Treatment Of Collagen Gene Related Diseases By Inhibition Of Natural Antisense Transcript To A Collagen Gene

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of a Collagen gene, in particular, by targeting natural antisense polynucleotides of a Collagen gene. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of Collagen genes.

Description

FIELD OF THE INVENTION

[0001] The present application claims the priority of U.S. provisional patent application 61/187,384 filed Jun. 16, 2009, No. 61/286,939 filed Dec. 16, 2009 and U.S. provisional patent application No. 61/286,965 filed Dec. 16, 2009 which are incorporated herein by reference in their entireties.

[0002] Embodiments of the invention comprise oligonucleotides modulating expression and/or function of a Collagen gene and associated molecules.

BACKGROUND

[0003] DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENETM (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.

SUMMARY

[0004] In one embodiment, the invention provides methods for inhibiting the action of a natural antisense transcript by using antisense oligonucleotide(s) targeted to any region of the natural antisense transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript can be achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention.

[0005] One embodiment provides a method of modulating function and/or expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 387 of SEQ ID SEQ ID NO: 4, 1 to 561 of SEQ ID NO: 5, 1 to 335 of SEQ ID SEQ ID NO: 6, 1 to 613 of SEQ ID NO: 7, 1 to 177 of SEQ ID NO: 8, and 1 to 285 of SEQ ID NO: 9 thereby modulating function and/or expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

[0006] In another embodiment, an oligonucleotide targets a natural antisense sequence of a Collagen gene polynucleotide, for example, nucleotides set forth in SEQ ID NO: 4 to 9, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 10 to 29.

[0007] Another embodiment provides a method of modulating function and/or expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of the an antisense of the Collagen gene polynucleotide; thereby modulating function and/or expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

[0008] Another embodiment provides a method of modulating function and/or expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to a Collagen gene antisense polynucleotide; thereby modulating function and/or expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

[0009] In one embodiment, a composition comprises one or more antisense oligonucleotides which bind to sense and/or antisense Collagen gene polynucleotides.

[0010] In another embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.

[0011] In another embodiment, the oligonucleotides comprise one or more modified bonds.

[0012] In yet another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acids, 2'-O-methyl, fluoro- or carbon, methylene or other locked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are locked nucleic acid molecules, including a-L-LNA.

[0013] In another embodiment, the oligonucleotides are administered to a patient subcutaneously, intramuscularly, intravenously or intraperitoneally.

[0014] In another embodiment, the oligonucleotides are administered in a pharmaceutical composition. A treatment regimen comprises administering the antisense compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies.

[0015] In another embodiment, the oligonucleotides are encapsulated in a liposome or attached to a carrier molecule (e.g. cholesterol, TAT peptide).

[0016] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a graph of real time PCR results showing the fold change+standard deviation in Col1a1 mRNA after treatment of HepG2 cells with phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of the Col1a1 mRNA in HepG2 cells are significantly increased 48 h after treatment with one of the oligos designed to Col1a1 antisense DW440457. Bars denoted as CUR-1361 to CUR-1364 correspond to samples treated with SEQ ID NOS: 10 to 13 respectively.

[0018] FIG. 2 is a graph of real time PCR results showing the fold change+standard deviation in Col1a2 mRNA after treatment of HUVEC cells with phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of the Col1a2 mRNA in HUVEC cells are significantly increased 48 h after treatment with one of the oligos designed to Col1a2 antisense Hs.571263. Bars denoted as CUR-0862, CUR-0864, CUR-0863, CUR-0865, CUR-0866 and CUR- CUR-0867 correspond to samples treated with SEQ ID NOS: 14 to 19 respectively.

[0019] FIG. 3 is a graph of real time PCR results showing the fold change+standard deviation in Col7a1 mRNA after treatment of HUVEC cells with phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of Col7a1 mRNA in HUVEC cells are significantly increased 48 h after treatment with one of the siRNAs designed to Col7a1 as CV425857 (CV425857.1.sub.--1, CV425857.12,) and one siRNA to BU615800.1 (BU615800.1.sub.--1). Bars denoted as CUR-0356, CUR-0358, CUR-0360, and CUR-0362, correspond to samples treated with SEQ ID NOS: 20 to 23 respectively.

[0020] FIG. 4 is a graph of real time PCR results showing the fold change+standard deviation in Col7a1 mRNA after treatment of HepG2 cells with phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of Col7a1 mRNA in HepG2 cells are significantly increased 48 h after treatment with two of the oligos designed to Col7a1 as be142537 and bg998538. Bars denoted as CUR-0360 and CUR-0856 to CUR-0860 correspond to samples treated with SEQ ID NO: 22 and SEQ ID NO: 24 to 28 respectively.

SEQUENCE LISTING DESCRIPTION

[0021] SEQ ID NO: 1: Homo sapiens collagen, type I, alpha 1 (COL1A1), mRNA (NCBI Accession No.: NM.sub.--000088.3). [0022] SEQ ID NO: 2: Homo sapiens collagen, type I, alpha 2 (COL1A2), mRNA (NCBI Accession No.: NM.sub.--000089.3). [0023] SEQ ID NO: 3: NM.sub.--024013.11 Homo sapiens collagen, type VII, alpha 1 (COL7A1), mRNA (NCBI Accession No.: NM.sub.--000094.3). [0024] SEQ ID NOs: 4 to 9: SEQ ID NO: 4: Natural COL1A1 antisense sequence (DW440457); SEQ ID NO: 5: Natural COL1A2 antisense sequence (Hs.571263); SEQ ID NO: 6: Natural COL7A1 antisense sequence (CV425857.1); [0025] SEQ ID NO: 7: Natural COL7A1 antisense sequence (BU615800.1); SEQ ID NO: 8: Natural COL7A1 antisense sequence (BE142537) and SEQ ID NO: 9: Natural COL7A1 antisense sequence (BG998538) [0026] SEQ ID NOs: 10 to 29 - Antisense oligonucleotides. `r` indicates RNA and * indicates phosphothioate bond. [0027] SEQ ID NO: 30 to 33 are the reverse complements of the antisense oligonucleotides SEQ ID NO: 20 to 23 respectively. `r` indicates RNA.

DETAILED DESCRIPTION

[0028] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[0029] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In embodiments, the genes or nucleic acid sequences are human.

Definitions

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0031] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0032] As used herein, the term "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.

[0033] By "antisense oligonucleotides" or "antisense compound" is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA. An antisense oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that 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, partially single-stranded, or circular oligomeric compounds.

[0034] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.

[0035] The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotides compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.

[0036] The oligonucleotide can be composed of regions that can be linked in "register" that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.

[0037] As used herein "Collagen genes" are inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.

[0038] As used herein, the words Collagen, type I, alpha 1; Collagen Alpha-1(I), COL1A1, Alpha-1 type I collagen, Collagen alpha-1(I) chain, O14, are considered the same in the literature and are used interchangeably in the present application.

[0039] As used herein, the words Collagen alpha-2(I), "collagen, type I, alpha 2", collagen alpha2(I), Alpha-2 type I collagen, Collagen alpha-2(I) chain and COL1A2 are considered the same in the literature and are used interchangeably in the present application.

[0040] As used herein, the words Collagen alpha-1(VII) chain, COL7A1, EBD1, EBDCT, EBR1, LC collagen, Long-chain collagen are considered the same in the literature and are used interchangeably in the present application.

[0041] As used herein, the term "oligonucleotide specific for" or "oligonucleotide which targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays. Exemplary assays for determining stability of hybridization complexes and duplexes are described in the Examples below.

[0042] As used herein, the term "target nucleic acid" encompasses DNA, RNA (comprising premRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense". The functions of DNA to be interfered include, for example, replication and transcription. The functions of RNA to be interfered, include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides.

[0043] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences. In certain embodiments of the present invention, the mediators are 5-25 nucleotide "small interfering" RNA duplexes (siRNAs). The siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer. siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion. Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt). In examples of non limiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

[0044] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

[0045] By "enzymatic RNA" is meant an RNA molecule with enzymatic activity. Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

[0046] By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

[0047] As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.

[0048] The term "nucleotide" covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides. It should be clear to the person skilled in the art that various nucleotides which previously have been considered "non-naturally occurring" have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidine heterocycles-containing molecules, but also heterocyclic analogues and tautomers thereof. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo- N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleotides described in U.S. Pat No. 5,432,272. The term "nucleotide" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleotides are those containing adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans. Nucleotides include the natural 2'-deoxy and 2'-hydroxyl sugars, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) as well as their analogs.

[0049] "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

[0050] As used herein, "hybridization" means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0051] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0052] As used herein, 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. In general, stringent hybridization conditions comprise low concentrations (<0.15M) of salts with inorganic cations such as Na++ or K++ (i.e., low ionic strength), temperature higher than 20.degree. C.-25.degree. C. below the Tm of the oligomeric compound:target sequence complex, and the presence of denaturants such as formamide, dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1% for each 1% formamide. An example of a high stringency hybridization condition is 0.1X sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60.degree. C. for 30 minutes.

[0053] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobase at a certain position of an antisense 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 oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides 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 nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

[0054] It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The oligomeric compounds of the present invention comprise at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. As such, an antisense compound which is 18 nucleotides in length having 4 (four) noncomplementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art. Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

[0055] As used herein, the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0056] As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.

[0057] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0058] The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

[0059] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group. Derivatives, e.g., derivative oligonucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0060] A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction/alkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.

[0061] As used herein, the term "animal" or "patient" is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

[0062] "Mammal" covers warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.

[0063] "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting it development; and/or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).

[0064] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancer manifests itself as a "tumor" or tissue comprising malignant cells of the cancer. Examples of tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Additional cancers which can be treated by the disclosed composition according to the invention include but not limited to, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.

[0065] "Neurological disease or disorder" refers to any disease or disorder of the nervous system and/or visual system. "Neurological disease or disorder" include disease or disorders that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Examples of neurological disorders include but are not limited to, headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuroopthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions. Addiction and mental illness, include, but are not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological disorder. The following is a list of several neurological disorders, symptoms, signs and syndromes that can be treated using compositions and methods according to the present invention: acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telegiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cytomegalic inclusion body disease; cytomegalovirus infection; dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawson disease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other "tauopathies"; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactic a polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HlVassociated dementia and neuropathy (also neurological manifestations of AIDS); holoprosencephaly; Huntington's disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile phytanic acid storage disease; infantile refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy disease Kinsboume syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e., motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease--neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; p muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae oflupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Neurodegenerative disease or disorder (Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis and other diseases and disorders associated with neuronal cell death); paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; post-polio syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; postural hypotension; Prader- Willi syndrome; primary lateral sclerosis; prion diseases; progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types 1 and 11); Rasmussen's encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome.

[0066] An "Inflammation" refers to systemic inflammatory conditions and conditions associated locally with migration and attraction of monocytes, leukocytes and/or neutrophils. Examples of inflammation include, but are not limited to, Inflammation resulting from infection with pathogenic organisms (including gram-positive bacteria, gram-negative bacteria, viruses, fungi, and parasites such as protozoa and helminths), transplant rejection (including rejection of solid organs such as kidney, liver, heart, lung or cornea, as well as rejection of bone marrow transplants including graft-versus-host disease (GVHD)), or from localized chronic or acute autoimmune or allergic reactions. Autoimmune diseases include acute glomerulonephritis; rheumatoid or reactive arthritis; chronic glomerulonephritis; inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and necrotizing enterocolitis; granulocyte transfusion associated syndromes; inflammatory dermatoses such as contact dermatitis, atopic dermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, and some forms of diabetes, or any other autoimmune state where attack by the subject's own immune system results in pathologic tissue destruction. Allergic reactions include allergic asthma, chronic bronchitis, acute and delayed hypersensitivity. Systemic inflammatory disease states include inflammation associated with trauma, burns, reperfusion following ischemic events (e.g. thrombotic events in heart, brain, intestines or peripheral vasculature, including myocardial infarction and stroke), sepsis, ARDS or multiple organ dysfunction syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques. Inflammation includes, but is not limited to, Non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto's thyroiditis, hepatocellular carcinoma, thymus atrophy, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn's disease, ulcerative colitis, acute cholecystitis, chronic cholecystitis, cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis, chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis, acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiple sclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura, primary IgA nephropathy, systemic lupus erythematosus, psoriasis, pulmonary emphysema, chronic pyelonephritis, and chronic cystitis.

Polynucleotide and Oligonucleotide Compositions and Molecules Targets

[0067] In one embodiment, the targets comprise nucleic acid sequences of a Collagen gene, including without limitation sense and/or antisense noncoding and/or coding sequences associated with a Collagen gene.

[0068] In one embodiment, the targets comprise nucleic acid sequences of COL1A1, including without limitation sense and/or antisense noncoding and/or coding sequences associated with COL1A1 gene.

[0069] In one embodiment, the targets comprise nucleic acid sequences of COL1A2, including without limitation sense and/or antisense noncoding and/or coding sequences associated with COL1A2 gene.

[0070] In one embodiment, the targets comprise nucleic acid sequences of COL7A1, including without limitation sense and/or antisense noncoding and/or coding sequences associated with COL7A1 gene.

[0071] Collagen type I is the most enriched ECM component in the vasculature and is found in abundance in the plaque, the levels of which affect the vulnerability of the plaque and correlate with the mortality and morbidity of atherosclerosis (Libby P., et al (2000) J. Intern. Med. 247:349-358; Rekhter Md., (1993) Am. J. Pathol. 143:1634-1648). It consists of two al (I) chains and one .alpha.2(I) chain that are produced by two fairly large genes that reside on different chromosomes in both the human and the mouse genome (Myllyharju, J., et al (2001) Ann. Med. 33, 7-21). Type I collagen is a member of a family of fibrillar collagens and accounts for 80 to 90% of the protein found in bone. It is also found in large amounts in tissues such as skin, ligaments and tendons. The COL1A1 gene is located on the long (q) arm of chromosome 17 between positions 21.3 and 22.1, from base pair 45,616,455 to base pair 45,633,991.

[0072] Collagen type I is the most enriched ECM component in the vasculature and is found in abundance in the plaque, the levels of which affect the vulnerability of the plaque and correlate with the mortality and morbidity of atherosclerosis (Libby P., et al (2000) J. Intern. Med. 247:349-358; Rekhter Md., (1993) Am. J. Pathol. 143:1634-1648). It consists of two a 1 (I) chains and one .alpha.2(I) chain that are produced by two fairly large genes that reside on different chromosomes in both the human and the mouse genome (Myllyharju, J., et al (2001) Ann. Med. 33, 7-21. The collagen alpha2(I) gene (COL1A2) is located on chromosome 7q22.1.

[0073] Collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive), also known as

[0074] COL7AI, is a human gene. This gene encodes the alpha chain of type VII collagen. The type VII collagen fibril, composed of three identical alpha collagen chains, is restricted to the basement zone beneath stratified squamous epithelia. It functions as an anchoring fibril between the external epithelia and the underlying stroma. Mutations in this gene are associated with all forms of dystrophic epidermolysis bullosa. In the absence of mutations, however, an autoimmune response against type VII collagen can result in an acquired form of this disease called epidermolysis bullosa acquisita.

[0075] Type VII collagen is also found in the retina; its function in this organ is unknown.

[0076] COL7A1 is located on the short arm of human chromosome 3, in the chromosomal region denoted 3p21.31. The gene is approximately 31,000 base pairs in size and is remarkable for the extreme fragmentation of its coding sequence into 118 exons. COL7A1 is transcribed into an mRNA of 9,287 base pairs. In the skin, the type VII collagen protein is synthesized by keratinocytes and dermal fibroblasts.

[0077] Dystrophic epidermolysis bullosa (DEB) is a clinically heterogeneous heritable skin disorder, characterized by blistering of the skin and mucous membranes following minor trauma, and with a broad range of clinical severity. All forms are caused by mutations in COL7AI, the gene coding for collagen VII. This collagen, the major component of the anchoring fibrils, is reduced or missing in DEB skin, and the anchoring fibrils are morphologically altered or absent and functionally defective. Notably, the full spectrum of gene mutations and the molecular mechanisms leading to these visual and functional alterations still remain elusive, despite significant scientific efforts.

[0078] Worldwide, DEB affects thousands of families. Therefore, efficient COL7A1 mutation detection is urgently needed for precise diagnosis, prognostication, genetic counseling and reliable prenatal diagnosis, and, importantly, for identification of suitable candidates for future gene therapy trials. COL7A1 is located within the 3p21 region and spans over 30.5 kb. It shares structural features with other collagen genes and consists of 118 small exons and small introns. To date, more than 200 different mutations have been reported, but very few recurrent mutations or hot spots are known.

[0079] In some embodiments, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with Collagen gene family members. Exemplary Collagen gene mediated diseases and disorders which can be treated with cell/tissues regenerated from stem cells obtained using the antisense compounds comprise: a collagen disorder, age related collagen degradation, Osteogenesis imperfecta, Otosclerosis (OTSC), Osteoporosis, Osteoarthritis, Oesophageal squamous cell cancer, chondrodysplasia, atypical Marfan syndrome, Ehlers-Danlos Syndrome (EDS), Dystrophic epidermolysis bullosa (DEB), Caffey disease, aneurysms (eg. intracranial aneurysms), idiopathic pulmonary fibrosis, liver cirrhosis, kidney fibrosis, liver fibrosis, heart fibrosis, scleroderma, hypertrophic scars, keloids, cancer, inflammation, a genetic disease (e.g. Duchenne muscular dystrophy), a neurological disease or disorder (e.g. Parkinson's, Alzheimer's, Huntington's, Gaucher disease), a metabolic disease (e.g. type I diabetes), an autoimmune disease or disorder, trauma (e.g. spinal cord injury, burns, etc), ischemia, and other blood vessel, heart, a skin disease or disorder, skin aging, a skin disease or disorder or condition requiring skin engineering, a liver or kidney disease requiring transplantation; tendon, bone or tissue regeneration; skeletal repair, cartilage and bone repair.

[0080] In another embodiment, the antisense oligonucleotides modulate the normal expression and/or normal function of a Collagen gene in patients suffering from or at risk of developing diseases or disorders associated with Collagen gene.

[0081] An embodiment of the present invention provides Collagen gene based matrix composition, said composition comprising a three dimensional purified collagen matrix comprised of Collagen fibrils, wherein function of and/or the expression of a Collagen gene polynucleotide in the cells of collagen fibrils is modulated in vivo or in vitro by contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide.

[0082] An embodiment of the present invention provides method of in vitro, ex vivo or in vivo wound healing or tissue regeneration or tissue engineering which comprises application of an extracellular matrix composition of the present invention to a wound.

[0083] An embodiment of the present invention provides method of preparing a Collagen gene based matrix composition, comprising contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

[0084] Another embodiment of the present invention provides a composition for supporting stem cells, the composition comprising an engineered, purified Collagen gene-based matrix comprising Collagen fibrils, and a population of stem cells. The engineered purified collagen based matrix compositions of the present invention can be used alone or in combination with cells as a tissue graft construct to enhance the repair of damaged or diseased tissues.

