Treatment Of Filaggrin (flg) Related Diseases By Modulation Of Flg Expression And Activity

Abstract

The present invention relates to antisense oligonucleotides and/or compounds that modulate the expression of and/or function of Filaggrin (FLG), in particular, by targeting natural antisense polynucleotides of Filaggrin (FLG). The invention also relates to the identification of these antisense oligonucleotides and/or compounds and their use in treating diseases and disorders associated with the expression of FLG.

Description

FIELD OF THE INVENTION

[0001] The present application claims the priority of U.S. provisional patent application No. 61/246,080 filed Sep. 25, 2009 and U.S. provisional patent application No. 61/307,654 filed Feb. 24, 2010 which is incorporated herein by reference in its entirety.

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

BACKGROUND

[0003] Filaggrin is a highly charged, cationic protein that aids aggregation and subsequent disulfide bonding of keratin filaments. It is derived from profilaggrin, a large (4400 kD) phosphorylated precursor expressed as keratohyalin granules in the granular layer of the epidermis. During the transition from the granular layer to the stratum corneum, profilaggrin is converted to filaggrin by site-specific proteolysis and dephosphoryiation. In addition to profilaggrin processing to filaggrin, the transition from a granular cell to a corneocyte is characterized by the degradation of the nucleus and other organelles, assembly of a cornified envelope, and reorganization of the keratin intermediate filament network into a two dimensional sheet. Fillagrin plays a critical role in the generation and maintenance of a flexible and hydrated stratum corneum and its hydrolysis is carefully regulated to generate free amino acids that form a major part of the natural moisturizing factors (NMF). The transition from a granular precursor, profilaggrin, to a diffusely distributed protein happens quickly at the granular to stratum corneum transition in response to an initiating signal which is not yet known. That profilaggrin is expressed as a precursor, rather than a mature protein, suggests that filaggrin expression must be regulated to prevent cytotoxic effects. Many inflammatory skin conditions are characterized by attenuation of the granular layer with concomitant parakeratosis, i.e. retained nuclei in the keratinocytes of the stratum corneum. While the signals that are disrupting terminal differentiation in these inflammatory conditions may be disparate, a common final theme is loss of the granular layer with subsequent incomplete terminal differentiation. In conditions where profilaggrin is decreased, such as atopic dermatitis, or essentially absent, as in ichthyosis vulgaris, the quality of the stratum corneum is compromised due to the inability of an NMF-depleted stratum corneum to remain hydrated under the desiccating action of the environment.

[0004] The natural moisturizing factors (NMF) perform an important function in maintaining the moisture content of the stratum corneum. It has been reported that amino acids forming the principal constituents of NMF are produced by the proteoliticaly cleaved filaggrin originating from keratohyalin granules. Filaggrin is a protein, composed of 317 amino acids. Since it was clarified that amino acids forming the principal constituents of NMF are derived from filaggrin, investigations on the relation of morbid states exhibiting a dry skirt to filaggrin have been carried forward. In recent years, it has been clarified that the amino acid content of the stratum, corneum is reduced in a dry skin as seen in senile xerosis, atopic diseases and the like, and that the expression of filaggrin in such a dry skin is decreased. Moreover, it is well known that skin troubles such as rough skin are caused by a dry environment.

[0005] Filaggrin gene plays a role in building up the barrier layers of the skin and mutations in this gene lead to conditions such as eczema. Filaggrin is an abundant protein in the outermost layers of the skin and is produced by the Filaggrin gene. Filaggrin's function is to help produce the impermeable skin barrier layers present at the skin's outermost surface and to keep these hydrated. The skin's inherent barrier function is akin to plastic or cling film--it acts to prevent water loss from the skin and importantly, to protect the body from foreign materials in the environment, such as allergens. Lack of an intact skin barrier leads to allergens entering the body where they produce a range of allergic responses that include eczema, asthma, hay fever and other allergies.

[0006] 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 ease for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENE.TM. (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.

SUMMARY

[0007] This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of file invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0008] 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.

[0009] One embodiment provides a method of modulating function and/or expression of an FLG 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 4629 of SEQ ID NO: 2 thereby modulating function and/or expression of the FLG polynucleotide in patient cells or tissues in vivo or in vitro.

[0010] In an embodiment, an oligonucleotide targets a natural antisense sequence of FLG polynucleotides, for example, nucleotides set forth in SEQ ID NOs: 2, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 3 to 13.

[0011] Another embodiment provides a method of modulating function and/or expression of an FLG 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 FLG polynucleotide; thereby modulating function and/or expression of the FLG polynucleotide in patient cells or tissues in vivo or in vitro.

[0012] Another embodiment provides a method of modulating function, and/or expression of an FLG 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 an FLG antisense polynucleotide; thereby modulating function and/or expression of the FLG polynucleotide in patient cells or tissues in vivo or in vitro.

[0013] In an embodiment, a composition comprises one or more antisense oligonucleotides which bind to sense and/or antisense FLG polynucleotides.

[0014] In an embodiment, a composition comprises one or morn of antisense oligonucleotides which bind to sense and/or antisense FLG polynucleotides, one or more FLG modulating molecule, a pharmaceutically acceptable carrier and combinations thereof.

[0015] In an embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.

[0016] In an embodiment, the oligonucleotides comprise one or more modified bonds.

[0017] 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 .alpha.-L-LNA.

[0018] In an embodiment, the oligonucleotides are administered to a patient subcutaneously, intramuscularly, intravenously or intraperitoneally.

[0019] In an 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.

[0020] In an embodiment, the oligonucleotides are administered in a pharmaceutical composition. A treatment regimen comprises administering at least once to a patient a composition comprising one or more of an antisense compound and one or more FLG modulating molecule; 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.

[0021] In an embodiment, the oligonucleotides are encapsulated in a liposome or attached to a carrier molecule (e.g. cholesterol, TAT peptide).

[0022] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a graph of real time PCR results fold change+standard deviation in FLG1 mRNA after treatment of RepG2 cells with phosphothioate oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of FLG1 mRNA in HepG2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431. Bars denoted as CUR-1157, CUR-1158, CUR-1159, and CUR-1160 and CUR-1161 correspond to samples treated with SEQ ID NOS: 3, 4, 5, 6 and 7 respectively.

[0024] FIG. 2 shows fold change+standard deviation in FLG1 mRNA after treatment of 518A2 cells with phosphodiester oligonucleotides with a 3' inverted T and 2' O Methyl gapmer phosphodiester oligos introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of FLG mRNA in 518A2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431. Bars denoted as CUR-1128, CUR-1129, CUR-1130, and CUR-1131 correspond to samples treated with SEQ ID NOS: 8, 9, 10 and 11 respectively.

[0025] FIG. 3 shows fold change+standard deviation in FLG1 mRNA after treatment of 518A2 cells with phosphodiester oligonucleotides introduced using Lipofectamine 2000, as compared to control. Real time PCR results show that the levels of FLG mRNA in 518A2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431. Bars denoted as CUR-1396 and CUR-1397 correspond to samples treated with SEQ ID NOS: 12, and 13 respectively.

[0026] FIG. 4 shows fold change in Filaggrin mRNA expression in 518A2 cells and primary keratinocytes treated with the compounds--Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine; as compared to untreated cells.

[0027] Sequence Listing Description--SEQ ID NO: 1: Homo sapiens filaggrin (FLG), mRNA. (NCBI Accession No.: NM.sub.--002016); SEQ ID NO: 2: Natural FLG antisense sequence AK056431; SEQ ID NOs: 3 to 13: Antisense oligonucleotides. * indicates phosphothioate bond and `m` indicates 2'O me modification.

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 an embodiment, 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 cart 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 Junction 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 alphanumeric 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 tee 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, DMA 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 ease 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 preferred 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 "FLG" and "Filaggrin" 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 `Filaggrin`, FLG, FLG1 and ATOD2 are considered same in the literature and used interchangeably in the present application.

[0039] 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.

[0040] 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.

[0041] 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 at, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

[0042] 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.

[0043] By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). 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 die target RNA.

[0044] 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.

[0045] 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.

[0046] 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, daminopurine, 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 Benner et al., 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.

[0047] "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl. Acid Res:, 25(22), 4429-4443, Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M, (1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213, Herdewin P., (2000)Antisense & Nucleic Acid Drug Dev., 10:297-310); 2'-O, 3'-C-linked [3.2.0]bicycloarabinonucleosides. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

[0048] 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.

[0049] 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 ease of in vitro assays.

[0050] 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 0.1.times. sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60.degree. C. for 30 minutes.

[0051] "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.

[0052] 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).

[0053] 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., 1.0 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0054] As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.

[0055] 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 snore times in a given sequence.

[0056] 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.

[0057] 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 radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0058] 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.

[0059] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic nontoxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N'-dibenzyl ethyl enediamine, diethylamide, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0060] When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from, pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[0061] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base, in a suitable solvent or solvent combination.

[0062] The compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers. All such isomers, including optical isomers, being included in the present invention.

[0063] Example of skin care products include but are not limited to moisturizers, fake tanning preparations, sun tan lotions, massage oils, bath oils, perfumes, balms, creams, face packs, shaving foams and gels. Examples of cosmetics include but are not limited to lipsticks, foundation, eye-shadow, eyeliner, blusher and concealer. Examples of cleansing products include but are not limited to shampoos (in particular antidandruff shampoos), soap, personal wash products including shower gel and bubble bath and fabric detergents and dishwashing detergents. Examples of hair care products include but are not limited to hair styling mousses, hair styling sprays, hair styling gels, hair conditioners or hair colourants.

[0064] By assessing the profilaggrin genotype of an individual it is possible to determine the individual's predisposition to a skin condition. By "profilaggrin genotype", is meant the identity of profilaggrin alleles in the genome of the individual. Individuals tested by a method of the invention are typically mammalian. In one embodiment the mammal may be a rodent. In another embodiment the mammal may be a human. Thus individuals tested by a method of the invention are diploid and so comprise two copies of the profilaggrin gene within their genome, If an individual has two identical copies of a profilaggrin gene then they are homozygous for that allele. If an individual has two different copies of a profilaggrin gene, i.e. one is polymorphic to the other, then the individual is heterozygous for that allele. By "predispositions is meant that the presence of an individual profilaggrin allele in the genome of an individual, or the combination of profilaggrin alleles present in the genome of an individual, are associated with, or are predictive of, a skin condition.

[0065] The term "skin conditions" as used heroin includes within its meaning all physical parameters of the skin, including the scalp, such as moisture retention, substance production or barrier formation. In one embodiment the term "skin conditions refers to the ability of the skin to maintain healthy levels of NMF production. Accordingly, the invention provides a method of determining the predisposition of an individual to maintain a healthy level of NMF production. To put it another way the invention provides a method of determining the individual's susceptibility to conditions related to aberrant NMF production. Typically skin conditions caused or i exacerbated by aberrant NMF production are caused by the production of less NMF than by healthy skin. Conditions associated with aberrant filaggrin and NMF production include ichthyosis Vulgaris. In another embodiment the term "skin conditions refers to dry skin. Dry skin conditions include senile/post-menopausal xerosis, surfactant: induced xerosis, winter xerosis, sunburn. In another embodiment the term "skin condition" refers to conditions of the scalp such as dandruff. In another embodiment the term "skin conditions refers to erythema, such as detergent-induced erythema.

[0066] The expression "care of the keratinous substrates" refers to all the actions intended to preserve or restore the healthy functioning of skin and/or hair and/or nails or any process providing the means to preserve or improve their appearance and/or texture. Thus, care includes hydration, appeasement, protection against all types of aggression, notably sun protection, and fighting against and preventing the signs of aging.

[0067] The phrase "signs of cutaneous aging" includes all of the modifications regarding external appearance of skin due to aging. Examples of these modifications include wrinkles and fine lines, limp skin, slackened skin, thin looking skin, loss of elasticity and/or skin tone, dull skin, and skin which lacks radiance, ft also includes internal skin, modifications that do not translate directly as changes in external skin appearance. An example of these internal modifications is the degradation that occurs internally in skin resulting from consecutive exposure to UV radiation. The expression "to enhance skin appearance" includes all the phenomena which are likely to have as consequences a visual improvement of skin appearance. The skirt will have a nicer look; it will be, for example, much more beautiful, firm, and/or smooth. All the small imperfections will be decreased or removed. The papery appearance of the skin, for example, will be attenuated. Moreover, the active ingredient according to the invention, or the composition containing it, can be intended to protect keratinous substrates and, particularly, the skin, hair, and nails from all types of external aggression. The use of these active agents, or the composition containing them, will allow the keratinous substrates to be protected and to better resist stress inflicted upon them by the environment.

[0068] The phrase "external aggression" refers to aggressions produced by the environment. These can be of chemical, physical, biological, or thermic origin.

[0069] The expression "dermatological disease or disorder" refers to all the diseases affecting the skin that may or may not have visible consequences. Therefore, by way of example: differentiation and cell proliferation disorders, keratinization disorders, signs of cutaneous aging, inflammatory or allergic reactions, disorders of sebaceous functions, dermal or epidermal proliferations (malignant or non-malignant), cutaneous disorders due to UV ray exposure, and pathologies associated with chronological or actinic aging can be mentioned.

[0070] 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.

[0071] "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.

[0072] "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing tire 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.).

[0073] 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 its 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, gastric 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.

[0074] "Neurological disease or disorder" refers to any disease or disorder of die 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 axe 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 myclinolysis; 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; Caching'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; HIV associated 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 Kinsbourne 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; myelinoelastic 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 multifocalleukoeneephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types I and II); 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.

[0075] 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; hepatitis; sepsis; alcoholic liver disease; non-alcoholic steatosis; 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.

[0076] A cardiovascular disease or disorder includes those disorders that can either cause ischemia or are caused by reperfusion of the heart. Examples include, but are not limited to, atherosclerosis, coronary artery disease, granulomatous myocarditis, chronic myocarditis (non-granulomatous), primary hypertrophic cardiomyopathy, peripheral artery disease (PAD), peripheral vascular disease, venous thromboembolism, pulmonary embolism, stroke, angina pectoris, myocardial infarction, cardiovascular tissue damage caused by cardiac arrest, cardiovascular tissue damage caused by cardiac bypass, cardiogenic shock, and related conditions that would be known by those of ordinary skill in the art or which involve dysfunction of or tissue damage to the heart or vasculature, especially, but not limited to, tissue damage related to FLG activation. CVS diseases include, but are not limited to, atherosclerosis, granulomatous myocarditis, myocardial infarction, myocardial fibrosis secondary to valvular heart disease, myocardial, fibrosis without infarction, primary hypertrophic cardiomyopathy, and chronic myocarditis (non-granulomatous).

[0077] "Neurodegenerative disease or disorder" refers to a wide range of diseases and disorders of the central and peripheral nervous system including, for example, 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.

Polynucleotide and Oligonucleotide Compactions and Molecules

[0078] Targets:

[0079] In one embodiment, the targets comprise nucleic acid sequences of Filaggrin (FLG), including without limitation sense and/or antisense noncoding and/or coding sequences associated with FLG.

[0080] Filaggrin gene plays a role in building up the barrier layers of the skin, and mutations in this gene lead to conditions such as eczema. Filaggrin is an abundant protein in the outermost layers of the skin and is produced by the filaggrin gene. Filaggrin's function is to help produce the impermeable skin barrier layers present at the skin's outermost surface and to keep these hydrated. The skin's inherent barrier function is akin, to plastic or cling film--it acts to prevent water loss from the skin and importantly, to protect the body from, foreign materials in the environment, such as allergens. Lack of an intact skin barrier leads to allergens entering the body where they produce a range of allergic responses that include eczema, asthma, hay fever and other allergies.

[0081] Lack of expression of the protein filaggrin has been shown to predispose individuals to the development of ichthyosis vulgaris and, more recently, atopic eczema or dermatitis. The filaggrin gene resides on human chromosome 1q21 within the epidermal differentiation complex, a region that also harbors genes for several other proteins that are important for the normal barrier function of the epidermis. The primary function of filaggrin seems to be to aggregate the epidermal cytoskeleton to form a dense protein-lipid matrix thereby regulating permeability of the skin to water and external particles such as allergens.

