actions of dioxin-type environmental contaminants.

[0008]The role of HIF1-beta in both hypoxia-induced and AHR signaling pathways makes it an attractive therapeutic candidate, as both of these pathways have been linked to various forms of malignancies (Harris, Nat. Rev. Cancer, 2002, 2, 38-47); (Safe, Toxicol Lett, 2001, 120, 1-7). The angiogenic promoting capabilities of HIF1-beta also mark this gene as a potential therapeutic target for a variety of angiogenic disorders, such as arthritis, cardiovascular diseases, skin conditions, aberrant wound healing and ocular conditions (e.g., macular degeneration, diabetic retinopathy, diabetic macular edema and retinopathy of prematurity).

[0009]PCT publication WO 02/053735 discloses the use of an oligonucleotide 35 nucleotides in length as a PCR primer for amplification of the HIF1-beta sequence.

[0010]U.S. Pat. No. 6,352,829 discloses the use of an oligonucleotide 26 nucleotides in length as a PCR primer for amplification of the HIF1-beta sequence.

[0011]U.S. pre-grant publication 2004-0152655 discloses antisense oligonucleotide compounds for inhibiting HIF1-alpha.

[0012]U.S. pre-grant publication 2004-0096848 discloses oligomeric compounds directed against HIF1-alpha.

[0013]U.S. pre-grant publication 2005-0163781 discloses compounds for use as inhibitors of hypoxia-induced genes, such as HIF1-alpha and HIF2-alpha, to treat adhesion formation.

[0014]U.S. pre-grant publication 2004-0180357 discloses HIF1-alpha siRNA compounds for downregulating expression of HIF1-alpha and VEGF and inhibiting angiogenesis.

[0015]U.S. pre-grant publication 2005-0148496 discloses methods of treating inflammatory disorders such as rheumatoid arthritis using compounds that inhibit HIF1-alpha activity.

[0016]U.S. pre-grant publication 2004-0086498 discloses methods for treating animals with advanced or large tumor burdens by administration of an immunotherapeutic agent and a tumor growth restricting agent, such as an expression vector encoding an antisense version of HIF1-alpha.

[0017]U.S. pre-grant publication 2005-0070474 discloses methods of treating tumors using an agent to increase B7-H3 in combination with an agent to inhibit HIF1-alpha, HIF2-alpha or HIF3-alpha.

[0018]Currently, there are no known therapeutic agents which effectively inhibit the synthesis of HIF1-beta and to date, investigative strategies aimed at modulating the function of HIF1-beta have involved the use of antibodies and inactive mutants. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting HIF1-beta function.

[0019]Antisense technology is 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 HIF1-beta expression. Provided herein are antisense compounds for inhibition of HIF1-beta expression. The disclosed compounds can used for treating or preventing conditions associated with HIF1-beta, such as cancer and angiogenic disorders.

SUMMARY OF THE INVENTION

[0020]The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding HIF1-beta, and which modulate the expression of HIF1-beta. Pharmaceutical and other compositions comprising the compounds of the invention and methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of HIF1-beta are also set forth herein.

[0021]Provided herein are antisense oligonucleotides which are specifically hybridizable with a nucleic acid molecule encoding human HIF1-beta. The HIF1-beta antisense oligonucleotides comprise at least one internucleoside, sugar or nucleobase modification. Contemplated are oligonucleotides 13 to 80, 13 to 50, 13 to 30, 20 to 30, 15 to 25 or 20 nucleobases in length. In one embodiment, the internucleoside modification is a phosphorothioate. In one embodiment, the modified sugar moiety is a 2'-O-(2-methoxyethyl). In one embodiment, the modified nucleobase in a 5-methyl cytosine. In some embodiments, the HIF1-beta antisense oligonucleotides further comprise a complementary strand. Also provided are antisense oligonucleotides which specifically hybridize to a splice variant of human HIF1-beta.

[0022]In one embodiment, the antisense oligonucleotides comprise at least an 8-nucleobase portion of one of the illustrative antisense oligonucleotides provided herein. In one embodiment, the antisense oligonucleotide is specifically hybridizable with at least a portion of a start codon region of human HIF1-beta. In another embodiment, the antisense oligonucleotide comprises at least an 8-nucleobase portion of SEQ ID NO: 30. In one embodiment, the antisense oligonucleotide is specifically hybridizable with at least of portion of nucleotides 1876-1895 of a coding region of human HIF1-beta. In another embodiment, the antisense oligonucleotide comprises at least an 8-nucleobase portion of SEQ ID NO: 77.

[0023]Further provided are chimeric antisense oligonucleotides which are specifically hybridizable with a nucleic acid molecule encoding human HIF1-beta. In one embodiment, the chimeric antisense oligonucleotides have a first region comprising deoxynucleotides and second and third regions flanking the first region comprising at least one 2'-O-(2-methoxyethyl) nucleotide. In some embodiments, the first region is 10 deoxynucleotides in length and the second and third regions are each 5 nucleotides in length. The chimeric antisense oligonucleotides provided herein may further comprise a phosphorothioate linkage at each position.

[0024]Also provided are pharmaceutical compositions comprising the antisense oligonucleotides of the invention and a pharmaceutically acceptable carrier or diluent. Pharmaceutical compositions further comprising a colloidal dispersion system are also provided.

[0025]The present invention also provides methods of inhibiting expression of human HIF1-beta in cells or tissues by contacting the cells or tissues with one or more of the compounds provided herein such that expression is inhibited. In one embodiment, HIF1-beta expression is inhibited by 20%. In another embodiment, HIF1-beta expression is inhibited by 40%. In another embodiment, HIF1-beta expression is inhibited by 50%. In another embodiment, HIF1-beta expression is inhibited by 60%. In another embodiment, HIF1-beta expression is inhibited by 70%. In another embodiment, HIF1-beta expression is inhibited by 80%. In one embodiment, the compounds used to inhibit expression of human HIF1-beta comprise at least an 8-nucleobase portion of SEQ ID NO: 30. In another embodiment, the compounds used to inhibit expression of human HIF1-beta comprise at least an 8-nucleobase portion of SEQ ID NO: 77.

[0026]Also provided are methods of inhibiting expression of HIF1-beta regulated genes in cells or tissues by contacting the cells or tissues with one or more of the compounds provided herein. In one embodiment, the HIF1-beta regulated gene is VEGF. In another embodiment, the HIF1-beta regulated gene is GLUT-1. In another embodiment, the HIF1-beta regulated gene is PGK-1. In another embodiment, the HIF1-beta regulated gene is PAI-1. In yet another embodiment, the HIF1-beta regulated gene is Epo.

[0027]Further provided are methods of treating an animal having a disease or condition associated with HIF1-beta comprising administrating to the animal a therapeutically or prophylactically effective amount of a composition comprising one of more of the antisense oligonucleotides provided herein. In one embodiment, the disease or condition associated with HIF1-beta is a hyperproliferative disorder. In one aspect, the hyperproliferative disorder is cancer. In another aspect, the hyperproliferative disorder is an angiogenic disorder. In further embodiments, the angiogenic disorder is an ocular disorder. Ocular disorders contemplated herein, include, but are not limited to macular degeneration, diabetic retinopathy, macular edema and retinopathy of prematurity. In one embodiment, the compounds used to treat the disease or disorder comprise at least an 8-nucleobase portion of SEQ ID NO: 30. In another embodiment, the compounds used to treat the disease or disorder comprise at least an 8-nucleobase portion of SEQ ID NO: 77.

[0028]Also provided are methods of treating an animal having a disease or condition associated with a HIF1-beta regulated gene by administrating to the animal a therapeutically or prophylactically effective amount of a composition comprising one or more of the HIF1-beta antisense oligonucleotides provided herein. In one embodiment, the disease or condition is a hyperproliferative disorder. In a further embodiment, the hyperproliferative disorder is an angiogenic disorder.

[0029]The present invention also provides methods of preventing or inhibiting aberrant angiogenesis in an animal, methods of inhibiting tumor growth in an animal and methods of preventing or inhibiting ocular neovascularization in an animal, comprising administering to said animal one or more of the antisense oligonucleotides provided herein.

[0030]In one embodiment of the methods, the antisense oligonucleotides comprise at least an 8-nucleobase portion of one of the illustrative antisense oligonucleotides provided herein.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031]HIF1-beta is known to play an important role in cellular signaling pathways that can contribute to a number of medically-important pathologic conditions such as cancer and disorders arising from aberrant angiogenesis. To date, there are no effective means for inhibition of HIF1-beta expression. Thus, disclosed herein are antisense compounds for modulation of HIF1-beta expression. The compounds of the invention can be used, for example, to inhibit or prevent aberrant angiogenesis, inhibit tumor growth, or inhibit expression of HIF1-beta regulated genes.

[0032]As used herein, "aberrant angiogenesis" refers to unwanted or uncontrolled angiogenesis.

[0033]As used herein, "targeting" or "targeted to" refer to the process of designing an oligomeric compound such that the compound hybridizes with a selected nucleic acid molecule or region of a nucleic acid molecule.

[0034]As used herein, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the context of the present invention, an oligomeric compound is "specifically hybridizable" when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences. One of skill in the art will be able to determine when an oligomeric compound is specifically hybridizable.

[0035]As used herein, a "HIF1-beta regulated gene" is a gene whose expression is modulated by HIF1-beta gene products.

[0036]The present invention employs antisense compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding HIF1-beta. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding HIF1-beta. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding HIF1-beta" have been used for convenience to encompass DNA encoding HIF1-beta, 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.

[0037]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 HIF1-beta. 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.

[0038]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.

[0039]An antisense compound is specifically hybridizable when 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.

[0040]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.

[0041]"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 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.

B. Compounds of the Invention

[0042]According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, siRNAs, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers 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.

[0043]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.

[0044]While the one 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.

[0045]The term "oligomeric compound" refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. An "antisense compound" or "antisense oligomeric compound" refers to an oligomeric compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and which modulates (increases or decreases) its expression. Consequently, while all antisense compounds can be said to be oligomeric compounds, not all oligomeric compounds are antisense compounds. An "antisense oligonucleotide" is an antisense compound that is a nucleic acid-based oligomer. An antisense oligonucleotide can be chemically modified. Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides and alternate splicers. In one embodiment, the oligomeric compound comprises an antisense strand hybridized to a sense strand. Oligomeric compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.

[0046]The oligomeric compounds in accordance with this invention comprise compounds 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 this comprehends antisense 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.

[0047]In one embodiment, the antisense compounds of the invention comprise 13 to 80 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 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.

[0048]In one embodiment, the antisense compounds of the invention comprise 13 to 50 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 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.

[0049]In one embodiment, the antisense compounds of the invention comprise 13 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases.

[0050]In one embodiment, the antisense compounds of the invention comprise 20 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases.

[0051]In one embodiment, the antisense compounds of the invention comprise 15 to 25 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0052]In one embodiment, the antisense compounds of the invention comprise 20 nucleobases.

[0053]In one embodiment, the antisense compounds of the invention comprise 19 nucleobases.

[0054]In one embodiment, the antisense compounds of the invention comprise 18 nucleobases.

[0055]In one embodiment, the antisense compounds of the invention comprise 17 nucleobases.

[0056]In one embodiment, the antisense compounds of the invention comprise 16 nucleobases.

[0057]In one embodiment, the antisense compounds of the invention comprise 15 nucleobases.

[0058]In one embodiment, the antisense compounds of the invention comprise 14 nucleobases.

[0059]In one embodiment, the antisense compounds of the invention comprise 13 nucleobases.

[0060]Antisense compounds 13-80 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. Antisense compounds 13-80 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.

[0061]Compounds of the invention include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative 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 13 to about 80 nucleobases). Other compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative 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 13 to about 80 nucleobases). It is also understood that compounds may be represented by oligonucleotide sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative compound, and may extend in either or both directions until the oligonucleotide contains about 13 to about 80 nucleobases.

[0062]One having skill in the art armed with the antisense compounds illustrated herein will be able, without undue experimentation, to identify further antisense compounds.

C. Targets of the Invention

[0063]"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 HIF1-beta.

[0064]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.

[0065]Suitable target regions include, for example, 3' untranslated region (3'UTR), start codon region, coding region, stop codon region, 5' untranslated region (5' UTR), 5' cap region, exons, introns, intron-exon junctions and exon-exon junctions.

[0066]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, regardless of the sequence(s) of such codons. The terms "start codon region" and "translation initiation codon region" refer to a portion of 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. The open reading frame (ORF) or "coding region" is known in the art to refer to the region between the translation initiation codon and the translation termination codon. The 5'UTR refers 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'UTR refers 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.

[0067]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, resulting in exon-exon junctions at the sites where exons are joined. Targeting exon-exon junctions can be useful in situations where the overproduction of a normal splice product is implicated in disease, or where the overproduction of an aberrant splice product is implicated in disease. 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. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0068]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.

[0069]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.

[0070]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.

[0071]The locations on the target nucleic acid to which the preferred antisense compounds hybridize are 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.

[0072]Target segments 13-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within a preferred target segment are considered to be suitable for targeting as well.

[0073]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 13 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 13 to about 80 nucleobases). It is also understood that preferred antisense target segments may be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred target segment, and may extend in either or both directions until the oligonucleotide contains about 13 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.

[0074]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.

[0075]The oligomeric antisense compounds can also be targeted to regions of a target nucleobase sequence, such as those disclosed herein.

D. Kits, Research Reagents, Diagnostics, and Therapeutics

[0076]The antisense 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.

[0077]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.

[0078]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.

[0079]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).

[0080]The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding HIF1-beta and inhibit their function. The primers and probes disclosed herein are useful in methods requiring the specific detection of nucleic acid molecules encoding HIF1-beta and in the amplification of said nucleic acid molecules for detection or for use in further studies of HIF1-beta. Hybridization of the primers and probes with a nucleic acid encoding HIF1-beta can be detected by means known in the art. Such means may include conjugation of an enzyme to the primer or probe, radiolabeling of the primer or probe or any other suitable detection means. Kits using such detection means for detecting the level of HIF1-beta in a sample may also be prepared.

[0081]The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds are 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 are useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0082]For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of HIF1-beta 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 HIF1-beta inhibitor. The HIF1-beta inhibitors of the present invention effectively inhibit the activity of the HIF1-beta protein or inhibit the expression of the HIF1-beta protein. In one embodiment, the activity or expression of HIF1-beta in an animal is inhibited by about 10%. Preferably, the activity or expression of HIF1-beta in an animal is inhibited by about 25%. More preferably, the activity or expression of HIF1-beta in an animal is inhibited by 40% or more. Thus, the oligomeric antisense compounds modulate expression of HIF1-beta mRNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.

[0083]For example, the reduction of the expression of HIF1-beta 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 HIF1-beta protein and/or the HIF1-beta protein itself.

[0084]The antisense 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.

E. Modifications

[0085]As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base sometimes referred to as a "nucleobase" or simply a "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)

[0086]Specific examples of oligomeric antisense compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. 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.

[0087]Oligomeric compounds of the invention can have one or more modified internucleoside linkages. One phosphorus-containing modified internucleoside linkage is the phosphorothioate internucleoside linkage. Other 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 phospho-nates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl-phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkyl-phosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950), 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. 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.

[0088]N3'-P5'-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3'-P5'-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44).

[0089]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, each of which is herein incorporated by reference.

[0090]In some embodiments of the invention, oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, 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 internucleotide linkage is represented as --O--P(═O)(OH)--O--CH₂--). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.

[0091]Some 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.

[0092]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, each of which is herein incorporated by reference.

Modified Sugar and Internucleoside Linkages (Mimetics)

[0093]Another group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein the furanose ring or the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.

[0094]One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). Nielsen et al., Science, 1991, 254, 1497-1500. PNAs have favorable hybridization properties, high biological stability and are electrostatically neutral molecules. In one recent study PNA compounds were used to correct aberrant splicing in a transgenic mouse model (Sazani et al., Nat. Biotechnol., 2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are bound directly or indirectly (--C(═O)--CH₂-- as shown below) to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA oligomeric 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. PNA compounds can be obtained commercially from Applied Biosystems (Foster City, Calif., USA). Numerous modifications to the basic PNA backbone are known in the art; particularly useful are PNA compounds with one or more amino acids conjugated to one or both termini. In particular, 1-8 lysine or arginine residues are useful when conjugated to the end of a PNA molecule.

[0095]Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds have been studied in zebrafish embryos (see: Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002, 243, 209-214). Further studies of morpholino-based oligomeric compounds have also been reported (see: Nasevicius et al., Nat. Genet., 2000, 26, 216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits. Linking groups can be varied from chiral to achiral, and from charged to neutral. U.S. Pat. No. 5,166,315 discloses linkages including --O--P(═O)(N(CH₃)₂)--O--; U.S. Pat. No. 5,034,506 discloses achiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 discloses phosphorus containing chiral intermorpholino linkages.

[0096]A further class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA or RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. coli RNase resulting in cleavage of the target RNA strand.

[0097]A further modification includes bicyclic sugar moieties such as "Locked Nucleic Acids" (LNAs) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (--CH₂--) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ENA® is used (Singh et al., Chem. Commun., 1998, 4, 455-456; ENA®: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3'-exonucleolytic degradation and good solubility properties. LNAs are commercially available from ProLigo (Paris, France and Boulder, Colo., USA).

[0098]An isomer of LNA that has also been studied is .A-inverted.-L-LNA which has been shown to have superior stability against a 3'-exonuclease (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). The .A-inverted.-L-LNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity.

[0099]Another similar bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3'-hydroxyl group via a single methylene group to the 4' carbon atom of the sugar ring thereby forming a 3'-C,4'-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).

[0100]The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

[0101]LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.

[0102]LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3'-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands. DNA•LNA chimeras have been shown to efficiently inhibit gene expression when targeted to a variety of regions (5'-untranslated region, region of the start codon or coding region) within the luciferase mRNA (Braasch et al., Nucleic Acids Research, 2002, 30, 5160-5167).

[0103]Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished. Further successful in vivo studies involving LNA's have shown knock-down of the rat delta opioid receptor without toxicity (Wahlestedt et al., Proc. Natl. Acad. Sci., 2000, 97, 5633-5638) and in another study showed a blockage of the translation of the large subunit of RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31, 953-962).

[0104]The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

[0105]The first analogs of LNA, phosphorothioate-LNA and 2'-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2'-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-Amino- and 2'-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0106]Another oligonucleotide mimetic amenable to the present invention that has been prepared and studied is threose nucleic acid. This oligonucleotide mimetic is based on threose nucleosides instead of ribose nucleosides. Initial interest in (3',2')-.A-inverted.-L-threose nucleic acid (TNA) was directed to the question of whether a DNA polymerase existed that would copy the TNA. It was found that certain DNA polymerases are able to copy limited stretches of a TNA template (reported in C&EN/Jan. 13, 2003). In another study it was determined that TNA is capable of antiparallel Watson-Crick base pairing with complementary DNA, RNA and TNA oligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).

[0107]In one study (3',2')-.A-inverted.-L-threose nucleic acid was prepared and compared to the 2' and 3' amidate analogs (Wu et al., Organic Letters, 2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNA and DNA with comparable strength to that of RNA/DNA.

[0108]Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002; and Renneberg et al., Nucleic acids res., 2002, 30, 2751-2757). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tms) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.

[0109]Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acids which incorporate a phosphorus group in the backbone. This class of oligonucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology. Further oligonucleotide mimetics amenable to the present invention have been prepared wherein a cyclobutyl ring replaces the naturally occurring furanosyl ring.

Modified Sugars

[0110]Oligomeric compounds may also contain one or more substituted sugar moieties. Suitable compounds can 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 suitable 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 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. One 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 modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE, as described in examples herein below, 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 herein below.

[0111]Other 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. One 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. Antisense compounds 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, each of which is herein incorporated by reference in its entirety.

[0112]In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3'-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3'-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3'-endo conformational geometry. There is an apparent preference for an RNA type duplex (A form helix, predominantly 3'-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2'-deoxy-2'-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3'-endo nucleosides include but are not limited to: modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3'-endo type conformation.

[0113]Nucleoside conformation is influenced by various factors including substitution at the 2', 3' or 4'-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2' position to favor the 3'-endo conformation can be achieved while maintaining the 2'-OH as a recognition element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O' kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3'-endo conformation can be achieved by deletion of the 2'-OH as exemplified by 2' deoxy-2'F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3'-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4'-position to give 4'-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3'-endo conformation. Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3'-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)

[0114]One conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA like conformations, A-form duplex geometry in an oligomeric context, are selected for use in the modified oligonucleotides of the present invention. The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press., and the examples section below.)

[0115]The terms used to describe the conformational geometry of homoduplex nucleic acids are "A Form" for RNA and "B Form" for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tms) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2' hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2' hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2' endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2'-endo pucker and O4'-endo pucker. This is consistent with Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4'-endo pucker contribution. DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as but not limited to antisense and RNA interference as these mechanisms require the binding of a synthetic oligomer strand to an RNA target strand. In the case of antisense, effective inhibition of the mRNA requires that the antisense DNA have a very high binding affinity with the mRNA. Otherwise the desired interaction between the synthetic oligomer strand and target mRNA strand will occur infrequently, resulting in decreased efficacy.

[0116]One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2'-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependant on the nature of the substituent at the 2'-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2'-halogens have been studied showing that the 2'-fluoro derivative exhibits the largest population (65%) of the C3'-endo form, and the 2'-iodo exhibits the lowest population (7%). The populations of adenosine (2'-OH) versus deoxyadenosine (2'-H) are 36% and 19%, respectively. Furthermore, the effect of the 2'-fluoro group of adenosine dimers (2'-deoxy-2'-fluoroadenosine-2'-deoxy-2'-fluoro-adenosine) is further correlated to the stabilization of the stacked conformation.

[0117]As expected, the relative duplex stability can be enhanced by replacement of 2'-OH groups with 2'-F groups thereby increasing the C3'-endo population. It is assumed that the highly polar nature of the 2'-F bond and the extreme preference for C3'-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and ¹HNMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2'-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2'-substituent on the 3'-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2'-substituted adenosine diphosphates. It is not clear whether the 3'-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3'-endo conformation.

[0118]Increasing the percentage of C3'-endo sugars in a modified oligonucleotide targeted to an RNA target strand should preorganize this strand for binding to RNA. Of the several sugar modifications that have been reported and studied in the literature, the incorporation of electronegative substituents such as 2'-fluoro or 2'-alkoxy shift the sugar conformation towards the 3' endo (northern) pucker conformation. This preorganizes an oligonucleotide that incorporates such modifications to have an A-form conformational geometry. This A-form conformation results in increased binding affinity of the oligonucleotide to a target RNA strand.

[0119]Representative 2'-substituent groups amenable to the present invention that give A-form conformational properties (3'-endo) to the resultant duplexes include 2'-O-alkyl, 2'-β-substituted alkyl and 2'-fluoro substituent groups. Suitable for the substituent groups are various alkyl and aryl ethers and thioethers, amines and monoalkyl and dialkyl substituted amines. It is further intended that multiple modifications can be made to one or more of the oligomeric compounds of the invention at multiple sites of one or more monomeric subunits (nucleosides are suitable) and or internucleoside linkages to enhance properties such as but not limited to activity in a selected application.

Natural and Modified Nucleobases

[0120]Antisense compounds may also include nucleobase (often referred to in the art as heterocyclic base or 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, 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',':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.

[0121]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

[0122]Another modification of the antisense compounds of the invention involves chemically linking to the antisense compound 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-S-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. Antisense compounds 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-triiodo-benzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, 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.

[0123]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.

[0124]Oligomeric compounds used in the compositions of the present invention can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of oligomeric compounds to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By "cap structure or terminal cap moiety" is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini. In non-limiting examples, the 5'-cap includes inverted abasic residue (moiety), 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).

[0125]Particularly preferred 3'-cap structures of the present invention include, for example 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

[0126]Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

Chimeric Compounds

[0127]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.

[0128]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. Chimeric antisense oligonucleotides are thus a form of antisense 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.

[0129]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. Chimeric antisense compounds 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". Such compounds have also been referred to in the art as hybrids. In a gapmer that is 20 nucleotides in length, a gap or wing can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. In one embodiment, a 20-nucleotide gapmer is comprised of a gap 8 nucleotides in length, flanked on both the 5' and 3' sides by wings 6 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 10 nucleotides in length, flanked on both the 5' and 3' sides by wings 5 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 12 nucleotides in length flanked on both the 5' and 3' sides by wings 4 nucleotides in length. In a further embodiment, a 20-nucleotide gapmer is comprised of a gap 14 nucleotides in length flanked on both the 5' and 3' sides by wings 3 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 16 nucleotides in length flanked on both the 5' and 3' sides by wings 2 nucleotides in length. In a further embodiment, a 20-nucleotide gapmer is comprised of a gap 18 nucleotides in length flanked on both the 5' and 3' ends by wings 1 nucleotide in length. Alternatively, the wings are of different lengths, for example, a 20-nucleotide gapmer may be comprised of a gap 10 nucleotides in length, flanked by a 6-nucleotide wing on one side (5' or 3') and a 4-nucleotide wing on the other side (5' or 3').

[0130]In a hemimer, an "open end" chimeric antisense compound, 20 nucleotides in length, a gap segment, located at either the 5' or 3' terminus of the oligomeric compound, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. For example, a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 5' end and a second segment of 10 nucleotides at the 3' end. Alternatively, a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 3' end and a second segment of 10 nucleotides at the 5' end.

[0131]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.

F. Formulations

[0132]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.

[0133]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.

[0134]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.

[0135]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.

[0136]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.

[0137]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.

[0138]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.

[0139]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.

[0140]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.

[0141]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.

[0142]One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0143]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.

G. Dosing

[0144]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. Such considerations are well known to those skilled in the art.

[0145]While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

Example 1

Synthesis of Nucleoside Phosphoramidites

[0146]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'-β-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-N⁴-benzoyl-5-methylcy- tidin-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-(dimethylaminooxy-ethyl) 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-methyluridine, 2'-O-(dimethylaminooxyethyl)-5-methyluridine, 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

[0147]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

[0148]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.

[0149]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.

[0150]Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0151]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.

[0152]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.

[0153]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.

[0154]3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0155]Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0156]Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides

[0157]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 oligonucleosides, 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.

[0158]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.

[0159]Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

RNA Synthesis

[0160]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.

[0161]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.

[0162]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.

[0163]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.

[0164]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.

[0165]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).

[0166]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 μM 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, or for diagnostic or therapeutic purposes.

Example 4

Synthesis of Chimeric Compounds

[0167]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

[0168]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

[0169][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

[0170][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.

[0171]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 HIF1-Beta

[0172]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 HIF1-beta. The nucleobase sequence of the antisense strand of the duplex comprises at least an 8-nucleobase portion of an oligonucleotide in Table 1 or Table 2. 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. The antisense and sense strands of the duplex comprise from about 17 to 25 nucleotides, or from about 19 to 23 nucleotides. Alternatively, the antisense and sense strands comprise 20, 21 or 22 nucleotides.

[0173]For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 187) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:

##STR00001##

[0174]Overhangs can range from 2 to 6 nucleobases and these nucleobases may or may not be complementary to the target nucleic acid. In another embodiment, the duplexes may have an overhang on only one terminus.

[0175]In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG may be prepared with blunt ends (no single stranded overhang) as shown:

##STR00002##

[0176]The RNA duplex can be unimolecular or bimolecular; i.e., the two strands can be part of a single molecule or may be separate molecules.

[0177]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 aliquotted and diluted to a concentration of 50 μM. Once diluted, 30 μl, of each strand is combined with 15 μL 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 μL. 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 μM. This solution can be stored frozen (-20° C.) and freeze-thawed up to 5 times.

[0178]Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate HIF1-beta expression.

[0179]When cells reach 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

[0180]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

[0181]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.

[0182]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

[0183]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

[0184]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.

[0185]T-24 Cells:

[0186]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 bovine 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.

[0187]A549 Cells:

[0188]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 bovine 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.

[0189]NHDF Cells:

[0190]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.

[0191]HEK Cells:

[0192]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. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0193]b.END Cells:

[0194]The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (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 #3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0195]MCF7 Cells:

[0196]The human breast carcinoma cell line MCF-7 was obtained from the American Type Culture Collection (Manassas, Va.). MCF-7 cells were routinely cultured in DMEM low glucose supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into poly-D-lysine coated 96-well plates (Falcon-Primaria #3872) at a density of 8000 cells/well for use in antisense oligonucleotide transfection.

[0197]PC3 Cells:

[0198]The human prostatic carcinoma cell line PC3 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). PC3 cells were routinely cultured in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.) and antibiotics (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of approximately 6000 cells/well for use in antisense oligonucleotide transfection.

[0199]Hep3B Cells:

[0200]The human hepatoma cell line Hep3B (Hep3B2.1-7) was obtained from the American Type Culture Collection (ATCC-ATCC Catalog # HB-8064) (Manassas, Va.). This cell line was initially derived from a hepatocellular carcinoma of an 8-yr-old black male. The cells are epithelial in morphology and are tumorigenic in nude mice. Hep3B cells are routinely cultured in Minimum Essential Medium (MEM) with Earle's Balanced Salt Solution, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (ATCC #20-2003, Manassas, Va.) and with 10% heat-inactivated fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.

[0201]HeLa Cells:

[0202]The human epithelioid carcinoma cell line HeLa was obtained from the American Tissue Type Culture Collection (Manassas, Va.). HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 24-well plates (Falcon-Primaria #3846) at a density of 50,000 cells/well or in 96-well plates at a density of 5,000 cells/well for use in RT-PCR analysis. For Northern blotting or other analyses, cells were harvested when they reached 90% confluence.

[0203]HuVEC Cells:

[0204]The human umbilical vein endothelial cell line HuVEC is obtained from Cascade Biologics (Portland, Oreg.). HuVEC cells are routinely cultured in EBM (Clonetics Corporation Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence and are maintained for up to 15 passages.

[0205]Treatment with Antisense Compounds:

[0206]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 LIPOFECTINT® (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.

[0207]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 ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (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 ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3), a 2'-β-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 ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) 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.

[0208]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.

Example 10

Analysis of Oligonucleotide Inhibition of HIF1-Beta Expression

[0209]Antisense modulation of HIF1-beta expression can be assayed in a variety of ways known in the art. For example, HIF1-beta 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.

[0210]Protein levels of HIF1-beta 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 HIF1-beta 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 for the Use of HIF1-Beta Inhibitors

Phenotypic Assays

[0211]Once HIF1-beta 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.

[0212]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 HIF1-beta 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.).

[0213]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 HIF1-beta 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.

[0214]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.

[0215]Analysis of the genotype 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 HIF1-beta 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.

Example 12

RNA Isolation

[0216]Poly(A)+ mRNA Isolation

[0217]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.

[0218]Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

[0219]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.

[0220]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 HIF1-beta mRNA Levels

[0221]Quantitation of HIF1-beta 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. 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.

[0222]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). 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.

[0223]Gene target quantities are obtained by real-time PCR. Prior to the real-time PCR, isolated RNA is subjected to a reverse transcriptase (RT) reaction, for the purpose of generating complementary DNA (cDNA). Reverse transcriptase and PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT, real-time 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). The method of obtaining gene target quantities by RT, real-time PCR is herein referred to as real-time PCR.

[0224]Gene target quantities obtained by real-time PCR were 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 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).

[0225]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) was pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate was read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0226]Probes and primers to human HIF1-beta were designed to hybridize to a human HIF1-beta sequence, using published sequence information (Genbank accession number BC028362.1, incorporated herein as SEQ ID NO: 4). For human HIF1-beta the PCR primers were:

forward primer: AGCAGAGGGTGTGGGTGTCT (SEQ ID NO: 5)reverse primer: TGGCGGTTGTTGAACATGTT (SEQ ID NO: 6) and the PCR probe was: FAM-CCAGCAGCCTCATCATCGTTCA-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 (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.

[0227]Probes and primers to mouse HIF1-beta were designed to hybridize to a mouse HIF1-beta sequence, using published sequence information (GenBank accession number BC012870.1, incorporated herein as SEQ ID NO: 11). For mouse HIF1-beta the PCR primers were:

forward primer: GGCATCTCCTCCAGCACTGT (SEQ ID NO: 12)reverse primer: GGTAAGACCACTATTCCTGAAATTCTCT (SEQ ID NO: 13) and the PCRprobe was: FAM-TCCCTCCTAACCCCCGGCCG-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 HIF1-Beta mRNA Levels

[0228]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 HYBONDT®-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 STRATALINKERT® 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.

[0229]To detect human HIF1-beta, a human HIF1-beta specific probe was prepared by PCR using the forward primer AGCAGAGGGTGTGGGTGTCT (SEQ ID NO: 5) and the reverse primer TGGCGGTTGTTGAACATGTT (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.).

[0230]To detect mouse HIF1-beta, a mouse HIF1-beta specific probe was prepared by PCR using the forward primer GGCATCTCCTCCAGCACTGT (SEQ ID NO: 12) and the reverse primer GGTAAGACCACTATTCCTGAAATTCTCT (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.).

[0231]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 HIF1-Beta Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0232]In accordance with the present invention, a series of antisense compounds was designed to target different regions of the human HIF1-beta RNA, using published sequences (GenBank accession number BC028362.1, incorporated herein as SEQ ID NO: 4, nucleotides 336578 to 404364 of Genbank accession number NT_--021907.12, the complement of which is incorporated herein as SEQ ID NO: 18, GenBank accession number N72808.1, incorporated herein as SEQ ID NO: 19, and GenBank accession number AL834279.1, 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'-β-methoxyethyl nucleotides, also known as 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 HIF1-beta mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which T-24 cells were treated with 100 nM of antisense oligonucleotides 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 HIF1-beta mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQUENCE % SEQ ID CONTROL SEQ ISIS # REGION NO SITE (5' to 3') INHIB NO ID NO 311073 Intron 1 18 1578 acacacatatctcaaggccc 65 21 2 311074 Intron 1 18 10523 aagggagcagaggactccct 6 22 2 311075 Intron 1 18 15791 caagatcaggctgggaaaca 25 23 2 311076 Intron 2 18 23071 cccctaatctggtcacctgt 71 24 2 311077 Intron 8: Exon 9 18 44694 actgccacacctgtttcaag 51 25 2 junction 311078 Intron 13 18 52827 taggaataataacttatttc 0 26 2 311079 Exon 21: Intron 18 63922 actctcttacctggaagacc 4 27 2 21 junction 311080 5'UTR 4 19 caagatggcggcttcagcag 3 28 2 311081 5'UTR 4 80 ggaaaagaaaggccactccc 0 29 2 311082 Start Codon 4 163 gccgccatggccgcagatgc 87 30 2 311083 Coding 4 199 ggtacatctgatgtcatttc 38 31 2 311084 Coding 4 281 cttaatagccctctggacaa 29 32 2 311085 Coding 4 308 atcatcaaaatccagccctg 42 33 2 311086 Coding 4 388 tccgacctggcaaaccgctc 52 34 2 311087 Coding 4 393 catcatccgacctggcaaac 17 35 2 311088 Coding 4 432 attttccctggcaagtctct 55 36 2 311089 Coding 4 472 ctgtcatcttgttccgtcgc 69 37 2 311090 Coding 4 494 tctgacagttctgtgatgta 82 38 2 311091 Coding 4 527 tttcgagccagggcactaca 83 39 2 311092 Coding 4 532 ctggttttcgagccagggca 76 40 2 311093 Coding 4 652 aatgtttcagttcctgatca 56 41 2 311094 Coding 4 658 agatcaaatgtttcagttcc 66 42 2 311095 Coding 4 663 ctccaagatcaaatgtttca 43 43 2 311096 Coding 4 668 gctgcctccaagatcaaatg 55 44 2 311097 Coding 4 673 catctgctgcctccaagatc 73 45 2 311098 Coding 4 678 aaagccatctgctgcctcca 80 46 2 311099 Coding 4 804 atcatctgggtgcacctgat 76 47 2 311100 Coding 4 810 atccacatcatctgggtgca 80 48 2 311101 Coding 4 815 agtttatccacatcatctgg 43 49 2 311102 Coding 4 892 cctttttcactgttccagtc 81 50 2 311103 Coding 4 900 ctgaccttcctttttcactg 68 51 2 311104 Coding 4 906 agactgctgaccttcctttt 63 52 2 311105 Coding 4 1029 tccattcctgcatctgttcc 53 53 2 311106 Coding 4 1036 agccaagtccattcctgcat 84 54 2 311107 Coding 4 1139 gcctctgggtcatcatctgg 79 55 2 311108 Coding 4 1200 gggagaactagttacctgca 48 56 2 311109 Coding 4 1205 cagttgggagaactagttac 43 57 2 311110 Coding 4 1210 ctgtacagttgggagaacta 74 58 2 311111 Coding 4 1215 catgtctgtacagttgggag 34 59 2 311112 Coding 4 1220 ttactcatgtctgtacagtt 84 60 2 311113 Coding 4 1251 tcgggagatgaactctgttg 65 61 2 311114 Coding 4 1256 ttgtgtcgggagatgaactc 49 62 2 311115 Coding 4 1261 caatgttgtgtcgggagatg 57 63 2 311116 Coding 4 1310 tagccaacagtagccacaca 48 64 2 311117 Coding 4 1315 gctggtagccaacagtagcc 51 65 2 311118 Coding 4 1320 ctgtggctggtagccaacag 52 66 2 311119 Coding 4 1343 acaatattctttcctaagag 27 67 2 311120 Coding 4 1405 atttcactacctgttggaag 67 68 2 311121 Coding 4 1418 acttggccttttaatttcac 55 69 2 311122 Coding 4 1423 acagcacttggccttttaat 63 70 2 311123 Coding 4 1434 gaacatgacagacagcactt 62 71 2 311124 Coding 4 1551 gttcttcacattggtgttgg 68 72 2 311125 Coding 4 1556 ctagagttcttcacattggt 56 73 2 311126 Coding 4 1706 ccatctcttcctggtaccat 77 74 2 311127 Coding 4 1862 ttactctgatccgcattgat 49 75 2 311128 Coding 4 1871 gagatgcctttactctgatc 67 76 2 311129 Coding 4 1876 tggaggagatgcctttactc 84 77 2 311130 Coding 4 1881 agtgctggaggagatgcctt 75 78 2 311131 Coding 4 1953 gaaattctctgccggccggg 70 79 2 311132 Coding 4 1958 ttcctgaaattctctgccgg 67 80 2 311133 Coding 4 1967 agaccactattcctgaaatt 15 81 2 311134 Coding 4 1990 ggacaatggttacaggaggg 68 82 2 311135 Coding 4 1995 tggctggacaatggttacag 80 83 2 311136 Coding 4 2276 gtctcaggagcaaagttaga 48 84 2 311137 Coding 4 2339 cactgtggccagacacccac 65 85 2 311138 Coding 4 2349 ctggccctgccactgtggcc 42 86 2 311139 Coding 4 2354 ggctgctggccctgccactg 85 87 2 311140 Coding 4 2507 ggaaacatagttagatcagg 66 88 2 311141 Stop Codon 4 2531 caatagttctattctgaaaa 21 89 2 311142 Stop Codon 4 2537 tcaccccaatagttctattc 27 90 2 311143 Stop Codon 4 2542 tatcctcaccccaatagttc 24 91 2 311144 3'UTR 4 2616 agaggaacttttattctgtt 51 92 2 311145 3'UTR 4 2621 aagggagaggaacttttatt 0 93 2 311146 3'UTR 4 2873 atccaaggcaaacagtggat 66 94 2 311147 3'UTR 4 3791 gtccaggccccatctatcat 57 95 2 311148 3'UTR 4 4429 tgaaaatctttgctacatgt 72 96 2 311149 5'UTR 19 231 ccaggtggtcacatctggtc 33 97 2 311150 Exon1: Exon1A 20 162 tgagtccaagatcaggcggg 18 98 2 junction

[0233]As shown in Table 1, the majority of antisense compounds targeting human HIF1-beta resulted in inhibition of HIF1-beta mRNA levels. Treatment of cells with antisense compounds represented by SEQ ID NOs: 21, 23-25, 30-34, 36-80, 82-92 and 94-97 resulted in at least 20% inhibition of HIF1-beta mRNA; SEQ ID NOs: 21, 24, 25, 30, 33, 34, 36-58, 60-66, 68-80, 82-88, 92 and 94-96 resulted in at least 40% inhibition of HIF1-beta mRNA; SEQ ID NOs: 21, 24, 25, 30, 34, 36-42, 44-48, 50-55, 58, 60, 61, 63, 65, 66, 68-74, 76-80, 82, 83, 85, 87, 88, 92 and 94-96 resulted in at least 50% inhibition of HIF1-beta mRNA; SEQ ID NOs: 21, 24, 30, 37-40, 42, 45-48, 50-52, 54, 55, 58, 60, 61, 68, 70-72, 74, 76-80, 82, 83, 85, 87, 88, 94 and 96 resulted in at least 60% inhibition of HIF1-beta mRNA; SEQ ID NOs: 24, 30, 38-40, 45-48, 50, 54, 55, 58, 60, 74, 77-79, 83, 87 and 96 resulted in at least 70% inhibition of HIF1-beta mRNA; and SEQ ID NOs: 30, 38, 39, 46, 48, 50, 54, 60, 77, 83 and 87 resulted in at least 80% inhibition of HIF1-beta mRNA.

Example 16

Antisense Inhibition of Mouse HIF1-Beta Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0234]In accordance with the present invention, a second series of antisense compounds was designed to target different regions of the mouse HIF1-beta RNA, using published sequences (GenBank accession number BC012870.1, incorporated herein as SEQ ID NO: 11, nucleotides 1145000 to 1210000 of GenBank accession number NW_--000199.1, incorporated herein as SEQ ID NO: 99, GenBank accession number AK040475.1, incorporated herein as SEQ ID NO: 100, GenBank accession number AK028546.1, incorporated herein as SEQ ID NO: 101, GenBank accession number BG083773.1, incorporated herein as SEQ ID NO: 102, and GenBank accession number AK049738.1, incorporated herein as SEQ ID NO: 103). 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'-O-methoxyethyl nucleotides, also known as 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 HIF1-beta mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which b.END cells were treated with 40 nM of antisense oligonucleotides 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 HIF1-beta mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQUENCE % SEQ ID CONTROL ISIS # REGION NO SITE (5' to 3') INHIB NO SEQ ID NO 347985 Intron 1 99 12686 gattccagcagaaacaagat 76 104 2 347986 Intron 2 99 20208 agtaccataaccaggaagag 82 105 2 347987 Intron 3: Exon 99 28425 atcatcgcatctgaaaagaa 51 106 2 4 junction 347988 Exon 11: 99 51989 tcatacttgcctgcagcctg 59 107 2 Intron 11 junction 347989 5'UTR 11 3 agattaggcaccttaccgcc 80 108 2 347990 Start Codon 11 98 gccgccatggtcgagatggc 73 109 2 347991 Start Codon 11 111 gttagctgtagtcgccgcca 88 110 2 347992 Coding 11 121 tcatttctgggttagctgta 75 111 2 347993 Coding 11 131 acatctgatgtcatttctgg 79 112 2 347994 Coding 11 163 ttccagaagcaatggtggga 69 113 2 347995 Coding 11 195 agctcctccaccttgaatcc 41 114 2 347996 Coding 11 221 cgtcgcttaatagccctctg 81 115 2 347997 Coding 11 231 cagccctgaccgtcgcttaa 83 116 2 347998 Coding 11 241 catcaaaatccagccctgac 29 117 2 347999 Coding 11 317 ctggcaaaccgctctttgtc 47 118 2 348000 Coding 11 327 atcatccgacctggcaaacc 85 119 2 348001 Coding 11 337 agctctgctcatcatccgac 41 120 2 348002 Coding 11 355 gtctctctttatccgcagag 89 121 2 348003 Coding 11 362 ctggcaagtctctctttatc 66 122 2 348004 Coding 11 367 tttccctggcaagtctctct 56 123 2 348005 Coding 11 372 atgattttccctggcaagtc 75 124 2 348006 Coding 11 414 gtaagctgtcatcttgttcc 73 125 2 348007 Coding 11 424 gttctgtgatgtaagctgtc 49 126 2 348008 Coding 11 477 tagcttgtctggttttcgag 63 127 2 348009 Coding 11 591 caaatgtttcagttcctgat 82 128 2 348010 Coding 11 622 taaacagaaagccatctgct 83 129 2 348011 Coding 11 686 tggttcaaaacgggagtcac 39 130 2 348012 Coding 11 763 gctgctctcgaagtttatcc 58 131 2 348013 Coding 11 858 gcacatcctcatggaagact 90 132 2 348014 Coding 11 929 ttcatggaaacagggtccac 84 133 2 348015 Coding 11 939 gctcagtctattcatggaaa 76 134 2 348016 Coding 11 949 tcctcaaaaagctcagtcta 56 135 2 348017 Coding 11 959 ctgcatctgttcctcaaaaa 86 136 2 348018 Coding 11 1015 tgcagtggactaccacaaag 68 137 2 348019 Coding 11 1081 ggccagcctctgggtcatca 89 138 2 348020 Coding 11 1097 cagaatttgctcccctggcc 73 139 2 348021 Coding 11 1107 ggccactaggcagaatttgc 79 140 2 348022 Coding 11 1119 cagcctgccaatggccacta 70 141 2 348023 Coding 11 1211 gtgaatatcccttcaatgtt 74 142 2 348024 Coding 11 1241 acagtagccacacaacgatg 79 143 2 348025 Coding 11 1291 gacaaaattctacaatattc 53 144 2 348026 Coding 11 1302 gtcttcaggatgacaaaatt 79 145 2 348027 Coding 11 1312 gaagttgttggtcttcagga 65 146 2 348028 Coding 11 1323 gctgtctcttagaagttgtt 25 147 2 348029 Coding 11 1333 cctgctgaaagctgtctctt 76 148 2 348030 Coding 11 1343 aatttcaccacctgctgaaa 56 149 2 348031 Coding 11 1408 tcatccacagccattctcgg 75 150 2 348032 Coding 11 1504 gtggttcctggctagagttc 58 151 2 348033 Coding 11 1550 gtcggacctagctgtgacct 80 152 2 348034 Coding 11 1579 ctgtacccatctctagggat 79 153 2 348035 Coding 11 1651 ccagcccatctcttcctggt 23 154 2 348036 Coding 11 1681 ggacagaaacctgggaatga 76 155 2 348037 Coding 11 1719 gggcttgctgtgttctgatc 70 156 2 348038 Coding 11 1742 aagagaccttctgacttctc 57 157 2 348039 Coding 11 1855 cctgggagaacagctgttgg 84 158 2 348040 Coding 11 1865 aatgagctgccctgggagaa 83 159 2 348041 Coding 11 1875 gttaggagggaatgagctgc 69 160 2 348042 Coding 11 1906 cactattcctgaaattctct 65 161 2 348043 Coding 11 1958 atctgccctgcagaagatga 58 162 2 348044 Coding 11 2083 aagaacgagtcttggctgta 70 163 2 348045 Coding 11 2110 tctgaaagttgttcacacca 51 164 2 348046 Coding 11 2219 gtctcaggaggaaagttgga 88 165 2 348047 Coding 11 2287 cctgccactgtggccagaca 77 166 2 348048 Coding 11 2379 ttcttgaaagacctcaggct 52 167 2 348049 Coding 11 2399 tctcccagcatggacagcat 68 168 2 348050 Stop Codon 11 2475 ccaatagttctattcggaaa 87 169 2 348051 3'UTR 11 2538 tctgtttacaaaagatttgc 78 170 2 348052 5'UTR 100 10 cggaatccaagatggcggac 88 171 2 348053 3'UTR 100 2358 ctccaaacaagcctgagacc 87 172 2 348054 Coding 101 352 atgattttccctggcaaacc 79 173 2 348055 Coding 101 2031 ggcacctgggcggcaaagcc 60 174 2 348056 Coding 102 608 agacttttcccccacatatc 67 175 2 348057 5'UTR 103 50 taacctatgtattcagtgat 34 176 2 311090 Coding 11 431 tctgacagttctgtgatgta 75 38 2 311091 Coding 11 464 tttcgagccagggcactaca 82 39 2 311112 Coding 11 1157 ttactcatgtctgtacagtt 82 60 2 311129 Coding 11 2819 tggaggagatgcctttactc 75 77 2 311130 Coding 11 1824 agtgctggaggagatgcctt 78 78 2

[0235]As shown in Table 1, the majority of antisense compounds targeting mouse HIF1-beta resulted in inhibition of HIF1-beta mRNA levels. Treatment of cells with antisense compounds represented by SEQ ID NOs: 38, 39, 60, 77, 78, 104-153 and 155-176 resulted in at least 25% inhibition of HIF1-beta mRNA; SEQ ID NOs: 38, 39, 60, 77, 78, 104-113, 115, 116, 119, 121-125, 127-129, 131-146, 148-153 and 155-175 resulted in at least 50% inhibition of HIF1-beta mRNA; 38, 39, 60, 77, 78, 104, 105, 108-113, 115, 116, 119, 121, 122, 124, 125, 127-129, 132-134, 136-143, 145, 146, 148, 150, 152, 153, 155, 156, 158-161, 163, 165, 166 and 168-resulted in at least 60% inhibition of HIF1-beta mRNA; SEQ ID NOs: 38, 39, 60, 77, 78, 104, 105, 108-112, 115, 116, 119, 121, 124, 125, 128, 129, 132-134, 136, 138-143, 145, 148, 150, 152, 153, 155, 156, 158, 159, 163, 165, 166 and 169-173 resulted in at least 70% inhibition of HIF1-beta mRNA; and SEQ ID NOs: 39, 60, 105, 108, 110, 115, 116, 119, 121, 128, 129, 132, 133, 136, 138, 152, 158, 159, 165, 169, 171 and 172 resulted in at least 80% inhibition of HIF1-beta mRNA.