[0085] In accordance with one embodiment an improved method for culturing stem cells is provided. The method comprises preparing a solubilized Collagen gene composition with desired characteristics as provided in the present invention, adding cells to the solubilized Collagen gene composition and polymerizing the collagen composition under controlled conditions to provide a matrix formed from collagen fibrils and having the desired microstructure. In one embodiment cells are added to the Collagen gene based matrix, and the cells are cultured under conditions suitable for proliferation of the cells.

[0086] In embodiments, Collagen gene -based matrix composition is used to prevent or treat diseases or disorders associated with loss of a particular cell type or tissue that can be replaced by new cells/tissues regenerated from the pluripotent stem cells.

[0087] In embodiments, the Collagen gene -based matrix composition is used in conjunction with at least one of the transcription factor selected from: OCT4, c-myc, KLF4 and/or Sox2, NANOG, LIN28 and other transcription factors required to induce pluripotent stem cells from a given adult tissue. These cells can be further introduced into a lesion and allowed to differentiate into a required cell type in vitro or in vivo.

[0088] Examples of diseases which can be treated with cells/tissues regenerated from Collagen gene-based matrix composition using the stem cells and the antisense compounds comprise cancer, genetic diseases (e.g. Duchenne muscular dystrophy), neurodegenerative diseases (e.g. Parkinson's, Alzheimer's, Huntington's, Gaucher disease), metabolic diseases (e.g. type I diabetes), trauma (e.g. spinal cord injury, burns, etc), ischemia, and other blood vessel, heart, liver or kidney diseases requiring transplantation.

[0089] In embodiments, the Collagen gene-based matrix composition may also be utilized in tendon, bone or tissue regeneration; skeletal repair, cartilage, bone repair processes, osteoporosis, prostheses for arthroplaties, etc. that can be injected in situ in small non-joining fractures or in cartilaginous lesions, in the systemic circulation of osteoporotic individuals or can be implanted adsorbed in the appropriate biomaterial (collagen, hydroxyapatite, etc.) to promote cartilage or bone formation in lesions. Furthermore, the composition can be adsorbed in the hydroxyapatite (periapatite) coating the prostheses for hip, knee arthroplasties to enhance biological integration of these prostheses in the host, prolonging their life. The composition can be also used in spinal arthrodesis for promoting spinal fusion and improve the efficiency of these procedures performed by the standard procedures with autologous graft or bone bank.

[0090] In embodiments of the present invention, therapeutic and/or cosmetic regimes and related tailored treatments are provided to subjects requiring skin treatments or at risk of developing conditions for which they would require skin treatments. Diagnosis can be made, e.g., based on the subject's Collagen gene status. A patient's Collagen gene expression levels in a given tissue such as skin can be determined by methods known to those of skill in the art and described elsewhere herein, e.g., by analyzing tissue using PCR or antibody-based detection methods.

[0091] A embodiment of the present invention provides a composition for skin treatment and/or a cosmetic application comprising Collagen gene antisense oligonucleotides, e.g., to upregulate expression of Collagen gene in the skin. Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 4 to 29. In embodiments, cells are treated in vivo with the oligonucleotides of the present invention, to increase cell lifespan or prevent apoptosis. For example, skin can be protected from aging, e.g., developing wrinkles, by treating skin, e.g., epithelial cells, as described herein. In an exemplary embodiment, skin is contacted with a pharmaceutical or cosmetic composition comprising a Collagen gene antisense compound as described herein. Exemplary skin afflictions or skin conditions include disorders or diseases associated with or caused by inflammation, sun damage or natural aging. For example, the compositions find utility in the prevention or treatment of contact dermatitis (including irritant contact dermatitis and allergic contact dermatitis), atopic dermatitis (also known as allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including penfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas (including erythema multiforme and erythema nodosum), damage caused by the sun or other light sources, discoid lupus erythematosus, dermatomyositis, skin cancer and the effects of natural aging.

[0092] In embodiments of the present invention, a composition comprising Collagen gene antisense oligonucleotides, e.g., to upregulate expression of Collagen gene in the scalp and inhibit androgen receptor signaling, thereby preventing androgenetic alopecia (hair loss). In embodiments, a patient suffering from alopecia is administered either a topical or systemic formulation.

[0093] In an embodiment, an antisense oligonucleotide described herein is incorporated into a topical formulation containing a topical carrier that is generally suited to topical drug administration and comprising any such material known in the art. The topical carrier may be selected so as to provide the composition in the desired form, e.g., as an ointment, lotion, cream, microemulsion, gel, oil, solution, or the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is preferable that the selected carrier not adversely affect the active agent or other components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Formulations may be colorless, odorless ointments, lotions, creams, microemulsions and gels.

[0094] Antisense oligonucleotides of the invention may be incorporated into ointments, which generally are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington's Pharmaceutical Sciences (Mack Pub. Co.), ointment bases may be grouped into four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Exemplary water-soluble ointment bases are prepared from polyethylene glycols (PEGs) of varying molecular weight (see, e.g., Remington's, supra).

[0095] Antisense oligonucleotides of the invention may be incorporated into lotions, which generally are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethylcellulose, or the like. An exemplary lotion formulation for use in conjunction with the present method contains propylene glycol mixed with a hydrophilic petrolatum such as that which may be obtained under the trademark Aquaphor.sup.RTM from Beiersdorf, Inc. (Norwalk, Conn.).

[0096] Antisense oligonucleotides of the invention may be incorporated into creams, which generally are viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in Remington's, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.

[0097] Antisense oligonucleotides of the invention may be incorporated into microemulsions, which generally are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). For the preparation of microemulsions, surfactant (emulsifier), co-surfactant (co-emulsifier), an oil phase and a water phase are necessary. Suitable surfactants include any surfactants that are useful in the preparation of emulsions, e.g., emulsifiers that are typically used in the preparation of creams. The co-surfactant (or "co-emulsifer") is generally selected from the group of polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier/co-emulsifier combinations are generally although not necessarily selected from the group consisting of: glyceryl monostearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol palmitostearate; and caprilic and capric triglycerides and oleoyl macrogolglycerides. The water phase includes not only water but also, typically, buffers, glucose, propylene glycol, polyethylene glycols, preferably lower molecular weight polyethylene glycols (e.g., PEG 300 and PEG 400), and/or glycerol, and the like, while the oil phase will generally comprise, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono-di- and triglycerides, mono- and di-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

[0098] Antisense oligonucleotides of the invention may be incorporated into gel formulations, which generally are semisolid systems consisting of either suspensions made up of small inorganic particles (two-phase systems) or large organic molecules distributed substantially uniformly throughout a carrier liquid (single phase gels). Single phase gels can be made, for example, by combining the active agent, a carrier liquid and a suitable gelling agent such as tragacanth (at 2 to 5%), sodium alginate (at 2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and mixing until a characteristic semisolid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose and gelatin. Although gels commonly employ aqueous carrier liquid, alcohols and oils can be used as the carrier liquid as well.

[0099] Various additives, known to those skilled in the art, may be included in formulations, e.g., topical formulations. Examples of additives include, but are not limited to, solubilizers, skin permeation enhancers, opacifiers, preservatives (e.g., anti-oxidants), gelling agents, buffering agents, surfactants (particularly nonionic and amphoteric surfactants), emulsifiers, emollients, thickening agents, stabilizers, humectants, colorants, fragrance, and the like. Inclusion of solubilizers and/or skin permeation enhancers is particularly preferred, along with emulsifiers, emollients and preservatives. An optimum topical formulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2 wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer; 2 wt. % to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the active agent and carrier (e.g., water) making of the remainder of the formulation.

[0100] A skin permeation enhancer serves to facilitate passage of therapeutic levels of active agent to pass through a reasonably sized area of unbroken skin. Suitable enhancers are well known in the art and include, for example: lower alkanols such as methanol ethanol and 2-propanol; alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO), decylmethylsulfoxide (C.sub.10 MSO) and tetradecylmethyl sulfboxide; pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone and N-(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-toluamide; C.sub.2-C.sub.6 alkanediols; miscellaneous solvents such as dimethyl formamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol; and the 1-substituted azacycloheptan-2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under the trademark Azone.sup.RTM from Whitby Research Incorporated, Richmond, Va.).

[0101] Examples of solubilizers include, but are not limited to, the following: hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available commercially as Transcutol.sup.RTM) and diethylene glycol monoethyl ether oleate (available commercially as Soficutol.sup.RTM); polyethylene castor oil derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, etc.; polyethylene glycol, particularly lower molecular weight polyethylene glycols such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8 caprylic/capric glycerides (available commercially as Labrasol.sup.RTM); alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act as absorption enhancers. A single solubilizer may be incorporated into the formulation, or a mixture of solubilizers may be incorporated therein.

[0102] Suitable emulsifiers and co-emulsifiers include, without limitation, those emulsifiers and co-emulsifiers described with respect to microemulsion formulations. Emollients include, for example, propylene glycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the like.

[0103] Other active agents may also be included in formulations, e.g., other anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunblock agents commonly found in sunscreen formulations including, but not limited to, anthranilates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoyl methanes (e.g, butyl methoxydibenzoyl methane), p-aminobenzoic acid (PABA) and derivatives thereof, and salicylates (e.g., octyl salicylate).

[0104] In one embodiment, the oligonucleotides are specific for polynucleotides of a Collagen gene, which includes, without limitation noncoding regions. The Collagen gene targets comprise variants of a Collagen gene; mutants of a Collagen gene, including SNPs; noncoding sequences of a Collagen gene; alleles, fragments and the like. Preferably the oligonucleotide is an antisense RNA molecule.

[0105] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to a Collagen gene polynucleotides alone but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of a Collagen gene.

[0106] In another embodiment, an oligonucleotide targets a natural antisense sequence (natural antisense to the coding and non-coding regions) of a Collagen gene targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense RNA or DNA molecule.

[0107] In another embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants may be produced which contain thymidine, guanosine, cytidine or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antisense compound.

[0108] In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0109] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays.

[0110] An antisense compound, whether DNA, RNA, chimeric, substituted etc, is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the antisense compound to non-target 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 in the case of in vitro assays, under conditions in which the assays are performed.

[0111] In another embodiment, targeting of a Collagen gene including without limitation, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NO: 4 to 9, and the like, modulate the expression or function of a Collagen gene. In one embodiment, expression or function is up-regulated as compared to a control. In another embodiment, expression or function is down-regulated as compared to a control.

[0112] In another embodiment, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOS: 10 to 29 including antisense sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or internucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In another embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.

[0113] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0114] In embodiments of the present invention oligomeric antisense compounds, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The functions of DNA to be interfered comprise, for example, replication and transcription. The functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The functions may be up-regulated or inhibited depending on the functions desired.

[0115] The antisense compounds, include, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that 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, partially single-stranded, or circular oligomeric compounds.

[0116] 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 a Collagen gene.

[0117] 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.

[0118] In one embodiment, the antisense oligonucleotides bind to the natural antisense sequences of a Collagen gene and modulate the expression and/or function of a Collagen gene (SEQ ID NO: 1 to 3). Examples of antisense sequences include SEQ ID NOS: 4 to 29.

[0119] In another embodiment, the antisense oligonucleotides bind to one or more segments of a Collagen gene polynucleotide and modulate the expression and/or function of a Collagen gene. The segments comprise at least five consecutive nucleotides of a Collagen gene sense or antisense polynucleotides.

[0120] In another embodiment, the antisense oligonucleotides are specific for natural antisense sequences of a Collagen gene wherein binding of the oligonucleotides to the natural antisense sequences of a Collagen gene modulate expression and/or function of a Collagen gene.

[0121] In another embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID NOS: 10 to 29, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or internucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In another embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.

[0122] Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes has a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG; and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a Collagen gene, regardless of the sequence(s) of such codons. A translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'- TAG and 5'-TGA, respectively).

[0123] The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions that may be targeted effectively with the antisense compounds of the present invention.

[0124] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0125] Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5' cap region.

[0126] 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. In one embodiment, targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, is particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0127] In another embodiment, the antisense oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.

[0128] In another embodiment, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule.

[0129] In another embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.

[0130] 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.

[0131] 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.

[0132] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more than 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 embodiments of target nucleic acids.

[0133] The locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5-nucleotide long portion of a target region to which an active antisense compound is targeted.

[0134] While the specific sequences of certain exemplary target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure.

[0135] Target segments 5-100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within the illustrative target segments are considered to be suitable for targeting as well.

[0136] Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5'-terminus of one of the illustrative target segments (the remaining nucleotides 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 5 to about 100 nucleotides). Similarly target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the illustrative target segments (the remaining nucleotides 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 5 to about 100 nucleotides). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further target segments.

[0137] 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.

[0138] In embodiments of the invention the oligonucleotides bind to an antisense strand of a particular target. The oligonucleotides are at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides are synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.

[0139] In one embodiment, specific nucleic acids are targeted by antisense oligonucleotides. Targeting an antisense compound to a particular nucleic acid, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This 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 non coding polynucleotide such as for example, non coding RNA (ncRNA).

[0140] RNAs can be classified into (1) messenger RNAs (mRNAs), which are translated into proteins, and (2) non-protein-coding RNAs (ncRNAs). ncRNAs comprise microRNAs, antisense transcripts and other Transcriptional Units (TU) containing a high density of stop codons and lacking any extensive "Open Reading Frame". Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci. ncRNAs are often rare and at least half of the ncRNAs that have been sequenced by the FANTOM consortium seem not to be polyadenylated. Most researchers have for obvious reasons focused on polyadenylated mRNAs that are processed and exported to the cytoplasm. Recently, it was shown that the set of non-polyadenylated nuclear RNAs may be very large, and that many such transcripts arise from intergenic regions. The mechanism by which ncRNAs may regulate gene expression is by base pairing with target transcripts. The RNAs that function by base pairing can be grouped into (1) cis encoded RNAs that are encoded at the same genetic location, but on the opposite strand to the RNAs they act upon and therefore display perfect complementarity to their target, and (2) trans-encoded RNAs that are encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.

[0141] Without wishing to be bound by theory, perturbation of an antisense polynucleotide by the antisense oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can either be discordant (antisense knockdown results in messenger RNA elevation) or concordant (antisense knockdown results in concomitant messenger RNA reduction). In these cases, antisense oligonucleotides can be targeted to overlapping or non-overlapping parts of the antisense transcript resulting in its knockdown or sequestration. Coding as well as non-coding antisense can be targeted in an identical manner and that either category is capable of regulating the corresponding sense transcripts--either in a concordant or disconcordant manner. The strategies that are employed in identifying new oligonucleotides for use against a target can be based on the knockdown of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the desired target.

[0142] Strategy 1: In the case of discordant regulation, knocking down the antisense transcript elevates the expression of the conventional (sense) gene. Should that latter gene encode for a known or putative drug target, then knockdown of its antisense counterpart could conceivably mimic the action of a receptor agonist or an enzyme stimulant.

[0143] Strategy 2: In the case of concordant regulation, one could concomitantly knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve knockdown, then this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense oligonucleotide to the corresponding antisense transcript, or a single energetically symmetric antisense oligonucleotide that simultaneously targets overlapping sense and antisense transcripts.

[0144] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, doublestranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.

[0145] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A-formlike structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.

[0146] In another embodiment, the desired oligonucleotides or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.

[0147] dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs).

[0148] Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing. However, in instances described in detail in the examples section which follows, oligonucleotides are shown to increase the expression and/or function of the Collagen gene polynucleotides and encoded products thereof. dsRNAs may also act as small activating RNAs (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa).

[0149] In a further embodiment, the "target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of a Collagen gene polynucleotide. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding a Collagen gene and which comprise at least a 5-nucleotide portion that is complementary to a target segment. The screening method comprises the steps of contacting a target segment of a nucleic acid molecule encoding sense or natural antisense polynucleotides of a Collagen gene with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding a Collagen gene polynucleotide, e.g. SEQ ID NOS: 10 to 29. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding a Collagen gene polynucleotide, the modulator may then be employed in further investigative studies of the function of a Collagen gene polynucleotide, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0150] Targeting the natural antisense sequence modulates the function of the target gene. For example, the Collagen gene (e.g. accession numbers NM.sub.--000088, NM.sub.--000089, NM.sub.--000094). In a embodiment, the target is an antisense polynucleotide of the Collagen gene. In a embodiment, an antisense oligonucleotide targets sense and/or natural antisense sequences of a Collagen gene polynucleotide (e.g. accession numbers NM.sub.--000088, NM.sub.--000089, NM.sub.--000094), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule and the targets include coding and noncoding regions of antisense and/or sense Collagen gene polynucleotides.

[0151] The target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0152] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications. For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target.

[0153] In a embodiment, an antisense oligonucleotide targets Collagen gene polynucleotides (e.g. accession numbers NM.sub.--000088, NM.sub.--000089, NM.sub.--000094), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule.

[0154] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to Collagen gene alone but extends to any of the isoforms, receptors, homologs and the like of a Collagen gene molecule.

[0155] In another embodiment, an oligonucleotide targets a natural antisense sequence of a Collagen gene polynucleotide, for example, polynucleotides set forth as SEQ ID NO: 4 to 9, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 10 to 29.

[0156] In one embodiment, the oligonucleotides are complementary to or bind to nucleic acid sequences of a Collagen gene antisense, including without limitation noncoding sense and/or antisense sequences associated with a Collagen gene polynucleotide and modulate expression and/or function of a Collagen gene molecule.

[0157] In another embodiment, the oligonucleotides are complementary to or bind to nucleic acid sequences of a Collagen gene natural antisense, set forth as SEQ ID NO: 4 to 9 and modulate expression and/or function of a Collagen gene molecule.

[0158] In a embodiment, oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS: 10 to 29 and modulate expression and/or function of a Collagen gene molecule.

[0159] The polynucleotide targets comprise Collagen gene, including family members thereof, variants of a Collagen gene; mutants of a Collagen gene, including SNPs; noncoding sequences of a Collagen gene; alleles of a Collagen gene; species variants, fragments and the like. Preferably the oligonucleotide is an antisense molecule.

[0160] In another embodiment, the oligonucleotide targeting Collagen gene polynucleotides, comprise: antisense RNA, interference RNA (RNAi), short interfering RNA (siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or, small activating RNA (saRNA).

[0161] In another embodiment, targeting of a Collagen gene polynucleotide, e.g. SEQ ID NO: 4 to 9 modulate the expression or function of these targets. In one embodiment, expression or function is up-regulated as compared to a control. In another embodiment, expression or function is down-regulated as compared to a control.

[0162] In another embodiment, antisense compounds comprise sequences set forth as SEQ ID NOS: 10 to 29. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.

[0163] In another embodiment, SEQ ID NOS: 10 to 29 comprise one or more LNA nucleotides.

[0164] The modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antisense oligonucleotides, siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript.

[0165] Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.

[0166] In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0167] Several approaches such as in vitro selection (evolution) strategies have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages.

[0168] The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min-1 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min-1 In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min-1 Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain selfcleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for

[0169] RNA phosphodiester cleavage.

[0170] Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1987. The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.

[0171] Catalytic RNAs designed based on the "hammerhead" motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences. This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the "hammerhead" model may possibly cleave specific substrate RNAs in vivo.

[0172] RNA interference (RNAi) has become a powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression.

[0173] In one embodiment, an oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof. This term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0174] According to the present invention, the oligonucleotides or "antisense compounds" include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.

