Treatment of filaggrin (flg) related diseases by modulation of flg expression and activity

ES 2 664 591 T3

DESCRIPTION

Treatment of filaggrin-related diseases (flg) by modulating the expression and activity of the FLG gene

FIELD OF THE INVENTION

The present application claims priority from United States Provisional Patent Application No. 61 / 246,080 filed September 25, 2009 and United States Provisional Patent Application No. 61 / 307,654 filed February 24 2010.

Embodiments of the invention comprise oligonucleotides that modulate the expression and / or function of the FLG gene and associated molecules.

BACKGROUND

Filaggrin is a highly charged cationic protein that aids in the aggregation and subsequent formation of disulfide bridges between keratin filaments. It is derived from profilaggrin, a large phosphorylated precursor (4400 kD) that is 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 dephosphorylation. In addition to the processing of profilaggrin to become filaggrin, the transition from a granular cell to a corneocyte is characterized by the degradation of the nucleus and other organelles, the assembly of a cornified envelope, and the reorganization of the network of intermediate keratin filaments into a two-dimensional sheet. Filaggrin plays a fundamental 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 natural hydration factors (NMF). The transition from a granular precursor, profilaggrin, to a diffusely distributed protein occurs rapidly in the granular transition to stratum corneum in response to an initiation signal that 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, that is, nuclei retained in the keratinocytes of the stratum corneum. Although the signals that are disrupting terminal differentiation in these inflammatory conditions may be disparate, a common end theme is loss of the granular layer with consequent incomplete terminal differentiation. In conditions where profilaggrin is diminished, such as atopic dermatitis, or is essentially absent, as in ichthyosis vulgaris, the quality of the stratum corneum is compromised due to the inability of an MFN-free stratum corneum to remain hydrated under the drying action of the environment .

Natural hydration factors (NMF) play an important role in maintaining the moisture content of the stratum corneum. The amino acids that form the major constituents of NMFs have been reported to be produced by proteolytically cleaved filaggrin originating from keratohyalin granules. Filaggrin is a protein made up of 317 amino acids. Since it became clear that the amino acids that make up the major constituents of NMFs are derived from filaggrin, research has been conducted on the relationship between dry skin conditions with filaggrin. In recent years, it has become clear that the amino acid content of the stratum corneum is reduced in dry skin as seen in senile xerosis, atopic diseases and the like, and that the expression of filaggrin in such dry skin decreases. Also, it is well known that skin problems like rough skin are caused by a dry environment.

The filaggrin gene plays a role in building the skin's barrier layers, 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 waterproof skin barrier layers present on the outermost surface of the skin and keep them hydrated. The inherent barrier function of the skin is similar to a plastic or adhesive film, it acts to prevent the loss of water from the skin and, more importantly, to protect the body from foreign materials in the environment, such as allergens. The lack of an intact skin barrier causes allergens to enter the body where they produce a variety of allergic responses including eczema, asthma, hay fever, and other allergies.

DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also critical to a variety of technologies that both detect a particular nucleic acid and alter its expression. Nucleotides

ES 2 664 591 T3 antisense, for example, alter gene expression by hybridizing with target RNA, thus interfering with RNA splicing, transcription, translation and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODN), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENE ™ (for the treatment of cytomegalovirus retinitis), which reflects that the antisense has therapeutic utility.

RESUME

This summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is presented with the understanding that it is not to be used to interpret or limit the scope or meaning of the claims. The invention is defined by the claims.

In one embodiment, the present disclosure provides methods of inhibiting the action of a natural antisense transcript using one or more antisense oligonucleotides directed to any region of the natural antisense transcript by upregulating the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript may be achieved by siRNA, ribozymes, and small molecules, which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating the function and / or expression of a polynucleotide of the FLG gene in cells or tissues of the patient in vivo or in vitro which comprises contacting said cells or tissues with an antisense oligonucleotide of 5 to 30 nucleotides. of length, wherein said oligonucleotide has at least 50% sequence identity with a reverse complement of a polynucleotide comprising from 5 to 30 consecutive nucleotides within nucleotides 1 to 4629 of SEQ ID NO: 2 thus modulating the function and / or the expression of the FLG gene polynucleotide in cells or tissues of the patient in vivo or in vitro.

In one embodiment, an oligonucleotide targets a natural antisense polynucleotide sequence of the FLG gene, for example, nucleotides set forth in SEQ ID NO: 2, and any variant, allele, homologue, mutant, derivative, fragment, and sequence complementary to those themselves. Examples of antisense oligonucleotides are set forth as SEQ ID NO: 3 to 13.

Another embodiment provides a method of modulating the function and / or expression of a polynucleotide of the FLG gene in cells or tissues of the patient in vivo or in vitro, which comprises contacting said cells or tissues with an antisense oligonucleotide from 5 to 30 nucleotides in length, where said oligonucleotide has at least 50% sequence identity with an antisense antisense complement of the FLG gene polynucleotide, thereby modulating the function and / or expression of the FLG gene polynucleotide in cells or tissues of the patient in vivo or in vitro.

Another embodiment provides a method of modulating the function and / or expression of a polynucleotide of the FLG family in cells or tissues of the patient in vivo or in vitro, which comprises contacting said cells or tissues with an antisense oligonucleotide of 5 at 30 nucleotides in length, wherein said oligonucleotide has at least 50% sequence identity with an antisense oligonucleotide for an antisense polynucleotide of the FLG family; thereby modulating the function and / or expression of the FLG family polynucleotide in cells or tissues of the patient in vivo or in vitro.

In one embodiment, a composition comprises one or more antisense oligonucleotides that bind to polynucleotides of the sense and / or antisense FLG gene.

In one embodiment, a composition comprises one or more antisense oligonucleotides that bind to polynucleotides of the sense and / or antisense FLG gene, one or more modulator molecules of the FLG gene, a pharmaceutically acceptable carrier, and combinations thereof.

In one embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.

In one embodiment, the oligonucleotides comprise one or more modified linkages.

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 blocked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are nucleic acid molecules

ES 2 664 591 T3 blocked, including α-L-LNA.

In one embodiment, the oligonucleotides are administered to a patient subcutaneously, intramuscularly, intravenously, or intraperitoneally.

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

In one 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 than one antisense compound and one or more FLG gene modulator molecules; this treatment can be modified to comprise multiple doses over a period of time. The treatment can be combined with one or more of the other types of therapies.

In one embodiment, the oligonucleotides are encapsulated in a liposome or attached to a carrier molecule (eg, cholesterol, TAT peptide).

Other aspects are described later.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of real-time PCR results showing the change in fold + standard deviation in FLG1 mRNA after treatment of HepG2 cells with introduced phosphothioate oligonucleotides using Lipofectamine 2000, compared to control. Real-time PCR results show that FLG1 mRNA levels in HepG2 cells are significantly increased with two of the oligonucleotides designed for FLG1 antisense AK056431. The bars indicated as CUR-1157, CUR-1158, CUR-1159, CUR-1160 and CUR-1161 correspond to samples treated with SEQ ID NO: 3, 4, 5, 6 and 7 respectively.

Figure 2 shows the change in fold + standard deviation in FLG1 mRNA after treatment of 518A2 cells with phosphodiester oligonucleotides with a 3 'inverted T and phosphodiester oligos with a 2'-Omethyl gapmer introduced using Lipofectamine 2000, compared to the control. Real-time PCR results show that FLG mRNA levels in 518A2 cells are significantly increased with two of the oligonucleotides designed for FLG1 antisense AK056431. The bars indicated as CUR-1128, CUR-1129, CUR-1130 and CUR-1131 correspond to samples treated with SEQ ID NO: 8, 9, 10 and 11 respectively.

Figure 3 shows the change in fold + standard deviation in FLG1 mRNA after treatment of 518A2 cells with introduced phosphodiester oligonucleotides using Lipofectamine 2000, compared to control. Real-time PCR results show that FLG mRNA levels in 518A2 cells are significantly increased with two of the oligonucleotides designed for FLG1 antisense AK056431. The bars indicated as CUR-1396 and CUR-1397 correspond to samples treated with SEQ ID NO: 12 and 13 respectively.

Figure 4 shows the change in times of Filaggrin mRNA expression in 518A2 cells and primary keratinocytes treated with the compounds: Pioglitazone, Lomerizine, Bupropion, Fenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Pyribedil, Oxaprozine, Glycopyrrolate Granisetron, Memantine, Nimodipine and Amlodipine; compared to untreated cells.

Description of the sequence listing: SEQ ID NO: 1: Homo sapiens Filaggrin (FLG), mRNA. (NCBI Accession No .: NM_002016); SEQ ID NO: 2: Natural antisense sequence of the FLG gene (AK056431); SEQ ID NO: 3-13: Antisense oligonucleotides. * indicates phosphothioate bond and 'm' indicates 2'-O-methyl modification.

DETAILED DESCRIPTION

Various aspects of the invention are described below with reference to illustrative application examples. It should be understood that the numerous specific details, relationships, and procedures are set forth to provide a complete understanding of the invention. One skilled 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 procedures. The present invention is not limited by the order of acts or events, since some acts can occur in different orders and / or simultaneously with other acts or events. In addition, not all illustrated acts or events are required to implement a methodology of

ES 2 664 591 T3 according to the present invention.

All genes, gene names, and gene products disclosed herein are intended to correspond to homologues of any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to, human and mouse genes and gene products. 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 construed as a limitation, unless the context in which it appears clearly indicates so. Thus, for example, for the genes disclosed herein, which in some embodiments refer 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 one embodiment, the genes or nucleic acid sequences are human.

Definitions

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

The term "approximately" means within an acceptable range of error 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, that is, the limitations of the measurement system. measurement. For example, about may mean 1 or more than 1 standard deviation, consistent with the practice of 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 may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value. In cases where particular values ​​are described in the application and claims, unless otherwise stated, the term "approximately" means that the value must be assumed to be within an acceptable range of error for the value. particular.

As used herein, the term "mRNA" means the currently known mRNA transcript (s) of a targeted gene, and any additional transcripts that may be elucidated.

By "antisense oligonucleotides" or "antisense compound" is meant an RNA or DNA molecule that binds to another RNA or DNA (RNA, target DNA). For example, if this is an RNA oligonucleotide, it binds to another RNA target through RNA-RNA interactions and alters the activity of the target DNA. An antisense oligonucleotide can up-regulate or down-regulate the expression and / or function of a particular polynucleotide. The definition is intended to encompass any foreign RNA or DNA molecule that is useful from a therapeutic, diagnostic or other point of view. 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, alternative splicing agents, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. Thus, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

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 bonds, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNAs), blocked nucleic acids (LNAs) , phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide through a regular pattern of monomer-to-monomer interactions, such as Watson-Crick base-pairing type, Hoogsteen or reverse Hoogsteen base-pairing types, or the like.

The oligonucleotide can be "chimeric", that is, it can be composed of different regions. In the context of this invention chimeric compounds are oligonucleotides, which contain two or more chemical regions, for

For example, one or more DNA regions, one or more RNA regions, one or more PNA regions, etc. Each chemical region is composed of at least one monomeric unit, that is, a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically comprise at least one region where the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited to, for example, increased resistance to degradation by nucleases, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide can 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.

The oligonucleotide can be composed of regions that can be "log" linked, that is, when monomers are linked consecutively, as in native DNA, or are linked by spacers. The spacers are intended to form a covalent bridge between the regions and have, in preferred cases, a length that does not exceed about 100 carbon atoms. Spacers can carry different functionalities, for example, have positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores, etc.), be lipophilic, induce special secondary structures such as, for example , alanine-containing peptides that induce alpha helices.

As used herein, FLG and Filaggrin are inclusive of all family members, mutants, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, etc.

As used herein, the words 'Filaggrin', FLG, FLGI and ATOD2, are considered the same in the literature and are used interchangeably in the present application.

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

As used herein, the term "target nucleic acid" encompasses DNA, RNA (comprising pre-mRNA and mRNA) transcribed from said DNA, and also DNA derived from said RNA, coding, non-coding sequences, sense or antisense polynucleotides. Specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of the function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense." The DNA functions with which they will interfere include, for example, replication and transcription. The RNA functions with which they will interfere comprise all vital functions such as, for example, translocation of RNA to the protein translation site, translation of protein from RNA, splicing of RNA to one or more species of mRNA, and catalytic activity that can be coupled to or facilitated by RNA. The overall effect of such interference with the functions of the target nucleic acid is the modulation of the expression of an encoded product or oligonucleotides.

RNAi interference RNA 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 (siRNA) duplexes. SiRNAs are derived from dcRNA processing by an RNase enzyme known as Dicer. The duplex products are recruited into a multiprotein siRNA called RISC (RNA-induced silencing complex). Without wishing to be bound by any particular theory, it is then believed that a RISC is guided to a target nucleic acid (suitably mRNA), in which the siRNA duplex interacts in a sequence-specific manner to mediate cleavage in a catalytic manner. . Small interfering RNAs that can be used in accordance with the present disclosure can be synthesized and used according to procedures that are well known in the art and will be familiar to one of ordinary skill in the art. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 and about 50 nucleotides (nt). In non-limiting examples of embodiments, the siRNAs may comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

Selection of the appropriate oligonucleotides is facilitated using computer programs that align

ES 2 664 591 T3 automatically indicate nucleic acid sequences and regions of identity or homology. Such programs are used to compare obtained nucleic acid sequences, 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 show 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 a rough measure of identity. These procedures allow the selection of oligonucleotides that show a high degree of complementarity with target nucleic acid sequences in a subject to be monitored and a lower degree of complementarity with 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.