[0082] Pioglitazone--ACTOS (pioglitazone hydrochloride) is an oral antidiabetic agent that acts primarily by decreasing insulin resistance. ACTOS is used in the management of type 2 diabetes mellitus (also known as non-insulin-dependent diabetes mellitus [NIDDM] or adult-onset diabetes). Pharmacological studies indicate that ACTOS improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis. ACTOS improves glycemic control while reducing circulating insulin levels. Pioglitazone (.+-.)-5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-2,4-]thiazolid- inedione monohydrochloride belongs to a different chemical class and has a different pharmacological action than the sulfonylureas, metformin, or the .alpha.-glucosidase inhibitors. The molecule contains one asymmetric carbon, and the compound is synthesized and used as the racemic mixture. The two enantiomers of pioglitazone interconvert in vivo. No differences were found in the pharmacologic activity between the two enantiomers.

[0083] Pioglitazone hydrochloride is an odorless while crystalline powder that has a molecular formula of C.sub.19H.sub.20N.sub.2O.sub.3S.HCl and a molecular weight of 392.90 daltons. It is soluble in N,N-dimethylformamide, slightly soluble in anhydrous ethanol, very slightly soluble in acetone and acetonitrile, practically insoluble in water, and insoluble in ether.

[0084] ACTOS is indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Pioglitazone has also been used to treat non-alcoholic steatohepatitis (fatty liver), but this use is presently considered experimental.

[0085] Pioglitazone, however, has not yet been investigated for use in the field of dermatology.

[0086] Lomerizine--is a calcium channel blocker with antimigraine properties and selectively inhibits the constriction of cerebral arteries. It has been known to be a neuroprotective and is in trials for glaucoma. Side effects of Lomerizine include minimal cardiovascular side effects, sleepiness and flushing.

[0087] Lomerizine, however, has not yet been investigated for use in the field of dermatology.

[0088] Bupropion--also known as Wellbutrin, Zyban, Voxra, Budeprion, or Aplenzin; formerly known as amfebutamone is an antidepressant of the aminoketone class, is chemically unrelated, to tricyclic, tetracyclic, selective serotonin re-uptake inhibitor, or other known antidepressant agents. Its structure closely resembles that of diethylpropion; it is related to phenylethylamines.

[0089] Bupropion is designated as (.+-.)-1-(3-chlorophenyl)-2-[(1,1-dimethylethyl)amino]-1-propanone hydrochloride. The molecular weight is 276.2. The molecular formula is C.sub.13H.sub.18ClNO.HCl. Bupropion hydrochloride powder is white, crystalline, and highly soluble in water. It has a bitter taste and produces the sensation of local anesthesia on the oral mucosa.

[0090] Bupropion is indicated for the treatment of major depressive disorder. A major depressive episode (DSM-IV) implies the presence of 1) depressed mood or 2) loss of interest or pleasure; in addition, at least 5 of the following symptoms have been present during the same 2 week period and represent a change from previous functioning: depressed mood, markedly diminished interest or pleasure in usual activities, significant change in weight and/or appetite, insomnia or hypersomnia, psychomotor agitation or retardation, increased fatigue, feelings of guilt or worthlessness, slowed thinking or impaired concentration, a suicide attempt, or suicidal ideation.

[0091] Bupropion has shown some success in treating social phobia and anxiety comorbid with depression, but not panic disorder with agoraphobia. Its anxiolytic potential has been compared to that of sertraline and doxepin. However, it can cause agitation in some patients, especially at higher doses, and often increases anxiety, much like methylphenidate. As a psychostimulant, it is inherently an anxiogenic compound and contrary benefits are poorly understood and seemingly paradoxical.

[0092] Bupropion reduces the severity of nicotine cravings and withdrawal symptoms. Other indications for Bupropion are obesity and Attention-deficit hyperactivity disorder (ADHD). Bupropion has been approved by the FDA for the prevention of seasonal affective disorder. According to several case studies and a pilot study, bupropion lowers the level of an inflammatory mediator TNF-alpha and may be useful in autoinflammatory conditions such as Crohn's disease and psoriasis.

[0093] Bupropion, however, has not yet been investigated for use in the field of dermatology.

[0094] Phenprobamate--is a centrally acting skeletal muscle relaxant, with additional sedative and anticonvulsant effects. Overdose is similar to barbiturate, its mechanism of action is probably similar to meprobamate. Phenprobamate was previously used in humans as an anxiolytic, and is still sometimes used in general anaesthesia and for treating muscle cramps and spasticity. Phenprobamate is still used in some European countries, but it has generally been replaced by newer drugs. Phenprobamate is metabolised by oxidative degradation of the amide group and ortho-hydroxylation of the benzene ring, and is eliminated in urine by the kidneys.

[0095] Phenprobamate, however, has not yet been investigated for use in the field of dermatology.

[0096] Benidipine--also known as Benidipinum or benidipine hydrochloride, is a dihydropyridine calcium channel blocker for the treatment of high blood pressure (hypertension). Benidipine is a dihydropyridine calcium channel blocker inhibiting not only L-type but also T-type calcium, channels. The chemical name for Benidipine is (4R)-rel-1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxy- lic acid 3-methyl 5-[(3R)-1-(phenylmethyl)-3-piperidinyl]ester hydrochloride. Orally active antihypertensive agent which displays a wide range of activities in vitro and in vivo, inhibits L-, N- and T-type Ca.sup.2+ channels. Also inhibits aldosterone-induced mineralocorticoid receptor activation. Exhibits cardioprotective and antiartherosclerotic effects.

[0097] Benidipine, however, has not yet been investigated for use in the field of dermatology.

[0098] Piroxicam--is a member of the oxicam group of nonsteroidal anti-inflammatory drags (NSAIDs). Each maroon and blue capsule contains 10 mg piroxicam, each maroon capsule contains 20 mg piroxicam for oral administration. The chemical name for piroxicam is 4-hydroxyl-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide. Piroxicam occurs as a white crystalline solid, sparingly soluble in water, dilute acid and most organic solvents, it is slightly soluble in alcohol and in aqueous solutions, it exhibits a weakly acidic 4-hydroxy proton (pKa 5.1) and weak basic pyridyl nitrogen (pKa 1.8). The molecular weight of piroxicam is 331.35. Its molecular formula is C.sub.15H.sub.13N.sub.3O.sub.4S.

[0099] Piroxicam is a non-steroidal anti-inflammatory drug used to relieve the symptoms of rheumatoid and osteoarthritis, primary dysmenorrhoea, postoperative pain; and act as an analgesic, especially where there is an inflammatory component. It is also used in veterinary medicine to treat certain neoplasias expressing cyclooxygenase (COX) receptors, such as bladder, colon, and prostate cancers.

[0100] Piroxicam, however, has not yet been investigated for use in the field of dermatology.

[0101] Topiramate--is a sulfamate-substituted monosaccharide. TOPAMAX.RTM. (topiramate) Tablets are available as 25 mg, 50 mg, 100 mg, and 200 mg round tablets for oral administration. TOPAMAX.RTM. (topiramate capsules) Sprinkle Capsules are available as 15 mg and 25 mg sprinkle capsules for oral administration as whole capsules or opened and sprinkled onto soft food.

[0102] Topiramate is a while crystalline powder with a bitter taste. Topiramate is most soluble in alkaline solutions containing sodium hydroxide or sodium phosphate and having a pH of 9 to 10. It is freely soluble in acetone, chloroform, dimethyl sulfoxide, and ethanol. The solubility in water is 9.8 mg/mL. Its saturated solution has a pH of 6.3. Topiramate has the molecular formula C.sub.12H.sub.21NO.sub.8S and a molecular weight of 339.36. Topiramate is designated chemically as 2,3:4,5-Di-O-isopropylidene-.beta.-D-fructopyranose sulfamate.

[0103] Topiramate is used alone or with other medications to treat certain types of seizures in people who have epilepsy. Topiramate is also used with other medications to control seizures in people who have Lennox-Gastaut syndrome (a disorder that causes seizures and developmental delays). Topiramate is used to treat patients who continue to have seizures even when they take other anti-seizure medications. Topiramate is also used to prevent migraine headaches, but not to relieve the pain of migraine headaches when they occur. Topiramate is in a class of medications called anticonvulsants, it works by decreasing abnormal excitement in the brain.

[0104] Topiramate treats epilepsy in children and adults and was originally marketed as an anticonvulsant. In children it is indicated for the treatment of Lennox-Gastaut syndrome, a disorder that causes seizures and developmental delay. It is also Food and Drug Administration (FDA) approved for, and most frequently prescribed for, the prevention of migraines. Psychiatrists have used topiramate to treat bipolar disorder, and often use topiramate to augment psychotrophics or counteract weight gain associated with numerous antidepressants.

[0105] Topiramate, has been investigated for use in treating alcoholism and obesity, especially to reduce binge eating.

[0106] Topiramate, is also used in clinical trials to treat posttraumatic stress disorder. A pilot study suggested that topiramate is effective against infantile spasms. Another study recommends topiramate as an effective treatment in the prevention of periventricular leukomalacia in preterm infants after a hypoxic-ischemic injury. Other off-label and investigational uses of topiramate include the treatment of essential tremor, bulimia nervosa, obsessive-compulsive disorder, alcoholism, smoking cessation, idiopathic intracranial hypertension, neuropathic pain, cluster headache, and cocaine dependence. Topiramate is also being studied with a mixture of phentermine to form a drug called Qnexa for the treatment of obesity.

[0107] Topiramate, however, has not yet been investigated for use in the field of dermatology.

[0108] Isradipine--is a calcium antagonist. Chemically, isradipine is 3,5-Pyridinedicarboxylic acid, 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethyl ester. Isradipine is a yellow, fine crystalline powder which is odorless or has a faint characteristic odor. Isradipine is practically insoluble in water (<10 mg/L at 37.degree. C.), but is soluble in ethanol and freely soluble in acetone, chloroform and methylene chloride.

[0109] Isradipine is indicated in the management of hypertension, it may be used alone or concurrently with thiazide-type diuretics. It is usually prescribed for the treatment of high blood pressure in order to reduce the risk of stroke and heart attack. More recent research in animal, models suggests that isradipine may have potential uses for treating Parkinson's disease.

[0110] Isradipine, however, has not yet been investigated for use in the field of dermatology.

[0111] Nicorandil--is one of the common drugs used in the treatment of Angina. The drug can be categorized as a vasodilator drug.

[0112] The action of Nicorandil is understood to be by the process of smoothing the smooth muscle of the blood vessels. The action is especially especially marked in case of the venous system.

[0113] Nicoradil acts by activating potassium channels, and by donating nitric oxide to activate the enzyme guanylate cyclase. The enzyme Guanylate cyclase causes activation of cGMP which in turn, leads to arterial and venous vasodilatation by de-phosphorylation of the myosin light chain. Being selective for vascular potassium channels, Nicorandil has no significant action on cardiac contractility and conduction.

[0114] Nicorandil can dilate the coronary vessels of a healthy individual, however, its effects on the coronary vessels of someone with ischaemic heart disease will be small as they will already be completely dilated. Instead, it dilates the venous system, reducing preload and the work of the heart.

[0115] Nicorandil, however, has not yet been investigated for use in the field of dermatology.

[0116] Piribedil--is the D2 agonist which is mainly used to treat Parkinson's disease. It acts by stimulating dopamine receptors thereby alleviating various symptoms like tremors. It is also used to treat other conditions like circulatory problems clue to its D2 antagonistic effects. The drug also comes under the brand name Trivastal that comes in the form of extended-release capsules which should be taken by mouth. Piribedil can be used as monotherapy or together with L-dopa therapy in early and advanced Parkinson's Disease. A lot of elderly patients have benefited because of its relative effects in cognition such as treating impaired memory, attention and focus.

[0117] Piribedil works by stimulating dopamine receptors present in the brain which in effect treats the deficit of the postsynaptic D2 and D3 receptors of the mesolimbic and mesocorticai pathways. The drug also has vasodilating effects thereby improving different cognitive symptoms and reinforces noradrenergic transmission resulting to improvement in focus, attention and memory.

[0118] Piribedil is also indicated in the treatment of pathological cognitive deficits in the elderly (impaired attention, motivation, memory, etc), treatment of dizziness in the elderly, treatment of retinal ischemic manifestations, adjuvant treatment in intermittent claudication due to peripheral vascular disease (PVD) of the lower limbs (stage 2), anhedonia and treatment-resistant depression in unipolar and bipolar depressives (off label).

[0119] Piribedil, however, has not yet been investigated for use in the field of dermatology.

[0120] Oxaprozin--is a member of the propionic acid group of nonsteroidal anti-inflammatory drugs (NSAIDs). The chemical name for oxaprozin potassium is 4,5-diphenyl-2-oxazolepropionic acid, potassium salt. Its empirical formula is C18H14NO3K and molecular weight is 331, Oxaprozin potassium is a white to off white powder with a melting point of 215.degree. C. It is slightly soluble in alcohol and very soluble in water. The PK in water is 9.7.

[0121] Oxaprozin is used to relieve the inflammation, swelling, stiffness, and joint pain associated, with osteoarthritis and rheumatoid arthritis.

[0122] Oxaprozin, however, has not yet been investigated for use in the field of dermatology.

[0123] Glycopyrrolate--is a quaternary ammonium salt with the chemical name: 3[(cyclopentylhydroxyphenylacetyl)oxy]-1,1-dimethyl pyrrolidinium bromide. The molecular formulas is C19H28BrNO3 and the molecular weight is 398.33.

[0124] Glycopyrrolate Injection is indicated for use as a preoperative antimuscarinic to reduce salivary, tracheobronchial, and pharyngeal secretions; to reduce the volume and free acidity of gastric secretions; and to block cardiac vagal inhibitory reflexes during induction of anesthesia and intubation. When indicated, Robinul Injection may be used intraoperatively to counteract surgically or drug-induced or vagal reflexes associated arrhythmias. Glycopyrrolate protects against the peripheral muscarinic effects (e.g., bradycardia and excessive secretions) of cholinergic agents such as neostigmine and pyridostigmine given to reverse the neuromuscular blockade due to non-depolarizing muscle relaxants.

[0125] Glycopyrrolate is indicated in Peptic Ulcer, for use in adults as adjunctive therapy for the treatment of peptic nicer when rapid anticholinergic effect is desired or when oral medication is not tolerated.

[0126] In anesthesia, glycopyrrolate injection can be used as a preoperative medication in order to reduce salivary, tracheobronchial, and pharyngeal secretions, as well as decreasing the acidity of gastric secretion. It is also used in conjunction with neostigmine, a neuromuscular blocking reversal agent, to prevent neostigmine's muscarinic effects such, as bradycardia. It is also used to reduce excessive saliva (sialorrhea), ft decreases acid secretion in the stomach and so may be used for treating stomach ulcers, in combination with other medications. Use in treating asthma and COPD has been described. It has been used, topically and orally to treat hyperhidrosis.

[0127] Glycopyrrolate, however, has not yet been investigated for use in the field of dermatology.

[0128] Granisetron--Granisetron hydrochloride, an antinauseant and antiemetic agent. Chemically it is endo-N-(9-methyl-9-azabicyclo [3.3.1]non-3-yl)-1-methyl-1H-indazole-3-carboxamide hydrochloride with a molecular weight of 348.9 (312.4 free base). Its empirical formula is C18H24N4.HCl.

[0129] Granisetron Indications: Granisetron hydrochloride is used for the prevention of nausea and vomiting associated with initial and repeat courses of emetogenic cancer therapy, including high-dose cisplatin. Chemotherapy-induced nausea and vomiting. 5-HT3 receptor antagonists are the primary drugs used to treat and prevent chemotherapy-induced nausea and vomiting. Many times they are given intravenously about 30 minutes before beginning therapy. Post-operative and post-radiation nausea and vomiting. Is a possible therapy for nausea and vomiting due to acute or chronic medical illness or acute gastroenteritis. Treatment of Cyclic vomiting syndrome although there are no formal trials to confirm efficacy. Nausea and vomiting associated with radiation, including total body irradiation and fractionated abdominal radiation.

[0130] Granisetron, however, has not yet been investigated for use in the field of dermatology.

[0131] Memantine--is an orally active NMDA receptor antagonist. The chemical name for memantine hydrochloride is 1-amino-3,5-dimethyladamantane hydrochloride.