[0175] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A-formlike structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.

[0176] The antisense compounds in accordance with this invention can comprise an antisense portion from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound. In other words, a single-stranded antisense compound of the invention comprises from 5 to about 80 nucleotides, and a double-stranded antisense compound of the invention (such as a dsRNA, for example) comprises a sense and an antisense strand or portion of 5 to about 80 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense portions of 5, 6, 7,8, 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 nucleotides in length, or any range therewithin.

[0177] In one embodiment, the antisense compounds of the invention have antisense portions of 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 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, or 50 nucleotides in length, or any range therewithin. In some embodiments, the oligonucleotides are 15 nucleotides in length.

[0178] In one embodiment, the antisense or oligonucleotide compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin.

[0179] In another embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense or dsRNA compounds. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

[0180] In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0181] In another embodiment, the antisense oligonucleotides, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 4 to 29 comprise one or more substitutions or modifications. In one embodiment, the nucleotides are substituted with locked nucleic acids (LNA).

[0182] In another embodiment, the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisense of coding and/or non-coding sequences associated with Collagen gene and the sequences set forth as SEQ ID NOS: 1 to 9. The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: 1 to 9.

[0183] Certain oligonucleotides of this invention are chimeric oligonucleotides. "Chimeric oligonucleotides" or "chimeras," in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is 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 antisense modulation of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In one embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H Affinity of an oligonucleotide for its target (in this case, a nucleic acid encoding ras) is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater is the affinity of the oligonucleotide for the target.

[0184] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotides mimetics as described above. Such; compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, 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, each of which is herein incorporated by reference.

[0185] In another embodiment, the region of the oligonucleotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2LOalkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide. In other embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance RNAi oligonucleotide inhibition of gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance. Some desirable modifications can be found in De Mesmaeker et al. (1995) Acc. Chem. Res., 28:366-374.

[0186] Specific examples of some oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 --NH--O--CH2, CH,--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 backbones, wherein the native phosphodiester backbone is represented as O--P--O--CH,). The amide backbones disclosed by De Mesmaeker et al. (1995) Acc. Chem. Res. 28:366-374 are also preferred. Also are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). In other embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. Oligonucleotides may also comprise one or more substituted sugar moieties. oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A modification includes 2'-methoxyethoxy [2'-O-CH2 CH2 OCH3, known as 2'-O-(2-methoxyethyl)]. Other modifications include 2'-methoxy (2'-O--CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (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 and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

[0187] Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me--C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. A "universal" base known in the art, e.g., inosine, may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently base substitutions.

[0188] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety, 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 . Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.

[0189] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.

[0190] In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.

[0191] The oligonucleotides 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 Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.

[0192] In accordance with the invention, use of modifications such as the use of LNA monomers to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides comprised of current chemistries such as MOE, ANA, FANA, PS etc. This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA monomers. The LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller. It is that such LNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and that their sizes are between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.

[0193] Modified oligonucleotide backbones comprise, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0194] Representative United States patents that teach the preparation of the above phosphorus containing linkages comprise, 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; and 5,625,050, each of which is herein incorporated by reference.

[0195] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise 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; 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.

[0196] Representative United States patents that teach the preparation of the above oligonucleosides comprise, 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; and 5,677,439, each of which is herein incorporated by reference.

[0197] In other oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. 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). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen, et al. (1991) Science 254, 1497-1500.

[0198] In another embodiment of the invention the oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH2-NH--O--CH2-, --CH2-N(CH3)--O--CH2-- known as a methylene (methylimino) or MMI backbone, --CH2-O--N(CH3)-CH2-, --CH2N(CH3)-N(CH3)CH2-- and--O--N(CH3)--CH2--CH2-- wherein the native phosphodiester backbone is represented as --O--P--O--CH2-- of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0199] Modified oligonucleotides may also contain one or more substituted sugar moieties. oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyl and alkynyl. Particularly are O(CH2)n OmCH3, O(CH2)n, OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 where n and m can be from 1 to about 10. Other oligonucleotides comprise one of the following at the 2' position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A modification comprises 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) i.e., an alkoxyalkoxy group. A further modification comprises 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-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2-O--CH2-N(CH2)2.

[0200] Other modifications comprise 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (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' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures comprise, 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; and 5,700,920, each of which is herein incorporated by reference.

[0201] Oligonucleotides may also comprise nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides comprise other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 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-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0202] Further, nucleotides comprise 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., `Angewandle Chemie, International Edition`, 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, `Antisense Research and Applications`, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleotides are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These comprise 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, `Antisense Research and Applications`, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.

[0203] Representative United States patents that teach the preparation of the above noted modified nucleotides as well as other modified nucleotides comprise, but are not limited to, 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; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.

[0204] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.

[0205] Such moieties comprise 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-t oxycholesterol moiety .

[0206] Representative United States patents that teach the preparation of such oligonucleotides conjugates comprise, 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, each of which is herein incorporated by reference.

[0207] Drug discovery: The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and target segments identified herein in drug discovery efforts to elucidate relationships that exist between a Collagen gene polynucleotide and a disease state, phenotype, or condition. These methods include detecting or modulating a Collagen gene polynucleotide comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of a Collagen gene polynucleotide and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression.

[0208] Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).

[0209] Expression of RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisense modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA. Based on sequence conservation, primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non coding region can be used. Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal. These plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region. The effectiveness of individual antisense oligonucleotides would be assayed by modulation of the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.

[0210] COL1A1, COL1A2, COL7A1 proteins and mRNA expression can be assayed using methods known to those of skill in the art and described elsewhere herein. For example, immunoassays such as the ELISA can be used to measure protein levels. Collagen gene antibodies for ELISAs are available commercially, e.g., from R&D Systems (Minneapolis, Minn.), Abcam, Cambridge, Mass.

[0211] In embodiments, COL1A1, COL1A2, COL7A1 expression (e.g., mRNA or protein) in a sample (e.g., cells or tissues in vivo or in vitro) treated using an antisense oligonucleotide of the invention is evaluated by comparison with Collagen gene expression in a control sample. For example, expression of the protein or nucleic acid can be compared using methods known to those of skill in the art with that in a mock-treated or untreated sample. Alternatively, comparison with a sample treated with a control antisense oligonucleotide (e.g., one having an altered or different sequence) can be made depending on the information desired. In another embodiment, a difference in the expression of the Collagen gene protein or nucleic acid in a treated vs. an untreated sample can be compared with the difference in expression of a different nucleic acid (including any standard deemed appropriate by the researcher, e.g., a housekeeping gene) in a treated sample vs. an untreated sample.

[0212] Observed differences can be expressed as desired, e.g., in the form of a ratio or fraction, for use in a comparison with control. In embodiments, the level of a Collagen gene mRNA or protein, in a sample treated with an antisense oligonucleotide of the present invention, is increased or decreased by about 1.25-fold to about 10-fold or more relative to an untreated sample or a sample treated with a control nucleic acid. In embodiments, the level of a Collagen gene mRNA or protein is increased or decreased by at least about 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

[0213] The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of 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.

[0214] For use in kits and diagnostics and in various biological systems, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, are useful 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.

[0215] As used herein the term "biological system" or "system" is defined as any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the Collagen genes. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.

[0216] As one non limiting 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 that affect expression patterns.

[0217] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480, 17-24; Celis, et al., (2000) FEBS Lett., 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., (2000) Drug Discov. Today, 5, 415- 425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, (1999) Methods Enzymol., 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., (2000) Proc. Natl. Acad. Sci. U.S.A., 97, 1976-81), protein arrays and proteomics (Celis, et al., (2000) FEBS Lett., 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Left., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., (2000) Anal. Biochem. 286, 91-98; Larson, et al., (2000) Cytometry 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, (2000) Curr. Opin. Microbiol. 3, 316-21), comparative genomic hybridization (Carulli, et al., (1998) J. Cell Biochem. Suppl., 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometry methods (To, Comb. (2000) Chem. High Throughput Screen, 3, 235-41).

[0218] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding a Collagen gene. For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective Collagen gene modulators are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding a Collagen gene and in the amplification of said nucleic acid molecules for detection or for use in further studies of a Collagen gene. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding a Collagen gene can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonucleotide, or any other suitable detection means. Kits using such detection means for detecting the level of a Collagen gene in a sample may also be prepared.

[0219] The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0220] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of a Collagen gene polynucleotide 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 Collagen gene modulator. The Collagen gene modulators of the present invention effectively modulate the activity of a Collagen gene or modulate the expression of a Collagen gene protein. In one embodiment, the activity or expression of a Collagen gene in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of a Collagen gene in an animal is inhibited by about 30%. More preferably, the activity or expression of a Collagen gene in an animal is inhibited by 50% or more. Thus, the oligomeric compounds modulate expression of a Collagen gene mRNA by at least 10%, by at least 50%, 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% as compared to a control.

[0221] In one embodiment, the activity or expression of a Collagen gene and/or in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of a Collagen gene in an animal is increased by about 30%. More preferably, the activity or expression of a Collagen gene in an animal is increased by 50% or more. Thus, the oligomeric compounds modulate expression of a Collagen gene mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 10%, 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% as compared to a control.

[0222] For example, the reduction of the expression of a Collagen gene may be measured in serum, blood, 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 Collagen gene peptides and/or the Collagen gene protein itself.

[0223] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0224] Conjugates: Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that 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. Typicalconjugate 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 No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

[0225] Representative United States patents that teach the preparation of such oligonucleotides 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.

[0226] Formulations: The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as forexample, 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; 5,543,165; 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.

[0227] Although, the antisense oligonucleotides do not need to be administered in the context of a vector in order to modulate a target expression and/or function, embodiments of the invention relates to expression vector constructs for the expression of antisense oligonucleotides, comprising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case.

[0228] In an embodiment, invention practice involves administering at least one of the foregoing antisense oligonucleotides with a suitable nucleic acid delivery system. In one embodiment, that system includes a non-viral vector operably linked to the polynucleotide. Examples of such nonviral vectors include the oligonucleotide alone (e.g. any one or more of SEQ ID NOS: 10 to 29) or in combination with a suitable protein, polysaccharide or lipid formulation.

[0229] Additionally suitable nucleic acid delivery systems include viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex. Preferably, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter.

[0230] Additionally vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector, Adenovirus Vectors and Adeno-associated Virus Vectors).

[0231] 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.

[0232] 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, examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0233] The present invention also includes pharmaceutical compositions and formulations that 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.

[0234] For treating tissues in the central nervous system, administration can be made by, e.g., injection or infusion into the cerebrospinal fluid. Administration of antisense RNA into cerebrospinal fluid is described, e.g., in U.S. Pat. App. Pub. No. 2007/0117772, "Methods for slowing familial ALS disease progression," incorporated herein by reference in its entirety.

[0235] When it is intended that the antisense oligonucleotide of the present invention be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier. Injection can be made, e.g., in the entorhinal cortex or hippocampus. Delivery of neurotrophic factors by administration of an adenovirus vector to motor neurons in muscle tissue is described in, e.g., U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference. Delivery of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is known in the art and described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference. Administration can be rapid as by injection or made over a period of time as by slow infusion or administration of slow release formulations.

[0236] The subject antisense oligonucleotides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, the antisense oligonucleotide can be coupled to any substance, known in the art to promote penetration or transport across the blood-brain barrier, such as an antibody to the transferrin receptor, and administered by intravenous injection. The antisense compound can be linked with a viral vector, for example, that makes the antisense compound more effective and/or increases the transport of the antisense compound across the blood-brain barrier. Osmotic blood brain barrier disruption can also be accomplished by, e.g., infusion of sugars including, but not limited to, meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids including, but not limited to, glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in U.S. Pat. No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier," 6,294,520, "Material for passage through the blood-brain barrier," and 6,936,589, "Parenteral delivery systems," all incorporated herein by reference in their entirety.

[0237] The subject antisense compounds may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. For example, cationic lipids may be included in the formulation to facilitate oligonucleotide uptake. One such composition shown to facilitate uptake is LIPOFECTIN (available from GIBCO-BRL, Bethesda, Md.).

[0238] 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.

[0239] 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.

[0240] 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 that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0241] 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.

[0242] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 um in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860.

[0243] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex.

[0244] Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0245] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomeslacking such specialized lipids.

[0246] Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

[0247] 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 by reference.

[0248] 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 nonsurfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0249] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0250] formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidyl ethanolamine DOTMA).

[0251] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860.

[0252] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference. Also are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0253] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0254] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0255] 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. For example, the first target may be a particular antisense sequence of a Collagen gene, and the second target may be a region from another nucleotide sequence. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same Collagen gene nucleic acid target. Numerous examples of antisense compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.

Dosing.

[0256] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 .mu.g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0257] In embodiments, a patient is treated with a dosage of drug that is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 mg/kg body weight. Certain injected dosages of antisense oligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884, "Antisense modulation of PTP1B expression," incorporated herein by reference in its entirety.

[0258] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

[0259] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

EXAMPLES

[0260] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example 1

Design of Antisense Oligonucleotides Specific for a Nucleic Acid Molecule Antisense to a Collagen Gene and/or a Sense Strand of a Collagen Gene Polynucleotide

[0261] As indicated above the term "oligonucleotide specific for" or "oligonucleotide targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of an mRNA transcript of the targeted gene.

[0262] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

[0263] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays

[0264] The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays as known in the art. For example, the properties of the oligonucleotides described herein can be obtained by determination of binding strength between the target natural antisense and a potential drug molecules using melting curve assay.

[0265] The binding strength between the target natural antisense and a potential drug molecule (Molecule) can be estimated using any of the established methods of measuring the strength of intermolecular interactions, for example, a melting curve assay.

[0266] Melting curve assay determines the temperature at which a rapid transition from double-stranded to single-stranded conformation occurs for the natural antisense/Molecule complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.

[0267] A melting curve assay can be performed using a cDNA copy of the actual natural antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule. Multiple kits containing all necessary reagents to perform this assay are available (e.g. Applied Biosystems Inc. MeltDoctor kit).

[0268] These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). The properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but are highly fluorescent when bound to dsDNA.

[0269] To perform the assay the cDNA or a corresponding oligonucleotide are mixed with Molecule in concentrations defined by the particular manufacturer's protocols. The mixture is heated to 95 .degree. C. to dissociate all pre-formed dsDNA complexes, then slowly cooled to room temperature or other lower temperature defined by the kit manufacturer to allow the DNA molecules to anneal. The newly formed complexes are then slowly heated to 95.degree. C. with simultaneous continuous collection of data on the amount of fluorescence that is produced by the reaction. The fluorescence intensity is inversely proportional to the amounts of dsDNA present in the reaction. The data can be collected using a real time PCR instrument compatible with the kit (e.g.ABI's StepOne Plus Real Time PCR System or LightTyper instrument, Roche Diagnostics, Lewes, UK).

[0270] Melting peaks are constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescence)/dT) on the y-axis) against temperature (x-axis) using appropriate software (for example LightTyper (Roche) or SDS Dissociation Curve, ABI). The data is analyzed to identify the temperature of the rapid transition from dsDNA complex to single strand molecules. This temperature is called Tm and is directly proportional to the strength of interaction between the two molecules. Typically, Tm will exceed 40 .degree. C.

Example 2

Modulation of a Collagen Gene Polynucleotide Treatment of HepG2 Cells with Antisense Oligonucleotides

[0271] HepG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat # MT-10-010-CV) +10% FBS (Mediatech cat# MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37.degree. C. and 5% CO2. One day before the experiment the cells were replated at the density of 1.5.times.105/ml into 6 well plates and incubated at 37.degree. C. and 5% CO2. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. All antisense oligonucleotides were diluted to the concentration of 20 .mu.M. Two .mu.l of this solution was incubated with 400 .mu.l of Opti-MEM media (Gibco cat#31985-070) and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HepG2 cells. A Similar mixture including 2 .mu.l of water instead of the oligonucleotide solution was used for the mock-transfected controls. After 3-18 h of incubation at 37.degree. C. and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) or High Capacity cDNA Reverse Transcription Kit (cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00164004_ml, Hs00164310 ml, by Applied Biosystems Inc., Foster City Calif.). The following PCR cycle was used: 50.degree. C. for 2 min, 95.degree. C. for 10 min, 40 cycles of (95.degree. C. for 15 seconds, 60.degree. C. for 1 min) using Mx4000 thermal cycler (Stratagene).

[0272] Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.

Results

[0273] Real time PCR results show that the levels of the Colla1 mRNA in HepG2 cells are significantly increased 48 h after treatment with one of the oligos designed to Colla1 antisense DW440457 (FIG. 1).

[0274] Real time PCR results show that the levels of Col7a1 mRNA in HepG2 cells are significantly increased 48 h after treatment with two of the oligos designed to Col7a1 as be142537 and bg998538 (FIG. 4).

[0275] Treatment of HUVEC cells with antisense oligonucleotides

[0276] HUVEC cells from ATCC (Promo Cell cat# C-12253) were grown in Epithelial Growth Media (Promo Cell cat #C-22010) at 37oC and 5% CO2. One day before the experiment the cells were replated using Promo Cell Detach Kit (cat#C-41200) at the density of 1.5.times.10 5/ml into 6 well plates and incubated at 27oC and 5% CO2. On the day of the experiment the media in the 6 well plates was changed to fresh Epithelial Growth Media. All antisense oligonucleotides were diluted to the concentration of 20 .mu.M. Two .mu.l of this solution was incubated with 400 .mu.l of Opti-MEM media (Gibco cat#31985-070) and 4 .mu.l of Lipofectamine 2000 (Invitrogen cat# 1168019) at room temperature for 20 min and applied to each well of the 6 well plates with HUVEC cells. Similar mixture including 2 .mu.l of water instead of the oligonucleotide solution was used for the mock-transfected controls. After 3-18 h of incubation at 37.degree. C. and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assays: Hs01028970_ml and Hs00164310_ml by Applied Biosystems Inc., Foster City Calif.). The following PCR cycle was used: 50.degree. C. for 2 min, 95.degree. C. for 10 min, 40 cycles of )95.degree. C. for 15 seconds, 60.degree. C. for 1 min) using StepOne Plus Real Time PCR Machine (Applied Biosystems Inc.) or Mx4000 thermal cycler (Stratagene).

[0277] Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.

Results.

[0278] Real time PCR results show that the levels of the Colla2 mRNA in HUVEC cells are significantly increased 48 h after treatment with one of the oligos designed to Colla2 antisense Hs.571263 (FIG. 2).

[0279] Real time PCR results show that the levels of Col7a1 mRNA in HUVEC cells are significantly increased 48 h after treatment with one of the siRNAs designed to Col7a1 as CV425857 (CV425857.1.sub.--1, CV425857.1.sub.--2,) and one siRNA to BU615800.1 (BU615800.1.sub.--1) (FIG. 3).