Example 17

Targeting of Individual Oligonucleotides to Specific Variants of Human HIF1-Beta

[0236]A search of the National Center for Biotechnology Information database revealed alternative mRNA variants of human HIF1-beta which are the result of alternative splicing. The sequence identified as Genbank accession number N72808.1 represents a variant of HIF1-beta designated herein as HIF1-beta-1 (incorporated herein as SEQ ID NO: 19). The sequence identified as Genbank accession number AL834279.1 represents a variant of HIF1-beta designated herein as HIF1-beta-2 (incorporated herein as SEQ ID NO: 20).

[0237]It is advantageous to selectively inhibit the expression of one or more variants of HIF1-beta. Consequently, in one embodiment of the present invention are oligonucleotides that selectively target, hybridize to, and specifically inhibit one or more, but fewer than all the variants of HIF1-beta. The oligonucleotides of the present invention that selectively target human HIF1-beta variants are presented in Table 4.

TABLE-US-00003 TABLE 4 Targeting of individual oligonucleotides to specific variants of human HIF1-beta SEQ ID Target SEQ ID ISIS # NO Target Site Target Variant NO 311149 97 231 HIF1-beta-1 19 331150 98 162 HIF1-beta-2 20

Example 18

Targeting of Individual Oligonucleotides to Specific Variants of Mouse HIF1-Beta

[0238]A search of the National Center for Biotechnology Information database revealed alternative mRNA variants of mouse HIF1-beta which are the result of alternative splicing. The sequence identified as Genbank accession number AK040475.1 represents a variant of HIF1-beta designated herein as HIF1-beta-3 (incorporated herein as SEQ ID NO: 100). The sequence identified as Genbank accession number AK028546.1 represents a variant of HIF1-beta designated herein as HIF1-beta-4 (incorporated herein as SEQ ID NO: 101). The sequence identified as Genbank accession number BG083773.1 represents a variant of HIF1-beta designated herein as HIF1-beta-5 (incorporated herein as SEQ ID NO: 102). The sequence identified as Genbank accession number AK049738.1 represents a variant of HIF1-beta designated herein as HIF1-beta-6 (incorporated herein as SEQ ID NO: 103).

[0239]It is advantageous to selectively inhibit the expression of one or more variants of HIF1-beta. Consequently, in one embodiment of the present invention are oligonucleotides that selectively target, hybridize to, and specifically inhibit one or more, but fewer than all the variants of mouse HIF1-beta. The oligonucleotides of the present invention that selectively target mouse HIF1-beta variants are presented in Table 5.

TABLE-US-00004 TABLE 5 Targeting of individual oligonucleotides to specific variants of mouse HIF1-beta SEQ ID Target Target Target SEQ ISIS # NO Site Variant ID NO 348052 171 10 HIF1-beta-3 100 348053 172 2358 HIF1-beta-3 100 348053 172 1543 HIF1-beta-6 103 348054 173 352 HIF1-beta-4 101 348054 173 149 HIF1-beta-5 102 348055 174 2031 HIF1-beta-4 101 348056 175 608 HIF1-beta-5 102 348057 176 50 HIF1-beta-6 103

Example 19

Expression of HIF1-Beta in Various Human Cell Lines

[0240]U87-MG human glioblastoma, PC-3 human prostate cancer, JEG-3 human choriocarcinoma, HeLa human cervical cancer, SK-N-BE(2) neuroblastoma, MCF-7 human breast cancer, 786-O human clear-cell renal cell carcinoma, Calu-1 human lung cancer, and Hep3B human hepatocellular carcinoma cells were purchased from American Type Culture Collection (ATCC; Manassas, Va.) and cultured according to ATCC directions. Human umbilical endothelial cells (HUVEC) were obtained from Cascade Biologics (Portland, Oreg.). Hypoxic treatments of cells (0.5-0.8×10⁶/60 mm dish or 1-2×10⁶/100 mm dish) were performed at 1% O₂ in a chamber controlled by ProOx oxygen sensor (BioSpherix, Redfield, N.Y.) for 18 h. To achieve the optimal hypoxic induction, 2 or 5 ml of medium was used for 60 mm and 100 mm culture dishes, respectively, during incubation. CoCl₂ (150 μM) was added to the cells to mimic hypoxic condition in some experiments.

[0241]After 18 h of culture at normoxia, hypoxia, or with CoCl₂, cells were harvested and whole cell lysates prepared with RIPA buffer containing protease inhibitor cocktails (Roche), 0.5 mM sodium orthovanadate, 10 mM β-glycerophophate, 250 ng/ml ubiquitin aldehyde (Sigma-Aldrich), and 400 nM epoxomicin (Alexis). Lysates were separated on 10% SDS-PAGE and transferred to PVDF membranes (Amersham Biosciences). Immunoblotting was performed with the following antibodies and dilutions: anti-HIF1-beta (BD Transduction Laboratories) at 1:1000; anti-VHL (BD Transduction Laboratories) at 1:500; anti-GLUT-1 (Alpha Diagnostic International) at 1:600, and anti-α-tubulin (Sigma) at 1:2000. Antibodies were diluted in 0.05% Tween-20/Tris-buffered saline (T-TBS) blocking buffer containing 5% nonfat skim milk and incubated with the PVDF membranes at 4° C. overnight, followed by washing with T-TBS for 30 min. Goat anti-mouse or rabbit IgGs coupled with HRP (BioRad) were used as secondary antibodies at 1:3000. Immunospecific bands were detected by enhanced chemiluminescence plus (ECL-Plus) detection kit (Amersham Biosciences).

[0242]Expression of HIF1-beta was detected under both normoxic and hypoxic conditions; however, levels of expression of HIF1-beta varied among cell lines. HIF1-beta expression was induced under hypoxic conditions and in the presence of CoCl₂ (which mimics hypoxia) in U87-MG human glioblastoma cells and MCF-7 human breast cancer cells. However, expression of HIF1-beta in JEG-3, PC-3, Hep3B, HeLa, 786-O, SK-N-BE(2), Calu-1 and HUVECs was not significantly altered by culturing under hypoxic conditions or in the presence of CoCl₂.

Example 20

Antisense Modulation of HIF1-Beta mRNA Expression in Cancer Cells (Dose Response)

[0243]Hep3B or U87-MG cells were plated in 96-well plates (8-10,000/well) 16 h prior to transfection. Control oligonucleotide ISIS 129688 (SEQ ID NO: 177) or HIF1-beta antisense oligonucleotides ISIS 311082 (SEQ ID NO: 30) and ISIS 311129 (SEQ ID NO: 77) at a concentration of 0, 6.25 or 25 nM were delivered into cells by lipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media (Invitrogen). Both control and HIF1-beta antisense oligonucleotides 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'-O-methoxyethyl nucleotides, also known as 2'-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.

[0244]The transfection medium (120 μl/well) was switched to low-serum medium (0.1% FBS) 4 h after transfection. Sixty microliters of medium was removed from the well 3 h after media switch and the cells were further incubated at normoxia or hypoxia for 16-20 h.

[0245]Total RNAs were isolated using RNeasy 96 BioRobot 9604 kit (Qiagen) according to the manufacturer's instructions. Quantitative real-time RT-PCR for detection of HIF1-beta mRNA was performed as described in other Examples herein by ABI Prism 7700 Sequence Detector (Applied Biosystems) in 25 or 50 μl reaction volumes. The level of mRNA for each gene was normalized to the amount of total RNA determined by Ribogreen® (Molecular Probes).

TABLE-US-00005 TABLE 6 HIF1-beta mRNA expression in hypoxic Hep3B cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells at normoxia) Percent expression of HIF1-beta mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 78 78 77 311082 79 50 20 311129 77 70 30

TABLE-US-00006 TABLE 7 HIF1-beta mRNA expression in hypoxic U87-MG cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells at normoxia) Percent expression of HIF1-beta mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 78 78 77 311082 79 50 20 311129 77 70 20

[0246]HIF1-beta antisense oligonucleotides ISIS 311082 and ISIS 311129 specifically inhibited mRNA expression of HIF1-beta in both Hep3B and U87-MG cells in a dose-dependent manner.

Example 21

Antisense Modulation of HIF1-Beta Protein Expression

[0247]U87-MG cells were plated in 10 cm dishes (1-2×10⁶ cells/dish) 16 h prior to transfection. 100 nM control oligonucleotide (ISIS 129688, SEQ ID NO: 177) or HIF1-beta antisense oligonucleotide (ISIS 311082, SEQ ID NO: 30) were delivered into cells by lipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media (Invitrogen).

[0248]The transfection medium (10 ml/dish) was switched to low-serum medium (0.1% FBS) 4 h after transfection. Five ml of medium was removed from the well 3 h after media switch and the cells were further incubated at normoxia or hypoxia for 16-20 h.

[0249]Following incubation at normoxia or hypoxia, transfected cells were harvested and whole cell lysates prepared with RIPA buffer containing protease inhibitor cocktails (Roche), 0.5 mM sodium orthovanadate, 10 mM β-glycerophophate, 250 ng/ml ubiquitin aldehyde (Sigma-Aldrich), and 400 nM epoxomicin (Alexis). Lysates were separated on 10% SDS-PAGE and transferred to PVDF membranes (Amersham Biosciences). Immunoblotting was performed with the following antibodies and dilutions: anti-HIF1-BETA (BD Transduction Laboratories) at 1:1000; anti-GLUT-1 (Alpha Diagnostic International) at 1:600, and anti-α-tubulin (Sigma) at 1:2000. Antibodies were diluted in 0.05% Tween-20/Tris-buffered saline (T-TBS) blocking buffer containing 5% nonfat skim milk and incubated with the PVDF membranes at 4° C. overnight, followed by washing with T-TBS for 30 min. Goat anti-mouse or rabbit IgGs coupled with HRP (BioRad) were used as secondary antibodies at 1:3000. Immunospecific bands were detected by enhanced chemiluminescence plus (ECL-Plus) detection kit (Amersham Biosciences).

[0250]The results demonstrated that transfection with HIF1-beta antisense oligonucleotide inhibited HIF1-beta protein expression. Furthermore, protein expression of GLUT-1, a HIF1-beta-responsive gene, was induced under hypoxia in the absence of HIF1-beta antisense oligonucleotide, but was nearly eliminated in hypoxic cells transfected with HIF1-beta antisense oligonucleotide.

Example 22

Expression HIF1-Beta-Regulated Genes in Cells Cultured Under Hypoxic Conditions

[0251]Genes whose products are significantly induced by hypoxia (or CoCl₂, a mimic of hypoxia) include erythropoietin (Epo), glucose transporter-1 (GLUT-1), vascular endothelial growth factor (VEGF), phosphoglycerate kinase-1 (PGK-1) and plasminogen activator inhibitor-1 (PAI-1). These genes are regulated by HIF1-beta and are induced under hypoxic conditions to varying extents in different cell lines. To determine mRNA expression levels of Epo, GLUT-1, VEGF, PGK-1 and PAI-1 at normoxia (21% O₂), hypoxia (1% O₂) and in the presence of 150 μM CoCl₂, in HeLa, Hep3B, U87-MG, PC-3 and 786-O cells, RT-PCR was performed. Total RNA was isolated after 18 h of culture using the RNeasy 96 BioRobot 9604 (Qiagen) according to the manufacturer's protocol. Quantitative real-time RT-PCR was performed as described in other Examples herein by ABI Prism 7700 Sequence Detector (Applied Biosystems) in 25 or 50 μl reaction volumes. The level of mRNA for each gene was normalized to the amount of total RNA determined by Ribogreen® (Molecular Probes).

TABLE-US-00007 TABLE 8 VEGF, GLUT-1, PGK-1, PAI-1 and Epo mRNA expression levels at normoxia, hypoxia and 150 μM CoCl₂ (shown as fold-induction relative to HeLa cells at normoxia) Gene Cell Type Normoxia Hypoxia CoCl₂ VEGF HeLa 1 3 2 Hep3B 4 24 11 U87-MG 17 46 28 PC-3 1 2 2 786-O 6 6 10 GLUT-1 HeLa 1 2.1 1.5 Hep3B 0.2 1 1 U87-MG 2.5 3.1 5.3 PC-3 1 4.1 3.7 786-O 2.9 3.4 5.7 PGK-1 HeLa 1 3 2 Hep3B 4 24 10 U87-MG 16 46 28 PC-3 1 2 2 786-O 6 6 9 PAI-1 HeLa 1 1.5 1 Hep3B 1.5 28 2 U87-MG 9 24 32 PC-3 3 4 3 786-O 2 3 16 Epo HeLa 1 1 1 Hep3B 7 260 15 U87-MG 1 1 1 PC-3 1 1 1 786-O 1 1 1

[0252]Although expression levels of the five genes under each condition varied widely among cell lines, VEGF, GLUT-1, PGK-1, PAI-1 and Epo exhibited a trend of increased expression when cells were cultured under hypoxia or in the presence of 150 μM CoCl₂. As previously reported, hypoxia-induced Epo expression occurred only in Hep3B cells.

Example 23

Antisense Inhibition of HIF1-Beta Target Genes

[0253]Hep3B or U87-MG cells were plated in 96-well plates (8-10,000/well) 16 h prior to transfection. Control oligonucleotide ISIS 129688 (SEQ ID NO: 177) or HIF1-beta antisense oligonucleotides ISIS 311082 (SEQ ID NO: 30) and ISIS 311129 (SEQ ID NO: 77) at a concentration of 0, 6.25 or 25 nM were delivered into cells by lipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media (Invitrogen).

[0254]The transfection medium (120 μl/well) was switched to low-serum medium (0.1% FBS) 4 h after transfection. Sixty microliters of medium was removed from the well 3 h after media switch and the cells were further incubated at normoxia or hypoxia for 16-20 h.

[0255]Total RNAs were isolated using RNeasy 96 BioRobot 9604 kit (Qiagen) according to the manufacturer's instructions. Quantitative real-time RT-PCR for detection of GLUT-1, VEGF or Epo mRNA was performed as described in other Examples herein by ABI Prism 7700 Sequence Detector (Applied Biosystems) in 25 or 50 μl reaction volumes. The level of mRNA for each gene was normalized to the amount of total RNA determined by Ribogreen® (Molecular Probes).

TABLE-US-00008 TABLE 9 GLUT-1 mRNA expression in hypoxic Hep3B cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells) Percent expression of GLUT-1 mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 100 85 83 311082 100 72 34 311129 100 74 79

TABLE-US-00009 TABLE 10 GLUT-1 mRNA expression in hypoxic U87-MG cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells) Percent expression of GLUT-1 mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 100 100 102 311082 100 76 28 311129 100 83 40

TABLE-US-00010 TABLE 11 VEGF mRNA expression in hypoxic U87-MG cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells) Percent expression of VEGF mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 100 101 82 311082 100 62 26 311129 100 71 28

TABLE-US-00011 TABLE 12 Epo mRNA expression in hypoxic Hep3B cells treated with antisense oligonucleotide to HIF1-beta (shown as percent of untreated control cells) Percent expression of Epo mRNA after treatment with oligonucleotide at the concentrations shown: Oligonucleotide 0 nM 6.25 nM 25 nM 129688 100 107 114 311082 100 95 38 311129 100 100 74

[0256]As shown in other Examples herein, GLUT-1, Epo and VEGF mRNA expression was induced under hypoxic conditions. HIF1-beta antisense oligonucleotides ISIS 311082 and ISIS 311129 specifically inhibited mRNA expression of GLUT-1, Epo and VEGF in both Hep3B cells (GLUT-1 and Epo) and U87-MG cells (GLUT-1 and VEGF).

Example 24

Antisense Inhibition of Protein Expression of HIF1-Beta Downstream Targets

[0257]U87-MG, HeLa or Hep3B cells were plated in 10 cm dishes (1-2×10⁶ cells/dish) 16 h prior to transfection. 100 nM control oligonucleotide (ISIS 129688, SEQ ID NO: 177) or HIF1-beta antisense oligonucleotide (ISIS 311082, SEQ ID NO: 30) were delivered into cells by lipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media (Invitrogen).

[0258]The transfection medium (10 ml/well) was switched to low-serum medium (0.1% FBS) 4 h after transfection. Five ml of medium was removed from the well 3 h after media switch and the cells were further incubated at normoxia or hypoxia.

[0259]After incubation at normoxia or hypoxia for 16 h, the media was removed and stored at -80° C. prior to use. Levels of VEGF protein in either U87-MG or HeLa cells was determined by Quantikine ELISA kit (R&D Systems) according to the manufacturer's protocol. To determine levels of Epo protein in Hep3B cells, cell culture media was concentrated up to 10% of the original volume by Amicon Ultra (Millipore) and Epo protein was quantitated by Quantikine IVD human Epo ELISA kit (R&D Systems). The obtained values were normalized to the number of cells (VEGF) or the amount of total protein (Epo) used for the assay.

[0260]Under hypoxic conditions, VEGF protein production in U87-MG cells treated with HIF1-beta antisense oligonucleotide was significantly reduced (approximately 4-fold) relative to cells treated with control oligonucleotide. In HeLa cells, VEGF protein levels were slightly reduced by treatment with HIF1-beta antisense oligonucleotide. In Hep3B cells treated with HIF1-beta antisense oligonucleotide, Epo protein levels were significantly reduced (approximately 15-fold) relative to cells treated with control oligonucleotide. Thus, treatment with HIF1-beta antisense oligonucleotides not only inhibits expression of HIF1-beta, but also results in downregulation of HIF1-beta target genes.

Example 25

Hypoxia-Induced Binding of HIF1-Beta to Hypoxia Response Element (HRE) In Vivo

[0261]To demonstrate binding of HIF1-beta to the hypoxia response element (HRE) on the promoter region of the VEGF gene under hypoxic conditions, chromatin immunoprecipitation (ChIP) assays were performed. U87-MG cells were plated in 10 cm dishes in complete growth media containing 10% FBS at a density of 1×10⁶ cells/dish. After 24 h, the media was replaced with low serum media (0.1% FBS) and the cells were incubated at either normoxia or hypoxia for 16 h. After incubation, cells were cross-linked with a 1% formaldehyde solution for 10 min at 37° C. After two washes with cold PBS, cells were processed following the ChIP assay kit protocol (Upstate). For immunoprecipitation, protein extracts were incubated with antibodies to HIF1-beta (10 μg) and mouse IgG (10 μg) at 4° C. for 40 h, followed by 2 h incubation with salmon sperm DNA/Protein A agarose slurry. After extensive washing, the immune/DNA complex was eluted in 500 μl of buffer (1% SDS, 0.1 M NaHCO₃), reverse-crosslinked at 65° C. for 4 h in the presence of 0.2 M NaCl and subjected to proteinase K (Ambion) digestion at 45° C. for 1 h. Samples were extracted with phenol-chloroform-isoamylalcohol, ethanol-precipitated overnight at -80° C. and the DNA was resuspended in water.

[0262]Samples were analyzed by PCR using Accuprime II (Invitrogen) Taq polymerase in the presence of [³²P-dCTP]. PCR products were separated on 6% Tris-borate-EDTA (TBE)-PAGE, dried and exposed to X-ray film at -80° C. The forward (F) and reverse (R) primers used for PCR are as follows:

TABLE-US-00012 (SEQ ID NO: 178) VEGF I (F): CCTGGCAACATCTGGGGTTGG (SEQ ID NO: 179) VEGF I (R): CAACAGGCTGGAGTGACTGGGCTCC (SEQ ID NO: 180) VEGF II (F): GTGGAGACAGGACTAGTGCACGAATG (SEQ ID NO: 181) VEGF II (R): CTGTGGAGGCATGGACTGAGAATGG (SEQ ID NO: 182) Epo (F): CTGGGAACCTCCAAATCCCCTGGC (SEQ ID NO: 183) Epo (R): CTGGGCAGGGTTGGCAGCTGCCTTAC

[0263]VEGF I primers amplify a region that includes the HRE. VEGF II primers amplify a promoter region upstream of the HRE, therefore this region serves as a negative control. The Epo gene also is a negative control since it is not expressed in U87-MG cells.

[0264]HIF1-beta was not detected on the VEGF HRE at normoxia. However, hypoxia induced the recruitment of HIF1-beta to the VEGF HRE (VEGF I). No binding of HIF1-beta was detected to the regions amplified by the VEGF II or Epo primers.

Example 26

Inhibition of HIF1-Beta Expression In Vivo

[0265]C57B1/6 mice are maintained on a standard rodent diet and are used as control animals. Seven-week old male C57B1/6 mice are injected subcutaneously with oligonucleotides at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, spleen, adipose and other tissues using RNA isolation and target mRNA expression level quantitation (RT-PCR) as described in other examples herein.

Example 27

Tube Formation Assay to Determine Effect of HIF1-Beta Antisense Inhibitors on Angiogenesis

[0266]Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This process can be reproduced in tissue culture by the formation of tube-like structures by endothelial cells. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet et al., (2000) Nature 407:249-257; and Zhang et al., (2002) Cancer Research 62:2034-42), which supports the use of in vitro tube formation as an endpoint for angiogenesis.

[0267]Angiogenesis, or neovascularization, is the formation of new capillaries from existing blood vessels. In adult organisms this process is typically controlled and short-lived, for example in wound repair and regeneration. However, aberrant capillary growth can occur and this uncontrolled growth plays a causal and/or supportive role in many pathologic conditions such as tumor growth and metastasis. In the context of this invention "aberrant angiogenesis" refers to unwanted or uncontrolled angiogenesis. Angiogenesis inhibitors are being evaluated for use as antitumor drugs. Other diseases and conditions associated with angiogenesis include arthritis, cardiovascular diseases, skin conditions (e.g., psoriasis), and aberrant wound healing. Aberrant angiogenesis can also occur in the eye, causing loss of vision. Examples of ocular conditions involving aberrant angiogenesis include macular degeneration, diabetic retinopathy, diabetic macular edema and retinopathy of prematurity.

[0268]The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.), or growth factor reduced Matrigel (BD Biosciences, Bedford, Mass.). HUVECs were plated at 4000 cells/well in 96-well plates. One day later, cells were transfected with antisense and control oligonucleotides according to standard published procedures (Monia et al., (1993) J Biol. Chem. 1993 Jul. 5; 268(19):14514-22) using 75 nM oligonucleotide in lipofectin (Gibco, Grand Island, N.Y.). Approximately fifty hours post-transfection, cells were transferred to 96-well plates coated with ECMatrix® (Chemicon Inter-national) or growth factor depleted Matrigel. Under these conditions, untreated HUVECs form tube-like structures. After an overnight incubation at 37° C., treated and untreated cells were inspected by light microscopy. Individual wells were assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network.

[0269]ISIS 29848 is a control oligonucleotide containing an equal mixture of the bases A, C, G and T at every position. ISIS 175510 (TGAGCTGTCTGTGATCCAGC; SEQ ID NO: 184) is targeted to HIF1α; ISIS 222035 (GCGCTGCTCCCAAGAACTCT; SEQ ID NO: 185) is targeted to HIF2α. ISIS 298697 (TCCTCATGGTCACATGGATG; SEQ ID NO: 186) is a cross-HIF1α/HIF2α oligonucleotide having perfect complementarity to HIF1α target and imperfect complementarity (and thus less inhibitory effect) for HIF2α; ISIS 311082 (SEQ ID NO: 30) is targeted to HIF1-beta.

TABLE-US-00013 TABLE 13 Effect of antisense oligonucleotides on angiogenic tube formation ISIS # Target Score Lipid control N/A 4.7 29848 Control 4.7 175510 HIF1α 2.0 222035 HIF2α 1.0 298697 HIF1α/2α 3.0 311082 HIF1-beta 2.0

[0270]As calculated from the assigned discrete scores, the results demonstrate that HUVEC tube formation is inhibited by treatment with antisense oligonucleotides targeting the Hif family. Thus, a reduction in HIF1-beta expression results in inhibition of angiogenic processes.

Example 28

HIF1-Beta Antisense Compounds in an Animal Model of Ocular Neovascularization

[0271]A pig model of ocular neovascularization, the branch retinal vein occlusion (BVO) model, is used to study ocular neovascularization. Male farm pigs (8-10 kg) are subjected to branch retinal vein occlusions (BVO) by laser treatment in both eyes. The extent of BVO is determined by indirect opthalmoscopy after a 2 week period. Intravitreous injections (10 μM) of HIF1-beta antisense oligonucleotides and control oligonucleotides are started on the day of BVO induction and are repeated at weeks 2, 6 and 10 after BVO (Right eye=vehicle, Left eye=antisense oligonucleotide). Stereo fundus photography and fluorescein angiography are performed at baseline BVO and at weeks 1, 6 and 12 following intravitreous injections to measure the neovascular response. In addition, capillary gel electrophoresis analysis of the eye sections containing sclera, choroid, and the retina are performed to determine antisense concentrations, and gross and microscopic evaluations are performed to determine eye histopathology.

Example 29

Matrix Metalloproteinase Activity Assay

[0272]During angiogenesis, endothelial cells need to be able to degrade the extracellular matrix (ECM). Endothelial cells secrete matrix metalloproteinases (MMPs) in order to accomplish this degradation. HIF1-beta antisense compounds of the invention are evaluated for their effects on MMP activity in HUVECs. MMP activity is measured using the EnzChek Gelatinase/Collagenase Assay Kit (Molecular Probes, Eugene, Oreg.). In this assay, HUVECs are plated at approximately 4000 cells per well in 96-well plates and transfected one day later. A 20-nucleotide oligomeric compound with a randomized sequence is used a negative control. An oligomeric compound targeted to integrin β3 is known to inhibit MMP activity and is used as a positive control.

[0273]Cells are transfected as described herein. Antisense compounds are mixed with LIPOFECTIN® in Opti-MEM to achieve a final concentration of 75 nM of antisense compound and 2.25 μg/mL LIPOFECTINT®. Antisense compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Untreated control cells received LIPOFECTINT® in Opti-MEM only.

[0274]Approximately 50 hours after transfection, a p-aminophenylmercuric acetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to each well of a Corning-Costar 96-well clear bottom plate (VWR International, Brisbane, Calif.). The APMA solution is used to promote cleavage of inactive MMP precursor proteins. Medium above the HUVECs is then transferred to the wells in the 96-well plate. After approximately 30 minutes, the quenched, fluorogenic MMP cleavage substrate is added, and baseline fluorescence is read immediately at 485 nm excitation/530 nm emission. Following an overnight incubation at 37° C. in the dark, plates are read again to determine the amount of fluorescence, which corresponds to MMP activity. Total protein from HUVEC lysates is used to normalize the readings, and MMP activity from cells treated with antisense compounds is normalized to that of untreated control cells. MMP activities above or below 100% are considered to indicate a stimulation or inhibition of MMP activity, respectively. HIF1-beta antisense compounds resulting in a decrease in MMP activity are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis.