[0132] Memantine hydrochloride is indicated for the treatment of moderate to severe dementia of the Alzheimer's type. Memantine is also being tested for generalized anxiety disorder, epilepsy, opioid dependence, systemic lupus erythematosus, depression, obsessive compulsive disorder, Tourette Syndrome, problem gambling, attention-deficit hyperactivity disorder (ADHD), glaucoma, tinnitus, neuropathic pain including Complex Regional Pain Syndrome, pervasive developmental disorders. HIV associated dementia, nystagmus, multiple sclerosis and autism.

[0133] Memantine, however, has not yet been investigated for use in the field of dermatology.

[0134] Nimodipine--belongs to the class of pharmacological, agents known as calcium, channel blockers. Nimodipine is isopropyl 2-methoxyethyl 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylate. It has a molecular weight of 418.5 and a molecular formula of C.sub.21H.sub.26N.sub.2O.sub.7.

[0135] Nimodipine is indicated for the improvement of neurological outcome by reducing the incidence and severity of ischemic deficits in patients with subarachnoid hemorrhage from ruptured intracranial berry aneurysms regardless of their post-ictus neurological condition (i.e., Hunt and Hess Grades I-V).

[0136] Nimodipine's main use is in the prevention of cerebral vasospasm and resultant ischemia, a complication of subarachnoid hemorrhage (a form of cerebral bleed), specifically from ruptured intracranial berry aneurysms irrespective of the patient's post-ictus neurological condition, its administration begins within 4 days of a subarachnoid hemorrhage and is continued for three weeks. If blood pressure drops by over 5%, dosage is adjusted. While nimodipine is not used in head injury currently, it has shown promise in clinical studies. A 2009 study (Asian A et al, February 2009 Pharmacol. Res. 59 (2): 120-4), found that patients with severe head trauma who were given nimodipine, via peripheral vein injection, along with the standard procedures had significantly higher cerebral perfusion pressure and jugular venous oxygen saturation, while intracranial pressure, jugular lactate and jugular glucose were lower. The study concluded that Glasgow outcome score values were higher, and that the cerebral metabolism was improved.

[0137] Nimodipine, however, has not yet been investigated for use in the field of dermatology.

[0138] Amlodipine--Amlodipine besylate is chemically described as 3-Ethyl-5-methyl(.+-.)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-d- ihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulphonate. Its empirical formula is C.sub.20H.sub.25ClN.sub.2O.sub.5.C.sub.6H.sub.6O.sub.3S.

[0139] Amlodipine Base (as besylate, mesylate or maleate) is a long-acting calcium channel blocker (dihydropyridine class) used as an anti-hypertensive and in the treatment of angina. Like other calcium channel blockers, amlodipine acts by relaxing the smooth muscle in the arterial wall, decreasing total peripheral resistance and hence reducing blood pressure.

[0140] Amlodipine is indicated for the treatment of hypertension. It may be used alone or in combination with, other antihypertensive agents. It is also indicated for Coronary Artery Disease (CAD). Amlodipine is indicated for the symptomatic treatment of chronic stable angina. Amlodipine may be used alone or in combination with, other antianginal agents.

[0141] Amlodipine is also indicated for the treatment of confirmed or suspected vasospastic angina. Amlodipine may be used as monotherapy or in combination with other antianginal agents.

[0142] In patients with recently documented Coronary Artery Disease (CAD) by angiography and without heart failure or an ejection fraction <40%, Amlodipine is indicated to reduce the risk of hospitalization due to angina and to reduce the risk of a coronary revascularization procedure.

[0143] Amlodipine, however, has not yet been investigated for use in the field of dermatology.

[0144] In an embodiment, antisense oligonucleotides and compositions of the present invention are used to prevent or treat diseases or disorders associated with FLG family members. Exemplary Filaggrin (FLG) mediated diseases and disorders which can be treated with cell/tissues regenerated from stem cells obtained using the antisense compounds comprise: a disease or disorder associated with abnormal function and/or expression of FLG, a dermatological disease or disorder, sign of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay fever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin.

[0145] In an embodiment, modulation of FLG by one or more antisense oligonucleotides and/or compositions of the present invention is administered to a patient in need thereof, to prevent or treat any disease or disorder related to FLG abnormal expression, function, activity as compared to a normal control.

[0146] In an embodiment, the composition of the present invention comprises one or more oligonucleotides specific for one or more Filaggrin (FLG) polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

[0147] In an embodiment the composition of the present invention comprises one or more oligonucleotides specific for one or more Filaggrin (FLG) polynucleotides and one or more FLG modulating molecule, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

[0148] One embodiment of the present, invention provides a composition, wherein the molecule is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

[0149] One embodiment of the present invention provides a composition for use in treatment of a dermatological disease or disorder, the composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable carrier.

[0150] One embodiment of the present invention provides a composition, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

[0151] One embodiment of the present invention provides a composition, wherein the composition further comprises art antisense oligonucleotide that modulates FLG expression or activity.

[0152] One embodiment of the present invention provides a composition, wherein the composition, further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

[0153] One embodiment of the present invention provides a composition, wherein the oligonucleotide comprises nucleotide sequences set form as SEQ ID NOS: 3 to 13.

[0154] One embodiment of the present invention provides a composition, wherein the oligonucleotide set forth as SEQ ID NOS: 3 to 13 comprise one or more modifications or substitutions.

[0155] One embodiment of the present invention provides a method of treating art FLG associated disease or disorder in a subject, the method comprises administering to the subject a composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable carrier.

[0156] One embodiment of the present invention provides a method, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

[0157] One embodiment of the present invention provides a method, wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity.

[0158] One embodiment of the present invention provides a method, wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

[0159] One embodiment of the present invention provides a method, wherein a disease associated with, the at least one Filaggrin polynucleotide is selected from: a dermatological disease or disorder, sign of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay fever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc.), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin.

[0160] One embodiment of the present invention provides a method of preventing or treating a skin condition associated with at least one Filaggrin (FLG) 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 a Filaggrin up-regulating compound, at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Filaggrin (FLG) polynucleotide and modulates expression of said at least one Filaggrin (FLG) polynucleotide and a pharmaceutically acceptable carrier; thereby preventing or treating the disease skin condition associated with the at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof.

[0161] One embodiment of the present invention provides a method, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

[0162] One embodiment of the present invention provides a method, wherein the skin condition is caused by caused by inflammation, light damage or aging.

[0163] One embodiment of the present invention provides a method, 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.

[0164] One embodiment of the present invention provides a use of the composition of claim 30, in the manufacture of a medicament for the treatment of a dermatological disease or disorder.

[0165] One embodiment of the present invention provides a use, wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity.

[0166] One embodiment of the present invention provides a use, wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

[0167] One embodiment of the present invention provides a use, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

[0168] One embodiment of the present invention provides a use wherein the dermatological disease or disorder is: 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.

[0169] 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 FLG status. A patient's FLG 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.

[0170] A preferred embodiment of the present invention provides a composition for skin treatment and/or a cosmetic application comprising the compounds of the present invention, e.g., to modulate expression of FLG in the skin. In embodiments, topical treatment by the compounds of the present invention, to increase cell lifespan of 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 of the present invention. 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.

[0171] In an embodiment of the present invention the composition 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.

[0172] The composition 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).

[0173] The composition 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..TM. from Beiersdorf, Inc. (Norwalk, Conn.).

[0174] The composition 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.

[0175] The composition 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 interracial 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.

[0176] The composition of the invention may be incorporated into gel formulations, which generally are semisolid systems consisting of either suspensions made rip 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 2-1.5%), 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.

[0177] 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.

[0178] 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..TM. from Whitby Research Incorporated, Richmond, Va.).

[0179] 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..TM.) and diethylene glycol monoethyl ether oleate (available commercially as Soficutol.sup..TM.); 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..TM.); 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.

[0180] 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.

[0181] 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).

[0182] The compositions, according to the present invention, can be applied most notably as a cosmetic or pharmaceutical composition for use on the skin, mucous membranes, and/or semi-mucous membranes. The compositions can be applied as skin protection and/or as skin care products, or as an anti-wrinkle and/or an anti-aging composition. We can also envision other applications in the domain of combined, compositions for example, with other active agents. We can also use the compounds according to the invention in the cosmetic compositions for body and hair health.

[0183] Moreover, the compounds according to the invention, such as previously defined, stimulate the metabolic functioning of skin cells. They allow protein synthesis to increase, which is essential, for its functioning, notably by increasing the synthesis of constitutive proteins of the extracellular matrix. The compounds, according to the invention, or the composition containing them, thus have a positive action on tissue regeneration. The compounds according to the invention are particularly efficient, in order to treat wound-healing disorders.

[0184] The compositions, intended to activate the endogenous synthesis of FLG proteins, previously defined, are used in or for the manufacture of pharmaceutical and/or cosmetic compositions, for topical use. They will be used, in a more general way, in order to treat, dermatological disorders.

[0185] Moreover, according to another aspect, compounds previously defined according to the invention, intended to activate the endogenous synthesis of FLG proteins in skin cells, are used for the manufacture of a medicament for the treatment of dermal conditions. The present invention also relates to the use of the compounds previously defined as medicaments.

[0186] Moreover, according to another aspect, the present invention relates to a cosmetic process of treatment for skin care and/or hair and nail care consisting of applying, to the surface of the skin, an effective amount of the active agent, such as previously defined, in order to obtain the desired action. The processes can notably be used in order to treat in a curative and/or preventive manner the signs of cutaneous aging, but also to protect the skin and/or hair and/or nails from external aggressions such as negative effects of radiation, and in particular UV radiation, or in order to combat the signs of cutaneous inflammation and irritation.

[0187] The process of cosmetic treatment related to the invention can be implemented notably by applying the cosmetic compositions defined above according to methods usually used for compositions, such as the application of creams, gels, serums, lotions, milks, shampoos, and sun protection creams, on skin or hair, and as a toothpaste applied to the gums. Particular modes of embodiment of this cosmetic treatment process also result from the preceding description.

[0188] The compounds of the present invention are useful in both therapeutic and non-therapeutic applications. In one embodiment compounds of the invention are used for therapeutic applications. In another embodiment compounds of the invention are used for non-therapeutic applications, such as cosmetic applications. Therapeutic applications of methods of the invention include means of diagnosing the cause of a medical skin condition. Accordingly the method of treatment for the medical skin condition can be tailored to complement the individual's phenotype. Therapeutic applications of methods of the invention also include means of determining whether an individual's skin is likely to react adversely to a pharmaceutical preparation, such as a topically administered pharmaceutical preparation. In that case the individual can be matched to a particular pharmaceutical preparation in order to provide maximum therapeutic benefit whilst minimizing or avoiding any undesirable effects on the condition of the individual's skin.

[0189] Non-therapeutic applications of methods of the invention include means of grouping individuals for the purposes of trials for agents, for example, cosmetics or any other form of preparation introduced to the body. This can be useful for interpreting the results obtained from such trials, for example where the reaction of the skin of different individuals during the trial is not uniform.

[0190] The heterogeneity of responses might be interpreted more clearly by grouping or stratifying individuals according to their predisposition to skin conditions. The skilled person, will appreciate that using this method it may be possible to develop agents that axe suitable for use with some individuals but not suitable with others. Accordingly a panel of agents can be built up, which panel includes different agents having suitability for use with, different individuals. Following the trials, individuals wishing to use such an agent can use a method of the invention to determine which agents are most suitable for use based on their own predisposition to skin conditions. Thus the method of the invention allows an individual to be matched with a personal care product such as those listed above.

[0191] Methods of identifying the profilaggrin genotype of an individual are performed, on biological material of the individual. Preferably the biological material is removed; from the individual prior to performing the method of identification. In other words, typically the biological material is ex vivo. The ex vivo material may be further cultured in vitro prior to performing the method.

[0192] An ex vivo sample may comprise tissue or cells taken from any part of the body. A preferred ex vivo sample comprises material taken from the circulatory system, or material taken from a bodily cavity, such as the oral cavity. A particularly preferred ex Vito sample is a saliva sample.

[0193] The alleles present in an individual can be determined from a saliva sample using methods known in the art, such as that described in Schie and Wilson (1997, Journal of Immunological Methods, 208, 91-101). Accordingly the ex vivo sample may be provided by an individual without need for specialized collection means. For example, a saliva sample or buccal swab can be simply provided, by the individual prior to testing.

[0194] The profilaggrin gene and protein are well known in the art and are described in Gan et al (1990, Biochemistry, 29, 9432-9440). Numerous profilaggrin sequences have been deposited in publicly accessible databases. A profilaggrin gene comprises multiple filaggrin repeals, usually 10, 11 or 12 repeats. The filaggrin repeats are typically of the same length (972 bp, 324 amino acids in humans) as each other, although this is less typical of filaggrin repeats at the 5'- and 3'-ends of the mRNA. The filaggrin repeats may display considerable sequence variation, typically of from 0-50%, more typically of from 2-30%, yet more typically of from 10-15%, between repeats on the same allele and between different alleles. Usually variations are attributable to a single-base change but may also involve a change in charge (Gan et al (1990) Biochemistry, 29, 9432-9440), A consensus amino acid sequence map of a human filaggrin repeat is known (Gan et al (1990) Biochemistry, 29, 9432-9440) and preferably a filaggrin repeat will have at least 50%, more preferably at least 75%, more preferably 90%, yet more preferably at least 95% sequence identity to that consensus sequence or a variant of the consensus sequence shown in Gan et al (1990, Biochemistry, 29, 9432-9440). Normally the amino acid sequences encoding the amino and carboxy termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene (Presland et al (1992) J Biol Chew, 267(33), 23772-23781).

[0195] In an embodiment, the oligonucleotides are specific for polynucleotides of FLG, which includes, without limitation noncoding regions. The FLG targets comprise variants of FLG; mutants of FLG, including SNPs; noncoding sequences of FLG: alleles, fragments and the like. Preferably the oligonucleotide is an antisense RNA molecule.

[0196] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to FLG polynucleotides alone but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of FLG.

[0197] In an embodiment, an oligonucleotide targets a natural antisense sequence (natural antisense to the coding and non-coding regions) of FLG targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense RNA or DNA molecule.

[0198] In an 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. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

[0199] 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%.

[0200] 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.

[0201] 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 complementarity 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 ease of in vitro assays, under conditions in which the assays are performed.

[0202] In an embodiment, targeting of FLG 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 NOS: 2, and the like, modulate the expression or function of FLG. In one embodiment, expression or function is up-regulated as compared to a control. In an embodiment, expression or function is down-regulated as compared to a control.

[0203] In an embodiment, oligonucleotides comprise nucleic acid sequences set form as SEQ ID NOS: 3 to 13 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 an embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative for 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.

[0204] 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, ft 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.

[0205] 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.

[0206] 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.

[0207] 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 Filaggrin (FLG).

[0208] 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.

[0209] In an embodiment, the antisense oligonucleotides bind to the natural antisense sequences of Filaggrin (FLG) and modulate the expression and/or function of FLG (SEQ ID NO: 1). Examples of antisense sequences include SEQ ID NOS: 2 to 13.

[0210] In an embodiment, the antisense oligonucleotides bind to one or more segments of Filaggrin (FLG) polynucleotides and modulate the expression and/or function of FLG. The segments comprise at least five consecutive nucleotides of the FLG sense or antisense polynucleotides.

[0211] In an embodiment, the antisense oligonucleotides are specific tor natural antisense sequences of FLG wherein binding of the oligonucleotides to the natural antisense sequences of FLG modulate expression and/or function of FLG.

[0212] In an embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID NOS: 3 to 13, 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 bands or internucleotide Linkages comprise phosphorothioate, phosphorodithioate or the like. In an 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.

[0213] 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 Filaggrin (FLG), regardless of the sequencers) 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).

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] In an 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.

[0219] In an embodiment, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule.

[0220] In an embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] Target segments 5-100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0227] 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 preferred 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 preferred 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 preferred 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 preferred target segments.

[0228] 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.

[0229] 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.

[0230] In one embodiment, it is preferred to target specific nucleic acids 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).

[0231] 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 so-called 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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 cm 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.

[0236] 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 "DMA-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.

[0237] In an 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.

[0238] 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). It is currently not known whether RNAa is conserved in other organisms.

[0239] 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 via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. However, in instances described in detail in the examples section which follows, oligonucleotides are shown to increase the expression and/or function of the Filaggrin (FLG) 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).