[0280] Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

[0281] The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210> SEQ ID NO 1 <211> LENGTH: 5927 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000088.3 <309> DATABASE ENTRY DATE: 2010-06-06 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(5927) <400> SEQUENCE: 1 tcgtcggagc agacgggagt ttctcctcgg ggtcggagca ggaggcacgc ggagtgtgag 60 gccacgcatg agcggacgct aaccccctcc ccagccacaa agagtctaca tgtctagggt 120 ctagacatgt tcagctttgt ggacctccgg ctcctgctcc tcttagcggc caccgccctc 180 ctgacgcacg gccaagagga aggccaagtc gagggccaag acgaagacat cccaccaatc 240 acctgcgtac agaacggcct caggtaccat gaccgagacg tgtggaaacc cgagccctgc 300 cggatctgcg tctgcgacaa cggcaaggtg ttgtgcgatg acgtgatctg tgacgagacc 360 aagaactgcc ccggcgccga agtccccgag ggcgagtgct gtcccgtctg ccccgacggc 420 tcagagtcac ccaccgacca agaaaccacc ggcgtcgagg gacccaaggg agacactggc 480 ccccgaggcc caaggggacc cgcaggcccc cctggccgag atggcatccc tggacagcct 540 ggacttcccg gaccccccgg accccccgga cctcccggac cccctggcct cggaggaaac 600 tttgctcccc agctgtctta tggctatgat gagaaatcaa ccggaggaat ttccgtgcct 660 ggccccatgg gtccctctgg tcctcgtggt ctccctggcc cccctggtgc acctggtccc 720 caaggcttcc aaggtccccc tggtgagcct ggcgagcctg gagcttcagg tcccatgggt 780 ccccgaggtc ccccaggtcc ccctggaaag aatggagatg atggggaagc tggaaaacct 840 ggtcgtcctg gtgagcgtgg gcctcctggg cctcagggtg ctcgaggatt gcccggaaca 900 gctggcctcc ctggaatgaa gggacacaga ggtttcagtg gtttggatgg tgccaaggga 960 gatgctggtc ctgctggtcc taagggtgag cctggcagcc ctggtgaaaa tggagctcct 1020 ggtcagatgg gcccccgtgg cctgcctggt gagagaggtc gccctggagc ccctggccct 1080 gctggtgctc gtggaaatga tggtgctact ggtgctgccg ggccccctgg tcccaccggc 1140 cccgctggtc ctcctggctt ccctggtgct gttggtgcta agggtgaagc tggtccccaa 1200 gggccccgag gctctgaagg tccccagggt gtgcgtggtg agcctggccc ccctggccct 1260 gctggtgctg ctggccctgc tggaaaccct ggtgctgatg gacagcctgg tgctaaaggt 1320 gccaatggtg ctcctggtat tgctggtgct cctggcttcc ctggtgcccg aggcccctct 1380 ggaccccagg gccccggcgg ccctcctggt cccaagggta acagcggtga acctggtgct 1440 cctggcagca aaggagacac tggtgctaag ggagagcctg gccctgttgg tgttcaagga 1500 ccccctggcc ctgctggaga ggaaggaaag cgaggagctc gaggtgaacc cggacccact 1560 ggcctgcccg gaccccctgg cgagcgtggt ggacctggta gccgtggttt ccctggcgca 1620 gatggtgttg ctggtcccaa gggtcccgct ggtgaacgtg gttctcctgg ccctgctggc 1680 cccaaaggat ctcctggtga agctggtcgt cccggtgaag ctggtctgcc tggtgccaag 1740 ggtctgactg gaagccctgg cagccctggt cctgatggca aaactggccc ccctggtccc 1800 gccggtcaag atggtcgccc cggaccccca ggcccacctg gtgcccgtgg tcaggctggt 1860 gtgatgggat tccctggacc taaaggtgct gctggagagc ccggcaaggc tggagagcga 1920 ggtgttcccg gaccccctgg cgctgtcggt cctgctggca aagatggaga ggctggagct 1980 cagggacccc ctggccctgc tggtcccgct ggcgagagag gtgaacaagg ccctgctggc 2040 tcccccggat tccagggtct ccctggtcct gctggtcctc caggtgaagc aggcaaacct 2100 ggtgaacagg gtgttcctgg agaccttggc gcccctggcc cctctggagc aagaggcgag 2160 agaggtttcc ctggcgagcg tggtgtgcaa ggtccccctg gtcctgctgg tccccgaggg 2220 gccaacggtg ctcccggcaa cgatggtgct aagggtgatg ctggtgcccc tggagctccc 2280 ggtagccagg gcgcccctgg ccttcaggga atgcctggtg aacgtggtgc agctggtctt 2340 ccagggccta agggtgacag aggtgatgct ggtcccaaag gtgctgatgg ctctcctggc 2400 aaagatggcg tccgtggtct gactggcccc attggtcctc ctggccctgc tggtgcccct 2460 ggtgacaagg gtgaaagtgg tcccagcggc cctgctggtc ccactggagc tcgtggtgcc 2520 cccggagacc gtggtgagcc tggtcccccc ggccctgctg gctttgctgg cccccctggt 2580 gctgacggcc aacctggtgc taaaggcgaa cctggtgatg ctggtgctaa aggcgatgct 2640 ggtccccctg gccctgccgg acccgctgga ccccctggcc ccattggtaa tgttggtgct 2700 cctggagcca aaggtgctcg cggcagcgct ggtccccctg gtgctactgg tttccctggt 2760 gctgctggcc gagtcggtcc tcctggcccc tctggaaatg ctggaccccc tggccctcct 2820 ggtcctgctg gcaaagaagg cggcaaaggt ccccgtggtg agactggccc tgctggacgt 2880 cctggtgaag ttggtccccc tggtccccct ggccctgctg gcgagaaagg atcccctggt 2940 gctgatggtc ctgctggtgc tcctggtact cccgggcctc aaggtattgc tggacagcgt 3000 ggtgtggtcg gcctgcctgg tcagagagga gagagaggct tccctggtct tcctggcccc 3060 tctggtgaac ctggcaaaca aggtccctct ggagcaagtg gtgaacgtgg tccccctggt 3120 cccatgggcc cccctggatt ggctggaccc cctggtgaat ctggacgtga gggggctcct 3180 ggtgccgaag gttcccctgg acgagacggt tctcctggcg ccaagggtga ccgtggtgag 3240 accggccccg ctggaccccc tggtgctcct ggtgctcctg gtgcccctgg ccccgttggc 3300 cctgctggca agagtggtga tcgtggtgag actggtcctg ctggtcccgc cggtcctgtc 3360 ggccctgttg gcgcccgtgg ccccgccgga ccccaaggcc cccgtggtga caagggtgag 3420 acaggcgaac agggcgacag aggcataaag ggtcaccgtg gcttctctgg cctccagggt 3480 ccccctggcc ctcctggctc tcctggtgaa caaggtccct ctggagcctc tggtcctgct 3540 ggtccccgag gtccccctgg ctctgctggt gctcctggca aagatggact caacggtctc 3600 cctggcccca ttgggccccc tggtcctcgc ggtcgcactg gtgatgctgg tcctgttggt 3660 ccccccggcc ctcctggacc tcctggtccc cctggtcctc ccagcgctgg tttcgacttc 3720 agcttcctgc cccagccacc tcaagagaag gctcacgatg gtggccgcta ctaccgggct 3780 gatgatgcca atgtggttcg tgaccgtgac ctcgaggtgg acaccaccct caagagcctg 3840 agccagcaga tcgagaacat ccggagccca gagggcagcc gcaagaaccc cgcccgcacc 3900 tgccgtgacc tcaagatgtg ccactctgac tggaagagtg gagagtactg gattgacccc 3960 aaccaaggct gcaacctgga tgccatcaaa gtcttctgca acatggagac tggtgagacc 4020 tgcgtgtacc ccactcagcc cagtgtggcc cagaagaact ggtacatcag caagaacccc 4080 aaggacaaga ggcatgtctg gttcggcgag agcatgaccg atggattcca gttcgagtat 4140 ggcggccagg gctccgaccc tgccgatgtg gccatccagc tgaccttcct gcgcctgatg 4200 tccaccgagg cctcccagaa catcacctac cactgcaaga acagcgtggc ctacatggac 4260 cagcagactg gcaacctcaa gaaggccctg ctcctccagg gctccaacga gatcgagatc 4320 cgcgccgagg gcaacagccg cttcacctac agcgtcactg tcgatggctg cacgagtcac 4380 accggagcct ggggcaagac agtgattgaa tacaaaacca ccaagacctc ccgcctgccc 4440 atcatcgatg tggccccctt ggacgttggt gccccagacc aggaattcgg cttcgacgtt 4500 ggccctgtct gcttcctgta aactccctcc atcccaacct ggctccctcc cacccaacca 4560 actttccccc caacccggaa acagacaagc aacccaaact gaaccccctc aaaagccaaa 4620 aaatgggaga caatttcaca tggactttgg aaaatatttt tttcctttgc attcatctct 4680 caaacttagt ttttatcttt gaccaaccga acatgaccaa aaaccaaaag tgcattcaac 4740 cttaccaaaa aaaaaaaaaa aaaaagaata aataaataac tttttaaaaa aggaagcttg 4800 gtccacttgc ttgaagaccc atgcgggggt aagtcccttt ctgcccgttg ggcttatgaa 4860 accccaatgc tgccctttct gctcctttct ccacaccccc cttggggcct cccctccact 4920 ccttcccaaa tctgtctccc cagaagacac aggaaacaat gtattgtctg cccagcaatc 4980 aaaggcaatg ctcaaacacc caagtggccc ccaccctcag cccgctcctg cccgcccagc 5040 acccccaggc cctgggggac ctggggttct cagactgcca aagaagcctt gccatctggc 5100 gctcccatgg ctcttgcaac atctcccctt cgtttttgag ggggtcatgc cgggggagcc 5160 accagcccct cactgggttc ggaggagagt caggaagggc cacgacaaag cagaaacatc 5220 ggatttgggg aacgcgtgtc aatcccttgt gccgcagggc tgggcgggag agactgttct 5280 gttccttgtg taactgtgtt gctgaaagac tacctcgttc ttgtcttgat gtgtcaccgg 5340 ggcaactgcc tgggggcggg gatgggggca gggtggaagc ggctccccat tttataccaa 5400 aggtgctaca tctatgtgat gggtggggtg gggagggaat cactggtgct atagaaattg 5460 agatgccccc ccaggccagc aaatgttcct ttttgttcaa agtctatttt tattccttga 5520 tatttttctt tttttttttt tttttttgtg gatggggact tgtgaatttt tctaaaggtg 5580 ctatttaaca tgggaggaga gcgtgtgcgg ctccagccca gcccgctgct cactttccac 5640 cctctctcca cctgcctctg gcttctcagg cctctgctct ccgacctctc tcctctgaaa 5700 ccctcctcca cagctgcagc ccatcctccc ggctccctcc tagtctgtcc tgcgtcctct 5760 gtccccgggt ttcagagaca acttcccaaa gcacaaagca gtttttcccc ctaggggtgg 5820 gaggaagcaa aagactctgt acctattttg tatgtgtata ataatttgag atgtttttaa 5880 ttattttgat tgctggaata aagcatgtgg aaatgaccca aacataa 5927 <210> SEQ ID NO 2 <211> LENGTH: 5411 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000089.3 <309> DATABASE ENTRY DATE: 2010-05-23 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(5411) <400> SEQUENCE: 2 gtgtcccata gtgtttccaa acttggaaag ggcgggggag ggcgggagga tgcggagggc 60 ggaggtatgc agacaacgag tcagagtttc cccttgaaag cctcaaaagt gtccacgtcc 120 tcaaaaagaa tggaaccaat ttaagaagcc agccccgtgg ccacgtccct tcccccattc 180 gctccctcct ctgcgccccc gcaggctcct cccagctgtg gctgcccggg cccccagccc 240 cagccctccc attggtggag gcccttttgg aggcacccta gggccaggga aacttttgcc 300 gtataaatag ggcagatccg ggctttatta ttttagcacc acggcagcag gaggtttcgg 360 ctaagttgga ggtactggcc acgactgcat gcccgcgccc gccaggtgat acctccgccg 420 gtgacccagg ggctctgcga cacaaggagt ctgcatgtct aagtgctaga catgctcagc 480 tttgtggata cgcggacttt gttgctgctt gcagtaacct tatgcctagc aacatgccaa 540 tctttacaag aggaaactgt aagaaagggc ccagccggag atagaggacc acgtggagaa 600 aggggtccac caggcccccc aggcagagat ggtgaagatg gtcccacagg ccctcctggt 660 ccacctggtc ctcctggccc ccctggtctc ggtgggaact ttgctgctca gtatgatgga 720 aaaggagttg gacttggccc tggaccaatg ggcttaatgg gacctagagg cccacctggt 780 gcagctggag ccccaggccc tcaaggtttc caaggacctg ctggtgagcc tggtgaacct 840 ggtcaaactg gtcctgcagg tgctcgtggt ccagctggcc ctcctggcaa ggctggtgaa 900 gatggtcacc ctggaaaacc cggacgacct ggtgagagag gagttgttgg accacagggt 960 gctcgtggtt tccctggaac tcctggactt cctggcttca aaggcattag gggacacaat 1020 ggtctggatg gattgaaggg acagcccggt gctcctggtg tgaagggtga acctggtgcc 1080 cctggtgaaa atggaactcc aggtcaaaca ggagcccgtg ggcttcctgg tgagagagga 1140 cgtgttggtg cccctggccc agctggtgcc cgtggcagtg atggaagtgt gggtcccgtg 1200 ggtcctgctg gtcccattgg gtctgctggc cctccaggct tcccaggtgc ccctggcccc 1260 aagggtgaaa ttggagctgt tggtaacgct ggtcctgctg gtcccgccgg tccccgtggt 1320 gaagtgggtc ttccaggcct ctccggcccc gttggacctc ctggtaatcc tggagcaaac 1380 ggccttactg gtgccaaggg tgctgctggc cttcccggcg ttgctggggc tcccggcctc 1440 cctggacccc gcggtattcc tggccctgtt ggtgctgccg gtgctactgg tgccagagga 1500 cttgttggtg agcctggtcc agctggctcc aaaggagaga gcggtaacaa gggtgagccc 1560 ggctctgctg ggccccaagg tcctcctggt cccagtggtg aagaaggaaa gagaggccct 1620 aatggggaag ctggatctgc cggccctcca ggacctcctg ggctgagagg tagtcctggt 1680 tctcgtggtc ttcctggagc tgatggcaga gctggcgtca tgggccctcc tggtagtcgt 1740 ggtgcaagtg gccctgctgg agtccgagga cctaatggag atgctggtcg ccctggggag 1800 cctggtctca tgggacccag aggtcttcct ggttcccctg gaaatatcgg ccccgctgga 1860 aaagaaggtc ctgtcggcct ccctggcatc gacggcaggc ctggcccaat tggcccagct 1920 ggagcaagag gagagcctgg caacattgga ttccctggac ccaaaggccc cactggtgat 1980 cctggcaaaa acggtgataa aggtcatgct ggtcttgctg gtgctcgggg tgctccaggt 2040 cctgatggaa acaatggtgc tcagggacct cctggaccac agggtgttca aggtggaaaa 2100 ggtgaacagg gtccccctgg tcctccaggc ttccagggtc tgcctggccc ctcaggtccc 2160 gctggtgaag ttggcaaacc aggagaaagg ggtctccatg gtgagtttgg tctccctggt 2220 cctgctggtc caagagggga acgcggtccc ccaggtgaga gtggtgctgc cggtcctact 2280 ggtcctattg gaagccgagg tccttctgga cccccagggc ctgatggaaa caagggtgaa 2340 cctggtgtgg ttggtgctgt gggcactgct ggtccatctg gtcctagtgg actcccagga 2400 gagaggggtg ctgctggcat acctggaggc aagggagaaa agggtgaacc tggtctcaga 2460 ggtgaaattg gtaaccctgg cagagatggt gctcgtggtg ctcctggtgc tgtaggtgcc 2520 cctggtcctg ctggagccac aggtgaccgg ggcgaagctg gggctgctgg tcctgctggt 2580 cctgctggtc ctcggggaag ccctggtgaa cgtggtgagg tcggtcctgc tggccccaat 2640 ggatttgctg gtcctgctgg tgctgctggt caacctggtg ctaaaggaga aagaggagcc 2700 aaagggccta agggtgaaaa cggtgttgtt ggtcccacag gccccgttgg agctgctggc 2760 ccagctggtc caaatggtcc ccccggtcct gctggaagtc gtggtgatgg aggcccccct 2820 ggtatgactg gtttccctgg tgctgctgga cggactggtc ccccaggacc ctctggtatt 2880 tctggccctc ctggtccccc tggtcctgct gggaaagaag ggcttcgtgg tcctcgtggt 2940 gaccaaggtc cagttggccg aactggagaa gtaggtgcag ttggtccccc tggcttcgct 3000 ggtgagaagg gtccctctgg agaggctggt actgctggac ctcctggcac tccaggtcct 3060 cagggtcttc ttggtgctcc tggtattctg ggtctccctg gctcgagagg tgaacgtggt 3120 ctaccaggtg ttgctggtgc tgtgggtgaa cctggtcctc ttggcattgc cggccctcct 3180 ggggcccgtg gtcctcctgg tgctgtgggt agtcctggag tcaacggtgc tcctggtgaa 3240 gctggtcgtg atggcaaccc tgggaacgat ggtcccccag gtcgcgatgg tcaacccgga 3300 cacaagggag agcgcggtta ccctggcaat attggtcccg ttggtgctgc aggtgcacct 3360 ggtcctcatg gccccgtggg tcctgctggc aaacatggaa accgtggtga aactggtcct 3420 tctggtcctg ttggtcctgc tggtgctgtt ggcccaagag gtcctagtgg cccacaaggc 3480 attcgtggcg ataagggaga gcccggtgaa aaggggccca gaggtcttcc tggcttaaag 3540 ggacacaatg gattgcaagg tctgcctggt atcgctggtc accatggtga tcaaggtgct 3600 cctggctccg tgggtcctgc tggtcctagg ggccctgctg gtccttctgg ccctgctgga 3660 aaagatggtc gcactggaca tcctggtaca gttggacctg ctggcattcg aggccctcag 3720 ggtcaccaag gccctgctgg cccccctggt ccccctggcc ctcctggacc tccaggtgta 3780 agcggtggtg gttatgactt tggttacgat ggagacttct acagggctga ccagcctcgc 3840 tcagcacctt ctctcagacc caaggactat gaagttgatg ctactctgaa gtctctcaac 3900 aaccagattg agacccttct tactcctgaa ggctctagaa agaacccagc tcgcacatgc 3960 cgtgacttga gactcagcca cccagagtgg agcagtggtt actactggat tgaccctaac 4020 caaggatgca ctatggatgc tatcaaagta tactgtgatt tctctactgg cgaaacctgt 4080 atccgggccc aacctgaaaa catcccagcc aagaactggt ataggagctc caaggacaag 4140 aaacacgtct ggctaggaga aactatcaat gctggcagcc agtttgaata taatgtagaa 4200 ggagtgactt ccaaggaaat ggctacccaa cttgccttca tgcgcctgct ggccaactat 4260 gcctctcaga acatcaccta ccactgcaag aacagcattg catacatgga tgaggagact 4320 ggcaacctga aaaaggctgt cattctacag ggctctaatg atgttgaact tgttgctgag 4380 ggcaacagca ggttcactta cactgttctt gtagatggct gctctaaaaa gacaaatgaa 4440 tggggaaaga caatcattga atacaaaaca aataagccat cacgcctgcc cttccttgat 4500 attgcacctt tggacatcgg tggtgctgac caggaattct ttgtggacat tggcccagtc 4560 tgtttcaaat aaatgaactc aatctaaatt aaaaaagaaa gaaatttgaa aaaactttct 4620 ctttgccatt tcttcttctt cttttttaac tgaaagctga atccttccat ttcttctgca 4680 catctacttg cttaaattgt gggcaaaaga gaaaaagaag gattgatcag agcattgtgc 4740 aatacagttt cattaactcc ttcccccgct cccccaaaaa tttgaatttt tttttcaaca 4800 ctcttacacc tgttatggaa aatgtcaacc tttgtaagaa aaccaaaata aaaattgaaa 4860 aataaaaacc ataaacattt gcaccacttg tggcttttga atatcttcca cagagggaag 4920 tttaaaaccc aaacttccaa aggtttaaac tacctcaaaa cactttccca tgagtgtgat 4980 ccacattgtt aggtgctgac ctagacagag atgaactgag gtccttgttt tgttttgttc 5040 ataatacaaa ggtgctaatt aatagtattt cagatacttg aagaatgttg atggtgctag 5100 aagaatttga gaagaaatac tcctgtattg agttgtatcg tgtggtgtat tttttaaaaa 5160 atttgattta gcattcatat tttccatctt attcccaatt aaaagtatgc agattatttg 5220 cccaaatctt cttcagattc agcatttgtt ctttgccagt ctcattttca tcttcttcca 5280 tggttccaca gaagctttgt ttcttgggca agcagaaaaa ttaaattgta cctattttgt 5340 atatgtgaga tgtttaaata aattgtgaaa aaaatgaaat aaagcatgtt tggttttcca 5400 aaagaacata t 5411 <210> SEQ ID NO 3 <211> LENGTH: 9169 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000094.3 <309> DATABASE ENTRY DATE: 2010-03-23 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(9169) <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000094.3 <309> DATABASE ENTRY DATE: 2010-03-21 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(9169) <400> SEQUENCE: 3 gatgacgctg cggcttctgg tggccgcgct ctgcgccggg atcctggcag aggcgccccg 60 agtgcgagcc cagcacaggg agagagtgac ctgcacgcgc ctttacgccg ctgacattgt 120 gttcttactg gatggctcct catccattgg ccgcagcaat ttccgcgagg tccgcagctt 180 tctcgaaggg ctggtgctgc ctttctctgg agcagccagt gcacagggtg tgcgctttgc 240 cacagtgcag tacagcgatg acccacggac agagttcggc ctggatgcac ttggctctgg 300 gggtgatgtg atccgcgcca tccgtgagct tagctacaag gggggcaaca ctcgcacagg 360 ggctgcaatt ctccatgtgg ctgaccatgt cttcctgccc cagctggccc gacctggtgt 420 ccccaaggtc tgcatcctga tcacagacgg gaagtcccag gacctggtgg acacagctgc 480 ccaaaggctg aaggggcagg gggtcaagct atttgctgtg gggatcaaga atgctgaccc 540 tgaggagctg aagcgagttg cctcacagcc caccagtgac ttcttcttct tcgtcaatga 600 cttcagcatc ttgaggacac tactgcccct cgtttcccgg agagtgtgca cgactgctgg 660 tggcgtgcct gtgacccgac ctccggatga ctcgacctct gctccacgag acctggtgct 720 gtctgagcca agcagccaat ccttgagagt acagtggaca gcggccagtg gccctgtgac 780 tggctacaag gtccagtaca ctcctctgac ggggctggga cagccactgc cgagtgagcg 840 gcaggaggtg aacgtcccag ctggtgagac cagtgtgcgg ctgcggggtc tccggccact 900 gaccgagtac caagtgactg tgattgccct ctacgccaac agcatcgggg aggctgtgag 960 cgggacagct cggaccactg ccctagaagg gccggaactg accatccaga ataccacagc 