[0275]Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each of the patents, applications, printed publications, and other published documents mentioned or referred to in this specification are herein incorporated by reference in their entirety. Those skilled in the art will appreciate that numerous changes and modifications may be made to the embodiments of the invention and that such changes and modifications may be made without departing from the spirit of the invention. It is therefore intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 190 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <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 Compound <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 Compound <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 4504 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (175)...(2542) <400> SEQUENCE: 4 agcgggggat gctgggggct gctgaagccg ccatcttgga ttccgcggta gcggaggcgg 60 cggtcaggcg ccgcttctgg ggagtggcct ttcttttccc ctccctcccg gttcggtggc 120 ggcggctcct cccactgggg ggggggtggc gcggcggcgg tggcatctgc ggcc atg 177 Met 1 gcg gcg act act gcc aac ccc gaa atg aca tca gat gta cca tca ctg 225 Ala Ala Thr Thr Ala Asn Pro Glu Met Thr Ser Asp Val Pro Ser Leu 5 10 15 ggt cca gcc att gcc tct gga aac tct gga cct gga att caa ggt gga 273 Gly Pro Ala Ile Ala Ser Gly Asn Ser Gly Pro Gly Ile Gln Gly Gly 20 25 30 gga gcc att gtc cag agg gct att aag cgg cga cca ggg ctg gat ttt 321 Gly Ala Ile Val Gln Arg Ala Ile Lys Arg Arg Pro Gly Leu Asp Phe 35 40 45 gat gat gat gga gaa ggg aac agt aaa ttt ttg agg tgt gat gat gat 369 Asp Asp Asp Gly Glu Gly Asn Ser Lys Phe Leu Arg Cys Asp Asp Asp 50 55 60 65 cag atg tct aac gat aag gag cgg ttt gcc agg tcg gat gat gag cac 417 Gln Met Ser Asn Asp Lys Glu Arg Phe Ala Arg Ser Asp Asp Glu His 70 75 80 ctc tgc gga taa aga gag act tgc cag gga aaa tca cag tga aat tga 465 Leu Cys Gly * Arg Glu Thr Cys Gln Gly Lys Ser Gln * Asn * 85 90 acg gcg gcg acg gaa caa gat gac agc cta cat cac aga act gtc aga 513 Thr Ala Ala Thr Glu Gln Asp Asp Ser Leu His His Arg Thr Val Arg 95 100 105 110 tat ggt acc cac ctg tag tgc cct ggc tcg aaa acc aga caa gct aac 561 Tyr Gly Thr His Leu * Cys Pro Gly Ser Lys Thr Arg Gln Ala Asn 115 120 125 cat ctt acg cat ggc agt ttc tca cat gaa gtc ctt gcg ggg aac tgg 609 His Leu Thr His Gly Ser Phe Ser His Glu Val Leu Ala Gly Asn Trp 130 135 140 caa cac atc cac tga tgg ctc cta taa gcc gtc ttt cct cac tga tca 657 Gln His Ile His * Trp Leu Leu * Ala Val Phe Pro His * Ser 145 150 gga act gaa aca ttt gat ctt gga ggc agc aga tgg ctt tct gtt tat 705 Gly Thr Glu Thr Phe Asp Leu Gly Gly Ser Arg Trp Leu Ser Val Tyr 155 160 165 170 tgt ctc atg tga gac agg cag ggt ggt gta tgt gtc tga ctc cgt gac 753 Cys Leu Met * Asp Arg Gln Gly Gly Val Cys Val * Leu Arg Asp 175 180 tcc tgt ttt gaa cca gcc aca gtc tga atg gtt tgg cag cac act cta 801 Ser Cys Phe Glu Pro Ala Thr Val * Met Val Trp Gln His Thr Leu 185 190 195 tga tca ggt gca ccc aga tga tgt gga taa act tcg tga gca gct ttc 849 * Ser Gly Ala Pro Arg * Cys Gly * Thr Ser * Ala Ala Phe 200 205 210 cac ttc aga aaa tgc cct gac agg gcg tat cct gga tct aaa gac tgg 897 His Phe Arg Lys Cys Pro Asp Arg Ala Tyr Pro Gly Ser Lys Asp Trp 215 220 225 aac agt gaa aaa gga agg tca gca gtc ttc cat gag aat gtg tat ggg 945 Asn Ser Glu Lys Gly Arg Ser Ala Val Phe His Glu Asn Val Tyr Gly 230 235 240 ctc aag gag atc gtt tat ttg ccg aat gag gtg tgg cag tag ctc tgt 993 Leu Lys Glu Ile Val Tyr Leu Pro Asn Glu Val Trp Gln * Leu Cys 245 250 255 gga ccc agt ttc tgt gaa tag gct gag ctt tgt gag gaa cag atg cag 1041 Gly Pro Ser Phe Cys Glu * Ala Glu Leu Cys Glu Glu Gln Met Gln 260 265 270 gaa tgg act tgg ctc tgt aaa gga tgg gga acc tca ctt cgt ggt ggt 1089 Glu Trp Thr Trp Leu Cys Lys Gly Trp Gly Thr Ser Leu Arg Gly Gly 275 280 285 cca ctg cac agg cta cat caa ggc ctg gcc ccc agc agg tgt ttc cct 1137 Pro Leu His Arg Leu His Gln Gly Leu Ala Pro Ser Arg Cys Phe Pro 290 295 300 305 ccc aga tga tga ccc aga ggc tgg cca ggg aag caa gtt ttg cct agt 1185 Pro Arg * * Pro Arg Gly Trp Pro Gly Lys Gln Val Leu Pro Ser 310 315 ggc cat tgg cag att gca ggt aac tag ttc tcc caa ctg tac aga cat 1233 Gly His Trp Gln Ile Ala Gly Asn * Phe Ser Gln Leu Tyr Arg His 320 325 330 gag taa tgt ttg tca acc aac aga gtt cat ctc ccg aca caa cat tga 1281 Glu * Cys Leu Ser Thr Asn Arg Val His Leu Pro Thr Gln His * 335 340 345 ggg tat ctt cac ttt tgt gga tca ccg ctg tgt ggc tac tgt tgg cta 1329 Gly Tyr Leu His Phe Cys Gly Ser Pro Leu Cys Gly Tyr Cys Trp Leu 350 355 360 cca gcc aca gga act ctt agg aaa gaa tat tgt aga att ctg tca tcc 1377 Pro Ala Thr Gly Thr Leu Arg Lys Glu Tyr Cys Arg Ile Leu Ser Ser 365 370 375 380 tga aga cca gca gct tct aag aga cag ctt cca aca ggt agt gaa att 1425 * Arg Pro Ala Ala Ser Lys Arg Gln Leu Pro Thr Gly Ser Glu Ile 385 390 395 aaa agg cca agt gct gtc tgt cat gtt ccg gtt ccg gtc taa gaa cca 1473 Lys Arg Pro Ser Ala Val Cys His Val Pro Val Pro Val * Glu Pro 400 405 410 aga atg gct ctg gat gag aac cag ctc ctt tac ttt cca gaa ccc tta 1521 Arg Met Ala Leu Asp Glu Asn Gln Leu Leu Tyr Phe Pro Glu Pro Leu 415 420 425 ctc aga tga aat tga gta cat cat ctg tac caa cac caa tgt gaa gaa 1569 Leu Arg * Asn * Val His His Leu Tyr Gln His Gln Cys Glu Glu 430 435 440 ctc tag cca aga acc acg gcc tac act ctc caa cac aat cca gag gcc 1617 Leu * Pro Arg Thr Thr Ala Tyr Thr Leu Gln His Asn Pro Glu Ala 445 450 455 aca act agg tcc cac agc taa ttt acc cct gga gat ggg ctc agg aca 1665 Thr Thr Arg Ser His Ser * Phe Thr Pro Gly Asp Gly Leu Arg Thr 460 465 470 gct ggc acc cag gca gca gca aca gca aac aga att gga cat ggt acc 1713 Ala Gly Thr Gln Ala Ala Ala Thr Ala Asn Arg Ile Gly His Gly Thr 475 480 485 agg aag aga tgg act ggc cag cta caa tca ttc cca ggt ggt tca gcc 1761 Arg Lys Arg Trp Thr Gly Gln Leu Gln Ser Phe Pro Gly Gly Ser Ala 490 495 500 tgt gac aac cac agg acc aga aca cag caa gcc cct tga gaa gtc aga 1809 Cys Asp Asn His Arg Thr Arg Thr Gln Gln Ala Pro * Glu Val Arg 505 510 515 tgg ttt att tgc cca gga tag aga tcc aag att ttc aga aat cta tca 1857 Trp Phe Ile Cys Pro Gly * Arg Ser Lys Ile Phe Arg Asn Leu Ser 520 525 530 caa cat caa tgc gga tca gag taa agg cat ctc ctc cag cac tgt ccc 1905 Gln His Gln Cys Gly Ser Glu * Arg His Leu Leu Gln His Cys Pro 535 540 545 tgc cac cca aca gct att ctc cca ggg caa cac att ccc tcc tac ccc 1953 Cys His Pro Thr Ala Ile Leu Pro Gly Gln His Ile Pro Ser Tyr Pro 550 555 560 ccg gcc ggc aga gaa ttt cag gaa tag tgg tct agc ccc tcc tgt aac 2001 Pro Ala Gly Arg Glu Phe Gln Glu * Trp Ser Ser Pro Ser Cys Asn 565 570 575 cat tgt cca gcc atc agc ttc tgc agg aca gat gtt ggc cca gat ttc 2049 His Cys Pro Ala Ile Ser Phe Cys Arg Thr Asp Val Gly Pro Asp Phe 580 585 590 ccg cca ctc caa ccc cac cca agg agc aac ccc aac ttg gac ccc tac 2097 Pro Pro Leu Gln Pro His Pro Arg Ser Asn Pro Asn Leu Asp Pro Tyr 595 600 605 610 tac ccg ctc agg ctt ttc tgc cca gca ggt ggc tac cca ggc tac tgc 2145 Tyr Pro Leu Arg Leu Phe Cys Pro Ala Gly Gly Tyr Pro Gly Tyr Cys 615 620 625 taa gac tcg tac ttc cca gtt tgg tgt ggg cag ctt tca gac tcc atc 2193 * Asp Ser Tyr Phe Pro Val Trp Cys Gly Gln Leu Ser Asp Ser Ile 630 635 640 ctc ctt cag ctc cat gtc cct ccc tgg tgc ccc aac tgc atc gcc tgg 2241 Leu Leu Gln Leu His Val Pro Pro Trp Cys Pro Asn Cys Ile Ala Trp 645 650 655 tgc tgc tgc cta ccc tag tct cac caa tcg tgg atc taa ctt tgc tcc 2289 Cys Cys Cys Leu Pro * Ser His Gln Ser Trp Ile * Leu Cys Ser 660 665 670 tga gac tgg aca gac tgc agg aca att cca gac acg gac agc aga ggg 2337 * Asp Trp Thr Asp Cys Arg Thr Ile Pro Asp Thr Asp Ser Arg Gly 675 680 685 tgt ggg tgt ctg gcc aca gtg gca ggg cca gca gcc tca tca tcg ttc 2385 Cys Gly Cys Leu Ala Thr Val Ala Gly Pro Ala Ala Ser Ser Ser Phe 690 695 700 aag ttc tag tga gca aca tgt tca aca acc gcc agc aca gca acc tgg 2433 Lys Phe * * Ala Thr Cys Ser Thr Thr Ala Ser Thr Ala Thr Trp 705 710 715 cca gcc tga ggt ctt cca gga gat gct gtc cat gct ggg aga tca gag 2481 Pro Ala * Gly Leu Pro Gly Asp Ala Val His Ala Gly Arg Ser Glu 720 725 730 caa cag cta caa caa tga aga att ccc tga tct aac tat gtt tcc ccc 2529 Gln Gln Leu Gln Gln * Arg Ile Pro * Ser Asn Tyr Val Ser Pro 735 740 745 ctt ttc aga ata g aactattggg gtgaggataa ggggtggggg agaaaaaatc 2582 Leu Phe Arg Ile actgtttgtt tttaaaaagc aaatctttct gtaaacagaa taaaagttcc tctcccttcc 2642 cttccctcac ccctgacatg tacccccttt cccttctggc tgttcccctg ctctgttgcc 2702 tctctaaggt aacatttata gaagaaatgg aatgaatctc caaggctttt aggactgtct 2762 gaaaatttga ggctgggtga agttaaaaca cctttcctta tgtctcctga cctgaaattg 2822 tatagtgttg atttgtgctg agatcaagag gcaggttaga agaacctgac atccactgtt 2882 tgccttggat agtatggctt gtttttggaa aggaattctg aagagagtgg aggagaggag 2942 aaatgtcctc atatttgagg accatgaaac attgtaggta tatatggggc tttagcaagt 3002 ttgagcctag gctctttttg ctgcctgtga gcagtccctc tggaaagaaa catgtgagta 3062 agtgagagag agtgtgtgtg tatgtgtgtg tgtgtgtgtg tgtgtgtgcg cacacatgct 3122 tctgtatttc actctttctc cctattaggg agttatgcaa aatttgtccc cgattttacc 3182 tttgtctttc tgtgtacttt tcaaagagtc ctaaggagtt aaatcttcca ggtattttcc 3242 acttagtatt gcagccaaag aatatttaaa taaacgtctt tgctgcgctt gcatccatgc 3302 ccagccaata tacaactgta aagcaaatat agaaagtcgg ctgttgatac gattgtctgt 3362 tatcgaacac attcagtgat aaagctgggt tactgctgct tttggtgctc tcaccttatc 3422 tggaagatct gcaaacatta cctaaatagg ctggcaagat aaacactttc tggaacccga 3482 gacttggcca taaagataat gctgcatttt tctgtcagaa tcacatatga tgtgtgttct 3542 gtagaggtta tttctgcatg gaaactcaac ttcttggatt agccgtccca gtgaaaatcc 3602 tcattgttgg agtgtaaacc aaatacgaag ccctcttgca aagtagcctc tttcatccca 3662 tactcaaaat acccagttta gcaagcaact gagatttaag tctctctggc cctaagaggt 3722 ttttcctctt tgctccctcc aatcttgaga ttgggttttg ctttagagtg caagtatcat 3782 aattccgtat gatagatggg gcctggacac ccatctcaac agggtcactt ggtaattaac 3842 aatagccata taaatgcgga tacaggttac taccctcacc ctttaccttc ctcaggtaac 3902 agtcgtagat accagctttt tttttttttt ttttaaattg gctttggcca gtagctaaag 3962 tgcaagactg agttaatgag aagatatatt aaatgtagtc ataggggact gaggagcaag 4022 ggtggccttg aagaggccaa aggaatgtcc atttgctgag tttcccttcc ttatgtctcc 4082 agtctggtgc caggtagtgg agtaaaaaag gagacagttt atttttttat tctatgtgca 4142 cacttacagt atacatatat atttatatca caatttacga aaccaaaaag ttgagtttcc 4202 aatggaaccc ttgtttttta ataatcgact ttttaaatgt gatcaagact ataatattgt 4262 acagttatta tagggctttt ggggaagggg aggatagcga gaagatgctc tgggggtttt 4322 gtttttgctt ttccttcagg gttttatttt tgactgtttt gttttcttgt tggccatttc 4382 tgtattgctg gcatctgtgc taagctttac agtggcaaaa ataatgacat gtagcaaaga 4442 ttttcaaaca aaatattttt tccttttgta aaaaaaaaaa aaaaagaaaa aaaaaaaaaa 4502 aa 4504 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 agcagagggt gtgggtgtct 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 tggcggttgt tgaacatgtt 20 <210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 ccagcagcct catcatcgtt ca 22 <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: 2691 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (110)...(2485) <400> SEQUENCE: 11 gcggcggtaa ggtgcctaat ctgcggagtg gctcttccct cccctccccc agctcggtgg 60 cggctgcccc tcccaccgag ggtggcgcag ggacggtgcc atctcgacc atg gcg gcg 118 Met Ala Ala 1 act aca gct aac cca gaa atg aca tca gat gta cca tcg ctg ggt ccc 166 Thr Thr Ala Asn Pro Glu Met Thr Ser Asp Val Pro Ser Leu Gly Pro 5 10 15 acc att gct tct gga aac cct gga cct ggg att caa ggt gga gga gct 214 Thr Ile Ala Ser Gly Asn Pro Gly Pro Gly Ile Gln Gly Gly Gly Ala 20 25 30 35 gtt gta cag agg gct att aag cga cgg tca ggg ctg gat ttt gat gat 262 Val Val Gln Arg Ala Ile Lys Arg Arg Ser Gly Leu Asp Phe Asp Asp 40 45 50 gaa gta gaa gtg aac act aaa ttt ttg aga tgc gat gat gac cag atg 310 Glu Val Glu Val Asn Thr Lys Phe Leu Arg Cys Asp Asp Asp Gln Met 55 60 65 tgt aat gac aaa gag cgg ttt gcc agg tcg gat gat gag cag agc tct 358 Cys Asn Asp Lys Glu Arg Phe Ala Arg Ser Asp Asp Glu Gln Ser Ser 70 75 80 gcg gat aaa gag aga ctt gcc agg gaa aat cat agt gaa ata gaa cgg 406 Ala Asp Lys Glu Arg Leu Ala Arg Glu Asn His Ser Glu Ile Glu Arg 85 90 95 cgg cga cgg aac aag atg aca gct tac atc aca gaa ctg tca gac atg 454 Arg Arg Arg Asn Lys Met Thr Ala Tyr Ile Thr Glu Leu Ser Asp Met 100 105 110 115 gta cct aca tgt agt gcc ctg gct cga aaa cca gac aag cta acc atc 502 Val Pro Thr Cys Ser Ala Leu Ala Arg Lys Pro Asp Lys Leu Thr Ile 120 125 130 tta cgc atg gcc gtt tct cac atg aag tcc ttg agg gga act ggc aac 550 Leu Arg Met Ala Val Ser His Met Lys Ser Leu Arg Gly Thr Gly Asn 135 140 145 aca tct act gat ggc tcc tac aag cca tct ttc ctc act gat cag gaa 598 Thr Ser Thr Asp Gly Ser Tyr Lys Pro Ser Phe Leu Thr Asp Gln Glu 150 155 160 ctg aaa cat ttg atc ttg gag gca gca gat ggc ttt ctg ttt att gtc 646 Leu Lys His Leu Ile Leu Glu Ala Ala Asp Gly Phe Leu Phe Ile Val 165 170 175 tcc tgt gag act gga cgg gtg gtg tat gtc tct gac tca gtg act ccc 694 Ser Cys Glu Thr Gly Arg Val Val Tyr Val Ser Asp Ser Val Thr Pro 180 185 190 195 gtt ttg aac cag cca cag tct gaa tgg ttc ggg agc aca ctg tat gat 742 Val Leu Asn Gln Pro Gln Ser Glu Trp Phe Gly Ser Thr Leu Tyr Asp 200 205 210 cag gtg cac cca gat gat gtg gat aaa ctt cga gag cag ctc tct aca 790 Gln Val His Pro Asp Asp Val Asp Lys Leu Arg Glu Gln Leu Ser Thr 215 220 225 tca gaa aat gcc cta aca ggg cgg gtc ctg gat ctg aag act gga aca 838 Ser Glu Asn Ala Leu Thr Gly Arg Val Leu Asp Leu Lys Thr Gly Thr 230 235 240 gtg aaa aag gaa ggc cag cag tct tcc atg agg atg tgc atg ggc tca 886 Val Lys Lys Glu Gly Gln Gln Ser Ser Met Arg Met Cys Met Gly Ser 245 250 255 cga agg tcg ttc atc tgc cgc atg agg tgt ggt act agc tcc gtg gac 934 Arg Arg Ser Phe Ile Cys Arg Met Arg Cys Gly Thr Ser Ser Val Asp 260 265 270 275 cct gtt tcc atg aat aga ctg agc ttt ttg agg aac aga tgc agg aat 982 Pro Val Ser Met Asn Arg Leu Ser Phe Leu Arg Asn Arg Cys Arg Asn 280 285 290 ggg ctt ggc tct gtg aag gaa gga gaa cct cac ttt gtg gta gtc cac 1030 Gly Leu Gly Ser Val Lys Glu Gly Glu Pro His Phe Val Val Val His 295 300 305 tgc aca ggc tac atc aag gcc tgg cca cca gca ggt gtc tcc ctc cca 1078 Cys Thr Gly Tyr Ile Lys Ala Trp Pro Pro Ala Gly Val Ser Leu Pro 310 315 320 gat gat gac cca gag gct ggc cag ggg agc aaa ttc tgc cta gtg gcc 1126 Asp Asp Asp Pro Glu Ala Gly Gln Gly Ser Lys Phe Cys Leu Val Ala 325 330 335 att ggc agg ctg cag gta act agt tct ccc aac tgt aca gac atg agt 1174 Ile Gly Arg Leu Gln Val Thr Ser Ser Pro Asn Cys Thr Asp Met Ser 340 345 350 355 aac att tgt cag cca aca gag ttc atc tcc cga cac aac att gaa ggg 1222 Asn Ile Cys Gln Pro Thr Glu Phe Ile Ser Arg His Asn Ile Glu Gly 360 365 370 ata ttc act ttt gta gac cat cgt tgt gtg gct act gtt ggc tac cag 1270 Ile Phe Thr Phe Val Asp His Arg Cys Val Ala Thr Val Gly Tyr Gln 375 380 385 cca cag gag ctc tta ggg aag aat att gta gaa ttt tgt cat cct gaa 1318 Pro Gln Glu Leu Leu Gly Lys Asn Ile Val Glu Phe Cys His Pro Glu 390 395 400 gac caa caa ctt cta aga gac agc ttt cag cag gtg gtg aaa tta aaa 1366 Asp Gln Gln Leu Leu Arg Asp Ser Phe Gln Gln Val Val Lys Leu Lys 405 410 415 ggt cag gtg ctg tcc gtc atg ttc cga ttc cga tct aag acc cga gaa 1414 Gly Gln Val Leu Ser Val Met Phe Arg Phe Arg Ser Lys Thr Arg Glu 420 425 430 435 tgg ctg tgg atg aga acg agc tcc ttt acc ttc caa aac cct tat tca 1462 Trp Leu Trp Met Arg Thr Ser Ser Phe Thr Phe Gln Asn Pro Tyr Ser 440 445 450 gat gaa att gag tat att atc tgc acc aac acc aat gtg aag aac tct 1510 Asp Glu Ile Glu Tyr Ile Ile Cys Thr Asn Thr Asn Val Lys Asn Ser 455 460 465 agc cag gaa cca cgg cct aca ctg tcc aac acc atc cca agg tca cag 1558 Ser Gln Glu Pro Arg Pro Thr Leu Ser Asn Thr Ile Pro Arg Ser Gln 470 475 480 cta ggt ccg aca gcc aat tta tcc cta gag atg ggt aca ggg cag ctg 1606 Leu Gly Pro Thr Ala Asn Leu Ser Leu Glu Met Gly Thr Gly Gln Leu 485 490 495 cca tcc agg cag cag cag cag cag cac aca gaa ctg gat atg gta cca 1654 Pro Ser Arg Gln Gln Gln Gln Gln His Thr Glu Leu Asp Met Val Pro 500 505 510 515 gga aga gat ggg ctg gcc agc tat aat cat tcc cag gtt tct gtc cag 1702 Gly Arg Asp Gly Leu Ala Ser Tyr Asn His Ser Gln Val Ser Val Gln 520 525 530 cct gtg gca agt gca gga tca gaa cac agc aag ccc ctt gag aag tca 1750 Pro Val Ala Ser Ala Gly Ser Glu His Ser Lys Pro Leu Glu Lys Ser 535 540 545 gaa ggt ctc ttt gca cag gac aga gat cca agg ttt cca gaa atc tat 1798 Glu Gly Leu Phe Ala Gln Asp Arg Asp Pro Arg Phe Pro Glu Ile Tyr 550 555 560 ccc agc atc act gca gat cag agt aaa ggc atc tcc tcc agc act gtc 1846 Pro Ser Ile Thr Ala Asp Gln Ser Lys Gly Ile Ser Ser Ser Thr Val 565 570 575 cct gcc acc caa cag ctg ttc tcc cag ggc agc tca ttc cct cct aac 1894 Pro Ala Thr Gln Gln Leu Phe Ser Gln Gly Ser Ser Phe Pro Pro Asn 580 585 590 595 ccc cgg ccg gca gag aat ttc agg aat agt ggt ctt acc cct cct gta 1942 Pro Arg Pro Ala Glu Asn Phe Arg Asn Ser Gly Leu Thr Pro Pro Val 600 605 610 acc att gtc cag cca tca tct tct gca ggg cag ata ctg gcc cag att 1990 Thr Ile Val Gln Pro Ser Ser Ser Ala Gly Gln Ile Leu Ala Gln Ile 615 620 625 tca cgt cac tcc aac cct gcc cag gga tca gcg ccg acc tgg acc tct 2038 Ser Arg His Ser Asn Pro Ala Gln Gly Ser Ala Pro Thr Trp Thr Ser 630 635 640 agc tcc cgc cca ggc ttt gcc gcc cag cag gtg ccc acc cag gct aca 2086 Ser Ser Arg Pro Gly Phe Ala Ala Gln Gln Val Pro Thr Gln Ala Thr 645 650 655 gcc aag act cgt tct tcc caa ttt ggt gtg aac aac ttt cag act tct 2134 Ala Lys Thr Arg Ser Ser Gln Phe Gly Val Asn Asn Phe Gln Thr Ser 660 665 670 675 tcc tcc ttc agt gct atg tct ctt ccg ggt gct ccc act gcc tca tct 2182 Ser Ser Phe Ser Ala Met Ser Leu Pro Gly Ala Pro Thr Ala Ser Ser 680 685 690 ggt act gct gcc tac cct gct ctc ccc aac cgt ggc tcc aac ttt cct 2230 Gly Thr Ala Ala Tyr Pro Ala Leu Pro Asn Arg Gly Ser Asn Phe Pro 695 700 705 cct gag act gga cag acc aca gga cag ttc cag gcc cgg aca gca gag 2278 Pro Glu Thr Gly Gln Thr Thr Gly Gln Phe Gln Ala Arg Thr Ala Glu 710 715 720 ggc gtg ggt gtc tgg cca cag tgg cag ggc cag cag ccc cat cat cgg 2326 Gly Val Gly Val Trp Pro Gln Trp Gln Gly Gln Gln Pro His His Arg 725 730 735 tct agt tcc agt gag cag cat gtt cag cag aca caa gca caa gca cct 2374 Ser Ser Ser Ser Glu Gln His Val Gln Gln Thr Gln Ala Gln Ala Pro 740 745 750 755 agc cag cct gag gtc ttt caa gaa atg ctg tcc atg ctg gga gac caa 2422 Ser Gln Pro Glu Val Phe Gln Glu Met Leu Ser Met Leu Gly Asp Gln 760 765 770 agc aac acc tac aac aat gaa gaa ttt cct gat cta act atg ttt ccc 2470 Ser Asn Thr Tyr Asn Asn Glu Glu Phe Pro Asp Leu Thr Met Phe Pro 775 780 785 ccc ttt tcc gaa tag aactattggg gtgaggataa gggtgggggg aaatcactgt 2525 Pro Phe Ser Glu 790 ttgtttttaa aagcaaatct tttgtaaaca gaataaaagt tcctctccct tcccttccct 2585 cacccctgat atgtaccctt tccacccctt gacttgctga agaaacgtta tagaagaaat 2645 taaatgaatt tcccaggcaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2691 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 12 ggcatctcct ccagcactgt 20 <210> SEQ ID NO 13 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 13 ggtaagacca ctattcctga aattctct 28 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 14 tccctcctaa cccccggccg 20 <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: 67787 <212> TYPE: DNA <213> ORGANISM: H. sapiens <400> SEQUENCE: 18 ttagtagaga cggggtttcg ccatgttggc caggctggtt tcgaacccct ggccacaggt 60 gatccggccg cctcggcctc ccaaagtgct gggattacag acgcgagcca ccgcgcccag 120 cctaaaatac ccattttaat tgcatttcaa tgacgttact cggtcattga cctcttacgc 180 aaggagggcg agattaggga gacagctgga cttcttcctc gccctccctt cactggactg 240 gctggcgcag tgagtagccc tgcccttgga cgatttggca ttttcattgg tcaatttttc 300 ttaggaggct ggccgtgtgt tgactccgcc tactatatag gcggggtctc cccgccgcag 360 gggctgggat gctgggggct gctgaagccg ccatcttgga ttccgcggta gcggaggcgg 420 cggtcaggcg ccgcttctgg ggagtggcct ttcttttccc ctccctcccg gttcggtggc 480 ggcggctcct cccactgggg gggggggtgg cgcggcggcg gtggcatctg cggccatggc 540 ggcgactact gccaaccccg gtgaggagac ggaggactgg ggcgcctcta aaggggaggg 600 gccgaagggg ctgacaacta aggagaccca gggctgaggg gacgcagctg gagctgaagg 660 cgccggggcg gggcgggggc cgcgagagac ggtgggggag cggtgacctg gggaggggcc 720 cgatcccggg gacttgggta gactcgagta tacccaaggg agcgaggggg caggtgggag 780 ttgagggggg ctccctgtcc tgacctccgt tggagcggca gccagtgcgg ggctggcccg 840 ccctcactcc tcggtcggcg tctggtctgt gatgtggtcc tcttttaacc ctctgcccgc 900 tggagccgct tgatgccgaa aactgtaggc agagtgtctc ggtttacttt aagagtagtt 960 tggattcata aaccctggac cacactgctc tttagttgag gcgttgcttc caaacattta 1020 aaggtttaga ttttaattta aaaaataaaa aatggacatt ctttgatata tacgacgttt 1080 ctttaggctg ttacctccca cccgcaaccc ccgtccccgt ctccgtcccc ggactatact 1140 ttgaaactta gatcttgtgt taagagggcc tgtaggtctt tgcctgctga gggcaggggt 1200 ttgtgtaatg gttatggttc cggacaggtc tttagttcca cagccccagg tttaaatctt 1260 gttaggccta cgtcgaggca atttccaaac tctttcagaa aagtttgatt ccacaccatt 1320 tctcaatttc aaaaaataat caaatatcaa aaaggtttgg tctgttttgt ctaagagatg 1380 gagtagggtg tagtgcagtt ctagagagat cagatcttca aattactagt cccagccctc 1440 tttcctctat gttacagtta ttcctcttta gaaggcattt cattttaggg gtctagttta 1500 tcttggagac cttggttatt cttgccaaat aaagtctatc cttttccaaa aaccaatata 1560 tttgatagcc ttaaaaaggg ccttgagata tgtgtgtgtg ttgcgtgggg cggagaattt 1620 cttgaaaata aaacagtagg agtgattacc cccatcccca cgttgaaatt taaattagat 1680 tttttttcat gttactgaaa ggcagtatga ttttgcaata tccagatttt gataggacag 1740 ttgaatcact gaaatttctt ttctaccgtg tttctgggaa tagctatttt aagaaatggt 1800 taatttgtca cgtcattttt ttttaatctt ttaaattttt tttgaaggaa agggaggtaa 1860 aattaatcgc aaaaattaca ttgcaggaag tgctgagtga tagaataaaa ctataaaaca 1920 ggaaaaatgc cgttatgtca aacctgacct tgtagtaata taacctatct gtgaactatg 1980 gaataaataa acaagatttt taactgatca cacattgcaa aatacttata taaccctagt 2040 taaattgcaa agcatttctc ttattaagaa gtggcctggg taaacaagtt ttagttcata 2100 tgtcagaagc ttttttccag ctgtgtgggt ggggttgctt tattttgttt tgttttttga 2160 acaacagcaa ccagaatgac agcctcttga ccatattttc ataagctgat caatatcagc 2220 ttaaatctta gcagaaaata gcatatgtta gtgtgtatgt gtgtgtgcag aatccttgtt 2280 aatgaaacag ctttgtttac tatgctttgg ctgcttgctt ctgcaatttt tagatttcct 2340 ttttcttttt ctttgttttc ctttctcctc tcccctctct cctgtcccct ctcccctctt 2400 tttcttttgc tacagggtct tgctccttgc tctgtcgccc agtctggagt gcagtggcaa 2460 gatcactgct cacggcagcc tccacctcag gggctcaagt gatcctctca cgatcagcct 2520 cccgaatagc tgggactaca ggtgccttgc caccatgcgc ggttaatttt taaatttttt 2580 tgtagagaca gatgccttgc tatgttgccc atgctgatct tgatctcctg gtctcaagca 2640 agccttgcct cagtctctcc aagttgggaa tacagatgtg agacactttg accagatttc 2700 ttttttgatg ttgaaaaatt attccatctc ttctgactta ttgatcccat gttcagttca 2760 atcaaatcag aagatatatt ttcttttttc ttttctccct ttgctcttgt atatcatgag 2820 gaagagatca gagatacatt gtggattttt tcagtagcct gttgcttttt gggaaatgag 2880 attaatgaat ggagggacat aggtggaaat gtactgtgct tagtgaaagt aaatgttttc 2940 aggtcagaat tctttgctac actgtcatac cttctttcct tagagccatt ttaaggacta 3000 gggacaccat gcctatttct ttgagttttt tctactttgt atttttgctg gagtactaat 3060 tttttttttt tttttttttt ttttacaatt cttgttagtt ttgtcaagat tttaaaaaat 3120 atttcactct ttaaccttca cttcagttaa tcctttttaa aaataatact tatttttatt 3180 tttcagacag gttcttgctc tgtctcccag actgtagtgc aatggtgtaa tcacagctca 3240 ctgtagcctt gaactcctga gctcaagtga tcttcccacc tcagcctcct gagtatctgg 3300 gtctacagac acacaccacc acacctggat acatttttta actttttgta gaaatgggat 3360 cttgctatgt tgcccaggct gatcttgaac tcctgtcctc aagcaatcct cccaccttag 3420 cctcctaaac tgctgggatt ataggtgtga gccactgacc cggccaagtt aatccttagg 3480 gttagtaata gtgcttgaac atgttaattg tgctggtggc cctgcagtgt ttttcaaggg 3540 gaccacgttc ttctgcattt ttcactgtgg tgctgcatta gaagaagagt aaaatatggt 3600 tggtactctc taagagttgt gttgaagtaa tagttctgtg atgatttttt ttctcgaatt 3660 gataattctt catattctat catttttcac tagttctact tatcactggc aagtagaact 3720 cggtgggtag gtggtccatg aaactttagg ctgtttctta tctatggact agacaagttt 3780 tgttttgttt tttttttgtt tgtttgtttg ttttgagaca gagtctcact ctgtcgccca 3840 ggctggagtg cagtggtgct atcttggctc actgcaagct ccgcctccca ggttcacgcc 3900 attctcctgc ctcagcctcc cgagtagctg ggactacagg tacccgccac cacgcccggc 3960 taattttttt ttttgtattt ttagtagaga cagggtttca ccctgttagc cagggtggtc 4020 tcgatctcct gaccttgtga tccgcctgcc tcggcctccc aaagtgctgg gattacaggc 4080 ttgagtcacc gtgcccggcc cggactagac aagttttgag gaacacattt tttttcccca 4140 taggagaggg tcttgctctg tcactgaggc tggagtgcag tgacacaatc atggctcact 4200 gcagccttga ccttctggga gcatgtgatc ctcccacttc agcctcctga gtaggtggga 4260 ccacaggtgt gcaccaccgt ggctggctaa ttttaaattt tttttagaga cagggtcttg 4320 ctatgttgcc caggctggtc tttaactcct ggcctcaagt gatcttcctg cgttgggcac 4380 tccccaagag ctgggattat aggcatgacc accatgccca gccaaggtgg aacacttttt 4440 aaattgaatg tgtcatttgt aatgcattgg ctgagggaac agttgggagg agattaggta 4500 tttgatttac aattccaaat tcaaactaga gatgttgaat gagtttgtag taagtgtagg 4560 tttttatctc tcttgattct ggtattctca ttagcagaga attctttttg tgctattatg 4620 tacaagttac tgtggatgaa gaatgtagaa ggaaaagggt aataccgttt gggtttgtaa 4680 ggctgtcatt taggaagaaa atttttttta gctaattttc acctatataa cttagtttta 4740 actgcgtcaa ttaacacaat tcttacctgt tacctttatt tacctttttc agatatggga 4800 aaataggaat tcatgcaatg taaagttaag atgtcaagat attcttttat ggccaggtgt 4860 ggtggctcgt gcctctaatc cctttgggag gccgaggcag gccgattgct tgagcccagg 4920 aatttgagac cagcccggac aacgtgacga aaccccttta ctacaaaaaa tacaaaaaat 4980 tttccgggca ctgtggtgca cttctatagt cccagctact tgggaggctg agatgggagt 5040 atcacctgag cctggaaagt tgaagctgca gtgagctgtg atcatgccac tgtactccag 5100 cctgggtgtt ggagtgagac cctgtccccc aacaaaaaaa gagatagtat tttattatat 5160 tgcaaaggcc tttttttttt tttttttttt tttttgagac agggtcttac tgtgtcgccc 5220 aggctggagt gcaatggcgt gatcttggct cactgcaacc tccgcctcct gggttcaagc 5280 tattctcccg cctcagcctc ccgagtagct gggattacag gcacctgcca tcacgccggc 5340 taatttttta tttttaatag agacagggtt tctccatgtt ggccaggctg gtcttgaact 5400 cctcacctca ggtgatccgc ccaccttggc ctcccaaagt gctgggatta caggcgtgag 5460 ccgccatgct ccgccgccat ctgtttttaa gaatgctgcc ttggccgggc atggtggctc 5520 atgcctgtaa tcctagcatt ttgggaggcc gaagtgggca gatcacctga ggtcaggagt 5580 tcaagaccag cctggccaac atggagaaac cctgtctcta ctaaaaatac aaaaattagc 5640 caggcatggt ggcaggtgcc tgtaatccca gctactcggg aggctgaggc gggagaatcg 5700 cttgaacctg gaaggtggtg gttgcagtga gctgagatcg taccactgca ctctagcctg 5760 ggcgacagag tgagactccg tctaaaaaaa aaaaaaaaaa aaaaaaaacc acgactgcct 5820 aatatgccat aatgacttta gtaagtagac atcagtgaat cttcaggttg gaaatatgac 5880 aacacctatc attttacact taaggagaca aacccaggga aaggagtgga taattaagtt 5940 ccttctatat gccaggctgc actttcctat tttgttgcta atctttatgt ctagtctgta 6000 gcaactgtct ccaacttttc acataaactc ttcaaattta tgtcactttt cataatttcg 6060 tcaatgtatt ttcttgtttt aaagttttta ttattttaga tagtgaccgg gtctcactat 6120 attgccccgg ctggtctcaa actcctgggc tcaagcaatc ctcctgcctc ggcctcccag 6180 agtgccgggg ttacaggtgt gagccaccct ctgaggccca ttttatattt tccctccacg 6240 ttttcattgt agttgaatgg acaagtaagt ttatggattc aacagtatac ttagggctgg 6300 acatggtggc tcatacctgt aatcctagca ctttgggagg ctgatgtagg tggattgctt 6360 gagcccaaaa gtttgagacc agcctgggca acgtggtgaa accccatctg tacaaaaaaa 6420 tacaaaaaat tagcagggtg tggtagcatt ccctgtagtc ccagctacgt gggagactga 6480 gatgcgagga tgacctgagc ctgggaggtt gaggctgcag tgagccatga ttgtgccact 6540 gcattcctgt ctgggtgact aggagacaga gtgagaccct gtctcaaaaa acacaacaaa 6600 accagcatac ttaggatata agttccatga gagcagaact tttgctttcc ttgtttacca 6660 gagcattgca gtgcttacag tgggtgccta gtatgttgga ttagagttca atgtatgttt 6720 gtggaataaa aatgaatgta aaacccactg ttaatatgaa atacctttga agggataaaa 6780 ctaattaagc cattgacatc atgaatttgt actaactata ataatatcta atatttatta 6840 gtaatactta ccagctgttc tcatttaatt tttataacaa tcgtattagg taagtattat 6900 tattcttgtt ttacagatga ggaaactaag gcacagagag tgaataagta acttgtctga 6960 agtcacatag ctactagaag ttagagacag aattttattg ttggcagtcc atctacacag 7020 tcatcattct taatctccaa gttatgattt tttccattgt gacttagagt caccttccta 7080 ctgaaatata gtacaatgga tatcttttga gaaggaggta ggtggagtat tgtctttttt 7140 gtagatgaaa agggtgaaat accacattag gcagcctggg ggcaaggcat tgcttggttg 7200 gagaaccaag tgcaaggtgc taaaaaaagg agctagaaca gtagtttcca ggctgagtgt 7260 ggtggctcat acctgtaatc tcagcacttt gagaggccag ggtgagagga ttgtttaagc 7320 ccaggagttg gagaccagcc tgagcaatat agggagacct tgtctccaca aaaatatata 7380 taaaaaaatt agctagatgt ggtggtggat gcctatagtc ccaactactt gggaggctga 7440 ggtggtggga ggatctctga gcttgctagg tcagggctgc agtgagccat gatcatgcca 7500 ctggactcta atctggttga cagagtgagc ccctgtctct aggaaaaaaa aaaaaaaagg 7560 agtagtttcc taaaatcttt tctgtaacat tctataattc ttcattttgt tttgttttgt 7620 ttttttgaga ctgagtgtcc ttctgtcacc caggctgggg tgcagtggca cgatctcagc 7680 tcactgcaag ctccgcctcc caggttcacg ccattctcct gcctcatcct cgcgagtagc 7740 tgggactaca ggcatccacc accatgccct gctaattttt tgtgttttta gtggagatgg 7800 ggtttcactg tgttagccag gatggcctcg atctcctgac cttgtgatcc gcccacctca 7860 gcctcccaaa gtgctgggat tataggcttg agctagcgcg cccagcctaa cattctatat 7920 ttctttctcc tagttctatg tttcaattct ttaaatactt ttaagggttt ttttgagaca 7980 aggtcctgtt gtgtcagcca ggctggagtg cagtagcgtg accatagctt cctgcaacac 8040 ctccaattcc taggctcaag cgattctcac tcctcagttt cctgaatagt tgggaccata 8100 ggtatgcacc atcatgccca gctaattttt taattttgta gagacaggat ctcgctatgt 8160 tgcccaggct ggtccttttt tttttaaatt ttattattat tttttgagac ggagtcttgc 8220 tttgtcaccc aggctggagt gcagtggcgt gatctcggct cactgcaacc tctgcctccc 8280 gggtttaagc gattctcctg cctcagcctc ctgtagctgg gactacaggc acgtgccacc 8340 gcgcctggct aatttttgtg tttttagtag agacggggtt tcaccatgtt ggccaggatg 8400 gtctcgatct cctgacctcg tgatctgccc acctcggcct cccaaagtgc tgggattaca 8460 ggcgtgagcc accgcgcccg gcctgcccag gctggtcttg aactactgtc ctcgagtgat 8520 cctcctgctg ttggcctccc agattgctga gattacaggc atgaatcatg cactgcagct 8580 tttaggattg ttattagaat cctctacaag ttcttgatac tccataattg agcctctcct 8640 agaaacttgg tttcagagac actgttgcta ggtctttatc actgattgtt ccttctttgt 8700 cctttttgct tatctttctg cttgtttctt tctcttgttc agtatgcttc tacttttctc 8760 ttctcaccct tgaggctagg actcttcttt gagacagggt cttgactctt ttttgagaca 8820 gggtcttgct atgttgccca cgttggtctt ggattcctga gctcaaatga tcctcccacc 8880 tctgcttcct gagtagctgg gattacaagt gtacaccaca gtgcccggct tggggctaga 8940 actaaatagt aaaccaagca gtttccttca tttcttccca tttgcccacc tacctgcctt 9000 tctagctgct tgccttccta cttcaatttg acattcactt ggtacctacc atagtcctct 9060 tccctttgta ttagtccatt ctcacactgc tataaataac tacctgagac tgggtaattt 9120 atgaagaaaa gaggtttaat tgactcatag ttccgcaggc tgtatgggag gcagggctgg 9180 ggaggcctca ggaaacttgg aatcatggcg gaaggcgaag gggaagcaag catgtcttca 9240 catggctggc aggatagagg gagagaaggg gaaggtgcta cacactttca aacaaccaga 9300 tctcttgaga actctatcat gagagagcat taggggaatg gtgctaaacc tttggaaacc 9360 acccctatga tccaataacc tcccaccaga ctgcaccttc aacactgggg attacaattt 9420 gacatgatat ttgggtggag acacacagag ccaaaccata tcacccttta tttatctttt 9480 atgtataaaa gactccaaaa atttcattcc tggcttctca cataaactac tttcctatat 9540 ttttatctat ttaaaagata tctctctctt tttcttttaa agttgagaca gggttttact 9600 atgttgccca ggctggtctt gaaatcctgg gctcaagcat tcctcccact tcagcaacct 9660 gagtagctgg actacaggtg tgtgccattg tgcctggctg catctctgtt tgaatagttt 9720 tttttttttt taatttatag aaatgaggtc tcactgtatt gcccagactg gtcttgaact 9780 gctgagctca agtagtcccc ccatcttggt ctccctagta gctgggatta taggtgtatg 9840 acacgatgcc cagctctatt tgagtaatct gttttttttt gttgttgttg ttttttttaa 9900 agacagagtc tttctgtgtt gcccaggctg gagtgcagtg gtgcgatctt ggctcaccgc 9960 agcctccgcc tcctaggttc aaatgatttt tgtgcctcag cctctcgagt agctgggact 10020 acagtgtgcc accacgcctg gctaattttt tgtatcttta gtagagatgg ggtttcacca 10080 tgttggccag gctggtcttg aactcccgtc ctcaagtgat ccacctgcct tggcctccca 10140 cagtgctggg attacaagtg tgagccattg cgcccagcct ctatttgaat aatctttaac 10200 atcctatata tgtatgtact caccagtatt tcctttaaat ctgctgttct tcctaccttt 10260 ctgtatcttt catcactatc acaattcagt ttttcatttc cccaggctaa ggaactataa 10320 gtgtttggat tttatttttt atgtgtttat ctgggttatt aactcttctc aggtcttttg 10380 ccaaatatac gtgtgtgtgt gtgtatatgt atgtgtgtat acatacatat ataaaagtta 10440 tttaaatcaa aactatacat ccatatatta atatttaaag acttagatag ttttataaaa 10500 tgtttgttac aaaaaaatta taagggagtc ctctgctccc tttctctcat ttccttctct 10560 ccagtggcat catttttatt tcttttaaca gaattctttg tttctgttta aataaagtgt 10620 cccagtgaaa gacattagtt tccagatcaa aggggctgtg cacacatcaa ggcccattat 10680 gctgaaattt cagcatcaga tagagaatat cctataagct tcagagagaa taagattatt 10740 cacattcaaa agattcaggt atcagattgg tcttttttct cagtagtcat gctggaaagt 10800 agaagacctt ggaacaggct gactgacctt ggtggctcac gcctgtaatc ccagcatttt 10860 gggaggctga ggcaggtgga tcacctgagg tcaggagttt gggaccagcc tggccaacat 10920 ggtgaaaccc cgtctctact aaaaatacaa agattagttg gacgtggtgg tgtgtgcctg 10980 tagtaccagc tacttgggag gctgaggcag gagaattgct tgaacctggg aagcagaggt 11040 tgcagtgagc cgagatcacg ccactgcaca ccagcctggg caaaagagcg aagactctgt 11100 ctcaaaaaaa aaaaaaaaaa ctttggagta atgccttcaa aattctgagg gaaaataaat 11160 tctaatttag aattccatag ctaggcaaac tatcaattag tgaaatagaa gaaatatttt 11220 catacatcca aggcatttct tacacccttt caggaatcca ctagaagata ggctctacca 11280 gtaggagata gtaaaccaag aaagtggaga tacgaaatat aggaaatagg cagtataaca 11340 cagaagaaaa atgaagcatg tagtatgcat acattggtgt tagttttcct gttttcagta 11400 cgggtccctc actttcacct ttacgtcgtg ttcactagac tgtaagacct ttgatttata 11460 ctctccagag agtaaatctg tcttttgaca gaatggagag gggtagttgc cttggttaga 11520 aggaatagag aaaaggatct aggaatccat ctctgtagta aacagatgct gaactaattc 11580 tccttatttt cacttcattc accctaccct gcatacccag agttacctgg actcaccagc 11640 ttctgagact ttggggaatt ccatagcgta aattgagttg gttttctgct ttttcccttg 11700 tcagtttagg atttagttct tgtcgatctc ccaaggcaat agtccatttt atctctagtt 11760 tcaaaaaaat tattgctctt cttctcttat tctttccatc cttgtggttt tatgtcttgt 11820 gtaaaaattt ttgcttttaa aaattttttt cctgtgtgtg agtgaagaga gaagagagta 11880 gaaatagata gatacattta attcactatc cttactcaga agttgcatag tttaaaattt 11940 tagaagtaac taccttttcg ttgtgccatc ctcatttagg cctttatcac cttgtttttt 12000 ttattaatct tgtcttccta cttctattac ttcaaacttt aaatctgtca gattaatttt 12060 aatttatatt ttcatcttgt tactcagact taaagttata tgtttgtctt tgttgaatct 12120 gatccaggtg tttcatcgtg gcattcaaag ctttctcttg gttagcactt gacatcttac 12180 cttttatagg tctttttctt tctttttttt tttttttttg atttaaagaa aaccattttg 12240 ttttcttcag gggcaatttt ttttttacat tccaccacat attatttata tgtttatcat 12300 atttctttta tttatttatt tatttattta tttatttttt attatacttt aagttttagg 12360 gtacatgtgc acattgtgca ggttagttac atatgtatac atgtgccatg ctggtgcgct 12420 gcacccacta actggtcatc tagcattagg tatatctccc aatgctatcc ctcccccctc 12480 cccccacccc accacagtcc ccagagtgtg atattcccct tcctgtgtcc atgtgatctc 12540 attgttcagt tcccacctat gagtgagaat atgcggtgtt tggttttttg atcttgcgat 12600 agtttactga gaatgatgat ttccaatttc gtccatgtcc ctacaaagga catgaactca 12660 tcatttttta tggctgcata gtattccatg gtgtatatgt gccacatttt cttaatccag 12720 tctatcattg ttggacattt gggttggttc caataggtct ttttctttac agctcttcac 12780 agttgctgtt atgtgtcacc taatgacact ttggtcatca aaggactgca tgtatgatgg 12840 tggtcccata agattataat ggagatgaaa aattcctatt gcccagtgac atcatagttg 12900 tcataaggtc ttagcacaac acattacttt ttctatgttt agatacataa atagttacca 12960 ctgagttata gttgcctaca gtattcagta tagtaacatg ttgtacaggt tgggagtaat 13020 aggctgttac tgggagtaat aggctacata taggtatgta ggctatacac ctacatacct 13080 aggtgtgtag tggctgtacc atctagattt gtgtaagtat actcaatgat attcacacaa 13140 tgttgaaatc tcctaatgac acatttctca gaacatatcc ctgttgttaa gtgatacata 13200 actgtatttt catatctccc tttagacttg ctcatgtact tttatctatg tggaatgcct 13260 ttttcttctt ctttactttt tttttttgag acggagtttt gctcttgttg cccaggctgg 13320 agttcagtgg ctcgattggc tcactgcaac ctctgcctcc tgggttcaag caattctcct 13380 atctcagcct cctgagtagc tgggattaca ggcgcctggt aattttttat atttttagta 13440 gagacggggt ttcaccatgt tggtcaggct ggtctcgaac tcctgacctt aggtgatcca 13500 cccacctcgg cctcccaaag tgttgggatt acaggcgtga gccactgtgc ctggtctcct 13560 ttactttttt taaggcccag tttgatttac ctttttgaaa cttccccatg ggctggttgc 13620 ctgccttctt tatttccttt agtttcctct tcatattgag catcttctct gcaagtacta 13680 tgccaggcct taatttaaac caggattatc ttctctgaat ttacctggca ctcttttttt 13740 tttttttgag acagagtctc attctgttgc ccaggctgga gtgcagtggc atgatctcgg 13800 ctcactgcag cctttgcctc ccaggcgatt ctcatgcctc agtctcccaa gtagctggga 13860 ttatagacgt gtaccaccac acctgactaa tttttgtatt tttagtagag acgggatttt 13920 gccgtgttgg tcaggctggt ctcaaacttg accttaagtg atctacccac ctcagcctcc 13980 taaagtgctg gggttacagg catcagccac cgtgcctggc ctccatggta atcttttttt 14040 tttttttttt ttgagacgga atcttgctct gtcacccagg ctagagtgca gtggcacgat 14100 cttggctcat tgcaacctcc gccttttgtg ttcaagcgat tcttctgcct cagcctcccg 14160 agtagctggg actacaggtg tgcgccacca tgcctggcta atttttgtat ttttagtaga 14220 cacggagttt caccatattg gccaggctgg tctcgaactc ctgatgtcgt gatctgcctg 14280 cctcggcctc tcaaagtgct aggattacag gtttgactcc atggtactct tatatcctat 14340 ataaacgtat tactaaaagt atgaactttt tttttttttt tttttttttt acagaggcag 14400 gatctcacta tgttgctcag gctagttttg aactcctgag ctcaagcaat cctcatgcct 14460 tgacctccca aagtgctggg attataggca tgagccactg cacttggccc tgaacttttt 14520 tttttaatgg aaaaagtgtt ttttcttagg aaagtaacat atgcttacta tttcaaattt 14580 ataggctgaa atagaaaaat tagcagtaac ataaaaatat actcagcctt aatgtatgtt 14640 aaactaacag tggtaattgt tttttaccaa gaaaatgacc acaaatttga aggattccca 14700 aaacacagtg ctgttggagg tatggctaaa ttgatgcagt ggatgaggct ttaaattggt 14760 ataacctcct tggcaggtaa tgggcaatca gagttttaaa tgtgtctacc cttaggccca 14820 gggagtcatt ttcagaaata atatggaagt actggcacct gtgcatgaag atacacaatg 14880 atgttccttg aaacattatt tataatagtg aaggtttgaa tgtcataact atacatttat 14940 agtacagagt actatgtaac tgttaaaaag aatgatctac gtgtgtattt attgccatag 15000 caatatattg ttacgtggaa gggaaaaagt tcagaataag tacatctgta cttatggtgg 15060 ggttatggct tgataaaccc attgtaaatt gaaaacatcc taagtcaaaa atacatttaa 15120 tacacttaac ctattcttag cctagactac cttaaatgtg tgtggaatac ttacattagc 15180 ctgcagtggg caaaataatc taacttaaat tctactttat aataaagtac tggatattat 15240 gtaatttaca gaacactgta cattacactg aaattgtaat ggtttcccac tatcataaag 15300 gtgaaaagcc ctacatggaa ccattataag tcaggactgt ttgtatagta tgaccctttt 15360 ttttgggtaa aaattaccga aaccctggct gggcgtggtg actcatgcct gtaatcaggc 15420 acataccaca tgtatggcca attttgttca ttttttttag agatagggtc tcactgtgtt 15480 gcccagggtg gtcttgaact tctggactca aatgatcctc ctccctcggc cttttaaagt 15540 gctagcctta caggtgtaag ccaccatgtt cagccccaca gtttctttat ccgttaacct 15600 gtagatagac acttgggttg ccctcaccct tgactactgt tgaatagtac ttctgtgaac 15660 gtgggtatac aaatgtttct ttgagaccct gctttcaatt ctttcaggta tatacccaga 15720 ggtggaattg ctggatcatg tggtaattct atttttaatt taattttttt tttgcgatgt 15780 tgtcttgctg tgtttcccag cctgatcttg gactcaagat tctcctgcct cagaccccga 15840 gtagctggga ctacaggtgc gcaccactgc acccagtttt atttttaatt tttggaggca 15900 tctccatact gttttctatt gctgttacgc cattttacat ttccactagc agtgcataag 15960 ggttttaatt tctccacatc cttgccaacc ctttatttat ttatatattt ttggataata 16020 gtcatcttaa tgagtagcaa atgctttctc attgtggttt tgatttgcat ttccctagcg 16080 attggttatg ttgagcactt tacatgtatt tattggccat tagtatatct tctttgtaga 16140 aatgcttgtt caagttcttt gcccatttta aaattgggtt tgctttttgt tgagttgtag 16200 gagctcttta tatattttgt tttatttttt atttgtttat ttttttgagg cagagtcttt 16260 ctctgtcgct caggctggag tgcagtggtg cgatcttggt tcactgcagt ctccgcctcc 16320 tgggttcaat taattctccc tgcctcagct tcccgagtag ctgggattac aggtgcccac 16380 caccattcct ggctaattgt tgtattttta gtagagatgg gatttcacca tgttgatcag 16440 gctggtctcg aactgctgac ctcaggtgat atgcccgcct tggcctccca aagtgctggg 16500 attacaggtg tgagccaccg tgcctggcca ctctttatat attttggata ttctgtgttg 16560 catatatgat tcgaaaattt ttttttctgc tgggcacggt ggctcatgcc tgtaatccta 16620 gcattttggg aggctgaggt gggcggatca cttgaggtca ggagttaaag accagcctgg 16680 ccaacatggt gaaaccccat ctctaataaa aatacaaaaa ttagccaggc gtggtggtgc 16740 gtgcctgtaa tcccagctat ttgggaggct gaggctcgag aatcacttga acccgagagt 16800 tggaggttgc agggagccaa gattgcgcca ctgcactcca gcctgggcga cagagtgaga 16860 ctctaactca aaaaaaaaaa aaaagaattt ttttttttct attccatggg ttgccttttc 16920 actctgttgg tagtgttatt tgatgcacaa aatattgtac actatacagt atgaactaac 16980 aaaaaacaat gagatgtgtg tagatagata ttcatgatgt atattgaaat gacgagcaag 17040 ttgaagatca ggcctccatt ttttactaag agaaaaatgc accctttttt tttttttaga 17100 caggatcttg ctctgttgcc cagggtggag tgcagtggtg caatcacgac tcactgcagc 17160 ctcgacttcc caggctcaag tgatcctccc acatcaacct cttgagtagc tgggactaca 17220 ggcatgggcc accatgctgg ctaacttgta ttttttgtac agatgaggtt tcactgtgtt 17280 gcaaaggcta gtctcgaact cctgggctca agtgatctgc ccaccttggt ctcccaaagc 17340 tctaggattc caggcatccg ccactgtgcc cagcctgcac ctctttttga ttacagagtt 17400 aggtatatat aaactgagat tgaaaaataa gagaaaatat actcagggct gggctcagtg 17460 gctcaaacct gtaattccag tgctctggga agctgaggtg ggagtattgc ttgagctcag 17520 gagtttgaga ccagcctggg gaacacagtg agaccctatc tctacaaaga aaaaaaaaga 17580 aaaaaaaaat caccgaggtg tggtggtacc catctgtagt cccagctact taggaagctg 17640 agacaggagg atcactggag cccgggaggt tgaggctgca gtaagccatg atcatgccac 17700 tgtactccag cctgggctgg acaaagtgag accctgtctt aaaaaaaaaa aaaaaaaagt 17760 actcagctgt tactaatggt tactgctggg ggatgagatt gaattggaag gagagaggag 17820 aggtacgggg ggcaggaaag ggagacaata atgagggact ttcagtttta ctttacataa 17880 ttttctttta agtattggaa tttaggtgat ttttcctttg gggttttctg tattttccaa 17940 tcacaataaa taaaataagt tataaatatt tgttgcatga atgaaatgta taaacccatt 18000 tatgtatgta tttttttaaa attagtatat tattaagtct atacaatatt agtatattgt 18060 tatgtatgta taagcttttt aacatgaagt ttgcagaata tagtacttct tccaaactct 18120 atgacatggg gggaactgaa gtatggggat atcttgtacc agtgtaagaa ttcaagaaga 18180 gaccgtgtgt ggtggctcat gcctataatc ccagcacttt gggatgccaa agcaggatga 18240 tctcttggag ctaggcgttc aagaccagcc tgggcaacat atcaagaccc catctctaaa 18300 aaaaaaaaaa aaaattaact gggtgtggta gtgcgggcct atagtcctag ctattccaga 18360 agctgaggtg ggaggattgc ttgagcccag gagtttgagg ctgtagtgac ctataccagt 18420 gattatacca gtgcactcta gcccaggcaa cagagtgaga cctggtctca aaaaaaaaaa 18480 aaaaaatcaa gaagagcaat ctggatatga gcatttggga tttttagcaa actgctgaga 18540 ttttgtctat agcttgaacc tttcttttag ctaacttgat gatactgatg ataagcaagg 18600 actgtctttt taaaaatgtt tacttcatta ttttcctgac agaaatgaca tcagatgtac 18660 catcactggg tccagccatt gcctctggaa actctggacc tggaattcaa ggtggaggag 18720 ccattgtcca gagggctatt aagcggcgac cagggtgagt ttgagtgtag tgtgttatga 18780 atatctctcc tataaaccaa ctttagttgc tgaatttatt tagttgctga actcacttcg 18840 ctattcctga ccatctcact tcaacttgat tacttactac actactgtca tataagtctc 18900 cttagtccct gtatttttgt ttatgaagaa tttgttttta tccaagtttc ccgtaagcag 18960 ttcttttttt aaaccaaaat ttccgtaaga taaaggactg tagctaagcc cagagatttt 19020 actatgcctg agaaaactgt ttgctgcttt cattaagctt tgtttctgtt ttcccaaaat 19080 cattttggta ggctgctttt ctgaatttag agaatgctga gctctagaat agctgtcttc 19140 taagttattg atatgttgct tggtttggaa tgcagagtcc attcagctcc aaaagtattt 19200 attaaattcc taattagtgt tatggcattg tgctgattcc aagtaggata caaagatgaa 19260 taagacacag tccttgtttc taagttggtt gtgttctgat agaaataatt attatacaca 19320 tgaatatatg atagaatatg cctagttttg ttaggaacaa atttgatact atgtttttct 19380 gtgtatggaa atatgcattt gttggtaaag actgagaaaa gcttcagaaa ggaggttgcc 19440 ttagaggggc ctggaagaat gggcactatt ttatttactt tttggtatta aattttcctt 19500 tttccttctt tttcttttta aaaattgtgc agctaattat aaccagtctt taagttttgt 19560 cccaccaata gtaatggaag agtgatgctt gggttcagtg gtaacatgag taacatatta 19620 agacttgtta tatgttcagt agctttacag attttaacca tttgctgaag cataagctga 19680 ataagtaact ttctaagatt gtaaagctag taagtgtcag atataggata tgaaccttag 19740 gggtttagct ttgtagctta ctctttaaac actgtgttat gtttatgtac agtaacaaac 19800 aaaagtatta taaatatcaa aattagggca tcacttgaga aagtcatagt atatttaaaa 19860 tggaatgcta tgtagttata agcgacaaat atgtttcagt gtagaaagat gttcacaatc 19920 tattaagaaa ggttacagta ttatagtatt tcactgtcaa aagatatgta taacatgtac 19980 ataggaaaaa actgaaagaa tatataccac atttctgggt aatagtatta tgggtgactt 20040 acggtttctt ttgtttttga tttttcagat ttttgaaatg aacatgtatg attggtaatc 20100 agaaaaatat attaaaggaa gtctttaaaa attttctttt tggttttttt agagacaggg 20160 tcttgtaccc tggctggagt gcagaggcac aatcatggcc cactgcagcc tcaaactcct 20220 gctcaagcag tcctcctgcc ttggcctccc aaagtactgg gattacaggc gtgagccact 20280 tcacctgtcc taaaagaagt cttttaatat ttcttttatt tttatttact tatttatttt 20340 cgaggcagag tcttgctctt ttgccaggct ggagtgcagt ggtgtgatct tggctcactg 20400 caatctccac ctctctggtt caagggattc tcctgcctca gcctcccaag cagctgggac 20460 tacaggcgtg cgccaccacg cccagctaat ttttgtattt ttagtagaga tgggatttca 20520 ccacgttggc caggctggtc tcgaactcct gacctcaggt gatatgccca ccttggcctc 20580 ccaaagtgct gggattacag gcatgagcca ccatgcccgg cctaatattt cttttattta 20640 tttattttat tttttatttt tttgagatgg agtctcgctc tgtcgcccag gctggagtgc 20700 agtagtgtga tctcagctca ctgtaacctc cacctcccag gttcacgcca ttctcctgtc 20760 tcagcctccc gagtagctgg gactacaggc gcccgccatg atgcccgact aatttttttg 20820 tattttcagt agagacgggg tttcaccgtg ttagccagga tggtctcgat ctcctgacct 