By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzyme nucleic acids (ribozymes) work by first binding to a target RNA. Such binding occurs through the target binding part of an enzymatic nucleic acid that is kept in close proximity to an enzymatic part of the molecule that acts to cleave the target RNA. In this way, the enzymatic nucleic acid first recognizes and then binds to target RNA by base pairing, and once attached to the correct site, it acts enzymatically to cut the target RNA.

By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. Decoy RNA therefore competes with the natural binding target for the binding of a specific ligand. For example, it has been shown that overexpression of activation response RNA in HIV trans (TAR) can act as a decoy and bind efficiently to the HIV tat protein, thereby preventing it from binding to TAR sequences. encoded by HIV RNA. This is indicated to be a specific example. Those skilled in the art will recognize that this is an example only, and other embodiments can easily be generated using techniques generally known in the art.

As used herein, the term "monomers" typically denotes monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides that range in size from a few monomer units, eg, between about 3-4, and about several hundred. of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methyl phosphonates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.

The term "nucleotide" covers naturally occurring nucleotides as well as non-naturally occurring nucleotides. It should be apparent to the person skilled in the art that various nucleotides that were previously considered "not naturally occurring" have subsequently been discovered in nature. Thus, nucleotides include not only the known purine and pyrimidine heterocycles containing molecules, but also heterocyclic analogs and tautomers thereof. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4, N4-ethanocytosine, N6, N6 -ethane-2,6-diaminopurine, 5-methylcytosine, 5-alkynyl (C3-C6) -cytosine, 5-fluorouracil, 3-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and the non-naturally occurring nucleotides described in Benner et al., US Patent No. 5,432,272. The term "nucleotide" is intended to cover each and every one of these examples, as well as analogs and tautomers thereof. Particularly interesting nucleotides are those containing adenine, guanine, thymine, cytosine and uracil, which are considered to be naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans. Nucleotides include the natural sugars 2'-deoxy and 2'-hydroxyl, for example, as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), in addition to their analogues.

Analogs, referring to nucleotides, include synthetic nucleotides having modified base residues and / or modified sugar residues (see, for example, generally described by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, ( 1997) Nucl Acid Res, 25 (22), 4429-4443, Toulmé, JJ, (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, eg, stability, duplex or triplex specificity, or the like.

As used herein, hybridization means the pairing of substantially complementary strands of oligomeric compounds. One mating mechanism involves the formation of bridges of

ES 2 664 591 T3 hydrogen, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds, between complementary nucleoside or nucleotide (nucleotide) bases of oligomeric compound chains. For example, adenine and thymine are complementary nucleotides that pair through hydrogen bonding. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when the 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 nonspecific binding of the compound. antisense to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo tests or therapeutic treatment, and under conditions under which tests are performed in the case of in vitro tests.

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 with a minimal number of other sequences. Stringent conditions are sequence dependent and will be different in different circumstances and in the context of this disclosure, stringent conditions in which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in the that are being investigated. In general, stringent hybridization conditions comprise low concentrations (<0.15 M) of salts with inorganic cations such as Na ++ or K ++ (i.e. low ionic strength), temperature greater than 20 ° C - 25 ° C below the Tm of the oligomeric compound: target sequence complex, and the presence of denaturants such as formamide, dimethylformamide, dimethylsulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1% for every 1% formamide. An example of a high stringency hybridization condition is 0.1X sodium chloride-sodium citrate (SSC) buffer / 0.1% (w / v) SDS at 60 ° C for 30 minutes.

"Complementary", as used herein, refers to the ability to precisely mate between two nucleotides in one or two oligomeric chains. 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 additional DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Thus, specifically hybridizable and complementarity are expressions that are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides so that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

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. In addition, an oligonucleotide can hybridize to one or more segments, so that intermediate or adjacent segments are not involved in the hybridization event (eg, a loop, 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 with a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of the 20 nucleotides of the antisense compound are complementary to a target region and therefore would specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleotides can be grouped or interspersed with complementary nucleotides and need not be contiguous with each other or with complementary nucleotides. Therefore, an antisense compound that is 18 nucleotides in length that is 4 (four) non-complementary nucleotides that 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 thus would be within the scope of the present disclosure. The percent complementarity of an antisense compound to a region of a target nucleic acid can be routinely determined 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 Package. Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), Using the default configuration, which uses the Smith-Waterman algorithm (Adv. Appl. Math., (1981) 2, 482-489).

ES 2 664 591 T3

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 they hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is the Na ion (or other salts) concentration of at least about 0.01 to 1.0 M at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short oligonucleotides (eg, 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

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

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

The resulting polypeptides will generally have significant amino acid identity to one another. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants can also 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 pathology, ie susceptibility to resistance.

Derived polynucleotides include nucleic acids undergoing chemical modification, eg, substitution of hydrogen for an alkyl, acyl, or amino group. Derivatives, for example derived oligonucleotides, can comprise non-naturally occurring parts, such as altered sugar moieties or inter-sugar linkages. By way of example, these include phosphorothioate and other sulfur-containing species that are known in the art. Derived nucleic acids can also contain tags, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogens, substrates, cofactors, inhibitors, magnetic particles, and the like.

A derived 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 can also be modified to contain a detectable label, both directly and indirectly, including, but not limited to, a radioisotope, fluorescent and enzymatic label.

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

ES 2 664 591 T3

When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isothionic, lactic, maleic, malic, mandelic, methanesulfonic, nitric, pantothenic, pamoic, phosphoric, succinic, sulfuric, tartaric, ptoluenesulfonic 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; alkaline or organic salts of acidic residues such as carboxylic acids and the like. 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 salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, maleic hydrox, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2 -acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic and the like.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical procedures. 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 combination of solvents.

The compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers. All of these isomers, including optical isomers, are included in the present invention.

Examples of skin care products include, but are not limited to, moisturizers, tanning simulation preparations, tanning lotions, massage oils, bath oils, perfumes, balms, creams, masks, foams, and gels for to shave. Examples of cosmetics include, but are not limited to, lipsticks, foundation, eyeshadow, eyeliner, blush, and concealer. Examples of cleaning products include, but are not limited to, shampoos (in particular anti-dandruff shampoos), soap, personal cleaning products including bubble bath and shower gel, and fabric detergents and dishwasher detergents. Examples of hair care products include, but are not limited to, hair setting mousses, hair setting sprays, hair setting gels, hair conditioners, or hair dyes.

By evaluating an individual's profilaggrin genotype, it is possible to determine the individual's predisposition to a skin condition. By "profilaggrin genotype" is meant the identity of the profilaggrin alleles in the genome of the individual. Individuals tested by a method of the invention are typically mammals. In one embodiment, the mammal can be a rodent. In another embodiment, the mammal can be a human. Thus, the individuals analyzed by a method of the invention are diploid and, therefore, 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, that is, one is polymorphic with respect to the other, then the individual is heterozygous for that allele. By predispositions it is meant that the presence of a single profilaggrin allele in an individual's genome, or the combination of profilaggrin alleles present in an individual's genome, is associated with or predictive of a skin condition.

The term "skin conditions" as used herein 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 an individual's predisposition to maintain a healthy level of NMF production. In other words, the invention provides a method of determining the susceptibility of the individual to conditions related to aberrant production of NMF. Typically, skin conditions caused or exacerbated by aberrant MFN production are caused by less MFN production than healthy skin. Conditions associated with aberrant filaggrin and MFN production include ichthyosis vulgaris. In another embodiment, the term "skin conditions" refers to dry skin. Dry skin conditions include senile / postmenopausal xerosis, surfactant-induced xerosis, winter xerosis, sunburn. In another embodiment, the term "skin condition" refers to scalp conditions such as dandruff. In another embodiment, the term "skin conditions" refers to erythema, such as detergent-induced erythema.

ES 2 664 591 T3

The expression care of keratinous substrates refers to all actions aimed at preserving or restoring the healthy functioning of the skin and / or hair and / or nails or any process that provides the means to preserve or improve their appearance and / or texture. In this way, care includes hydration, soothing action, protection against all kinds of aggressions, especially sun protection, and the fight against the signs of aging and its prevention.

The phrase signs of skin aging includes all changes related to the external appearance of the skin due to aging. Examples of these modifications include fine lines and wrinkles, sagging skin, loose skin, thin-appearing skin, loss of elasticity and / or skin tone, dull skin, and dull skin. Also included are internal skin modifications that do not directly translate into changes in the external appearance of the skin. An example of these internal modifications is the degradation that occurs internally in the skin as a result of subsequent exposure to UV radiation. The term "to improve the appearance of the skin" includes all phenomena that are likely to result in a visual improvement of the appearance of the skin. The skin will look more pleasant; it will be, for example, much more beautiful, firm and / or smooth. All small blemishes will be reduced or eliminated. For example, stiff skin will fade. Furthermore, the active principle according to the invention, or the composition that contains it, can be intended to protect keratinous substrates and, particularly, the skin, hair and nails from any type of external aggression. The use of these active agents, or the composition that contains them, will allow to protect the keratinous substrates and to better resist the stress caused by the environment.

The phrase external aggression refers to the aggressions produced by the environment. These can be of chemical, physical, biological or thermal origin.

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

As used herein, the term animal or patient is intended to encompass, for example, humans, sheep, elk, deer, mule deer, mink, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats. , rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

Mammal covers warm-blooded mammals that are normally under medical care (eg, humans and domesticated animals). Examples include felines, canines, equines, bovines, and humans, as well as just humans.

Treating or "treatment" covers the treatment of a pathology in a mammal, and includes: a) preventing the pathology from occurring in a mammal, in particular when said mammal is predisposed to the pathology, but has not yet been diagnosed as having it; (b) inhibiting the pathology, eg, arresting development; and / or (c) alleviating the pathology, for example by causing regression of the pathology until a desired endpoint is reached. Treat also includes amelioration of a symptom of a disease (eg, reducing pain or discomfort), where such improvement may or may not directly affect the disease (eg, cause, transmission, expression, etc.).

As used herein, "cancer" refers to all types of cancer or neoplasia or malignant tumors in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas, and sarcomas. The cancer itself manifests as a tumor or tissue comprising malignant cancer cells. Examples of tumors include sarcomas and carcinomas as such, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomisarcoma, rhabdomyosarcoma, colon carcinoma, breast cancer, prostate cancer, cancer of the colon , 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, ependymone, pinealoma, hemangioblastoma, acoustic neuroma, oligondendroglioma, meningioma,

ES 2 664 591 T3 melanoma, neuroblastoma, and retinoblastoma. Other cancers that can be treated with the disclosed composition according to the invention include, but are 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, pancreatic tumors, malignant carcinoid insulanoma, urinary bladder, gastric cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, cortical adrenal cancer, and prostate cancer.

Neurological disease or disorder refers to any disease or disorder of the nervous system and / or visual system. Neurological disease or disorder includes diseases or disorders that involve the central nervous system (brain, brainstem, and cerebellum), the peripheral nervous system (including the cranial nerves), and the autonomic nervous system (parts of which are located in the nervous system both central and peripheral). Examples of neurological disorders include, but are not limited to, headache, stupor and coma, dementia, seizures, sleep disorders, trauma, infections, neoplasms, neuropathology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders. of the peripheral nerves, muscles and neuromuscular junctions. Addictions and mental illnesses, including, but not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological disorder. The following is a list of various neurological disorders, symptoms, signs and syndromes that can be treated using the 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; alternate hemiplegia; vascular dementia; Amyotrophic Lateral Sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxemia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Chiari Anronl malformation; arteriovenous malformation; Asperger syndrome; telegiectasia ataxia; attention deficit hyperactivity disorder ; autism; autonomic dysfunction; Back pain; Batten's 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); medullary tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; brain disorder; brain aneurysm; cerebral arteriosclerosis; Brain atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; Korea; chronic inflammatory demyelinating polyneuropathy; chronic pain; regional chronic pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cyotomegalic inclusion body disease; cytomegalovirus infection; dancing eyes and dancing feet syndrome; DandyWalker syndrome; Dawson's disease; Morsier syndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty chair syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry disease; Fahr syndrome; Fainting; familial spastic paralysis; Feverish convulsions; Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other tauopathies; Gaucher disease; Gerstmann syndrome; giant cell arteritis; giant cell inclusion disease; globose cell leukodystrophy; Guillain Barre syndrome; HTLV-1 associated myelopathy; Hallervorden-Spatz disease; head trauma; headache; hemifacial spasm; spastic paraplegia; atactic polyneuritiform inheritance; otic herpes zoster; 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; immunity-mediated encephalomyelitis; inclusion body myositis; pigmentary incontinence; infant phytanic acid storage disease; Childhood Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease, Kinsboume syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora's disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; medullary lateral syndrome (Wallenberg); learning difficulties; Leigh's disease; Lennox-Gustaut syndrome; LeschNyhan syndrome; leukodystrophy; Lewy body dementia; lissencephaly; locked-in syndrome; Lou Gehrig's disease (ie, motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease - neurological sequelae; Machado-Joseph disease; macrocephaly; megalocephaly; Melkelsson-Rosenthal syndrome; Meniere's disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini spills; mitochondrial myopathies; Moebius syndrome; monomelic amyotrophy; motor neuron disease; disease of

ES 2 664 591 T3

Moyamoya; mucopolysaccharidosis; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating diseases; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; diffuse myelinoclastic sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; congenital myotonia; narcolepsy; neurofibromatosis; Neuroleptic Malignant Syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; Ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; McLeod-O'Sullivan syndrome; occipital neuralgia; spina bifida occult 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); congenital paramyotonia; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; PelizaeusMerzbacher disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; profound 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 multifocal leukoencephalopathy; progressive sclerosing polyodystrophy; progressive supranuclear palsy; brain pseudotumor; RamsayHunt syndrome (types I and 11); Rasmussen encephalitis; reflex sympathetic dystrophy syndrome; Refsum's disease; repetitive movement disorders; repetitive stress injuries; Restless Leg Syndrome; retrovirus associated myelopathy; Rett syndrome; Reye's syndrome; dance of San Vito; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; herpes; ShyDrager syndrome; Sjogren's syndrome; Sleep apnea; Soto syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; muscle atrophy in the spine; Stuff-Person syndrome; cerebrovascular accident; 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; shaking; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau disease; Wallenberg syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome: Wallenberg syndrome; Werdnig-Hoffman disease; West syndrome; lash; Williams syndrome; Wildon's disease; and Zellweger syndrome.