1020 ccacagcctc ctggtggcct ggcggagtgt gccaggtgcc actggctacc gtgtgacatg 1080 gcgggtcctc agtggtgggc ccacacagca gcaggagctg ggccctgggc agggttcagt 1140 gttgctgcgt gacttggagc ctggcacgga ctatgaggtg accgtgagca ccctatttgg 1200 ccgcagtgtg gggcccgcca cttccctgat ggctcgcact gacgcttctg ttgagcagac 1260 cctgcgcccg gtcatcctgg gccccacatc catcctcctt tcctggaact tggtgcctga 1320 ggcccgtggc taccggttgg aatggcggcg tgagactggc ttggagccac cgcagaaggt 1380 ggtactgccc tctgatgtga cccgctacca gttggatggg ctgcagccgg gcactgagta 1440 ccgcctcaca ctctacactc tgctggaggg ccacgaggtg gccacccctg caaccgtggt 1500 tcccactgga ccagagctgc ctgtgagccc tgtaacagac ctgcaagcca ccgagctgcc 1560 cgggcagcgg gtgcgagtgt cctggagccc agtccctggt gccacccagt accgcatcat 1620 tgtgcgcagc acccaggggg ttgagcggac cctggtgctt cctgggagtc agacagcatt 1680 cgacttggat gacgttcagg ctgggcttag ctacactgtg cgggtgtctg ctcgagtggg 1740 tccccgtgag ggcagtgcca gtgtcctcac tgtccgccgg gagccggaaa ctccacttgc 1800 tgttccaggg ctgcgggttg tggtgtcaga tgcaacgcga gtgagggtgg cctggggacc 1860 cgtccctgga gccagtggat ttcggattag ctggagcaca ggcagtggtc cggagtccag 1920 ccagacactg cccccagact ctactgccac agacatcaca gggctgcagc ctggaaccac 1980 ctaccaggtg gctgtgtcgg tactgcgagg cagagaggag ggccctgctg cagtcatcgt 2040 ggctcgaacg gacccactgg gcccagtgag gacggtccat gtgactcagg ccagcagctc 2100 atctgtcacc attacctgga ccagggttcc tggcgccaca ggatacaggg tttcctggca 2160 ctcagcccac ggcccagaga aatcccagtt ggtttctggg gaggccacgg tggctgagct 2220 ggatggactg gagccagata ctgagtatac ggtgcatgtg agggcccatg tggctggcgt 2280 ggatgggccc cctgcctctg tggttgtgag gactgcccct gagcctgtgg gtcgtgtgtc 2340 gaggctgcag atcctcaatg cttccagcga cgttctacgg atcacctggg taggggtcac 2400 tggagccaca gcttacagac tggcctgggg ccggagtgaa ggcggcccca tgaggcacca 2460 gatactccca ggaaacacag actctgcaga gatccggggt ctcgaaggtg gagtcagcta 2520 ctcagtgcga gtgactgcac ttgtcgggga ccgcgagggc acacctgtct ccattgttgt 2580 cactacgccg cctgaggctc cgccagccct ggggacgctt cacgtggtgc agcgcgggga 2640 gcactcgctg aggctgcgct gggagccggt gcccagagcg cagggcttcc ttctgcactg 2700 gcaacctgag ggtggccagg aacagtcccg ggtcctgggg cccgagctca gcagctatca 2760 cctggacggg ctggagccag cgacacagta ccgcgtgagg ctgagtgtcc tagggccagc 2820 tggagaaggg ccctctgcag aggtgactgc gcgcactgag tcacctcgtg ttccaagcat 2880 tgaactacgt gtggtggaca cctcgatcga ctcggtgact ttggcctgga ctccagtgtc 2940 cagggcatcc agctacatcc tatcctggcg gccactcaga ggccctggcc aggaagtgcc 3000 tgggtccccg cagacacttc cagggatctc aagctcccag cgggtgacag ggctagagcc 3060 tggcgtctct tacatcttct ccctgacgcc tgtcctggat ggtgtgcggg gtcctgaggc 3120 atctgtcaca cagacgccag tgtgcccccg tggcctggcg gatgtggtgt tcctaccaca 3180 tgccactcaa gacaatgctc accgtgcgga ggctacgagg agggtcctgg agcgtctggt 3240 gttggcactt gggcctcttg ggccacaggc agttcaggtt ggcctgctgt cttacagtca 3300 tcggccctcc ccactgttcc cactgaatgg ctcccatgac cttggcatta tcttgcaaag 3360 gatccgtgac atgccctaca tggacccaag tgggaacaac ctgggcacag ccgtggtcac 3420 agctcacaga tacatgttgg caccagatgc tcctgggcgc cgccagcacg taccaggggt 3480 gatggttctg ctagtggatg aacccttgag aggtgacata ttcagcccca tccgtgaggc 3540 ccaggcttct gggcttaatg tggtgatgtt gggaatggct ggagcggacc cagagcagct 3600 gcgtcgcttg gcgccgggta tggactctgt ccagaccttc ttcgccgtgg atgatgggcc 3660 aagcctggac caggcagtca gtggtctggc cacagccctg tgtcaggcat ccttcactac 3720 tcagccccgg ccagagccct gcccagtgta ttgtccaaag ggccagaagg gggaacctgg 3780 agagatgggc ctgagaggac aagttgggcc tcctggcgac cctggcctcc cgggcaggac 3840 cggtgctccc ggcccccagg ggccccctgg aagtgccact gccaagggcg agaggggctt 3900 ccctggagca gatgggcgtc caggcagccc tggccgcgcc gggaatcctg ggacccctgg 3960 agcccctggc ctaaagggct ctccagggtt gcctggccct cgtggggacc cgggagagcg 4020 aggacctcga ggcccaaagg gggagccggg ggctcccgga caagtcatcg gaggtgaagg 4080 acctgggctt cctgggcgga aaggggaccc tggaccatcg ggcccccctg gacctcgtgg 4140 accactgggg gacccaggac cccgtggccc cccagggctt cctggaacag ccatgaaggg 4200 tgacaaaggc gatcgtgggg agcggggtcc ccctggacca ggtgaaggtg gcattgctcc 4260 tggggagcct gggctgccgg gtcttcccgg aagccctgga ccccaaggcc ccgttggccc 4320 ccctggaaag aaaggagaaa aaggtgactc tgaggatgga gctccaggcc tcccaggaca 4380 acctgggtct ccgggtgagc agggcccacg gggacctcct ggagctattg gccccaaagg 4440 tgaccggggc tttccagggc ccctgggtga ggctggagag aagggcgaac gtggaccccc 4500 aggcccagcg ggatcccggg ggctgccagg ggttgctgga cgtcctggag ccaagggtcc 4560 tgaagggcca ccaggaccca ctggccgcca aggagagaag ggggagcctg gtcgccctgg 4620 ggaccctgca gtggtgggac ctgctgttgc tggacccaaa ggagaaaagg gagatgtggg 4680 gcccgctggg cccagaggag ctaccggagt ccaaggggaa cggggcccac ccggcttggt 4740 tcttcctgga gaccctggcc ccaagggaga ccctggagac cggggtccca ttggccttac 4800 tggcagagca ggacccccag gtgactcagg gcctcctgga gagaagggag accctgggcg 4860 gcctggcccc ccaggacctg ttggcccccg aggacgagat ggtgaagttg gagagaaagg 4920 tgacgagggt cctccgggtg acccgggttt gcctggaaaa gcaggcgagc gtggccttcg 4980 gggggcacct ggagttcggg ggcctgtggg tgaaaaggga gaccagggag atcctggaga 5040 ggatggacga aatggcagcc ctggatcatc tggacccaag ggtgaccgtg gggagccggg 5100 tcccccagga cccccgggac ggctggtaga cacaggacct ggagccagag agaagggaga 5160 gcctggggac cgcggacaag agggtcctcg agggcccaag ggtgatcctg gcctccctgg 5220 agcccctggg gaaaggggca ttgaagggtt tcggggaccc ccaggcccac agggggaccc 5280 aggtgtccga ggcccagcag gagaaaaggg tgaccggggt ccccctgggc tggatggccg 5340 gagcggactg gatgggaaac caggagccgc tgggccctct gggccgaatg gtgctgcagg 5400 caaagctggg gacccaggga gagacgggct tccaggcctc cgtggagaac agggcctccc 5460 tggcccctct ggtccccctg gattaccggg aaagccaggc gaggatggca aacctggcct 5520 gaatggaaaa aacggagaac ctggggaccc tggagaagac gggaggaagg gagagaaagg 5580 agattcaggc gcctctggga gagaaggtcg tgatggcccc aagggtgagc gtggagctcc 5640 tggtatcctt ggaccccagg ggcctccagg cctcccaggg ccagtgggcc ctcctggcca 5700 gggttttcct ggtgtcccag gaggcacggg ccccaagggt gaccgtgggg agactggatc 5760 caaaggggag cagggcctcc ctggagagcg tggcctgcga ggagagcctg gaagtgtgcc 5820 gaatgtggat cggttgctgg aaactgctgg catcaaggca tctgccctgc gggagatcgt 5880 ggagacctgg gatgagagct ctggtagctt cctgcctgtg cccgaacggc gtcgaggccc 5940 caagggggac tcaggcgaac agggcccccc aggcaaggag ggccccatcg gctttcctgg 6000 agaacgcggg ctgaagggcg accgtggaga ccctggccct caggggccac ctggtctggc 6060 ccttggggag aggggccccc ccgggccttc cggccttgcc ggggagcctg gaaagcctgg 6120 tattcccggg ctcccaggca gggctggggg tgtgggagag gcaggaaggc caggagagag 6180 gggagaacgg ggagagaaag gagaacgtgg agaacagggc agagatggcc ctcctggact 6240 ccctggaacc cctgggcccc ccggaccccc tggccccaag gtgtctgtgg atgagccagg 6300 tcctggactc tctggagaac agggaccccc tggactcaag ggtgctaagg gggagccggg 6360 cagcaatggt gaccaaggtc ccaaaggaga caggggtgtg ccaggcatca aaggagaccg 6420 gggagagcct ggaccgaggg gtcaggacgg caacccgggt ctaccaggag agcgtggtat 6480 ggctgggcct gaagggaagc cgggtctgca gggtccaaga ggcccccctg gcccagtggg 6540 tggtcatgga gaccctggac cacctggtgc cccgggtctt gctggccctg caggacccca 6600 aggaccttct ggcctgaagg gggagcctgg agagacagga cctccaggac ggggcctgac 6660 tggacctact ggagctgtgg gacttcctgg accccccggc ccttcaggcc ttgtgggtcc 6720 acaggggtct ccaggtttgc ctggacaagt gggggagaca gggaagccgg gagccccagg 6780 tcgagatggt gccagtggaa aagatggaga cagagggagc cctggtgtgc cagggtcacc 6840 aggtctgcct ggccctgtcg gacctaaagg agaacctggc cccacggggg cccctggaca 6900 ggctgtggtc gggctccctg gagcaaaggg agagaaggga gcccctggag gccttgctgg 6960 agacctggtg ggtgagccgg gagccaaagg tgaccgagga ctgccagggc cgcgaggcga 7020 gaagggtgaa gctggccgtg caggggagcc cggagaccct ggggaagatg gtcagaaagg 7080 ggctccagga cccaaaggtt tcaagggtga cccaggagtc ggggtcccgg gctcccctgg 7140 gcctcctggc cctccaggtg tgaagggaga tctgggcctc cctggcctgc ccggtgctcc 7200 tggtgttgtt gggttcccgg gtcagacagg ccctcgagga gagatgggtc agccaggccc 7260 tagtggagag cggggtctgg caggcccccc agggagagaa ggaatcccag gacccctggg 7320 gccacctgga ccaccggggt cagtgggacc acctggggcc tctggactca aaggagacaa 7380 gggagaccct ggagtagggc tgcctgggcc ccgaggcgag cgtggggagc caggcatccg 7440 gggtgaagat ggccgccccg gccaggaggg accccgagga ctcacggggc cccctggcag 7500 caggggagag cgtggggaga agggtgatgt tgggagtgca ggactaaagg gtgacaaggg 7560 agactcagct gtgatcctgg ggcctccagg cccacggggt gccaaggggg acatgggtga 7620 acgagggcct cggggcttgg atggtgacaa aggacctcgg ggagacaatg gggaccctgg 7680 tgacaagggc agcaagggag agcctggtga caagggctca gccgggttgc caggactgcg 7740 tggactcctg ggaccccagg gtcaacctgg tgcagcaggg atccctggtg acccgggatc 7800 cccaggaaag gatggagtgc ctggtatccg aggagaaaaa ggagatgttg gcttcatggg 7860 tccccggggc ctcaagggtg aacggggagt gaagggagcc tgtggccttg atggagagaa 7920 gggagacaag ggagaagctg gtcccccagg ccgccccggg ctggcaggac acaaaggaga 7980 gatgggggag cctggtgtgc cgggccagtc gggggcccct ggcaaggagg gcctgatcgg 8040 tcccaagggt gaccgaggct ttgacgggca gccaggcccc aagggtgacc agggcgagaa 8100 aggggagcgg ggaaccccag gaattggggg cttcccaggc cccagtggaa atgatggctc 8160 tgctggtccc ccagggccac ctggcagtgt tggtcccaga ggccccgaag gacttcaggg 8220 ccagaagggt gagcgaggtc cccccggaga gagagtggtg ggggctcctg gggtccctgg 8280 agctcctggc gagagagggg agcaggggcg gccagggcct gccggtcctc gaggcgagaa 8340 gggagaagct gcactgacgg aggatgacat ccggggcttt gtgcgccaag agatgagtca 8400 gcactgtgcc tgccagggcc agttcatcgc atctggatca cgacccctcc ctagttatgc 8460 tgcagacact gccggctccc agctccatgc tgtgcctgtg ctccgcgtct ctcatgcaga 8520 ggaggaagag cgggtacccc ctgaggatga tgagtactct gaatactccg agtattctgt 8580 ggaggagtac caggaccctg aagctccttg ggatagtgat gacccctgtt ccctgccact 8640 ggatgagggc tcctgcactg cctacaccct gcgctggtac catcgggctg tgacaggcag 8700 cacagaggcc tgtcaccctt ttgtctatgg tggctgtgga gggaatgcca accgttttgg 8760 gacccgtgag gcctgcgagc gccgctgccc accccgggtg gtccagagcc aggggacagg 8820 tactgcccag gactgaggcc cagataatga gctgagattc agcatcccct ggaggagtcg 8880 gggtctcagc agaaccccac tgtccctccc cttggtgcta gaggcttgtg tgcacgtgag 8940 cgtgcgtgtg cacgtccgtt atttcagtga cttggtcccg tgggtctagc cttcccccct 9000 gtggacaaac ccccattgtg gctcctgcca ccctggcaga tgactcactg tgggggggtg 9060 gctgtgggca gtgagcggat gtgactggcg tctgacccgc cccttgaccc aagcctgtga 9120 tgacatggtg ctgattctgg ggggcattaa agctgctgtt ttaaaaggc 9169 <210> SEQ ID NO 4 <211> LENGTH: 387 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 ggccattacg gccgggcagc caggccccag ctgagaataa tgcactggat gggggtggtt 60 gttgtgttag gggaccccag ttctcactgc tgctctctgg tagtcagaag tgaggggtca 120 gccccatctt tcctgctgga gtgagctccc agctgggtag ctggcagcct ttctgtcata 180 tccctccctg ctcctgatgt ctggaggcag tgtagttctt cttaaaagtt ggcttggcag 240 ggcgcggtgg ctcacacctg taatcccaac attttgggag gctgaggcag tcggatcacc 300 tgaggtcgag agtttgagac cccagcctat ccaacatggt gaaactccat ctctactaaa 360 aatacaaaaa ttagcccggc atggtgg 387 <210> SEQ ID NO 5 <211> LENGTH: 561 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 attttggtgg gaggccctgg acctagaaac agaaaatgaa agtggagaaa accaaacaag 60 aatctttctt tcttaaagta aacatcggac tgacatctcc tttcaaacat tcattgagga 120 tctactctgt gagaagaagc aataccatgt tatctcattc tagttccagt tctagttgac 180 ttgattgtca aacgactgct aagaagtaga gaggaggctt caccatgttt gccaggctag 240 tctggaactc ttgacctcag gtgatccacc cacctcggcc tcccaaagtg ctaggattac 300 aggcatgagc cacctcgccc agctgtttct gtgtcacgtt ttgatacttg aataaaaatg 360 acactttgga gccttgaaaa atggactttc cttctgaagt acccaggaga gtaggatgga 420 cttcttcctc tctccctaca ctatcccttg gagaaaacct tttatcaaga agataccgaa 480 tatcatcact gatataatgt cagaggaggt cactctctat agcccaacct ccaggatttc 540 agcacttcgt ggacatggag g 561 <210> SEQ ID NO 6 <211> LENGTH: 335 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 gggaccagag agctgcgata tggctgaaaa aagtgagtgc aagacaagga cataggatga 60 aaggggccag tgatggtcag ggacactgga ggacaggagt ctgtgggatc ttagcatgta 120 ggatgacagg agtcagtgga ctgcagtgag aactgatgag ccattgacag acactgatgt 180 gggatggcag aggtcagtgc tggatgggga catgcaatga gaggttggtg tacagtggtc 240 accactaggg taacagaaag acaggtgatt gagcttgtgg ctgtgcagag gtgaaagccg 300 aggatggtgt gtgggcagat aggagattgg ttgtt 335 <210> SEQ ID NO 7 <211> LENGTH: 613 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 tttttttttt ttttttttaa gagaagatgt ctcgctccgt cgtccaggct ggagcgcagt 60 ggcgcaatca tggatcactg cagccttgac cttcccgggc tcaagcgatc ctcccggctc 120 acccccagta gctggaacca caggcgcgct tccacaccgg aaagcccatt ttctagaggc 180 ggaaaccgaa gcgcccagtg ggaaaggcga cccgccgggg atgcggggtg ctcaacgcgc 240 tgccacctgg ggcccaacgc gttgacctcg cggtcaggtt gcttccgcgg actacggttc 300 tggctcgcta gctctggaac aggcaggaag gagtggggct atctgatagg ggaagatgat 360 gggagtctaa caggagacgg gaatttgata gaggagatgg tgaataggtc cagtggagga 420 gtggggaggt ggaggtttta ggaagcagtg agcaggtctg atggaggaga aagtgtaaat 480 ctcagaggag atttgggtcc agcaaaaaag ggatggggta gggtaggggc agacacaccc 540 tgttgacagt tcagggctca gtgccatctt gggaagcaga tggataacga gacagggagg 600 agactatagg gac 613 <210> SEQ ID NO 8 <211> LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 tcccaccggg tccaggtcag gcccaagggg accagctctg ctcccaccat catcccactc 60 cccacacagc caagctggac atctgagagc actgcatggc aatcctcatc tgcctgtgtg 120 tctccctcca ctggggacac atgtcatgtg tcagtcctgc agcacatgtg tccttct 177 <210> SEQ ID NO 9 <211> LENGTH: 285 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 gaactcagac atgcgaccaa gaattggctc acaggagctc agacatgacc atggcctatc 60 tcaggacagc acagacagag ggacgctcag atactaccat agacaggtgg gagttcaggc 120 atggcacagg cacagggagc ccacacgcga gtgcagacat ctggctccac agacgtgagt 180 gcggacacac gggcgctcag aggggaaccc caacacgtcc actcccgggt agagcaggca 240 tggccacagg cttgaacgct gggagccaga ggcagggaca agcag 285 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 10 acatcaggag cagggaggga 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 11 ccctaactca acaacatccc 20 <210> SEQ ID NO 12 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 12 tctcaaactc tcgacctcag g 21 <210> SEQ ID NO 13 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 13 gtttcaccat gttggctagg c 21 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 14 gcctcctctc tacttcttag 20 <210> SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 15 gggatagtgt agggagagag g 21 <210> SEQ ID NO 16 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 16 cctactctcc tgggtacttc a 21 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 17 ggtgtctgtc ttccttaggg 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 18 aattagctgg gcgtggtggc 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 19 aatcccagct gctcaggagg 20 <210> SEQ ID NO 20 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 20 rgrcrurcra rarurcrarc rcrurgrurc rurururcru rgrururarc rcrc 54 <210> SEQ ID NO 21 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 21 rcrurgrcrc rarurcrcrc rarcrarurc rargrurgru rcrurgrurc rara 54 <210> SEQ ID NO 22 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 22 rcrurcrcru rcrcrarurc rargrarcrc rurgrcrurc rarcrurgrc ruru 54 <210> SEQ ID NO 23 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 23 rgrurcrcrc rurarurarg rurcrurcrc rurcrcrcru rgrurcrurc rgru 54 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 24 ggagtgggat gatggtggga 20 <210> SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 25 gcagtgctct cagatgtcca g 21 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 26 tggagggaga cacacaggca 20 <210> SEQ ID NO 27 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 27 tccctctgtc tgtgctgtcc t 21 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 28 gcctgaactc ccacctgtct 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 29 ctctggctcc cagcgttcaa 20 <210> SEQ ID NO 30 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 20 <400> SEQUENCE: 30 rgrurararc rargrarara rgrarcrarg rgrurgraru rurgragc 48 <210> SEQ ID NO 31 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 21 <400> SEQUENCE: 31 rgrarcrarg rarcrarcru rgrarurgru rgrgrgraru rgrgrcag 48 <210> SEQ ID NO 32 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 22 <400> SEQUENCE: 32 rgrcrargru rgrargrcra rgrgrurcru rgrarurgrg rargrgag 48 <210> SEQ ID NO 33 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 23 <400> SEQUENCE: 33 rgrargrarc rargrgrgra rgrgrargra rcrurarura rgrgrgac 48