20880 cgtgatccac ccgccttggc ctcccgaagt gcgggattac aggcacgagc cactgcaccc 20940 ggcccatatt tcttttaaag aaagattgga aaatacagaa agttagaaag aacaataaaa 21000 aggccaaaat ctactaccat gttttttagt gcatgtcctt cagtctttat atgtaaattg 21060 tttttaatag ttatgtaatt atatagtttt acatggccta gtcttttcac cttatataaa 21120 taataagcaa tacacacgca cacacacatt ttggcacctt atataaataa taagcaatac 21180 acacacacac acatttcgga gacagagtct tgctctgttg cccaggctgg agtgcagtgg 21240 catgatcatg gctcattgta gcctcaactt cttgggccca ggaagcaatc ctcctacttc 21300 agttctccga gtagctggga ccacaggcac atggcaccat acctggctat tttttttttt 21360 ttttttaaag acatggtctc actacgttga ccaggctggt ctaaaactcc taggctcaag 21420 cagccctccc atctcgacgt cctaaagtgt tgggattaca gacatgacct actgtacctg 21480 gccctttaaa aaaatattgt tacatattct atataaacat aatttttatt tatttttttt 21540 tgagacggag tctcgctttg ttgtccaggc tggagtgcgg tgatgcgatc ttggctcact 21600 gcaagctccg cctcctgggt tcatgctatt ctcctgcctc agcctcccga gtagctggga 21660 ctacaggcgt ccaccaccac gtctggctaa tttttttttt ttttgtattt ttagtagaga 21720 cggggtttta ccatattagc caggatggtc tccatctcct gacttcgtga tccgcctgcc 21780 ttggcttctc aaagtgctgg gattacaggc atgagccact gcgcacagcc ataaacataa 21840 tttttaatgg ttgcgtgaaa ggatgtactt aacttcctat tttgggacat ctaaattgtt 21900 ttgaagattt tgctgttaca tatgatgcta aaaagaactt ctttgtacct aaactttttt 21960 ttcctatttc atattatttc tttagattct tagaaataga gttattgggc tgagcacgga 22020 ggctcatacc tgtagtgcca gcactttggg aggctgaggt aggaggatta cttgagccca 22080 ggaattcaag accagcctgg ggaaaatggc gagacttttt tttctttgac ttagcaatta 22140 tctttctttt ccttccttcc ttcatttttt tcctttgact tagcaattac ctttccctcc 22200 ctccttccct ctctttcccc ttcccttttt tttttttttt tttttgagat gtagtttcgc 22260 tcttgttgcc taggctggag tgcagtggcg caattttggc tcactgcaac ctctgcctcc 22320 cgggttcagg caattctcat gcctcagcct cccgagtagc tgggattaca ggtgcccgcc 22380 accacaccca gctaattttt gtatttttag tagagacgag gtttcaccat gttgactggg 22440 ctggtcttga actcctgacc tcaagtgatc cgcctgcctc agcttcccaa agtgctggga 22500 ttgcagatgt gagccacagt ggctggcccc ttttcttttt tgagacaggg tcttgccatg 22560 tcactgaggt tggagtgcag tggcccaatc tcagctcact gcagccttga cctcccaggc 22620 tcaaggcctg cagcccctcc cgccccccca acccaagtag ctaggactac acatgcgcca 22680 ccatgcctgg ttagtttttg tatgttttgt agagacggga tttcaccgtg ttgcccaggc 22740 tggtcttaaa ctcctgagtt caagtagtct gctcgccttg gcctcccaaa gtgcgtggac 22800 tacaggtgtg agcaaccatg cctggctgag attttttaaa ataaaaaaat ttagttgagt 22860 gcagtggtgg gctcctatag ttccagctaa ttgggaggcc aagatgggag gatcccttga 22920 gcccaggagc tcaaggtggc agtgagctat gatcatgcca ctgtactcca gcctgagtaa 22980 cagagtgaga gcttgtctct taaaaagaaa gaaaggaaga gatagagaga aagaaagaat 23040 ttgagttact gggtagatag ataggatttt acaggtgacc agattagggg attcaggaag 23100 gaggaagagg agcaaacact ttcaggattg atgctgtatg tgtacttcaa atgcgaccca 23160 tctagaggcc cataatatca aggtatccca atagtagaag taaaaagagt gatcactagg 23220 ttaaggtggt aacatgagca atattttagt tcaatagtgt acagagatta taaggggatt 23280 ggtctatttt attgtttata atctttccta atctccttta aagaaattca cttcttcttt 23340 ccctgaatgt ctgtagcatg ttgattgtac cccttatgtg acactttcct attctcactt 23400 gttttatatt tgtatttctc taggtgaaga gccccttgag ggcaaggttc ttgtttttac 23460 ctcacctagc acagtgtctt gaatgaagta tatattacat gtttattaga tcaatgaagg 23520 aaagaaacat tatctaacaa tcttcgtagg tattaagtca ctcctttatg taagatcact 23580 gctttgaaag atgtttcaga aatttggtaa cacggcttag agcagactct agaaatgaaa 23640 catggacctg aattatttac gttaattttt tcttattttt tctgagtgga ttcctgctcc 23700 ctttacagag gttgtagtct gattgaaaac tctggcaaag attgactgct actctaggaa 23760 agtgttaagg tagcagaagg tacttttgtt tctattgccc agttttgtac tttttttttt 23820 tgagatggag tcttgcagtt gtcgcccagg cttgagtgca atggtgcgat cttggctcac 23880 tgaaacctcc acctcccggg ttccagcaat tctcctgcct cagcctcctg agtagctgag 23940 attacaggca cccgccacca tgcccagcta atttttgtat ttttagtaga gatggggttt 24000 cagcatgttg gccaggctga tctcgaactc ctgacctcag gtgatccacc cacctcagcc 24060 ttccaaagtg ctgggattac aggcgtgagc caccactcct ggccccagtt ttgtacttct 24120 ttgcctagtt tgggactatg aacaagagga aatgtagctt tgtttgactt ctgccacttc 24180 ctctttccat tcttccattt gggtgggtgt tccggtagct tgtgttgaga aattttaact 24240 tcttaatgtt ttgtattatc agcaggctta aagtatttat tgttggcttt cctcaggctg 24300 gattttgatg atgatggaga agggaacagt aaatttttga ggtaagagac tgaaaaactt 24360 tccttagatg tctgatatta aaaattagtt tatgatcttt atacttctga cttgtaaatt 24420 tttgtcctta ggtctaagga gagtacttca tcctaaaact ataaatattc atatatctca 24480 gaaaattttt aagcattccg ttaatatctc tagagaaagg acctcagtga ggagaggacg 24540 gcatttacaa accctctgta tcagttctct gctacttcat agcctaaaaa gaacaacagt 24600 tcgttatttc tcaattctgt ggattgactg catgtttatt ctgcttatct cacctagact 24660 cactcataag actgcattct cagctggtca gctgagaact ggactcagct gggatggctg 24720 ggtatttctg tctatgtgtt tttttatcct caaggaggcc agactgaatt ttttcatgtg 24780 gtgatggcta caatctacag cacaaatccc agtgtgcaag tgcttatcaa gcctctgctt 24840 atatgacatt tgcttatgtc ccattggcca aagcaagtca tatgttccag gtgtgagtgt 24900 gtgagggggt tacacaagga agtacatact gagaggtgta attcattggt tgaggtgtca 24960 ttaatctgtg actctgtcat accctctgac acctgttgac acctcttata ggtagcagga 25020 attggaattt gtattttttg tttttgtttt tttttttttt gagacggagt cttgctctgt 25080 cgccaggagt gcagtggtgc aatctcggct cactgcaacc tccgcctcct gggttctagc 25140 gattctcctg cctcagcctc ccaagtagcc gggactacag gtgcgtgcca ccatgcccag 25200 ctaatttttg tatttttatt tttttatttt ttattaattt tcttttttat tgatcattct 25260 tgggtgtttc tcgcagaggg ggatttggca gggtcatagg acaatagtgg agggaaggtc 25320 agcagataaa caagtgaaca aaggtctctg gttttcctag gcagaggacc ctgcggcctt 25380 ccacagtgtt tgtgtccctg ggtacttgag attagggagt ggtgatgact cttaatgcgc 25440 atgctgcctt caagcatctg tttaacaaag cacatcttgc accgccctta atccatttaa 25500 ccctgagtgg acacagcaca tgtttcagag agcactgggt tgggggtaag gtcatagatc 25560 aacagcatcc caaggcagaa gaatttttct tagtacagaa caaaatagag tctcctatgt 25620 ctacttcttt ctacacagac acagcaacaa tctgatttct ctatcttttc cccacatttc 25680 ccccttttct attcgacaaa accgccatcg tcatcatggc ccgttctcaa tgagctgttg 25740 ggtacacctc ccagacgggg tggcggccgg gcagaggggc tcctcacttc ccagaagggg 25800 tggccgggca gaggcgcccc ccacctcccg gacggggcgg cggctgggcg gaggcgcccc 25860 caccaccctc ccggatgggg cggctggccg ggcgggggct ggcccccgcc tctctcctgg 25920 acggggtggc tggccaggcg ggggctgccc cccacctccc ggacggggcg gctgccgggc 25980 ggagatgctc ctcacttccc ggacggggcg gctgcggggc ggaggggctc ctcacttctc 26040 agacggggcg gctgccgggc ggaggggctc ctcacttctc agacggggtg gctgccgggc 26100 agaggggctc ctcaattctc agacggggcg gctgccgggc ggaggggctc ctcacctccc 26160 agacggggtc gtggccgggc agaggcgctc ctcacctccc agacggggtg gcggggcaga 26220 ggcgctcccc acatctcaga ggatgggctg cggggcagag accctcctca cttcctagac 26280 gggatggcgg ccgggaagag gcgctcctca gttcccagac tgggcagccg ggcagagggg 26340 ctcctcacat cccagacgat gggcggccag gcagagatgc tcctcacttc ccagacgggg 26400 tggcggccag gcagaggctg caatcctggc actttgggaa gccaaggcag gcggctggga 26460 ggtggaggtt gtagcgagcc gagatcacgc cactgcactc cagcctgggc aacattgagc 26520 actgagtgaa cgagactcag tctgcaatcc cggcacctcg ggaggccgag gctggcggat 26580 cactcacggt taggagctgg agaccagccc ggccaaccca gcgaaacccc gtctccacca 26640 aaaaaatacg aaaaccagtc aggcgtggcg gcgcgcgact gcaatcgcag gcactcggca 26700 ggctgaggca ggagaatcag gcagggaggt tgcagtgagc ggagatggca gcagtacagt 26760 ccagcttcgg ctcggcatca gagggagacc gtggaaagag agggagaggg agaccgtggg 26820 gagagggaga ccgtggggag agggagatgg agagggagag ggctaatttt tgtattttta 26880 gtagaaacag ggtttcacca tgttgggcag gatggtctcg atctcttgac ctcgtgatcc 26940 gccctcctcg gcctcccaaa gtgctgggat tacaggtgtg aggaatttgt atttttgagt 27000 tgttaatatt ctggagcttt taaaatggac tatttatttg tttgtttttt tgagacagag 27060 tcttcctctg ttgcccaggc tggagtgcag tgctgcagtc ttggctcact acaacttctg 27120 ccttccaggt tcaagcgatt ctagtgcctc agcctcctga gtagctggga ctaccacacc 27180 tggctaattt ttgtattttt agtagagacg gggtttcacc atgttagcca gactggtctt 27240 gaactcttgg ccttaagtgg tccacctgcc tcagcttccc aaagggctgg ggttataggc 27300 ataagccacc atgtccagcc tattttcttc ttattttttt gagacagggt cttactctgt 27360 cacccagact ggagtgcagt ggcacagtct cggctcacta cagcctcgaa ctccagggct 27420 caagcgatcc tcccacctca gtctcccaag tagctgggtc tacaggtgtg agccataata 27480 cctggctaat tttaaaatga tgttgcccag gctggtctta aactcctgtg ctcaagcaat 27540 cctcccacct tggccttcca aagtgttggg attacaggca tgagccactg tacccggcct 27600 gaaaatggac cttttaatat attgatgaag gagttctttc agaaaagggg gatattcttg 27660 ctgaagacca attgcttgtc ttcttttcaa gtaagaaaaa cagtaagact caaaaggaag 27720 agaactttga ccgcaatctg ctttttttct ttccagagtt gaaaatatta cccaggtagt 27780 ctgtacgttg ctgagtaaca gacaatttga taaaggagcc caatgaaaaa aaaatgattt 27840 gattgtgtgg gtgcccagat ttaatatcat ttatttattt tctttctttc tttttttttg 27900 agactgattt tcactcttgt tgcccaggct ggagtgcaat ggtatgatct cggctcaccg 27960 caacctctgc ctcccaggtt caagcgattc tcctgcctca gcctcccgag tagctgggat 28020 tacaggcatg cactaccatg cccggctaat tttgtatttt tagtagagat ggggtttctc 28080 catgttggtc aggctggtct tgaactgccg acctcaggtg atccgcccac ctcggcctcc 28140 caaagtgctg ggattatagg catgagccac cacacccggc ccatttcttt ttcttatttg 28200 tttgttttgt taactaagtt ttttctttaa ttgggaaagt aatataagtg tattttatta 28260 cagaaatttc aggctgggcc tggtggctta cacctgtaat cccagcactt taggaggctg 28320 aggtgggtgg atcgcttaag ctctggaatt caggaccagt ctgggcaaca tggcaaaact 28380 ccatctgtac aaaaaatgtt acaaaaatta gctggaagtg ctggtgtgtg cctgtagtct 28440 cagctactcg ggaagctgag gtgggagggt ggtttgagtc ctggaagcag agattgcagt 28500 gagccgaggt tgcgccactg ctctccaacc tgggcgacct tgcctcaaaa aagaaaaaca 28560 acaaaatttc aaacagtgca gagttatata tagtgaaagc acatcttcgt tttactttgg 28620 accttcagaa ctcctttttt tttttttgag aggagtctcg ctcttgttgc ccacgttgga 28680 gtgcagtggt tcgatctctg ctcattgcgt cctccgcctc ctgagttcaa gctattctcc 28740 tgcctcagcc tcctgagtag ctgggattac aggcgcctgc caccatgccc ggctaatttt 28800 tgtactttta gtagcgacaa ggtttcgcca tgttggccag gctggtctca aactcctgat 28860 ccactcggct cggcctccca aagtgctggg attacaagtg tgggccactg cccccagcct 28920 ggaccttcaa aactctttag acaactacag tttccagttt gttgagtatc cttccaaaaa 28980 tagtatatat agaattgtat ataaatgtgt atgtgtgagc atacacacac ttatctcctc 29040 agtttttttt acacaaagga tgtccatatt gtaaattgct tgccattttt tagttttata 29100 ttgcttcata cagttaatag catctatctt ctttttttat agtaacattt aaagttaagg 29160 ctcatatttc tggctactca ctagatgaac tttgccaaat actcttgaaa acaacaaccg 29220 tgacttggcc atcataaaga aatagttgca agtggaagta taattctcta agaggtctct 29280 tgagacttaa tgagtctcag taaatgtgaa gaagggaaga gatttcaatt tctggagaag 29340 atagactttt tcaaacagct ttattgagaa ttcctcctat tgagaatcct gaattataat 29400 gatacatgct attagtggaa cttcactgtg tgtatgaaag atatgagggt aaccactagt 29460 ctttttttat acccgtgaat tagctctaac cctgagtcat tgcttccata aaatccagta 29520 gtcacaacta catgcagatc caaagagagg ttcgtttgtc cttttcctaa ccataaaaaa 29580 agactatcgt agtttatctt accaagtcgg gttgttgtgc ctgagaaaag cactgccgaa 29640 ttccctttcc cccttctttt tttttttttt tttttttgag acggagtctc gctctgttgc 29700 cacaaatgca gtggtgtgat ctcagctcac tgcaacctcc gcctcccagg ttcaagtggt 29760 tctgctgcct cagcctccga agtagctggg actataagca cgtgccacca cacccagcta 29820 atttttgtat ttttagtaga gacggggttt caccatgttg gccagagtgg tctggatctc 29880 ttgacttcgt gatccgccca gtgttggcct cccaaagtgc tgggattaca ggcgtgagcc 29940 actgcgccca gcctccccct tctttttctg ggtatatata tacaaaataa tcgaaggcag 30000 aatcttgaag agatatttgc acactcatgt ttattggccc attttgcgca atagataaga 30060 ggtcgaagta accgaaatgt ccactgacag atgaatggtt acagaaaatg tagtatgtac 30120 atacaaggga atattattca gccttaaaaa gaaagaacct gtcatatgct gcaagatgga 30180 tgaatcttaa ggacattata ctgaaagaat aagccaataa caaaaagaca attactgtat 30240 gattccactt acatgaggta tctacaagta gtcaaattca tagacacaga aagtaaaatg 30300 gtggttgcta ggggttgggg tgaaggagaa atgagaaaat ggtgtttgat gagtatagag 30360 tttcagtttt gcaagatgaa aaagttctag atatctgttg cacaacaatg tgaatatggt 30420 tagcactact caactgtaca cttaaaaatg gtatacagta aattttatgt gttttttacc 30480 agaattaaaa aaaaacccaa aactaacccc ttactttaga attgtgctga caggccagtc 30540 agctgtgttg tcattagatc atcatctttt tttggtgtgt ctggtaaggg taatggaaat 30600 accagaaacc tgacaaataa tagttgtggg tcttttaagt tctatggggt gctgctgtta 30660 tttctatcac tttgtgatgc ttttccattg gctttttttc tattgaatat tttcacccct 30720 ttctagttta cttttcagag tgaaatagat ataacaagtg taatgctttg aaacaatcct 30780 ttttctctcc ttcaggtgtg atgatgatca gatgtctaac gataaggagc ggtttgccag 30840 gtaatattgt agtaggtaat atattgtaat atataatatg atccatgttg tagaaccaga 30900 cagtcctagc atattgactt aattttttct ggatgagacg gaatttctct gtttaatatc 30960 tttcctattt ggaagtatgt gaaacttagt attataacta tcatttatgt tcaggtgaca 31020 tggcttcaaa ctggcggtat attttataca gtgtttttct gtgtatgtga taactaaagc 31080 aatgtgcttg caaggtttcc ataggagcac aaattatgga ttttgtgctt gcatttatta 31140 ttaaatggat ctacaaaaat aggaatacag ataatggttc tgtaattaat ttatttattt 31200 tgagacagag tcttgctctg ttgcccaggc tggagtgcaa tggcgccatc tcggctcact 31260 gcaaccttca cctcctgggt tcaagcgatt ctcctgcctc agcctcctga gtagctggga 31320 ttataggccc ctgccaccac gcccagctaa tttttgtatt tttagtagag atggggtttc 31380 accatgttgg tcagggtgat cttgaactcc tgaccttgtg atccgcccgc ctcggcctcc 31440 caaagtgctg ggattatagg tgtgagccac cgcacccggc ctattttatt tttttgagac 31500 agagtctcac tccatcaccc atgctgtagt acagtggtgt aatctcggct cactgtaacc 31560 tctgcctcct gggttcaagc tgttcttcca cctcagcctc cctagtagct gggaatatgg 31620 gcatttgcca ccatgcctag ctaatttttg taataatttt ttttagcaga gatggggttt 31680 caccatgttg gccaggcttg tctcgaactc ctcacctcaa gcgattcacc cacctcagcc 31740 tcccaaagtg ctgggattac aggtgtgagc cattgtgcct ggcctattat tttattttaa 31800 gatatgtata ttttttagag acattgtttt cattgtgttt cccaggctgg agtacagtgg 31860 catgatcata gctcactgca gcctcaaact ctggggtttc agtgatcctc ctacctcagc 31920 ttcccaaata ttgggattat atgcatagcc accatgcctg gttggtcctg tttttttaaa 31980 aatgacagta agaggcgggg agtggtggca tatgcctgta attccagcac tttgggaggc 32040 agatgcaggt ggatcacttg aggtcaggag ttcaaaacca gcctggccaa catggtgaaa 32100 ccccatctct actaaaaata taaaaattag ccgggcatca tggtgggcac tcataatccc 32160 agctactcta gaggctgagg catgagaatt gcatgagccc gggaggtgga ggttgcagtg 32220 agcagagatg gcaccattgc actccagcct gggtgacagc aagattttgt gtcagaaaaa 32280 aaaaaaaaag acagtaagga aacagttttt gtgacaagta gagttttgat tgaaaaaaac 32340 ttaaatttgt ttaaattacc tatcaagatg atgaaatata ctttttttta ttaaattctt 32400 aaatgtcagt tttcttttta gaaagttttt attaaatatt aggcaataaa ttatttcttt 32460 tttgaaaatt aagtttgtag ctacctcaga aagatgaata attcgttatt tcaaaatcca 32520 gtgattaact gagcacttag cacttagtat ttgttcgttg ctgatgctcc tggtctcggg 32580 aacatacttt aagaaccgtt gatgtagaga gattagaaat atcaggggaa gtagttaaaa 32640 actattctgg agtggtgaga tgcaatctca ggctttgaat tagagggtat tacataaaat 32700 gcattgtagg gatatttctc tggtagcagt tatagaattg attaaaggga tggactgtta 32760 gactgtaggg aggtagatag gaagctgttg aaataataag acagctataa aatcatgagg 32820 gcctgggtta cagtggcagt ggtaacagga aaggagtgaa gttaagaggt ttttgaagaa 32880 taatttgttt atcgaaacta attgaaataa atatttgtaa agtctttagg atgtgaacca 32940 tctctaaaat gagaagttaa tcatagatat ttggaggata gtttttcaag cttcattgaa 33000 aagtcaggcc atcagttatt attggcagta tcttgatgaa atttcaaaaa gccatgaaaa 33060 catcgcatga atgattttgg ttttcatttg tcctgcttaa tgtgcatata tttcattcag 33120 aaatactgag gtggattagg gattgtggat ctgtagtagt agtagtagta gaaataggaa 33180 ttttagatgc ttaacttttt tttaaataac agaattcatt catatgatgt gttaaggtag 33240 tgcccctcac atatcctcct tgggatatag agggtcttaa aagctgaaaa cttcttgaaa 33300 aacttttggg attatatgca tagccaccat gcctggttag tcctgttctt ttaaaaatga 33360 cagtaagagg ccgggagtgg tggcttatgc ctgtaattcc agcactttgg gaggcagagg 33420 caaaagttgc tcaacttttt gaaaaaaaag aagtttgtag tttgttaagg aactatctag 33480 aagaaataac ccaaggaatg taaaactcta aactgctgaa tatcactcag ttctccttct 33540 cttgtcatca gaatatatgc gtaaattttt acattcttct tcattgttac tgtgttattt 33600 tctgcctatt gaccatttta taaaaacatt cccatatatt gataaggtta ttgtatttgt 33660 catttttaat agctaaataa tcttttagct tgttaacctg tcatacttag acattttatt 33720 cagggcctct ttatctatat aaatatattt aaaatggaat tgacacattc ctgacacaat 33780 ggcctgccaa caccttgcta tttcctcagt tgccacccat cattacagta tctcagtttt 33840 ctagaaaaat ttagtgatgg ttctttgtat ccttctccag tggagaggga attgttactt 33900 ttgatccatt cctgtgtggc tatactgcag agaaatggca aaaggaccga atcaaagtta 33960 ataattattt taggaacaaa tcaataatag aagatatgcc agaaacctac ctcttagagt 34020 tatttatata atttctccag tgaaatctgg ttggttattt tgtcattgtg tggcacgtgc 34080 atgtgttgtt tgtgtgagag ggagagagat tatatttgtc atcacttgtt tgatagtatc 34140 atctttaatg tactctgtct ttaatttctt cattatagaa tagcacatgt ggatatcatt 34200 ttctttccag attgggagca gtgcatgaaa atggtattcc tgaattccct tggttggttc 34260 ttgttcagac tctgtatatc tttggtccct acagagatcg attggcaaaa tgctttctgt 34320 gtttagatca tgttaattta ctatatattg gctttgcttt tatgttgacc tttatcttgt 34380 aagttacttt ttctttatcc taacagatgg ctttgtagag ttacaggcaa ggttcctgcc 34440 tataattcca tttccctcct ctcttctctg catctgttta gttctatatc cttttctctc 34500 ttctttctcc ttttcttttt ttttctccct ccatttcctc tccctccctc cttccttttt 34560 tcccttcctc cctcccccct tccttccttc ttttttattt taattttagc ttagttcatt 34620 aattctattt ttaggtcgga tgatgagcag agctctgcgg ataaagagag acttgccagg 34680 taggagaaca gtgtctttta gcatgatgaa gcagatgatg ctgctttttc tatccttttt 34740 cttactcttt cttttcttcc cctttctctt tgtatttttc cttatctgtg gcaagagagg 34800 acaagatttt ttagaagttt gagtgtaaca ggaactttgg cttcccccat cagaaagtgg 34860 gtgagttgag ggaactttgc ttagggattt aagaaattgc tattagtttt aagttttttt 34920 ttctttttct ctttatgtca gtactaagtt tcacagaaca aaaagctctt agaaggaatg 34980 caaccgtgcc agttggtgct ttaacaggga aatactcttt ttatcagaaa accaataaat 35040 atatctgtat ttgtgattag ttcccagtat ttaggcctca gcatttactc cacacctcta 35100 ggaaactcac acctattttc ctatgaagac tcacagccta gattattctc acaacagaac 35160 tagtgttgct ttggtgaccg aatcctttct tgcggtagtt ttctagaaaa gttttagttt 35220 ccttgatgtg gctatttaaa agaccaggtt tctgtactta cgtgtcagaa atctgtcaga 35280 tactaggaag atgagtgctt tatgtttgag aatagaattt tatgtcttag gcaaagtgta 35340 actaaatatg ggcctatgtg gtgagaccct ttttgccatt tagaaaggag actctagaat 35400 tctcttggga gactgttgtt tgtaatgtag aaatgctgca gaagaaatat gcatagactt 35460 ttctgttttc tagtatctgt gatttggggg tgacttagga aatagatcat tgatgccaat 35520 acccattttt actatattcc cccttttttc tactatttcc tctttaatct gggtcacaaa 35580 ctcattttgc tgccagttta actcgagctt cttactaacc ctttcactgt tcagaaaatg 35640 gatttggcat gatgtggtgg agaaaacatt gtattgggaa gagagcagcc tggggaaagt 35700 cacttaccta cttgacctcc ctttgctctt ctcagaaaaa agagatttat gttagatttt 35760 taattttctt actttctttt ttttttggac aaagaacttt tgttcaagta gaattcttaa 35820 gtggtaacag aaataaataa aagagataaa gcaggccggg cgcagtggct catgcttgta 35880 accctagcac tttgggaggc cgaggcaggc agatcacgag gtcaagagat ggagaatatc 35940 ctggccaact tggtgaaacc gcgtctctac taaaaataaa aaaaaaaaaa actgggcgtg 36000 gtggtgtgcg cctgtagtcc cagctacctg ggaggctgag atagcagaat tgcttgaacc 36060 cagaaggcgg aggttgcagt gagccgagat cccgccactg tactccagcc tgggtgacag 36120 agcaagactc catctcaaaa aaaaaaaaaa aaaaaaagaa gaagagagat aaggcaaata 36180 tttgagtaga agcagaaatg cagcatgttg catgattatc tccttgaggc atccccatgg 36240 aggacactga gaaacttaat gggcttttaa aaattcctgt tggaaaactg ctggattatt 36300 cctgttaaca gtgatatctt tctgtcttaa ttttgaggaa gtcagtgttg gagctgtggt 36360 ctatttacct gggtgagatt caaattgtct tgtcagacct ttaatcatcc tcctctccat 36420 tccactcctc cagttaactt cgtcccagac tggggaccca tatgggactt ttagtagatg 36480 gtgtatctta agtcttgtaa gaagtttagt gcactggcag cacacccaga taaagaaggt 36540 aggactttgt gcattaatgg gccaaataaa acttcaaaat cttcaaattc tgccttttaa 36600 tgttgcaaat aagagagagg cttaccatat tttatagacc aaggaaatct gtactatcaa 36660 ttcttgtatc agctatggag ccacatactt gagttggcaa aaattggtcc ttttattttc 36720 tggcctttaa atagttgaat tagtaagcat gggagttaac caagctgagg ttatatgttc 36780 cataggaact taagtgagta aaatcagcat ttaaaaatac tatctttttt tttctcttgt 36840 tttttgtttt ttgttttttt ttgaaatgga gtcttgctct gccacccagg ctgaagtgca 36900 gtggtgtgat ctcagctcac tgcaacctcc acctcccaga ttcaagtgat tctcctgtct 36960 cagcctccca agtagctggg attacagatg catgccactg tgccctgcta atttttgtat 37020 ttttagtaga gacaggattt caccatgttg gccaggccgg tctcaaactc ctggcctcaa 37080 gtaatccacc tgcctccatc tcccaaagtg ctgggattac aggcatgagc caccatgcct 37140 gacctgtcgt ttcttaaaac agcttttgtt ctgagggagt ggtaatttac aaaggatgtg 37200 aagtttccag gaaatagggg gaagggaatt acattatctt cttgttctct gtctgcctta 37260 ttagttctgt ttcatgcttg ctttgcatga gaaggttggc aaaccttatt ttaactgctg 37320 agacttaagc atcactaaat ctgaatacca cattcttcag cagcacactt ggtatccata 37380 tcactctccc tgctaccaaa tgaccagatg tgaccacctg gatggggctt ctctttcttt 37440 ccatgcaggg aaaatcacag tgaaattgaa cggcggcgac ggaacaagat gacagcctac 37500 atcacagaac tgtcagatat ggtacccacc tgtagtgccc tggctcgaaa accagacaag 37560 ctaaccatct tacgcatggc agtttctcac atgaagtcct tgcggggaac tggcaacaca 37620 tccactgatg gctcctataa gccgtctttc ctcactgatc aggtctctgg gacttatagt 37680 tctgagagag tctggaatct gggtgaatct cttgaaagtt ttcgtttttt ggacaagaat 37740 tcagcttttc aggaagaagt cagacaatgg gaaaacgaat ttcaatcctt ggctataaca 37800 ttaattagca ttgggacaat gagaagtaga gaagagttgt gaaaactatt taataagcta 37860 ataagtatta atatttgaga acttgactca tgaatatagc atataggatg gaagaagaac 37920 agtggaatca cagaggaaat gactatgtcc atggaaccaa ttttctttct tgcctttagg 37980 gttatagaag atggaagaaa tctatttctt atccctgaag cagcttctag ttttagtaat 38040 agaatgaatc tgtcccacct ttggtgatag aagaactgag agtctaattg ttgcttaggg 38100 atgtgctctg ttacatgtga tcactatgaa aaaaagaagg cgtaaacatt ttctgccttt 38160 caggaacttc atctgaatat aagtatgtga gtggcaggat atcacagaaa ataacaggaa 38220 aatgcataaa gagaggaatt gtatttttta attagtaatt ttatgtggga ctagatagac 38280 atactgaagg gatggctaaa gtgaatagaa tggctagact tgagtgagga tggttaggga 38340 agacttctga gggtaaggaa gccatgttct gttttggtta ttaaaataac atgatcattg 38400 cagaaaaatt tggaaaatgt aggaggtata aggaagaaaa aaatttactt cagtatcaat 38460 caagtattcc cttaatgcca ccaatttaat caaatgatta gaaagaagga gagaatatag 38520 tttgagaaaa tggaataaga attttccaaa taggatggtc tacttaaaac tacatacttt 38580 gtagctatat acattgaaat agttaatatg ttctaacagt acatgtgcaa gtattcaaca 38640 gactccagtt atgcaccttt tgtgggcaaa ccaggtgtgt tgtgctgtga gaaatagaaa 38700 gaatggtgag acaaatggtt ttctggtgga aacagacatg taaataaata aattaaacat 38760 agaactagtt ctataataga agtgctgtaa tgaatcctgt aaaatgcaga tatggaaaat 38820 gagttgggga gtagtgttgt ggattttggg aagcacttga gcaaaaacct agaagtgtgg 38880 aataattggg ttatgcaaag aaagtcaagt ggtttagcat gtttttggta gataatagga 38940 aggtaggctg ggatctaatg atggaatgtt taggtgttaa agaatttaga ttttaatttt 39000 tatgcagtgg ggagacataa aaaatgtatt agatctggta gcattttaag gattgattga 39060 aagcagggcg actacttaat tagttttggt aaaagatgac taggacagtg acaaagcatt 39120 ggaaagtaga atcgataaaa ctgaattatc actggaatgt gagagaatag ttagattttg 39180 aggcttctag cttaggagga tgctgttaag aatattggaa gagcacggca ggtttttttt 39240 tttaagaggg aaataatgat tcaggttttg ggatgttgat gttgagttgc tggtagaata 39300 tttataaata ttttacagat acttgaaatt caagtctgct gaaagctcag gaaaaaacgt 39360 tagtcatgtc tagggctata gacttggtta ttatttcgta gtggggaaga gtgaatatgg 39420 tttcccagga agaaagtatg gtattaataa agagggctta agatgaactt tggaaatgtc 39480 tacatttaag acttgaacaa aggaaaggaa gtctgaaaca gaagaggaag caaaaattgg 39540 agtacagtct catagaagaa ggtagggaaa aataaaattt aaaggataag atggacgaca 39600 ttgtcacatt ctgcagagag gttgaataaa gtgatgaaga cccaggaaaa gggacttgaa 39660 ttggtaatta ggaggacatt agtaacctca ttaaaaatat atgtatgctg ttcctggcag 39720 aacaaaaacc aaaccaaaca ggaaaacagt agtttagagt gagagtgaag tgggattgag 39780 aaataattga aaggtaagag gataaagcca gtgaatataa cattattctt agtataagct 39840 tgctgctgaa aaagagagat gaggtgggtc aaactgaggg aagatttatc tagaattgag 39900 aaaacttgat catttttata ggcctgaagg gaaagagaga aagtgggaat atttgtcaag 39960 caagatccta aaaagagacc agagaggatg gaattaagaa gtcaattatt gttcatggta 40020 agcctttttt tttttgagac agggtctctt gctctgtcgc tcaggctgga gtgtggtggt 40080 atgatctcgg ctcactgcaa cctctgcttc ccaggctcag gtgatcctcc cacctcagcc 40140 tccggagtag ctgggactac aggcgtgtgc caccacacct ggctagtttt gtttttgttt 40200 ttgttttgtt ttgttttgtt tgtttgtaaa gatggagttt cgccccattg cctaggctgg 40260 tcttgaactc ctggactcag tgaccctccc actttgacct cccaaagtgc tgggattacc 40320 ggcgggagcc gctgtgccta gcccaagcct ttttattctt cttgaatcct gagatagaga 40380 ggaagaggtg gatagtgaca tagagaaagt gaggaaacat gtattagaaa aaactttctt 40440 atcgatgaac tacatttagg gtggaaacct gtggctgtgg atcaggtgtg aaccagcagt 40500 tgcttacgga gagatgcatg tggcctgaag tgtcttactt cttcctgtga atagaaatac 40560 ttgttttttc agagtaaaat attaacttct atttcttttt cttgcgcagg aactgaaaca 40620 tttgatcttg gaggcagcag atggctttct gtttattgtc tcatgtgaga caggcagggt 40680 ggtgtatgtg tctgactccg tgactcctgt tttgaaccag ccacagtctg aatggtttgg 40740 cagcacactc tatgatcagg tgcacccaga tgatgtggat aaacttcgtg agcagctttc 40800 cacttcagaa aatgccctga caggtgagag ttatgtgtat gggaaatgaa tgagaagtcc 40860 tttcttgttt ttttcctgag acttaagaga tgttttagct gttaaattgg tttgttgact 40920 ctggcaaggc ttcaagaatt ttctacttta atgaatatag tcagttcttt ttatccatat 40980 gagattatct actttgtggc tcagccttag aaaatatttc attggtgata atattttaca 41040 tttatcttaa tattggtata aatagaacag taaaagccaa acctacaata cttttttttt 41100 tccgttctaa aagaattatc catgttttta tctcatttgt atggataatt atctggtatt 41160 ttttctacct cctggtgctt ggctttgtgc taggttcaat gataacagct ttttattcta 41220 tagatatggt tattggtcaa tgtataaggt gttttctgtt gttgttgttg ttgttgtttg 41280 tatctgtact gttgttcttt ttttctcccc tattttatta tgttcagtct tttggccaga 41340 gtttggctag aggaaacaag tcatatctat tcttgagcaa ctctagaaaa aaatttaaag 41400 tggaagcaga taaaaaaact ggtagttaaa atgcaagaaa tttcaatata ctcatattag 41460 tgttgttgat ctttagtttt cctccttttt tcccacccaa aaaagagaca gggtctctct 41520 cttgcctagg ctggagtacg gtggcacatc atagcttact gtaatcttga actcttctgg 41580 gctcagtaat ctgcctgctt acagcttctt gagtagctag gagtagttca tgtcaccaca 41640 cttgaccaat ttttaaattt tttgtagaga caggttctgt ctgttgccca gactggtctc 41700 aaactcctgg ccttcagcat tcctcctccc atcttggctt ctcaaagggc tgggattatc 41760 ggcatgagcc accacacttg accagttttc ccctccttta tgtttttatg atttcatttt 41820 tctagttctt ccttttcccc aaaagttgtt cttcgtttct gtataataaa gaagacaaac 41880 agatctatat gtttctataa catataaaat tacttggttt ttttcttttt aaaatttttt 41940 ctttttattc tttttttttt ttttgagaca aggtcttgct ttattgctga ggctagagta 42000 cagtgactct tcacaggcac agtcatagca cactacagcc tcaaactcct gccctcaagc 42060 agtcctcctg cctcagcctc ctgattagct gggactacag aaaagtactt gtttttcaac 42120 caatgacatt tactctgtat gtatgtctgt atgtgtatac agataatcag ctatgagaat 42180 atagccttgc ctcttgtttt ctactactac tttccactcc tacttttcct tgcacaatgt 42240 tattttcaat gctgcctttg aacttaagag tgagattcat tgatgataat tgaagtattt 42300 taggcttgaa aaaaaattca tctcctgctt ggtcagttct gttataagca aggagattaa 42360 gggcatgaat aggatgctta cttatctttg ccttcagtat ctctccccct cttccccaca 42420 cacaaaaatg cactccagac tgctcttcac atcttccttc agggcgtatc ctggatctaa 42480 agactggaac agtgaaaaag gaaggtcagc agtcttccat gagaatgtgt atgggctcaa 42540 ggagatcgtt tatttgccga atgaggtgag tgtcaagctg aggattgtga tttggtatag 42600 gaaggatcaa gagctgagag ttttatttct gtcagagtta agttggatta gctccagtgg 42660 attaaattta actctccata cccagatgga ttgtaacaca gaataaagta tttggaaagg 42720 gaactaacgt ttctgaactt gccagacact atgataggtg ctttatatct gtcatcttat 42780 tttatcctca caattgcctt gtagtgtaag attgatggtt accattttgc agatggaaaa 42840 acagatataa agaaatgaac ttggccaggt gcagtggctc aaggctgtaa tcccagcact 42900 ttaggaggct gaggcgagtg gatcacctga ggtcaggagt ttgagaccag cctggccaac 42960 atggtgaaac cccatctcta ctaaaaaaaa acaacaacaa aattagacgg gcgtggtggc 43020 gtgcgcccat aatcccagcc acttggaggc tgaggcagga caattgcttg aacccaggag 43080 gtggaggttg cagtgagccg agattgtgcc attacactcc agcctaggca aaaagagtga 43140 aactctgtct caaaaaaaaa aaaaagaaaa agaaaaagaa atgaatttcc cactgttaca 43200 tactgtttga tacaggattt tgttttaatt catagtagtc tgactacaaa acctctactt 43260 tttccctgtt acaacacaag gcaatatcca tttactcaga ccatttcttc tttttttttt 43320 tggttagaaa tttgagactt cctatgtctt tcagtaggtg tttagtgttt ataaattata 43380 tactgtacgt tttaggattc tgtagaaaat atggtggtcc tttctataca ggtacaaaag 43440 gcatctcagg gtcacaaagt tcaggctata taatggaaat tgactacatt gtactgagag 43500 gatagttgct agaaattatg ggtaggatat taaaggtttg cttggagagg cacaaaattg 43560 aacattatgt ggtttagtga tttattttta tttttattta tttatttttt tgagacagag 43620 tcttgctctg ttgcccaggc tggagtgcag tggcatgatc ttgggtcatt acaacctcta 43680 cctcctgggt tcaagcgatt ctcatgcctc agtttcctga gtagctggat tgccaccaca 43740 gctggctaat ttttgtattt ttagtaaaac agtgtttcac catgttggcc aggctggcct 43800 caaactcttg acctcaagtg attcgcctgc gtcagcctcc caaagtgctg agattacagg 43860 cctgaaccac tgcacccagc gtgtaattta gagtagcttc tagacccaga ctgctggatt 43920 ttgttttaat ccatattcta tggatttgaa ttctagcttt gatgctatct tctgaaacct 43980 tggatgatta catgactaca ttgtgctttg atttcatcat ctcacattgg cgataatgtt 44040 aatactgact ttataaagtt gttatgaaga ttagatgaat taatatatgt aaagatattt 44100 agaacagagc atgacacata ttaaccctat gtaagtttta tttttgtttt aaaggatagg 44160 gagagggaaa gtagcattgg caggagtatc ccaatatgtg gacatggcta atgcaaagac 44220 ataggcaaga gcaagataat aatgaactgt agcaattaca ttaagttgtg gttaatgtag 44280 agcaggagta agcaaaccac agccctttat ttgtaaataa agttttattg gaacatagcc 44340 atgcccatat ttttacatat tatctatggc tattttcatg ctataatgct agagttgact 44400 agttgcaaca gactttctgg cccgcaaagc tgaaaatatg tactatctgg tcctttacag 44460 aaaaagcttg ccaactcttg atgttgagaa tgtttgcata tgaagaacat atggaacatt 44520 ttgacttcaa attctaaaag ttttagaaat actaaacttg acctatcttt atccttcatt 44580 attagtagca ttaccaattt tctatgtctg gttgtatcca gagcatgtta ttctgctatt 44640 actgtggaaa gttctttgat agggcagtct gattgctttt aatctcttta ttccttgaaa 44700 caggtgtggc agtagctctg tggacccagt ttctgtgaat aggctgagct ttgtgaggaa 44760 cagatgcagg tgagatccta agtggtgaaa accaaaggga tggccaaata cctgcagaga 44820 tcatcacatt tttacctgtc ttactgtagt cgttccttca gcagctctca cttgcatccc 44880 ttacctccca cttaacatcc cttacctccc acttactttt tttctggcaa tattttccta 44940 aacttctaaa acttctcttg aaaatcctgt ttaaggaagt cgctatgcta ttttacctac 45000 tttcctccta ctgcatacct tttggttact ttactttggc aagggtaaaa atgtggcggt 45060 catttttggg tgggaaagat gattatcctg ttttctaaac tcctaagagc ataaacttaa 45120 aagtactaag gcagcattgc cctttgagtt ttacgggtag attttttttt tttttttcaa 45180 actcctgtaa ctcttctagg aatggacttg gctctgtaaa ggatggggaa cctcacttcg 45240 tggtggtcca ctgcacaggc tacatcaagg cctggccccc agcaggtaag aaagtgaaat 45300 agtaaatatt tccccttggt acagttggtt cctcacagag tccatgaaag ctaatattta 45360 ttatatacct ggtatatgaa atgtactttt gtgtaagatg aaagaaaata ggaaaagaaa 45420 atgtacaatc cttcccttcc attattgagc ttttattcca gttgaggaga tagataactc 45480 aggctggaaa atgattcagt attggctgtg tcacagaatg tggtttttat gtgaacaaat 45540 ttatactgaa catatgtatt ctaagcattt gttgcaaaga aacttagaca ttgaatgcag 45600 ttaatttgag aaagatttct aaagtaggaa caagactttg agagaaaagg ggaaaatgcc 45660 tttattgtaa taacttatca agaggatatt ctctgcaaag actttaaatc aagctttgag 45720 cagattagct ttaccagaac ttgaggtcaa acaaggaatg tgagaaaggt gattgggttg 45780 caggatcaaa gttttaagtt ggcttgtcag agtttccaaa tcttagcaac tttattactt 45840 ccctgctgcc tgggtattat tggaaagtag gggttttggg gagacagaaa ctaagagaaa 45900 agagaagcaa ggtgatgtgt tttggaaaaa ggttaaactt tggatgtgga gaaacctgga 45960 tgtgattcct gtcattgtta cttattagtg gcatcaccta gggtaagttg cttgaccttt 46020 ataaagctca gttttctcat ctgtaattca gagttagtac atcctgtata gggtttttgt 46080 gaggattaga tttaatgtaa ggaaagcatc cagcccagtg cctggcatat ggcaggtaac 46140 ccaataaaag taattaatgt aatttaaaaa aatttaactg aagtagtaat gacatttgaa 46200 ctacttagtc tatatactat ataagccaca cagttaaagt atgtgatctt tcatacctct 46260 atgtagcatc aaggaatact atttttctgg ataaaaagag tataactatg caaaaaacag 46320 gggagaaatg cagtcttctt ccctttctgt gtaaaacatt ggtttttctc ttttccaagg 46380 gacatgaata actattgatg gttggtataa cttcattttg ggttgcttgc taactttaaa 46440 agttacagat taggcaaagc ataaattttc tgcctataac atggtcatag aatggatgtc 46500 ttcatatgtg ccatatttgg ccagcatagt tttttagagt actctgggta ggacttgtat 46560 tttccagttt actataatta acatgggtaa aatgtaggaa ttaatatata tgtaaatact 46620 taaaacaatg actggcatat atggtaagtt ttatatactt gtttattctt atttatcatt 46680 ctctattgct ttatgcttag cctcttcata actagatgta ttttgttttg ttttgctttt 46740 tggttttttt tttgagaggg agtctcactc tgtctgtcgc ccaggctgga gtgcagtagt 46800 gcgatctcgg ctcactgcat cctccgcctc ctgggttcaa gtgattctct cacctcagcc 46860 tcccaagtag ctgggattac aggtgcatgc caccatgcct ggctaatttt tgtattttta 46920 gtagagatgg ggtttcactg tgtcggccag gctggtcctc aagtgatccg cccaccttgg 46980 cttcccaaag tgttgggatt acaggcgtca gccacggtgc ctggcccata aataggtcta 47040 ttttgaatct ttacttgtct gagttttgaa ggcatttgag ttggaggtcc ccgttaaacc 47100 ttttaacgtc acgtttctga aggtgtttcc ctcccagatg atgacccaga ggctggccag 47160 ggaagcaagt tttgcctagt ggccattggc agattgcagg caagtatgaa ttttccacat 47220 ctatattccc gttcaattag agcagatctt caggactcat tcctgttaat tttcttttac 47280 tttctgaata caaatgaaga attccataaa actctcaaaa tttgaaggaa tatggcattt 47340 atagtgacca ttgctattct tggatttaag taaagttgaa aagtatgaga ggagggagat 47400 ctttttcccc ttgtcttaat tttagcttta ctatgcttaa ttttctattt ccagttaatt 47460 tcctttgccc ctatacaaaa gaagaaagat ccttttcatt gtatcattac ctgactaaca 47520 atagaaaagt ggaattattt tgattttttc ataagtatag ataagtttct tggttacttg 47580 taccatatca acctgagtaa tgagttcagc atagccagta tgtggatttt agattgaata 47640 aactttattc ttactttact aacttggtaa agtgtaaatg tatgggagca gagctagacc 47700 ttatgccttg tctgattgtg attgtcattt ttttttcctt tttggataaa atgtgaaagt 47760 ttagaaagtc ctaaaactgg gaatcttatg tctatgcaaa agaccatgag gagataggaa 47820 atacatctgt aaataatggt atcattttac ctcattttta tctcttcact ctcaggtaac 47880 tagttctccc aactgtacag acatgagtaa tgtttgtcaa ccaacagagt tcatctcccg 47940 acacaacatt gagggtatct tcacttttgt ggatcaccgc tgtgtggcta ctgttggcta 48000 ccagccacag gtgaggagct ggagctccat taggcctcca ttttcctttg gctatgttga 48060 cattatgtaa tcatgtagtt cctaagacag ccaaaacata tcaacctcag ttaagaaaaa 48120 gagatcatca tattctgtta gtacctaaca ttattttcag cttcctatta ggactgtcat 48180 ctcatgtaga gaaatatggc ttgtcaaacc aggtgggagc agcaggtaca aatatgtatt 48240 tattttttgt tgttgatatt aatacagatg attcaaaggt actcatatta attagttata 48300 ccagtatagc tacatttaga taattcatgt aattacctaa atgaataatg gcccataaaa 48360 catgcagatt tagcaccagt tattataatt tactcatgca acagaccagt tagccatctc 48420 tgaattgacg catcatataa acttttaaaa ctgttgtggg tcggaaggac ttctggctgt 48480 ggctatgtga aagaggttgg tgaaaaagag gtcttgaaaa caaagaacaa agagaattta 48540 cactacctga ttcaacacta actataaagc tattaccaag acactgtggt gttggtgtaa 48600 ggatagatat atagatcaat agaccagaat aaggtctatt cttatacttg tcaactaatt 48660 ttcagcaaag gtgacaagac aattcaatgg ataaaataaa tatttctaac aaatggaaca 48720 attggatatc tgtatgcaaa aaaaaaaaaa aacaaaaaaa aaaccacacc caaaaatgaa 48780 aacacataga tcttacctca tacaatttac aaaaatcagc ttagaggcct aaatgtgtaa 48840 gagctaaagt tacaaataaa ctcctaggag aaaatctttg tgattttgag ttaggcaaaa 48900 gatttcttac actaaaagca tgattcacag aagaaaaaaa attataaatt ggatttaatt 48960 gtaattaaaa tttgccctct ttaaaggata ttattaagaa aatgaaaaga ccagacataa 49020 atggagagaa aatagttaca agtcatatac ctgaaagagg atttgtacca ggaatatata 49080 aacaactcat taagacaaac agctggtaaa aaagagcata agacttgaca tttgactgaa 49140 gaataaatat gcatttatgc acatgaaaag atgctcaaca tctttttacc attaggaaag 49200 tgcaaattaa aatcacaatg agataccact atatacccac tagaatggct gtaatcaaaa 49260 agtattggtg aaaatgtgta gaagctggaa ggaaccctca tacattgctg atagacatgt 49320 aaaatggtat agctactagc tttgcaaaag cattttggca gtttcctaca aagttaaaca 49380 tactcttagc ctataaccta gcaattttat tcctgagtat ctacctaaga gaaatgaaaa 49440 catgttcacc catagatttg tacacagttc atatctgtat tattcataat agccaaaaaa 49500 atgaaaacta tttaaacgtc cattaacatt ttgtaaatga atacacaact gtgttgtatc 49560 catgtgagaa tactactgag cataaaaagg aataaactac tgataatgca gccatgtaga 49620 tgaacttcaa aaataccatg ctcaatgaaa gaagccagac ccaaaagacc acatattatg 49680 ttgttttatt tatatgaaat ttgtagaaat agcagaacta gagaggcaga aagcagattt 49740 gtggttggct ggggagttgg agtgggagca gagattgact gcagatggca caagggaaca 49800 tcttggggca gtgaatgtgt tctgaaactg gattgtggta atcattgcac aactataaat 49860 ttagtagaca tcatcaaatc atacacttag aatggctgaa ttatgaatgt aaattttatc 49920 taaaatttat aatctcatta aaataaatgt ataatattct gagaaagaaa aatgttttag 49980 aagccagctc cttaacagat tctgcctttt tttagtagat ttcatctttt gtttattgtc 50040 tttttttttt ctcctcctca cttaactata atcttaggat taaaacagaa gaaataaaat 50100 ccaggtcccc agctgatgga ccaggccagt tagatgacca taaaattata tatgttggct 50160 gggcacggtg gctcacacct gtaatcccag cgctttggga ggccgaggcg ggtggatcac 50220 ttgaggtcag gagttcgaga ccagcctgac caacatggtg aaaccctgtc tctatttaaa 50280 aaaatacaaa attagccagg tgtggtagca cacccctgta atcccagcta cttgggaggt 50340 tgagacagga aaattgcttg aactcaggag gtggaggttg cagtgagcca agatcgcgcc 50400 atcgtactcc agcctgggca acaagagcga aactccatct taaaaaaaaa aaagtatata 50460 tcttactctt ctttctgtat tctaggaact cttaggaaag aatattgtag aattctgtca 50520 tcctgaagac cagcagcttc taagagacag cttccaacag gtaacttttt tcctggtttg 50580 gttctgaata aatatttgtc atattcactc cataaatatt gactactgat taactgaaca 50640 ctgtggcagg cactacagtt ttatgttctt tagtagttaa tctgcatttt taaggaatag 50700 aaaaggacta atactttgaa attatggata atgcccaagg tatttctgtt tggctttggc 50760 tatttactgt cttgtattca attaactgta tccaaggagc tgtctttaag gtatttaaac 50820 tattgcgcca ggcatggtgg ctcatgcgcc caacctctgt agatgctgtg aaaatagatg 50880 ttttcctcgt ctgggcatgg tggctcacgc ctataatccc agcactctgg gaggctgagg 50940 caggtggatc acttgaggcc aggagttcaa gcccagcctg gccaacacag tgaaacccca 51000 tgtctactaa aaatacaaaa aattagcctg gtatggtggt gcatgcctgt aatcccaggt 51060 actcgggagg ctgaggcacg agaatcactt gaacctggga ggcagaggtt gctgtgaact 51120 gagatcatgc cactgtactc tagcctggat gacagagcta gactctgtct caaaaaaaaa 51180 aagataaaaa agaaaattgt atacttcact aagcttgtag tagaaaaatt cattttatat 51240 agtttttttt tttttttaga aggagtctag ctctgtcgcc agggtggagt gtagtgtgca 51300 atctcagctc attgtaacct ctgcctctta ggttcaagcg attctcctgt ttcagccccc 51360 cgagtagctg ggattatagg cacatgctgc cacgcccagc taatttttgt atttttagta 51420 gaggcggggt ttcaccacgt tggccaggat ggtctcgatc tcctgacctc gtgatccacc 51480 cacctcagcc tcccaaggtg ctaggattac aggcatgagc cattgcgccc agcctagact 51540 gttcttttat ggatgagtga gagtcgtaat gaattatata agctgactgt taattgtcat 51600 tctcaggctc cagctcctga aaatatctgg tgaattttat agacatggct tttgataacg 51660 gtttttactt tgtattagac aagttaatta acctctttaa gtctcagtag tgtcgttatt 51720 gatacaatga atatattaat agtacctaaa ttcagacggt tgttgggaag attaaataag 51780 gtaatgaata taaaacacat cacccagtat ttgatacgta gtattacaaa ataagtggtt 51840 agcttctaat actgtttatt tttatttttt taatttttag gaatatagag ttaaaagatt 51900 attttctatt ccatgagact agtatctaaa ataacctaaa attggctggg catggtcgct 51960 catgcctgta atcccagcac tttgggaggc tgaggcaggt gatcacttga agccaggagt 52020 ttgaaccagc ctggccaaca tcttgaaacc ctgtctctac taaaaataca aaaattagcc 52080 gggtatggtg gctcatgcct gtagtcccag ctacttggga ggctgaggca tgagaattgc 52140 ttgaacccag gaggcagagg ttgcagtgac ccaagattgc cccactgcac tccagcctgg 52200 gcgatagagc aagactgtct aaaaataaaa taaaataaaa aataaaataa ctaaaattac 52260 ttttaaaaaa taaaagcaaa acaagactaa agccaactta attttattta tggaaacctc 52320 tgtagatgct gtgaaaacag atgctctcat ctgggtgcag tggctcacac ctataatccc 52380 agcactttgg gaggccaagg caggcggatc atttgaggtc aggagtttga gaccagccta 52440 gccaacatgg tgaaaacccg tctctactaa aaatacaaaa attagctggg cgtgatggtg 52500 cacgcctcta gtccccagct actcaggagg ctgaggcagg agaatcactt gaaccctgga 52560 ggcgaggttg cagtgagcca agattgcacc actgcactcc agcctggcga cagagcgaga 52620 ctccatctca aaaaaaaaaa aaaagaaaaa gaaaagaaaa cagatgttct caggtttcgg 52680 ggaaaaaata ggattgaaga gcaatatata agctatattc tgtgtcctta aacttaccaa 52740 atttctggta tagacttgta aagctaggtc agagtatctt taatggattt cccaagggaa 52800 gtagggaaac agtcttttcc ttcctggaaa taagttatta ttcctatttg actagaatag 52860 tattaggttg gtgcaaaaga aattgtgatt ttttgccatt tttttaaatg gcaaaaaatg 52920 caattacttt tgcaccaacc taataagaaa gcttgagtct ctggccgggc tcagtggctc 52980 acgcctgtaa tcctagcact ttgggaggcc gaggcaggcg gatcccgagg tcgggaaatc 53040 gagaccatcc tggccaacat ggtgaaaccc cgtctatact aaaaatacaa aaattagctg 53100 ggcgtggtgg cacgtgccta taatctcagc tacttgggag gctgaggcag gagaatcgct 53160 tgaaccaggg agtcggaggt tgtagtgagc cgagattgcg ccactgcact ctagcctggt 53220 gacagagcga gactccgtct caaaaaaaaa aaagtctggg cacggtggct cacacttgta 53280 atcccagcac tttgggaggc cgaggcgggc ggatcacaag gtcaggagat caagaccatc 53340 ctggctaaca tggtgaaacc ccgtctctac taaaaataca aaaaattagc tgggcgtggt 53400 ggcacgcgcc tgtagtccca gccactcggg aggctgaggc aagaaaatcg cttgaacccg 53460 aggggtggtg gttgcagtga gcagagatcg tgccactgca ctccagcctg ggcgacagag 53520 ggagactccg tctcaaaaaa aaaaaaaaaa gcttaagtct ctgaaaggaa gcatgagaaa 53580 tatgcttcca tgtttaatca cttagttttt actctcattt tgttttaata ttgaaaaata 53640 ttggtgcctc aaggacaatg acaagagttt tagggttata gaaattggaa aatttttatt 53700 tatttttgta atgaaaattt tctatgagtt ccactgatgg catgaaaact ttttcaggta 53760 gtgaaattaa aaggccaagt gctgtctgtc atgttccggt tccggtctaa gaaccaagaa 53820 tggctctgga tgagaaccag ctcctttact ttccagaacc cttactcaga tgaaattgag 53880 tacatcatct gtaccaacac caatgtgaag tatgtattat acaggagtgt gaaaaaactg 53940 tttttcctct gttctcacaa cagaaaacac ttctgatgcc ctatgtgggg ggtaaacaat 54000 caagcaacaa cacaaaaatt tagccgggcg tggtggcatg tgcctgtagt cccagctact 54060 ctggaggctg agacaggaga atcacttgaa cctgagaggt ggaggttgca gtgagctggg 54120 atcatgccac tgcactccag cctgggctac agagcgagac tccatttcaa aaaaagaaaa 54180 agaaaaaaga aatcaagcaa tcagtagtgg acaccagctg ggtgtccttc cattcaattc 54240 agttcactat ctacttggag atagcatcag atcccccaat ttgtgtatgc agtaccacaa 54300 gactgctccc acttctgatg ccagttgcaa gccccaggtt gttttacctg tgcatctgac 54360 tgaccagctg tctcccatga ccccctactt gggttcagtc aatttgcttg aatggctcag 54420 ggaacattta cctatgttta ccagtttatt ataaaggata ttacaaagga tactttgtac 54480 atcagatgaa gagatagata gggcaaggta aggaggaagg agtgcagagc tttcagaccc 54540 tttctgggtg ggctaccctc cggggatctc catgtgttta cctatcaaga agctcctcaa 54600 acccagtcct tttgggtttt aatggaaatt tcattatgta gccatgagtg attaaatcat 54660 tggccattgg taatcaactt aaccttaggt accggctccc ctccatgagg ttgagggtta 54720 gggctaaaag tcccagccct ctaattttac cttgatcttt ccagagatga gcccccatct 54780 tgaagctacc taggggttgc cagccctcag tcaactcatt agcagacaaa aagacactta 54840 tcacactgaa gattccaaag attttaattg gtaaaaatcc aggtccactt atacatggat 54900 ttttttcagt aaatatatta gaaaattctt ttgatatttg tgacaatttg aaaaacccaa 54960 aaataagcta catagcctgg atatattgaa aaaattagaa aaagttagcc atgtcataca 55020 tgaatgtatt aaatatatat aaattctagt ctattttatc atttactacc atacaacata 55080 tacaaatcta ttataaaaag taaaaatgct ggacaggtgc agtggctcac acctgtaatc 55140 ccatcacttt atcactttgg gataccaagt tgggatacca agttgagcag atcacttgag 55200 gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ctgtctctac tgaaaaaaaa 55260 aaaaaaaaaa aaaagaaaat acaaaaatta gctgggcatg gtggcacacg cctatagtcc 55320 cagctacttg ggaggttaag gtatgagaat cacttgaacc tgggaggtgg aggttgcagt 55380 gagctgagat cgcaccactg cactccagcc tgggtgacag agggagactc catctcaaaa 55440 aaaataaaaa taaaaatgta tcacaacgta tacatacaca ccgtttgtac aatggcacca 55500 tttgtagttg agagaaatgt aaacaaatgt aaagatgcag ttttaaatca taaccgcgta 55560 aagttaacta tagtatatac tgtactgttg taataatttg gtagccacct attgctcaat 55620 tgttgccagt ttgcttaaaa tgctgtgtga tgctaatcat ctcttcatga gcaattcact 55680 ccagtaaatt gcatattgca gtaaacagtg aaatctcatg gttcttgcat atttttcatc 55740 gtgttgagtg caatactgaa accttgaata acatttggga acagtatgaa gtgccactag 55800 tgatgctgga agtcttccca agaagcaggg aaaagtcatg acattataag aaaaaggtga 55860 attgcttgat atgtactata gaatgaagtc tgcagctgtg gttgcttgcc acttcatata 55920 gatgactcat cttgtaagtc atcttgtaag atgatataag cttatgataa cgataaatac 55980 agtacagtgc tgtaaatgca ttttctcttc cttatgattt tcttaataac attttctctt 56040 tagctttatt gtaagaatac agtacataat acgtacaaca tacaaaacgt gttcaactgc 56100 ttatgttatt ggtaaggctt ctggccaaca gtaggctatt agtagtaacg ttttgggaga 56160 gtcagaagtt tacagtcgaa ttttcgatgg cacaggggtc agtgccccta gcctccatgt 56220 tgagcaatca gttgtatttc ataatatcac ataggcattt tccaatgttg ggatttctaa 56280 tgtaccaact cttcttagtc ttatcaagct atccctttct cctctacttt tctagtatta 56340 aacaccatta gtttgaagtt cttcattttc cagcttatag gatttgggat agttgatcag 56400 agtagagtaa ggtttttttg ttttgttttg ttttgttttg ttttttcaga cagggtctcg 56460 ctctggcacc caggctggaa tgcagtggtg tgatcatggc tcactgcagc ttgaacctcc 56520 caggctcaag tgatcctccc acctcagtcc cctgagtagc tgggactaca gacatctacc 56580 aagacgcctg gctaattttt gtattttttg tagagatggg atttttccat gttgcagagt 56640 ctggtcttaa actcctgggc acaagcagtc catcgtcctc ccaaagtgct aggatcacag 56700 gcgtaagcca ctgcacctgg ccagagtaag attttacaaa caataattca gagactaggt 56760 cctgagatgg aagggtttag tttcaattct atgtatcaac tctttctgga aaggactatg 56820 ttttgtacac cagtttattg ctctagtctg gcaggatttt cctctggagc aggggtaggc 56880 aagctctttc tataagtggg gctagatagt aaatatttta ggctttgtgg gccatgtggt 56940 ctttattcca cctactcaac tctgctgtag catgaaaaca gtcatagaca aatgaaagaa 57000 tggggctgtg ttccagtaaa tctttattta gaaaaacagg cagtgggcct gatttggtca 57060 gttttcttta gagcatagct acatcctcct ctttcattag ttactgttcc tctcttttgc 57120 acttttctat ttgagttttc tttctcagtt tcttttttaa aaagttttat gagatatatt 57180 tcacatacca tatacctcac tcatttaaag tatacagtgt tttttagtat atcacatagt 57240 tatgtaactc ttactgcagt ctaatttcag