[0240] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen, for additional compounds that modulate the expression of Filaggrin (FLG) polynucleotides. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding FLG and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding sense or natural antisense polynucleotides of FLG 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 FLG polynucleotides, e.g. SEQ ID NOS: 3 to 13, 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 FLG polynucleotides, the modulator may then be employed in further investigative studies of the function of FLG polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0241] Targeting the natural antisense sequence preferably modulates the function of the target gene. For example, the FLG gene (e.g. accession number NM.sub.--002016). In an embodiment, the target is an antisense polynucleotide of the FLG gene. In an embodiment, an antisense oligonucleotide targets sense and/or natural antisense sequences of FLG polynucleotides (e.g. accession number NM.sub.--002016), 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 FLG polynucleotides.

[0242] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0243] 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, (hereby triggering enzymatic degradation of the target.

[0244] In an embodiment, an antisense oligonucleotide targets Filaggrin (FLG) polynucleotides (e.g. accession number NM.sub.--002016), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule.

[0245] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to FLG alone but extends to any of the isoforms, receptors, homologs and the like of FLG molecules.

[0246] In an embodiment, an oligonucleotide targets a natural antisense sequence of FLG polynucleotides, for example, polynucleotides set forth as SEQ ID NOS: 2, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 3 to 13.

[0247] In one embodiment, the oligonucleotides are complementary to or bind to nucleic acid sequences of FLG antisense, including without limitation noncoding sense and/or antisense sequences associated with FLG polynucleotides and modulate expression and/or function of FLG molecules.

[0248] In an embodiment, the oligonucleotides are complementary to or bind to nucleic acid sequences of FLG

[0249] natural antisense, set forth as SEQ ID NOS: 2 and modulate expression and/or function of FLG molecules.

[0250] In an embodiment, oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS: 3 to 13 and modulate expression and/or function of FLG molecules.

[0251] The polynucleotide targets comprise FLG, including family members thereof, variants of FLG; mutants of FLG, including SNPs; noncoding sequences of FLG; alleles of FLG; species variants, fragments and fire like. Preferably the oligonucleotide is an antisense molecule.

[0252] In an embodiment, the oligonucleotide targeting FLG ribonucleotides, 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).

[0253] In an embodiment, targeting of Filaggrin (FLG) polynucleotides, e.g. SEQ ID NOS: 2 modulate the expression or function of these targets. In one embodiment, expression or function is up-regulated as compared to a control. In an embodiment, expression or function is down-regulated as compared to a contro.

[0254] In an embodiment, antisense compounds comprise sequences set forth as SEQ ID NOS: 3 to 13. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.

[0255] In an embodiment, SEQ ID NOS: 3 to 13 comprise one or more LNA nucleotides.

[0256] 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.

[0257] Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman & McSwiggen, (1995) Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, (1995) J. Med. Chem. 38, 2023-2037). 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.

[0258] 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 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 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.

[0259] Several approaches such as in vitro selection (evolution) strategies (Orgel, (1979) Proc. R. Soc. London, B 205, 435) 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.

[0260] The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for die purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (keat) 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 DMA 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 RNA phosphodiester cleavage.

[0261] Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1987 (Uhlenbeck, O. C. (1987) Nature, 328; 596-600). The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.

[0262] 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.

[0263] 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.

[0264] In an 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] In one embodiment, the antisense compounds of tire invention have antisense portion 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.

[0269] 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.

[0270] In an 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.

[0271] 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%.

[0272] In an embodiment, the antisense oligonucleotides, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 2 to 13 comprise one or more substitutions or modifications, in one embodiment, the nucleotides are substituted with locked nucleic acids (LNA).

[0273] In an 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 FLG and the sequences set forth as SEQ ID NOS: 1 and 2. The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: 1 and 2.

[0274] Certain preferred 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 an 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.

[0275] 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.

[0276] In an 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 2''-Oalkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide. In other an embodiment, RNA modifications include 2'-fluoro, 2-amino and 2' O-methyl modifications on the ribose of pyrimidines, a basic 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 an 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.

[0277] Specific examples of some preferred oligonucleotides envisioned tor 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 preferred 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) Ace. Chem. Res. 28:366-374 are also preferred. Also preferred are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). In other an embodiment, 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. Preferred oligonucleotides comprise one of die following at the 2' position; OH, SB, SCH3, F, OCN, OCH3 OCH3, OCH3O(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; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkykimino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2-methoxyethoxy [2-O--CH2 CH2OCH3, also known as 2, --O-(2-methoxyethyl)]. Other preferred 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.

[0278] 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. and are presently preferred base substitutions.

[0279] 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, an aliphatic chain, e.g., dodecandiol or undecyl residues, 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.

[0280] 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.

[0281] In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited, to a basic 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.

[0282] 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.

[0283] 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 preferred 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.

[0284] Preferred modified oligonucleotide backbones comprise, but 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, thionoalkylphospbonates, 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 fee acid forms are also included.

[0285] 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.

[0286] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These 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.

[0287] Representative United States patents that teach the preparation of the above oligonucleotides 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.

[0288] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The 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.

[0289] In an 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--CH2- of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0290] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F.sup.-; 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 preferred 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. Oilier preferred 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, SB, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification comprises 2-methoxyethoxy(2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) i.e., an alkoxyalkoxy group. A further preferred 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--CH2OCH2-N(CH2)2.

[0291] Other preferred modifications comprise 2'-methoxy (2'-O CH3), 2'-aminopropoxy (2'-O CH2CH2CH2NH2) 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' position of 5' terminal nucleotide, Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures 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.

[0292] 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, S-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-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0293] 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. L, 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.about.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 preferred base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.

[0294] 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. Nos. 3,687,808, as well as 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.

[0295] 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.

[0296] Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-1 oxycholesterol moiety.

[0297] 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.

[0298] Drug Discovery

[0299] The compounds of the present invention can also be applied in the areas of drug discovery and target, validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between Filaggrin (FLG) polynucleotides and a disease state, phenotype, or condition. These methods include detecting or modulating FLG polynucleotides comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of FLG polynucleotides 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:

[0300] 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).

[0301] 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 glucuronidase (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.

[0302] FLG protein 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. FLG ELISA assay kits are available commercially, e.g., from R&D Systems (Minneapolis, Minn.).

[0303] In embodiments, FLG 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 FLG 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 FLG 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.

[0304] 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 FLG 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 FLG 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-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

[0305] 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.

[0306] 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.

[0307] 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 Filaggrin (FLG) genes. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.

[0308] 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.

[0309] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays, SAGE (serial analysis of gene expression), READS (restriction enzyme amplification of digested cDNAs), TOGA (total gene expression analysis), protein arrays and proteomics, expressed sequence tag (EST) sequencing, subtractive RNA fingerprinting (SuRF), subtractive cloning, differential display (DD), comparative genomic hybridization, FISH (fluorescent in situ hybridization) techniques and mass spectrometry methods.

[0310] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Filaggrin (FLG). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective FLG 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 FLG and in the amplification of said nucleic acid molecules for detection or for use in further studies of FLG. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding FLG 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 FLG in a sample may also be prepared.

[0311] 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.

[0312] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of FLG polynucleotides 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 FLG modulator. The FLG modulators of the present invention effectively modulate the activity of the FLG or modulate the expression of the FLG protein. In one embodiment, the activity or expression of FLG in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of FLG in an animal is inhibited by about 30%. More preferably, the activity or expression of FLG in an animal is inhibited by 50% or more. Thus, the oligomeric compounds modulate expression of Filaggrin (FLG) 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.

[0313] In one embodiment, the activity or expression of Filaggrin (FLG) and/or in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of FLG in an animal is increased by about 30%. More preferably, the activity or expression of FLG in an animal is increased by 50% or more. Thus, the oligomeric compounds modulate expression of FLG 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.

[0314] For example, the reduction of the expression of Filaggrin (FLG) 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 FLG peptides and/or the FLG protein itself.

[0315] 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.

Conjugates

[0316] 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. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in fee 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-3Hphosphonate, 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 drag, an antidiabetic, an antibacterial or an antibiotic.

[0317] 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.

Formulations

[0318] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,165; 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.

[0319] 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.

[0320] 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: 3 to 13) or in combination with a suitable protein, polysaccharide or lipid formulation.

[0321] 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 hemagglutination 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.

[0322] Additionally preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One preferred HIV-based viral vector comprises at least two vectors wherein the gag and pot 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.

[0323] 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.

[0324] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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 myoinositol, 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,` U.S. Pat. No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, `Parenteral delivery systems,` all incorporated herein by reference in their entirety.

[0329] 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, canonic 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.).

[0330] 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.

[0331] 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 carriers) 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.

[0332] 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.

[0333] 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.

[0334] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m 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.

[0335] 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. 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.

[0336] Liposomes also include "statically 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 liposome slacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are farther described in U.S. Pat. No. 6,287,860.

[0337] 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 tire art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0338] 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 farther described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0339] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0340] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. 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).

[0341] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof and their uses are further described in U.S. Pat. No. 6,287,860.

[0342] Compositions and formulations for oral administration, include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0343] 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.

[0344] 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 drags are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0345] 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 snore additional antisense compounds targeted to a second nucleic acid target. For example, the first target may be a particular antisense sequence of Filaggrin (FLG), 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 Filaggrin (FLG) 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:

[0346] 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 seventy 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 tire relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective 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 dally, 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.

[0347] In embodiments, a patient is treated with a dosage of drug that is at least about I, 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.

[0348] 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.

[0349] 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

[0350] The following non-limiting Examples serve to illustrate selected embodiments of the invention, ft 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 Filaggrin (FLG) and/or a Sense Strand of FLG Polynucleotide

[0351] 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 (it) capable of forming a stable duplex with a portion of an mRNA transcript of the targeted gene.

[0352] 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 fee present invention.

[0353] 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 ease of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the ease of in vitro assays.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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). 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.

[0358] To perform toe 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 LighyTyper instrument, Roche Diagnostics, Lewes, UK).

[0359] 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 FLG Polynucleotides

[0360] Treatment of HEPG2 Cell with Antisense Oligonucleotides

[0361] HepG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediated) cat #MT-10-010-CV)+10% FBS (Mediatech cat #MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-C1)) at 37.degree. C. and 5% CO.sub.2. 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% CO.sub.2 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; Hs00856927_g1 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). 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:

[0362] Real time PCR results show that the levels of FLG1 mRNA in HepG2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431 (FIG. 1).

Treatment of 518A2 Cells with Antisense Oligonucleotides

[0363] 518A2 cells obtained from Albert Einstein-Montefiore Cancer Center, NY 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 518A2 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.times. 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 ft 23105) 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: Hs00856927_g1 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). 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:

[0364] Real time PCR results show that the levels of FLG mRNA in 518A2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431 (FIG. 2). Another set of Real time PCR results show that the levels of FLG mRNA in 518A2 cells are significantly increased with two of the oligos designed to FLG1 antisense AK056431 (FIG. 3).

Example 3

Modulation of FLG Expression and Activity

[0365] Treatment of 518A2 Cells with Small Compounds

[0366] 518A2 cells were grown in a growth media [DMEM (Mediatech cat #10-013-CV)+5% FBS (Mediatech cat# MT35-011-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)] at 37.degree. C. with 5% CO.sub.2. One day before the experiment the cells were replaced at approximately 1.times.10 4/ml (or about 1/5 dilution from 90% confluency) into 6-well plates and incubated at 37.degree. C. and 5% CO.sub.2 overnight. On the day of the experiment the media in the 6-well plates was changed to 2 ml fresh growth media. Small compounds were diluted in DMSO to the concentration of 1000 uM. On the day of the experiment this solution was diluted 1:100 in fresh growth media. Pure DMSO was diluted in media at the same ratio (1:100) to treat vehicle control samples. To dose one well, 200 ul of the compound or pure DMSO solution was added directly to well of a 6-well plate. The final concentration of compounds was 1 uM. Dosing volume was adjusted if a different concentration of compound was desired. After dosing the plates were incubated overnight at 37.degree. C., 5% CO.sub.2. 24 h after addition, of small compounds the media was replaced with fresh growth media and the dosing was repeated as described above. 24 h after second dosing RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) following the manufacturers' instructions. 600 ng of total RNA was added to the reverse transcription reaction performed using High Capacity cDNA kit from Applied Biosystems (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 (for example, assay ID#Hs00856927_g1 for FLG). The following PCR cycle was used; 50.degree. C. for 2 mm, 95.degree. C. for 10 min, 40 cycles of (95.degree. C. for 15 seconds, 60.degree. C. for 1 min) using StepOne thermal cycler (ABI). The assay for 18S used to normalize the mRNA levels was manufactured by ABI (cat#4319413E), Fold change in gene expression after treatment with small compounds was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.

Results:

[0367] Real Time PCR results shows fold change in Filaggrin mRNA expression in 518A2 cells treated with the small molecules (FIG. 4)

Treatment of Primary Keratinocytes with Small Compounds

[0368] Primary keratinocytes (from Promocell) were grown in a growth media (Keratinocyte Growth Media, Promocell cat # C-20011) at 37.degree. C. with 5% CO2. One day before the experiment the cells were replated at approximately 5.times.10 4/ml (or about 1/3 dilution from 90% confluency) into 24-well collagen-coated plates (Beckton Dickinson BioCoat plates cat #35 6408) and incubated at 37.degree. C. and 5% CO2 overnight. On the day of the experiment the media in the 24-well plates was changed to 1 ml fresh growth media. Small compounds were diluted in DMSO to the concentration of 1000 uM. On the day of the experiment this solution was diluted 1:100 in fresh growth media. Pure DMSO was diluted in media at the same ratio (1:100) to treat vehicle control samples. To dose one well, 100 ul of the compound or pure DMSO solution was added directly to well of a 24-well plate. The final concentration of compounds was 1 uM. Dosing volume was adjusted if a different concentration of compound was desired. After dosing the plates were incubated overnight at 37.degree. C., 5% CO2. 24 h after addition of small compounds the media was replaced with fresh growth media and the dosing was repeated as described above, 24 h after second dosing RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) following the manufacturers' instructions. 600 ng of total RNA was added to the reverse transcription reaction performed using High Capacity cDNA kit from Applied Biosystems (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 (for example, assay ID#Hs00856927_g1 for FLG). 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 thermal cycler (ABI). The assay for 18S used to normalize the mRNA levels was manufactured by ABI (cat#4319413E). Fold change in gene expression after treatment with small compounds was calculated based on the difference in. 18S-normalized dCt values between treated and mock-transfected samples.

Results:

[0369] Real Time PCR results shows fold change in Filaggrin. mRNA expression in primary keratinocytes treated with the small molecules (FIG. 4). Another set of Real Time PCR results show that the levels of FLG Natural Antisense Transcripts in primary keratinocytes are significantly decreased after treatment with small molecules Bupropion, Bendipine and Topiramate (FIG. 5).

[0370] 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.