An inflammation refers to systemic inflammatory conditions and conditions associated with local migration and attraction of monocytes, leukocytes and / or neutrophils. Examples of inflammation include, but are not limited to, inflammation resulting from infection with pathogenic microorganisms (including gram-positive and gram-negative bacteria, viruses, fungi, and parasites such as protozoa and helminths), transplant rejection (including rejection to 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 by acute or chronic localized autoimmune allergic reactions. Autoimmune diseases include acute glomerulonephritis; arthritis or reactive arthritis; chronic glomerulonephritis; inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, and necrotizing enterocolitis; hepatitis; sepsis; alcoholic liver disease, nonalcoholic hepatic steatosis; granulocyte transfusions associated syndromes; inflammatory dermatoses such as contact dermatitis, atopic dermatitis, psoriasis, systemic lupus erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, and some forms of autoimmune diabetes, or any other state where the attack of the subject's own immune system results in the destruction of pathological tissue. Allergic reactions include allergic asthma, chronic and acute bronchitis of delayed hypersensitivity. Systemic inflammatory disease states include inflammation associated with trauma, burns, reperfusion following ischemic events (eg, thrombotic events in the heart, brain, intestine, or peripheral vessels, including myocardial infarction and stroke), sepsis, distress syndrome acute respiratory tract (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 disease, Hashimoto's thyroiditis, hepatocellular carcinoma, thymic atrophy, chronic pancreatitis, rheumatoid arthritis, osteoarthritis, reactive lymphoid hyperplasia, papillary carcinoma, ulcerative colitis, Crohn's disease, colitis ulcerative, 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,

A cardiovascular disease or disorder includes those disorders that can cause ischemia or are caused

ES 2 664 591 T3 by reperfusion of the heart. Examples include, but are not limited to, atherosclerosis, coronary artery disease, granulomatous myocarditis, chronic (non-granulomatous) myocarditis, primary hypertrophic cardiomyopathy, peripheral arterial 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 to those skilled in the art or that involve dysfunction of or tissue damage to the heart or vasculature, especially, but not limited to, tissue damage related to PONI 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 (non-granulomatous) myocarditis.

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.

Polynucleotides and compositions of oligonucleotides and molecules

Targets: In one embodiment, the targets include Filaggrin nucleic acid (FLG) sequences, including without limitation non-coding sense and / or antisense sequences and / or coding sequences associated with fLg.

The filaggrin gene plays a role in building 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 waterproof skin barrier layers present on the outermost surface of the skin and keep them hydrated. The inherent barrier function of the skin is similar to a plastic or adhesive film, it acts to prevent the loss of water from the skin and, more importantly, to protect the body from foreign materials in the environment, such as allergens. The lack of an intact skin barrier causes allergens to enter the body where they produce a variety of allergic responses including eczema, asthma, hay fever, and other allergies.

Lack of expression of the filaggrin protein 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 hosts genes for many other proteins that are important for normal barrier function of the epidermis. The main function of filaggrin appears to be to aggregate the epidermal cytoskeleton to form a dense protein and lipid matrix that regulates the permeability of the skin to water and external particles such as allergens.

Pioglitazone - ACTOS (pioglitazone hydrochloride) is an oral antidiabetic agent that works mainly by reducing insulin resistance. ACTOS is used to treat type 2 diabetes mellitus (also known as non-insulin dependent diabetes mellitus [NIDDM] or adult-onset diabetes). Pharmacological studies indicate that ACTOS improves insulin sensitivity in muscles 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-] thiazolidinedione monohydrochloride belongs to a different chemical class and has an action different pharmacological than sulfonylureas, metformin or α-glucosidase inhibitors. The molecule contains a symmetric 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 pharmacological activity of the two enantiomers.

Pioglitazone Hydrochloride is an odorless white crystalline powder that has a molecular formula of C1gH20N2O3S'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.

ACTOS is indicated as a supplement to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Pioglitazone has also been used to treat nonalcoholic steatohepatitis (fatty liver), but this use is currently considered experimental.

However, pioglitazone has not yet been investigated for use in the field of dermatology.

Lomerizine: It is a calcium channel blocker with anti-migraine properties and selectively inhibits the constriction of the cerebral arteries. It is known to be a neuroprotective and is in trials for glaucoma. The

ES 2 664 591 T3 side effects of Lomerizine include minimal cardiovascular side effects, drowsiness and flushing.

However, lomerizine has not yet been investigated for use in the field of dermatology.

Bupropion, also known as Wellbutrin, Zyban, Voxra, Budeprion, or Aplenzin; formerly known as amfebutamone is an antidepressant of the aminoketone class, chemically it is not related to the tricyclic, tetracyclic, selective serotonin reuptake inhibitor or other known antidepressant agents, its structure closely resembles that of diethylpropion; It is related to phenylethylamines.

Bupropion is designated as (±) -1- (3-chlorophenyl) -2 - [(1,1-dimethylethyl) amino] -1-propanone hydrochloride. Its molecular weight is 276.2. The molecular formula is C13H18ClNO * HCl. Bupropion hydrochloride powder is white, crystalline, and highly soluble in water. It has a bitter taste and produces the sensation of local anesthesia in the oral mucosa.

Bupropion is indicated for the treatment of acute depressive disorder. An acute depressive episode (DSM-IV) involves the presence of 1) depressed mood or 2) loss of interest or pleasure; Additionally, 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 decreased interest or pleasure in usual activities, significant change in weight and / or appetite, insomnia or hypersomnia, agitation or psychomotor retardation, increased fatigue, feelings of guilt or worthlessness, decreased thinking or concentration altered, suicide attempt or suicidal ideation.

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, as does methylphenidate. As a psychostimulant, it is inherently an anxiogenic compound and the opposite benefits are poorly understood and apparently paradoxical.

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 facility studies and a pilot study, bupropion reduces the level of an inflammatory mediator TNF-alpha and may be useful in autoinflammatory conditions such as Crohn's disease and psoriasis.

However, bupropion has not yet been investigated for use in the field of dermatology.

Fenprobamate is a centrally acting muscle relaxant with additional sedative and anticonvulsant effects. The overdose is similar to that of the barbiturate. Its mechanism of action is probably similar to meprobamate. Fenprobamate was previously used in humans as an anxiolytic, and is still sometimes used in general anesthesia and to treat muscle cramps and spasticity. Fenprobamate is still used in some European countries, but has generally been replaced by newer drugs. Fenprobamate is metabolized by oxidative degradation of the amide group and ortho-hydroxylation of the benzene ring, and the kidneys eliminate it in the urine.

However, fenprobamate has not yet been investigated for use in the field of dermatology.

Benidipine, also known as benidipine or benidipine hydrochloride, is a dihydropyridine calcium channel blocker for the treatment of high blood pressure (hypertension). Benidipine is a dihydropyridine calcium channel blocker that inhibits not only L-type but also T-type calcium channels. The chemical name for Benidipine is 3-methyl-5 - [(3R) -1- (phenylethyl) -3-piperidinyl] acid (4R) -rel-1,4-dihydro-2,6-dimethyl-4 acid hydrochloride ester - (3-nitrophenyl) -3,5-pyridinedicarboxylic. Orally active antihypertensive agent showing a wide range of in vitro and in vivo activities. Inhibits Ca channels² + L- and T-type. It also inhibits aldosterone-induced mineralocorticoid receptor activation. It exhibits cardioprotective and anti-atherosclerotic effects.

However, benidipine has not yet been investigated for use in the field of dermatology.

Piroxicam is a member of the oxicam group of non-steroidal anti-inflammatory drugs (NSAIDs). Each maroon and blue capsule contains 10 mg of piroxicam, each maroon capsule contains 20 mg of piroxicam for oral administration. The chemical name for piroxicam is 4-hydroxyl-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamid-1,115

ES 2 664 591 T3 dioxide. Piroxicam occurs as a white crystalline solid, moderately soluble in water, dilute acid, and most organic solvents. It is poorly soluble in alcohol and in aqueous solutions. It has a weakly acid 4-hydroxy proton (pKa 5.1) and a weak basic pyridyl nitrogen (pKa 1.8). The molecular weight of piroxicam is 331.35. The molecular formula is C15H13N3O4S.

Piroxicam is a non-steroidal anti-inflammatory drug used to relieve the symptoms of rheumatoid arthritis and osteoarthritis, primary dysmenorrhea, postoperative pain; and it acts as a pain reliever, especially when there is an inflammatory component. It is also used in veterinary medicine to treat certain neoplasms that express cyclooxygenase (COX) receptors, such as cancers of the bladder, colon, and prostate.

However, piroxicam has not yet been investigated for use in the field of dermatology.

Topiramate is a sulphamate substituted monosaccharide. TOPAMAX® (topiramate) tablets are available as 25 mg, 50 mg, 100 mg and 200 mg round tablets for oral administration. TOPAMAX® (topiramate capsules) Sprinkle Capsules are available as 15 mg and 25 mg sprinkle capsules for oral administration as whole capsules, or they are opened and sprinkled on soft foods.

Topiramate is a white crystalline powder with a bitter taste. Topiramate is more soluble in alkaline solutions containing sodium hydroxide or sodium phosphate and has a pH of 9 to 10. It is easily 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 C12H21NO8S and a molecular weight of 339.36. Topiramate is designated chemically as 2,3: 4,5-di-O-isopropylidene-pD-fructopyranose sulfamate.

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 taking other anti-seizure medications. Topiramate is also used to prevent migraine headaches, but not to relieve the pain of migraines when they occur. Topiramate belongs to a class of medications called anticonvulsants. It works by reducing abnormal arousal in the brain.

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 delayed development. It is also approved by the Food and Drug Administration (FDA) and, most often, prescribed for the prevention of migraines. Psychiatrists have used topiramate to treat bipolar disorder and often use topiramate to increase psychotrophics or counteract the weight gain associated with numerous antidepressants.

Topiramate has been investigated for use in the treatment of alcoholism and obesity, especially to reduce binge eating.

Topiramate is also used in clinical trials to treat post-traumatic 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 hypoxic ischemic injury. Other unapproved and investigational uses include treatment of essential tremor, bulimia nervosa, obsessive compulsive disorder, alcoholism, smoking cessation, idiopathic intracranial hypertension, neuropathic pain, cluster headache, and drug dependence. cocaine. Topiramate is also being studied with a mixture of phentermine to form a drug called Qnexa for the treatment of obesity.

However, topiramate has not yet been investigated for use in the field of dermatology.

Isradipine is a calcium antagonist. Chemically, isradipine is 4- (4-benzofurazanyl) -1,4-dihydro-2,6-dimethyl, 3,5-pyridinedicarboxylic acid methyl-1-methylethyl ester. Isradipine is a fine, crystalline, yellow powder that is odorless or has a faint, characteristic odor. Isradipine is practically insoluble in water (<10 mg / l at 37 ° C), but it is soluble in ethanol and easily soluble in acetone, chloroform and methylene chloride.

Isradipine is indicated for the treatment of hypertension. It can be used alone or at the same time as thiazide-type diuretics. It is usually prescribed for the treatment of high blood pressure to reduce the risk of stroke and heart attack. More recent research in animal models

ES 2 664 591 T3 suggests that isradipine may have potential uses to treat Parkinson's disease.

However, isradipine has not yet been investigated for use in the field of dermatology.

Nicorandil is one of the common medications used in the treatment of angina pectoris. The drug can be classified as a vasodilator drug.

The action of nicorandil is understood by the process of smoothing the smooth muscle of the blood vessels. The action is especially marked especially in the case of the venous system.

Nicorandil works by activating potassium channels and 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 vasodilation by dephosphorylation of the myosin light chain. Being selective for vascular potassium channels, nicorandil does not have a significant action on cardiac contractility and conduction.

Nicorandil can dilate the coronary vessels of a healthy individual, however, its effects on the coronary vessels of a person with ischemic heart disease will be small as they will be fully dilated. Instead, it dilates the venous system, reducing the preload and work of the heart.

However, nicorandil has not yet been investigated for use in the field of dermatology.

Piribedil is the D2 agonist that is used primarily to treat Parkinson's disease. It works by stimulating dopamine receptors, which relieves various symptoms such as tremors. It is also used to treat other conditions, such as circulatory problems, due to its D2 antagonistic effects. The drug also comes under the Trivastal brand name that comes in the form of extended-release capsules to be taken by mouth. Piribedil can be used as monotherapy or in conjunction with L-dopa therapy in early and advanced Parkinson's disease. A large number of elderly patients have benefited from its relative effects on cognition, such as treating impaired memory, attention, and focus.

Piribedil works by stimulating the dopamine receptors present in the brain which, in fact, treats the deficit of postsynaptic D2 and D3 receptors of the mesolimbic and mesocortical pathways. The drug also has vasodilator effects thereby improving different cognitive symptoms and enhances noradrenergic transmission resulting in improved focus, attention and memory.

Piribedil is also indicated in the treatment of pathological cognitive deficits in older people (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).