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210> SEQ ID NO 1 <211> LENGTH: 5927 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000088.3 <309> DATABASE ENTRY DATE: 2010-06-06 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(5927) <400> SEQUENCE: 1 tcgtcggagc agacgggagt ttctcctcgg ggtcggagca ggaggcacgc ggagtgtgag 60 gccacgcatg agcggacgct aaccccctcc ccagccacaa agagtctaca tgtctagggt 120 ctagacatgt tcagctttgt ggacctccgg ctcctgctcc tcttagcggc caccgccctc 180 ctgacgcacg gccaagagga aggccaagtc gagggccaag acgaagacat cccaccaatc 240 acctgcgtac agaacggcct caggtaccat gaccgagacg tgtggaaacc cgagccctgc 300 cggatctgcg tctgcgacaa cggcaaggtg ttgtgcgatg acgtgatctg tgacgagacc 360 aagaactgcc ccggcgccga agtccccgag ggcgagtgct gtcccgtctg ccccgacggc 420 tcagagtcac ccaccgacca agaaaccacc ggcgtcgagg gacccaaggg agacactggc 480 ccccgaggcc caaggggacc cgcaggcccc cctggccgag atggcatccc tggacagcct 540 ggacttcccg gaccccccgg accccccgga cctcccggac cccctggcct cggaggaaac 600 tttgctcccc agctgtctta tggctatgat gagaaatcaa ccggaggaat ttccgtgcct 660 ggccccatgg gtccctctgg tcctcgtggt ctccctggcc cccctggtgc acctggtccc 720 caaggcttcc aaggtccccc tggtgagcct ggcgagcctg gagcttcagg tcccatgggt 780 ccccgaggtc ccccaggtcc ccctggaaag aatggagatg atggggaagc tggaaaacct 840 ggtcgtcctg gtgagcgtgg gcctcctggg cctcagggtg ctcgaggatt gcccggaaca 900 gctggcctcc ctggaatgaa gggacacaga ggtttcagtg gtttggatgg tgccaaggga 960 gatgctggtc ctgctggtcc taagggtgag cctggcagcc ctggtgaaaa tggagctcct 1020 ggtcagatgg gcccccgtgg cctgcctggt gagagaggtc gccctggagc ccctggccct 1080 gctggtgctc gtggaaatga tggtgctact ggtgctgccg ggccccctgg tcccaccggc 1140 cccgctggtc ctcctggctt ccctggtgct gttggtgcta agggtgaagc tggtccccaa 1200 gggccccgag gctctgaagg tccccagggt gtgcgtggtg agcctggccc ccctggccct 1260 gctggtgctg ctggccctgc tggaaaccct ggtgctgatg gacagcctgg tgctaaaggt 1320 gccaatggtg ctcctggtat tgctggtgct cctggcttcc ctggtgcccg aggcccctct 1380 ggaccccagg gccccggcgg ccctcctggt cccaagggta acagcggtga acctggtgct 1440 cctggcagca aaggagacac tggtgctaag ggagagcctg gccctgttgg tgttcaagga 1500 ccccctggcc ctgctggaga ggaaggaaag cgaggagctc gaggtgaacc cggacccact 1560 ggcctgcccg gaccccctgg cgagcgtggt ggacctggta gccgtggttt ccctggcgca 1620 gatggtgttg ctggtcccaa gggtcccgct ggtgaacgtg gttctcctgg ccctgctggc 1680 cccaaaggat ctcctggtga agctggtcgt cccggtgaag ctggtctgcc tggtgccaag 1740 ggtctgactg gaagccctgg cagccctggt cctgatggca aaactggccc ccctggtccc 1800 gccggtcaag atggtcgccc cggaccccca ggcccacctg gtgcccgtgg tcaggctggt 1860 gtgatgggat tccctggacc taaaggtgct gctggagagc ccggcaaggc tggagagcga 1920 ggtgttcccg gaccccctgg cgctgtcggt cctgctggca aagatggaga ggctggagct 1980 cagggacccc ctggccctgc tggtcccgct ggcgagagag gtgaacaagg ccctgctggc 2040 tcccccggat tccagggtct ccctggtcct gctggtcctc caggtgaagc aggcaaacct 2100 ggtgaacagg gtgttcctgg agaccttggc gcccctggcc cctctggagc aagaggcgag 2160 agaggtttcc ctggcgagcg tggtgtgcaa ggtccccctg gtcctgctgg tccccgaggg 2220 gccaacggtg ctcccggcaa cgatggtgct aagggtgatg ctggtgcccc tggagctccc 2280 ggtagccagg gcgcccctgg ccttcaggga atgcctggtg aacgtggtgc agctggtctt 2340 ccagggccta agggtgacag aggtgatgct ggtcccaaag gtgctgatgg ctctcctggc 2400 aaagatggcg tccgtggtct gactggcccc attggtcctc ctggccctgc tggtgcccct 2460 ggtgacaagg gtgaaagtgg tcccagcggc cctgctggtc ccactggagc tcgtggtgcc 2520 cccggagacc gtggtgagcc tggtcccccc ggccctgctg gctttgctgg cccccctggt 2580 gctgacggcc aacctggtgc taaaggcgaa cctggtgatg ctggtgctaa aggcgatgct 2640 ggtccccctg gccctgccgg acccgctgga ccccctggcc ccattggtaa tgttggtgct 2700 cctggagcca aaggtgctcg cggcagcgct ggtccccctg gtgctactgg tttccctggt 2760 gctgctggcc gagtcggtcc tcctggcccc tctggaaatg ctggaccccc tggccctcct 2820 ggtcctgctg gcaaagaagg cggcaaaggt ccccgtggtg agactggccc tgctggacgt 2880 cctggtgaag ttggtccccc tggtccccct ggccctgctg gcgagaaagg atcccctggt 2940 gctgatggtc ctgctggtgc tcctggtact cccgggcctc aaggtattgc tggacagcgt 3000 ggtgtggtcg gcctgcctgg tcagagagga gagagaggct tccctggtct tcctggcccc 3060 tctggtgaac ctggcaaaca aggtccctct ggagcaagtg gtgaacgtgg tccccctggt 3120 cccatgggcc cccctggatt ggctggaccc cctggtgaat ctggacgtga gggggctcct 3180 ggtgccgaag gttcccctgg acgagacggt tctcctggcg ccaagggtga ccgtggtgag 3240 accggccccg ctggaccccc tggtgctcct ggtgctcctg gtgcccctgg ccccgttggc 3300 cctgctggca agagtggtga tcgtggtgag actggtcctg ctggtcccgc cggtcctgtc 3360 ggccctgttg gcgcccgtgg ccccgccgga ccccaaggcc cccgtggtga caagggtgag 3420 acaggcgaac agggcgacag aggcataaag ggtcaccgtg gcttctctgg cctccagggt 3480 ccccctggcc ctcctggctc tcctggtgaa caaggtccct ctggagcctc tggtcctgct 3540 ggtccccgag gtccccctgg ctctgctggt gctcctggca aagatggact caacggtctc 3600 cctggcccca ttgggccccc tggtcctcgc ggtcgcactg gtgatgctgg tcctgttggt 3660 ccccccggcc ctcctggacc tcctggtccc cctggtcctc ccagcgctgg tttcgacttc 3720 agcttcctgc cccagccacc tcaagagaag gctcacgatg gtggccgcta ctaccgggct 3780 gatgatgcca atgtggttcg tgaccgtgac ctcgaggtgg acaccaccct caagagcctg 3840 agccagcaga tcgagaacat ccggagccca gagggcagcc gcaagaaccc cgcccgcacc 3900 tgccgtgacc tcaagatgtg ccactctgac tggaagagtg gagagtactg gattgacccc 3960 aaccaaggct gcaacctgga tgccatcaaa gtcttctgca acatggagac tggtgagacc 4020 tgcgtgtacc ccactcagcc cagtgtggcc cagaagaact ggtacatcag caagaacccc 4080 aaggacaaga ggcatgtctg gttcggcgag agcatgaccg atggattcca gttcgagtat 4140 ggcggccagg gctccgaccc tgccgatgtg gccatccagc tgaccttcct gcgcctgatg 4200 tccaccgagg cctcccagaa catcacctac cactgcaaga acagcgtggc ctacatggac 4260 cagcagactg gcaacctcaa gaaggccctg ctcctccagg gctccaacga gatcgagatc 4320 cgcgccgagg gcaacagccg cttcacctac agcgtcactg tcgatggctg cacgagtcac 4380 accggagcct ggggcaagac agtgattgaa tacaaaacca ccaagacctc ccgcctgccc 4440 atcatcgatg tggccccctt ggacgttggt gccccagacc aggaattcgg cttcgacgtt 4500 ggccctgtct gcttcctgta aactccctcc atcccaacct ggctccctcc cacccaacca 4560 actttccccc caacccggaa acagacaagc aacccaaact gaaccccctc aaaagccaaa 4620 aaatgggaga caatttcaca tggactttgg aaaatatttt tttcctttgc attcatctct 4680 caaacttagt ttttatcttt gaccaaccga acatgaccaa aaaccaaaag tgcattcaac 4740 cttaccaaaa aaaaaaaaaa aaaaagaata aataaataac tttttaaaaa aggaagcttg 4800 gtccacttgc ttgaagaccc atgcgggggt aagtcccttt ctgcccgttg ggcttatgaa 4860 accccaatgc tgccctttct gctcctttct ccacaccccc cttggggcct cccctccact 4920 ccttcccaaa tctgtctccc cagaagacac aggaaacaat gtattgtctg cccagcaatc 4980 aaaggcaatg ctcaaacacc caagtggccc ccaccctcag cccgctcctg cccgcccagc 5040 acccccaggc cctgggggac ctggggttct cagactgcca aagaagcctt gccatctggc 5100 gctcccatgg ctcttgcaac atctcccctt cgtttttgag ggggtcatgc cgggggagcc 5160 accagcccct cactgggttc ggaggagagt caggaagggc cacgacaaag cagaaacatc 5220 ggatttgggg aacgcgtgtc aatcccttgt gccgcagggc tgggcgggag agactgttct 5280 gttccttgtg taactgtgtt gctgaaagac tacctcgttc ttgtcttgat gtgtcaccgg 5340 ggcaactgcc tgggggcggg gatgggggca gggtggaagc ggctccccat tttataccaa 5400 aggtgctaca tctatgtgat gggtggggtg gggagggaat cactggtgct atagaaattg 5460 agatgccccc ccaggccagc aaatgttcct ttttgttcaa agtctatttt tattccttga 5520 tatttttctt tttttttttt tttttttgtg gatggggact tgtgaatttt tctaaaggtg 5580 ctatttaaca tgggaggaga gcgtgtgcgg ctccagccca gcccgctgct cactttccac 5640 cctctctcca cctgcctctg gcttctcagg cctctgctct ccgacctctc tcctctgaaa 5700 ccctcctcca cagctgcagc ccatcctccc ggctccctcc tagtctgtcc tgcgtcctct 5760 gtccccgggt ttcagagaca acttcccaaa gcacaaagca gtttttcccc ctaggggtgg 5820 gaggaagcaa aagactctgt acctattttg tatgtgtata ataatttgag atgtttttaa 5880 ttattttgat tgctggaata aagcatgtgg aaatgaccca aacataa 5927 <210> SEQ ID NO 2 <211> LENGTH: 5411 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000089.3 <309> DATABASE ENTRY DATE: 2010-05-23 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(5411) <400> SEQUENCE: 2 gtgtcccata gtgtttccaa acttggaaag ggcgggggag ggcgggagga tgcggagggc 60 ggaggtatgc agacaacgag tcagagtttc cccttgaaag cctcaaaagt gtccacgtcc 120 tcaaaaagaa tggaaccaat ttaagaagcc agccccgtgg ccacgtccct tcccccattc 180 gctccctcct ctgcgccccc gcaggctcct cccagctgtg gctgcccggg cccccagccc 240 cagccctccc attggtggag gcccttttgg aggcacccta gggccaggga aacttttgcc 300 gtataaatag ggcagatccg ggctttatta ttttagcacc acggcagcag gaggtttcgg 360 ctaagttgga ggtactggcc acgactgcat gcccgcgccc gccaggtgat acctccgccg 420 gtgacccagg ggctctgcga cacaaggagt ctgcatgtct aagtgctaga catgctcagc 480 tttgtggata cgcggacttt gttgctgctt gcagtaacct tatgcctagc aacatgccaa 540 tctttacaag aggaaactgt aagaaagggc ccagccggag atagaggacc acgtggagaa 600 aggggtccac caggcccccc aggcagagat ggtgaagatg gtcccacagg ccctcctggt 660 ccacctggtc ctcctggccc ccctggtctc ggtgggaact ttgctgctca gtatgatgga 720