[0371] 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: 13 <210> SEQ ID NO 1 <211> LENGTH: 12747 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_002016 <309> DATABASE ENTRY DATE: 2010-08-08 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(12747) <400> SEQUENCE: 1 cttttggtga acaaggttca catttattgc caaaagatgt ctactctcct ggaaaacatc 60 tttgccataa ttaatctttt caagcaatat tcaaaaaaag ataaaaacac tgacacattg 120 agtaaaaaag agctgaagga acttctggaa aaggaatttc ggcaaatcct gaagaatcca 180 gatgacccag atatggttga tgtcttcatg gatcacttgg atatagacca caacaagaaa 240 attgacttca ctgagtttct tctgatggta ttcaagttgg ctcaagcata ttatgagtct 300 accagaaaag agaatttacc gatatcagga cacaagcaca gaaagcacag tcatcatgat 360 aaacatgaag ataataaaca ggaagaaaac aaagaaaaca gaaaaagacc ctcaagtctg 420 gaaagaagaa acaatagaaa agggaataag ggaagatcca agagcccaag agaaacaggg 480 gggaaaaggc atgaatctag ttctgaaaaa aaagaaagaa aaggatattc acctactcat 540 agagaagaag aatatggaaa aaaccatcat aactcaagta aaaaagagaa aaacaagact 600 gaaaatacta gattaggaga caataggaag aggctaagtg aaagacttga agagaaagaa 660 gacaatgaag aaggagtata tgattatgaa aatacaggaa gaatgactca aaaatggata 720 caatcaggcc atattgccac atattacaca atccaggatg aagcctatga caccactgat 780 agtctattag aagaaaacaa aatatatgaa agatcaaggt catctgatgg caaatcatca 840 tctcaagtga acaggtcaag acatgaaaat acaagccagg taccattgca ggagtccagg 900 acaagaaagc gtaggggatc cagagttagc caggacaggg acagtgaggg acactcagaa 960 gactctgaga ggcactctgg gtcggcttcc agaaaccatc atggatctgc gtgggagcag 1020 tcaagagatg gctccagaca ccccaggtcc catgatgaag acagagccag tcatgggcac 1080 tctgcagaca gctccagaca atcaggcact cgtcacgcag agacttcctc tcgtggacag 1140 actgcatcat cccatgaaca ggcaagatca agtccaggag aaagacatgg atccggccac 1200 cagcagtcag cagacagctc cagacactca gccactgggc gcgggcaagc ttcatctgca 1260 gtcagcgatc gtggacaccg ggggtctagc ggtagtcagg ccagtgacag tgagggacat 1320 tcagaaaact cagacacaca atcagtgtca ggccacggaa aggctgggct gagacagcag 1380 agccaccaag agtccacacg tggccggtca ggggaacggt ctggacgttc agggtcttcc 1440 ctctaccagg tgagcactca tgaacagcct gactctgccc atggacggac cgggaccagc 1500 actggaggaa gacaaggatc gcaccacgag caggcacgag acagctccag gcattcagcg 1560 tcccaagagg gtcaggacac cattcgtgga cacccggggt caagcagagg aggaaggcag 1620 ggatcccacc acgagcaatc ggtaaatagg tctggacact caggttccca tcacagccac 1680 accacatccc agggaaggtc tgatgcctcc catgggcagt caggatccag aagtgcaagc 1740 agacaaacac gaaatgagga acaatcagga gacggcacca ggcactcagg gtcacgtcat 1800 catgaagctt cctctcaggc tgacagctct agacactcac aggtgggcca gggacaatca 1860 tcggggccca ggacaagtag gaaccaggga tccagtgtta gccaggacag tgacagtcag 1920 ggacactcag aagactctga gaggtggtct gggtctgctt ccagaaacca tcatggatct 1980 gctcaggagc agtcaagaga tggctccaga caccccaggt cccatcacga agacagagct 2040 ggtcatgggc actctgcaga cagctccaga aaatcaggca ctcgtcacac acagaattcc 2100 tctagtggac aggctgcgtc atcccatgaa caggcaagat caagtgcagg agaaagacat 2160 ggatcccgcc accagctcca gtcagcagac agctccagac actcaggcac tgggcacgga 2220 caagcttcat ctgcagtcag agacagtgga caccgagggt ccagtggtag tcaggccact 2280 gacagtgagg gacattcaga agactcagac acacagtcag tgtcaggcca tggacaggct 2340 ggtcaccatc agcagagcca ccaagagtcc gcacgtgacc ggtcagggga aaggtctcga 2400 cgttcagggt ctttcctcta ccaggtgagc actcataaac agtctgagtc ctcccatgga 2460 tggacagggc ccagcactgg agtaagacaa ggatcccacc atgagcaggc acgagacaac 2520 tccaggcact cagcatccca agatggtcag gacaccattc gtggacaccc ggggtcaagc 2580 agaagaggaa ggcaggggtc ccaccacgag caatcggtag ataggtctgg acactcaggg 2640 tcccatcaca gccacaccac atcccaggga aggtctgatg cctcccgtgg gcagtcagga 2700 tccagaagtg caagcagaac aacacgtaat gaggaacaat caagagacgg ctccaggcac 2760 tcagggtcac gtcaccatga agcttcctct catgccgaca tctctagaca ctcacaggca 2820 ggccagggac aatcagaggg gtccaggaca agcaggcgcc agggatccag tgttagccag 2880 gacagtgaca gtgagggaca ttcagaagac tctgagaggt ggtctgggtc tgcttccaga 2940 aaccatcgtg gatctgctca ggagcagtca agacatggct ccagacaccc caggtcccat 3000 cacgaagaca gagccggtca cgggcactct gcagacagct ccagacaatc aggaactcct 3060 cacgcagaga cttcctctgg tggacaggct gcgtcatccc atgaacaggc aagatcaagt 3120 ccaggagaaa gacacggatc ccgccaccag cagtcagcag acagctccag acactcaggc 3180 attccgcgca gacaagcttc atctgcagtc agagacagtg gacactgggg gtccagtggt 3240 agtcaggcca gtgatagtga gggacattca gaggagtcag acacacagtc agtgtcaggc 3300 catggacagg atgggcccca tcagcagagc caccaagagt ccgcacgtga ctggtcaggg 3360 ggaaggtctg gacgttcagg gtctttcatc taccaggtga gcactcatga acagtctgag 3420 tctgcccatg ggcggaccag gaccagcact ggacgaagac aaggatccca ccacgagcag 3480 gcacgagaca gctccaggca ctcagcgtcc caagagggtc aggacaccat tcgtgcacac 3540 ccggggtcaa ggagaggagg aaggcaggga tcccaccatg agcaatcggt agatagatct 3600 ggacactcag ggtcccatca cagccacacc acatcccagg gaaggtctga tgcctcccat 3660 gggcagtcag gatccagaag tgcaagcaga caaactcgta aggacaaaca atcaggagac 3720 ggctccaggc actcagggtc acgtcaccat gaagctgcct cttgggctga cagctctaga 3780 cactcacagg tgggacagga acaatcatcg gggtccagga caagcaggca ccagggatcc 3840 agtgttagcc aggacagtga cagtgagaga cactcagacg actccgagag gttgtctggg 3900 tctgcttcca gaaaccatca tggatcttct cgggagcagt caagagatgg ctccagacac 3960 cctgggttcc atcaagaaga cagagccagt cacgggcact ctgcagacag ctccagacaa 4020 tcaggcactc atcacacaga gtcttcctct catggacagg ctgtgtcatc ccatgaacag 4080 gcaagatcaa gtccaggaga aagacatgga tcccgccacc agcagtcagc agacagctcc 4140 agacactcag gcattgggca cagacaagct tcatctgcag tcagagacag tggacaccga 4200 gggtccagtg gtagtcaggt cactaacagt gagggacatt cagaagactc agacacacag 4260 tcagtgtcag cccacggaca agctgggccc catcagcaga gccacaaaga gtccgcacgt 4320 ggccagtcag gggaaagctc tggacgttca aggtctttcc tctaccaggt gagctctcat 4380 gaacagtctg agtccacaca cggacagact gcacccagca ctggaggaag acaaggatcc 4440 cgccatgagc aggcacgaaa cagctctagg cactcagcat cccaagacgg tcaggacacc 4500 attcgtggac acccggggtc aagcagagga ggaaggcagg gatcctacca cgagcaatca 4560 gtagataggt ctggacactc agggtaccat cacagccaca ccacacccca gggaaggtct 4620 gatgcctccc atgggcagtc aggacccaga agtgcaagca ggcaaacaag aaatgaggaa 4680 caatcaggag acggctccag gcactcaggg tcacgtcacc atgaaccttc cactcgggcc 4740 ggcagctcta gacactcaca ggtgggccag ggagaatcag cggggtccaa gacaagcagg 4800 cgccagggat ccagtgttag tcaggacagg gacagtgagg gacactcaga agactctgag 4860 aggcggtctg agtcggcttc cagaaaccat tatggatctg ctcgggagca gtcaagacat 4920 ggctccagga accccaggtc ccatcaagaa gatagagcca gtcatgggca ctctgcagag 4980 agctccagac aatcaggcac tcgtcatgca gagacttcct ctggtggaca ggctgcatca 5040 tcccaggaac aggcaaggtc aagtccagga gaaagacatg gatcccgcca ccagcagtca 5100 gcagacagct ccacagactc aggcactggg cgcagacaag attcatctgt agtcggagac 5160 agtggaaacc gagggtccag tggtagccag gccagtgaca gcgagggaca ctcagaagag 5220 tcagacacac agtcagtgtc agcccacgga caggctgggc cccatcagca gagccaccaa 5280 gagtccacac gtggccagtc aggggaaagg tctggacgtt cagggtcttt cctctaccag 5340 gtgagcactc atgaacagtc tgagtccgcc catggacgca cagggcccag cactggagga 5400 agacaaagat cccgccacga gcaggcacga gacagctcca ggcactcagc gtcccaagag 5460 ggtcaggaca ccattcgtgg acacccaggg tcaagcagag gaggaaggca gggatcccac 5520 tatgagcaat cggtagatag ttctggacac tcagggtctc atcacagcca caccacgtcc 5580 caggaaaggt ctgatgtctc ccgtgggcag tcaggatcca gaagtgtcag cagacaaaca 5640 cgtaatgaga aacaatcagg agacggctcc aggcactcag ggtcgcgtca ccatgaagct 5700 tcctctcggg ccgacagctc tagacactcg caggtgggcc agggacaatc atcagggccc 5760 aggacaagca ggaaccaggg atccagtgtt agccaggaca gtgacagtca gggacactca 5820 gaagactctg agaggtggtc tgggtctgct tccagaaacc atcttggatc tgcttgggag 5880 cagtcaagag atggctccag acaccctggg tcccatcacg aagacagagc cggtcacggg 5940 cactctgcag acagctccag acaatcaggc actcgtcaca cagagtcttc ctctcgtgga 6000 caggctgcgt catcccatga acaggcaaga tcaagtgcag gagaaagaca tggatcccac 6060 caccagctcc agtcagcaga cagctccaga cactcaggca ttgggcatgg acaagcttca 6120 tctgcagtca gagacagtgg acaccgaggg tacagtggta gtcaggccag tgacagtgag 6180 ggacattcag aagactcaga cacacagtca gtgtcagcac agggaaaagc tgggccccat 6240 cagcagagcc acaaagagtc cgcacgtggc cagtcagggg aaagctctgg acgttcaggg 6300 tctttcctct accaggtgag cactcatgaa cagtctgagt ccacccatgg acagtctgcg 6360 cccagcactg gaggaagaca aggatcccat tatgatcagg cacaagacag ctccaggcac 6420 tcagcatccc aagagggtca ggacaccatt cgtggacacc cggggccaag cagaggagga 6480 agacaggggt cccaccaaga gcaatcggta gataggtctg gacactcagg gtctcatcac 6540 agccacacca catcccaggg aaggtctgat gcctcccgtg ggcagtcagg atccagaagt 6600 gcaagcagaa aaacatatga caaggaacaa tcaggagatg gctctaggca ctcagggtcg 6660 catcatcatg aagcttcctc ttgggccgac agctctagac actcactggt gggccaggga 6720 caatcatcag ggcccaggac aagcaggccc cggggatcca gtgttagcca ggacagtgac 6780 agtgagggac actcagaaga ttctgagagg cggtctgggt ctgcgtccag aaaccatcat 6840 ggatctgctc aggagcagtc aagagatggc tccagacacc ccaggtccca tcacgaagac 6900 agagccggtc atgggcactc tgcagagagc tccagacaat caggcactca tcatgcagag 6960 aattcctctg gtggacaggc tgcatcatcc catgaacagg caagatcaag tgcaggagag 7020 agacacggat cccaccacca gcagtcagca gacagctcca gacactcagg cattgggcac 7080 ggacaagctt catctgcagt cagagacagt ggacaccgag ggtccagtgg tagtcaggcc 7140 agtgacagtg agggacattc agaagactca gacacacagt cagtgtcagc ccacggacag 7200 gctgggcccc atcagcagag ccaccaagag tccacacgtg gccggtcagc aggaaggtct 7260 ggacgttcag ggtctttcct ctaccaggtg agcactcatg aacagtctga gtccgcccat 7320 ggacggaccg ggaccagcac tggaggaaga caaggatccc accacaagca ggcacgagac 7380 agctccaggc actcaacgtc ccaagagggt caggacacca ttcatggaca cccggggtca 7440 agcagtggag gaaggcaggg atcccactac gagcaattgg tagatagatc tggacactca 7500 gggtctcatc acagccacac cacatcccag ggaaggtctg atgcctccca tgggcactca 7560 ggatccagaa gtgcaagcag acaaactcgt aacgatgaac aatcaggaga cggctccagg 7620 cactcagggt cgcgtcacca tgaagcttcc tctcgggccg acagctctgg acactcgcag 7680 gtgggccagg gacaatcaga ggggcccagg acaagcagga actggggatc cagttttagc 7740 caggacagtg acagtcaggg acactcagaa gactctgaga ggtggtctgg gtctgcttcc 7800 agaaaccatc atggatctgc tcaggagcag ctaagagatg gctccagaca ccccaggtcc 7860 catcaagaag acagagctgg tcatgggcac tctgcagaca gctccagaca atcaggcact 7920 cgtcacacac agacttcctc tggtggacag gctgcatcat cccatgaaca ggcaagatca 7980 agtgcaggag aaagacatgg atcccaccac cagcagtcag cagacagctc cagacactca 8040 ggcattgggc acggacaagc ttcatctgca gtcagagaca gtggacaccg agggtacagt 8100 ggtagtcagg ccagtgacaa tgagggacat tcagaagact cagacacaca gtcagtgtca 8160 gcccacggac aggctgggtc ccatcagcag agccaccaag agtccgcacg tggccggtca 8220 ggggaaacgt ctggacattc aggatctttc ctctaccagg tgagcactca tgaacagtct 8280 gagtcctccc atggatggac ggggcccagc actagaggaa gacaaggatc ccgccatgag 8340 caggcacaag acagctccag gcactcagca tcccaagacg gtcaggacac cattcgtgga 8400 cacccggggt caagcagagg aggaaggcag gggtaccacc acgagcattc ggtagatagc 8460 tctggacact cagggtccca tcacagccac accacatccc agggaaggtc tgatgcctcc 8520 cgtgggcagt caggatccag aagtgcaagc agaacaacac gtaatgagga acaatcagga 8580 gacggctcca ggcactcagg gtcgcgtcac catgaagctt ccactcatgc cgacatctct 8640 agacactcac aggcagtcca gggacaatca gaggggtcca ggagaagcag gcgccaggga 8700 tccagtgtga gccaggacag tgacagtgag ggacattcag aagactctga gaggtggtct 8760 gggtctgctt ccagaaacca tcatggatct gctcaggagc agctaagaga tggctccaga 8820 caccccaggt cccatcaaga agacagagct ggtcatgggc actctgcaga cagctccaga 8880 caatcaggca ctcgtcacac acagacttcc tctggtggac aggctgcatc atcccatgaa 8940 caggcaagat caagtgcagg agaaagacat ggatcccacc accagcagtc agcagacagc 9000 tccagacact caggcattgg gcacggacaa gcttcatctg cagtcagaga cagtggacac 9060 cgagggtaca gtggtagtca ggccagtgac aatgagggac attcagaaga ctcagacaca 9120 cagtcagtgt cagcccacgg acaggctggg tcccatcagc agagccacca agagtccgca 9180 cgtggccggt caggggaaac gtctggacat tcaggatctt tcctctacca ggtgagcact 9240 catgaacagt ctgagtcctc ccatggatgg acggggccca gcactagagg aagacaagga 9300 tcccgccatg agcaggcaca agacagctcc aggcactcag catcccaata cggtcaggac 9360 accattcgtg gacacccggg gtcaagcaga ggaggaaggc aggggtacca ccacgagcat 9420 tcggtagata gctctggaca ctcagggtcc catcacagcc acaccacatc ccagggaagg 9480 tctgatgcct cccgtgggca gtcaggatcc agaagtgcaa gcagaacaac acgtaatgag 9540 gaacaatcag gagacagctc caggcactca gtgtcacgtc accatgaagc ttccactcat 9600 gccgacatct ctagacactc acaggcagtc cagggacaat cagaggggtc caggagaagc 9660 aggcgccagg gatccagtgt gagccaggac agtgacagtg agggacattc agaagactct 9720 gagaggtggt ctgggtctgc ttccagaaac catcgtggat ctgttcagga gcagtcaagg 9780 cacggctcca gacaccccag gtcccatcac gaagacagag ccggtcacgg gcactctgca 9840 gaccgctcca gacaatcagg cactcgtcac gcagagactt cctctggtgg acaggctgca 9900 tcatcccatg aacaggcaag atcaagtcca ggagagagac acggatcccg ccaccagcag 9960 tcagcagaca gctccagaca ctcaggcatt ccgcgtggac aagcttcatc tgcagtcaga 10020 gacagtagac actgggggtc cagtggtagt caggccagtg atagtgaggg acattcagaa 10080 gagtcagaca cacagtcagt gtcaggccat ggacaggctg ggccccatca gcagagccac 10140 caagagtccg cacgtgaccg gtcaggggga aggtctggac gttcagggtc tttcctctac 10200 caggtgagca ctcatgaaca gtctgagtct gcccatgggc ggaccaggac cagcactgga 10260 cgaagacaag gatcccacca cgagcaggca cgagacagct ccaggcactc agcgtcccaa 10320 gagggtcagg acaccattcg tggacacccg gggtcaagca gaagaggaag gcagggatcc 10380 cactacgagc aatcggtaga taggtctgga cactcagggt cccatcacag ccacaccaca 10440 tcccagggaa ggtctgatgc ctcccgtggg cagtcaggat ccagaagtgc cagcagacaa 10500 actcgtaatg acgaacaatc aggagatggc tccaggcact catggtcgca tcaccatgaa 10560 gcttccactc aggcggacag ctctagacac tcacagtccg gccagggaca atcagcgggg 10620 cccaggacaa gcaggaacca gggatccagt gttagccagg acagtgacag tcagggacac 10680 tcagaagact ctgagaggtg gtctgggtct gcttccagaa accatcgtgg atctgctcag 10740 gagcagtcaa gagatggctc cagacacccc acgtcccatc acgaagacag agccggtcac 10800 gggcactctg cagagagctc cagacaatca ggcactcatc atgcagagaa ttcctctggt 10860 ggacaggctg catcatccca tgaacaggca agatcaagtg caggagagag acatggatcc 10920 caccaccagc agtcagcaga cagctccaga cactcaggca ttgggcacgg acaagcttca 10980 tctgcagtca gagacagtgg acaccgaggg tccagtggta gtcaggccag tgacagtgag 11040 ggacattcag aagactcaga cacacagtca gtgtcagccc acggacaggc tgggccccat 11100 cagcagagcc accaagagtc cacacgtggc cggtcagcag gaaggtctgg acgttcaggg 11160 tctttcctct accaggtgag cactcatgaa cagtctgagt ctgcccatgg acgggctggg 11220 cccagtactg gaggaagaca aggatcccgc cacgagcagg cacgagacag ctccaggcac 11280 tcagcgtccc aagagggtca ggacaccatt cgtggacacc cggggtcaag gagaggagga 11340 agacagggat cctaccacga gcaatcggta gataggtctg gacactcagg gtcccatcac 11400 agccacacca catcccaggg aaggtctgat gcctcccatg ggcagtcagg atccagaagt 11460 gcaagcagag aaacacgtaa tgaggaacag tcaggagacg gctccaggca ctcagggtcg 11520 cgtcaccatg aagcttccac tcaggctgac agctctagac actcacagtc cggccagggt 11580 gaatcagcgg ggtccaggag aagcaggcgc cagggatcca gtgttagcca ggacagtgac 11640 agtgaggcat acccagagga ctctgagagg cgatctgagt ctgcttccag aaaccatcat 11700 ggatcttctc gggagcagtc aagagatggc tccagacacc ccggatcctc tcaccgcgat 11760 acagccagtc atgtacagtc ttcacctgta cagtcagact ctagtaccgc taaggaacat 11820 ggtcacttta gtagtctttc acaagattct gcgtatcact caggaataca gtcacgtggc 11880 agtcctcaca gttctagttc ttatcattat caatctgagg gcactgaaag gcaaaaaggt 11940 caatcaggtt tagtttggag acatggcagc tatggtagtg cagattatga ttatggtgaa 12000 tccgggttta gacactctca gcacggaagt gttagttaca attccaatcc tgttgttttc 12060 aaggaaagat ctgatatctg taaagcaagt gcgtttggta aagatcatcc aaggtattat 12120 gcaacgtata ttaataagga cccaggttta tgtggccatt ctagtgatat atcgaaacaa 12180 ctgggattta gtcagtcaca gagatactat tactatgagt aagaaattaa tggcaaagga 12240 attaatccaa gaatagaaga atgaagcaag ttcactttca atcaagaaac ttcataatac 12300 tttcagggaa gttatctttt cctgtcaatc tgtttaaaat atgctatagt atttcattag 12360 tttggtggta gcttattttt attgtgtaat gatctttaaa cgctatattt cagaaatatt 12420 aaatggaaga aatcaatatc atggagagct aactttagaa aactagctgg agtattttag 12480 gagattctgg gtcaagtaat gttttatgtt tttgaaagtt taagttttag acactcccca 12540 aatttctaaa ttaatctttt tcagaaatat cgaaggagcc aaaaatataa aacagttctg 12600 tataccaaag tggctatatc aacatcaggg ctagcacatc tttctctatt atccttctat 12660 tggaattcta gtattctgta ttcaaaaaat catcttggac ataattaata ttatagtaag 12720 ctgcatctaa attaaaaata aactatt 12747 <210> SEQ ID NO 2 <211> LENGTH: 4629 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 aaatgctcca catggcccca gtcaccctgc agaaaagaaa tcatcagctt tctgcagcat 60 cagacttagc agcatcttcc tgtcccaaga gaagagggga tgggaggatg gcatggttca 120 gggcttaggg gaggataggg gagaaaaagt ggaagcaaag ggagaggccc aggaaggatc 180 ttgttgcgac atgttacctg ggctgcacct gctgtatgca agagaagagg ctgggagtcc 240 cctctgcagg gtgttcatgc cccatctctg tgccacgagg agttgggctg ctgaaggata 300 tatcagcaca acttctctgc tccaactagt ctacattgca aagcatctcc tagtgtcttt 360 ggaagaagga tctatgtcat gagtgctcac ctggtagagg gaagaccctg aacgtccaga 420 ccgttcccct gaccggccac gtgtggactc ttggtggctc tgctgtctca gcccagcctt 480 tccgtggcct gacactgatt gtgtgtctga gttttctgaa tgtccctcac tgtcactggc 540 ctgactaccg ctagaccccc ggtgtccacg atcgctgact gcagatgaag cttgcccgcg 600 cccagtggct gagtgtctgg agctgtctgc tgactgctgg tggccggatc catgtctttc 660 tcctggactt gatcttgcct gttcatggga tgatgcagtc tgtccacgag aggaagtctc 720 tgcgtgacga gtgcctgatt gtctggagct gtctgcagag tgcccatgac tggctctgtc 780 ttcatcatgg gacctggggt gtctggagcc atctcttgac tgctcccacg cagatccatg 840 atggtttctg gaagccgacc cagagtgcct ctcagagtct tctgagtgtc cctcactgtc 900 cctgtcctgg ctaactctgg atcccctacg ctttcttgtc ctggactcct gcaatgactc 960 ataatatgct tgagccaact tgaataccat cagaagaaac tcagtgaagt caattttctt 1020 gttgtggtct atatccaagt gatccatgaa gacatcaacc atatctgggt catctggatt 1080 ctgtacagag ggaagtcaca gagggagact gcatcagaca gaatcacatt atcagccaat 1140 taattcaaag ttaatttaag gaacctgatc tttgagtatt aaaaagtggg acaaaatctt 1200 ttttttttta agactttttt gtctcatcct cattcaggtg ttatatgtga acaaagatgt 1260 agactagtaa ggtatcaaga tttgatgggg atatccagac aacttggagc caagtcccca 1320 cagctggatc agacaacttt tggtgacatc tcacagtgtg gattatcaag ccagtgtatc 1380 agtctggagc ctaggttttt ttgcctttag ctttgctcag gatgacttct gctcttctat 1440 ggcaatactt gagaagctac agctttgatt agacgagagg ctatttcaat aactttctcc 1500 tgataagaag accaccgacc gtggacggat tctggccagt ttacaaaggc tgcataccat 1560 agggcagggc accccactat ggcaaacact aataatgata ttgctcctga ttttgtttct 1620 gaagggaatg cctctaagat ttcttcatta attataagag actgggtctc actctgttgc 1680 ccaagctgga gaacagtggc accatcatag ctcactgcag ccttcaactt ctgggctcaa 1740 gtgcttctcc cacctcagcc tcccaagtag ctgggacaac aggtgaaatt gaaggccagc 1800 ttttggaact caaccttcaa aggaacttaa aatttccatt atgtagagtc cctgaggaaa 1860 ctggatccac tagctacact ggctgagaat gctctcaccc caggatttca tttttatttt 1920 accacatcat tgttggtatc ttaagatcaa tgtactggag cagcggggtc tcacaaagag 1980 gatacctggt gacacgccca ggctttcaga atgcactata gggctttagt gacttgtcca 2040 ggttcagctg aaatgtgcca tctgggacct actaatgctt gttgaacttt actggatctc 2100 agaagctggg tctcagaagc tcagatgttc ctcagagcta cgatatcaac aacctgtgat 2160 gacagagaga aggttgctcc atgatggatt tggaaactta ctggctagct aaacctgact 2220 gaatgggaag gaatgtggat agctttggaa ctctagtttc actagatgag ctggaatttg 2280 tattttgaca aattgcacgt tatgattatt aagtaatgca actgattttt ttttccctta 2340 aaacaaacaa tctagaatct gtgtaatcaa aataatttct ctaaaaggct gcaagtatat 2400 gcttaaagtg ttggggcatt cagagcattt ggaacattac attcttttga atgtcaattg 2460 gtagatgaaa ataccagctt ttaagtcata catttgattt tttgaaacaa tatgcattta 2520 gagtttgtaa gtcaagtgaa taactgataa ggtaaaaaaa agggggagtt cattgttgag 2580 tatgaattta aagtaaccag actgcctttt gtccagtggc tgtcagtaat ttacttcagc 2640 aggcattttt tttttttgag gctgttctat gatatcatga cccttcttgt aggaatgtgc 2700 ttccagtggt gaggcagtct gagaatgtgt gaagcagtat aatgaagcca gaccaagatg 2760 gaagcttggc ctgggatttg agcatcagga aaactgttga agggttatgt atacatcaca 2820 cacacacaca cacacacaca cacacacaca cacacacaca ctctcttcca tactcctata 2880 acaacgtata gtatttacta tacctggtga caggtattta ctatacattg ttgaagatct 2940 ataacatgga atgcctagtg ctaagtgcag tgtccttagt gaaattgtat tggtttggaa 3000 ttatttatga gtttgggatt atttgtcact acccttaaat gatcttgaca ctcacctatt 3060 tgaaaagata ttgaggattt gccatttgat attgaccagg gtgtattgtg cacaaatatt 3120 gtgaatatac atctgtctgt ccttaaatca ctgtaagttt taactggaat agatttgctc 3180 cacattactc ggtagggctg atatttcatg cctcatggat gagaaaagaa taggcaaaaa 3240 ttatatctcg ggctgcctca cacatctttt aacaggataa gggaaaaata aatataagac 3300 ctatgagtta tggcatcagg cttgaacttt aatttatgaa ttaaaccaac aatattattg 3360 attattgcaa ttatctatct taatttcatt tgttctcctt ttaaaaatta attatgttta 3420 ttttacctat tgtgaataaa gtcactcctc ccatgtgctt atttcttgac tctggcacta 3480 attattcttc agtgcctaga tttcctggga ctgctcagtt taagtaactt ctgaacattg 3540 gtctctgaga gaaaagacag tgaagtggga tctgtggaaa aacacatctg caaatgtctc 3600 atttttttct tcaggggtaa gttttcttcc ttggagactc ttgtcttgaa aaagtgatac 3660 tagatcacca tttggcaaga taattacaaa aggcagatag agtaattatt ttacatagct 3720 gttatagggt gactaagatc tcagggaatg actgtgttct gtaatccact tgccccactc 3780 aataccccaa gttttataca agccaaagat gcttggagca gaatctgcta cctgaggctc 3840 cagggccttg gcctagggaa actaatactt aactttttaa gtggtcattg atgacatccc 3900 actcctttct gagatgggta tgatttgaaa ggcaggtgta ttcatggaga gcagggcttg 3960 ggacctttgc tgcaagagga ggaactatta gcctagactt ttctaataag gactttctca 4020 tacctgtttc ctagatctgg tccacataga agaaatcagg gaacacacag ggtaggactc 4080 ttaaaggagt atgtttcttg gctttttcct aaaaactaaa acaaaaacat tcatagcaaa 4140 gcagtttagc ccagtgacta agagtataga cattggaatc tgaaagatct gggttggaat 4200 tctgcttcag ccatttacta attatgtaac ttcacatctc tgagaatggg ggaaattttc 4260 tttgcagggt tgttacaaag cttaaaagag ttaagtcaag tgcctggaac agaacagcca 4320 ttcaataaat catagaaatt tttgttttta cacaaatttt taaaaagtaa tattttttgc 4380 atttgttaga aagactatat tttctgttta gtggatgaca tcatcatcat aaaaatcact 4440 gttacaagtt ttgttttgag aattaaaatc atatattaat cccaatcaca ttgtgttggg 4500 attaatcaac atctctgggc atctaatact gtaaaaacat ggcctgtgat tggtctgctt 4560 ggaagtgcat cttggttctg gctcagttgg ctagtgggcc acattgatta aagatgaata 4620 caatgccaa 4629 <210> SEQ ID NO 3 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 3 ctccctctgt gacttccctc t 21 <210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 4 gctatccaca ttccttccca 20 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 5 gcagagccac caagagtcca 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 6 gtgtgtgtgt gtgtgtgtgt 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 7 tccacattcc ttcccattca 20 <210> SEQ ID NO 8 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 8 ccctctgtga cttccctc 18 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 9 ccacattcct tcccattcag 20 <210> SEQ ID NO 10 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 10 mumcmcmcmu ctgtgacttc mcmcmumcmu 30 <210> SEQ ID NO 11 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 11 mcmumcmcmc tctgtgactt cmcmcmumcm u 31 <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 ctccctctgt gacttccctc t 21 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 13 tccacattcc ttcccattca 20