However, piribedil has not yet been investigated for use in the field of dermatology.

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

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

However, oxaprozin has not yet been investigated for use in the field of dermatology.

Glycopyrrolate is a quaternary ammonium salt with the chemical name: 3 [(cyclopentylhydroxyphenylacetyl) oxy] -1,1-dimethylpyrrolidinium bromide. Its molecular formula is C19H28BrNO3 and its molecular weight is 398.33.

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 incubation. When indicated, Robinul injection can be used intraoperatively to counteract

ES 2 664 591 T3 arrhythmias associated with vaginal or drug-induced or vagal reflexes. Glycopyrrolate protects against the peripheral muscarinic effects (eg, bradycardia and excessive secretions) of cholinergic agents such as neostigmine and pyridostigmine given to reverse neuromuscular blockade due to non-depolarizing muscle relaxants.

Glycopyrrolate is indicated in peptic ulcer, for use in adults as adjunctive therapy for the treatment of peptic ulcer when a rapid anticholinergic effect is desired or when oral medication is not tolerated.

In anesthesia, glycopyrrolate injection can be used as a preoperative medication to reduce salivary, tracheobronchial, and pharyngeal secretions, as well as to decrease the acidity of gastric secretion. It is also used in conjunction with neostigmine, a neuromuscular agent that blocks the reversal agent, to prevent the muscarinic effects of neostigmine, such as bradycardia. It is also used to reduce excessive saliva (hypersalivation). It decreases the acid secretion in the stomach and therefore can be used to treat stomach ulcers, along with other drugs. Its use in the treatment of asthma and COPD has been described. It has been used topically and orally to treat hyperhidrosis.

However, glycopyrrolate has not yet been investigated for use in the field of dermatology.

Granisetron, granisetron hydrochloride, an antiemetic and antiemetic agent. Chemically it is endoN- (9-methyl-9-azabicyclo- [3.3.1] -non-3-yl) -1-methyl-1H-indazole-3-carboxamide hydrochloride with a molecular weight of 348.9 (free base of 312.4). Its empirical formula is C18h24N4O * HCI.

Granisetron Indications: Granisetron hydrochloride is used for the prevention of nausea and vomiting associated with initial and repeated courses of emetogenic cancer therapy, including high doses of cisplatin. Chemotherapy-induced nausea and vomiting. 5-HT3 receptor antagonists are the main drugs used to treat and prevent chemotherapy-induced nausea and vomiting. They are often given intravenously about 30 minutes before starting therapy. Postoperative and post-diagnostic nausea and vomiting. It is a possible therapy for nausea and vomiting due to acute or chronic medical conditions or acute gastroenteritis. Treatment of cyclic vomiting syndrome although there are no formal trials to confirm efficacy. Nausea and vomiting associated with radiation, including whole body radiation and fractionated abdominal radiation.

However, granisetron has not yet been investigated for use in the field of dermatology.

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

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, opiate dependence, systemic lupus erythematosus, depression, obsessive compulsive disorder, Tourette syndrome, gambling problems, 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.

However, memantine has not yet been investigated for use in the field of dermatology.

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 C21H26N2O7.

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-stroke neurological condition (i.e., Hunt and Hess Grades IV).

The main use of nimodipine is the prevention of cerebral vasospasm and the resulting ischemia, a complication of subarachnoid hemorrhage (a form of cerebral hemorrhage), specifically ruptured intracranial berry aneurysms, regardless of the patient's post-stroke neurological condition. Its administration begins within 4 days after a subarachnoid hemorrhage and is continued for three weeks. If the blood pressure drops more than 5%, the dosage is adjusted. While nimodipine is not currently used in head injury, it has shown promise in clinical studies. A 2009 study (Asian A and

ES 2 664 591 T3 cois., February 2009 Pharmacol. Res. 59 (2): 120-4), found that patients with severe head injury given nimodipine via peripheral venous injection, along with standard procedures, had significantly higher cerebral perfusion pressure and venous oxygen saturation. jugular, while intracranial pressure, jugular lactate and jugular glucose were lower. The study concluded that the Glasgow outcome score values ​​were higher and that brain metabolism improved.

However, nimodipine has not yet been investigated for use in the field of dermatology.

Amlodipine, amlodipine besylate is chemically described as 3-ethyl-5-methyl (±) -2 - [(2-aminoethoxy) methyl] -4- (2-chlorophenyl) -1,4-dihydro-6-methyl-3, 5-pyridinedicarboxylate, monobenzenesulfonate. The empirical formula is C20H25CIN2O₅- C6H8O3S.

Amlodipine base (as besylate, mesylate, or maleate) is a long-acting calcium channel blocker (dihydropyridine class) used as an antihypertensive and in the treatment of angina pectoris. Like other calcium channel blockers, amlodipine works by relaxing the smooth muscle of the arterial wall, lowering total peripheral resistance, and therefore lowering blood pressure.

Amlodipine is indicated for the treatment of hypertension. It can 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 can be used alone or in combination with other antianginal agents.

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

In patients with coronary artery disease (CAD) recently documented by angiography and without heart failure or an ejection fraction <40%, amlodipine is indicated to reduce the risk of hospitalization for angina and to reduce the risk of a coronary artery bypass procedure.

However, amlodipine has not yet been investigated for use in the field of dermatology.

In one embodiment, antisense oligonucleotides and compositions of the present invention are used to prevent or treat diseases or disorders associated with members of the FLG family. Exemplary filaggrin-mediated (FLG) diseases and disorders that can be treated with cells / tissues regenerated from stem cells obtained using the antisense compounds comprising: a disease or disorder associated with abnormal function and / or expression of the FLG gene, a dermatological disease or disorder, signs of skin aging, a skin condition caused by external aggression, an allergy, psoriasis, asthma, eczema, fever hay, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immunity-mediated disease or disorder, a hyperimmune or hypoimmune disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact allergy, food allergy, etc.), cehac disease, a disease or neurological disorder, a neurodegenerative disease or disorder (eg. g., Alzheimer's disease, Parkinson's disease, ALS, etc.), AIDS-related wasting syndrome, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin .

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

In one embodiment, the composition of the present invention comprises one or more oligonucleotides specific for one or more polynucleotides for filaggrin (FLG), said polynucleotides comprise antisense sequences, complementary sequences, alleles, homologues, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

In one embodiment, the composition of the present invention comprises one or more oligonucleotides specific for one or more polynucleotides for filaggrin (FLG) and one or more modulator molecules of the FLG gene, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologues, isoforms ,

ES 2 664 591 T3 variants, derivatives, mutants, fragments or combinations thereof.

An embodiment of the present invention provides a composition, where the molecule is selected from the group of Pioglitazone, Lomerizine, Bupropion, Fenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Pyribedil, Oxaprozine, Glycopyrrolate, Granisetron, Memantine, Nimodipine, and Amlodipine

One embodiment of the present invention provides a composition for use in treating a dermatological disease or disorder, the composition comprising one or more FLG gene modulator molecules and a pharmaceutically acceptable carrier.

An embodiment of the present invention provides a composition, where the compound is selected from the group of Pyoglitazone, Lomerizine, Bupropion, Fenprobarnate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Pyribedil, Oxaprozine, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

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

One embodiment of the present invention provides a composition, wherein the composition further comprises an antisense oligonucleotide for a natural Filaggrin antisense sequence, where the antisense oligonucleotide modulates the expression of the FLG gene in a subject.

An embodiment of the present invention provides a composition, wherein the oligonucleotide comprises nucleotide sequences established as SEQ ID NOS: 3 to 13.

An embodiment of the present invention provides a composition, where the oligonucleotide stated as SEQ ID NOS: 3 to 13 comprise one or more modifications or substitutions.

One embodiment of the present invention provides a method of treating an FLG-associated disease or disorder in a subject, the method comprising administering to the subject a composition comprising one or more FLG gene modulator molecules and a pharmaceutically acceptable carrier.

An embodiment of the present invention provides a process, where the compound is selected from the group of Pyoglitazone, Lomerizine, Bupropion, Fenprobarnate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Pyribedil, Oxaprozine, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

One embodiment of the present invention provides a method, wherein the compositions further comprise an antisense oligonucleotide that modulates the expression or activity of the FLG gene.

An embodiment of the present invention provides a method, where the composition further comprises an antisense oligonucleotide for a natural Filaggrin antisense sequence, where the antisense oligonucleotides modulate the expression of the FLG gene in a subject.

An embodiment of the present invention provides a method, wherein a disease associated with at least one Filaggrin polynucleotide is selected from: a dermatological disease or disorder, a sign of skin aging, a skin condition caused by external aggression, an allergy, psoriasis, asthma, eczema, hay fever, ichthyosis vulgaris, atopic dermatitis (AD), herpetic eczema, rheumatoid arthritis , a cardiovascular disease or disorder, cancer, an inflammatory disease, a disease or disorder of medium immunity, a disease of hyperimmunity or hypoimmunity or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (eg, allergic rhinitis, contact-type allergy, food allergy, etc.), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (eg, Alzheimer's disease, Parkinson's disease, ALS, etc.), AIDS-related wasting syndrome, a disease or disorder associated with the barrier function of the skin, a chronic inflammatory skin disease, clinical dry skin.

An embodiment of the present invention that provides a method of preventing or treating a skin condition associated with at least one Filaggrin polynucleotide (FLG) and / or at least one encoded product thereof, comprising: administering to a patient who has a skin condition or risk of developing a skin condition a therapeutically effective dose of a Filaggrin regulatory compound, at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Filaggrin polynucleotide (FLG) and modulates the expression of said at least one Filaggrin polynucleotide (FLG) and a pharmaceutically carrier

ES 2,664,591 T3 acceptable; thereby preventing or treating skin disease associated with at least one Filaggrin polynucleotide (FLG) and / or at least one encoded product thereof,

One embodiment of the present invention provides a process, where the compound is selected from the group of Ploglitazone, Lomerizine, Bupropion, Fenprobarnate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Pyribedil, Oxaprozine, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

An embodiment of the present invention provides a method, where the skin condition is caused by inflammation, light damage or aging.

An embodiment of the present invention provides a method, where the skin condition is the development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullous disease, exfoliative dermatitis, seborrheic dermatitis, an erythema , discoid lupus erythematosus, dermatomyositis, skin cancer or an effect of natural aging.

An 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.

An embodiment of the present invention provides a use, where the composition further comprises an antisense oligonucleotide that modulates the expression or activity of the FLG gene.

An embodiment of the present invention provides a use, where the composition further comprises an antisense oligonucleotide for a natural antisense sequence of Filaggrin, where the antisense oligonucleotide modulates the expression of the FLG gene in a subject.

An embodiment of the present invention provides a use, where the compound is selected from the group of Ploglitazone, Lomerizine, Bupropion, Fenprobarnate, Benidipine, Piroxicam. Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine.

An embodiment of the present invention provides a use, where the skin disease or disorder is the development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullous disease, exfoliative dermatitis, seborrheic dermatitis, an erythema, discoid lupus erythematosus, dermatomyositis, skin cancer, or an effect of natural aging.

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

A preferred embodiment of the present invention provides a composition for skin treatment and / or cosmetic application comprising the compounds of the present invention, e.g. g., to modulate the expression of the FLG gene in the skin. In embodiments, topical treatment by the compounds of the present invention, to increase cell life expectancy or prevent apoptosis. For example, the skin can be protected from aging, eg, the development of wrinkles, by treating the skin, eg, epithelial cells, as described herein. In an exemplary embodiment, the skin is contacted with a pharmaceutical or cosmetic composition of the present invention. Exemplary skin lesions 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 pemphigus), exfoliative dermatitis, seborrheic dermatitis, erythema (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.

In one embodiment of the present invention the composition is incorporated into a topical formulation containing a topical carrier which is generally suitable for the topical administration of drugs and which comprises any such material known in the art. The topical vehicle can be selected to provide the composition in the desired form, for example, in the form of an ointment, lotion, cream, microemulsion, gel, oil,

ES 2 664 591 T3 solution, or the like, and may be composed of a material of natural or synthetic origin, it is preferable that the selected carrier does not adversely affect the active agent or other components of the topical formulation. Examples of suitable topical vehicles for use herein include water, alcohols, and other non-toxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. The formulations can be colorless and odorless ointments, lotions, creams, microemulsions and gels.

The composition of the invention can be incorporated into ointments, which are generally semi-solid preparations that are typically based on petroleum jelly 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 optimal drug delivery and preferably provide other desired characteristics as well, eg, emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, non-irritating, and non-sensitive. As explained in Remington's Pharmaceutical Sciences (Mack Pub. Co.), ointment bases can be grouped into four classes: oil bases; emulsifiable bases; emulsion bases; and water soluble bases. Oil bases for ointments 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 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, eg, Remington's, supra).

The composition of the invention can be incorporated into lotions, which are generally frictionless preparations to be applied to the surface of the skin, and are typically liquid or semi-liquid preparations in which solid particles, including the active agent, are present in a base of water or alcohol. Lotions are generally suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations for treating large areas of the body, due to 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 locating and maintaining the active agent in contact with the skin, eg, methyl cellulose, sodium carboxymethyl cellulose, or the like. An exemplary lotion formulation for use in conjunction with the present method contains propylene glycol in admixture with a hydrophilic petrolatum such as that available under the Aquaphor® trademark from Beiersdorf, Inc. (Norwalk, CT).

The composition of the invention can be incorporated into creams, which are generally liquid or semi-solid viscous emulsions, either oil-in-water or water-in-oil. Cream bases are washable with water, and contain an oil phase, an emulsifier and a water phase. The oil phase is generally composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, though not necessarily, exceeds the oil phase by volume, and generally contains a humectant. The emulsifier in a cream formulation, as discussed in Remington's, above, is generally a nonionic, anionic, cationic or amphoteric surfactant.