aatattttca ttcccccaaa agaaattcta 57300 gatcagttaa cagtcattcc ctgttctttc ccccggctct gaaattctgt ataactatga 57360 atctgttgtc tttataaatt tgtctattct agacttttcg tataaatgga atcatagact 57420 ttgtaattcc tttgtaactg gcctatttta cttagtataa tgtttacaga ggttatctgt 57480 actgatacat gttgtagcat gtatcagtac ttcatttctt tttattgcca aataagattt 57540 cattgtatgg atataccaca tactgtctgt atgttcgtta atggacattt ggttgtttct 57600 acttcttggt tatcaaaaat gcttccatat gtaaaatagg gataaggaac tgaaaaaatg 57660 ctttcatgaa tatttgttta ctagttttta tgtggatata tgtttttatt ctcttgaatt 57720 ccacccagga gtggaattgc tggccacata gtaactctgt tttacctttt gaggaactgc 57780 caaactgttt tccaaagctg ccccatcatt ttgcattccc aggagctctg attcctccac 57840 atccttgtta acacttgtta ttgccttttt ttttttattg tagccatcta gtaggtatca 57900 attgtcaggt taccaccttg ccatttaatc ttttctttct gtagaattgg ccttgaatcc 57960 acctcttgaa gatcagatta tgactactta gcaaatatga ataataccta gtgacaagtt 58020 ttgtccaatt cagtctttag aacttgtaaa gtttaattct ttgctaacta acttagtata 58080 acagagtgga ttgagttaga aatttttatt aacttaggac tcaagtggat ggtcatggtc 58140 attgtagttc tttatttttg tctctttgca tctttatatt acacaacagc cttcattttt 58200 gtgtttccat tttgtttaaa tttttaaaaa tttaagttgt ttgaaggact cgagtttctg 58260 gaaataattg ctttctccat gtttatgtat cattttccct accatgatta attttaatta 58320 gcttttaggg atttgtttgt ttgtttgttt cattgagaca gtgtctcact ctgtcaccca 58380 ggctggagtg cggtggcgct atctcagctc actgcaacct ccgcctccca ggctcaagca 58440 atcctcccat ctgtctcctg agtatctggg actacaggag cccgccacca cgcctgctaa 58500 tttttgtatt ttttgtagag acggagcttc accatgttgc ccaggctggt ctcaaactcc 58560 tgagctcaag caatccaccc tgcctcagcc tcccaaagtg ccaggattac aggtgtaagc 58620 caccgtgcct ggccagtatt ttatttttaa ataaatattc tttatagaaa atgtggaaag 58680 tattgaaaaa tacaaagaga aggaataaaa ttctcacact ccagatagtc tttgttgatg 58740 tacagtgtat gtactttcat ttttttacct atgcttttaa aaaatacctt atatgaatat 58800 atacacacat tcacaataac caggttttga atacctagaa ggagttagaa cccaacaaga 58860 tgacccctgt gtcatagaac atggtttctg tttgctgaaa ctgacaccct agattaacta 58920 taggatagga ctttaatgaa ggatttattg attgttccac ctacagatga atctatagag 58980 ctttacatac agaatcttat tcttctcttt ctctctctct ccccccttcc tccctctcca 59040 ccccctcttt actgtctact ctggtttcct aggaactcta gccaagaacc acggcctaca 59100 ctctccaaca caatccagag gccacaacta ggtcccacag ctaatttacc cctggagatg 59160 ggctcaggac agctggcacc caggtaaaaa agggtgaaat aatcatctgt tgagcagtca 59220 cccagggggt ggtcatttgc aatcccatat attttttgtt cggttggtta attttttttt 59280 tttttttttg agaaggagtt ttgctctcat tgctcaggct ggatggaata caatggcacg 59340 atctaggctc actgtgacct ctgtctcctg ggttcaagtg attcacctgc ctcagcctcc 59400 caaatagctg ggattacagg tgcctgccac cacccccggc taattttttg tatttttagt 59460 agagacgggg ttttgccatg ttggccaggc tggtgtcgaa ctcctgacct caggtgatcc 59520 acctgcttcg gcctcccaaa gtgttgggat tacaggcatg agccaccatg cccggcctgt 59580 ttaatttgtt ttaaggttct ttctccagat tcttttttaa aaaaaatttt tttttctatt 59640 tgtcttgtca actggccttt gacatatagg cagcagcaac agcaaacaga attggacatg 59700 gtaccaggaa gagatggact ggccagctac aatcattccc aggtgagttg tgtcctcttc 59760 gttgaagagg gtagggagta tttacttagg aagtgttctc cggtactagt tagaatgtac 59820 atatgttgta tatgaatttt agggttattg aattgtcatg ttaaatcttt aatggttatt 59880 tttatcattg tattccacag gtggttcagc ctgtgacaac cacaggacca gaacacagca 59940 agccccttga gaagtcagat ggtttatttg cccaggatag agatccaaga ttttcagaaa 60000 tctatcacaa catcaatgcg ggtatgtttc tttctcatta tccttttaaa ttctcattta 60060 gatcacttac tgatgggcat gccactgccc agtcagtaat cttccagtgt ttttccactt 60120 aatcataata ccacctgagt aaataggaac ttgctgaact aatatactac agccccttga 60180 ctggcccttc cccaactcct tttggtccac agatcagagt aaaggcatct cctccagcac 60240 tgtccctgcc acccaacagc tattctccca gggcaacaca ttccctccta ccccccggcc 60300 ggcagagaat ttcaggtgag ccccgtatat atgtgctgct ttacagggcc ctgagggatt 60360 cagctgctga atccaaattt tattcttccc ttgctttctc tggttacttc agaaaaagca 60420 gtgaagcttg tagggcctag cgtgaggcaa acaagctgct tttcttcctc ctatttcttt 60480 gcacctgtcc tattgccatg ttctaggctc catctctgtg tgtcctggtc agtgtgtgac 60540 tgtcagtctt tcttgtcttt tccaaattgt tatcaaattt tccttaacct gcaggaagtc 60600 aaggggatct agggatagca ctagattgtc ctttgattcc tagcttctgt gataaatcta 60660 tccttttaat cttttacctc atttattcac tcctaggaat agtggcctag cccctcctgt 60720 aaccattgtc cagccatcag cttctgcagg acagatgttg gcccagattt cccgccactc 60780 caaccccacc caaggagcaa ccccaacttg gacccctact acccgctcag gcttttctgc 60840 ccaggtaaaa cttatcatct gtgtgttccc tgtgtattat tttttgtttg tttgggcttt 60900 tttccgtatg taaaatcagt gttttctatt ttaaatacct tctccccaac ccctgttctc 60960 cggtttccaa tttccatctt tgttgagagt agctaattaa aaatcacaga taataaaaaa 61020 aaatctcagt agagtctgtg gttttcacct taaatcagaa ttgctcactt ggaacgtttt 61080 gagcacgtct gattttcaga tttgttttgg agtaaattct aagatgtttc ctctttgttt 61140 aggactccat aaggcaggag caaaggagaa aattaatgac taacttacag tgatgtctgt 61200 ttacaaaaaa gttgaaaaat tctttttttt tttttttttg agacagagtc ttgctctgtc 61260 gcccaggcta gagtgcagtg gtgcgatctt ggctcactgc aagctctgcc tcccgggttc 61320 acgccattct cctgcctcag ccttccaagt agctgggact tgtacaggcg cccgccactg 61380 cgcctaattt tttttttttg tatttttagt agagatgggg tttcaccatg ttagccagga 61440 tggtctcaac ctcctgacct tgtgatccgc ccacctcagc ctcccaaagt gctgggatta 61500 caggcgtgag ccaccgcgcc tggctgaaaa attctttttt tttttttttc ttgagacaga 61560 ctgtcacttt gttgcccagg ctctggagtg cagtggcgcg atctcggctc actgcaagct 61620 ccgcttcctg ggttcatgcc attctcctgc ctcagcctcc cgagtagctg ggactacagg 61680 gtgctcgcca ccacgcctgg ctaatttttt gtatttttag tagagacggc atttcaccgt 61740 gttagccaag atggtcttga tctcttgacc tcgtgatccg ccctcctcgg cctcccaaag 61800 tgctgggatt acaggcatga gccaccgcat ccagccgaaa aattctttta taatattcat 61860 atatataata taacgcacaa taaatacact gtctaaagaa agattcttta atattactat 61920 atatttatgt tatacaagta atagttttaa aaagtcaaaa ccaaaagcag gttccagaat 61980 gttatatgca atacgatctc aattgtgtac aaaatgcatg agaaaataga aactggaaga 62040 aattatccaa acatgttaac catggaatta tgaatgattc ttattttctt tatattttcc 62100 ttcactttct gaatattcta taatgcatat acagaactct catgagaaaa tagtttttat 62160 aaaaaataca tcattaggaa caaatgaatg cagaacagac agaataatgg tgcagagtag 62220 tttttctctg catatggtac tttttttgtt gtttgttttt ttgagacaga gtctcgctgt 62280 tgcccaggct agagtgcagt ggcgcgatct tggctcactg caacctctgt ctcccgggtt 62340 caagtgattc tcctgcctca gcctcctggg ttgctgggat tacaggcacg tgccaccatg 62400 cccagctaat ttttgtattt ttagtagaga cggggatttc accatgttgg cccggctggt 62460 ctggaactcc tcacctcagg tggtctgcct gccttggcct cccaaagtgc tgggattata 62520 ggcatgagcc atgagccacc ccgcctggcc tgcatataat actttactgt tatgaatgcc 62580 tctagtttta tataacttca cagtttataa gatgctttca tttaattctt acaattttta 62640 ttaatcccat agttcattgc ttttttgtaa ttttatctca gctgcctaaa aaatagtgtc 62700 aagagagatt gagagttaat tggaagaaat atacaatagg aaataagtga tgagcttggt 62760 tcagaaggat gcagtgattg acagtgttga ctctcatagg catggtatgt gcaatgatgt 62820 taatgctgta tttgttctat atcccctctc catctctctt tagcaggtgg ctacccaggc 62880 tactgctaag actcgtactt cccagtttgg tgtgggcagc tttcagactc catcctcctt 62940 cagctccatg tccctccctg gtgccccaac tgcatcgcct ggtgctgctg cctaccctag 63000 tctcaccaat cgtggatcta actttggtga gtccagacca taaggagagt aacaggaaaa 63060 tcgcaccact aaagagaaag gatttggtag ttaaagttgt ttgcctgtgt tgtgggtaca 63120 ctgacctgat tgtagggaaa tgcaaggtga caatctattt agaatttaaa acctaccagc 63180 tgggtgcggt ggctcacgcc tgtaatacca gcactttggg aggctgaggc aggcggatca 63240 cttgaggttg ggagttcaac cccagcctga ccaacatgga gaaaccctgt ctctaccggg 63300 tgtggtaccg catgcctgta attccagcta ctcgggcggc tgaggcagga gaatcgcttg 63360 aacccaggag gcagaggttg cggtgagctg agatcgcgcc tttgcactcc agcctgggca 63420 acaagagtga aactccgtct caaaaaaata aaaaataaaa aaaaaaaact acccacatga 63480 aaaatacttt agcacatata acaaaaatca tgtgaatttt tatacattta atagtatgca 63540 catttaacct aaatgagtaa agaactctat ggaaaggctg cctggagaag aagaattaat 63600 ttagggctga gttttgagat agaaaaggca ttgattggca gagagaagga cagagttatc 63660 ctaggtaaaa ttaatggctt acctatagtt gtttacttgt ggcattagta cacaatggaa 63720 ttgtgtagat tggagttgtt tattcttcct tgctgtattt ctagctcctg agactggaca 63780 gactgcagga caattccaga cacggacagc agagggtgtg ggtgtctggc cacagtggca 63840 gggccagcag cctcatcatc gttcaagttc tagtgagcaa catgttcaac aaccgccagc 63900 acagcaacct ggccagcctg aggtcttcca ggtaagagag tgaaaagact ttcaaaaatt 63960 agaagctggg agagaaaggg tccaggagga ggagagacag tgaaggaagc atgcctggat 64020 tgaggtgttt ggttgggggt atatgtgaga agacagagag ggataaatgt agggatcact 64080 gtcagttatt gaaaagattg cagaagctag atgcagtggt gcttgtgtat atgatgtcag 64140 ccccttagaa ggctgaagca ggggatcact tgaggccatg agttcaaagc caggctgagc 64200 aactagcctg atcctgtccc tgtcaaacaa acaaaaaagg agtatgaatt gagtgtgata 64260 cataccattt aaccagaaca gacaaattta gcaccatagg aagatgccaa agaaagttac 64320 tttagctcat tcaaatagct ccatataccc aagtcacagt agctttgggt ttaaaagaga 64380 cagaatgatt aaaaataaaa agtagtgctc gcttcagcag catatatact aaaattggaa 64440 tgaatataga gaagattagc atggcccctg cgtaaggatg acacacaaat tcatgaagtg 64500 ttccatttaa aaaattataa aaagtaaatg aaatagaaca taatgattat agccataatg 64560 gtctatttac acaagtcctg agggactgca agagtgaatg gagtaatctt aggcaggaca 64620 aaggaagagc tggtttaaag caaagattga aagaaagcaa aacaggtctt ggtggaaaac 64680 aaataggata agagactcca tatatgtcta tagggggtta tatgaaatac agcaagcaga 64740 tttttctccc tttgaaaata ttgagaacta ggaaaaggaa aaaggtggaa ctgtaggagg 64800 aagacagaag ggattaggaa aaaaggctgc gatctaaagg agtcaaagtt gttggaagta 64860 aggaaggcta agagctcagc acagcaaaga ctcggggtca gggatggtag tgcaggggaa 64920 tggtggagta gaacttggta agtgtaagag atcaaggtgt gtgacccaaa cttaatcttt 64980 ttcttttatc aggagatgct gtccatgctg ggagatcaga gcaacagcta caacaatgaa 65040 gaattccctg atctaactat gtttcccccc ttttcagaat agaactattg gggtgaggat 65100 aaggggtggg ggagaaaaaa tcactgtttg tttttaaaaa gcaaatcttt ctgtaaacag 65160 aataaaagtt cctctccctt cccttccctc acccctgaca tgtaccccct ttcccttctg 65220 gctgttcccc tgctctgttg cctctctaag gtaacattta tagaagaaat ggaatgaatc 65280 tccaaggctt ttaggactgt ctgaaaattt gaggctgggt gaagttaaaa cacctttcct 65340 tatgtctcct gacctgaaat tgtatagtgt tgatttgtgc tgagatcaag aggcaggtta 65400 gaagaacctg acatccactg tttgccttgg atagtatggc ttgtttttgg aaagaaattc 65460 tgaagagagt ggaggagagg agaaatgtcc tcatatttga ggaccatgaa acattgtagg 65520 tatatatggg gctttagcaa gtttgagcat aggctctttt tgctgcctgt gagcagtccc 65580 tctggaaaga aacatgtgag taagtgagag agagtgtgtg tgtatgtgtg tgtgtgtgtg 65640 tgtgcgcaca catgcttctg tatttcactc tttctcccta ttagggagtt atgcaaaatt 65700 tgtccccgat tttacctttg tctttctgtg tacttttcaa agagtcctaa ggagttaaat 65760 cttccaggta ttttccactt agtattgcag ccaaagaata tttaaataaa cgtctttgct 65820 gcgcttgcat ccatgcccag ccaatataca actgtaaagc aaatatagaa agtcggctgt 65880 tgatacgatt gtctgttatc gaacacattc agtgataaag ctgggttact gctgcttttg 65940 gtgctctcac cttatctgga agatctgcaa acattaccta aataggctgg caagataaac 66000 actttctgga acccgagact tggccataaa gataatgctg catttttctg tcagaatcac 66060 atatgatgtg tgttctgtag aggttatttc tgcatggaaa ctcaacttct tggattagcc 66120 gtcccagtga aaatcctcat tgttggagtg taaaccaaat acgaagccct cttgcaaagt 66180 agcctctttc atcccatact caaaataccc agtttagcaa gcaactgaga tttaagtctc 66240 tctggcccta agaggttttt cctctttgct ccctccaatc ttgagattgg gttttgcttt 66300 agagtgcaag tatcataatt ccgtatgata gatggggcct ggacacccat ctcaacaggg 66360 tcacttggta attaacaata gccatataaa tgcggataca ggttactacc ctcacccttt 66420 accttcctca ggtaacagtc gtagatacca gctttttttt tttttttttt aaattggctt 66480 tggccagtag ctaaagtgca agactgaatt aatgagaaga tatattaaat gtagtcatag 66540 gggactgagg agcaagggtg gccttgaaga ggccaaagga atgtccattt gctgagtttc 66600 ccttccttat gtctccagtc tggtgccagg tagtggagta aaaaaggaga cagtttattt 66660 ttttattcta tgtgcacact tacagtatac atatatattt atatcacaat ttacgaaacc 66720 aaaaagttga gtttccaatg gaacccttgt tttttaataa tcgacttttt aaatgtgatc 66780 aggactataa tattgtacag ttattatagg gcttttgggg aaggggagga tagcgagaag 66840 atgctctggg ggttttgttt ttgcttttcc ttcagggttt tatttttgac tgttttgttt 66900 tcttgttggc catttctgta ttgctggcat ctgtgctaag ctttacagtg gcaaaaataa 66960 tgacatgtag caaagatttt caaacaaaat attttttcct tttgtaaaat ttcttgtgtt 67020 gtgtgatctt gattgcggct ttatcattcc tttccagttc ataaacaaca ggcacccaca 67080 accagaggaa tctatagttt aagctccaga catacaaaca taaggcacat tgtgtcttta 67140 atttcaggaa tcagaaatca tagggttctg atcacattgc acgcctcccc cctcacttgt 67200 cctcctgatc ctgacacatt ctgagtaaca tcagcaggaa tgctctgacc atgaggtggg 67260 ggttttgggg tgggcgttgc ctgggttctt gggagagagg ggaagagtcg ggacttgaaa 67320 accactaggg cacatctgga tgccttcccc cagtatgtcc ttttctggat taaaatgagt 67380 gaaatttaaa ctgttcaagt ctggacctgg tttccctcta ggagactatg ttggttcatt 67440 agcaactttt tttttttttt ttttttttgt gtgtgtgtgt gtgtgtgtgt gtgagagatg 67500 gagtctcgct ctgttaccca ggctggagtg cagtggcacg atcttggctc actgcaacct 67560 ctgccccttg ggttcaagca attctcctgc ctcagcttcc caagtagctg gcactacagg 67620 tgtgtgccac catgcccagc taatttttgt gttttttttt tttagtagag atggggtttc 67680 actatatgtt ggccaggcta gtctcgaact cctgacctta ggtgatccac atgctttggc 67740 ctcccaaagg cctgggatta caggcgtgaa ccactgcgcc tagcctg 67787 <210> SEQ ID NO 19 <211> LENGTH: 501 <212> TYPE: DNA <213> ORGANISM: H. sapien <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 217, 441, 498 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 217, 441, 498 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 19 ggagggagtg gtaatttaca aaggatgtga agtttccagg aaataggggg aagggaatta 60 cattatcttc ttgttctctg tctgccttat tagttctgtt tcatgcttgc tttgcatgag 120 aaggttggca aaccttattt taactgctga gacttaagca tcactaaatc tgaataccac 180 attcttcagc agcacacttg gtatccatat cactctncct gctaccaaat gaccagatgt 240 gaccacctgg atgggggctt ctctttcttt ccatgcaggg aaaatcacag tgaaaattga 300 acggcggcga cggaacaaag atgacagcct acatcacaga actgtcagat atggtaccca 360 cctgtagttg ccctgggctc ggaaaaccag accaagctaa ccatcttacg catggcaagt 420 ttctcacatg aagtccttgc nggggaactt ggcaacacat ccactgatgg gtcctataag 480 ccggctttcc tcactganta a 501 <210> SEQ ID NO 20 <211> LENGTH: 2908 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 20 cttggattcc gcggtagcgg aggcggcggt caggcgccgc ttctggggag tggcctttct 60 tttcccctcc ctcccggttc ggtggcggcg gctcctccca ctgggggggg ggtggcgcgg 120 cggcggtggc atctgcggcc atggcggcga ctactgccaa ccccgcctga tcttggactc 180 aagattctcc tgcctcagac cccgagtagc tgggactaca gaaatgacat cagatgtacc 240 atcactgggt ccagccattg cctctggaaa ctctggacct ggaattcaag gtggaggagc 300 cattgtccag agggctatta agcggcgacc agggctggat tttgatgatg atggagaagg 360 gaacagtaaa tttttgaggt gtgatgatga tcagatgtct aacgataagg agcggtttgc 420 caggtcggat gatgagcaga gctctgcgga taaagagaga cttgccaggg aaaatcacag 480 tgaaattgaa cggcggcgac ggaacaagat gacagcctac atcacagaac tgtcagatat 540 ggtacccacc tgtagtgccc tggctcgaaa accagacaag ctaaccatct tacgcatggc 600 agtttctcac atgaagtcct tgcggggaac tggcaacaca tccactgatg gctcctataa 660 gccgtctttc ctcactgatc aggaactgaa acatttgatc ttggaggcag cagatggctt 720 tctgtttatt gtctcatgtg agacaggcag ggtggtgtat gtctctgact ccgtgactcc 780 tgttttgaac cagccacagt ctgaatggtt tggcagcaca ctctatgatc aggtgcaccc 840 agatgatgtg gataaacttc gtgagcagct ttccacttca gaaaatgccc tgacagggcg 900 tatcctggat ctaaagactg gaacagtgaa aaaggaaggt cagcagtctt ccatgagaat 960 gtgtatgggc tcaaggagat cgtttatttg ccgaatgagg tgtggcagta gctctgtgga 1020 cccagtttct gtgaataggc tgagctttgt gaggaacaga tgcaggaatg gacttggctc 1080 tgtaaaggat ggggaacctc acttcgtggt ggtccactgc acaggctaca tcaaggcctg 1140 gcccccagca gatgatgacc cagaggctgg ccagggaagc aagttttgcc tagtggccat 1200 tggcagattg caggtaacta gttctcccaa ctgtacagac atgagtaatg tttgtcaacc 1260 aacagagttc atctcccgac acaacattga gggtatcttc acttttgtgg atcaccgctg 1320 tgtggctact gttggctacc agccacagga actcttagga aagaatattg tagaattctg 1380 tcatcctgaa gaccagcagc ttctaagaga cagcttccaa caggtagtga aattaaaagg 1440 ccaagtgctg tctgtcatgt tccggttccg gtctaagaac caagaatggc tctggatgag 1500 aaccagctcc tttactttcc agaaccctta ctcagatgaa attgagtaca tcatctgtac 1560 caacaccaat gtgaagaact ctagccaaga accacggcct acactctcca acacaatcca 1620 gaggccacaa ctaggtccca cagctaattt acccctggag atgggctcag gacagctggc 1680 acccaggcag cagcaacagc aaacagaatt ggacatggta ccaggaagag atggactggc 1740 cagctacaat cattcccagg tggttcagcc tgtgacaacc acaggaccag aacacagcaa 1800 gccccttgag aagtcagatg gtttatttgc ccaggataga gatccaagat tttcagaaat 1860 ctatcacaac atcaatgcgg atcagagtaa aggcatctcc tccagcactg tccctgccac 1920 ccaacagcta ttctcccagg gcaacacatt ccctcctacc ccccggccgg cagagaattt 1980 caggaatagt ggcctagccc ctcctgtaac cattgtccag ccatcagctt ctgcaggaca 2040 gatgttggcc cagatttccc gccactccaa ccccacccaa ggagcaaccc caacttggac 2100 ccctactacc cgctcaggct tttctgccca gcaggtggct acccaggcta ctgctaagac 2160 tcgtacttcc cagtttggtg tgggcagctt tcagactcca tcctccttca gctccatgtc 2220 cctccctggt gccccaactg catcgcctgg tgctgctgcc taccctagtc tcaccaatcg 2280 tggatctaac tttgctcctg agactggaca gactgcagga caattccaga cacggacagc 2340 agagggtgtg ggtgtctggc cacagtggca gggccagcag cctcatcatc gttcaagttc 2400 tagtgagcaa catgttcaac aaccgccagc acagcaacct ggccagcctg aggtcttcca 2460 ggagatgctg tccatgctgg gagatcagag caacagctac aacaatgaag aattccctga 2520 tctaactatg tttcccccct tttcagaata gaactattgg ggtgaggata aggggtgggg 2580 gagaaaaaat cactgtttgt ttttaaaaag caaatctttc tgtaaacaga ataaaagttc 2640 ctctcccttc ccttccctca cccctgacat gtaccccctt tcccttctgg ctgttcccct 2700 gctctgttgc ctctctaagg taacatttat agaagaaatg gaatgaatct ccaaggcttt 2760 taggactgtc tgaaaatttg aggctgggtg aagttaaaac acctttcctt atgtctcctg 2820 acctgaaatt gtatagtgtt gatttgtgct gagatcaaga ggcaggttag aagaacctga 2880 catccactgt taaaaaaaaa aaaaaaaa 2908 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 21 acacacatat ctcaaggccc 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 22 aagggagcag aggactccct 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 23 caagatcagg ctgggaaaca 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 24 cccctaatct ggtcacctgt 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 25 actgccacac ctgtttcaag 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 26 taggaataat aacttatttc 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 27 actctcttac ctggaagacc 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 28 caagatggcg gcttcagcag 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 29 ggaaaagaaa ggccactccc 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 30 gccgccatgg ccgcagatgc 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 31 ggtacatctg atgtcatttc 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 32 cttaatagcc ctctggacaa 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 33 atcatcaaaa tccagccctg 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 34 tccgacctgg caaaccgctc 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 35 catcatccga cctggcaaac 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 36 attttccctg gcaagtctct 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 37 ctgtcatctt gttccgtcgc 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 38 tctgacagtt ctgtgatgta 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 39 tttcgagcca gggcactaca 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 40 ctggttttcg agccagggca 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 41 aatgtttcag ttcctgatca 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 42 agatcaaatg tttcagttcc 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 43 ctccaagatc aaatgtttca 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 44 gctgcctcca agatcaaatg 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 45 catctgctgc ctccaagatc 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 46 aaagccatct gctgcctcca 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 47 atcatctggg tgcacctgat 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 48 atccacatca tctgggtgca 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 49 agtttatcca catcatctgg 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 50 cctttttcac tgttccagtc 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 51 ctgaccttcc tttttcactg 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 52 agactgctga ccttcctttt 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 53 tccattcctg catctgttcc 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 54 agccaagtcc attcctgcat 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 55 gcctctgggt catcatctgg 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 56 gggagaacta gttacctgca 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 57 cagttgggag aactagttac 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 58 ctgtacagtt gggagaacta 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 59 catgtctgta cagttgggag 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 60 ttactcatgt ctgtacagtt 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 61 tcgggagatg aactctgttg 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 62 ttgtgtcggg agatgaactc 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 63 caatgttgtg tcgggagatg 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 64 tagccaacag tagccacaca 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 65 gctggtagcc aacagtagcc 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 66 ctgtggctgg tagccaacag 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 67 acaatattct ttcctaagag 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 68 atttcactac ctgttggaag 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 69 acttggcctt ttaatttcac 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 70 acagcacttg gccttttaat 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 71 gaacatgaca gacagcactt 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 72 gttcttcaca ttggtgttgg 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 73 ctagagttct tcacattggt 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 74 ccatctcttc ctggtaccat 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 75 ttactctgat ccgcattgat 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 76 gagatgcctt tactctgatc 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 77 tggaggagat gcctttactc 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 78 agtgctggag gagatgcctt 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 79 gaaattctct gccggccggg 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 80 ttcctgaaat tctctgccgg 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 81 agaccactat tcctgaaatt 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 82 ggacaatggt tacaggaggg 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 83 tggctggaca atggttacag 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 84 gtctcaggag caaagttaga 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 85 cactgtggcc agacacccac 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 86 ctggccctgc cactgtggcc 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 87 ggctgctggc cctgccactg 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 88 ggaaacatag ttagatcagg 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 89 caatagttct attctgaaaa 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 90 tcaccccaat agttctattc 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 91 tatcctcacc ccaatagttc 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 92 agaggaactt ttattctgtt 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 93 aagggagagg aacttttatt 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 94 atccaaggca aacagtggat 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 95 gtccaggccc catctatcat 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 96 tgaaaatctt tgctacatgt 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 97 ccaggtggtc acatctggtc 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 98 tgagtccaag atcaggcggg 20 <210> SEQ ID NO 99 <211> LENGTH: 65001 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 14965, 14966, 14967, 14968, 14969, 14970, 14971, 14972, 14973, 14974, 14975, 14976, 14977, 14978, 14979, 14980, 14981, 14982, 14983, 14984, 14985, 14986, 14987, 14988, 14989, 14990, 14991, 14992, 14993, 14994, 14995, 14996, 14997 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 14998, 14999, 15000, 15001, 15002, 15003, 15004, 15005, 15006, 15007, 15008, 15009, 15010, 15011, 15012, 15013, 15014, 15015, 15016, 15017, 15018, 15019, 15020, 15021, 15022, 15023, 15024, 15025, 15026, 15027, 15028, 15029, 15030 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 15031, 15032, 15033, 15034, 15035, 15036, 15037, 15038, 15039, 15040, 15041, 15042, 15043, 15044, 15045, 15046, 15047, 15048, 15049, 15050, 15051, 15052, 15053, 15054, 15055, 15056, 15057, 15058, 15059, 15060, 15061, 15062, 15063 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 15064 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 14965, 14966, 14967, 14968, 14969, 14970, 14971, 14972, 14973, 14974, 14975, 14976, 14977, 14978, 14979, 14980, 14981, 14982, 14983, 14984, 14985, 14986, 14987, 14988, 14989, 14990, 14991, 14992, 14993, 14994, 14995, 14996, 14997 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 14998, 14999, 15000, 15001, 15002, 15003, 15004, 15005, 15006, 15007, 15008, 15009, 15010, 15011, 15012, 15013, 15014, 15015, 15016, 15017, 15018, 15019, 15020, 15021, 15022, 15023, 15024, 15025, 15026, 15027, 15028, 15029, 15030 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 99 agataagggt cactggtgag ccttccaatt ccggattgta gttcattcca aatattgtca 60 agttgacaac caggaatagc cactacacat ggcttttttt actacatgat tataggtgta 120 atcaacatgt ctattatata ctaaaaaaaa aaagttttta accatgactt tttgtttggt 180 ttttcaacac atagtttctt tgtgttaatc atcctggctg ctttagatct taatttgtag 240 acctggctgg catccaactc aaagagatcc attggcctct acctcccaag tactgggatt 300 aaaagcatgt aatgccagtg aactacaatt tcttatatta aagtgtgcca tatagatgtc 360 atgttttgtt ttgttttgtt ttgtttttgt aaatttgaca caagctaaga tcatctgaga 420 ggaggaaaag ccattaaggc aatgcctcca tcttggcata gtggtgcccc cctttaatgc 480 cagcaaatga aagtgtgaaa gcaggtggat ttctatctgt tcacaactgt ttgaaactcc 540 aggttaatgg gatctttctt gtctgtctaa ttgtgttcca atactcaggt gtactgactt 600 aaatgcagac acattcacat aattaaaaat aaaactattc aaaaccccaa gaaaatgtct 660 aaacaaacca accaaaccaa ccaaataacc taaagcaagc aagcaaccaa caaacgagaa 720 gccagcatga tgtcactgcg cctctcactg cacctctttt tagtgaatgg gagttagttc 780 tctcctcgaa ccattgtgtg gcttttggag aatccaactt caagtacctt tagctcttga 840 gccatcttgt cagttctact tctaaatgtt tttaaaattt ttgtgtatgt gtgagtgcac 900 atgttttggt gtgaggaaag ttgaagataa tttgcaggag tcctctcctt gtgtaagttc 960 agagcattgg atcttgggat gttaggtgta gctgtgtagt ggagtcgtct ttatggctaa 1020 ttaaaaaaat tttttttggt gaatcagact gacaacaagc caaagagtag tgccatatgc 1080 ttatagttct aacattggaa ggagagggtg aattcaagac cattttcatc ctcatagtga 1140 atttggggtc agtctaggct acatgagacc ttgtttcaaa gaacaaaatt accctcctcc 1200 ccccagtcca catatacatg gtgatgaata taatagactt tgggtgttag tttcattcac 1260 tttgcttctc actactggat taatttgcaa ggtggccttt gtttcatgtc tttggttttt 1320 gtttgttttt gagatagtgt ctttctatat atagctcctg ctgtcttaga actcactatg 1380 tagaccaggc tggcattgaa ctcagagaaa ttggccagtc tctgcctcct gagtactagg 1440 attaaaggca tgcatcacta tgcctggatc tgtttgtagt tctttttttt ttttaaatat 1500 atatttttaa gatttattta tttattttat gtatatgagt acactgtagc tgtacagatg 1560 gctgtgagcc ttcatgtggt tattgggtat tgactttagg acctctgctt gctccaatca 1620 acctcgccca ctccagttgg tcggctcact cagttcctgc tttctctggc ccaaagattt 1680 atttattatt ctaaataagt acactgtaat tgtcttcaaa cacaccagac gagggcgtta 1740 gatctcatta ggggtggttg tgggccacca tgtggttgct gggatttgaa cgcaggacct 1800 ctgaaagagc agtcagtgct cttacccgct gagccatctc tccagcccct gcttgtagtt 1860 cttgatttgt aaaattatag aatttatctc acagtgtttt gagtcgacat tgtctgttct 1920 gaattgctca gttcttataa ttagacatta cgtctgtgtg tttactacct tgtcatttta 1980 aaaaaacttg tgacatagga cacctcactt ttggagggca gagaaaggga ctagtttcat 2040 tttgtagtac agactggtct ttgcattctg agttctggaa tacttgttta ataaaaattc 2100 tttgttcgtt gtctccattt caaatgttgg catctttggt accacgtaat cacccagctt 2160 tttcctacca gcttcagagc caactcttaa gaattagtta tcggactttt actagtgaca 2220 aaagttcata aagaaaaaac aacctagaat ggattagtga tccctgtgtt agaacctata 2280 aagagggttt gcataacaaa tcgtctgtag gaagcatatg ctgatgagcc cactaagaaa 2340 gaaaacattt tagtaaaatg ctcgttttct ttgcatttca ctgacgtcaa tttggttatg 2400 aatctctgac gccgagaagg ccggattagg gaaacagctg gttggcttcc tagctcaggc 2460 ttcctagcac tcctcgggtc accggactca ctggccgagt cactagcccg gcctctatgg 2520 cgacttactg tttctattgg ccagttctcc cagggaggct gtctggagac tgaccgcgcc 2580 catagtttgg ggcgtgtctt ctgcccagga tggggagggg ggtgggcgtc cgccatcttg 2640 gattccgcgg tagcggtggc ggcggtaagg tgcctaatct gcggagtggc tcttccctcc 2700 cctcccccag ctcggtggcg gctgcccctc ccaccgaggg tggcgcaggg acggtgccat 2760 ctcgaccatg gcggcgacta cagctaaccc aggtgaggcg gcagaaagga ctgacaagtg 2820 cggagagcca gagcagagca gcagggccgt ggcgttcggg tgttggggga ggggtgtcga 2880 ggaggcgccc tgcccggagg acctggggaa cgggtgacgg gtgtttgggg gctccgtgcc 2940 ccaacttctg gagtagtggt ggcgcttggc cagcgtctcc actctgcctg cggtgttagg 3000 gagtgttgtg gtcttttaac cctctgccag ctgatgttcc cctgatgcct agtaagtaac 3060 tgggcaaact gacttggtgt aactgcgagc gtaaactggg ctatagacgg ggccacgttg 3120 acgttttgca aagtcttgct tgattctaca cttggtttac atttaaatgg aaaacttagt 3180 tctttgagaa tctgctcaga ttcttgtaac tcctatactt tgaaatttag ggttttagcg 3240 tagttattag tgtcttgctg ctgaaggcag aggtttgggt aatggtaata gttcaggaca 3300 ggtctttagt ccagaaccca ggtttaaatc ttgttaggtc gaggtggagc aactcctgtt 3360 ctttcagaaa aatgtcccgc gccatttgtc tgtggacgtc ctcttgcttg tttgtctgtc 3420 tgtctctgtc tctcgttcat cttattttga gtcttgtctc attccagccc ccggttgact 3480 ttggatcact gcagtctccc tctcagtcct ttctactgta ttcagttaca tcttaggaag 3540 tatttcattt tagttgggct aatttacctt gttattttcc aacaaagtaa atccatcatt 3600 tcccagaagt ctgtatcatc gaagagtttg gagggagggt tgtgtaatat tggtctctgc 3660 tatttcacat tgaactttta aattttatct tgttaatgta ctatattaca gtaacagggt 3720 acacacacgt atgtatatac atacacacaa tagctgaaac actgggaaat gtattgtttt 3780 cccattattt ttggaaacag cctttttttt taagagagag agagagagag agagagagag 3840 agtcctagaa cttatatgta gcccaggttg gcttcaaatt catgtgtcaa tatcaacttt 3900 aaaaaaaata tttttaagct ttagaaatta tgtatatgat aatgtctact tgtgagtgca 3960 ggaatgggag gagtccagaa tagggtgttg gatcccctgc agcaagagtt tcaggcagtt 4020 gaaagccccg tgatgtggat gctaggaact gacattaggt cctagaagag taacacatac 4080 tcacaactac tgaaccttcc tttcagcccc tgaatatcag ttttttaaat cctaaattat 4140 tatttttttt acatgaagat gaagtaaatt gtcaggaaac tattggaata tttgatgaat 4200 tatagaataa aactatacaa ctgaaaaatg ttagagaaaa tcagaccttg tagcaaaata 4260 gcccatgtct gattagagta aacaagattt caaactgatc ccatgttgca aaatccttat 4320 ataactcttc agactgcaaa gtgtctctct tgttaaggcc gtagagacac gttagtaagt 4380 cagaagcttt tctagctgcc ctgttctgat tttaaaccaa ttactaaaac aacactttct 4440 tgaccaaata gtcatttcct tatcaatatt agcttaagtc tgtaagcagg aaatagtaat 4500 ttttttctat ttttttttct gagtggggaa ttttgagagg ctggcctgca gtgttctgtg 4560 aagatcaggc tggccttgaa cttgcagtgg tcttcttgct tctatcttca agtgctggaa 4620 tcactcgcat gtgccatcct atataaacta tcaaaacttt ttaaaaagta cgatttaaga 4680 atctttttta atgaaacagt tgtttgtttg tttgtttgtt tgttttgact ttggtttttc 4740 aaggcagggc ttctatatgt atccctggct gtcatgaaac tcactctgta gaccaggatg 4800 gcctccaact caagatctgc ttgctcctgc ctcctgagtg ctggattaaa gctgtgagtg 4860 tcatcaccag tagaaataat tgattttatt tattgttatt tgattgcttg cttcttcaat 4920 tttgaggtgg gcagagtcat cataggtact taccaaacag agggagaata ggctggaatt 4980 cctttaatct ttgcagaagc agggggatat cttagttcat ggccatcttg gtctacaaag 5040 tcagttccat gctacatagt gagaccttgt ttcaaaatca aaagccagac caaaccaagc 5100 aaaaacctca aaaggtgagg tgtaggttct tagcaaacct ttgaaaatta acctgagaat 5160 gtaagagttt tcttaaattc tagtctgctg ttagttatgg catagcaccc agtggttcct 5220 gctgtagctg acatccctgc gtagcctcag cctcttatga gatgggatgg taggtctgag 5280 cggagaacag agaacgacag acccagtgag actaaccccc tgctctcttt accatctgcc 5340 ttaacttcct tttccggggg ctacattcct agctcaagat ttcttttttc catgtggaga 5400 attcttccat gtcttctaat ttattgatcc catgttcagt acatgcatga tacttaagtc 5460 ataaaataga gccttttgtt gttgcttttt cctccttcct ttttgtgtca aacaggatat 5520 gttttggcct gttgaatttt ggaaaattag attcaccact ggaggtatat acagtgggaa 5580 attactatgt tcagtgtgca cttgtacaca ggccagaaga caccggatat ccacctctgt 5640 aactttctgt cttattcctt tgaggcagaa tttcttctta actgtgggca tcttactgtg 5700 tcactctgcc ttatttcttc caggtcaggt gtctccctta acctgaggct tgtgtgtttt 5760 gctcggctga cagtcagcaa ggcctagcag tcttcctgcc ttggatcctc agagtgcatg 5820 gattcctggt ggatgcagga tgatgccaga cttgttatgt gggttctggg atccaaactc 5880 aggtctccag attgtgtagc aagtgctctt aaccactgga tcatgtcttc attccacatc 5940 ccacctccgc tttttgagac aggatctgtt actggcctgg agtgggccaa agaggataaa 6000 cttctggtta gagagcccca agctcactgg gtttcggctt taccaatgct ggaaatacaa 6060 gtaaacgcta ccatgccttg tcttttcatg tgggttctgg agattaactc aggaccttat 6120 tgcttgattg gcaattcttt tttgattgag ctaccaagcc ctttattatt aattagtcag 6180 tgttcttaat tgctggtcat atctccagcc ccactttccc cccacccccc cccaaaaaaa 6240 gattttaatt aaattatgca cattggtgta gtagttactt ttctgtggcc atgaagagac 6300 accatgacca tgggaactta gagaagcatc gctttattag acttgagttt tccagaggtg 6360 agtccatgat cactgtgact ggaagcatgg cagcagctgt attacttcca acaaagccat 6420 acctcttaat tcttcccaag acactaacac caactgtgga ccaagaatta aaatatagga 6480 gcctgtgggg accattctca ttcaaaagac cgtaattact gcatgtatgt ttgttcatgt 6540 gagtgcaggt gcagaagagt gtgttgattc tcttgtagtg caattacagg tggttgtgag 6600 ccatctaatg tgggttctca gaactgagct catgtgttat gcaggaatgc caagttgtct 6660 taacaactga gccatctctc cagcccccca aatcaatcaa tttatctctt ttttttttct 6720 ttctttttaa aggaattgaa ccattttact aactcagcag ttaaaataca ataaactttt 6780 tgtttttgag gcaagtctca tttagactag gctaggttga aactcactat gtctctgagg 6840 gtgaccttga atttcttttt ttcatttttt aagatttctt tctttttttg tttttttgtt 6900 tttcgagaca gggtttctct gtatagctct ggctgtcctg gaactcactc tgtagaccag 6960 gctggcctcg aacttagaaa tccgcttgcc tcttatttct tgatattgag tacactgtag 7020 ctgttttcag agacaccaga agagggcatc agatcccatt acagatggtt gtgagccacc 7080 atgtggttgg tgggaattaa acccaggacc tctagaagag cagtcaatac tcttaaccgc 7140 tgaactatca tctctccagc cctgatctta aatttctgat ccaagtgcta tgatcttata 7200 gttacaacca tgcctgtctt accatgtgct atggatggag ccagggcttg gtgcatgcct 7260 ggcaagcatt ctaccaactg aactgcatac tcagtggata acagttttgg aacgaaactt 7320 cagttggtgt acttttatgc agtagacttg tggtaccaag tttgactcta ttttcctaga 7380 atttaagaag taaagactta cgttgagaat ggtagccgtg tatcagtaaa caaaaatggc 7440 tggagagtgg ttctgccatt agaagcaagc actggctgct cttggagttg actgaggttt 7500 ggttccagca catactggtg ctactaccca cagctataat tccagtagat ctattgccac 7560 cttctggcct ccatggacaa caagcctgca tgtgttgcat atatacacac aggcaaaaca 7620 tacacataaa gtcgtttaaa atacaaaata gagaaaagtt ctctgatgtt acaatattat 7680 aaagaatctt gaaaaatgtt tccattattt gtccagtgtt ttgtcagcta tataactgat 7740 ttcatccaga tgttgtaata ataaaagaat gtctttaaca cccctttgaa aatgtaggct 7800 tgaactcagc agagtctgag ccaactcccc aaccctcagg tagtaggcac tgcttgtata 7860 acctggttga attgcttgac ttgctgtttt gacagggtct gggtgtgtgg ttcaggctgg 7920 acttaggctc tccatctctc ctgagtgttt aagagatgga catgagaacc tcagctgggt 7980 agtgagttca aggccacttc tgggctacat gagactgcct cagcttcccc agtactagga 8040 ttataggcat gacctaccat gccttgctaa atccatctat ttttaggatt gctgtcctat 8100 tttccatgat gacttcagta ggtaaatatc agggagtgag tccattgtaa ataaaacagc 8160 actgtcctaa agggcttcag tgccagcaaa gatctgtgaa ttctgagggc agacaaaact 8220 atacagcaag agagaccatg tctcaaacaa ataaaaacaa aggatgataa ctctttgatt 8280 ttatagcaag gaaacaaacc tagagaaaga aatgaacata tttattaagt atttattatt 8340 tttctctgtg tccaggctat actttagtat tttgttggtt actcattata ttatttattt 8400 ttaaaatatt tatttgcttt atgtgtatat gcgtgcctga gtgtatgtct gtaccacatg 8460 ggtttagaag acttcagagg tcaaaagagg aggacttttt aagtcccctg gaactggagt 8520 tacagatgtt tgtgagccac catgtgggtg ctgggaattg aacctgggtc tctgcaagaa 8580 cagtcagttt cttaacccct gagatatctc tgtagttctt attctctgta tcataaagtc 8640 tcttctattt actccctcca caatttttaa aactttatac cttccttcta agcatggcgg 8700 tgcacaccat taatccgagc acttgggagg caaaggcggg tggatcagtg cgagttcaag 8760 gctagcctgg tctacaaatc cagtctagga cagccagggc tgttacacag agaaaccctg 8820 actccaaaaa caaaattata ctttaacttt tatatttctc cttttcattt ttacagtagt 8880 tgggtgacca cagtaagttt ttcctgagac aggctctcaa ataccatgct ggccttaatc 8940 ttactatcct aagctagggc ttgaacttcc agaccctact gcttatacct ctgtagtggt 9000 gggattagag ttaggtgcca tcactccagg gctattgggc tcaagatagg ggcttcatgc 9060 ctgccaggca aatacctagc tgagctacat gggttccagg tcagtcagaa ctagggtgag 9120 gtcctgtctt aaaaaaaaat aataataata aaattgggcc agggatgcag cttagttaga 9180 agaacccttg tgtgtcagat acaagatcct aagtctcatt cctagtacat tgcaaacccc 9240 acctccctcc aaacaccaaa gggtttctaa gctggataag gtggtggcct atatctgttt 9300 tttctttgtt ttttcctttg agaacgagtg taatttagct ggcctaaaat ttcatgtgaa 9360 cttcctgtga ttctcctacc gtctcctcag tgctggaatt acaggctaca ctactgacaa 9420 agcaagtccg ttctgtcaga agattccttc agttgactgc ttttttcata tatatttagt 9480 ttttcatttc ccatgaaggg ggagtcagta ttttccattt ttttccatgt gagttgatat 9540 cagtaaacct taagttcaga tatgtaaatg catatatatg tgtgtagaca aacaactcca 9600 tcaactttgg tgtctgtttt tgagacaggg tcttaccatg tagctttagc tatcctcgat 9660 cttgctatgt ggaccaggct ggtctagaac ttaaattaac ctgcttttgc ctcccatgtg 9720 ctgatattaa aggtatccac caccctatct agtaattttg ttttttagac ttatttttta 9780 attgtatata tgtatatgtg agttcaggtg cccttgaagg tcagaagagg tgttgggtcc 9840 tctggtgctg gagttccaga ttgagcttcc aggtgtaggg aacagaggaa gtcctgagta 9900 actgcattgt tgacatgcac gtggccatat tgaggtctcc aaggccacag acccatggga 9960 gattgacaaa gccattaccc atatggccag gctaaagaga gtgacaaagc tcttctgaat 10020 tctagtatga gtgtgttagt gtgtgcttac atgtgtataa atgtatgtgt gtgtatgtgc 10080 gcatatgtgc gcgtgtggag tctggctttc cctcatccgt ctacctctct tacccaagtc 10140 tggggtttta tatgtgtgcc actatacctg ccttgtagat gactaactcc tatttggtaa 10200 tcttttgtaa agtattttag tatcagtttt cagaaagaat attggtttat aattggcata 10260 ttttttactg tttgttttgt tgtgatatca gattaatact agaatcatca gtaaaatgag 10320 gccaggagag gatatcacat ccaaatattt tcttgaagag actatgtaga actcatagta 10380 aagtaatttg aacctggata tttctttttg gagtacagtt gtagaactat tattattatt 10440 attattatta ttgagacagg gtttctcttt gtagccctgg ctgtcctaga aattactctg 10500 tagaccaggc tggtcttgaa ttcaagagat ccacctatct ctgcctctgg gcttaaagtc 10560 gtgcaccact gctgcatggc tattacattt gtttatttat ttttatattt agtcagttat 10620 aattgtatgg gcttttccta gtgttgttgg tcttgaagta aaagcttaat gattgatttt 10680 gttttgtttt ttaagatgta tgtgtgtgtg tgtgtctgtg tgtgtctgtg ttattatata 10740 tatgtgccct tggaggccag aagagaaact ctggaactag agtttcaggt gactggatta 10800 tctgccgtgg tgctagggat agaactttgg gcttttggaa gggagccatc ttgctaaccc 10860 tttgctttgt tgctttttgt tttctgtgat acagcagtgt tgtaaattgc ttaccaggta 10920 gagttttaat tgtctcccgt aattttaatt ttgctatatt gtatttaaat tttttgttta 10980 ttgttgttat tattcctcct cctctccctc ctcttcttct tctctctctc tctctctctc 11040 tctctcttgc tctctcgctc tctcgctctc tctgtatgcg tgtgacacat ttttctaagt 11100 cagttttctc ctactgtggg ttcaagggca gaactttgat cagatttgca tggctggtac 11160 tttttctctc tgagtcacac tgtcagctct ctagtatatt tttgttttat gcacttgtgc 11220 atatctgtgt atgtgcttgg agtgcagtgc ccttagagac caaacaagga tgtaggatcc 11280 tctagagcaa gaggtacctg tgtttgtgag cagcccaaca tggtgccttg aagaactctg 11340 caagagcaga gcttgtgcct cacttacatt tagttaagct tgtgacagtt ttcttttgag 11400 actttgactc attcattaat tgtattgtat ttttttcatg tttgtagaga ttttcctatc 11460 tttttccttc cttctttcct tccttccttc cttccttcct tccttccttc cttcctttct 11520 ttctttcttt ctttttttga gacagggttt ctctgtgtag ccctggctgt tctgaaactc 11580 actctgtaga ccaggctggc ctcaaactca gaaattcacc tgcctctgcc tcccaagtgc 11640 taggattaaa ggcgtgcgcc accatgcccg gctttttttt tttttttttt aagatttatt 11700 tatttatttt atgtatttga gtcagatgat cgtgatccat catgtggttg ctgggaattg 11760 aactcagacc tctgctcgct ctaacccaaa gatttattta ttgttatatg taagtacact 11820 gtagctgact tcagacactc cagaagaggg caccagattc cattacggat ggtggtgaga 11880 caccatgtgg ttgctgggaa ttgaactcag gacctctgga agagcagtta tatctccaga 11940 tataatgttt ttcttttagt tgtgtcagat tttacaacac attttgccct gtcttgttta 12000 gtatatgtgc attgctgtgt tactggtggt ggattctcac tctgtaattt cttcaggtaa 12060 attttatttg ctgttttttt ggtttttttg ttttttttgg tttttcgaaa cagggtttct 12120 ctgtatagcc ctggctgtcc tggagctcac tctgtacacc aggcaggcct cgaactcaga 12180 aatctgcctg cctctgcctc ctgagtgctg ggattaaagg cgtgcgccac cacgcccagc 12240 ttatttgctg ttttatcggg tttatttgta tatataacaa ttcctataac cacagcttta 12300 ggaaaaaaag cttggtctct ctgtgtcccc tggctaggct cagtcaccta acccaattaa 12360 ttgacaacta atgatgaaat acagagattt actcattgtg gtcacattgt cctaggaaca 12420 gatagaaggg tccagtgacc cctaagtccc tttaactata ttgacttcag ctttaggttt 12480 aagtagaaca gttagggcag caggccggag gtttagccag tcctgaacac agtgtctcat 12540 tgtctcgggt cctgctaggt ggatgaatta aattgttgct gacaggacca ctgtggctcc 12600 tacaagatag tgcattctgg gtgtagcctc ttcatcacag gtaattgttc tcactgtctc 12660 agccgaggca tagcttcagt tctttatctt gtttctgctg gaatccaagg tgacaagaag 12720 acagattaaa gacagtgacc tagctctgtg tctccagaag taaaggagac agacagacaa 12780 aacggaggcg gagatgccag cctttcatct tactttagta aagcaaaggc tcacattggt 12840 ctttgcaaga gtaacttctg tcttagttag gttttctggt gctttgatga aaactttata 12900 tgtgaccaaa acatgttggg gaggaaggat ttatttcagc ttatacttcc acatacacat 12960 cacagtccat cacttaagag agtccaggca ggaactcagc agggcaggat ttggggacag 13020 gaactcaagc agaaatgact tggtttactg gcttcgtctc ccatggcttg ctcaacctgc 13080 cttcttatag cacccaggac caccagccca gggatggcat tacctacagt gaactgggcc 13140 ctcccatatc aatcactaat taagaaaact ttccacaggc ttgcctgcat accaatctag 13200 tggggcattt tctcagttga tgtccctgtt ttcagtgact cctttgtcca gttgacatga 13260 agcatagcct ctaaggctgt tgcagtccca cttaactttg gttaatgtgt gcatgatgtt 13320 cctttccttt cccctttctt tctttttttt ttttttaaag atttatttat taatatatgt 13380 aagtacactg tagctgtctt tagacactcc agaagaggga gtcagatctt gttgcggatg 13440 gttgtgagcc accatgtggt tgccgggatt tgaactctgg accttcggaa gagcattcgg 13500 gtgctcttac ccactgagcc atctcaccag ccctcctttc ccctttcctt tcccctcctt 13560 tcctttcctt tcctgaggtg gagcaccgag gatgactttg accccccagc cccgagtgct 13620 ggagttatac acccagagcc tctagcatgc taggcccata ttctactggc tgagccatac 13680 tccaagcccc atgctgtgac acagggtggg tccctttagc aagttcttga tctcatggat 13740 aaaagattga agaggctaga gagatggctc agtggttaag agcctgcatg actcgtccag 13800 aggatctgag cttggtttca agtgcccatt atcagggcaa ctcccagctg actgtgcagt 13860 gccctctttg gcatccgtag gcagcagcac tcacatgtgc acacatcctc ctcactactc 13920 cccaccacaa taaaaataaa tccttttaaa aaaatgtgct ttgaagggta tattttacta 13980 gagtcagtaa gaaaaggcag ggcaggcaag ctagagatct cagcattgtc atgaggaagg 14040 aaatgtaaag aagacaaaaa ggaaaaagtc acatccttaa atagaacagg aggaaaacga 14100 cctcttgggg gacaggtgac tcacggaaga actggggtat tcagagccca tcaccaaact 14160 gccctccttg gtagacgtac tttatggggc ggtctcactg cagcccaggt ggcctgggat 14220 tagcagccat cctgaacgct gcttttatag accagaagct ctacacttgg atggaaatag 14280 taatactact tggtttaggt ttgtaatcag tcaagagttg ctctatgtgg cctaggactc 14340 agatgtgttt aagattagaa agaataataa ctgtatgtag tgttgttagc ttagtgtcta 14400 gttttattag ctttggatca ggaagggatg aataacagtt ttgaactatg tcaatttacc 14460 tttaaaatgg tttacaaatt tgtttatggg agttcttaca tgtgtgtgca cacatacatc 14520 agaggacaac ctcaaatatt gttccttaga gtagttcacc ttgttatttg agacagtctc 14580 tcattggcct tgaacgtata acaagatggc cagcttgtct acttgttttt acctcccagc 14640 ttctacatca gtgctgggca tctgacctca ggtccttatg ctattatggt aaacactttg 14700 ctgtctgaga catgtctctc taatcccagt ttacctgttg gcaaaggcta gttttggact 14760 aatttgaagc tcctgtcatt ggctcatttt aaattttcct tagtttttgt tttgttttgt 14820 tttgagagtg tgtatgtgcc catcttgatg aaggtacaga ggtcagagga cagcttgcct 14880 gagttaggtg atcagggtcc tgctcagtct tggcaggaag ctcccgcatc tgttaagcca 14940 ttttcctagc cctcaaaacc tgagnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15060 nnnntcttgc ttctctggtg ctctggttca ttctcattga tgcccatgat ctattgataa 15120 gatcatatga catttttatt tgaattatac atatttctat acaatacagt ccctactttc 15180 ttccctccca tccacacttg ttttccccat cctgttaaga tggtcttgct atgctggcct 15240 taaactaggt agctagcata ggttgacctc aaacatgcag gccctttgcc agtctcccga 15300 gtgtcaacat tatagggatg caccgttatg cctggttcac ttacagcttt tattcccaaa 15360 gttcccattt ttcccccata ttctatgttt ttatttgcta agactgttta acatttgttt 15420 caaatgttct tctaaatact tgctgaagaa tttttatcag ggctacttgc aaaacttttg 15480 ttagataatt ctaacatctg tattgtctta gtgttggcat ctattagctg atgctccccc 15540 acccccgccc acagctctct tgagacctgc ctgattcttt gtatgaccag tgattttctt 15600 ttaaattaaa aaccttgata attttgtatt ttaaatgacc tgttctgaat atgtaattct 15660 tctaatacat acatgattaa gccatagctt aaacatttct ttctttcttt ttctttcttc 15720 aagacagggt ttctctgtat agtccctctg gctgtcctgg aacttgatct aaagaccagg 15780 ctagcctgga actcagggat ctgcctctct ttgtccccaa gtactaaaag gcgtgcagta 15840 ccactcttgg ctttttgtgg gggttttgag tcaatttcat gtggcctgga tagtgtggct 15900 ccaaatccac tttgtataat aagatgactt tgaactttgt tttgttttgt attttttgag 15960 acaggatatc tctgtgaaat