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 13 <210> SEQ ID NO 1 <211> LENGTH: 12747 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: NM_002016 <309> DATABASE ENTRY DATE: 2010-08-08 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(12747) <400> SEQUENCE: 1 cttttggtga acaaggttca catttattgc caaaagatgt ctactctcct ggaaaacatc 60 tttgccataa ttaatctttt caagcaatat tcaaaaaaag ataaaaacac tgacacattg 120 agtaaaaaag agctgaagga acttctggaa aaggaatttc ggcaaatcct gaagaatcca 180 gatgacccag atatggttga tgtcttcatg gatcacttgg atatagacca caacaagaaa 240 attgacttca ctgagtttct tctgatggta ttcaagttgg ctcaagcata ttatgagtct 300 accagaaaag agaatttacc gatatcagga cacaagcaca gaaagcacag tcatcatgat 360 aaacatgaag ataataaaca ggaagaaaac aaagaaaaca gaaaaagacc ctcaagtctg 420 gaaagaagaa acaatagaaa agggaataag ggaagatcca agagcccaag agaaacaggg 480 gggaaaaggc atgaatctag ttctgaaaaa aaagaaagaa aaggatattc acctactcat 540 agagaagaag aatatggaaa aaaccatcat aactcaagta aaaaagagaa aaacaagact 600 gaaaatacta gattaggaga caataggaag aggctaagtg aaagacttga agagaaagaa 660 gacaatgaag aaggagtata tgattatgaa aatacaggaa gaatgactca aaaatggata 720 caatcaggcc atattgccac atattacaca atccaggatg aagcctatga caccactgat 780 agtctattag aagaaaacaa aatatatgaa agatcaaggt catctgatgg caaatcatca 840 tctcaagtga acaggtcaag acatgaaaat acaagccagg taccattgca ggagtccagg 900 acaagaaagc gtaggggatc cagagttagc caggacaggg acagtgaggg acactcagaa 960 gactctgaga ggcactctgg gtcggcttcc agaaaccatc atggatctgc gtgggagcag 1020 tcaagagatg gctccagaca ccccaggtcc catgatgaag acagagccag tcatgggcac 1080 tctgcagaca gctccagaca atcaggcact cgtcacgcag agacttcctc tcgtggacag 1140 actgcatcat cccatgaaca ggcaagatca agtccaggag aaagacatgg atccggccac 1200 cagcagtcag cagacagctc cagacactca gccactgggc gcgggcaagc ttcatctgca 1260 gtcagcgatc gtggacaccg ggggtctagc ggtagtcagg ccagtgacag tgagggacat 1320 tcagaaaact cagacacaca atcagtgtca ggccacggaa aggctgggct gagacagcag 1380 agccaccaag agtccacacg tggccggtca ggggaacggt ctggacgttc agggtcttcc 1440 ctctaccagg tgagcactca tgaacagcct gactctgccc atggacggac cgggaccagc 1500 actggaggaa gacaaggatc gcaccacgag caggcacgag acagctccag gcattcagcg 1560 tcccaagagg gtcaggacac cattcgtgga cacccggggt caagcagagg aggaaggcag 1620 ggatcccacc acgagcaatc ggtaaatagg tctggacact caggttccca tcacagccac 1680 accacatccc agggaaggtc tgatgcctcc catgggcagt caggatccag aagtgcaagc 1740 agacaaacac gaaatgagga acaatcagga gacggcacca ggcactcagg gtcacgtcat 1800 catgaagctt cctctcaggc tgacagctct agacactcac aggtgggcca gggacaatca 1860 tcggggccca ggacaagtag gaaccaggga tccagtgtta gccaggacag tgacagtcag 1920 ggacactcag aagactctga gaggtggtct gggtctgctt ccagaaacca tcatggatct 1980 gctcaggagc agtcaagaga tggctccaga caccccaggt cccatcacga agacagagct 2040 ggtcatgggc actctgcaga cagctccaga aaatcaggca ctcgtcacac acagaattcc 2100 tctagtggac aggctgcgtc atcccatgaa caggcaagat caagtgcagg agaaagacat 2160 ggatcccgcc accagctcca gtcagcagac agctccagac actcaggcac tgggcacgga 2220 caagcttcat ctgcagtcag agacagtgga caccgagggt ccagtggtag tcaggccact 2280 gacagtgagg gacattcaga agactcagac acacagtcag tgtcaggcca tggacaggct 2340 ggtcaccatc agcagagcca ccaagagtcc gcacgtgacc ggtcagggga aaggtctcga 2400 cgttcagggt ctttcctcta ccaggtgagc actcataaac agtctgagtc ctcccatgga 2460 tggacagggc ccagcactgg agtaagacaa ggatcccacc atgagcaggc acgagacaac 2520 tccaggcact cagcatccca agatggtcag gacaccattc gtggacaccc ggggtcaagc 2580 agaagaggaa ggcaggggtc ccaccacgag caatcggtag ataggtctgg acactcaggg 2640 tcccatcaca gccacaccac atcccaggga aggtctgatg cctcccgtgg gcagtcagga 2700 tccagaagtg caagcagaac aacacgtaat gaggaacaat caagagacgg ctccaggcac 2760 tcagggtcac gtcaccatga agcttcctct catgccgaca tctctagaca ctcacaggca 2820 ggccagggac aatcagaggg gtccaggaca agcaggcgcc agggatccag tgttagccag 2880 gacagtgaca gtgagggaca ttcagaagac tctgagaggt ggtctgggtc tgcttccaga 2940 aaccatcgtg gatctgctca ggagcagtca agacatggct ccagacaccc caggtcccat 3000 cacgaagaca gagccggtca cgggcactct gcagacagct ccagacaatc aggaactcct 3060 cacgcagaga cttcctctgg tggacaggct gcgtcatccc atgaacaggc aagatcaagt 3120 ccaggagaaa gacacggatc ccgccaccag cagtcagcag acagctccag acactcaggc 3180 attccgcgca gacaagcttc atctgcagtc agagacagtg gacactgggg gtccagtggt 3240 agtcaggcca gtgatagtga gggacattca gaggagtcag acacacagtc agtgtcaggc 3300 catggacagg atgggcccca tcagcagagc caccaagagt ccgcacgtga ctggtcaggg 3360 ggaaggtctg gacgttcagg gtctttcatc taccaggtga gcactcatga acagtctgag 3420 tctgcccatg ggcggaccag gaccagcact ggacgaagac aaggatccca ccacgagcag 3480 gcacgagaca gctccaggca ctcagcgtcc caagagggtc aggacaccat tcgtgcacac 3540 ccggggtcaa ggagaggagg aaggcaggga tcccaccatg agcaatcggt agatagatct 3600 ggacactcag ggtcccatca cagccacacc acatcccagg gaaggtctga tgcctcccat 3660 gggcagtcag gatccagaag tgcaagcaga caaactcgta aggacaaaca atcaggagac 3720 ggctccaggc actcagggtc acgtcaccat gaagctgcct cttgggctga cagctctaga 3780 cactcacagg tgggacagga acaatcatcg gggtccagga caagcaggca ccagggatcc 3840 agtgttagcc aggacagtga cagtgagaga cactcagacg actccgagag gttgtctggg 3900 tctgcttcca gaaaccatca tggatcttct cgggagcagt caagagatgg ctccagacac 3960 cctgggttcc atcaagaaga cagagccagt cacgggcact ctgcagacag ctccagacaa 4020 tcaggcactc atcacacaga gtcttcctct catggacagg ctgtgtcatc ccatgaacag 4080 gcaagatcaa gtccaggaga aagacatgga tcccgccacc agcagtcagc agacagctcc 4140 agacactcag gcattgggca cagacaagct tcatctgcag tcagagacag tggacaccga 4200 gggtccagtg gtagtcaggt cactaacagt gagggacatt cagaagactc agacacacag 4260 tcagtgtcag cccacggaca agctgggccc catcagcaga gccacaaaga gtccgcacgt 4320 ggccagtcag gggaaagctc tggacgttca aggtctttcc tctaccaggt gagctctcat 4380 gaacagtctg agtccacaca cggacagact gcacccagca ctggaggaag acaaggatcc 4440 cgccatgagc aggcacgaaa cagctctagg cactcagcat cccaagacgg tcaggacacc 4500 attcgtggac acccggggtc aagcagagga ggaaggcagg gatcctacca cgagcaatca 4560 gtagataggt ctggacactc agggtaccat cacagccaca ccacacccca gggaaggtct 4620 gatgcctccc atgggcagtc aggacccaga agtgcaagca ggcaaacaag aaatgaggaa 4680 caatcaggag acggctccag gcactcaggg tcacgtcacc atgaaccttc cactcgggcc 4740 ggcagctcta gacactcaca ggtgggccag ggagaatcag cggggtccaa gacaagcagg 4800 cgccagggat ccagtgttag tcaggacagg gacagtgagg gacactcaga agactctgag 4860 aggcggtctg agtcggcttc cagaaaccat tatggatctg ctcgggagca gtcaagacat 4920 ggctccagga accccaggtc ccatcaagaa gatagagcca gtcatgggca ctctgcagag 4980 agctccagac aatcaggcac tcgtcatgca gagacttcct ctggtggaca ggctgcatca 5040 tcccaggaac aggcaaggtc aagtccagga gaaagacatg gatcccgcca ccagcagtca 5100 gcagacagct ccacagactc aggcactggg cgcagacaag attcatctgt agtcggagac 5160 agtggaaacc gagggtccag tggtagccag gccagtgaca gcgagggaca ctcagaagag 5220 tcagacacac agtcagtgtc agcccacgga caggctgggc cccatcagca gagccaccaa 5280 gagtccacac gtggccagtc aggggaaagg tctggacgtt cagggtcttt cctctaccag 5340 gtgagcactc atgaacagtc tgagtccgcc catggacgca cagggcccag cactggagga 5400 agacaaagat cccgccacga gcaggcacga gacagctcca ggcactcagc gtcccaagag 5460 ggtcaggaca ccattcgtgg acacccaggg tcaagcagag gaggaaggca gggatcccac 5520 tatgagcaat cggtagatag ttctggacac tcagggtctc atcacagcca caccacgtcc 5580 caggaaaggt ctgatgtctc ccgtgggcag tcaggatcca gaagtgtcag cagacaaaca 5640 cgtaatgaga aacaatcagg agacggctcc aggcactcag ggtcgcgtca ccatgaagct 5700 tcctctcggg ccgacagctc tagacactcg caggtgggcc agggacaatc atcagggccc 5760 aggacaagca ggaaccaggg atccagtgtt agccaggaca gtgacagtca gggacactca 5820 gaagactctg agaggtggtc tgggtctgct tccagaaacc atcttggatc tgcttgggag 5880 cagtcaagag atggctccag acaccctggg tcccatcacg aagacagagc cggtcacggg 5940 cactctgcag acagctccag acaatcaggc actcgtcaca cagagtcttc ctctcgtgga 6000 caggctgcgt catcccatga acaggcaaga tcaagtgcag gagaaagaca tggatcccac 6060 caccagctcc agtcagcaga cagctccaga cactcaggca ttgggcatgg acaagcttca 6120 tctgcagtca gagacagtgg acaccgaggg tacagtggta gtcaggccag tgacagtgag 6180 ggacattcag aagactcaga cacacagtca gtgtcagcac agggaaaagc tgggccccat 6240 cagcagagcc acaaagagtc cgcacgtggc cagtcagggg aaagctctgg acgttcaggg 6300 tctttcctct accaggtgag cactcatgaa cagtctgagt ccacccatgg acagtctgcg 6360 cccagcactg gaggaagaca aggatcccat tatgatcagg cacaagacag ctccaggcac 6420 tcagcatccc aagagggtca ggacaccatt cgtggacacc cggggccaag cagaggagga 6480 agacaggggt cccaccaaga gcaatcggta gataggtctg gacactcagg gtctcatcac 6540 agccacacca catcccaggg aaggtctgat gcctcccgtg ggcagtcagg atccagaagt 6600 gcaagcagaa aaacatatga caaggaacaa tcaggagatg gctctaggca ctcagggtcg 6660 catcatcatg aagcttcctc ttgggccgac agctctagac actcactggt gggccaggga 6720 caatcatcag ggcccaggac aagcaggccc cggggatcca gtgttagcca ggacagtgac 6780 agtgagggac actcagaaga ttctgagagg cggtctgggt ctgcgtccag aaaccatcat 6840 ggatctgctc aggagcagtc aagagatggc tccagacacc ccaggtccca tcacgaagac 6900 agagccggtc atgggcactc tgcagagagc tccagacaat caggcactca tcatgcagag 6960 aattcctctg gtggacaggc tgcatcatcc catgaacagg caagatcaag tgcaggagag 7020