The composition of the invention can be incorporated into microemulsions, which are generally thermodynamically stable, isotopically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker , 1992), volume 9). For the preparation of microemulsions, a surfactant (emulsifier), cosurfactant (co-emulsifier), an oil phase and an aqueous phase are needed. Suitable surfactants include any surfactant that is useful in the preparation of emulsions, e.g. For example, emulsifiers that are typically used in the preparation of creams, the co-surfactant (or co-emulsifier) ​​is generally selected from the group of polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier / co-emulsifier combinations are generally, but not necessarily selected from the group consisting of glyceryl monostearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol palmitostearate; and caprylic and capric triglycerides and olcoyl macrogolglycerides, the aqueous phase includes not only water but also typically buffers, glucose, propylene glycol polyethylene glycols, preferably low 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, mono-, di- and triglyceride mixtures, mono- and diesters of PEG (eg, oleoyl-macrogol-glycerides), etc.

The composition of the invention can be incorporated into gel formulations, which are generally semi-solid systems consisting of suspensions formed by small inorganic particles (two-phase systems)

ES 2 664 591 T3 or large organic molecules substantially uniformly distributed throughout a carrier liquid (single phase gels). Monophasic layers can be prepared, for example, by combining the active agent, a carrier liquid, and a suitable gelling agent such as tragacanth (2-5%), sodium alginate (2-10%), gelatin (2-15%). %), methylcellulose (3-5%), sodium carboxymethylcellulose (2-5%), carbomer (0.3-5%) or polyvinyl alcohol (10-20%) together and mixed until produced a characteristic semi-solid product. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose, and gelatin. Although gels commonly employ an aqueous carrier fluid, alcohols and oils can also be used as the carrier fluid.

Various additives, known to those skilled in the art, can be included in the formulation, e.g. eg, topical formulations. Examples of additives include, but are not limited to, solubilizers, skin permeation enhancers, opacifiers, preservatives (eg, antioxidants), gelling agents, buffering agents, surfactants (particularly nonionic and amphoteric surfactants), emulsifiers, emollients, thickening agents, stabilizers, humectants, colorants, fragrances, and the like. The inclusion of solubilizers and / or skin permeation enhancers is particularly preferred, along with emulsifiers, emollients, and preservatives. An optimal topical formulation comprises about: 2% by weight to 60% by weight, preferably 2% by weight% to 50% by weight of skin permeation enhancer and / or solubilizer; 2% by weight to 50% by weight, preferably 2% by weight% to 20% by weight of emulsifiers; 2% by weight to 20% by weight of emollient; and from 0.01 to 0.2% by weight of preservative, with the active agent and vehicle (eg water) constituting the remainder of the formulation.

A skin permeation enhancer serves to facilitate the passage of therapeutic levels of active agent to pass through a reasonably sized area of ​​intact 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 tetradecylmethylsulfoxide; 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; various solvents such as dimethylformamide (DMF), 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® from Whitby Research Incorporated, Richmond, VA).

Examples of solubilizers include, but are not limited to, the following: hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, commercially available as Transcutol®) and diethylene glycol oleate monoethyl ether (commercially available as Soficutol®); 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 caprylic / capric glycerides PEG-8 (commercially available as Labrasol®); alkylmethylsulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DNA. Many solubilizers can also act as absorption enhancers. A single solubilizer can be incorporated into the formulation, or a mixture of solubilizers can be incorporated into the formulation.

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

Other active agents can also be included in formulations, for example other anti-inflammatory agents, pain relievers, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunblocks that are commonly found in sunscreen formulations including, but not limited to, anthranlates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (eg octyl methoxycinnamate), dibenzoyl methanes (eg, butyl methoxydibenzoyl methane), paminobenzoic acid (PABA) and derivatives thereof, and salicylates (eg, octyl salicylate).

The compositions according to the present invention can most notably be applied 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 anti-aging composition. We can also imagine 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 cosmetic compositions for body and hair health.

Furthermore, the compounds according to the invention, as previously defined, stimulate the metabolic functioning of skin cells. They allow protein synthesis to increase, which is essential for your

ES 2 664 591 T3 function, especially 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 treating wound healing disorders.

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, more generally, to treat dermatological disorders.

Furthermore, according to another aspect, the previously defined compounds 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 previously defined compounds as medicaments.

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

The cosmetic treatment process related to the invention can be implemented notably by applying the cosmetic compositions defined above according to the procedures generally used for compositions, such as for example the application of creams, gels, serums, lotions, milks, shampoos and creams. sunscreen, on the skin or hair, and as a toothpaste applied to the gums. The particular modes of carrying out this cosmetic treatment process also appear from the preceding description.

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

Non-therapeutic applications of the methods of the invention include means of grouping individuals for the purposes of testing for agents, eg, cosmetics or any other form of preparation introduced into the body. This can be useful to interpret the results obtained from such tests, for example, where the reaction of the skin of different individuals during the test is not uniform.

The heterogeneity of responses can be more clearly interpreted by grouping or stratifying individuals according to their predisposition to skin conditions. The skilled person will appreciate that by using this procedure, it is possible to develop agents that are suitable for use with some individuals but are not suitable for others. Consequently, an agent panel can be built, whose panel includes different agents who have the suitability for use with different individuals. After testing, 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. Therefore, the method of the invention allows individuals to match a personal care product such as those listed above.

Procedures to identify the prophylaggrin genotype of an individual are performed on biological material from the individual. Preferably, the biological material is removed; of the individual before performing the identification procedure. In other words, typically the biological material is ex vivo. Ex vivo material can be further cultured in vitro prior to performing the procedure.

ES 2 664 591 T3

An ex vivo sample can comprise tissue or cells taken from anywhere in the body. A preferred ex vivo sample comprises material taken from the circulatory system, or material taken from a body cavity, such as the oral cavity. A particularly preferred ex vivo sample is a saliva sample.

Alleles present in an individual can be determined from a saliva sample using procedures known in the art, such as those described in Schie and Wilson (1997, Journal of Immunological Methods, 208, 91-101). Consequently, an individual can provide the sample ex vivo without the need for specialized collection means. For example, an individual may simply provide a saliva sample or a buccal swab prior to testing.

The prophylaggrin 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 repeats, generally 10, 11, or 12 repeats. The filaggrin repeats are typically the same length (972 bp, 324 amino acids in humans) to each other, although this is less typical of the filaggrin repeats at the 5 'and 3' ends of the mRNA. Filaggrin repeats can show considerable sequence variation, typically 0-50%, more typically 2-30%, still more typically 10-15%, between repeats on the same allele and between different alleles. Variations are generally attributable to a single base change but can 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%, even more preferably at least 95% sequence identity with 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 carboxyl ends are more conserved, as are the 5 'and 3' DNA sequences that flank the coding parts of the gene (Presland et al. (1992) J Biol Chew, 267 ( 33), 2377223781).

In one embodiment, the oligonucleotides are specific for polynucleotides of the NEUFLG gene, including, without limitation, noncoding regions (matrix). The FLG gene targets comprise FLG gene variants; FLG gene mutants, including SNPs; non-coding sequences of the FLG gene; alleles, fragments and the like. Preferably, the oligonucleotide is an antisense RNA molecule.

According to embodiments of the invention, the target nucleic acid molecule is not limited to polynucleotides of the FLG gene alone, but extends to any of the isoforms, receptors, homologues, non-coding regions, and the like of the FLG gene.

In one embodiment, an oligonucleotide is directed to a natural antisense sequence (natural antisense to the coding and noncoding regions) of FLG gene targets, including, without limitation, variants, alleles, homologues, mutants, derivatives, fragments, and sequences complementary to these. Preferably the oligonucleotide is an antisense RNA or DNA molecule.

In one 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 can be produced that contain thymidine, guanosine, cytidine, or other natural or non-natural nucleotides at this position. This can be done at any of the antisense compound positions. These compounds are then tested using the procedures described herein to determine their ability to inhibit the expression of a target nucleic acid.

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

An antisense compound is specifically hybridizable when the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to produce a loss of activity, and there is a degree of

ES 2 664 591 T3 sufficient complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired. Such conditions include, ie, physiological conditions in the case of in vivo tests or therapeutic treatment, and conditions in which the tests are performed in the case of in vitro tests.

An antisense compound, be it DNA, RNA, chimeric, substituted, etc., is specifically hybridizable when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to produce a loss of utility. , and there is a degree of complementarity sufficient to prevent nonspecific binding of the antisense compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo tests or therapeutic treatment, and in the case of in vitro tests, under conditions in which the tests are performed.

In one embodiment, targeting the FLG gene, including without limitation, antisense sequences that are identified and expanded, using, for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NO: 2 , and the like, modulate the expression or function of the FLG gene. In one embodiment, expression or function is up-regulated compared to a control. In one embodiment, expression or function is down-regulated compared to a control.

In one embodiment, the oligonucleotides comprise sequences set forth as SEQ ID NO: 3 to 13 including antisense sequences that are identified and expanded, using, for example, PCR, hybridization, etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like. Examples of modified linkages or internucleotide linkages include phosphorothioate, phosphorodithioate, or the like. In one embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention can be a monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphate, phosphorothioate and the like. The preparation of the phosphate analogs indicated above, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, is also known per se and need not be described here.

Antisense specificity and sensitivity is also employed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been used as therapeutic moieties in the treatment of pathologies in animals and humans. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are currently underway. Thus, it is established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimens for the treatment of cells, tissues and animals, especially humans.

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 DNA functions with which they will interfere include, for example, replication and transcription. The RNA functions with which they will interfere include all vital functions such as, for example, translocation of DNA to the protein translation site, translation of protein from RNA, splicing of RNA to one or more species of mRNA, and catalytic activity that can be coupled to or facilitated by RNA. The functions can be upregulated or inhibited depending on the desired functions.

Antisense compounds include oligomeric antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicing agents, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. Thus, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

The targeting of an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process normally begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid can be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or pathology, or a nucleic acid molecule of an infectious agent. In the present invention, the target nucleic acid encodes the filaggrin gene (FLG).

The targeting process typically also includes determining at least one target region, segment, or site within the target nucleic acid so that the antisense interaction occurs in a manner that results in the desired effect, eg, modulation of expression. Within the context of the present invention,

ES 2 664 591 T3 the term "region" is defined as a part of the target nucleic acid that has at least one identifiable structure, function or characteristic. Within the target nucleic acid regions are segments. Segments are defined as smaller parts or sub-parts of regions within a target nucleic acid. Sites, as used in the present invention, are defined as positions within a target nucleic acid.

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

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

In one embodiment, the antisense oligonucleotides are specific for natural antisense sequences of the FLG gene where the binding of the oligonucleotides to the natural antisense sequences of the FLG gene modulate the expression and / or function of the FLG gene.

In one embodiment, the oligonucleotide compounds comprise sequences set forth as SEQ ID NO: 3 to 13, antisense sequences that are identified and expanded using, for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like. Examples of modified linkages or internucleotide linkages include phosphorothioate, phosphorodithioate, or the like. In one embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) that can be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present disclosure can be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the phosphate analogs indicated above, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, is also known per se and need not be described here.

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 it is also called the AUG codon, the start codon, or the AUG start codon. A minority of genes have a translation initiation codon that has 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 initiation codon can encompass many codon sequences, even though the initiator amino acid in each case is usually methionine (in eukaryotes) or formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes can have two or more alternative start codons, any one of which can be preferentially used for translation initiation in a particular type of cell or tissue, or under a particular set of conditions. In the context of the invention, "initiation codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of a transcribed mRNA of a gene encoding filaggrin (FLG), regardless of the one or more sequences of said codons. A translation stop codon (or stop codon) of a gene can 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).

The terms "initiation codon region" and "translation initiation codon region" refer to a portion of said mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in any direction (i.e., 5 'or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "stop codon region of translation" refer to a part of said mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5 'or 3' ) from a translation stop codon. Accordingly, the initiation codon region (or translation initiation codon region) and the stop codon region (or translation termination codon region) are all regions that can be effectively targeted with the antisense compounds of the present invention.

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 that can be effectively chosen as Diana. Within the context of the present invention, a targeted region is the intragenic region that encompasses the translation initiation or termination codon of the open reading frame (ORF) of a gene.

ES 2 664 591 T3

Another target region includes the 5 'untranslated region (5'UTR), known in the art to refer to the part of an mRNA in the 5' direction from the translation initiation codon, and therefore includes nucleotides between the 5 'cap site (5' cap) and the translation initiation codon of an mRNA (or corresponding nucleotides in the gene). Yet another target region includes the 3 'untranslated region (3'UTR), known in the art to refer to the part of an mRNA downstream from the translation stop codon, and therefore includes , nucleotides between the translation stop codon and the 3 'end of an mRNA (or corresponding nucleotides in the gene). The 5 'cap site of an mRNA comprises an N7-methylated guanosine residue linked 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, in addition to the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5 'cap region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as introns, that 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 splicing sites, i.e., intron-exon splicing or exon-intron splicing, is particularly useful in situations where aberrant splicing is involved in disease, or where excess production of a particular splicing product participates in the disease. An aberrant fusion splice due to rearrangement or deletion is another embodiment of a target site. The transcribed mRNAs produced by the process of splicing two (or more) mRNAs from different gene sources are known as fusion transcripts. Introns can be efficiently targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

In one embodiment, 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.

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

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

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 their start or stop position and contain both intronic and exonic sequence.