tctgactgtc ttggaactct atctgtagac cagactggcc 16020 ttgaactcag agatctgcct gcctttgact cctgaatgct gctgggatta aaggcataaa 16080 ccaccattcc tgattttcct ctttccagtc tttcaagtgg tggaatcaca gatgtttgcc 16140 accaggcttg gcttgagcca gactgccttc ctgcctgcct tatttccttt cttccttcct 16200 tcttttcata tttaaattgt atgtatttac ttgtgagcaa gtgcatgtct ggaggtatat 16260 gcctacctat gagtcagttc tctctttcca ccatgtgggt gtgaaggatt gaattaaggt 16320 catcagaatt ggcagcaagg ggttttgatg acccctggtg tgagtatttc ttttagccag 16380 catcatgaca ttgattgctg agaatatgga cgatattctt ttacgacaga aatgacatca 16440 gatgtaccat cgctgggtcc caccattgct tctggaaacc ctggacctgg gattcaaggt 16500 ggaggagctg ttgtacagag ggctattaag cgacggtcag ggtaagtttg aatgtttatc 16560 atccgtgtgt ttctcttgta ccatcaactt ttgagttagt tgctgttggc tcttgtttat 16620 tcctgaccac acatctaatt gattacttac tatactgctg tcacataact aaaactaact 16680 cttagtccct ggattattcc ttatccaggc tctctatttt aagtaaattt ctttaatttt 16740 ttttgtataa tttgaatata aatatagctg taaagaaaaa aatgaggact ggagagatgg 16800 ctcagtggtt aagagcactg aggtcctgag ttcaattccc agcacccaca tggtggctca 16860 caaccatctg taatagaatc ggatgccctc gtctggtatg tttgaagaca gctacagtgt 16920 gctcatacat aaataaatgt atcttaaagg aaagaaaagg atgaaacggt cacacctatt 16980 acttatcatc cgacctcact cacagtgcct gtatgtcacg tagggttttg gcttttatta 17040 cttacatcgt tttcacataa aaatttctat acaggttaaa aattgttttc tagtatcttt 17100 attgataagc ctgtttaaaa aaaaaaaaaa cgaagccaaa ccaaaccaaa attttctgaa 17160 aggtaaaggg actgtggcaa agaagagttt cactgttggt aaaaacaaac aaacaaaacc 17220 aaaccaaaaa aaaccccaaa aatctgttgg ctttccttaa gctctggtct gcattctcaa 17280 aatcattttg ttaagcttat ttctttttta atttattaat actttctttg acttggagtg 17340 cacagttcat ttagtttcaa aagtctttat tgaagtacca gatttgaatg tgccattgaa 17400 gtatgattgc tgtcttagtc aggatttcta ttcctgcaca aacatcatga ccaagaagca 17460 agttagggag gaaagggttt attcggctta cacttccatg ctgctgttca tcaccaaagg 17520 aagtcaggac tggaactcaa acaggccatg gagggatgtt ctttactggc ttgtttcccc 17580 tggcttgctc agcctgctct tctatagaac ccaagactac cagccctgag atggtcccac 17640 ccacaagggg cctttccccc ttggtcacta attgagaaaa tgccttacag ctggatctca 17700 tggaggcatt tctccaactg aagctccttt ctctgtgata actccagctg tgtcaagttg 17760 acacaaaact agccagtaca attgctaata ggctctaaag atagatagga aaagttccta 17820 aagttctgat ggtcgttata tttatgtata aatatacaag acacagtatg acaaaagtct 17880 gcaatacctg aagttctagc agtcagtagg gctgagtcag gatggagact gagtccatcc 17940 tgagttacat agtgatactc ttatctcaaa acaaagcgaa aaacaaatta attttaaaat 18000 attatttttt aaatatatag aaacactgct tttggacggt aaagattgag aaaagttttt 18060 aaaagaagtt tgctttaggg cagtggttct caactttctt aatcctgtga ccttttaata 18120 tagttcctca tgttgtggtg actacaacca taaagttatt attattttta tttttatttt 18180 tttggttttt cgagacaggg tttctatgta tagtcctggc tatcctggaa ctcattttgt 18240 agaccaggct ggtctcgagc tcagaaatcc gcctgcctct gcctcccaag tgccgggatt 18300 aaaggtgtgt gccaccacgc ccggcttcat aaaattattt tagttctact taataactgt 18360 aatcttgcta ctgttaataa ctgtaatgta ggatatcaga tatgcaactt ccaaaggggt 18420 cgttgtgact cacaggttga gaacagctgc tttagggcct gtgaggtagc ttgttaaaag 18480 cctagttagt tacctttaat tgattcccag aacccacaca caagtagaag gagaaaacta 18540 actctaccaa attgccctct gacctctaca tgagtgtggt acctatatgc acacactact 18600 aaagcacaca aaaatattaa acttaaaaat gaatgaatga gcagcaacca catggtggct 18660 cccagccatt tgtaatgtga tatggtgccc tcttctggcc tgctgacata tttgcaagca 18720 gaacacttaa agcagtcaac actctcaatc tctgactttt ctccagccca aagagtgcac 18780 gctactcttc cagagaatct gaattcaggt cctagaaccc atgtcagtga ctcacatctg 18840 tgatctagtt ttggggttct gccacccctt ttctgggtac ctgtattcac atgggcatat 18900 atccctatac agacacatca taaaataaaa tcattttaaa aatcataaaa gatgctctct 18960 tcagtattaa aaacacattt caaatagtgt ttgctttttt tgttttttaa caaagtctgt 19020 ttaatttgat tttattattt tgacatagct ttcttgtaga ctcagctggc ctgcgttctt 19080 cctggttcca cttcccaagt gcttggaaac aaaaacatgt gaccaccaca cctagtttca 19140 gacagtcctg ctgtgtagcc atgttggtct ggaattcagt atgtacacaa agctggcctc 19200 ccactcatga ttctcctgcc ttagcttcct atgtcgcagg actacagtca agtgccacta 19260 catctgggtt ttgtggcacg tctttaaggc cagcccttga gagacaaagg cagtgtgggt 19320 gggagggcag cttggtgtac atggaggttt ctgggacagt cagagctatg tagagagatc 19380 ttgtctccaa acccaaaccc aacctcattc tcacttatta tatatattaa catgcattta 19440 tatgtttatt tactcactct gtgtttgtgt aggttagaaa acaagttgtg ggaaatcttt 19500 ctttaccatt tgggtcccag ggatccaatt tatgcctgta ggcttggtgg caggtttttt 19560 aatttgctga gctgtctatc tctctggcct cttgctttat agttgctgtt gtctgggctg 19620 aaaagctccc tgagggtatt ttttttcttt taccacgggt cccacactat tttgtgtggc 19680 gtgtgatctt gctttcttgc tttcttcttt gacaggatct ggctatgtag cttaagctgg 19740 ccttggattt atcatccttt tgcttctact tgatgaatac tttgagctaa aggtacgtgc 19800 caccaaggct ggccacttag cacagtgatt tgaatatggt atatatttta tttttagact 19860 aacaactttt tgtgtcttct ttcctctatg taatattgct ttgaaagatg tttcagaaaa 19920 ctgacaaggc ttagattaga tcataaaaat gaaaactaga tctcagggat tttctgtttt 19980 tcaggcaggg tctcacgtag ctcaccctaa ccttgaactc agtggtatag ccaaggttga 20040 ccttgagctc ccgatcatcc tgctgccacg tcccttgtgg cagaattcca ggtgtgtagg 20100 tgtgtgccac catgtccagt tttacgctgt actggaattg aaccggggct tcctgactcc 20160 tcagctgagc tgcattctta gactctgggc gctatttata ctcgtatctc ttcctggtta 20220 tggtactggc ctcctgtatg gaatccctgg tggaggggga gggggtcggt gctgctgctg 20280 ctgctccctc acttcttcat tttgcttttt gaaacagggt ctgattatgt agccaaggct 20340 agcttagaat tcattatgtg tctgaggata gccttgaact cccagaaccc accacaggct 20400 ctggacggtg ggttctatat aggcaccgct acggctgcct tttactgcct aacttctgcc 20460 cagttgtaat actatgaaaa gcatgtatta tagacatgtt tttgttttgc ttctgtccac 20520 ttttccttcc attaaggtga gtacataggc atcttttgtt aatatgtttt gctttcttat 20580 ggtgttatct ttactgtatt atcatcaata aacttagaat attgtttgtt gcctttcccc 20640 aggctggatt ttgatgatga agtagaagtg aacactaaat ttttgaggta agatttaaaa 20700 atatttttag agacttctta gtggggtgga gggctggtgg ctggctcagc aggtaacagc 20760 actcctgcca agcaggatgg tctgagtttg atacccaaag ttactgctga aggagagagc 20820 tgactgctgt aaacagccct ctgatctcca catgctggct gaggcgtgag cacccatcac 20880 cctcacagat aactaaatgt catcagatta ttttcagatc accagctggg aatgtagctt 20940 ggtgggatgc ttgcctccta tgcttcaggt tcaatccata gtaccaagaa ggttatttaa 21000 gtatgtttat actttgggca ttgtcctaaa gaatatgctt catcctaaaa ctatgttttc 21060 ctacatcata ggatagtttc aggcatttta ttatttttag agagaatact tcagggagga 21120 gggcacagaa aatataagcc agtcatgttg attcatggct taagagaagt aacacactag 21180 ggagattgct cagtggttaa gaccttggcg ttgcaaatct aaggctaata gttcagatca 21240 ccaaatctca cataaattct gagtgggcat agcagcctgc ttcctgtctt ggaaggtaga 21300 gacaggagag caagctggca ttgagaataa ttctatctgc aacctgtgaa tttgattgag 21360 aaattttgtc taattgaata aagtagtaga gtgatagagg aggattcctg acattaacct 21420 tggacctcta cctgtatgtc cactcacgtg taatgcaccc acacgtgtga gtgctgagtt 21480 tgttattatt ttgttcccat ctgtcccatg agccctgggt gagattaaat gaggctgcct 21540 catcaagtaa ggtagaagag agatgaaggg ttatatcaac tttgggcttc ttcatacttg 21600 ttcacccaca cacatgagca cccacacaga cctgtgccta tacccataca aacatgcgta 21660 tacatacaaa tggaaaataa aaaaaaaaag ttgattcgtt ttcacaggtc tataattgac 21720 taaatcttgc ttgggttcat ggtaagattg tgctcagcca cctggtctat taaaagttat 21780 actcaactgg cacagttgca cctctgtctg tctgtctgtc tgtctgtcta tctgtctctg 21840 tctgtctctc tctctcttgc tctctctctc tcatgttctc tctctctcac acacacactt 21900 gcactctcac ttaaactctt tctccatctc tccctcctta tttctcctat ccccattgtc 21960 ttgtcttcaa acagaggagt gtggtgatgg ttgtaacaca aagggcaagt ctccgtgtgc 22020 ttaacaagcc cctgcttctg tgcttttgtc cttcactgat gtctcctagg ccagagcgct 22080 tgtgtatccg tgtcctgagt cactgctgta gagggctaca gaaggacgta gatgttagag 22140 ggaataattt gataatgctc tactctgtca tgctgcagta cctgttggcc ccttttttag 22200 gtagcaggaa ttagaggttc tgtatgtata aatggtgatg gttgggcagg gtctcatgta 22260 atccaggcct gcctcaaact ttatatagcc tgagatggga tggcggcttg aatgagaatg 22320 ctcacggtaa ggttgtgtgt ttgagtgctt gctccccagt tggtagacct ttttttgaag 22380 ggttaggtgt ggccttggtt ggttgtgtgg cccaagtgga tgtctctagg actacctgcg 22440 caggggaggt agtgagatag actttcctca tcaatcaata atcaaaataa tattacactg 22500 gcttgcccac aggccactct gatagagaca ttgtctcagt tgggattccc tcttctcaga 22560 tgtctaggtt tgtgtcaaat tgatagaaac gaaccagcac agcaattaaa cgtaattctt 22620 ctgatactta gtggatccta ataaatgtga agaagaaaaa agatttcact ttttgcccta 22680 ggcaagcttt cattgttact gctggattta tctattttta tcttaagtgt tttacctgta 22740 agtgtgtata tcgtgctggc agaggccaga agagggagtt gggtcctcta gtactaggag 22800 ttatagatga ttgtgagctg ccatatgggt gttggtgtta tggagtatcc accagaggat 22860 gcttggatat ggatttaatc caatagaaag ttactattag cctgccagca actacactgg 22920 tgtttgggat tccaatgcag cactcagcct ttctcagggt gagcttttaa acacgacagt 22980 atgttctgtg ttgttggcat acttcagtta acaagaacag ttagccagaa gcagaactgc 23040 agaagccaaa gagactttcc cagaactgca gactttgatg gattaggcct ttgttttagt 23100 tttggtaggt agtgctgtat acatactgaa tttcacaacc tgaatggttc ttctatcaag 23160 ggatcaattg tgttaaggtc tggagctatg ttagtactgt gaactgaacc caggtcctct 23220 gcaaaagcag caactgttct taataactga gccatttttc cacccctaag atagcatttt 23280 cacacaggac tattgggaat gtcttcatta gagaattctt aattacagtc acatgatgtt 23340 actggaccta tgttgtatga atgctacagg ggtaactaga gtatttgtca ctgtcaaata 23400 gttttgacct tgagttattc cacaaaatca agtagtcata agttttgtaa cattttggtt 23460 cttatcagac tactatatga ccgtgggttc aaacaattat aatgggagtt ataaaagtct 23520 aattacatag gacatagcta ttataacttc agagtgcaaa gaattcatca ttcatgtttg 23580 tgatgatatt cacataataa tgttgacatg atggtgttgc tggttgtatt taaatgtagc 23640 acatatgtgt acagttaata acacttgagt ataaaaaaat cccttgggta tgtatttact 23700 atgttacaat gttcattcat gtttgcactg ttagtaatca ggcacaggga ttactgaatg 23760 ctggggaagc actgtgccac tgagtcagcc ctggccccta ttgttgttat tttagagctg 23820 cccctttctt actgaatgac aaagtttact ttaacacatt gtatcatgta attgccagtt 23880 tgtgaaactt acttaagtcc taatagttct ttttgcttgt cctgaagaag taaaatgtag 23940 aattgggaaa aaatacttgc aaatcatgga tcttagcagc agtcggtatc tagaatatgt 24000 aataaactac aattctgata caacctttga aaagtgattt aatatttaag atgaagaact 24060 gtgggaattg gagagatggc tcagctatta agagtgcttg ctgctctttg agagtactag 24120 agttttgtac ctggaaccca ctttaggcaa cttacaactg cctataacta cagcgccaaa 24180 ggatctagtg ccctcttctg gcttccacag tctcccaaac acatggcaaa gcacacactc 24240 acaaatagaa atagaaataa atgtttattt attttaagta gaatttggta caacttaatt 24300 cagtgcttgt ctagagtgca tgaagctctg ggttcccttc ccagcactgc ataagccagg 24360 catggtggga tacacagcct tggggagact taatcaggac gattataggt tcaagtttga 24420 ggtcaccctg cactgtatag ccagacctta tctatacaag attaaaaaaa ccaaacaaaa 24480 caataatacc cccagccaag tatggtacct gtaattccag cactggggta atggcagcag 24540 cttagtgagt tctaccttaa tcaagttaca taggaaacag tttcaaaaag ataagacaga 24600 aatgacagca gctgtgctct tgatgctgca cagtggttaa aatagggcta cagtgtagct 24660 cagtggtgaa gcgtgtgctt agcgtgtgca cggcctgggc ttcattccca gcactactct 24720 gcgtgtgtgt actaagaagg aaattctgaa ttttattcat ccagttcagt tagtgtccat 24780 ggatagacag cgatagtaac tgtataggca tgcctttgtt gctagtgtag gggaagaaag 24840 accttccttt tcttctaagc attttgatga ccaagttgat aagacaaact gactggaggc 24900 aggtaaacag aagaggtaga tagactaata tgtggtctaa gagtagccta gctgcccaaa 24960 gggaaaacag taagtgtgca gtcagtgaaa ttggagaatt tatatggtct cataggaatg 25020 gcaacatagg caactcagta gaagattcac agatacgatg aatgggcatt tgagtaatgg 25080 gtgatagttg tggcacagtt tgccttgatc tatagttgtc tcttgacctc ctgtcctttg 25140 gttgagtact ctttccaggt tgataaatct tcctagaggg tatttgtggc tataagagaa 25200 cttaatgaat gtgtgtgtgt gtcattgtgt gttttaaaca ttttgcgtgt gtgtgtgtgt 25260 gtatcttgac acatgtgcta cacatgtatg aaggtcagag aaaacttgga ggaataggta 25320 tccgcgccct cccccttcac cgcccccccc ccctcccatg taggttctgg gaactcagat 25380 cagcaggctt ggcagcaaat gttttatcta ttgaaacatc tcaccactta cctccatttt 25440 gtttgacaga atcccacttg gtaatccaat caagccttgt atagcccagg ctttacacaa 25500 atgtacggag agcctctttt ctcagaggct actgtaggct caagccacta caccctgctt 25560 agcacttgtt tttgttttgt tgtaaatagg tctgactctg tagctttggc tggcctgaaa 25620 cttccagagg tctgcctgtc tctgccttct aattctggga ataaagttgt gacccctaac 25680 acagtattgt tttttcacat tctttctaat ctgaataatc agtagactag agcattgagg 25740 tattttctga ctcctttgag ttaaagtcta tctcttcatg tgctgcgcct cttgtgggga 25800 ggacagtaat cagctagaca gatgagagca cttcaatagg acaggggtat tcacactact 25860 gctgtgtaac tgaggcccat attagactga agacctcaag actttggggg aggacacatt 25920 tcatctttag gctgacgtgg tgaaaaggaa gtgaaatggg atgttagcta gagaggcttc 25980 ctagaaaagg aaagggaagt acaaagaccg gaagcaagac gggaagaatg tagctgctgt 26040 tgggaacaca gagtccttgc ttgtaggcca cagttaggag ggaagtagga atccccagaa 26100 ggcagtagcc aggagcttta ccatcttgga gaacctttct cagaggaaga gttagggagt 26160 agtcttgttt caatttaatg cttttgtttg tttgttttac accaaacata gattactttc 26220 aaagttggta aaagcaaatt taaaaatgaa agggggaaaa atgccaaaag acatggagag 26280 gcatttctcc aaagaaaaga tacaaatggc caagaagcat gtgaaaggtt gacattagta 26340 atcattcaga gatatttagg aggtatacaa gtcaaagata acatttatat ccaatccaga 26400 gaccactatc aaaaccaaaa caaaatcgga gctaagtctg gagactgata cccaccagcc 26460 tcccatgggt agagggaacc cagggctgca cagggagctc ctgtctcaaa aagacacaag 26520 taacccccca gaaaataaca aaattgctag agaagtggag gaatcggaac agttatacac 26580 tgtgatattg taaaatggta cagtcacaat ggaagagaat atggtgccac tttacaagtt 26640 acagctcagg gtcacttgcc tcacaagctt aaaagattcg attgctcata ctaaaacttt 26700 gtatattagc ttttttctga agactttaga aatttttatt tttatatgtg tgttttgcct 26760 acatatgtat cttgcacagt atgtgtgcct ggttcctaga cctggagtta cagacaattg 26820 taagctgcca ggtgggttct gagaactgaa catgggctct ctgcatgaac aacaagtgct 26880 tgtaatcatt gagctctctc tctagcccct aaatgaatta gttttgagct aggtatgatg 26940 gcacatacat ataatcccaa cacttgaggc ttatgtaaaa ggatcgctgt gaattcaagt 27000 ctgagctgct ctcaataatt acacacagag acctaacgca tagagtgctt ttgaggtgac 27060 tgatctcgga acccacactg tgaaaggata gagttgttct tgaaagttgt tctccacctc 27120 catatctgcc ctgtggctgc aaacacaagt ttggttttga tggcctgaaa cttgctgtct 27180 agaccatgcc gacctccaac ttgaactgat cctctttgaa cttgcattct gcatgcaggg 27240 gttcagttgt gcacaattta tattttcata catatatata aggcattgta taccccaccc 27300 ctcaccctgg gacatcctct tctcgatagt ccccttcctt ttcccaacta gttcctcttc 27360 tactttcgtg accttttttt ttttttcttt ttcttttgga gtgggatgtt tcattagggt 27420 tgcttaccag tagtatgtgt gagtgattac actttcctcg gggctacaac aatgaagaaa 27480 agctctccca gcaactaact gcttatagat cctcaagggg gtttgtggct ttgtgaactc 27540 ctcccccctt ctatgctagg atgttgtgta gtttttgtag tcacagctgt tgtgaattca 27600 agtatgcatt gctcatgtca tttcagaagg cagtgttcca caacagtctg ccctttctct 27660 taactcagag tgttaactct tatgcaatgg tccctgaccc ttggaggctt tggctatctc 27720 tttcccatcc cccattcctt gtggagtgtt gctgtgttgc tcagaccagc ctcctacacc 27780 tggctcaaat gttcttttct ctcagctgcc taagtaggcc taaagaactg caggcacatg 27840 ctaccacacc cagcttaaag cagggtcaaa atatgggtat tagatgaata cccatattta 27900 tgatcctgtt tttcacagaa gaatgaaaac atgaaaaata tacccacacg ctgttctatt 27960 ggttttttga tttttgtttt tgtttttgtt tttttaaagc tacaattaaa aaacaaacaa 28020 actgagtcag gcttagtgat tgacacctgt aataagcaat tgggaggcag aggcaaaggg 28080 tctgtaagtt gagagtgaat gccagactag ccagtgctac atagctgtcc ccatctcaaa 28140 aaaacacccc ccccccggaa aaaaaccaaa caaaaacccc aaatatattc ccaactttag 28200 agaaatcaca agacatttca tgttttcatc ataccatctt tctgtgtgtg gtcactgaga 28260 catgatagtc atcggctctc agtgttctct cagatgctat tagtgtctgc tgctttgtgg 28320 tgctggtcca ttatctttcc ctgttgagta gttttattta tttctagttt ttatttcaga 28380 gcggagtaga tataagtaca agggtttgaa ttaatccctg ccctttcttt tcagatgcga 28440 tgatgaccag atgtgtaatg acaaagagcg gtttgccagg taatgtacta gaattcttgt 28500 cctggttatg ggatcaagca gtctaaatgt tggatttaaa ttttatctag gtgagagtga 28560 atttctctgg ttagtctcct cctcttctga agtttgaaac agcctggaat ttatgcatgc 28620 tcattgactt gaagctaggt atatttaaca taatgtttct ctcatgcatg caagttttag 28680 taatgtgtat attgcatcta caggatcaca aactgtagac ttcttgggca tattatattt 28740 gtacaaatac agaagaatct tttatagaaa atggtttatt catttaaatg agtttttatt 28800 ctggatacca aacttaaatt tgtttaagta actgtcagaa gattatgaaa tatactgatt 28860 ttactttttt tttctcctcc cacccctgat tttacttttt attaaagagt aatcattctc 28920 aactcatata ctggttttac ttttagaaaa ttcttgctgt aagttactct tttaaaaaga 28980 ggggagggtg tcatggggct ggagagatgg ctctatgggt aaaagtgctt gttgctctga 29040 cagaggacct gaatttggtt tctagcatgc acaaccaggg agtctgacat cctcttttgg 29100 cttctgttga cgtctacatg tacatgatca tacatatata tagatgtaca cgtatacaca 29160 tacatacatg catgcataca tacatacata catacataca tgctttattt attatcatta 29220 agtgttcatg cttacttggg gagccaagat gttgggttcc aagtataagc ctgtgtccct 29280 gaccactctc cccagtatct tcaccccagc tgagtatctc tggagagaga cccagaaggc 29340 acaagtagaa ggtttgggca taagggtgaa gctcagcggg agtacttgct tcagtacagt 29400 ggccccaaac accaaatgca caataaagac cctgccttga ttcattgaac ttattcagga 29460 tcaacaaatg tttaatctaa tttcagaacc actcccaaga atttactcat tttttaggag 29520 ctctgctggt atttgggccc ctcatcttct ggtattatgt tttcaaaaca gatagggaca 29580 gaaaatatag gaaaattgaa ctgaacattt aacctctcat ttaatttggc aagggacttt 29640 tattgctata taaatagatc tactaattta aacactggaa ataaataagt aaataaatca 29700 atactttaag ctcaaatcct gaaagttttt cccttacaca cacacacaca cacacacaca 29760 cacacacaca cacacacaca cacatataca cagtggtact catgtgcctg tacttatata 29820 tatacattgt atagacacac ttaatgataa taaataatta aaaatttaga agatactgaa 29880 tttttggctg gggagatgac tcaggattta aagtgcttaa ccccagaacc cacataaaat 29940 cttgatacag agtatcccaa gtctatgatc ctagtgagtc tttattgggc gatagaagac 30000 agagatggga atagccacaa actcatggct cagctaactt ggtgtgtgca gcagtgaaca 30060 ataagagtga ctctgggctg gagagatggc gcagtggtga agactgctct tctagaggtc 30120 ctgagttcaa ttaccagcga ccacatggtg gctcacaacc atctgaaatg ggatctgatg 30180 ccctcttctg gtgtgtctga agacagctat agtacagttg tatacataaa ataaatattt 30240 tttaaaaaac ttaaaaaaaa agagtgtctg tctcagacta agtggaaggc atcgaacaac 30300 atctgaggct gccttttaac tttcccttgc atttcatgca cacgcatgca cgtgcacgca 30360 cacacataaa attgagtttg tgatcctctc aaaaattagt aactttattt ttaaatttgt 30420 atgtaattga ttactttaaa aatgatctat ttcattttta attatgtgtt tgtatagatt 30480 atacagtttt tatagataaa tggttatata catatgaatt tggataccaa aagaagacat 30540 caggctccta gaaacgcagc aagcccattt acccaccaag ctgatcctat agcccaatga 30600 atacttactt aaggtttgat tacattgatt gattgattga gaaagtatgt gtgtttgtga 30660 catgccaaca tgctaagctc agaggacaac ttctaggagc agattcttgc ctttcacccg 30720 tgagtcccag tgttgaaact cagatttagc agcaagcacc tttacctatt aagcctgtct 30780 gctatgtctt ctgtgagtac tgatcaacag tgtttactca ttgctcagct gtctgaatgg 30840 gatctacttt gaggaccact gatcgagaga accaagaaat atcaggaaga atagttaact 30900 cttagacaca gtgttggcag tatatagagt tgactgaaaa gagcaggtag gtaggtaggt 30960 agtaggtaag taataggtag aaagagggag ggtagataga atctgctgaa gtattaggat 31020 agctatgaaa tcatgagtac ttgggcccag aaatgttgcc gtgacactag tgaaagaatg 31080 aatataggtt gttttccaag agcagtgtat caaaacttct tgaaataaat gaaatctaaa 31140 ggatacagta atctttaaag tgaaagttaa ttattgattt tgggtaataa tctttcacat 31200 tctattaaaa agtctagcta taaaactgga ggtgatcctg taacacttgt aatggaggtg 31260 gaggtaagag attagaaatt ctaggttagg gactagagag atggctcagt agttaagagc 31320 actggctact ccctgtagtg gacctgagtt tgattcccag cacttactgg tggcgcacaa 31380 ccatgtatga ttcagttctt gggggagggt ggggagtcca atgccgcctt catacatata 31440 tacatacata catacataca tacatacaca catacataca tacgagtaaa atacttacac 31500 acctaaaaat aaaaataggt aaatctttat ttttttaggt tactgggcag cagggcagtg 31560 gtggcacatg actttaatcc cagcacttag gaggcagagg caggcaggat agccagcctg 31620 ttctatagag tgaattccag gacagccagg actagagaga gaaactgttt tgaaaaacag 31680 aacaaaagaa aagttatttg gctatgtagt tcaagaccag cgttggcttc atgtgatcct 31740 aaaaaccaaa ggtcagatca tcattagggc agctaataca ggtccagtcc tcagtctttg 31800 atcgacactc tggaaagcca taaaacacaa cctggagtat ctaatctgcc tatgtcctat 31860 tctgtttaga aatattggag tagagagggg gttgtgtgac tctactatta attttaaagg 31920 aattaattaa aggaatttta aatgattaac ctcaaacaaa aatgcatttc ttagagttac 31980 agtttgttat gatagtttac ctcatacacc accagaaatt cagtaggtga aaagctaaaa 32040 ataggtgaat ttacttatct tttgaaaatg aatgttttgt tgcttgttaa aaactatcta 32100 ggataaggaa caggcaggtg tgactactct gttatctgtc ttctcttcag catgtgtgca 32160 tacattttat agtattcatg actatctgca gttctaactt tttttatttc ttatttttaa 32220 cattcaacaa ttttattcca gtttaaaaga agaacagaac aggtaagctg taaaatgtca 32280 gccagtggct ggaggaagag agtggaggaa agaggaagca aggccacgcc attttaagct 32340 ggcgtggaga tgagacattt gaggggcgct ttagggaaag agtagggaag cccaaaacgc 32400 tggtgagacg catgcttgga aaagacacag agagttgttt tgggtgatca aatgaccctc 32460 tcacaggggt ctcatatcag acatcctgca tatcagatgt ttatattaca aaaataattt 32520 atgattgggg tcaccacagc atgagggctg ataaagggtt gcagcattag gaaggctgag 32580 aagaaccact gtgctagaga aaggagaaag gagaaggtga cagataatga tccttacatg 32640 gctgaggcct gttcaggttc tgctattaga cattagctga ttgtgtttat gtatgtgagt 32700 atgtgcatat gaaggcatat aggattttcc agttgaattt acaggcagct gtaagctatc 32760 cagcatggat gctggggatc agactccgat cctgtgccag aacagtgtgt gcttttaact 32820 cctgagccat ctttccttct cctttttttg agttaagatc tcaccaggcc aggctttcct 32880 ggaacctgca gaaaataatt ttacctttgt ttctgactgt tggtattaaa taaagttgag 32940 caccaccaca gccagttgtt ctttttctac tgatcatttg tgtatatatg tgcatggaga 33000 ggacagagga caactccagg tgtcatttct cagccccacc cccacccccg ccgagatgga 33060 gcccttctct gtcttggatt gcactaggtc gactaggatg ctgttcagtg agcccatagg 33120 atcctcctat ctctgccttt cctgtgccaa gattgtaagg attgtagttt ccactttatt 33180 tttacatata ttgataaaat tattattatt aagctcaagt tttactctgt tgcaaatccc 33240 taggctcaag cagttcttcc atttcatcat caccagtagc taggactaca aggtccctgc 33300 ttcctgctgt atccatttta aggcagagtc ttaggaagtt attttatttg cgtgtctggc 33360 tttattattt tggtttatgt gagacagtct tggcctgtca cccatgctca tgctctcagg 33420 ggatgattga gattatgctc catggaggac aggagagggc gtcaggtctt ctagagttag 33480 agctagagac tgtcatgaat tgctggacat tgaacccagt ttctctggag gagtagccag 33540 tgttccttat ctactcacct catctctcca gccccatcat ctttaatagt gaagtctttt 33600 tggactatta atatacctta ctagataatt tatttgaggc ttatctctct taaaatgagc 33660 acttataata ctgagttagc tagtaccttg ttttggtttg tttgcgtttt tgtgtagcct 33720 ggctgtcctg gaactcactt tgtagaccag gctggcctca agctcagaaa tccacctgcc 33780 tctgcctccc aagtgctggg attaaaggcg tgcgccacca cacccagccc ttgttttttt 33840 tcagttgctt ctctctcccc tttttgtgcg gtgggtattg agccagtcct tccacattgc 33900 cctggttatc ctggaactct ctatctctgt gtgtaccaat ctggacttga actcctcaag 33960 tgctaggatc ggaggcacca ccaccatgcc tagcccctgc cacccattct taaagtgttt 34020 tgtttttctg gaagagttta atgaaaacct ttgtctttct tcgagggagg agggattgac 34080 atactggtta aaccatagaa gaaatgctaa ggtgcctaaa ccagagttca ttaaattagg 34140 agtgtgtcag tgatagagaa tatgcaggaa acctagctct tgatttttat catgtgatca 34200 tgtcaagcta cacggctcgg tgtatggctt tcactccaag cctgagaact caggttcttc 34260 ctcacagggt agaaggagag agccaactcc cgcagtcgcc ttctgacctg tggcacacag 34320 acacactaag tactacaatg ggaaaaataa atcttgtcat aatttgtgtg tgtgaatgtg 34380 tgtgtgtgag gaaattatat ttctaatccg ttgtttgaag ctgtgtaatc tagtcactag 34440 tagacaatag cgaatgtgaa taagtgtttc ttcccagatt ggcagcagtt tataagaatg 34500 ataattctga taattctgaa tttcctggtt ggttcttgtt cacactctgt atctttggtt 34560 tctgtagaga ttgtttgaca aagtgttttt tgtgttgaga ttatgttagt ttctaattgt 34620 ccttgacttt cgacttataa tttacttgtt acaaaaatac agttctatag gttctcaagg 34680 ttcttccttg tatggccttt cctcactttt tctgtatcca tttcatttct tcctttcttt 34740 ccttttcatt ttgcatttca ttttccctcc ctcgctttca tctgcctttt cttctttcct 34800 ttcttccttt ttattttaat tttcgcttag ttcatcaatt ctgtttttag gtcggatgat 34860 gagcagagct ctgcggataa agagagactt gccaggtagg agctgtgctg tttagcatga 34920 agcacatact gccttcttac cctttgtcta acccatcttt ttttcccttt ctgtattttt 34980 ctcttacctg tggcaaagga ggacatgagg ttttacaagt tggggtacaa taagaacttt 35040 ggcttcccca acaggaagtg ggtgaattaa gccaggtatg gtggctcact cttttgatcc 35100 ctgcatttgg gaagcagagt ggagttctgt aagattgagg ccagcctggt ttacaaagaa 35160 attctaagac agagaaaccc tacctgaggg tgatggaaga ataaattata aattactgtt 35220 catctttaag atttttattt tttctttact tcatcactaa gttccacaga taatgattta 35280 gaagtaatgt ggctgtgtcc acagatgctt taacaggcgc atactctctc agaataccga 35340 taattaccat agttctatca gaataccgat aattaccata gctcccagaa tttaggccat 35400 agcacatact ctgacttccc tatgccgatt cacagtctag attactatga tggtagaact 35460 gattgaatcc ttataatttc tgaggaaagt tttagctact ttggtatgac tatttaaagg 35520 atggattttc tagggtggag agatagtctg taaaatactt gctctgcagg tatgattacc 35580 agagtttgat cctcagaacc cattgaatag aagggtgcgg gtttataact gcacatgttt 35640 atgattctag tacaataggg aagcaggggc agacctctca ctgggcagct gcactggctt 35700 aggagaagag ccctgggtcc ccatgagaga tgctgtctca gaaaacaacg tgggaagtgg 35760 atatcttaaa aatgacattt taaattgacc tttggatttg acatacatgc acatgcatgc 35820 tcacacagtg tgtatttgca cacatagttt tctggacttc agtctcagaa atttgtcaga 35880 tactaagaag atgggtactt caaaattaag agtagatttt tttaaatgtc tgagagtagc 35940 taaatattaa cctataaagt aggttctttt ttctttcgga aagagaccac ttttcctttc 36000 cttcaggaca gggtctcatg tactttatgc aggccctaac gtcctgatcc tcctgccggg 36060 gtgctggatt gcaagggtgt gccgtcatgt ccagataaac atctccatag gcagatttcc 36120 ttctgaggga ctcccagctt tgtgtgttgt gggtgacact gtgacactca ctgagctaca 36180 ttcagggacc tgttttagtt ctctttagag tgttgtccat gatacagaaa tagtgctttc 36240 ctgctctggc ctttgagttg gtgtacctgg atcattataa aaatactttt accactgtcc 36300 ccttgtgtta cttcttcaag ctaggccaca cattcattct gtgactgtta acccagggtt 36360 gctactgcga tgcttccagc cttcagagaa ctggaaggcc ttacactggg gaggagggca 36420 gctctgagaa agccagctaa ctcttgcttc atttggctca ttaaacacaa ggatgtcagt 36480 gttagggttt gtggttttgt ttttctcttc agaagagatc cataagtggc gagagagata 36540 cataaagaca gtaaagccaa gctgttcctt cgaggcgtcc tcgtgggaga ccccaaagag 36600 tccatttgta aactcttagg ttattccttt tgacagttgt attgcaatcc tgcctcacgt 36660 ttgcggtctt tgttaccctg gtgagatgcg ggttctcttt cagtgctttt gatgttcatt 36720 tccccagcta ctcctcttcc cacaatacct tttgcccagg ctgggaaacc acatgataac 36780 cagcactctt ttaagttcag cgcactgcca aacccagaga agaattttgt gcattagtat 36840 aacaaataaa attccaacat accccaattc tgtatcttaa tgccataagc aactttgaca 36900 aggcagcatt taatatacta aaagaattta cactatcatg tcttcttttt ctttcctttt 36960 ttccttttgg gataagatct cactatgtag cgctggctgt tctagaactc agagattccc 37020 ctgcctccac ctctgagtac tgcaattaaa ggtgggcgcc accaccctgg cgcgcgctcg 37080 ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct cacacacaca 37140 cacacacaca cacacacaca cacgctctcc attttaaagg cagtcttatg tattccagga 37200 ggtgagaagg aattatccac tcaagtgctg gactcacagg ttctcctgac tgctagcgat 37260 cagacccaga gccttggcct tgctagacaa gcactccaac acctgagcta aattaccagg 37320 ccaaggtggc tgctgctttt cttcttcctt aaaaaggagg cattgggtag ttttgagatc 37380 catcttgctt catagcccag gctggcttca aactcacctt gaagttgatg gtgattctct 37440 tgcctcagcc tcccagtagc agggattata gacaggcatg tgccagcatg cctggcctca 37500 tttggatatt tggaacaggg tatcactatg tagtccatgc tgccctgtat agcccaggct 37560 ggcctcaaac ccaaaactct tgtgctttat agtcttcgga gtgctgaatt gtagtatttg 37620 ctaccagctc tggcctttat actattattt ctttcataag ctactggttc atttacttaa 37680 cttttcaaaa gttggtcatt ttattttata gccttcaaat gttttaacta gaaaacatgt 37740 ggtcagtgaa gctgagattt atccccttag gaacttaaat gagtgaaatc aacatttaaa 37800 aaaacactca ctctcaacct tacccctgag acaggatttc tgtgtgttgc ccttgctgtt 37860 actttgtaga ccaggctggc ttagaactca gtgatccatc tgcctgcctc tgcctcatga 37920 gtgctgggat taaaggcatg caccaccaac accttgtttt aattatttct taaactattc 37980 actctgaagt ggtgatttaa tggaagtggt tgactggaga ggcgatatgt acttgttctt 38040 tgtctagact ttgttagttt gtttcattct tactctgcat gaggttggcc tgcttggctt 38100 ctgagatttg aacatcatca gacctaagtt actgcatcct tcagcagctg accaggtatc 38160 catatcactc tcctgatacc aaaggaacag atgtgaccac ctagaatgga gcctctttct 38220 ctttttttat gcagggaaaa tcatagtgaa atagaacggc ggcgacggaa caagatgaca 38280 gcttacatca cagaactgtc agacatggta cctacatgta gtgccctggc tcgaaaacca 38340 gacaagctaa ccatcttacg catggccgtt tctcacatga agtccttgag gggaactggc 38400 aacacatcta ctgatggctc ctacaagcca tctttcctca ctgatcaggt ctctggatct 38460 taaaactgac tctgggtata tctgttgaaa gttttggact aatttaatgg ttttgtttgg 38520 ttgcttgctt ggtttttttt gggagggtct catgtagccc aggctgacat ccaactcagt 38580 atattgccaa catgtgccac catgtccagc ttcaggagag tgattcttca ggagacatca 38640 gatgtagaaa agtgaattca gcccttagct ataagtgatc aagtattggg ggagtgagaa 38700 tagataaatt tagttgtgag aacttattta gcaaggtgtc aggtatttta gaacttggct 38760 catacaaatg acctgtgaag tgaaagaagg gtgtgtcccc agagaggttg ccacactcat 38820 ggatcagtgt tccttgggtt atatttctag agcatggaag aaatctgcct cacccttgaa 38880 gcaactttta gtcttaacga tgagagacgt gtagtaaaca acccctgttg ctccatgata 38940 tgttttgtta cacttgatat atcatgagga aaacagaaag ggcagaaata cttgccatca 39000 ttcaggagcc ccatctaaga gttatctcac tattgccagg agagagaact aaatgtgagt 39060 agagtaaacg ggtgagtagt aagatgccta agtgaaaaac gatgggaaga ggcatgcgga 39120 aaagagccgg tgtctttgtt aatagtttgt gtgtgataga tatacaatgg tggcatgtct 39180 aactttattt agtctctcta gacttgactg agaatgacta ggaaagacaa gttacatttc 39240 acttaaaaat ccacgattgg gtagggcata gtggtgaatg cctttaatcc cagtactcag 39300 gaagctaagg ccagcttgtt ctacagaggt ccaggccagc cagagctaca tagcaagacc 39360 ctgtttcaaa aataaaaaga catatgattg ttcataccta gcctatgcaa agccctgaat 39420 ttggtcccaa cactatatgc agttgtgtat ggtaactgga tatggctgct cataacccgt 39480 ctttcagcac tgaggcacaa gtaggaggtt gagggttaga gttattctca aggccagcct 39540 gttaacatag gagactgtct aaaatagata catacacaca taagatggtc atataggaga 39600 ctgtctaaaa tagatacata cacacataag atggtcatat aggagactgt ctaaaataga 39660 tacatacaca cataagatgg taatatagga gactgtctaa aatagataca cacataagat 39720 ggtaatatag gagactgtct aaaatagata aatacacaca taagatggta acataggaga 39780 ctgtctaaaa tagatacata cacacataag atggtaacat aggagactgt ctaaaataga 39840 tacatacaca cataagatgg taatatagga gactgtctaa aatagataca tacacacata 39900 agatggtaat ataggagact gtctaaaata gatacacaca taagatggta atataggaga 39960 ctgtctaaaa tagatacata cacacataag atggtcatat aggagactgt ctaaaataga 40020 tacatacaca cataagatgg tcatatagga gactgtctaa aatagataca tacacacata 40080 agatggtcat ataggagact gtctaaaata gatacataca cacataagat ggtaatatag 40140 gagactgtct aaaatagata catacacaca taagatggtc atataggaga ctgtctaaaa 40200 tagatacata cacacataag atggtaatat aggagactgt ctaaaataga tacacacata 40260 agatggtaat ataggagact gtctaaaata gataaataca cacataagat ggtaacatag 40320 gagactgtct aaaatagata catacacaca taagatggta acataggaga ctgtctaaaa 40380 tagatacata cacacataag atggtaatat aggagactgt ctaaaataga tacatacaca 40440 cataagatgg taatatagga gactgtctaa aatagataca cacataagat ggtaatatag 40500 gagactgtct aaaatagata catacacaca taagatggta atataggaga ctgtctaaaa 40560 tagatacata cacacataag atggtcatat aggagactgt ctaaaataga tacatacaca 40620 cataagatgg tcatatagga gactgtctaa aatagataca tacacacata agatggtaat 40680 ataggagact gtctaaaata gatacataca cacataagat ggtaatatag gagactgtct 40740 aaaatagata catacacaca taagatggta acataggaga ctgtctaaaa tagatacata 40800 cacacataag atggtaacat aggagactgt ctaaaataga tacatacaca tataagatgg 40860 taatatagga gactgtctaa aatagataca tacacatata agatggtaat ataggagact 40920 gtctaaagta agtgtcaaca tataaaatgt gtgagtagtg tgggaagttt tagaaaatac 40980 aggaaatgac acttaatgta atgtcaccat tgctgtctga gaattatagg aatacttctt 41040 gagagaaagg gataaatttt ccaaataggg cagtctgttt gctagccatg gagtacattt 41100 aatagctatg tacatagaaa tagtcaattc atgtcctcag aatacgtcag gtatttatta 41160 aactccagtc atgctccttt tgtggcaaac caggtacttg cgctatgaga aatccaaaga 41220 atttaaaaag gaaataaagg tcactggtgg aatcagataa tataaattga taactaataa 41280 tacgaaacta gagttgtaac aggaacagta acaaggctaa tagagtatag gtatagaaga 41340 ggaactaggt gctggccatg gtgacccact actgtagtcc cagcagtcaa ggtatctgag 41400 ttacattgca attcagtgat acgtggaagt gggggaggag tgtggtggta tggagggtaa 41460 gtacagggta tacttggatt ttattttatg atgtggaagc atgtgtatgg tttctaagaa 41520 gagaggatga ataggatcta ggaagtgttt acatgtaaca tacgaacaaa gggaagagta 41580 tctgggcaga aaataaagca gaaatgaatg gaatttcaga agatactaag caaaaatgaa 41640 atttctaggg ttccatgggc agtattaaca ggttgtgtag agagattgaa tttaaaaaat 41700 gatgagaatc caggagaagg gacttgaatc atcaattagg aggatgtaat tgaatcctca 41760 attacagaag tagtgggtta cagtgaagat ggagggataa ttgaaaagca agagagtgta 41820 aaatatggtc tcacccatct taccatcttt tatctaccct caaaatgaca gtgatctaat 41880 taggtttttg tagtgctgac ttagttaaac ctttggctcc tggtgagcat ccgctcaaga 41940 gctctctgga tttgtggatg aaagacgtgt gccagcttaa ttttgatatg ccttgactag 42000 ttcaatggct gggcagttct aaaactccca aagccagcaa acactcccct tgggtattcc 42060 tgggttaata tcttttaaaa tctgtatttt ctttgcttcc ctggctcctt tggggcagcc 42120 ctttcttttt ccccctttgt tttcccccag ggaacctaga agtcccgcct ctttcatcct 42180 gcccatcctg gctctttatt gatcaatcaa aaaccagttt gggtgaccaa aattagcatt 42240 ataacacaag cagcattaga ccaaacccac ttcatgtttt tatggttact tcctaagcat 42300 taaaacctag atcctttaat aacatattct gaacaagttg agggaggcta tgtttgtgtt 42360 tataagttgg agtggaaagg gggaaagtga gaggctccta accctaaccc ctgccccctc 42420 cagaagtctt atcatttaat gtaagccttt ttattttcct ttgagtgcta agaggcaaat 42480 agcaacttag agaaattgca ggaatactta ctgtaaaaag tttgccagtg gatatttagg 42540 gtaggaaccc atggcttata tcgtggtata agcctgtgat tcctagtgga gcagtgcata 42600 tgtacccaag gatcttgctg cttcccatga tcagttgtgt atgttttttc agagttcact 42660 attaactttg gtttattttc ttgaccagga actgaaacat ttgatcttgg aggcagcaga 42720 tggctttctg tttattgtct cctgtgagac tggacgggtg gtgtatgtct ctgactcagt 42780 gactcccgtt ttgaaccagc cacagtctga atggttcggg agcacactgt atgatcaggt 42840 gcacccagat gatgtggata aacttcgaga gcagctctct acatcagaaa atgccctaac 42900 aggtgagagc tggctggaca gcagatatgt gggggaaaag tctttatttc actcaagtag 42960 ttaagatatt taagcccgaa aaaccaaaaa aaaaaaaaaa aaaaaaaaga tatttaagcc 43020 tgtacattgg ttggttgaat ctagcaaagc ttctaaaatt tttttcctta atgactataa 43080 ttgtttttaa ttcacgtgca aaatatccac tctgtagctt attcttagaa aatatttcat 43140 cagtgcttgt gttcttataa aagctcattt ctatcttaat aatatcttaa aataggatgg 43200 agaggctgtt ggttagtgta taagggactt gtttgttgtc actgtatctc tattaggtca 43260 tttcccctcc accagtaggt gagactcagt cctttaccat cgtttggaga ataagtgcca 43320 cctgttctca gcaactgtac aggaagtaac aaaatggaaa caaataacta ggagttaaaa 43380 taggagaaac tagggctgga gaggtggctt agtggttaag agcactgact gctttttcag 43440 aggtcttgag ttcaattctc agcatggtga gatggtggct cacaaccatc tgtaatgaga 43500 tctgatgcct tcctctggtg tgtctgaaga tactacaatg tactcataaa catgaaataa 43560 ataaataagt ctaaaaaatt taaaagaaaa agaaactcca atatctttgc agtgggaatg 43620 ttcatattaa tgttgtttgt ttgtttgttt gtttgttttc cccctctttt gtgattttgt 43680 acatttcatt tctataattt tccttttccc caaatgttac ttacattttt aaaaaaataa 43740 ataaaggaga gagactgttt tgtttttgga cacatgcatg tttcttagca tgtaaaatta 43800 cttgttttta aaccagacac tgtccctacc cgaagcgtga cgatagttag tgactggcct 43860 aatggctcgg tggtgaagga gcatgccacg taagcccagc agtctacgct cagtctgcag 43920 actgcataac agtggaagca gtgacagctc cacatagttg tcttctgacc tccacataca 43980 ggagtgggct cacatgcaca atagttctag tagtttccac tcctgcttta ctcagttctt 44040 gtgccgtttt ggagctaata gtgagattcc tagatgagga cgcaagtagt ttaggcttca 44100 agaataaagc atctctgtca taaacacgga atctggaagg cttggctgga cccaagcttt 44160 gccgcggtgt ctccttcctt tcccatccac aagaagtaac ttgagattgc tctctacctc 44220 cttgaccagg gcgggtcctg gatctgaaga ctggaacagt gaaaaaggaa ggccagcagt 44280 cttccatgag gatgtgcatg ggctcacgaa ggtcgttcat ctgccgcatg aggtgtggtg 44340 cttgggattg tgttgggtgg gaataagagc agctaaggct tccatttctg tcagaggcat 44400 tagttagtat ctagtatcta gagctacgtt ccttgccaca gttggattac atttaacttt 44460 caagaaccag ttgttttacc atacttataa tatgtaagag aataacagtg aattacagtt 44520 aaggcacagt tggtaggtcc tttgtgttga cttcttactt ctgtcttagc ttcactactg 44580 ttcatagggt aagagtcaac gttcattatt ttgttcaggg gctagagaga tggctcagca 44640 gttaagagca ccttttgctc ttccagacga cctcagtttg aattccaaca ccacatggca 44700 gctcacaaca tctgtaactc cagttccagg ggaatctgat ttctctcttc tcgattctgt 44760 gggcatcagg catgcatgtg gtgcacagtc atgcaggcaa aaacccatac gcattaaaaa 44820 tgttttaact ggatttaaga tgtgggagag ttgtcttact agttaagagc acttgttttg 44880 ttcgtctaaa ggacttaggt ttcttttgta gtacccatgt ggtggctcac agccacttgt 44940 ggctccagtt ctaaggggtc tgatatcctc ttgggccagc aggccatata catggtgcat 45000 atatttgcat gcaggcaaaa cattcataca cataaaataa aaataaatct ttttttcctt 45060 tttaaagtga atgcagaaat tggagagata gcccagtact aagtgtgtga tgcacaaaca 45120 tgaagaacct gagctcaatc cccagcacct atggaaagtt agatagtccc atttctggag 45180 aggcctagcc aaattaatga gcctcaggtc ctagtgagag accctgtctc aaaattcagg 45240 gtaggagggg ctggagagat ggctcagcag ttaagaataa tagctgctct tccagaggac 45300 tggctcaatt cccagcatct gcatggttgc tcacagccat ctatgacccc agttccaggg 45360 catcggatac ctttttctgg ttcctgtgta tccaggcgtg cacgtaatgc acttagagac 45420 aggtagagca ttaatacaca tttcaaaaga attacgatgg agagctcttg aggaactaca 45480 tgtgaggctt gtctctggcc tccgcatgcc tgtgcgtgta cagcatgaac atatacatac 45540 atatacatgg atcaagaaac cggctgcaga gatggctatg cagggataag agattatatt 45600 gctcttgcag aggactagct ggggttagtt tcccagcatt cttatcagat agcttgtaac 45660 catctgtgct tctagctgca aggagattca aagcctccag cctccacagg tagctcaaca 45720 tgcacatact cccctccttt tccatgcata cagctaaata aaataaatct tacaaaacag 45780 aacagatgtg ctagaaagat ggctcatcag ttaagggccc taactgctct tctagaagtc 45840 ctgggttcaa atcccatcac ctaatggcag ctcgaaactg taactacaag atctgacgct 45900 ttgacactga catacatgca ggcaaaacac caatgcatat aaagtaaaaa caatagacaa 45960 accgagtgtg acattgcttg tcttaaatca tagtgctcag gaggcagagg ccggcatatg 46020 tgagtgaagc attaggttct cgagatcacg agactgatag agaatcttgt tttagttcat 46080 agcagcagaa ctccaccatt gatgtgtatt cagacttaaa actcaagact gacctgcttc 46140 atcatccaag tgctgagatt ataggcggtt tttgtttgtt ttgtttttca gattaaaaaa 46200 aattatgtgt gcatgatata tgtggatgtg gactcgtgtg tggtgtttgg ggtgcaagtt 46260 gttggttctt tcctacaaca ctgtgggttg aggtaatcaa atttaggtca tgaggcttgt 46320 cacaagcacc tttacctgat ggccaatctc cctggctacg ttaaaaaaaa aaaaaaaaaa 46380 aaagagtttc tggcagaggt acaaaattga ctattccata gtttaatagt ttagaggagc 46440 cgctggaatc aggctactct ggtttatttt gagggggtgg ggtcttatta tgttgtccag 46500 gctcatttaa agatcctcgg ctcaagttat cctccatttg agtttcctaa gtagcttggg 46560 tctacagaag gtatatcact gtagttgact gaatgttggt atttgtttag ggatggggtc 46620 tcactgtgta cccctggctg ttctagaatt ggctatgtag gacaggctga ccttgaaatc 46680 acagagacct gtctgactgc ctcccaagta agttgtttga atccatagta ttccctggat 46740 ttgaattcta atttgatact ataagacttg tgttgctttg atgtcatcat ccactattgg 46800 aggtgagact gtcaaacgta aaacattgtt atcaggatta gctcgattga tagtatgaaa 46860 atactttaaa aagaacgttg aggcaggagg attcctacaa ggccagtatg gcctatatag 46920 caagttctgg atctcctggg ctatatagtg aaaccttgtc tcaaacaatc aatctagatt 46980 tggtggtgca tgcctgtaat cccagaacct tggaggctaa ggtaggaaga tcatgaattc 47040 caggctagaa tgaactacat agtaagacct tatctctaaa aaaaaaaaaa aaatagagaa 47100 agaatagaag taggattgga gggcgtatct tgtggacatg gtaagcctgg agtaaggtga 47160 ataataatga aatatagcac acagagatga tatgcttaga gcagaagtca gagaacagaa 47220 gttcttattt gtgactaaag cttcattgca ccatagctct tcctgtgtga acacttgtca 47280 tctttggctg cttttatgct tgattgctca gaattgtcta gttaaaagac tgtgtgacca 47340 tcagactaaa accacttact gtctagtcat tcacagaaaa cagtaaaaca gtcaccaact 47400 cctgatgtaa aaagatggca tttgaagagt gtatgcaatt gttttagttg gagttttagg 47460 ccaattaaaa gttttaaagg agtccaaacc tttcatcttt gctcctcttg aggctaagga 47520 aagaggatct caatcaagat caaggccagt gtgggcaact tggtgagctc cagtccaaag 47580 ttagggaaat aaaacataaa aggaggactt actgctcgga ggccagaggc tcgcctggca 47640 agcacagaaa cagctcaata ctcagttttt aagtaatcta ctaactctgt tggcctggtg 47700 gtactttcta tatctcatta cttgggactt aaaggcaagc agacagtttg tgttcattct 47760 gtgttgtatg agatcctgtt tcaaaaagtg gtctgggttg gaaaccagag atagatagct 47820 tagtgtaagg aaacttggca gaaagcctga taatacacgg ttgattactg gaacctacat 47880 agggcatgca cagagacaga gacagagaga gagaccgaca gacagacaga cagacagaca 47940 gacagacaga cagacagaca gacacacaat cagccagggt cgggaatata atatggcttg 48000 gttggtagag tgcttgtcta aaatgcatgg agtcatgggt tcaataccta acaccgactg 48060 gtatgttgat acctgcctgt agtgccatca ctcaggtgct gttgatcaaa gggtcagaag 48120 ttcaaggtct tccctggcca catagaaagt ttcaaaccag cctaagcctg aaggtgctgc 48180 ctcaaattaa acaaaacaac aagcaaacaa aataatagaa ggtttttcca tctcagcctg 48240 acttttcttg ttacttacta gttttcagtg tgtagttgga tcttcttatt ctgttgttac 48300 tgtttcccat agcccagcaa tttgcatctt ctgtttgttg acaggtgtgg tactagctcc 48360 gtggaccctg tttccatgaa tagactgagc tttttgagga acagatgcag gtaagatctt 48420 aggcagtgga gatcaagaga tgggattttt accttctcta ctggctctca ccttctttta 48480 cctcacggtc acttaccgtc tcacaatgtg tttctgaact tactgaatag atcaatccat 48540 ccatcaattg attgactgat tggtttttct gtgacagggt ttctctgtgt agccctggct 48600 gtcctgaaac ttaatctgta gaccaggctg gtcttgaact cagagatctt gagtgctggg 48660 atcaaaggca tgcaccaccc tgcctgtatt gtttctgaac tttaaaagct tctcttgttc 48720 aaggaagttg cttactatgt tacctatttt tcttgtgtgt tttatttcaa caagggctgg 48780 tgcagcatcc acacacgtac cagtaataaa tggataaata aataaataaa taaataaata 48840 aatgctttta aaacacaggt ttcctttgta gaatttcacc ccggattcct ataccttttc 48900 taggaatggg cttggctctg tgaaggaagg agaacctcac tttgtggtag tccactgcac 48960 aggctacatc aaggcctggc caccagcagg tagggaaaat atacagtaat ttctctctgg 49020 tatatttggt tacataaaca attggcaata tgttactaac agttagcaat actatgtacc 49080 cctttatgaa atgtgcttaa gaataattca aaagatagat agacaaataa aacagactcc 49140 ctctcatctg ttaccaatgt tatagtctat ttgagcatat ggattactaa aacataaaaa 49200 tgactcaata ttagctttat tacaaaatgc tgtaggtttg ttttcgaagc aggtttctct 49260 gtagccctgg ctgctttgga gctgactttg tagaccaagc ctcatttcca ctgcctctgt 49320 ctccccagag ctggattaac taaagcctac accacaactg ctcaaccttt tttttttttt 49380 tttttttttt aaatgtttta ttgggagctg gaaagagggc tcagcagcag ttaagaacac 49440 ttactgctct tgcagaaaat ctgggttcag ttctcagaac ccacatgttg gcttaaaact 49500 gcctataact ccagtttctt gtgatctgtc actctcttct ggcctctgca ggttcctgta 49560 cttatattgt gtccataaat tcacacaggc aaatacacaa aaacaaatgt attactgatg 49620 cttatcccta tatttatagt agataatatc atatgtaaag tagatacttt tcatctctgg 49680 cctggaactc ctattttcag caattttctc ttcaatacct tctctgggaa ttgcaagcat 49740 gagctgctgt agttggtgta gaattcagtt attagtgcta agaatatagc tcagtaatag 49800 agtacttgcc aggtttatgt caggaccaaa acaacagaac aaaacaagcc agtgtgtggt 49860 ttaaaataat tctcaagtat aatagtagta tacactgaat actgtgtctt taagtagctg 49920 tggcaaaaca aacaaacaaa aaaaaaacca ctggagtttt attttatttt ttgggtctta 49980 gtgtattcct ggctgccctg gagctggcct cagattcaca gagatccacc tgcctttatc 50040 gtgctaaggg ctaggataga aggtgtgtgc cactatgccc agcttgaaac attggtattt 50100 aatgcaatta atttgggcaa gctgaaatgg aagagaaact ttaagcaatg aatgcaatgg 50160 ctttcttgca gtaactttaa aagggaggtt tctcttgaaa tgttagtcgt gagtagagta 50220 gctttatttt aatttgagat taactaaggg atgtagcaaa agaggtaggc tgtggggtca 50280 gacttggttt ttagatcatg gccatgaact ccttacctca gtctaacaaa ttctgaatta 50340 cgggctggtt ctccatgccc agcaaggcca gctttttaaa ctgactgtca actttccaga 50400 tctcacccta ttactttcct actataaaag gattgctggg aaatggggat ttacagaaga 50460 cagaaatgca gagaaaagag agacaggttt ttaatactta gttgttttgt gtgtggtagt 50520 gtttcgtctg catatatgta tgtctgtgca ccacttgttt gtctggcgcc cacagaggtc 50580 agaagaggat gttgaatccc ctggaactgg aattacaggc ggttgtgagc tgccatgtgg 50640 gtaatgggat ttgagcctag tctggaagag cagccagtgc tcttaaccac tgagcctttt 50700 ctcagcttcc ttttctcagc tgatatattt taaaaggaaa ttaacattga tttgtgtctt 50760 agggtttcta ttcgtgcaca aacatatgac caagaagcaa gttggggagg aaagggttta 50820 ttcagcgtat actttccaca ttgctgttta tcacggaagt caggcctgga actcaagcag 50880 gtcaggaagc aggagctgat gtagaggcca tggagggatg ttccttactg gcttgcttcc 50940 cctggcttgc tcagcctgct ctcttataga accaagacta ccagcccaga gatggtccca 51000 cccaccaggg gcctttcccc cttgatcact aattgagaaa atgccttaca gctggatctc 51060 atggaggcat ttccccaact gaagctcctt tctctgtgat aactccagct gtgtcaagtt 51120 gacacaaaac tagccagtac gatttgggaa aaacttaata tttgctctct ggcatttgtg 51180 aatagtttaa cctatggcaa gtttcttgac ctttacatgt ttttccttat ctgcagtcca 51240 ggtcatacat cctgtatatc atcatcgtga gttagattta gtgtaaggaa agaacacccc 51300 agtgcctggc acatgctcta tagccaagaa atgggaatta ataattgagg aaatattatg 51360 tatagtggca catgaactgt tgagttaact tcctatgtaa atcatacctt tactgtatgt 51420 atgatctttg atacttctgc atagctgtcc tagataagaa tgagagataa agacattgtg 51480 agggatgaac tcttattccc tttttctgtg gaaatgaata tcttttggtg ttgggacaat 51540 ttaatttttc tttactttaa aaatcactgg ttaggcagga atatttactt ctctgtgtat 51600 gatatagtca catagcataa gtaaagttta tatttttaga ctcctttgat tgggacttgc 51660 attttcaaat ttcttaaaat ggttaaagcc aggtgtgctc atatgcctga aatcctgcat 51720 tgaaaagatg aagagtccag gttacacaca cacacacaca cacacacgca cacactcaca 51780 cgtgtgtcac atgaaacagt aactggttat tttattaaat cttagcatct tcatagggaa 51840 atagtgagtc ttcagtgact tgcccttaaa gggatttgaa tttgtgatct ctctgagtca 51900 ttctcctttt tcttcatgaa ggtgtctccc tcccagatga tgacccagag gctggccagg 51960 ggagcaaatt ctgcctagtg gccattggca ggctgcaggc aagtatgagt cttcacatct 52020 atgtcctttc agtttaggga aaaaaaactt ttcagatttt atttgtggta attatctttt 52080 gctctgtgaa tacatagaaa aattccataa aactcaagat ctaaaataat gtgatattta 52140 taataaacat tgctattctc agacttagct aaaatcaaaa gcatgagaga gggtggtaat 52200 cttgttgttg ttgatcttgg taatgactcg tccctgttac ctaggcctcg agctccttgg 52260 ttcaagggat cctcccccta aagcctcctg catgttggga gagaggcagg cgtccgtcac 52320 catgccaggc tcccctggac attgttagtt ttagctttac agtacttact tttctatttc 