agacacggat cccaccacca gcagtcagca gacagctcca gacactcagg cattgggcac 7080 ggacaagctt catctgcagt cagagacagt ggacaccgag ggtccagtgg tagtcaggcc 7140 agtgacagtg agggacattc agaagactca gacacacagt cagtgtcagc ccacggacag 7200 gctgggcccc atcagcagag ccaccaagag tccacacgtg gccggtcagc aggaaggtct 7260 ggacgttcag ggtctttcct ctaccaggtg agcactcatg aacagtctga gtccgcccat 7320 ggacggaccg ggaccagcac tggaggaaga caaggatccc accacaagca ggcacgagac 7380 agctccaggc actcaacgtc ccaagagggt caggacacca ttcatggaca cccggggtca 7440 agcagtggag gaaggcaggg atcccactac gagcaattgg tagatagatc tggacactca 7500 gggtctcatc acagccacac cacatcccag ggaaggtctg atgcctccca tgggcactca 7560 ggatccagaa gtgcaagcag acaaactcgt aacgatgaac aatcaggaga cggctccagg 7620 cactcagggt cgcgtcacca tgaagcttcc tctcgggccg acagctctgg acactcgcag 7680 gtgggccagg gacaatcaga ggggcccagg acaagcagga actggggatc cagttttagc 7740 caggacagtg acagtcaggg acactcagaa gactctgaga ggtggtctgg gtctgcttcc 7800 agaaaccatc atggatctgc tcaggagcag ctaagagatg gctccagaca ccccaggtcc 7860 catcaagaag acagagctgg tcatgggcac tctgcagaca gctccagaca atcaggcact 7920 cgtcacacac agacttcctc tggtggacag gctgcatcat cccatgaaca ggcaagatca 7980 agtgcaggag aaagacatgg atcccaccac cagcagtcag cagacagctc cagacactca 8040 ggcattgggc acggacaagc ttcatctgca gtcagagaca gtggacaccg agggtacagt 8100 ggtagtcagg ccagtgacaa tgagggacat tcagaagact cagacacaca gtcagtgtca 8160 gcccacggac aggctgggtc ccatcagcag agccaccaag agtccgcacg tggccggtca 8220 ggggaaacgt ctggacattc aggatctttc ctctaccagg tgagcactca tgaacagtct 8280 gagtcctccc atggatggac ggggcccagc actagaggaa gacaaggatc ccgccatgag 8340 caggcacaag acagctccag gcactcagca tcccaagacg gtcaggacac cattcgtgga 8400 cacccggggt caagcagagg aggaaggcag gggtaccacc acgagcattc ggtagatagc 8460 tctggacact cagggtccca tcacagccac accacatccc agggaaggtc tgatgcctcc 8520 cgtgggcagt caggatccag aagtgcaagc agaacaacac gtaatgagga acaatcagga 8580 gacggctcca ggcactcagg gtcgcgtcac catgaagctt ccactcatgc cgacatctct 8640 agacactcac aggcagtcca gggacaatca gaggggtcca ggagaagcag gcgccaggga 8700 tccagtgtga gccaggacag tgacagtgag ggacattcag aagactctga gaggtggtct 8760 gggtctgctt ccagaaacca tcatggatct gctcaggagc agctaagaga tggctccaga 8820 caccccaggt cccatcaaga agacagagct ggtcatgggc actctgcaga cagctccaga 8880 caatcaggca ctcgtcacac acagacttcc tctggtggac aggctgcatc atcccatgaa 8940 caggcaagat caagtgcagg agaaagacat ggatcccacc accagcagtc agcagacagc 9000 tccagacact caggcattgg gcacggacaa gcttcatctg cagtcagaga cagtggacac 9060 cgagggtaca gtggtagtca ggccagtgac aatgagggac attcagaaga ctcagacaca 9120 cagtcagtgt cagcccacgg acaggctggg tcccatcagc agagccacca agagtccgca 9180 cgtggccggt caggggaaac gtctggacat tcaggatctt tcctctacca ggtgagcact 9240 catgaacagt ctgagtcctc ccatggatgg acggggccca gcactagagg aagacaagga 9300 tcccgccatg agcaggcaca agacagctcc aggcactcag catcccaata cggtcaggac 9360 accattcgtg gacacccggg gtcaagcaga ggaggaaggc aggggtacca ccacgagcat 9420 tcggtagata gctctggaca ctcagggtcc catcacagcc acaccacatc ccagggaagg 9480 tctgatgcct cccgtgggca gtcaggatcc agaagtgcaa gcagaacaac acgtaatgag 9540 gaacaatcag gagacagctc caggcactca gtgtcacgtc accatgaagc ttccactcat 9600 gccgacatct ctagacactc acaggcagtc cagggacaat cagaggggtc caggagaagc 9660 aggcgccagg gatccagtgt gagccaggac agtgacagtg agggacattc agaagactct 9720 gagaggtggt ctgggtctgc ttccagaaac catcgtggat ctgttcagga gcagtcaagg 9780 cacggctcca gacaccccag gtcccatcac gaagacagag ccggtcacgg gcactctgca 9840 gaccgctcca gacaatcagg cactcgtcac gcagagactt cctctggtgg acaggctgca 9900 tcatcccatg aacaggcaag atcaagtcca ggagagagac acggatcccg ccaccagcag 9960 tcagcagaca gctccagaca ctcaggcatt ccgcgtggac aagcttcatc tgcagtcaga 10020 gacagtagac actgggggtc cagtggtagt caggccagtg atagtgaggg acattcagaa 10080 gagtcagaca cacagtcagt gtcaggccat ggacaggctg ggccccatca gcagagccac 10140 caagagtccg cacgtgaccg gtcaggggga aggtctggac gttcagggtc tttcctctac 10200 caggtgagca ctcatgaaca gtctgagtct gcccatgggc ggaccaggac cagcactgga 10260 cgaagacaag gatcccacca cgagcaggca cgagacagct ccaggcactc agcgtcccaa 10320 gagggtcagg acaccattcg tggacacccg gggtcaagca gaagaggaag gcagggatcc 10380 cactacgagc aatcggtaga taggtctgga cactcagggt cccatcacag ccacaccaca 10440 tcccagggaa ggtctgatgc ctcccgtggg cagtcaggat ccagaagtgc cagcagacaa 10500 actcgtaatg acgaacaatc aggagatggc tccaggcact catggtcgca tcaccatgaa 10560 gcttccactc aggcggacag ctctagacac tcacagtccg gccagggaca atcagcgggg 10620 cccaggacaa gcaggaacca gggatccagt gttagccagg acagtgacag tcagggacac 10680 tcagaagact ctgagaggtg gtctgggtct gcttccagaa accatcgtgg atctgctcag 10740 gagcagtcaa gagatggctc cagacacccc acgtcccatc acgaagacag agccggtcac 10800 gggcactctg cagagagctc cagacaatca ggcactcatc atgcagagaa ttcctctggt 10860 ggacaggctg catcatccca tgaacaggca agatcaagtg caggagagag acatggatcc 10920 caccaccagc agtcagcaga cagctccaga cactcaggca ttgggcacgg acaagcttca 10980 tctgcagtca gagacagtgg acaccgaggg tccagtggta gtcaggccag tgacagtgag 11040 ggacattcag aagactcaga cacacagtca gtgtcagccc acggacaggc tgggccccat 11100 cagcagagcc accaagagtc cacacgtggc cggtcagcag gaaggtctgg acgttcaggg 11160 tctttcctct accaggtgag cactcatgaa cagtctgagt ctgcccatgg acgggctggg 11220 cccagtactg gaggaagaca aggatcccgc cacgagcagg cacgagacag ctccaggcac 11280 tcagcgtccc aagagggtca ggacaccatt cgtggacacc cggggtcaag gagaggagga 11340 agacagggat cctaccacga gcaatcggta gataggtctg gacactcagg gtcccatcac 11400 agccacacca catcccaggg aaggtctgat gcctcccatg ggcagtcagg atccagaagt 11460 gcaagcagag aaacacgtaa tgaggaacag tcaggagacg gctccaggca ctcagggtcg 11520 cgtcaccatg aagcttccac tcaggctgac agctctagac actcacagtc cggccagggt 11580 gaatcagcgg ggtccaggag aagcaggcgc cagggatcca gtgttagcca ggacagtgac 11640 agtgaggcat acccagagga ctctgagagg cgatctgagt ctgcttccag aaaccatcat 11700 ggatcttctc gggagcagtc aagagatggc tccagacacc ccggatcctc tcaccgcgat 11760 acagccagtc atgtacagtc ttcacctgta cagtcagact ctagtaccgc taaggaacat 11820 ggtcacttta gtagtctttc acaagattct gcgtatcact caggaataca gtcacgtggc 11880 agtcctcaca gttctagttc ttatcattat caatctgagg gcactgaaag gcaaaaaggt 11940 caatcaggtt tagtttggag acatggcagc tatggtagtg cagattatga ttatggtgaa 12000 tccgggttta gacactctca gcacggaagt gttagttaca attccaatcc tgttgttttc 12060 aaggaaagat ctgatatctg taaagcaagt gcgtttggta aagatcatcc aaggtattat 12120 gcaacgtata ttaataagga cccaggttta tgtggccatt ctagtgatat atcgaaacaa 12180 ctgggattta gtcagtcaca gagatactat tactatgagt aagaaattaa tggcaaagga 12240 attaatccaa gaatagaaga atgaagcaag ttcactttca atcaagaaac ttcataatac 12300 tttcagggaa gttatctttt cctgtcaatc tgtttaaaat atgctatagt atttcattag 12360 tttggtggta gcttattttt attgtgtaat gatctttaaa cgctatattt cagaaatatt 12420 aaatggaaga aatcaatatc atggagagct aactttagaa aactagctgg agtattttag 12480 gagattctgg gtcaagtaat gttttatgtt tttgaaagtt taagttttag acactcccca 12540 aatttctaaa ttaatctttt tcagaaatat cgaaggagcc aaaaatataa aacagttctg 12600 tataccaaag tggctatatc aacatcaggg ctagcacatc tttctctatt atccttctat 12660 tggaattcta gtattctgta ttcaaaaaat catcttggac ataattaata ttatagtaag 12720 ctgcatctaa attaaaaata aactatt 12747 <210> SEQ ID NO 2 <211> LENGTH: 4629 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 aaatgctcca catggcccca gtcaccctgc agaaaagaaa tcatcagctt tctgcagcat 60 cagacttagc agcatcttcc tgtcccaaga gaagagggga tgggaggatg gcatggttca 120 gggcttaggg gaggataggg gagaaaaagt ggaagcaaag ggagaggccc aggaaggatc 180 ttgttgcgac atgttacctg ggctgcacct gctgtatgca agagaagagg ctgggagtcc 240 cctctgcagg gtgttcatgc cccatctctg tgccacgagg agttgggctg ctgaaggata 300 tatcagcaca acttctctgc tccaactagt ctacattgca aagcatctcc tagtgtcttt 360 ggaagaagga tctatgtcat gagtgctcac ctggtagagg gaagaccctg aacgtccaga 420 ccgttcccct gaccggccac gtgtggactc ttggtggctc tgctgtctca gcccagcctt 480 tccgtggcct gacactgatt gtgtgtctga gttttctgaa tgtccctcac tgtcactggc 540 ctgactaccg ctagaccccc ggtgtccacg atcgctgact gcagatgaag cttgcccgcg 600 cccagtggct gagtgtctgg agctgtctgc tgactgctgg tggccggatc catgtctttc 660 tcctggactt gatcttgcct gttcatggga tgatgcagtc tgtccacgag aggaagtctc 720 tgcgtgacga gtgcctgatt gtctggagct gtctgcagag tgcccatgac tggctctgtc 780 ttcatcatgg gacctggggt gtctggagcc atctcttgac tgctcccacg cagatccatg 840 atggtttctg gaagccgacc cagagtgcct ctcagagtct tctgagtgtc cctcactgtc 900 cctgtcctgg ctaactctgg atcccctacg ctttcttgtc ctggactcct gcaatgactc 960 ataatatgct tgagccaact tgaataccat cagaagaaac tcagtgaagt caattttctt 1020 gttgtggtct atatccaagt gatccatgaa gacatcaacc atatctgggt catctggatt 1080 ctgtacagag ggaagtcaca gagggagact gcatcagaca gaatcacatt atcagccaat 1140 taattcaaag ttaatttaag gaacctgatc tttgagtatt aaaaagtggg acaaaatctt 1200 ttttttttta agactttttt gtctcatcct cattcaggtg ttatatgtga acaaagatgt 1260 agactagtaa ggtatcaaga tttgatgggg atatccagac aacttggagc caagtcccca 1320 cagctggatc agacaacttt tggtgacatc tcacagtgtg gattatcaag ccagtgtatc 1380 agtctggagc ctaggttttt ttgcctttag ctttgctcag gatgacttct gctcttctat 1440 ggcaatactt gagaagctac agctttgatt agacgagagg ctatttcaat aactttctcc 1500