Upon cleavage of one or more exon or intron regions, or parts thereof during splicing, the pre-mRNA variants produce smaller mRNA variants. Accordingly, 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 splicing variants. If splicing of the pre-mRNA variant does not occur, then the pre-mRNA variant is identical to the mRNA variant.

Variants can be produced by using alternative signals for 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 using alternative start codons are known as alternative start variants of that pre-mRNA or mRNA. Those transcripts that use an alternate stop codon are known as alternate stop variants of that pre-mRNA or mRNA. A specific type of alternative stop variant is the poly A variant, in which the multiple transcripts produced result from alternative selection of one of the polyA stop signals by the transcription machinery, thereby producing transcripts that end in unique polyA sites. Within the context of the invention, the types of variants described herein are also embodiments of target nucleic acids.

Locations in the target nucleic acid to which 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.

Although the specific sequences of certain exemplary target segments are set forth herein, one of ordinary 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 easily identifiable by a

ES 2 664 591 T3 skilled in the art in view of this disclosure.

Target segments 5-100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within exemplary preferred target segments are also considered suitable for targeting.

The target segments may comprise DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5 'end of one of the exemplary preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA starting immediately upstream of the 5 'end of the target segment and continuing until the DNA or RNA contains from about 5 to about 100 nucleotides). Similarly preferred target segments are represented by DNA or RNA sequences comprising at least the 5 consecutive nucleotides from the 3 'end of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA immediately beginning strand downstream of the 3 'end of the target segment and continuing until the DNA or RNA contains from about 5 to about 100 nucleotides). One skilled in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify additional preferred target segments.

Once one or more target regions, segments or sites have been identified, antisense compounds are chosen that are sufficiently complementary to the target, that is, they hybridize sufficiently well and with sufficient specificity, to give the desired effect.

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

In one embodiment, it is preferred to target specific nucleic acids by antisense oligonucleotides. The targeting of an antisense compound to a particular nucleic acid is a multistep process. The process normally begins with the identification of a nucleic acid sequence whose function is to be modulated. This can be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or pathology, or a non-coding polynucleotide such as, for example, non-coding RNA (cRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which are translated into proteins, and (2) non-coding protein RNAs (cRNA). CRNAs comprise microRNAs, antisense transcripts, and other transcriptional units (TUs) that contain a high density of stop codons and that lack any wide open reading frames. Many cRNAs appear to start from initiation sites in 3 'untranslated regions (3'UTRs) of protein-coding loci. CRNAs are frequently rare and at least half of the cRNAs that have been sequenced by the FANTOM consortium do not appear to be polyadenylated. Most researchers have relied, for obvious reasons, on polyadenylated mRNAs that are processed and exported to the cytoplasm. Recently, it was shown that the pool of non-polyadenylated nuclear RNAs can be very large, and that many of these transcripts arise from so-called intergenic regions. The mechanism by which cRNAs can regulate gene expression is by base pairing with target transcripts. RNAs that function by base pairing can be grouped into (1) cis-encoded RNAs that are encoded in the same genetic location, but on the opposite strand to the RNAs on which they act and therefore show perfect complementarity with their target, and (2) trans-encoded RNAs that are encoded in a chromosomal location other than the RNAs on which they act and generally do not exhibit perfect base-pairing potential with their targets.

Without wishing to be bound by theory, disruption of an antisense polynucleotide by the antisense oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can be both discordant (antisense inactivation produces elevation of messenger RNA) and concordant (antisense inactivation produces concomitant reduction of messenger RNA). In these cases, the antisense oligonucleotides can be targeted to overlapping or non-overlapping parts of the antisense transcript causing its inactivation or sequestration. The coding antisense, in addition to noncoding, can be targeted in an identical way and that any category is capable of regulating the corresponding sense transcripts - both in a concordant and a discordant way. The strategies employed in identifying new oligonucleotides for use against a target can be based on the inactivation of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the target.

ES 2 664 591 T3 desired.

Strategy 1 In the case of discordant regulation, inactivation of the antisense transcript elevates the expression of the conventional (sense) gene. If the latter gene is to encode a known or putative target drug, then inactivation of its antisense counterpart could possibly mimic the action of a receptor agonist or stimulating enzyme.

Strategy 2 In the case of concordant regulation, both antisense and sense transcripts could be concomitantly inactivated and thus achieve a synergistic reduction of conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve inactivation, then this strategy can be used to apply an antisense oligonucleotide targeting 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.

In accordance with 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 others. oligomeric compounds that hybridize with at least a part of the target nucleic acid and modulate its function. Thus, they can be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or they can 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 protrusions, mismatches, or loops. Antisense compounds are routinely prepared linearly, but can be linked or otherwise prepared to be circular and / or branched. Antisense compounds may comprise constructs such as, for example, two strands hybridized to form a fully or partially double-stranded compound or a single strand with sufficient self-complementarity to allow hybridization and formation of a fully or partially double-stranded compound. The two chains can be linked internally, leaving the 3 'or 5' ends free, or they can be linked to form a continuous hairpin or loop structure. The hairpin structure may contain a protruding nucleotide at either the 5 'or 3' ends that produces an extension of the single-stranded character. Double-stranded compounds may optionally comprise end-protruding nucleotides. Additional modifications can comprise conjugated groups attached to one of the ends, 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 linking group. When formed from just one strand, dsRNA can take the form of a self-complementary hairpin molecule that folds back on itself to form a duplex. Thus, the dsRNA 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, gene expression or function is up-regulated. When formed from two chains, or a single chain that takes the form of a self-complementary hairpin-type molecule that folds back on itself to form a duplex, the two chains (or single-chain duplex-forming regions ) are complementary RNA strands that are base-paired in the Watson-Crick mode.

Once introduced into a system, the compounds of the invention can elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or they can work by occupation-based mechanisms. In general, nucleic acids (including oligonucleotides) can be described as DNA-like (i.e. generally having one or more 2'deoxysugars and generally T bases instead of U) or RNA-like (i.e. generally they have one or more 2-hydroxylsugars or 2 'modified sugars and, generally bases U instead of T). Nucleic acid helices can take on more than one type of structure, most commonly forms A and B. Oligonucleotides having similar structure to form B are generally believed to be DNA-like and those having similar structure to form A are similar to RNA. In some (chimeric) embodiments, an antisense compound can contain both A and B-form regions.

In one embodiment, the desired antisense oligonucleotides or compounds comprise at least one antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (siRNA), small interference RNA (siRNA); an interference microRNA (miRNA); a small temporary RNA (tRNA); or a short hairpin RNA (shRNA); small RNA (RNA) induced gene activation; Small activating RNA (pRNA), or combinations thereof.

DsRNAs can also activate gene expression, a mechanism that has been called small RNA or RNA-induced gene activation. Gene promoters that target dsRNA induce potent transcriptional activation of associated genes. RNA was demonstrated in human cells using synthetic dsRNA, called small activating RNA (ssRNA). It is not currently known whether ANA is conserved in other organisms.

ES 2 664 591 T3

Small double-stranded RNA (dsRNA), such as small interference RNA (siRNA) and microRNA (miRNA), have been found to trigger an evolutionarily conserved mechanism known as interference RNA (RNAi). RNAi invariably leads to gene silencing through chromatin remodeling to thereby suppress transcription, breakdown of complementary mRNA, or block protein translation. However, in the aspects described in detail in the examples section below, the oligonucleotides are shown to increase the expression and / or function of filaggrin polynucleotides (FLG) and products encoded by them. DsRNAs can also act as small activating RNAs (apRNAs). Without wishing to be bound by theory, by targeting sequences to gene promoters, apRNAs will induce expression of target genes in a phenomenon called dsRNA-induced transcriptional activation (RNA).

In a further embodiment, the preferred target segments identified herein can be employed in a screen for additional compounds that modulate the expression of filaggrin family (FLG) polynucleotides. Modulators are those compounds that decrease or increase the expression of a nucleic acid molecule encoding the FLG gene and that 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 natural sense or antisense polynucleotides of the FLG gene with one or more candidate modulators, and selecting one or more candidate modulators that decrease or increase the expression of a nucleic acid molecule encoding polynucleotides of the FLG gene, eg, SEQ ID NO: 3 to 13. Once it is demonstrated that the candidate modulator (s) are capable of modulating (e.g., both decreasing and increasing) the expression of a nucleic acid molecule encoding polynucleotides of the FLG gene, the modulator can then be employed in further research studies of the polynucleotide function of the FLG gene, or for use as an investigational, diagnostic or therapeutic agent in accordance with the present disclosure.

Targeting the natural antisense sequence preferentially modulates the function of the target gene. For example, the FLG gene (for example, accession number NM_002016). In one embodiment, the target is an antisense polynucleotide from the FLG gene. In one embodiment, an antisense oligonucleotide targeted to natural sense and / or antisense sequences of FLG gene polynucleotides (eg, accession number NM_002016), variants, alleles, isoforms, homologues, mutants, derivatives, fragments, and corresponding complementary sequences. Preferably, the oligonucleotide is an antisense molecule and the targets include coding and non-coding regions of antisense and / or sense polynucleotides of the FLG gene.

The preferred target segments of the present invention may also combine with their respective complementary antisense compounds of the present invention to form stabilized double-stranded oligonucleotides (duplexes).

Such double-stranded oligonucleotide residues have been shown in the art to modulate target expression and regulate translation, in addition to RNA processing through an antisense mechanism. Additionally, double-stranded moieties can undergo chemical modifications. For example, such double-stranded residues have been shown to inhibit the target by classical hybridization of the antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target.

In one embodiment, an antisense oligonucleotide targets filaggrin polynucleotides (FLG) (eg, accession number: NM_002016), variants, alleles, isoforms, homologues, mutants, derivatives, fragments, and sequences complementary thereto. Preferably, the oligonucleotide is an antisense molecule.

According to embodiments of the invention, the target nucleic acid molecule is not limited to FLG alone, but extends to any of the isoforms, receptors, homologues, and the like of FLG gene molecules.

In one aspect, an oligonucleotide targets a natural antisense sequence of polynucleotides of the NEU4 gene, for example, the polynucleotides set forth in SEQ ID NOS: 2, and any variant, allele, homologous, mutant, derivative, fragment, and sequence complementary to those themselves. Examples of antisense oligonucleotides are set forth as SEQ ID NO: 3 to 13. The oligonucleotide comprising sEq ID No. 6 is not part of the invention.

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

In one embodiment, the oligonucleotides are complementary to, or bind to, nucleic acid sequences of the

ES 2 664 591 T3 natural antisense of the FLG gene, set forth as SEQ ID NO: 2 and modulates the expression and / or the function of molecules of the FLG gene.

In one embodiment, the oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NO: 3 to 13 and modulate the expression and / or function of FLG gene molecules.

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

In one embodiment, the oligonucleotides that target polynucleotides of the FLG gene comprise: antisense RNA, interference RNA (RNAi), small interference RNA (siRNA); interference microRNA (miRNA); a small temporary RNA (tRNA); or a short hairpin RNA (shRNA); small RNA (RNA) induced gene activation; or, small activating RNA (pRNA).

In one embodiment, targeting Filaggrin polynucleotides (FLG), eg, SEQ ID: 2, modulates the expression or function of these targets. In one embodiment, expression or function is up-regulated compared to a control. In one embodiment, expression or function is down-regulated compared to a control.

In one embodiment, the antisense compounds comprise sequences set forth as SEQ ID NO: 3 to 13. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like.

In one embodiment, SEQ ID NO: 3 to 13 comprise one or more LNA nucleotides.

Modulation of a desired target nucleic acid can be accomplished in various ways known in the art. For example, antisense oligonucleotides, siRNAs, etc. Enzymatic nucleic acid molecules (eg, ribozymes) are nucleic acid molecules capable of catalyzing one or more of several reactions, including the ability to repeatedly cleave other separated nucleic acid molecules in a specific nucleotide base sequence mode. . Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript.

Due to their sequence specificity, trans-cleaved 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 nonfunctional and abrogates the expression of proteins from that RNA. In this way, the synthesis of a protein associated with a pathology can be selectively inhibited.

In general, enzymatic nucleic acids with RNA cleavage activity act by first binding to a target RNA. Such binding occurs through the target binding part of an enzymatic nucleic acid that is kept in close proximity to an enzymatic part of the molecule that acts to cleave the target RNA. In this way, the enzymatic nucleic acid is first recognized and then bound to target RNA by complementary base pairing, and once bound to the correct site, it acts enzymatically to cut the target RNA. Strategic cleavage of said target RNA will destroy its ability to direct the synthesis of an encoded protein. After an enzymatic nucleic acid binds and cleaves its target RNA, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

Various strategies such as in vitro selection (evolution) (Orgel, (1979) Proc. R. Soc. London, B 205, 435) to develop new nucleic acid catalysts capable of catalyzing various reactions, such as cleavage and ligation of phosphodiester bonds and amide bonds.

The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy employing ribozymes that cleave RNA in order to regulate gene expression. Hammerhead ribozyme, for example, operates with a catalytic rate (kcat) of approximately 1 min-1 in the presence of saturating concentrations (10 mM) of Mg2 + cofactor. An artificial RNA ligase ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of approximately 100 min-1. Furthermore, certain modified hammerhead ribozymes having substrate-binding arms made of DNA are known to catalyze RNA cleavage with multiple rates of recovery.

ES 2 664 591 T3 approaching 100 min-1. Finally, substitution of a specific residue within the hammerhead catalytic core with certain nucleotide analogs gives modified ribozymes that show up to a 10-fold improvement in catalytic rate. These discoveries demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly higher than those exhibited in vitro by most naturally occurring self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes can be optimized to give maximum catalytic activity, or that entirely new RNA motifs can be prepared showing significantly faster rates for RNA phosphodiester cleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that conforms to the hammerhead model was first demonstrated in 1987 (Uhlenbeck, OC (1987) Nature, 328: 596-600). RNA and reacted with multiple RNA molecules, showing that it was truly catalytic.