52380 caattaattt cttttgttcc tatgcaaagt aagaacaatc atttttatta tgtcattaca 52440 ttctttataa tagatctaaa atatcttaaa atttttattt tatgtgtatg ggtgttttgc 52500 tttcatgtgt gtctgtgcac ttgcatgcct ggtacccgag gaggctagaa gaggttattg 52560 aatcccctgg aactggagtt ccagaaagtt gtgacttgac gtatgggttc tgggaatgga 52620 acgcgggttc tctggaaagc agtcagtgct cttaacactg agccgtctct ccagcccttg 52680 attttgatta ttttcatgaa ccctgttcag tttctcagaa acttgaatca ctctgaataa 52740 tgaatacggg ctaaccaaat aaaaagtagg tggtttaggg taacattcac tattttctta 52800 ctttaccaac ttggtaagag aacttacata ggagtagagt tagagtttat gcctgtcgtt 52860 aggattgaca tttttgcatt tctgggtaaa acttctaaga agtttctaaa tctggtagtt 52920 tcatatctac acagatactc atgaaatatt aaaagtattt atgtaagtaa tggtgttatt 52980 tgggctcatt ttatctctcc tttcttaggt aactagttct cccaactgta cagacatgag 53040 taacatttgt cagccaacag agttcatctc ccgacacaac attgaaggga tattcacttt 53100 tgtagaccat cgttgtgtgg ctactgttgg ctaccagcca caggtgagga gcgtagcttc 53160 tgcattagta acacgtaggc ctctgttttc tgttaccagt gttgaggtta catggctaat 53220 tccagagatg accaaaatat agcagtccag ttcagaaaaa gagatgctcc tattttgcga 53280 gtagcaaaat atagtctcaa cttcctttca agactatcac tccatataga gaaatgtagt 53340 tggtaaaacc atatggaaat agtaggtata aatatatagt ttttattact aatgtcaata 53400 cagatcattt aaaactcctt attttaatta ttctagtata actagaattt atataattac 53460 tatattttag taatttatat aattactagt agaataagcc tataataatt ataagtgtag 53520 cactgcttat taaaattctc atacagtagt atatcagtta actacccctg acttacatgg 53580 acatggtctc tgttatttat gagcgtggct atgagtgtgt gaagtggtca gtcaagatgt 53640 catcttgggg gctggagaga tggcttagtg gttaagagtt caaatcccag caaccacatg 53700 gtggcttata accatctgta atagatctaa tgccctcttc tggtgcgtct gaagacagct 53760 acagtgtacg tatattaaat aaataaataa ataaataaat ctttaaaaaa aaaaagaagt 53820 tgccttgaaa atgaagaagg tcgggcagtg gtggtgtatg cctttaatcc cagcacttgg 53880 gaggcagagg caagccgatt tctgagttca aggccagcct agtctataga gtgagttcca 53940 ggacagccag ggctatacag agaaaccctg tgtcaacccc cccccccaaa aaaaaaaaaa 54000 gaaaatgaag aagaaagttg gagaatttac ttacacccag atccaacact ccctgtaaag 54060 gtgctgttat taaggctgtg tggtgttaat gtactgatct attctcatga gtaagctaaa 54120 gagtcttttt tgcattggta attggatttt cagcaaagat gcttagacat ttcaagggga 54180 ttctatatag accacagcca ccacccctaa aaatcatcat accatagaca aaaatcgact 54240 caaagcccta aatgtataag tactaattgg aaacaccttt tagaagaaaa tctttatgct 54300 cataggtgag caggccgcgg cacaccttgg gagggtggga ctcaccgggt gctattcatc 54360 tttttgtttt gaggcaagat ctcccattgg actagaactt gcccagtagt atagacttgc 54420 tggccactga gctcccataa tccactgctc taagattatg agaatatcct acacttgact 54480 ttttgttttt tggaaacagt gtctcactcc attgctttgg gtactggagc tctttataga 54540 gaccaggctg gcctcaattt cagaggtctg cctgcctctg cctcctaagt gctgagatta 54600 aaaggcctgg ctatttttta tttttatttt atgtatatat gtgagtgcct aagtatatga 54660 atgtgtgcca tgtgtttact tagtaccaga aagaccagaa aaatgcgttg gttctcctga 54720 aactggattc taggtggtta taacattctt gataacaact gctctacagg aagtgttggg 54780 tcagggattg taaatttttc aacttagttc ttcattttta acgtctggtg tgtgatcttt 54840 gcaaatgcag gaagtgcttt taagggatga gccatctctt ctgctctgcc atacccctac 54900 ctttttctat ggtttttgaa aatgtattct aaggaccaca ctcagctcct ctaatgttat 54960 ctacaggctt agctctttcc atccctccgt ccctcctaac tcccatcttt ttctaggcta 55020 gcctcaaact ggaaatcttt ctgtcctagc ctgtgatgct gagattagag tatgccacta 55080 tgcctggcaa aaattttctc taaaatgata tcattaaaag agctggtaaa gtgattttgt 55140 gagaaaggcc tctttgtttc tagttgttcc attacattct tcaagcacac acgtgaccca 55200 tagagtccaa gtgtgttttg gaggacactg agcaggagtc agctccccga gttgggttcc 55260 agagatggac tcactgaacc atctactggc cctgcttctc cacacaccta tttatagtag 55320 gataaagaga agaagtaaca tccatatctc taacagatgg gaaggcgggt ttattttacg 55380 tctctccttc ccttctagga gctcttaggg aagaatattg tagaattttg tcatcctgaa 55440 gaccaacaac ttctaagaga cagctttcag caggtaacat ttttcctggt ttgatctgag 55500 tacatatttt gatcaattct tcaagtttat tctagatact tactgataag tgttcagttg 55560 agcactgtgg tggatactat ctacctatct taatttctaa aaaatataaa atgataatac 55620 tttgaaacta gacaaagtcc aaggtatttc tgctttggtt ttatccaata aattctcttc 55680 aaagcattta aaatattatc tgcttaacta ctttatttta gattatagca gaaaaattgt 55740 ctcagaattc ttttctctat aattgaaagt taactcacta gtaactcact ggtaacttgc 55800 tgtagttctc aggcacgagc tcttgaaaac actcggagac gataaatggc cttggcttta 55860 gataacaggt ctttatattt tgtaagttaa ttaagtcttg atactgtgct tcctgataca 55920 atggctatag taatacttaa ggtagaagct tgtgggagag tcaagtatgg taaagccata 55980 ggcacatata tacatatggc ccagcctttg acacatgttc attgctaata tctttattgt 56040 tagagatgga taactaagag attgtcttct gttccataag acagaaacta aaacattaac 56100 atagtacttc tcttaggggc tttgtggaaa ccaaaattgt taagttttga gttgaagagc 56160 tgggattgct aagtaaggtg taaagtgtat ttagtgtcac ccacctgggg ttcctggtat 56220 taacctagaa agccagtttg ttgagaatgt ctgcatcaaa cttcaaggaa acatggagac 56280 actcgcttct gtgttccctc ttcactgtac agtcatacag gctgcagctc tgggaagata 56340 cctgatagat agatgccccc cttaatttcc tcttcctcgt ttgaacgttg aaaaagactg 56400 gtgcttcagg ggcaacaaca cgagtcctaa ggctgtaaac atggcaagat tattacttaa 56460 ttttcttttt ttttaagtcc actgatggat gaaacccctt tcaggtggtg aaattaaaag 56520 gtcaggtgct gtccgtcatg ttccgattcc gatctaagac ccgagaatgg ctgtggatga 56580 gaacgagctc ctttaccttc caaaaccctt attcagatga aattgagtat attatctgca 56640 ccaacaccaa tgtgaagtat gtacccttca gtctgcccct gtgttgggtt cttcatcctg 56700 tagccactcc tagggtgggg tgggggtgca gcagggcaag tcttagcttt atcatgctgt 56760 cttctcttca gcttgaactt actaaataac atcagtttga gattcctcat ttaacattta 56820 aatttaacat taattgaaat tcctttctat aggagttgat gtagttgagg tagagaacaa 56880 ggtgggtttt agttgctgtt ggtatctact ttctaatttg tgtatgttga atgggcccgt 56940 aatagcatgt gtgaacatca gagcacagct tgcagtagtg ggtccccctt tatcaagtag 57000 gtcgcaggca tggaattcaa gttgtcagct gttggctggc acttttccta ccaggccatc 57060 tcattggctc acggtggaat aagtgttaat aagcagcagt agttggtgct gtgagcagag 57120 ctttcactgc tgcccagcct tgttgcccac tgcagtgtgc tcagccaact tgttttctaa 57180 aaggctgcaa gtaaaatttc caaagattag atttttttgt tttttcttct ttttggtttt 57240 acgagacagg gtttctctgt gtagccttgg ctgtcctgga actcactttg tagaccaggc 57300 tggcctccaa ctcagagatc cccctgcctc tgcctcctga gttctgggat taaaggcatg 57360 gaccagcact gcctttcttg ttgtaaattt ttatataata tattctgatc acactttccc 57420 cttctagctc cttccagatc ctcccaccta ctaaactcca tgacctacac acacccgaga 57480 gagacagaga cacagagaga gacagacaga cagacagaga gaccaaaata tataaacaaa 57540 agacccaata agacaaaaaa aatgcctgaa cagagcaaga tgagataaaa accctgtaaa 57600 aatatcactg aatacatagg ttacctatgg gactggacta agatgttagt aattgttcac 57660 ctttagtcaa tatatggagt ttattgtatt gagtaattcc ttgtttcccc ttttgtgttc 57720 ttgtctactc tattttccta ggaactctag ccaggaacca cggcctacac tgtccaacac 57780 catcccaagg tcacagctag gtccgacagc caatttatcc ctagagatgg gtacagggca 57840 gctgccatcc aggtaagaaa tggtgaaata gttagctttt caagtaaaaa ctagctgggt 57900 gtagtgatgt gcaatcacat atttttgttg ttgctgcatg tggtttattt tttaaagtct 57960 ttttctccaa attctttgtt aaaagtctaa ttttccccta tcgctttcca cactgatgtt 58020 tgacgtatag gcagcagcag cagcagcaca cagaactgga tatggtacca ggaagagatg 58080 ggctggccag ctataatcat tcccaggtga gtgtaccttt ttcctataag gagacaatga 58140 acatttcttt aaaatgtata ttgaagctac cagaatggtt tagtggttaa gagagtactg 58200 ctcttggaga aagcccaaat tcaattccta gtaaccatag tgggtggctc acaatcacct 58260 gtagacacta tgttcatata cacagacaca gacacaacaa tttaaaacaa aaatgaatct 58320 taaaaacaat atttatcagt gccagtgaga atgttctata taaaattgaa cattattgat 58380 tgttttcact attttattcc ataggtttct gtccagcctg tggcaagtgc aggatcagaa 58440 cacagcaagc cccttgagaa gtcagaaggt ctctttgcac aggacagaga tccaaggttt 58500 ccagaaatct atcccagcat cactgcaggt atgggtttct tccgcaggct ccttttacag 58560 gctgagtttc attattactg atgggactgc ccagtcagca tacttctact tcagttacag 58620 tacttcatga ataagtagga attgattaaa ctatgtgcta cagccccttg atggccctcc 58680 cccaactcct tttggtctgc agatcagagt aaaggcatct cctccagcac tgtccctgcc 58740 acccaacagc tgttctccca gggcagctca ttccctccta acccccggcc ggcagagaat 58800 ttcaggtgag ccccatatgt gtgtgctgct tgacagggct ctgcagggtt cagttgctgg 58860 atccgtgtgt catcttccct tgctttctct ggtcacctca gacaaagcag tagaacttac 58920 tggacctagg gtgagacaac gaagctgctt ttcctcctgt ttttgcacct attctattgc 58980 cttgctctag gttccaaacg tctctgtgtg gtcagtgtgt gacagtcagt ctttcttgtg 59040 tttttaaatt tatcaggttt tccttaactc caagaaatca gaggaatcta gggatagaat 59100 gtgtcctttg attcatagct tctgtgatgg agtcaacctt ttaaccttca ctttctattt 59160 gttccttcct aggaatagtg gtcttacccc tcctgtaacc attgtccagc catcatcttc 59220 tgcagggcag atactggccc agatttcacg tcactccaac cctgcccagg gatcagcgcc 59280 gacctggacc tctagctccc gcccaggctt tgccgcccag gtagcgcctc ttacctgtct 59340 gaccctgtgt gttgttgttt gtgggatctt ttttttttcc tccaggtaaa tttattattg 59400 tcagatacct ccttttcagt tcctgttctt catcttttaa ttttcatctt ttttgcgagg 59460 agctagataa ttaaaatacc agagataaca tccagaagtc tctttagaac ctatgttttc 59520 atatgctcac cttaagacag aactgctcat tcacagcagt ttgaacagct ttggtctcag 59580 gcttgtttgg agtaaattca aagagtttgt ctttgtttag gactatgtaa tatagtacca 59640 aagtagaaaa ttagtaatta tattcattta tttatgaaaa tatagtatcc atctatataa 59700 tatatagtaa atatgttaaa tttatttatt actatgtatt tctcttgtct atataataat 59760 ttagaaaagt ttaaaaaaaa atgaaaactc aaacttgact ccagaattgt tctctatagc 59820 ttttttgtgt atgtgtaaaa atactcagga atgttgccca tgactcttgg tcaggatgtg 59880 ccgtgatgtt gatgacagct gctaccatgg ttttcatact ggtgacataa tcaagcatgc 59940 atatattatc tcccttggag ttacttttta aattgtggat ttaaaacggt ctctagaaat 60000 tattgtatct cacaaaaaat gttagaggca ataactagtt tcttaatcaa tatacagtaa 60060 atgggtgtga gaaaggacag ctttaatttc aacaaagaga ctgaggcagg agcatgggcc 60120 tgggccacat agtgagagat aaactgtctc ataaaagaaa gaacagaaca aaaccaaaat 60180 agcataatgg aaaataaaag aggacattgt ccactgtgcc aagtaacagt ctcttctggt 60240 ggggttaaca gtgaggtttc atttctagtt tattttttcc aatttttttc tattcttttt 60300 gaggcagatt tagaatttat attaaacaac cctttgcaga tttacactta aagtcaagag 60360 caaaagagaa gcactcagaa ctgagtgtgg gtgtgaactc cctaagatgc caccaaattc 60420 ctctgtaaca catagggatt ccagtaaaaa agaaaatggt tgtgccttta ggaaaaggat 60480 ctagaaacgg gtgcagtgtg gagtgagtgg tttctctttt ataacactct accattggga 60540 cccgagagat gacgccattg ggaaagtgtt ttgtcacttg agcctgacct gatgacctga 60600 gtttaatccc caatctcctt ggggggaaga gagaaccaat tcctgcatgt tgtcctctaa 60660 catgcgtgcc agagcatgca tgtgttctca tgtgcataca tactcatgca cactaaaaat 60720 aaggtggaaa cctttaatac caccacttgg gaggccaatt caggtagatt tctgagtttg 60780 agttcagcct ggtcttcaga gtgagttcca tgacctcaga gaaatcctga ctccaaagac 60840 aatagaaaaa agaaataata ataataatgt ggaaaataat aaaggaaaac acccagtgtt 60900 attgacatct gtgtgtacat gcaaccatta tacagagaca tgcacatgaa cacacacaaa 60960 tgaagtgaag acatgttgat ctctggttcc cattaatttg tgcacaatag cctgtacatg 61020 tatgttgaac ttggacacac atggatgtta tgctttctat taattgccat aatttatgag 61080 ctgattatat ataggaatag tttttttttt taagatttat ttacttatta tatgtaagta 61140 cactgtagct gtcctcagac acaccagacg agggcgtcag atctcatttc gggtggttgt 61200 gagccaccat gtggttgctg ggatttgaac tctggacctt tcggaagagc agtcgggtgc 61260 tcttacccac tgagccatct caccagcccc aggaatgggt ttttaataat gttttcatgt 61320 atttatatag tgttgttttg ggttttttgg tctttttgaa atagagtttc tctgtataac 61380 agagtcctgg ctgtccttaa cctgacttaa tagaccaggc tgaccttgaa ctcacaagag 61440 gtctatctgc cttcatctct ccttcccgag tgctggctgg gattaaaggc atgtgccacc 61500 acactgaccc gtatacagtg tattttgatc ataacgtgtt ctactccaga ccagtgatgc 61560 ctctccctct taccaaccag tccctcttgt atgtcagtgt ctctttccca tttcatgatc 61620 cagtgagctt aattatcaca atttgtaatt ctttgttttt ttctttttca cttttatttt 61680 agtttcctaa aacatttagt tttaggaaga attgggagtt aatcagaagt atacattaga 61740 agcaggtaat gggttgggtt tggaagaagg tagtggttga tgagtatcga cttacaggaa 61800 tgcagtacac gattaacaat cctttgttct gtaccccctt cctttagcag gtgcccaccc 61860 aggctacagc caagactcgt tcttcccaat ttggtgtgaa caactttcag acttcttcct 61920 ccttcagtgc tatgtctctt ccgggtgctc ccactgcctc atctggtact gctgcctacc 61980 ctgctctccc caaccgtggc tccaactttc gtaagtgcag acaagggaga taacaggaaa 62040 atcaaactac taaagaagga ggacttggca gttatgattg ttttccctgc gtcctgcatg 62100 tgctgaccga attataagga aatgcatggt ccaggtctgt ttgattttaa acagccttgc 62160 atagggcctg ttgaaatggc tcattgtgca aagtgctcga cacacaaacc gggtaacctg 62220 agttcatatc caggagagag ctgacgccac catgagctca catgcaccat acacaagaca 62280 gtaagaatct taagaatcat tataaaaaca tggtatagga aaaccattta actcctgtag 62340 ttaaagacca tgtgaatcat tttaatatgt ataacaatct tacttaatag actgcatatg 62400 taaatgaaca aggaattctt cagaaatgct atctaggaaa tgaaacttac tgtgagactg 62460 agttttgaaa atagaagcag ccctgaccag cagagagaag agcagaaggt agcatatgga 62520 gactactgac ttacacgtag ttgtgtgctg gtttctcctc tccattacaa ctataaatgc 62580 ttattcttat ttctagctcc tgagactgga cagaccacag gacagttcca ggcccggaca 62640 gcagagggcg tgggtgtctg gccacagtgg cagggccagc agccccatca tcggtctagt 62700 tccagtgagc agcatgttca gcagacacaa gcacaagcac ctagccagcc tgaggtcttt 62760 caagtgagtg ggtaaagact tcagagagag atcggtcagg gaagagagga agaaagaagc 62820 acatggtatt ggttagggct gaacggaatg agacagagga tgcatgtcag cagttgaagc 62880 aattataaac atagcctgta accagaccag gcagaggtac tgatgggaag caacatgcaa 62940 agatgctaaa gaaaggtatt ttaattttat acagagattt acatctcaaa gtagtagtta 63000 tggatttaaa tagaagaata cgaaaatata tggagtagaa catactgatc atagctataa 63060 gtatctgttt cctttgaggg gcctgcaaga ttgaatgtgg tcatctcaga gaagccagtc 63120 agaaagacca aaggaaggca acatagggtt tgattgagaa cagatgtgat taggcacatt 63180 tcagatgtgc ctgtaggagt taaatgaaat gttggcatgc atatttcttc agttaaaaat 63240 attgagtaca agaaaagagg aaaataaatg atctcagagg aagatagaac agagaatgag 63300 gaaagagttc tcagaagagg gaggttgaga gaactcgcca tgcagacttg tgcggctcag 63360 gaatggtggt ggtgtgggtg ggggaagggg gtcctggaag ggaactcact aaaataaggg 63420 tatgtgaccc aagtctaccc tttttctcat taggaaatgc tgtccatgct gggagaccaa 63480 agcaacacct acaacaatga agaatttcct gatctaacta tgtttccccc cttttccgaa 63540 tagaactatt ggggtgagga taagggtggg gggaaatcac tgtttgtttt taaaagcaaa 63600 tcttttgtaa acagaataaa agttcctctc ccttcccttc cctcacccct gatatgtacc 63660 ctttccaccc cttgacttgc tgaagaaacg ttatagaaga aattaaatga atttcccagg 63720 cttttaggat cctctgaaat tttgaggata ggtgaggcct gaattcctgt ccttatttct 63780 tctgaccaga agttgcacag acatgatttg tgctggagtc aaggggcaga acagaagaat 63840 ctgacaggca ctaattggat actgtggctt gtttggatag aaattctgaa tggagtggag 63900 gaaaggagaa atgccctcat cactgaggat catgaaacac agtaggggtg tgtggagcct 63960 tgggacgtga gcagtctcca gaaagaggcc tgagagagag catgaacact tatctttgtg 64020 agtgagtgag tgagtgagtg agtgagtgtg tgtgtgtgtg tgtgtgtgtg tgagagagag 64080 agagagagag taagagacag agacagatgg ggtgaggggg gcatttgagc tcctatgttt 64140 tgttccctat tatagagtat atgcaaaatt tgtcccagat cttctttgac tttgtgcttg 64200 ctctttaaag tgtcctaaga aaattaattt tttcaggtat ttttctattt agtgttgcag 64260 ccaaagagta tttaaattaa gtctttgctg cacttaaatt catacccagc caaaatggaa 64320 ctttaggcca acccccagcc ttctgttgct agggttggtc tcctacagac acagtgatca 64380 agctggatga ctcctgctct ttggtgcttt caactcattg ggaagagctg cagatattac 64440 caaaataggc tggctacatg aacactgtca gaaatcccag acttgcccac aaggataatg 64500 ctgcattttt ctgtcagagt cacacatgtt tttctggaga ggttatttct gcatggaaac 64560 tcaacttctt ggattagcta tcttgagtga aagtcctcac tgacgagtat gcaaaccaaa 64620 tagcctcctg cacagtagcc tctccttcct gtcaccaaaa cagttttagg tctgctgaag 64680 tctggtgttc tttgctcctt ctgcaatctt gaaattgggg tttgctttag agcacaaaca 64740 taagtctgtg ttaggtggac ttaaatccca acagggtcac ttgataatta tagccataga 64800 aatgcagatg caggtaactg cttttaccct ttaccgtcct caggtgagtc tcctagatca 64860 acagcctttt tttttttttt tttccttaaa ctggctcctg tcaaagatta agttaatatg 64920 gaaaagacct cttatgtgta ttgatggggc atgaggagcc caggcaagga gaggctcgtg 64980 gagaggctga gggaatgtta c 65001 <210> SEQ ID NO 100 <211> LENGTH: 2557 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (150)...(2174) <400> SEQUENCE: 100 ggggggggcg tccgccatct tggattccgc ggtagcggtg gcggcggtaa ggtgcctaat 60 ctgcggagtg gctcttccct cccctccccc agctcggtgg cggctgcccc tcccaccgag 120 ggtggcgcag ggacggtgcc atctcgacc atg gcg gcg act aca gct aac cca 173 Met Ala Ala Thr Thr Ala Asn Pro 1 5 gaa atg aca tca gat gta cca tcg ctg ggt ccc acc att gct tct gga 221 Glu Met Thr Ser Asp Val Pro Ser Leu Gly Pro Thr Ile Ala Ser Gly 10 15 20 aac cct gga cct ggg att caa ggt gga gga gct gtt gta cag agg gct 269 Asn Pro Gly Pro Gly Ile Gln Gly Gly Gly Ala Val Val Gln Arg Ala 25 30 35 40 att aag cga cgg tca ggg ctg gat ttt gat gat gaa gta gaa gtg aac 317 Ile Lys Arg Arg Ser Gly Leu Asp Phe Asp Asp Glu Val Glu Val Asn 45 50 55 act aaa ttt ttg aga tgc gat gat gac cag atg tgt aat gac aaa gag 365 Thr Lys Phe Leu Arg Cys Asp Asp Asp Gln Met Cys Asn Asp Lys Glu 60 65 70 cgg ttt gcc agg tcg gat gat gag cag agc tct gcg gat aaa gag aga 413 Arg Phe Ala Arg Ser Asp Asp Glu Gln Ser Ser Ala Asp Lys Glu Arg 75 80 85 ctt gcc agg gaa aat cat agt gaa ata gaa cgg cgg cga cgg aac aag 461 Leu Ala Arg Glu Asn His Ser Glu Ile Glu Arg Arg Arg Arg Asn Lys 90 95 100 atg aca gct tac atc aca gaa ctg tca gac atg gta cct aca tgt agt 509 Met Thr Ala Tyr Ile Thr Glu Leu Ser Asp Met Val Pro Thr Cys Ser 105 110 115 120 gcc ctg gct cga aaa cca gac aag cta acc atc tta cgc atg gcc gtt 557 Ala Leu Ala Arg Lys Pro Asp Lys Leu Thr Ile Leu Arg Met Ala Val 125 130 135 tct cac atg aag tcc ttg agg gga act ggc aac aca tct act gat ggc 605 Ser His Met Lys Ser Leu Arg Gly Thr Gly Asn Thr Ser Thr Asp Gly 140 145 150 tcc tac aag cca tct ttc ctc act gat cag gaa ctg aaa cat ttg atc 653 Ser Tyr Lys Pro Ser Phe Leu Thr Asp Gln Glu Leu Lys His Leu Ile 155 160 165 ttg gag gca gca gat ggc ttt ctg ttt att gtc tcc tgt gag act gga 701 Leu Glu Ala Ala Asp Gly Phe Leu Phe Ile Val Ser Cys Glu Thr Gly 170 175 180 cgg gtg gtg tat gtc tct gac tca gtg act ccc gtt ttg aac cag cca 749 Arg Val Val Tyr Val Ser Asp Ser Val Thr Pro Val Leu Asn Gln Pro 185 190 195 200 cag tct gaa tgg ttc ggg agc aca ctg tat gat cag gtg cac cca gat 797 Gln Ser Glu Trp Phe Gly Ser Thr Leu Tyr Asp Gln Val His Pro Asp 205 210 215 gat gtg gat aaa ctt cga gag cag ctc tct aca tca gaa aat gcc cta 845 Asp Val Asp Lys Leu Arg Glu Gln Leu Ser Thr Ser Glu Asn Ala Leu 220 225 230 aca ggg cgg gtc ctg gat ctg aag act gga aca gtg aaa aag gaa ggc 893 Thr Gly Arg Val Leu Asp Leu Lys Thr Gly Thr Val Lys Lys Glu Gly 235 240 245 cag cag tct tcc atg agg atg tgc atg ggc tca cga agg tcg ttc atc 941 Gln Gln Ser Ser Met Arg Met Cys Met Gly Ser Arg Arg Ser Phe Ile 250 255 260 tgc cgc atg agg tgt ggt act agc tcc gtg gac cct gtt tcc atg aat 989 Cys Arg Met Arg Cys Gly Thr Ser Ser Val Asp Pro Val Ser Met Asn 265 270 275 280 aga ctg agc ttt ttg agg aac aga tgc agg aat ggg ctt ggc tct gtg 1037 Arg Leu Ser Phe Leu Arg Asn Arg Cys Arg Asn Gly Leu Gly Ser Val 285 290 295 aag gaa gga gaa cct cac ttt gtg gta gtc cac tgc aca ggc tac atc 1085 Lys Glu Gly Glu Pro His Phe Val Val Val His Cys Thr Gly Tyr Ile 300 305 310 aag gcc tgg cca cca gca ggt gtc tcc ctc cca gat gat gac cca gag 1133 Lys Ala Trp Pro Pro Ala Gly Val Ser Leu Pro Asp Asp Asp Pro Glu 315 320 325 gct ggc cag ggg agc aaa ttc tgc cta gtg gcc att ggc agg ctg cag 1181 Ala Gly Gln Gly Ser Lys Phe Cys Leu Val Ala Ile Gly Arg Leu Gln 330 335 340 gta act agt tct ccc aac tgt aca gac atg agt aac att tgt cag cca 1229 Val Thr Ser Ser Pro Asn Cys Thr Asp Met Ser Asn Ile Cys Gln Pro 345 350 355 360 aca gag ttc atc tcc cga cac aac att gaa ggg ata ttc act ttt gta 1277 Thr Glu Phe Ile Ser Arg His Asn Ile Glu Gly Ile Phe Thr Phe Val 365 370 375 gac cat cgt tgt gtg gct act gtt ggc tac cag cca cag gag ctc tta 1325 Asp His Arg Cys Val Ala Thr Val Gly Tyr Gln Pro Gln Glu Leu Leu 380 385 390 ggg aag aat att gta gaa ttt tgt cat cct gaa gac caa caa ctt cta 1373 Gly Lys Asn Ile Val Glu Phe Cys His Pro Glu Asp Gln Gln Leu Leu 395 400 405 aga gac agc ttt cag cag gtg gtg aaa tta aaa ggt cag gtg ctg tcc 1421 Arg Asp Ser Phe Gln Gln Val Val Lys Leu Lys Gly Gln Val Leu Ser 410 415 420 gtc atg ttc cga ttc cga tct aag acc cga gaa tgg ctg tgg atg aga 1469 Val Met Phe Arg Phe Arg Ser Lys Thr Arg Glu Trp Leu Trp Met Arg 425 430 435 440 acg agc tcc ttt acc ttc caa aac cct tat tca gat gaa att gag tat 1517 Thr Ser Ser Phe Thr Phe Gln Asn Pro Tyr Ser Asp Glu Ile Glu Tyr 445 450 455 att atc tgc acc aac acc aat gtg aag aac tct agc cag gaa cca cgg 1565 Ile Ile Cys Thr Asn Thr Asn Val Lys Asn Ser Ser Gln Glu Pro Arg 460 465 470 cct aca ctg tcc aac acc atc cca agg tca cag cta ggt ccg aca gcc 1613 Pro Thr Leu Ser Asn Thr Ile Pro Arg Ser Gln Leu Gly Pro Thr Ala 475 480 485 aat tta tcc cta gag atg ggt aca ggg cag ctg cca tcc agg cag cag 1661 Asn Leu Ser Leu Glu Met Gly Thr Gly Gln Leu Pro Ser Arg Gln Gln 490 495 500 cag cag cag cac aca gaa ctg gat atg gta cca gga aga gat ggg ctg 1709 Gln Gln Gln His Thr Glu Leu Asp Met Val Pro Gly Arg Asp Gly Leu 505 510 515 520 gcc agc tat aat cat tcc cag gtt tct gtc cag cct gtg gca agt gca 1757 Ala Ser Tyr Asn His Ser Gln Val Ser Val Gln Pro Val Ala Ser Ala 525 530 535 gga tca gaa cac agc aag ccc ctt gag aag tca gaa ggt ctc ttt gca 1805 Gly Ser Glu His Ser Lys Pro Leu Glu Lys Ser Glu Gly Leu Phe Ala 540 545 550 cag gac aga gat cca agg ttt cca gaa atc tat ccc agc atc act gca 1853 Gln Asp Arg Asp Pro Arg Phe Pro Glu Ile Tyr Pro Ser Ile Thr Ala 555 560 565 gat cag agt aaa ggc atc tcc tcc agc act gtc cct gcc acc caa cag 1901 Asp Gln Ser Lys Gly Ile Ser Ser Ser Thr Val Pro Ala Thr Gln Gln 570 575 580 ctg ttc tcc cag ggc agc tca ttc cct cct aac ccc cgg ccg gca gag 1949 Leu Phe Ser Gln Gly Ser Ser Phe Pro Pro Asn Pro Arg Pro Ala Glu 585 590 595 600 aat ttc agg aat agt ggt ctt acc cct cct gta acc att gtc cag cca 1997 Asn Phe Arg Asn Ser Gly Leu Thr Pro Pro Val Thr Ile Val Gln Pro 605 610 615 tca tct tct gca ggg cag ata ctg gcc cag att tca cgt cac tcc aac 2045 Ser Ser Ser Ala Gly Gln Ile Leu Ala Gln Ile Ser Arg His Ser Asn 620 625 630 cct gcc cag gga tca gcg ccg acc tgg acc tct agc tcc cgc cca ggc 2093 Pro Ala Gln Gly Ser Ala Pro Thr Trp Thr Ser Ser Ser Arg Pro Gly 635 640 645 ttt gcc gcc cag gta gcg cct ctt acc tgt ctg acc ctg tgt gtt gtt 2141 Phe Ala Ala Gln Val Ala Pro Leu Thr Cys Leu Thr Leu Cys Val Val 650 655 660 gtt tgt ggg atc ttt ttt ttt tcc tcc agg taa atttattatt gtcagatacc 2194 Val Cys Gly Ile Phe Phe Phe Ser Ser Arg 665 670 tccttttcag ttcctgttct tcatctttta attttcatct tttttgcgag gagctagata 2254 attaaaatac cagagataac atccagaagt ctctttagaa cctatgtttt catatgctca 2314 ccttaagaca gaactgctca ttcacagcag tttgaacagc tttggtctca ggcttgtttg 2374 gagtaaattc aaagagtttg tctttgttta ggactatgta atatagtacc aaagtagaaa 2434 attagtaatt atattcattt atttatgaaa atatagtatc catctatata atatatagta 2494 aatatgttaa atttatttat tactatgtat ttctcttgtc tatataataa tttagaaaag 2554 ttt 2557 <210> SEQ ID NO 101 <211> LENGTH: 4259 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (135)...(2417) <400> SEQUENCE: 101 gatcttggat tccgcggtag cggtggcggc ggtaaggtgc ctaatctgcg gagtggctct 60 tccctcccct cccccagctc ggtggcggct gcccctccca ccgagggtgg cgcagggacg 120 gtgccatctc gacc atg gcg gcg act aca gct aac cca gaa atg aca tca 170 Met Ala Ala Thr Thr Ala Asn Pro Glu Met Thr Ser 1 5 10 gat gta cca tcg ctg ggt ccc acc att gct tct gga aac cct gga cct 218 Asp Val Pro Ser Leu Gly Pro Thr Ile Ala Ser Gly Asn Pro Gly Pro 15 20 25 ggg att caa ggt gga gga gct gtt gta cag agg gct att aag cga cgg 266 Gly Ile Gln Gly Gly Gly Ala Val Val Gln Arg Ala Ile Lys Arg Arg 30 35 40 tca ggg ctg gat ttt gat gat gaa gta gaa gtg aac act aaa ttt ttg 314 Ser Gly Leu Asp Phe Asp Asp Glu Val Glu Val Asn Thr Lys Phe Leu 45 50 55 60 aga tgc gat gat gac cag atg tgt aat gac aaa gag cgg ttt gcc agg 362 Arg Cys Asp Asp Asp Gln Met Cys Asn Asp Lys Glu Arg Phe Ala Arg 65 70 75 gaa aat cat agt gaa ata gaa cgg cgg cga cgg aac aag atg aca gct 410 Glu Asn His Ser Glu Ile Glu Arg Arg Arg Arg Asn Lys Met Thr Ala 80 85 90 tac atc aca gaa ctg tca gac atg gta cct aca tgt agt gcc ctg gct 458 Tyr Ile Thr Glu Leu Ser Asp Met Val Pro Thr Cys Ser Ala Leu Ala 95 100 105 cga aaa cca gac aag cta acc atc tta cgc atg gcc gtt tct cac atg 506 Arg Lys Pro Asp Lys Leu Thr Ile Leu Arg Met Ala Val Ser His Met 110 115 120 aag tcc ttg agg gga act ggc aac aca tct act gat ggc tcc tac aag 554 Lys Ser Leu Arg Gly Thr Gly Asn Thr Ser Thr Asp Gly Ser Tyr Lys 125 130 135 140 cca tct ttc ctc act gat cag gaa ctg aaa cat ttg atc ttg gag gca 602 Pro Ser Phe Leu Thr Asp Gln Glu Leu Lys His Leu Ile Leu Glu Ala 145 150 155 gca gat ggc ttt ctg ttt att gtc tcc tgt gag act gga cgg gtg gtg 650 Ala Asp Gly Phe Leu Phe Ile Val Ser Cys Glu Thr Gly Arg Val Val 160 165 170 tat gtc tct gac tca gtg act ccc gtt ttg aac cag cca cag tct gaa 698 Tyr Val Ser Asp Ser Val Thr Pro Val Leu Asn Gln Pro Gln Ser Glu 175 180 185 tgg ttc ggg agc aca ctg tat gat cag gtg cac cca gat gat gtg gat 746 Trp Phe Gly Ser Thr Leu Tyr Asp Gln Val His Pro Asp Asp Val Asp 190 195 200 aaa ctt cga gag cag ctc tct aca tca gaa aat gcc cta aca ggg cgg 794 Lys Leu Arg Glu Gln Leu Ser Thr Ser Glu Asn Ala Leu Thr Gly Arg 205 210 215 220 gtc ctg gat ctg aag act gga aca gtg aaa aag gaa ggc cag cag tct 842 Val Leu Asp Leu Lys Thr Gly Thr Val Lys Lys Glu Gly Gln Gln Ser 225 230 235 tcc atg agg atg tgc atg ggc tca cga agg tcg ttc atc tgc cgc atg 890 Ser Met Arg Met Cys Met Gly Ser Arg Arg Ser Phe Ile Cys Arg Met 240 245 250 agg tgt ggt act agc tcc gtg gac cct gtt tcc atg aat aga ctg agc 938 Arg Cys Gly Thr Ser Ser Val Asp Pro Val Ser Met Asn Arg Leu Ser 255 260 265 ttt ttg agg aac aga tgc agg aat ggg ctt ggc tct gtg aag gaa gga 986 Phe Leu Arg Asn Arg Cys Arg Asn Gly Leu Gly Ser Val Lys Glu Gly 270 275 280 gaa cct cac ttt gtg gta gtc cac tgc aca ggc tac atc aag gcc tgg 1034 Glu Pro His Phe Val Val Val His Cys Thr Gly Tyr Ile Lys Ala Trp 285 290 295 300 cca cca gca ggt gtc tcc ctc cca gat gat gac cca gag gct ggc cag 1082 Pro Pro Ala Gly Val Ser Leu Pro Asp Asp Asp Pro Glu Ala Gly Gln 305 310 315 ggg agc aaa ttc tgc cta gtg gcc att ggc agg ctg cag gta act agt 1130 Gly Ser Lys Phe Cys Leu Val Ala Ile Gly Arg Leu Gln Val Thr Ser 320 325 330 tct ccc aac tgt aca gac atg agt aac att tgt cag cca aca gag ttc 1178 Ser Pro Asn Cys Thr Asp Met Ser Asn Ile Cys Gln Pro Thr Glu Phe 335 340 345 atc tcc cga cac aac att gaa ggg ata ttc act ttt gta gac cat cgt 1226 Ile Ser Arg His Asn Ile Glu Gly Ile Phe Thr Phe Val Asp His Arg 350 355 360 tgt gtg gct act gtt ggc tac cag cca cag gag ctc tta ggg aag aat 1274 Cys Val Ala Thr Val Gly Tyr Gln Pro Gln Glu Leu Leu Gly Lys Asn 365 370 375 380 att gta gaa ttt tgt cat cct gaa gac caa caa ctt cta aga gac agc 1322 Ile Val Glu Phe Cys His Pro Glu Asp Gln Gln Leu Leu Arg Asp Ser 385 390 395 ttt cag cag gtg gtg aaa tta aaa ggt cag gtg ctg tcc gtc atg ttc 1370 Phe Gln Gln Val Val Lys Leu Lys Gly Gln Val Leu Ser Val Met Phe 400 405 410 cga ttc cga tct aag acc cga gaa tgg ctg tgg atg aga acg agc tcc 1418 Arg Phe Arg Ser Lys Thr Arg Glu Trp Leu Trp Met Arg Thr Ser Ser 415 420 425 ttt acc ttc caa aac cct tat tca gat gaa att gag tat att atc tgc 1466 Phe Thr Phe Gln Asn Pro Tyr Ser Asp Glu Ile Glu Tyr Ile Ile Cys 430 435 440 acc aac acc aat gtg aag aac tct agc cag gaa cca cgg cct aca ctg 1514 Thr Asn Thr Asn Val Lys Asn Ser Ser Gln Glu Pro Arg Pro Thr Leu 445 450 455 460 tcc aac acc atc cca agg tca cag cta ggt ccg aca gcc aat tta tcc 1562 Ser Asn Thr Ile Pro Arg Ser Gln Leu Gly Pro Thr Ala Asn Leu Ser 465 470 475 cta gag atg ggt aca ggg cag ctg cca tcc agg cag cag cag cag cag 1610 Leu Glu Met Gly Thr Gly Gln Leu Pro Ser Arg Gln Gln Gln Gln Gln 480 485 490 cac aca gaa ctg gat atg gta cca gga aga gat ggg ctg gcc agc tat 1658 His Thr Glu Leu Asp Met Val Pro Gly Arg Asp Gly Leu Ala Ser Tyr 495 500 505 aat cat tcc cag gtt tct gtc cag cct gtg gca agt gca gga tca gaa 1706 Asn His Ser Gln Val Ser Val Gln Pro Val Ala Ser Ala Gly Ser Glu 510 515 520 cac agc aag ccc ctt gag aag tca gaa ggt ctc ttt gca cag gac aga 1754 His Ser Lys Pro Leu Glu Lys Ser Glu Gly Leu Phe Ala Gln Asp Arg 525 530 535 540 gat cca agg ttt cca gaa atc tat ccc agc atc act gca gat cag agt 1802 Asp Pro Arg Phe Pro Glu Ile Tyr Pro Ser Ile Thr Ala Asp Gln Ser 545 550 555 aaa ggc atc tcc tcc agc act gtc cct gcc acc caa cag ctg ttc tcc 1850 Lys Gly Ile Ser Ser Ser Thr Val Pro Ala Thr Gln Gln Leu Phe Ser 560 565 570 cag ggc agc tca ttc cct cct aac ccc cgg ccg gca gag aat ttc agg 1898 Gln Gly Ser Ser Phe Pro Pro Asn Pro Arg Pro Ala Glu Asn Phe Arg 575 580 585 aat agt ggt ctt acc cct cct gta acc att gtc cag cca tca tct tct 1946 Asn Ser Gly Leu Thr Pro Pro Val Thr Ile Val Gln Pro Ser Ser Ser 590 595 600 gca ggg cag ata ctg gcc cag att tca cgt cac tcc aac cct gcc cag 1994 Ala Gly Gln Ile Leu Ala Gln Ile Ser Arg His Ser Asn Pro Ala Gln 605 610 615 620 gga tca gcg ccg acc tgg acc tct agc tcc cgc cca ggc ttt gcc gcc 2042 Gly Ser Ala Pro Thr Trp Thr Ser Ser Ser Arg Pro Gly Phe Ala Ala 625 630 635 cag gtg ccc acc cag gct aca gcc aag act cgt tct tcc caa ttt ggt 2090 Gln Val Pro Thr Gln Ala Thr Ala Lys Thr Arg Ser Ser Gln Phe Gly 640 645 650 gtg aac aac ttt cag act tct tcc tcc ttc agt gct atg tct ctt ccg 2138 Val Asn Asn Phe Gln Thr Ser Ser Ser Phe Ser Ala Met Ser Leu Pro 655 660 665 ggt gct ccc act gcc tca tct gct cct gag act gga cag acc aca gga 2186 Gly Ala Pro Thr Ala Ser Ser Ala Pro Glu Thr Gly Gln Thr Thr Gly 670 675 680 cag ttc cag gcc cgg aca gca gag ggc gtg ggt gtc tgg cca cag tgg 2234 Gln Phe Gln Ala Arg Thr Ala Glu Gly Val Gly Val Trp Pro Gln Trp 685 690 695 700 cag ggc cag cag ccc cat cat cgg tct agt tcc agt gag cag cat gtt 2282 Gln Gly Gln Gln Pro His His Arg Ser Ser Ser Ser Glu Gln His Val 705 710 715 cag cag aca caa gca caa gca cct agc cag cct gag gtc ttt caa gaa 2330 Gln Gln Thr Gln Ala Gln Ala Pro Ser Gln Pro Glu Val Phe Gln Glu 720 725 730 atg ctg tcc atg ctg gga gac caa agc aac acc tac aac aat gaa gaa 2378 Met Leu Ser Met Leu Gly Asp Gln Ser Asn Thr Tyr Asn Asn Glu Glu 735 740 745 ttt cct gat cta act atg ttt ccc ccc ttt tcc gaa tag aactattggg 2427 Phe Pro Asp Leu Thr Met Phe Pro Pro Phe Ser Glu 750 755 760 gtgagaataa gggtgggggg aaatcactgt ttgtttttaa aagcaaatct tttgtaaaca 2487 gaataaaagt tcctctccct tcccttccct cacccctgat atgtaccctt tccacccctt 2547 gacttgctga agaaacgtta tagaagaaat taaatgaatt tcccaggctt ttaggatcct 2607 ctgaaatttt gaggataggt gaggcctgaa ttcctgtcct tatttcttct gaccagaagt 2667 tgcacagaca tgatttgtgc tggagtcaag gggcagaaca gaagaatctg acaggcacta 2727 attggatact gtggcttgtt tggatagaaa ttctgaatgg agtggaggaa aggagaaatg 2787 ccctcatcac tgaggatcat gaaacacagt aggggtgtgt ggagccttgg gacgtgagca 2847 gtctccagaa agaggcctga gagagagcat gaacacttat ctttgtgagt gagtgagtga 2907 gtgagtgagt gagtgtgtgt gtgtgtgtgt gtgtgtgaga gagagagaga gagagtaaga 2967 gacagagaca gatggggtga ggggggcatt tgagctccta tgttttgttc cctattatag 3027 agtatatgca aaatttgtcc cagatcttct ttgactttgt gcttgctctt taaagtgtcc 3087 taagaaaatt aattttttca ggtatttttc tatttagtgt tgcagccaaa gagtatttaa 3147 attaagtctt tgctgcactt aaattcatac ccagccaaaa tggaacttta ggccaacccc 3207 cagccttctg ttgctagggt tggtctccta cagacacagt gatcaagctg gatgactcct 3267 gctctttggt gctttcaact cattgggaag agctgcagat attaccaaaa taggctggct 3327 acatgaacac tgtcagaaat cccagacttg cccacaagga taatgctgca tttttctgtc 3387 agagtcacac atgtttttct ggagaggtta tttctgcatg gaaactcaac ttcttggatt 3447 agctatcttg agtgaaagtc ctcactgacg agtatgcaaa ccaaatagcc tcctgcacag 3507 tagcctctcc ttcctgtcac caaaacagtt ttaggtctgc tgaagtctgg tgttctttgc 3567 tccttctgca atcttgaaat tggggtttgc tttagagcac aaacataagt ctgtgttagg 3627 tggacttaaa tcccaacagg gtcacttgat aattatagcc atagaaatgc agatgcaggt 3687 aactgctttt accctttacc gtcctcaggt gagtctccta gatcaacagc cttttttttt 3747 tttttttcct taaactggct cctgtcaaag attaagttaa tatggaaaag acctcttatg 3807 tgtattgatg gggcatgagg agcccaggca aggagaggct cgtggagagg ctgagggaat 3867 gttactaagt ttccctccgt ttgtctccag tctggtgcca ggcagtagag tggaaaagga 3927 ggctattttt ttattctatg tgcacacata cagtatacat atatatttat atcacatttt 3987 actgaaccaa aaagttgggt tttccaataa aatacttgtt ttttaataac cgacttgttt 4047 ttaactgtga tctgaactat aacgtacagt tattacaggg cttctgaaga agggggggcg 4107 gggagaagct tctctgaggg gctcgctctg cttttccttc acggttttat ttttgattgt 4167 tttttcttgt tgcccatctg tgctaagcct taactgtggc aaaaataatg acatgtagca 4227 aagattttaa aacaaagtat tttttctttt at 4259 <210> SEQ ID NO 102 <211> LENGTH: 701 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 670 <223> OTHER INFORMATION: unknown <400> SEQUENCE: 102 ggaaaccctg gacctgggat tcaaggtgga ggagctgttg tacagagggc tattaagcga 60 cggtcagggc tggattttga tgatgaagta gaagtgaaca ctaaattttt gagatgcgat 120 gatgaccaga tgttttatga caaagagcgg tttgccaggg aaaatcatag tgaaatagaa 180 cggcggcgac ggaacaagat gacagcttac atcacagaac tgtcagacat ggtacctaca 240 tgtagtgccc tggctcgaaa accagacaag ctaaccatct tacgcatggc cgtttctcac 300 atgaagtcct tgaggggaac tggcaacaca tctactgatg gctcctacaa gccatctttc 360 ctcactgatc aggaactgaa acatttgatc ttggaggcag cagatggctt tctgtttatt 420 gtctcctgtg agactggacg ggtggtgtat gtctctgact cagtgactcc cgttttgaac 480 cagccacagt ctgaatggtt cgggagcaca ctgtatgatc aggtgcaccc agatgatgtg 540 gataaacttc gagagcagct ctctacatca gaaaatgccc taacaggtga gagctggctg 600 gacagcagat atgtggggga aaagtcttta tttcactcaa gtagttaaga tattttaagc 660 ccgaaaccan aaaaaaaaaa aaaaaaaaaa agggcggccg c 701 <210> SEQ ID NO 103 <211> LENGTH: 2412 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (120)...(551) <400> SEQUENCE: 103 aaaaaaaatg cctgaacaga gcaagatgag ataaaaaccc tgtaaaaata tcactgaata 60 cataggttac ctatgggact ggactaagat gttagtaatt gttcaccttt agtcaatat 119 atg gag ttt att gta ttg agt aat tcc ttg ttt ccc ctt ttg tgt tct 167 Met Glu Phe Ile Val Leu Ser Asn Ser Leu Phe Pro Leu Leu Cys Ser 1 5 10 15 tgt cta ctc tat ttt cct agg aac tct agc cag gaa cca cgg cct aca 215 Cys Leu Leu Tyr Phe Pro Arg Asn Ser Ser Gln Glu Pro Arg Pro Thr 20 25 30 ctg tcc aac acc atc cca agg tca cag cta ggt ccg aca gcc aat tta 263 Leu Ser Asn Thr Ile Pro Arg Ser Gln Leu Gly Pro Thr Ala Asn Leu 35 40 45 tcc cta gag atg ggt aca ggg cag ctg cca tcc agg cag cag cag cag 311 Ser Leu Glu Met Gly Thr Gly Gln Leu Pro Ser Arg Gln Gln Gln Gln 50 55 60 cag cac aca gaa ctg gat atg gta cca aga aga gat ggg ctg gcc agc 359 Gln His Thr Glu Leu Asp Met Val Pro Arg Arg Asp Gly Leu Ala Ser 65 70 75 80 tat aat cat tcc cag gtt tct gtc cag cct gtg gca agt gca gga tca 407 Tyr Asn His Ser Gln Val Ser Val Gln Pro Val Ala Ser Ala Gly Ser 85 90 95 gaa cac agc aag ccc ctt gag aag tca gaa ggt ctc ttt gca cag gac 455 Glu His Ser Lys Pro Leu Glu Lys Ser Glu Gly Leu Phe Ala Gln Asp 100 105 110 aga gat cca agg ttt cca gaa atc tat ccc agc atc act gca ggt atg 503 Arg Asp Pro Arg Phe Pro Glu Ile Tyr Pro Ser Ile Thr Ala Gly Met 115 120 125 ggt ttc ttc cgc agg ctc ctt tta cag gct gag ttt cat tat tac tga 551 Gly Phe Phe Arg Arg Leu Leu Leu Gln Ala Glu Phe His Tyr Tyr 130 135 140 tgggactgcc cagtcagcat acttctactt cagttacagt acttcatgaa taagtaggaa 611 ttgattaaac tatgtgctac agccccttga tggccctccc ccaactcctt ttggtctgca 671 gatcagagta aaggcatctc ctccagcact gtccctgcca cccaacagct gttctcccag 731 ggcagctcat tccctcctaa cccccggccg gcagagaatt tcaggtgagc cccatatgtg 791 tgtgctgctt gacagggctc tgcagggttc agttgctgga tccgtgtgtc atcttccctt 851 gctttctctg gtcacctcag acaaagcagt agaacttact ggacctaggg tgagacaacg 911 aagctgcttt tcctcctgtt tttgcaccta ttctattgcc ttgctctagg ttccaaacgt 971 ctctgtgtgg tcagtgtgtg acagtcagtc tttcttgtgt ttttaaattt atcaggtttt 1031 ccttaactcc aagaaatcag aggaatctag ggatagaatg tgtcctttga ttcatagctt 1091 ctgtgatgga gtcaaccttt taaccttcac tttctatttg ttccttccta ggaatagtgg 1151 tcttacccct cctgtaacca ttgtccagcc atcatcttct gcagggcaga tactggccca 1211 gatttcacgt cactccaacc ctgcccaggg atcagcgccg acctggacct ctagctcccg 1271 cccaggcttt gccgcccagg tagcgcctct tacctgtctg accctgtgtg ttgttgtttg 1331 tgggatcttt tttttttcct ccaggtaaat ttattattgt cagatacctc cttttcagtt 1391 cctgttcttc atcttttaat tttcatcttt tttgcgagga gctagataat taaaatacca 1451 gagataacat ccagaagtct ctttagaacc tatgttttca tatgctcacc ttaagacaga 1511 actgctcatt cacagcagtt tgaacagctt tggtctcagg cttgtttgga gtaaattcaa 1571 agagtttgtc tttgtttagg actatgtaat atagtaccaa agtagaaaat tagtaattat 1631 attcatttat ttatgaaaat atagtatcca tctatataat atatagtaaa tatgttaaat 1691 ttatttatta ctatgtattt ctcttgtcta tataataatt tagaaaagtt taaaaaaaaa 1751 tgaaaactca aacttgactc cagaattgtt ctctatagct tttttgtgta tgtgtaaaaa 1811 tactcaggaa tgttgcccat gactcttggt caggatgtgc cgtgatgttg atgacagctg 1871 ctaccatggt tttcatactg gtgacataat caagcatgca tatattatct cccttggagt 1931 tactttttaa attgtggatt taaaacggtc tctagaaatt attgtatctc acaaaaaatg 1991 ttagaggcaa taactagttt cttaatcaat atacagtaaa tgggtgtgag aaaggacagc 2051 tttaatttca acaaagagac tgaggcagga gcatgggcct gggccacata gtgagagata 2111 aactgtctca taaaagaaag aacagaacaa aaccaaaata gcataatgga aaataaaaga 2171 ggacattgtc cactgtgcca agtaacagtc tcttctggtg gggttaacag tgaggtttca 2231 tttctagttt attttttcca atttttttct attctttttg aggcagattt agaatttata 2291 ttaaacaacc ctttgcagat ttacacttaa agtcaagagc aaaagagaag cactcagaac 2351 tgagtgtggg tgtgaactcc ctaagatgcc accaaattcc tctgtaacac atagggattc 2411 c 2412 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 104 gattccagca gaaacaagat 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 105 agtaccataa ccaggaagag 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 106 atcatcgcat ctgaaaagaa 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 107 tcatacttgc ctgcagcctg 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 108 agattaggca ccttaccgcc 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 109 gccgccatgg tcgagatggc 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 110 gttagctgta gtcgccgcca 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 111 tcatttctgg gttagctgta 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 112 acatctgatg tcatttctgg 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 113 ttccagaagc aatggtggga 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 114 agctcctcca ccttgaatcc 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 115 cgtcgcttaa tagccctctg 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 116 cagccctgac cgtcgcttaa 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 117 catcaaaatc cagccctgac 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 118 ctggcaaacc gctctttgtc 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 119 atcatccgac ctggcaaacc 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 120 agctctgctc atcatccgac 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 121 gtctctcttt atccgcagag 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 122 ctggcaagtc tctctttatc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 123 tttccctggc aagtctctct 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 124 atgattttcc ctggcaagtc 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 125 gtaagctgtc atcttgttcc 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 126 gttctgtgat gtaagctgtc 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 127 tagcttgtct ggttttcgag 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 128 caaatgtttc agttcctgat 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 129 taaacagaaa gccatctgct 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 130 tggttcaaaa cgggagtcac 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 131 gctgctctcg aagtttatcc 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 132 gcacatcctc atggaagact 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 133 ttcatggaaa cagggtccac 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 134 gctcagtcta ttcatggaaa 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 135 tcctcaaaaa gctcagtcta 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 136 ctgcatctgt tcctcaaaaa 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 137 tgcagtggac taccacaaag 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 138 ggccagcctc tgggtcatca 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 139 cagaatttgc tcccctggcc 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 140 ggccactagg cagaatttgc 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 141 cagcctgcca atggccacta 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 142 gtgaatatcc cttcaatgtt 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 143 acagtagcca cacaacgatg 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 144 gacaaaattc tacaatattc 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 145 gtcttcagga tgacaaaatt 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 146 gaagttgttg gtcttcagga 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 147 gctgtctctt agaagttgtt 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 148 cctgctgaaa gctgtctctt 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 149 aatttcacca cctgctgaaa 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 150 tcatccacag ccattctcgg 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 151 gtggttcctg gctagagttc 20 <210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 152 gtcggaccta gctgtgacct 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 153 ctgtacccat ctctagggat 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 154 ccagcccatc tcttcctggt 20 <210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 155 ggacagaaac ctgggaatga 20 <210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 156 gggcttgctg tgttctgatc 20 <210> SEQ ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 157 aagagacctt ctgacttctc 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 158 cctgggagaa cagctgttgg 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 159 aatgagctgc cctgggagaa 20 <210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 160 gttaggaggg aatgagctgc 20 <210> SEQ ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 161 cactattcct gaaattctct 20 <210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 162 atctgccctg cagaagatga 20 <210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 163 aagaacgagt cttggctgta 20 <210> SEQ ID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 164 tctgaaagtt gttcacacca 20 <210> SEQ ID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 165 gtctcaggag gaaagttgga 20 <210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 166 cctgccactg tggccagaca 20 <210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 167 ttcttgaaag acctcaggct 20 <210> SEQ ID NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 168 tctcccagca tggacagcat 20 <210> SEQ ID NO 169 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 169 ccaatagttc tattcggaaa 20 <210> SEQ ID NO 170 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 170 tctgtttaca aaagatttgc 20 <210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 171 cggaatccaa gatggcggac 20 <210> SEQ ID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 172 ctccaaacaa gcctgagacc 20 <210> SEQ ID NO 173 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 173 atgattttcc ctggcaaacc 20 <210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 174 ggcacctggg cggcaaagcc 20 <210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 175 agacttttcc cccacatatc 20 <210> SEQ ID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 176 taacctatgt attcagtgat 20 <210> SEQ ID NO 177 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 177 ttcgcggctg gacgattcag 20 <210> SEQ ID NO 178 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 178 cctggcaaca tctggggttg g 21 <210> SEQ ID NO 179 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 179 caacaggctg gagtgactgg gctcc 25 <210> SEQ ID NO 180 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 180 gtggagacag gactagtgca cgaatg 26 <210> SEQ ID NO 181 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 181 ctgtggaggc atggactgag aatgg 25 <210> SEQ ID NO 182 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 182 ctgggaacct ccaaatcccc tggc 24 <210> SEQ ID NO 183 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 183 ctgggcaggg ttggcagctg ccttac 26 <210> SEQ ID NO 184 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 184 tgagctgtct gtgatccagc 20 <210> SEQ ID NO 185 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 185 gcgctgctcc caagaactct 20 <210> SEQ ID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 186 tcctcatggt cacatggatg 20 <210> SEQ ID NO 187 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 187 cgagaggcgg acgggaccg 19 <210> SEQ ID NO 188 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 188 cgagaggcgg acgggaccgt t 21 <210> SEQ ID NO 189 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 189 ttgctctccg cctgccctgg c 21 <210> SEQ ID NO 190 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Compound <400> SEQUENCE: 190 gctctccgcc tgccctggc 19