tgataagaag accaccgacc gtggacggat tctggccagt ttacaaaggc tgcataccat 1560 agggcagggc accccactat ggcaaacact aataatgata ttgctcctga ttttgtttct 1620 gaagggaatg cctctaagat ttcttcatta attataagag actgggtctc actctgttgc 1680 ccaagctgga gaacagtggc accatcatag ctcactgcag ccttcaactt ctgggctcaa 1740 gtgcttctcc cacctcagcc tcccaagtag ctgggacaac aggtgaaatt gaaggccagc 1800 ttttggaact caaccttcaa aggaacttaa aatttccatt atgtagagtc cctgaggaaa 1860 ctggatccac tagctacact ggctgagaat gctctcaccc caggatttca tttttatttt 1920 accacatcat tgttggtatc ttaagatcaa tgtactggag cagcggggtc tcacaaagag 1980 gatacctggt gacacgccca ggctttcaga atgcactata gggctttagt gacttgtcca 2040 ggttcagctg aaatgtgcca tctgggacct actaatgctt gttgaacttt actggatctc 2100 agaagctggg tctcagaagc tcagatgttc ctcagagcta cgatatcaac aacctgtgat 2160 gacagagaga aggttgctcc atgatggatt tggaaactta ctggctagct aaacctgact 2220 gaatgggaag gaatgtggat agctttggaa ctctagtttc actagatgag ctggaatttg 2280 tattttgaca aattgcacgt tatgattatt aagtaatgca actgattttt ttttccctta 2340 aaacaaacaa tctagaatct gtgtaatcaa aataatttct ctaaaaggct gcaagtatat 2400 gcttaaagtg ttggggcatt cagagcattt ggaacattac attcttttga atgtcaattg 2460 gtagatgaaa ataccagctt ttaagtcata catttgattt tttgaaacaa tatgcattta 2520 gagtttgtaa gtcaagtgaa taactgataa ggtaaaaaaa agggggagtt cattgttgag 2580 tatgaattta aagtaaccag actgcctttt gtccagtggc tgtcagtaat ttacttcagc 2640 aggcattttt tttttttgag gctgttctat gatatcatga cccttcttgt aggaatgtgc 2700 ttccagtggt gaggcagtct gagaatgtgt gaagcagtat aatgaagcca gaccaagatg 2760 gaagcttggc ctgggatttg agcatcagga aaactgttga agggttatgt atacatcaca 2820 cacacacaca cacacacaca cacacacaca cacacacaca ctctcttcca tactcctata 2880 acaacgtata gtatttacta tacctggtga caggtattta ctatacattg ttgaagatct 2940 ataacatgga atgcctagtg ctaagtgcag tgtccttagt gaaattgtat tggtttggaa 3000 ttatttatga gtttgggatt atttgtcact acccttaaat gatcttgaca ctcacctatt 3060 tgaaaagata ttgaggattt gccatttgat attgaccagg gtgtattgtg cacaaatatt 3120 gtgaatatac atctgtctgt ccttaaatca ctgtaagttt taactggaat agatttgctc 3180 cacattactc ggtagggctg atatttcatg cctcatggat gagaaaagaa taggcaaaaa 3240 ttatatctcg ggctgcctca cacatctttt aacaggataa gggaaaaata aatataagac 3300 ctatgagtta tggcatcagg cttgaacttt aatttatgaa ttaaaccaac aatattattg 3360 attattgcaa ttatctatct taatttcatt tgttctcctt ttaaaaatta attatgttta 3420 ttttacctat tgtgaataaa gtcactcctc ccatgtgctt atttcttgac tctggcacta 3480 attattcttc agtgcctaga tttcctggga ctgctcagtt taagtaactt ctgaacattg 3540 gtctctgaga gaaaagacag tgaagtggga tctgtggaaa aacacatctg caaatgtctc 3600 atttttttct tcaggggtaa gttttcttcc ttggagactc ttgtcttgaa aaagtgatac 3660 tagatcacca tttggcaaga taattacaaa aggcagatag agtaattatt ttacatagct 3720 gttatagggt gactaagatc tcagggaatg actgtgttct gtaatccact tgccccactc 3780 aataccccaa gttttataca agccaaagat gcttggagca gaatctgcta cctgaggctc 3840 cagggccttg gcctagggaa actaatactt aactttttaa gtggtcattg atgacatccc 3900 actcctttct gagatgggta tgatttgaaa ggcaggtgta ttcatggaga gcagggcttg 3960 ggacctttgc tgcaagagga ggaactatta gcctagactt ttctaataag gactttctca 4020 tacctgtttc ctagatctgg tccacataga agaaatcagg gaacacacag ggtaggactc 4080 ttaaaggagt atgtttcttg gctttttcct aaaaactaaa acaaaaacat tcatagcaaa 4140 gcagtttagc ccagtgacta agagtataga cattggaatc tgaaagatct gggttggaat 4200 tctgcttcag ccatttacta attatgtaac ttcacatctc tgagaatggg ggaaattttc 4260 tttgcagggt tgttacaaag cttaaaagag ttaagtcaag tgcctggaac agaacagcca 4320 ttcaataaat catagaaatt tttgttttta cacaaatttt taaaaagtaa tattttttgc 4380 atttgttaga aagactatat tttctgttta gtggatgaca tcatcatcat aaaaatcact 4440 gttacaagtt ttgttttgag aattaaaatc atatattaat cccaatcaca ttgtgttggg 4500 attaatcaac atctctgggc atctaatact gtaaaaacat ggcctgtgat tggtctgctt 4560 ggaagtgcat cttggttctg gctcagttgg ctagtgggcc acattgatta aagatgaata 4620 caatgccaa 4629 <210> SEQ ID NO 3 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 3 ctccctctgt gacttccctc t 21 <210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 4 gctatccaca ttccttccca 20 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 5 gcagagccac caagagtcca 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 6 gtgtgtgtgt gtgtgtgtgt 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 7 tccacattcc ttcccattca 20 <210> SEQ ID NO 8 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 8 ccctctgtga cttccctc 18 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 9 ccacattcct tcccattcag 20 <210> SEQ ID NO 10 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 10 mumcmcmcmu ctgtgacttc mcmcmumcmu 30 <210> SEQ ID NO 11 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 11 mcmumcmcmc tctgtgactt cmcmcmumcm u 31 <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 ctccctctgt gacttccctc t 21 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense oligonucleotide <400> SEQUENCE: 13 tccacattcc ttcccattca 20

Claims

1. A method of modulating a function of and/or the expression of a Filaggrin (FLG) 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 4629 of SEQ ID NO: 2; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Filaggrin (FLG) 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: 3 to 13.

14. A method of modulating a function of and/or the expression of a Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; and, modulating a function of and/or the expression of Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide.

16. A method of modulating a function of and/or the expression of Filaggrin (FLG) 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 Filaggrin (FLG) 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 and 2; and, modulating the function and/or expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Filaggrin (FLG) 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: 3 to 13.

30. A composition comprising one or more oligonucleotides specific for one or more Filaggrin (FLG) 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 composition further comprises one or more FLG modulating molecules and a pharmaceutically acceptable carrier.

32. The composition of claim 31, wherein the molecule is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

33. 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: 3 to 1.3.

34. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 3 to 13.

35. The composition of claim 34, wherein the oligonucleotides set forth as SEQ ID NOS: 3 to 1.3 comprise one or more modifications or substitutions.

36. The composition of claim 35, wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

37. A composition for use in treatment of a dermatological disease or disorder, the composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable earner.

38. The composition of claim 37, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

39. The composition of claim 37, wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity.

40. The composition of claim 37, wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

41. The composition of claim 40, wherein the oligonucleotide comprises nucleotide sequences set forth as SEQ ID NOS: 3 to 13.

42. The composition of claim 40, wherein the oligonucleotide set forth as SEQ ID NOS: 3 to 13 comprise one or more modifications or substitutions.

43. A method of preventing or treating a disease associated with at least one Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide and modulates expression of said at least one Filaggrin (FLG) polynucleotide; thereby preventing or treating the disease associated with the at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof.

44. The method of claim 43, wherein a disease associated with the at least one Filaggrin (FLG) polynucleotide is selected from: a disease or disorder associated with abnormal function and/or expression of FLG, a dermatological disease or disorder, sign of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay lever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin.

45. 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 rise 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.

46. A method of treating an FLG associated disease or disorder in a subject, the method comprises administering to the subject a composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable carrier.

47. The method of claim 46, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

48. The method of claim 46, wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity.

49. The method of claim 46, wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

50. The method of claim 46, wherein a disease associated with the at least one Filaggrin polynucleotide is selected from: a dermatological disease or disorder, sign, of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay fever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc.), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin.

53. A method of preventing or treating a skin condition associated with at least one Filaggrin (FLG) 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 close, of a Filaggrin up-regulating compound, at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Filaggrin (FLG) polynucleotide and modulates expression of said at least one Filaggrin (FLG) polynucleotide and a pharmaceutically acceptable carrier; thereby preventing or treating the disease skin condition associated with the at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof.

52. The method of claim 51, wherein the compound, is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

53. The method of claim 51, wherein the skin condition is caused by caused by inflammation, light damage or aging.

54. The method of claim 51, 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.

55. A use of the composition of claim 30, in the manufacture of a medicament for the treatment of a dermatological disease or disorder.

56. The use of claim 55, wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity.

57. The use of claim 55, wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject.

58. The use of claim 55, wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

59. The use of claim 55, wherein the dermatological disease or disorder is: 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.