aaaggagttg gacttggccc tggaccaatg ggcttaatgg gacctagagg cccacctggt 780 gcagctggag ccccaggccc tcaaggtttc caaggacctg ctggtgagcc tggtgaacct 840 ggtcaaactg gtcctgcagg tgctcgtggt ccagctggcc ctcctggcaa ggctggtgaa 900 gatggtcacc ctggaaaacc cggacgacct ggtgagagag gagttgttgg accacagggt 960 gctcgtggtt tccctggaac tcctggactt cctggcttca aaggcattag gggacacaat 1020 ggtctggatg gattgaaggg acagcccggt gctcctggtg tgaagggtga acctggtgcc 1080 cctggtgaaa atggaactcc aggtcaaaca ggagcccgtg ggcttcctgg tgagagagga 1140 cgtgttggtg cccctggccc agctggtgcc cgtggcagtg atggaagtgt gggtcccgtg 1200 ggtcctgctg gtcccattgg gtctgctggc cctccaggct tcccaggtgc ccctggcccc 1260 aagggtgaaa ttggagctgt tggtaacgct ggtcctgctg gtcccgccgg tccccgtggt 1320 gaagtgggtc ttccaggcct ctccggcccc gttggacctc ctggtaatcc tggagcaaac 1380 ggccttactg gtgccaaggg tgctgctggc cttcccggcg ttgctggggc tcccggcctc 1440 cctggacccc gcggtattcc tggccctgtt ggtgctgccg gtgctactgg tgccagagga 1500 cttgttggtg agcctggtcc agctggctcc aaaggagaga gcggtaacaa gggtgagccc 1560 ggctctgctg ggccccaagg tcctcctggt cccagtggtg aagaaggaaa gagaggccct 1620 aatggggaag ctggatctgc cggccctcca ggacctcctg ggctgagagg tagtcctggt 1680 tctcgtggtc ttcctggagc tgatggcaga gctggcgtca tgggccctcc tggtagtcgt 1740 ggtgcaagtg gccctgctgg agtccgagga cctaatggag atgctggtcg ccctggggag 1800 cctggtctca tgggacccag aggtcttcct ggttcccctg gaaatatcgg ccccgctgga 1860 aaagaaggtc ctgtcggcct ccctggcatc gacggcaggc ctggcccaat tggcccagct 1920 ggagcaagag gagagcctgg caacattgga ttccctggac ccaaaggccc cactggtgat 1980 cctggcaaaa acggtgataa aggtcatgct ggtcttgctg gtgctcgggg tgctccaggt 2040 cctgatggaa acaatggtgc tcagggacct cctggaccac agggtgttca aggtggaaaa 2100 ggtgaacagg gtccccctgg tcctccaggc ttccagggtc tgcctggccc ctcaggtccc 2160 gctggtgaag ttggcaaacc aggagaaagg ggtctccatg gtgagtttgg tctccctggt 2220 cctgctggtc caagagggga acgcggtccc ccaggtgaga gtggtgctgc cggtcctact 2280 ggtcctattg gaagccgagg tccttctgga cccccagggc ctgatggaaa caagggtgaa 2340 cctggtgtgg ttggtgctgt gggcactgct ggtccatctg gtcctagtgg actcccagga 2400 gagaggggtg ctgctggcat acctggaggc aagggagaaa agggtgaacc tggtctcaga 2460 ggtgaaattg gtaaccctgg cagagatggt gctcgtggtg ctcctggtgc tgtaggtgcc 2520 cctggtcctg ctggagccac aggtgaccgg ggcgaagctg gggctgctgg tcctgctggt 2580 cctgctggtc ctcggggaag ccctggtgaa cgtggtgagg tcggtcctgc tggccccaat 2640 ggatttgctg gtcctgctgg tgctgctggt caacctggtg ctaaaggaga aagaggagcc 2700 aaagggccta agggtgaaaa cggtgttgtt ggtcccacag gccccgttgg agctgctggc 2760 ccagctggtc caaatggtcc ccccggtcct gctggaagtc gtggtgatgg aggcccccct 2820 ggtatgactg gtttccctgg tgctgctgga cggactggtc ccccaggacc ctctggtatt 2880 tctggccctc ctggtccccc tggtcctgct gggaaagaag ggcttcgtgg tcctcgtggt 2940 gaccaaggtc cagttggccg aactggagaa gtaggtgcag ttggtccccc tggcttcgct 3000 ggtgagaagg gtccctctgg agaggctggt actgctggac ctcctggcac tccaggtcct 3060 cagggtcttc ttggtgctcc tggtattctg ggtctccctg gctcgagagg tgaacgtggt 3120 ctaccaggtg ttgctggtgc tgtgggtgaa cctggtcctc ttggcattgc cggccctcct 3180 ggggcccgtg gtcctcctgg tgctgtgggt agtcctggag tcaacggtgc tcctggtgaa 3240 gctggtcgtg atggcaaccc tgggaacgat ggtcccccag gtcgcgatgg tcaacccgga 3300 cacaagggag agcgcggtta ccctggcaat attggtcccg ttggtgctgc aggtgcacct 3360 ggtcctcatg gccccgtggg tcctgctggc aaacatggaa accgtggtga aactggtcct 3420 tctggtcctg ttggtcctgc tggtgctgtt ggcccaagag gtcctagtgg cccacaaggc 3480 attcgtggcg ataagggaga gcccggtgaa aaggggccca gaggtcttcc tggcttaaag 3540 ggacacaatg gattgcaagg tctgcctggt atcgctggtc accatggtga tcaaggtgct 3600 cctggctccg tgggtcctgc tggtcctagg ggccctgctg gtccttctgg ccctgctgga 3660 aaagatggtc gcactggaca tcctggtaca gttggacctg ctggcattcg aggccctcag 3720 ggtcaccaag gccctgctgg cccccctggt ccccctggcc ctcctggacc tccaggtgta 3780 agcggtggtg gttatgactt tggttacgat ggagacttct acagggctga ccagcctcgc 3840 tcagcacctt ctctcagacc caaggactat gaagttgatg ctactctgaa gtctctcaac 3900 aaccagattg agacccttct tactcctgaa ggctctagaa agaacccagc tcgcacatgc 3960 cgtgacttga gactcagcca cccagagtgg agcagtggtt actactggat tgaccctaac 4020 caaggatgca ctatggatgc tatcaaagta tactgtgatt tctctactgg cgaaacctgt 4080 atccgggccc aacctgaaaa catcccagcc aagaactggt ataggagctc caaggacaag 4140 aaacacgtct ggctaggaga aactatcaat gctggcagcc agtttgaata taatgtagaa 4200 ggagtgactt ccaaggaaat ggctacccaa cttgccttca tgcgcctgct ggccaactat 4260 gcctctcaga acatcaccta ccactgcaag aacagcattg catacatgga tgaggagact 4320 ggcaacctga aaaaggctgt cattctacag ggctctaatg atgttgaact tgttgctgag 4380 ggcaacagca ggttcactta cactgttctt gtagatggct gctctaaaaa gacaaatgaa 4440 tggggaaaga caatcattga atacaaaaca aataagccat cacgcctgcc cttccttgat 4500 attgcacctt tggacatcgg tggtgctgac caggaattct ttgtggacat tggcccagtc 4560 tgtttcaaat aaatgaactc aatctaaatt aaaaaagaaa gaaatttgaa aaaactttct 4620 ctttgccatt tcttcttctt cttttttaac tgaaagctga atccttccat ttcttctgca 4680 catctacttg cttaaattgt gggcaaaaga gaaaaagaag gattgatcag agcattgtgc 4740 aatacagttt cattaactcc ttcccccgct cccccaaaaa tttgaatttt tttttcaaca 4800 ctcttacacc tgttatggaa aatgtcaacc tttgtaagaa aaccaaaata aaaattgaaa 4860 aataaaaacc ataaacattt gcaccacttg tggcttttga atatcttcca cagagggaag 4920 tttaaaaccc aaacttccaa aggtttaaac tacctcaaaa cactttccca tgagtgtgat 4980 ccacattgtt aggtgctgac ctagacagag atgaactgag gtccttgttt tgttttgttc 5040 ataatacaaa ggtgctaatt aatagtattt cagatacttg aagaatgttg atggtgctag 5100 aagaatttga gaagaaatac tcctgtattg agttgtatcg tgtggtgtat tttttaaaaa 5160 atttgattta gcattcatat tttccatctt attcccaatt aaaagtatgc agattatttg 5220 cccaaatctt cttcagattc agcatttgtt ctttgccagt ctcattttca tcttcttcca 5280 tggttccaca gaagctttgt ttcttgggca agcagaaaaa ttaaattgta cctattttgt 5340 atatgtgaga tgtttaaata aattgtgaaa aaaatgaaat aaagcatgtt tggttttcca 5400 aaagaacata t 5411 <210> SEQ ID NO 3 <211> LENGTH: 9169 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000094.3 <309> DATABASE ENTRY DATE: 2010-03-23 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(9169) <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_000094.3 <309> DATABASE ENTRY DATE: 2010-03-21 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(9169) <400> SEQUENCE: 3 gatgacgctg cggcttctgg tggccgcgct ctgcgccggg atcctggcag aggcgccccg 60 agtgcgagcc cagcacaggg agagagtgac ctgcacgcgc ctttacgccg ctgacattgt 120 gttcttactg gatggctcct catccattgg ccgcagcaat ttccgcgagg tccgcagctt 180 tctcgaaggg ctggtgctgc ctttctctgg agcagccagt gcacagggtg tgcgctttgc 240 cacagtgcag tacagcgatg acccacggac agagttcggc ctggatgcac ttggctctgg 300 gggtgatgtg atccgcgcca tccgtgagct tagctacaag gggggcaaca ctcgcacagg 360 ggctgcaatt ctccatgtgg ctgaccatgt cttcctgccc cagctggccc gacctggtgt 420 ccccaaggtc tgcatcctga tcacagacgg gaagtcccag gacctggtgg acacagctgc 480 ccaaaggctg aaggggcagg gggtcaagct atttgctgtg gggatcaaga atgctgaccc 540 tgaggagctg aagcgagttg cctcacagcc caccagtgac ttcttcttct tcgtcaatga 600 cttcagcatc ttgaggacac tactgcccct cgtttcccgg agagtgtgca cgactgctgg 660 tggcgtgcct gtgacccgac ctccggatga ctcgacctct gctccacgag acctggtgct 720 gtctgagcca agcagccaat ccttgagagt acagtggaca gcggccagtg gccctgtgac 780 tggctacaag gtccagtaca ctcctctgac ggggctggga cagccactgc cgagtgagcg 840 gcaggaggtg aacgtcccag ctggtgagac cagtgtgcgg ctgcggggtc tccggccact 900 gaccgagtac caagtgactg tgattgccct ctacgccaac agcatcgggg aggctgtgag 960 cgggacagct cggaccactg ccctagaagg gccggaactg accatccaga ataccacagc 1020 ccacagcctc ctggtggcct ggcggagtgt gccaggtgcc actggctacc gtgtgacatg 1080 gcgggtcctc agtggtgggc ccacacagca gcaggagctg ggccctgggc agggttcagt 1140 gttgctgcgt gacttggagc ctggcacgga ctatgaggtg accgtgagca ccctatttgg 1200 ccgcagtgtg gggcccgcca cttccctgat ggctcgcact gacgcttctg ttgagcagac 1260 cctgcgcccg gtcatcctgg gccccacatc catcctcctt tcctggaact tggtgcctga 1320 ggcccgtggc taccggttgg aatggcggcg tgagactggc ttggagccac cgcagaaggt 1380 ggtactgccc tctgatgtga cccgctacca gttggatggg ctgcagccgg gcactgagta 1440 ccgcctcaca ctctacactc tgctggaggg ccacgaggtg gccacccctg caaccgtggt 1500 tcccactgga ccagagctgc ctgtgagccc tgtaacagac ctgcaagcca ccgagctgcc 1560 cgggcagcgg gtgcgagtgt cctggagccc agtccctggt gccacccagt accgcatcat 1620 tgtgcgcagc acccaggggg ttgagcggac cctggtgctt cctgggagtc agacagcatt 1680 cgacttggat gacgttcagg ctgggcttag ctacactgtg cgggtgtctg ctcgagtggg 1740 tccccgtgag ggcagtgcca gtgtcctcac tgtccgccgg gagccggaaa ctccacttgc 1800 tgttccaggg ctgcgggttg tggtgtcaga tgcaacgcga gtgagggtgg cctggggacc 1860 cgtccctgga gccagtggat ttcggattag ctggagcaca ggcagtggtc cggagtccag 1920 ccagacactg cccccagact ctactgccac agacatcaca gggctgcagc ctggaaccac 1980 ctaccaggtg gctgtgtcgg tactgcgagg cagagaggag ggccctgctg cagtcatcgt 2040 ggctcgaacg gacccactgg gcccagtgag gacggtccat gtgactcagg ccagcagctc 2100 atctgtcacc attacctgga ccagggttcc tggcgccaca ggatacaggg tttcctggca 2160 ctcagcccac ggcccagaga aatcccagtt ggtttctggg gaggccacgg tggctgagct 2220 ggatggactg gagccagata ctgagtatac ggtgcatgtg agggcccatg tggctggcgt 2280