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

RNA interference (RNAi) has become a powerful tool to modulate gene expression in mammals and mammalian cells, this approach requires the administration of small interfering RNA (siRNA) either as RNA itself or as DNA, using a expression plasmid or virus and the coding sequence for small hairpin RNA that is processed to siRNA. This system allows the efficient transport of pre-siRNAs to the cytoplasm in which they are active and allows the use of tissue-specific and regulated promoters for gene expression.

In one embodiment, an antisense oligonucleotide or compound comprises an oligomer or polymer of ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homologue thereof. This term includes oligonucleotides composed of natural nucleotides, sugars. and covalent internucleoside linkages (backbones) as well as oligonucleotides that have unnatural parts that function similarly. Such modified or substituted oligonucleotides are frequently desired over native forms because of desirable properties such as, for example, enhanced cell uptake, enhanced affinity for a target nucleic acid, and high stability in the presence of nucleases.

According to the present invention, antisense oligonucleotides or compounds include antisense oligonucleotides (eg, RNA, DNA, mimetic, chimera, analog or homologue thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds , single- or double-stranded RNA interference compounds (RNAi) such as siRNA, apRNA, RNA compounds, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid and modulate its function. Thus, they can be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or they can 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 protrusions, mismatches, or loops. Antisense compounds are routinely prepared linearly, but can be linked or otherwise prepared to be circular and / or branched. Antisense compounds may comprise constructs such as, for example, two strands hybridized to form a fully or partially double-stranded compound or a single strand with sufficient self-complementarity to allow hybridization and formation of a fully or partially double-stranded compound. The two chains can be linked internally, leaving the 3 'or 5' ends free, or they can be linked to form a continuous hairpin or loop structure. The hairpin structure may contain a protruding nucleotide at either the 5 'or 3' ends that produces an extension of the single-stranded character. Double-stranded compounds may optionally comprise end-protruding nucleotides. Additional modifications can comprise conjugated groups attached to one of the ends, 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 linking group. When formed from just one strand, dsRNA can take the form of a self-complementary hairpin molecule that folds back on itself to form a duplex. Thus, the dsRNA 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 chains, or a single chain that takes the form of a self-complementary hairpin-type molecule that folds back on itself to form a duplex, the two chains (or single-chain duplex-forming regions ) are strands of RNA

Complementary ES 2,664,591 T3 base-pairing in Watson-Crick mode.

Once introduced into a system, the compounds of the invention can elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or they can work by occupation-based mechanisms. In general, nucleic acids (including oligonucleotides) can be described as DNA-like (i.e. generally having one or more 2'deoxysugars and generally T bases instead of U) or RNA-like (i.e. generally they have one or more 2'-hydroxyl or 2 'modified sugars, and generally bases U instead of T). Nucleic acid helices can take on more than one type of structure, most commonly forms A and B. Oligonucleotides having similar structure to form B are generally believed to be DNA-like and those having similar structure to form A are similar to RNA. In some (chimeric) embodiments, an antisense compound can contain both A and B-form regions.

Antisense compounds according to this invention may comprise an antisense portion of about 5 to about 80 nucleotides (ie, about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense chain or part 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 antisense strand or part of 5 to about 80 nucleotides. of length. One of ordinary skill in the art will appreciate that it comprises 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 interval in between.

In one embodiment, the antisense compounds of the invention have antisense portions 10 to 50 nucleotides in length. One of ordinary skill in the art will appreciate that these integrate 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 of length, or any interval in between. In some embodiments, the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of the invention have antisense portions 12 or 13 to 30 nucleotides in length. One skilled in the art will appreciate that these integrate 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 interval in between.

In one 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 can be produced that contain thymidine, guanosine, or cytidine at this position. This can be done at any of the antisense compound or dsRNA positions. These compounds are then tested using the procedures described herein to determine their ability to inhibit the expression of a target nucleic acid.

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

In one embodiment, antisense oligonucleotides, such as, for example, the nucleic acid molecules set forth in SEQ ID NO: 2 to 13 comprise one or more substitutions or modifications. In one embodiment, the nucleotides are substituted with blocked nucleic acids (LNAs).

In one embodiment, the oligonucleotides target one or more regions of the sense and / or antisense nucleic acid molecules of coding and / or non-coding sequences associated with the FLG gene and the sequences set forth as SEQ ID NO: 1 and 2. The oligonucleotides also target overlapping regions of SEQ ID NO: 1 and 2.

ES 2 664 591 T3

Certain preferred oligonucleotides of this invention are chimeric oligonucleotides. Chimeric oligonucleotides or chimeras, in the context of this invention, are oligonucleotides that contain two or more chemically distinct regions, each consisting of at least one nucleotide. These oligonucleotides typically contain at least one modified nucleotide region that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased cell uptake, increased target binding affinity) 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 that clears the RNA strand of an RNA: DNA duplex. RNase H activation therefore produces cleavage of the target RNA, thus 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 that hybridize to the same target region. Cleavage of the target RNA can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In one embodiment, a chimeric oligonucleotide comprises at least one region modified to increase the binding affinity of the target, and typically a region that acts as a substrate for RNase H. The 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 the target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention can be formed as structures composed of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and / or oligonucleotide mimetics, as described above. As such, these compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents teaching the preparation of these hybrid structures include, but are not limited to, US patents issued to us. 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.

In one embodiment, the region of the oligonucleotide to be modified comprises at least one modified nucleotide at the 2 'position of the sugar, most preferably a 2'-O-alkyl, 2'-O-alkyl-O- modified nucleotide. alkyl or 2'-fluorine. In another embodiment, RNA modifications include 2'-fluoro, 2'-amino, and 2'-O-methyl modifications on pyrimidine ribose, abasic residues, or an inverted base at the 3 'end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (ie, higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance the RNAi oligonucleotide inhibition of gene expression. RNase H is a cellular endonuclease that cleaves the RNA strand of RNA: DNA duplexes; therefore, activation of this enzyme results in cleavage of the target RNA, and thus can greatly enhance the efficiency of RNA inhibition. Cleavage of the target RNA can be routinely demonstrated by gel electrophoresis. In one embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain various exo- and endo-nucleases that can degrade nucleic acids. Various modifications of nucleotides and nucleosides have been shown to render 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 cell extracts or isolated nuclease solutions and measuring the degree of intact oligonucleotide that remains over time, usually by gel electrophoresis. Oligonucleotides that have been modified to enhance nuclease resistance survive intact longer than unmodified oligonucleotides. Various oligonucleotide modifications have been shown to enhance or confer nuclease resistance. Oligonucleotides containing at least one phosphorothioate modification are currently more preferred. In some cases, oligonucleotide modifications that enhance target binding affinity are also independently capable of enhancing nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned by this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methylphosphonates, linkages between short chain alkyl or cycloalkyl sugars, or linkages between short chain heteroatomic or heterocyclic sugars. The most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH, --N (CH3) -O-CH2 backbones [known as a methylene (methylimino) or MMI], CH2-O-N (CH3) -CH2, CH2-N (CH3) -N (CH3) -CH2 and O-- N (CH3) -CH2-- , where the native phosphodiester backbone is represented as O-P-O-CH,). The amide backbones disclosed by De Mesmaeker et al., (1995) Acc. Chem. Res. 28: 366-374 are also preferred. Oligonucleotides having morpholino backbones are also preferred (Summerton and Weller, US Patent No. 5,034,506). In other embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the

ES 2 664 591 T3 oligonucleotide is replaced by a polyamide backbone, the nucleotides being attached directly or indirectly to the azo nitrogen atoms of the polyamide backbone. The oligonucleotides can also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 'position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O (CH2) n CH3, O (CH2) n NH2 or O (CH2) n CH3, where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, alkyl, alkaryl or substituted lower aralkyl: CI; Br; CN; CF3; OCF3; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; SOCH3; SO2 CH3; ONO2: NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavage group: a reporter group; an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide; or a group to improve the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Preferred modifications include 2'-methoxyethoxy [2'-O-CH2 CH2 OCH3, 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 can also be made at other positions on the oligonucleotide, particularly the 3 'position of the sugar at the nucleotide at the 3' end and the 5 'position of the nucleotide at the 5' end. The oligonucleotides can also have sugar mimetics such as cyclobutyl in place of the pentofuranosyl group.

The oligonucleotides may also comprise, additionally or alternatively, nucleobase modifications or substitutions (often referred to in the art simply as "base"). 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, for example, hypoxanthine, 6-methyladenine, 5-Me-pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'-deoxycytosine and frequently referred to in the 5-Me-C technique), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, in addition to synthetic nucleotides, for example, 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) and 2,6-diaminopurine. A universal base known in the art can be included, for example, inosine. 5-ME-C substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C, and are currently the preferred base substitutions.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as cholesterol moieties, cholesteryl moieties, an aliphatic chain, e.g. ex. dodecandiol or undecyl moieties, a polyamine or a polyethylene glycol chain, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties, and procedures for preparing such oligonucleotides, are known in the art, for example, US Pat. 5,138,045, 5,218,105 and 5,459,255.

It is not necessary that all positions in a given oligonucleotide be uniformly modified, and in fact more than one of the modifications mentioned above can be incorporated into a single oligonucleotide or even within a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides that are chimeric oligonucleotides, as defined hereinbefore.

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

The oligonucleotides used in accordance with this invention can be conveniently and routinely prepared by the well known technique of solid phase synthesis. Equipment for such syntheses is marketed by several vendors including Applied Biosystems. Any other means for said synthesis can also be used; the actual synthesis of the oligonucleotides is well within the skills of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as 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 and / or CPG modified amidites (available from Glen Research, Sterling VA) to synthesizing biotinylated, fluorescently labeled oligonucleotides or other modified oligonucleotides such as cholesterol modified oligonucleotides.

In accordance with the invention, the use of modifications such as the use of LNA monomers to enhance the

ES 2 664 591 T3 potency, specificity and duration of action and broadening the oligonucleotide administration routes comprises current chemistries such as MOE, ANA, FANA, PS, etc. This can be accomplished by substituting some of the monomers in the current oligonucleotides for LNA monomers. The LNA-modified oligonucleotide can be similar in size to the parent compound or it can 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% of LNA monomers and that their sizes are between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.

Preferred modified oligonucleotide skeletons include, but are not limited to, chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates comprising 3'-alkylene phosphonates and chiral phosphorothioates, phosphoramida'amide phosphonates, phosphoramida-amino compounds comprising 3 phosphoramidaphins aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates that have normal 3'-5 'bonds, 2'-5' linked analogs of these, and those that have reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5 'to 5'- 3 'or 2'-5' to 5'2 '. Also included are various salts, mixed salts, and free acid forms.

Representative United States patents teaching the preparation of the phosphorus-containing linkages above include, but are not limited to, United States Patent 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.

Preferred modified oligonucleotide backbones that do not include a phosphorous atom in them 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 heteroatomic or heterocyclic internucleoside linkages of short chain. These include those that have morpholino bonds (formed in part from the sugar part of a nucleoside); siloxane skeletons; sulfur, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl backbones; skeletons containing alkene; sulphamate skeletons; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide skeletons; amide skeletons; and others that have mixed N, O, S, and CH2 component parts.

Representative United States patents teaching the preparation of oligonucleosides include, but are not limited to, United States patents. No. 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.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, ie the backbone, of the nucleotide units are replaced by novel groups. Base units are maintained for hybridization to an appropriate target nucleic acid compound. Such an oligomeric compound, a mimetic oligonucleotide that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to azo nitrogen atoms of the amide part of the backbone. Representative United States patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262.

Additional teachings of PNA compounds can be found in Nielsen, et al. (1991) Science 254, 14971500.

In one embodiment of the invention, oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones, and in particular -CH2-NH-O-CH2-, -CH2-N (CH3) -O-CH2-, known as a backbone of methylene (methylimino) or MMI, -CH2-ON (CH3) -CH2-, -CH2N (CH3) -N (CH3) CH2- and -ON (CH3) -CH2- CH2- where the native phosphodiester backbone is represented such as -OPO-CH2- of US Patent No. 5,489,677, cited above, and the amide skeletons of US Pat. No. 5,602,240. Oligonucleotides having the morpholino backbones of US Patent No. 5,034,506, cited above, are also preferred.

Modified oligonucleotides can also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 'position: OH; F; O-, S- or N-alkyl; O-, S-, or N-alkenyl; O-, S37

ES 2 664 591 T3 or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl and alkynyl can 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. Other 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, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a reporter group, an intercalator, a group to improve the pharmacokinetic properties of an oligonucleotide, or a group to improve the pharmacodynamic properties of an oligonucleotide, and other substituents that have similar properties. A preferred modification comprises 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O- (2-methoxyethyl) or 2'-MOE), ie an alkoxyalkoxy group. Other preferred modifications comprise 2'-dimethylaminooxyethoxy, i.e., an O (CH2) 2ON (CH3) 2 group, also known as 2-DMAOE, as described in the examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-Odimethylaminoethoxyethyl or 2'-DMAEOE), that is, 2'-O-CH2-O-CH2-N (CH2) 2.

Other preferred modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the oligonucleotide, particularly the 3 'position of the sugar at the 3' end nucleotide or 2'-5 'linked oligonucleotides and the 5' position of the 5 'end nucleotide. The oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent 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; 5.76.427; 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.