Claims

1. An antisense oligonucleotide 13 to 50 nucleobases in length which is specifically hybridizable with a nucleic acid molecule encoding human HIF1-beta, said antisense oligonucleotide comprising at least one modification selected from the group consisting of a modified internucleoside linkage, a modified sugar moiety and a modified nucleobase, wherein said antisense oligonucleotide inhibits expression of human HIF1-beta.

2. The antisense oligonucleotide of claim 1 comprising 13 to 30 nucleobases in length.

3. The antisense oligonucleotide of claim 1, wherein the modified internucleoside linkage is a phosphorothioate linkage.

4. The antisense oligonucleotide of claim 1, wherein the modified sugar moiety is a 2'-O-(2-methoxyethyl) moiety.

5. The antisense oligonucleotide of claim 1, wherein the modified nucleobase is a 5-methyl cytosine.

6. The antisense oligonucleotide of claim 1 which is a chimeric oligonucleotide.

7. The chimeric antisense oligonucleotide of claim 6 having a first region comprising 2'-deoxynucleotides and second and third regions flanking said first region, comprising at least one 2'-O-(2-methoxyethyl) nucleotide.

8. The chimeric antisense oligonucleotide of claim 7, wherein said first region is ten 2'-deoxynucleotides in length and said second and third regions are each five nucleotides in length.

9. The chimeric antisense oligonucleotide of claim 8 further comprising a phosphorothioate internucleoside linkage at each position.

10. A pharmaceutical composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

11. A method of inhibiting expression of human HIF1-beta in cells or tissues, comprising contacting said cells or tissues with the antisense oligonucleotide of claim 1, such that expression of human HIF1-beta is inhibited.

12. A method of inhibiting expression of a HIF1-beta regulated gene in a cell or tissue comprising contacting said cells or tissues with the antisense oligonucleotide of claim 1, such that expression of the HIF1-beta regulated gene is inhibited.

13. The method of claim 12, wherein the HIF1-beta regulated gene is selected from the group consisting of VEGF, GLUT-1, PGK-1, PAI-1 and Epo.

14. A method of treating an animal having a disease or condition associated with HIF1-beta comprising administrating to said animal a therapeutically or prophylactically effective amount of the composition of claim 10 so that expression of HIF1-beta is inhibited.

15. The method of claim 14, wherein the disease or condition is a hyperproliferative disorder.

16. The method of claim 15, wherein the hyperproliferative disorder is cancer.

17. The method of claim 15, wherein the hyperproliferative disorder is an angiogenic disorder.

18. The method of claim 17, wherein the angiogenic disorder is an ocular disorder.

19. The method of claim 18, wherein the ocular disorder is selected from the group consisting of macular degeneration, diabetic retinopathy, macular edema and retinopathy of prematurity.

20. A method of preventing or inhibiting aberrant angiogenesis in an animal, comprising administering to said an animal an antisense oligonucleotide of claim 1 such that aberrant angiogenesis is prevented or inhibited.

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