ggatgggccc cctgcctctg tggttgtgag gactgcccct gagcctgtgg gtcgtgtgtc 2340 gaggctgcag atcctcaatg cttccagcga cgttctacgg atcacctggg taggggtcac 2400 tggagccaca gcttacagac tggcctgggg ccggagtgaa ggcggcccca tgaggcacca 2460 gatactccca ggaaacacag actctgcaga gatccggggt ctcgaaggtg gagtcagcta 2520 ctcagtgcga gtgactgcac ttgtcgggga ccgcgagggc acacctgtct ccattgttgt 2580 cactacgccg cctgaggctc cgccagccct ggggacgctt cacgtggtgc agcgcgggga 2640 gcactcgctg aggctgcgct gggagccggt gcccagagcg cagggcttcc ttctgcactg 2700 gcaacctgag ggtggccagg aacagtcccg ggtcctgggg cccgagctca gcagctatca 2760 cctggacggg ctggagccag cgacacagta ccgcgtgagg ctgagtgtcc tagggccagc 2820 tggagaaggg ccctctgcag aggtgactgc gcgcactgag tcacctcgtg ttccaagcat 2880 tgaactacgt gtggtggaca cctcgatcga ctcggtgact ttggcctgga ctccagtgtc 2940 cagggcatcc agctacatcc tatcctggcg gccactcaga ggccctggcc aggaagtgcc 3000 tgggtccccg cagacacttc cagggatctc aagctcccag cgggtgacag ggctagagcc 3060 tggcgtctct tacatcttct ccctgacgcc tgtcctggat ggtgtgcggg gtcctgaggc 3120 atctgtcaca cagacgccag tgtgcccccg tggcctggcg gatgtggtgt tcctaccaca 3180 tgccactcaa gacaatgctc accgtgcgga ggctacgagg agggtcctgg agcgtctggt 3240 gttggcactt gggcctcttg ggccacaggc agttcaggtt ggcctgctgt cttacagtca 3300 tcggccctcc ccactgttcc cactgaatgg ctcccatgac cttggcatta tcttgcaaag 3360 gatccgtgac atgccctaca tggacccaag tgggaacaac ctgggcacag ccgtggtcac 3420 agctcacaga tacatgttgg caccagatgc tcctgggcgc cgccagcacg taccaggggt 3480 gatggttctg ctagtggatg aacccttgag aggtgacata ttcagcccca tccgtgaggc 3540 ccaggcttct gggcttaatg tggtgatgtt gggaatggct ggagcggacc cagagcagct 3600 gcgtcgcttg gcgccgggta tggactctgt ccagaccttc ttcgccgtgg atgatgggcc 3660 aagcctggac caggcagtca gtggtctggc cacagccctg tgtcaggcat ccttcactac 3720 tcagccccgg ccagagccct gcccagtgta ttgtccaaag ggccagaagg gggaacctgg 3780 agagatgggc ctgagaggac aagttgggcc tcctggcgac cctggcctcc cgggcaggac 3840 cggtgctccc ggcccccagg ggccccctgg aagtgccact gccaagggcg agaggggctt 3900 ccctggagca gatgggcgtc caggcagccc tggccgcgcc gggaatcctg ggacccctgg 3960 agcccctggc ctaaagggct ctccagggtt gcctggccct cgtggggacc cgggagagcg 4020 aggacctcga ggcccaaagg gggagccggg ggctcccgga caagtcatcg gaggtgaagg 4080 acctgggctt cctgggcgga aaggggaccc tggaccatcg ggcccccctg gacctcgtgg 4140 accactgggg gacccaggac cccgtggccc cccagggctt cctggaacag ccatgaaggg 4200 tgacaaaggc gatcgtgggg agcggggtcc ccctggacca ggtgaaggtg gcattgctcc 4260 tggggagcct gggctgccgg gtcttcccgg aagccctgga ccccaaggcc ccgttggccc 4320 ccctggaaag aaaggagaaa aaggtgactc tgaggatgga gctccaggcc tcccaggaca 4380 acctgggtct ccgggtgagc agggcccacg gggacctcct ggagctattg gccccaaagg 4440 tgaccggggc tttccagggc ccctgggtga ggctggagag aagggcgaac gtggaccccc 4500 aggcccagcg ggatcccggg ggctgccagg ggttgctgga cgtcctggag ccaagggtcc 4560 tgaagggcca ccaggaccca ctggccgcca aggagagaag ggggagcctg gtcgccctgg 4620 ggaccctgca gtggtgggac ctgctgttgc tggacccaaa ggagaaaagg gagatgtggg 4680 gcccgctggg cccagaggag ctaccggagt ccaaggggaa cggggcccac ccggcttggt 4740 tcttcctgga gaccctggcc ccaagggaga ccctggagac cggggtccca ttggccttac 4800 tggcagagca ggacccccag gtgactcagg gcctcctgga gagaagggag accctgggcg 4860 gcctggcccc ccaggacctg ttggcccccg aggacgagat ggtgaagttg gagagaaagg 4920 tgacgagggt cctccgggtg acccgggttt gcctggaaaa gcaggcgagc gtggccttcg 4980 gggggcacct ggagttcggg ggcctgtggg tgaaaaggga gaccagggag atcctggaga 5040 ggatggacga aatggcagcc ctggatcatc tggacccaag ggtgaccgtg gggagccggg 5100 tcccccagga cccccgggac ggctggtaga cacaggacct ggagccagag agaagggaga 5160 gcctggggac cgcggacaag agggtcctcg agggcccaag ggtgatcctg gcctccctgg 5220 agcccctggg gaaaggggca ttgaagggtt tcggggaccc ccaggcccac agggggaccc 5280 aggtgtccga ggcccagcag gagaaaaggg tgaccggggt ccccctgggc tggatggccg 5340 gagcggactg gatgggaaac caggagccgc tgggccctct gggccgaatg gtgctgcagg 5400 caaagctggg gacccaggga gagacgggct tccaggcctc cgtggagaac agggcctccc 5460 tggcccctct ggtccccctg gattaccggg aaagccaggc gaggatggca aacctggcct 5520 gaatggaaaa aacggagaac ctggggaccc tggagaagac gggaggaagg gagagaaagg 5580 agattcaggc gcctctggga gagaaggtcg tgatggcccc aagggtgagc gtggagctcc 5640 tggtatcctt ggaccccagg ggcctccagg cctcccaggg ccagtgggcc ctcctggcca 5700 gggttttcct ggtgtcccag gaggcacggg ccccaagggt gaccgtgggg agactggatc 5760 caaaggggag cagggcctcc ctggagagcg tggcctgcga ggagagcctg gaagtgtgcc 5820 gaatgtggat cggttgctgg aaactgctgg catcaaggca tctgccctgc gggagatcgt 5880 ggagacctgg gatgagagct ctggtagctt cctgcctgtg cccgaacggc gtcgaggccc 5940 caagggggac tcaggcgaac agggcccccc aggcaaggag ggccccatcg gctttcctgg 6000 agaacgcggg ctgaagggcg accgtggaga ccctggccct caggggccac ctggtctggc 6060 ccttggggag aggggccccc ccgggccttc cggccttgcc ggggagcctg gaaagcctgg 6120 tattcccggg ctcccaggca gggctggggg tgtgggagag gcaggaaggc caggagagag 6180 gggagaacgg ggagagaaag gagaacgtgg agaacagggc agagatggcc ctcctggact 6240 ccctggaacc cctgggcccc ccggaccccc tggccccaag gtgtctgtgg atgagccagg 6300 tcctggactc tctggagaac agggaccccc tggactcaag ggtgctaagg gggagccggg 6360 cagcaatggt gaccaaggtc ccaaaggaga caggggtgtg ccaggcatca aaggagaccg 6420 gggagagcct ggaccgaggg gtcaggacgg caacccgggt ctaccaggag agcgtggtat 6480 ggctgggcct gaagggaagc cgggtctgca gggtccaaga ggcccccctg gcccagtggg 6540 tggtcatgga gaccctggac cacctggtgc cccgggtctt gctggccctg caggacccca 6600 aggaccttct ggcctgaagg gggagcctgg agagacagga cctccaggac ggggcctgac 6660 tggacctact ggagctgtgg gacttcctgg accccccggc ccttcaggcc ttgtgggtcc 6720 acaggggtct ccaggtttgc ctggacaagt gggggagaca gggaagccgg gagccccagg 6780 tcgagatggt gccagtggaa aagatggaga cagagggagc cctggtgtgc cagggtcacc 6840 aggtctgcct ggccctgtcg gacctaaagg agaacctggc cccacggggg cccctggaca 6900 ggctgtggtc gggctccctg gagcaaaggg agagaaggga gcccctggag gccttgctgg 6960 agacctggtg ggtgagccgg gagccaaagg tgaccgagga ctgccagggc cgcgaggcga 7020 gaagggtgaa gctggccgtg caggggagcc cggagaccct ggggaagatg gtcagaaagg 7080 ggctccagga cccaaaggtt tcaagggtga cccaggagtc ggggtcccgg gctcccctgg 7140 gcctcctggc cctccaggtg tgaagggaga tctgggcctc cctggcctgc ccggtgctcc 7200 tggtgttgtt gggttcccgg gtcagacagg ccctcgagga gagatgggtc agccaggccc 7260 tagtggagag cggggtctgg caggcccccc agggagagaa ggaatcccag gacccctggg 7320 gccacctgga ccaccggggt cagtgggacc acctggggcc tctggactca aaggagacaa 7380 gggagaccct ggagtagggc tgcctgggcc ccgaggcgag cgtggggagc caggcatccg 7440 gggtgaagat ggccgccccg gccaggaggg accccgagga ctcacggggc cccctggcag 7500 caggggagag cgtggggaga agggtgatgt tgggagtgca ggactaaagg gtgacaaggg 7560 agactcagct gtgatcctgg ggcctccagg cccacggggt gccaaggggg acatgggtga 7620 acgagggcct cggggcttgg atggtgacaa aggacctcgg ggagacaatg gggaccctgg 7680 tgacaagggc agcaagggag agcctggtga caagggctca gccgggttgc caggactgcg 7740 tggactcctg ggaccccagg gtcaacctgg tgcagcaggg atccctggtg acccgggatc 7800 cccaggaaag gatggagtgc ctggtatccg aggagaaaaa ggagatgttg gcttcatggg 7860 tccccggggc ctcaagggtg aacggggagt gaagggagcc tgtggccttg atggagagaa 7920 gggagacaag ggagaagctg gtcccccagg ccgccccggg ctggcaggac acaaaggaga 7980 gatgggggag cctggtgtgc cgggccagtc gggggcccct ggcaaggagg gcctgatcgg 8040 tcccaagggt gaccgaggct ttgacgggca gccaggcccc aagggtgacc agggcgagaa 8100 aggggagcgg ggaaccccag gaattggggg cttcccaggc cccagtggaa atgatggctc 8160 tgctggtccc ccagggccac ctggcagtgt tggtcccaga ggccccgaag gacttcaggg 8220 ccagaagggt gagcgaggtc cccccggaga gagagtggtg ggggctcctg gggtccctgg 8280 agctcctggc gagagagggg agcaggggcg gccagggcct gccggtcctc gaggcgagaa 8340 gggagaagct gcactgacgg aggatgacat ccggggcttt gtgcgccaag agatgagtca 8400 gcactgtgcc tgccagggcc agttcatcgc atctggatca cgacccctcc ctagttatgc 8460 tgcagacact gccggctccc agctccatgc tgtgcctgtg ctccgcgtct ctcatgcaga 8520 ggaggaagag cgggtacccc ctgaggatga tgagtactct gaatactccg agtattctgt 8580 ggaggagtac caggaccctg aagctccttg ggatagtgat gacccctgtt ccctgccact 8640 ggatgagggc tcctgcactg cctacaccct gcgctggtac catcgggctg tgacaggcag 8700 cacagaggcc tgtcaccctt ttgtctatgg tggctgtgga gggaatgcca accgttttgg 8760 gacccgtgag gcctgcgagc gccgctgccc accccgggtg gtccagagcc aggggacagg 8820 tactgcccag gactgaggcc cagataatga gctgagattc agcatcccct ggaggagtcg 8880 gggtctcagc agaaccccac tgtccctccc cttggtgcta gaggcttgtg tgcacgtgag 8940 cgtgcgtgtg cacgtccgtt atttcagtga cttggtcccg tgggtctagc cttcccccct 9000 gtggacaaac ccccattgtg gctcctgcca ccctggcaga tgactcactg tgggggggtg 9060 gctgtgggca gtgagcggat gtgactggcg tctgacccgc cccttgaccc aagcctgtga 9120 tgacatggtg ctgattctgg ggggcattaa agctgctgtt ttaaaaggc 9169 <210> SEQ ID NO 4 <211> LENGTH: 387 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 ggccattacg gccgggcagc caggccccag ctgagaataa tgcactggat gggggtggtt 60 gttgtgttag gggaccccag ttctcactgc tgctctctgg tagtcagaag tgaggggtca 120 gccccatctt tcctgctgga gtgagctccc agctgggtag ctggcagcct ttctgtcata 180 tccctccctg ctcctgatgt ctggaggcag tgtagttctt cttaaaagtt ggcttggcag 240 ggcgcggtgg ctcacacctg taatcccaac attttgggag gctgaggcag tcggatcacc 300 tgaggtcgag agtttgagac cccagcctat ccaacatggt gaaactccat ctctactaaa 360 aatacaaaaa ttagcccggc atggtgg 387

<210> SEQ ID NO 5 <211> LENGTH: 561 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 attttggtgg gaggccctgg acctagaaac agaaaatgaa agtggagaaa accaaacaag 60 aatctttctt tcttaaagta aacatcggac tgacatctcc tttcaaacat tcattgagga 120 tctactctgt gagaagaagc aataccatgt tatctcattc tagttccagt tctagttgac 180 ttgattgtca aacgactgct aagaagtaga gaggaggctt caccatgttt gccaggctag 240 tctggaactc ttgacctcag gtgatccacc cacctcggcc tcccaaagtg ctaggattac 300 aggcatgagc cacctcgccc agctgtttct gtgtcacgtt ttgatacttg aataaaaatg 360 acactttgga gccttgaaaa atggactttc cttctgaagt acccaggaga gtaggatgga 420 cttcttcctc tctccctaca ctatcccttg gagaaaacct tttatcaaga agataccgaa 480 tatcatcact gatataatgt cagaggaggt cactctctat agcccaacct ccaggatttc 540 agcacttcgt ggacatggag g 561 <210> SEQ ID NO 6 <211> LENGTH: 335 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 gggaccagag agctgcgata tggctgaaaa aagtgagtgc aagacaagga cataggatga 60 aaggggccag tgatggtcag ggacactgga ggacaggagt ctgtgggatc ttagcatgta 120 ggatgacagg agtcagtgga ctgcagtgag aactgatgag ccattgacag acactgatgt 180 gggatggcag aggtcagtgc tggatgggga catgcaatga gaggttggtg tacagtggtc 240 accactaggg taacagaaag acaggtgatt gagcttgtgg ctgtgcagag gtgaaagccg 300 aggatggtgt gtgggcagat aggagattgg ttgtt 335 <210> SEQ ID NO 7 <211> LENGTH: 613 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 tttttttttt ttttttttaa gagaagatgt ctcgctccgt cgtccaggct ggagcgcagt 60 ggcgcaatca tggatcactg cagccttgac cttcccgggc tcaagcgatc ctcccggctc 120 acccccagta gctggaacca caggcgcgct tccacaccgg aaagcccatt ttctagaggc 180 ggaaaccgaa gcgcccagtg ggaaaggcga cccgccgggg atgcggggtg ctcaacgcgc 240 tgccacctgg ggcccaacgc gttgacctcg cggtcaggtt gcttccgcgg actacggttc 300 tggctcgcta gctctggaac aggcaggaag gagtggggct atctgatagg ggaagatgat 360 gggagtctaa caggagacgg gaatttgata gaggagatgg tgaataggtc cagtggagga 420 gtggggaggt ggaggtttta ggaagcagtg agcaggtctg atggaggaga aagtgtaaat 480 ctcagaggag atttgggtcc agcaaaaaag ggatggggta gggtaggggc agacacaccc 540 tgttgacagt tcagggctca gtgccatctt gggaagcaga tggataacga gacagggagg 600 agactatagg gac 613 <210> SEQ ID NO 8 <211> LENGTH: 177 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 tcccaccggg tccaggtcag gcccaagggg accagctctg ctcccaccat catcccactc 60 cccacacagc caagctggac atctgagagc actgcatggc aatcctcatc tgcctgtgtg 120 tctccctcca ctggggacac atgtcatgtg tcagtcctgc agcacatgtg tccttct 177 <210> SEQ ID NO 9 <211> LENGTH: 285 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 gaactcagac atgcgaccaa gaattggctc acaggagctc agacatgacc atggcctatc 60 tcaggacagc acagacagag ggacgctcag atactaccat agacaggtgg gagttcaggc 120 atggcacagg cacagggagc ccacacgcga gtgcagacat ctggctccac agacgtgagt 180 gcggacacac gggcgctcag aggggaaccc caacacgtcc actcccgggt agagcaggca 240 tggccacagg cttgaacgct gggagccaga ggcagggaca agcag 285 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 10 acatcaggag cagggaggga 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 11 ccctaactca acaacatccc 20 <210> SEQ ID NO 12 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 12 tctcaaactc tcgacctcag g 21 <210> SEQ ID NO 13 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 13 gtttcaccat gttggctagg c 21 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 14 gcctcctctc tacttcttag 20 <210> SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 15 gggatagtgt agggagagag g 21 <210> SEQ ID NO 16 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 16 cctactctcc tgggtacttc a 21 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 17 ggtgtctgtc ttccttaggg 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 18 aattagctgg gcgtggtggc 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 19 aatcccagct gctcaggagg 20 <210> SEQ ID NO 20 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 20 rgrcrurcra rarurcrarc rcrurgrurc rurururcru rgrururarc rcrc 54 <210> SEQ ID NO 21 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQUENCE: 21 rcrurgrcrc rarurcrcrc rarcrarurc rargrurgru rcrurgrurc rara 54 <210> SEQ ID NO 22 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 22 rcrurcrcru rcrcrarurc rargrarcrc rurgrcrurc rarcrurgrc ruru 54 <210> SEQ ID NO 23 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 23 rgrurcrcrc rurarurarg rurcrurcrc rurcrcrcru rgrurcrurc rgru 54 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 24 ggagtgggat gatggtggga 20 <210> SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 25 gcagtgctct cagatgtcca g 21 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 26 tggagggaga cacacaggca 20 <210> SEQ ID NO 27 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 27 tccctctgtc tgtgctgtcc t 21 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 28 gcctgaactc ccacctgtct 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 29 ctctggctcc cagcgttcaa 20 <210> SEQ ID NO 30 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 20 <400> SEQUENCE: 30 rgrurararc rargrarara rgrarcrarg rgrurgraru rurgragc 48 <210> SEQ ID NO 31 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 21 <400> SEQUENCE: 31 rgrarcrarg rarcrarcru rgrarurgru rgrgrgraru rgrgrcag 48 <210> SEQ ID NO 32 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 22 <400> SEQUENCE: 32 rgrcrargru rgrargrcra rgrgrurcru rgrarurgrg rargrgag 48 <210> SEQ ID NO 33 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Reverse complement of the antisense oligonucleotide SEQ ID NO: 23 <400> SEQUENCE: 33 rgrargrarc rargrgrgra rgrgrargra rcrurarura rgrgrgac 48

Claims

1. A method of modulating a function of and/or the expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 387 of SEQ ID SEQ ID NO: 4, 1 to 561 of SEQ ID NO: 5, 1 to 335 of SEQ ID SEQ ID NO: 6, 1 to 613 of SEQ ID NO: 7, 1 to 177 of SEQ ID NO: 8, and 1 to 285 of SEQ ID NO: 9; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

2. A method of modulating a function of and/or the expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

3. A method of modulating a function of and/or the expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of a Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of the Collagen gene is increased in vivo or in vitro with respect to a control.

6. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a Collagen gene polynucleotide.

7. The method of claim 4, wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a Collagen gene polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Collagen gene polynucleotide.

9. The method of claim 4, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from: at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

10. The method of claim 9, wherein the one or more modifications comprise at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

11. The method of claim 9, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from: a phosphorothioate, 2'- Omethoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

12. The method of claim 9, wherein the one or more modifications comprise at least one modified nucleotide selected from: a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof.

13. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS: 10 to 29.

14. A method of modulating a function of and/or the expression of a Collagen gene in mammalian cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 5 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for an antisense polynucleotide of a Collagen gene polynucleotide, wherein said at least one siRNA oligonucleotide has at least 50% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense nucleic acid molecule of the Collagen gene polynucleotide; and, modulating a function of and/or the expression of a Collagen gene in mammalian cells or tissues in vivo or in vitro.

15. The method of claim 14, wherein said oligonucleotide has at least 80% sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the Collagen gene polynucleotide.

16. A method of modulating a function of and/or the expression of a Collagen gene in mammalian cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide of about 5 to 30 nucleotides in length specific for noncoding and/or coding sequences of a sense and/or natural antisense strand of a Collagen gene polynucleotide wherein said at least one antisense oligonucleotide has at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS: 1 to 9; and, modulating the function and/or expression of the Collagen gene in mammalian cells or tissues in vivo or in vitro.

17. A synthetic, modified oligonucleotide comprising at least one modification wherein the at least one modification is selected from: at least one modified sugar moiety; at least one modified internucleotide linkage; at least one modified nucleotide, and combinations thereof; wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a Collagen gene in vivo or in vitro as compared to a normal control.

18. The oligonucleotide of claim 17, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

19. The oligonucleotide of claim 17, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

20. The oligonucleotide of claim 17, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.

21. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from: a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.

22. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

23. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.

24. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

25. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a Collagen gene polynucleotide wherein said oligonucleotide has at least about 20% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the Collagen gene polynucleotide.

27. The oligonucleotide of claim 17, wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the Collagen gene polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Collagen gene polynucleotide in vivo or in vitro, as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS: 10 to 29.

30. A composition comprising one or more oligonucleotides specific for one or more Collagen gene polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

31. The composition of claim 30, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS: 10 to 29.

32. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 10 to 29.

33. The composition of claim 32, wherein the oligonucleotides set forth as SEQ ID NOS: 10 to 29 comprise one or more modifications or substitutions.

34. The composition of claim 33, wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

35. A method of preventing or treating a disease associated with at least one Collagen gene polynucleotide and/or at least one encoded product thereof, comprising: administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Collagen gene polynucleotide and modulates expression of said at least one Collagen gene polynucleotide; thereby preventing or treating the disease associated with the at least one Collagen gene polynucleotide and/or at least one encoded product thereof.

36. The method of claim 35, wherein a disease associated with the at least one Collagen gene polynucleotide is selected from: a collagen disorder, age related collagen degradation, Osteogenesis imperfecta, Otosclerosis (OTSC), Osteoporosis, Osteoarthritis, Oesophageal squamous cell cancer, chondrodysplasia, atypical Marfan syndrome, Ehlers-Danlos Syndrome (EDS), Dystrophic epidermolysis bullosa (DEB), Caffey disease, aneurysms (eg. intracranial aneurysms), idiopathic pulmonary fibrosis, liver cirrhosis, kidney fibrosis, liver fibrosis, heart fibrosis, scleroderma, hypertrophic scars, keloids, cancer, inflammation, a genetic disease (e.g. Duchenne muscular dystrophy), a neurological disease or disorder (e.g. Parkinson's, Alzheimer's, Huntington's, Gaucher disease), a metabolic disease (e.g. type I diabetes), an autoimmune disease or disorder, trauma (e.g. spinal cord injury, burns, etc), ischemia, and other blood vessel, heart, a skin disease or disorder, skin aging, a skin disease or disorder or condition requiring skin engineering, a liver or kidney disease requiring transplantation; tendon, bone or tissue regeneration; skeletal repair, cartilage and bone repair.

37. A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising: selecting a target polynucleotide associated with a disease state; identifying at least one oligonucleotide comprising at least five consecutive nucleotides which are complementary to the selected target polynucleotide or to a polynucleotide that is antisense to the selected target polynucleotide; measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and selecting at least one oligonucleotide for in vivo administration based on the information obtained.

38. A method of preventing or treating a skin condition associated with at least one Collagen gene polynucleotide and/or at least one encoded product thereof, comprising: administering to a patient having a skin condition or at risk of developing a skin condition a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Collagen gene polynucleotide and modulates expression of said at least one Collagen gene polynucleotide; thereby preventing or treating the disease skin condition associated with the at least one Collagen gene polynucleotide and/or at least one encoded product thereof.

39. The method of claim 38, wherein the skin condition is caused by caused by inflammation, light damage or aging.

40. The method of claim 39, wherein the skin condition is the development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullosa disease, exfoliative dermatitis, seborrheic dermatitis, an erythema, discoid lupus erythematosus, dermatomyositis, skin cancer, or an effect of natural aging.

41. A Collagen gene based matrix composition, said composition comprising a three dimensional purified collagen matrix comprised of Collagen fibrils, wherein function of and/or the expression of a Collagen gene polynucleotide in the cells of collagen fibrils is modulated in vivo or in vitro by contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide.

42. The composition of claim 41, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS: 10 to 29.

43. The composition of claim 41, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 10 to 29.

44. The composition of claim 41, wherein the composition supports stem cells.

45. A method of in vitro, ex vivo or in vivo wound healing or tissue regeneration or tissue engineering which comprises application of an extracellular matrix composition as defined in claim 41 to a wound.

46. A method of preparing a Collagen gene based matrix composition, comprising contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.