The oligonucleotides can also comprise nucleobase modifications or substitutions (often referred to in the art simply as "base"). 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). The modified nucleotides include other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, derivatives of 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo-uracil) , 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8,5-halogen substituted adenines and guanines, particularly 5-bromo, 5-trifluoromethyl and other 5,7-methylquanine substituted uracils and cytosines and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Furthermore, nucleotides comprise those disclosed in US Patent No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke , ST and Lebleu, B. ca., CRC Press, 1993. Some 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 O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability around 0.6-1.2 ° C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds, 'Antisense Research and Applications ', CRC Press, Boca Raton, 1993, pp. 276-278) and are currently the preferred base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.

Representative United States patents teaching the preparation of the modified nucleotides listed above, as well as other modified nucleotides, include, but are not limited to, U.S. Patent 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.

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.

Such moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, eg, hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, eg, dodecanediol residues or

ES 2 664 591 T3 undecyl, a phospholipid, for example di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate of triethylammonium, a polyamine or a polyethylene glycol chain , or adamantane acetic acid, a palmityl residue, or an octadecylamine or hexylaminocarbonyltoxycholesterol residue.

Representative United States patents teaching the preparation of these oligonucleotide conjugates include, but are not limited to, U.S. Patent 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.

The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention encompasses the use of the compounds and preferred target segments identified herein in a drug discovery effort to elucidate relationships that exist between filaggrin polynucleotides (FLG) and a pathology, phenotype, or condition. These methods include detecting or modulating the FLG gene polynucleotides which comprise contacting a sample, tissue, cell or organism with the compounds of the present invention, measuring the nucleic acid or protein level of the FLG gene polynucleotides and / or a related chemical or phenotypic endpoint sometime after treatment, and optionally comparing the measured value with an untreated sample or sample treated with another compound of the disclosure. These procedures can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the target validation process or to determine the validity of a particular gene product as a target for the treatment or prevention of a disease, condition or particular phenotype.

Evaluation of upregulation or inhibition of gene expression:

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 accomplished by various procedures well known in the art. For example, the presence of exogenous nucleic acid can be detected by Southern blotting or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Exogenous nucleic acid expression can also be measured using standard procedures including gene expression analysis. For example, mRNA produced from exogenous nucleic acid can be detected and quantified using Northern blot and reverse transcription PCR (RT-PCR).

Exogenous nucleic acid RNA expression can also be detected by measuring enzyme activity or 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 of each gene can be used to construct 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 upstream of the gene and a possible RNAi target in the 3 'noncoding region. The efficacy of individual antisense oligonucleotides would be tested by modulation of the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxy acid 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, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Procedures for determining the modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorimetric procedures (eg, fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy). , determination of antibiotic resistance.

The expression of the FLG protein and mRNA can be controlled using procedures known to those of skill in the art and described herein. For example, immunoassays such as ELISA can be used

ES 2 664 591 T3 to measure protein levels. ELISA test kits for FLG are commercially available, e.g. eg, at R&D Systems (Minneapolis, MN).

In some embodiments, the expression of the FLG gene (eg mRNA or protein) in a sample (eg cells or tissues in vivo or in vitro) treated with an antisense oligonucleotide of the invention is evaluated by comparison with the expression of the FLG gene in a control sample. For example, the expression of the protein or nucleic acid can be compared, using procedures known to those of skill in the art, with a mock-treated and untreated sample. Alternatively, a comparison can be made to a sample treated with a control antisense oligonucleotide (eg, one having a different or modified sequence) depending on the information desired. In another embodiment, a difference in the expression of the FLG protein or its nucleic acid in a treated versus an untreated sample can be compared to the difference in the expression of a different nucleic acid (including whatever standard the investigator deems appropriate, eg a housekeeping gene) in a treated versus an untreated sample.

The observed differences can be expressed as desired, e.g. ex. in proportion or fraction form, for use in comparison with a control. In embodiments, the level of FLG gene mRNA or protein, in a sample treated with an antisense oligonucleotide of the present invention, increases or decreases between 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 FLG gene mRNA or protein level increases or decreases 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 times, at least about 1.7 times, at least about 1.8 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, or at least about 10 times or more.

Kits, research, diagnostic and therapeutic reagents

The compounds of the present invention can be used for diagnostic, therapeutic and prophylactic agents, and as research reagents and kit components. Furthermore, antisense oligonucleotides, which are capable of inhibiting gene expression with exquisite specificity, are frequently used by those of skill in the art to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

For use in kits and diagnostics and in various biological systems, the compounds of the present invention, both alone and in combination with other compounds or therapeutics, are useful as tools in differential and / or combinatorial analysis to clarify patterns of expression of a part or of the entire complement of genes expressed within cells and tissues.

As used herein, the term "biological system or system" is defined as any organism, cell, cell culture, or tissue that expresses, or has become competent to express, filaggrin family (FLG) gene products. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants, and combinations thereof.

As a 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 are related, for example, to disease association, signaling pathway, cell location, expression level, size, structure or function of the genes examined. These analyzes can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.

Examples of gene expression analysis procedures known in the art include DNA arrays or microarrays, SAGE (Serial Gene Expression Analysis), LEE (Restriction Enzyme Digested cDNA Amplification), TOGA (Total Gene Expression Analysis), protein and proteomics arrays, expressed sequence tag (EST), sequencing, subtractive RNA fingerprinting (SuRF), subtractive cloning, differential display (DD), comparative genomic hybridization, FISH (fluorescent in situ hybridization), mass spectrometry techniques and procedures.

ES 2 664 591 T3

The compounds of the invention are useful for research and diagnosis, because these compounds hybridize with nucleic acids encoding the filaggrin family (FLG). For example, oligonucleotides that hybridize with such efficiency and under such conditions that have been described herein as effective modulators of the FLG gene are effective primers or probes under conditions that favor gene amplification or detection, respectively. These primers and probes are useful in procedures that require the specific detection of nucleic acid molecules encoding the FLG gene and in the amplification of such nucleic acid molecules for detection or for use in further studies of the FLG gene. Hybridization of antisense oligonucleotides, particularly primers and probes of the invention with a nucleic acid encoding the FLG gene, can be detected by means known in the art. Such means may comprise conjugation of an enzyme to the oligonucleotide, radiolabeled the oligonucleotide, or any other suitable detection means. Kits using such detection means can also be prepared to detect the level of the FLG gene in a sample.

Antisense specificity and sensitivity are also employed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been used as therapeutic moieties in the treatment of pathologies in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are currently underway. Thus, it is established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimens for the treatment of cells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, that is suspected of or has a disease or disorder that can be treated by modulating the expression of the FLG gene is treated by administering antisense compounds in accordance with this invention. For example, in a non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment a therapeutically effective amount of the FLG gene modulator. The FLG gene modulators of the present invention effectively modulate the activity of the FLG gene or modulate the expression of the FLG gene protein. In one embodiment, the activity or expression of the FLG gene in an animal is inhibited by about 10% compared to a control. Preferably, the activity or expression of the FLG gene in an animal is inhibited by about 30%. More preferably, the activity or expression of the FLG gene in an animal is inhibited 50% or more. Thus, oligomeric compounds modulate the expression of filaggrin mRNA (FLG) by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99%, or 100% compared to a control.

In one embodiment, the activity or expression of the filaggrin family (FLG) in an animal is increased by approximately 10% 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 the expression of the FLG gene mRNA by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%. , at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least less 99%, or 100% compared to a control

For example, reduced expression of the filaggrin family (FLG) can 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 to be analyzed contain a nucleic acid molecule encoding peptides of the FLG gene and / or the FLG protein itself.

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

Conjugated

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can comprise conjugated groups covalently linked to functional groups such as primary or secondary hydroxyl groups. Conjugated groups of the invention include intercalators, reporter gene molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance

ES 2 664 591 T3 pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugated groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance pharmacodynamic properties, in the context of this invention, include groups that enhance uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that enhance the uptake, distribution, metabolism, or elimination of the compounds of the present invention. Representative conjugated groups are described in International Patent Application No. PCT / US92 / 09196, filed 23, 1992, and US Patent No. 6,287,860. Conjugated moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, eg, hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, eg, dodecanediol or undecyl residues, a phospholipid, for example di-hexadecylrac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate of triethylammonium, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl residue , or an octadecylamine or hexylaminocarbonyloxycholesterol moiety. The oligonucleotides of the invention can also be conjugated with active principles, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S) - (+) - pranoprofen, carprofen, dansylsarcosine, 2,3,5- acid triiodobenzoic, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethacin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial, or an antibiotic.

Representative US patents that teach the preparation of these oligonucleotide conjugates include, but are not limited to, US Pat. No. 4,828,979; 4,948,882; 5,218,105

5.525.465

5.138.045

4.789.737

5.112.963

5.371.241

5.585.481

5.541.313; 5.545.730; 5.552.538; 5.578.717,5.580.731; 5.380.731; 5.591.584; 5.109.124

5.414.077; 5.486.603; 5.512.439; 5.578.718; 5.608.046; 4.587.044; 4.605.735; 4.667.025

4.824.941; 4.835.263; 4.876.335; 4.904.582; 4.958.013; 5.082.830; 5.112.963; 5.214.136

5.214.136; 5.245.022; 5.254.469; 5.258.506; 5.262.536; 5.272.250; 5.292.873;

5,391,723; 5,416,203.5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

5.118.802

4.762.779

5.082.830

5.317.098

5.574.142

Formulations

The compounds of the invention can also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, such as, for example, liposomes, receptor-targeting molecules, oral, rectal, topical or other formulations, to help in the uptake, distribution and / or absorption. Representative United States patents that teach the preparation of these uptake, distribution, and / or absorption aid formulations include, but are not limited to, U.S. Pat. No. 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.

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

In one embodiment, the practice of the invention involves administering at least one of the above 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 non-viral vectors include the oligonucleotide alone (eg, any one or more of SEQ ID NO: 3 to 13) or in combination with a suitable protein, polysaccharide or lipid formulation.

Additional suitable nucleic acid delivery systems include viral vector, typically sequence of at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or Japan hemagglutinating virus-liposome complex (JVH). Preferably, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide, eg, a cytomegalovirus (CMV) promoter.

Additional preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. A preferred HIV-based viral vector comprises at least two vectors in which the gag and pol genes are from an HIV genome and the gene

ES 2 664 591 T3 env is from another virus. Viral DNA vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpes virus vectors such as a herpes simplex virus I (HSV) vector, adenovirus vectors, and adenovirus associated virus vectors.

The antisense compounds of the invention encompass any pharmaceutically acceptable salt, ester or salt of said esters, or any other compound which, after administration to an animal, including a human being, is capable of providing (directly or indirectly) the metabolite biologically. asset or residue thereof.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: that is, salts that retain the desired biological activity of the parent compound and do not confer unwanted toxicological effects thereon. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in US Patent No. 6,287,860.

The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the disclosure. The pharmaceutical compositions of the present invention can be administered in various forms depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal administration), pulmonary, for example, 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 administration, eg, intrathecal or intraventricular.

For the treatment of tissues in the central nervous system, administration can be carried out, e.g. ex. by injection or infusion into the cerebrospinal fluid. Administration of antisense RNA into cerebrospinal fluid is described, e.g. ex. in the US patent application. Pub. No. 2007/0117772, Methods for slowing familial ALS disease progression. '

When it is intended that the antisense oligonucleotides of the present invention can be administered to cells of the central nervous system, the administration can be with one or more agents capable of facilitating the penetration of the antisense oligonucleotides through the blood-brain barrier of the subject. Injection can be made, for example, into the entorhinal cortex or the hippocampus. The application of neurotrophic factors by administration of an adenovirus vector to motor neurons in muscle tissue is described in, for example, US Patent No. 6,632,427, Adenoviral-vector-mediated gene transfer into medullary motor neurons. ». Delivery of vectors directly to the brain, eg, the striatum, the thalamus, the hippocampus, or the substantia nigra, is known in the art and is described, for example, in US Pat. No. 6,756,523, Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain. Administration can be rapid such as by injection or over a period of time either by slow infusion or by administration of slow release formulations.

The subject antisense oligonucleotides can also be bound 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 to a viral vector, for example, which makes the antisense compound more effective and / or increases the transport of the antisense compound across the blood-brain barrier. Alteration of the osmotic blood-brain barrier can also be accomplished by perfusion, e.g. ex. of sugars including, but not limited to, mesoerythritol, 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 (-) lixose, L (+) - lixose and L (-) - lixose or amino acids, including, but not limited to, glutamine, lysine, arginine, asparagine, cysteine, Aspartic Acid, Glutamic Acid, Glycine, Histidine, Leucine, Methionine, Phenylalanine, Proline, Serine, Threonine, Valine, Tyrosine, and Taurine. Procedures and materials to increase penetration into the blood brain barrier are described, e.g. ex. in US Patents No. 4,866,042, Method for the delivery of genetic material across the blood brain barrier, 6,294,520, Material for passage through the blood-brain barrier, and 6,936,589, Parenteral delivery systems

The subject antisense compounds may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecule structures, or mixtures of compounds, such as, for example, liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations, to help in the acquisition, distribution and / or

ES 2 664 591 T3 absorption. For example, cationic lipids can be included in the formulation to facilitate oligonucleotide absorption. One such composition that has been shown to facilitate absorption is LIPOFECTIN (available from GIBCO BRL, Bethesda, MD).

Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Compositions and pharmaceutical formulations for topical administration can comprise transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

The pharmaceutical formulations of the present invention, which may be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Said techniques include the step of associating the active principles with the pharmaceutical vehicle (s) or excipient (s). In general, formulations are prepared by uniformly and intimately associating the active ingredients with liquid vehicles or finely divided solid vehicles, or both, and then, if necessary, molding the product.

The composition of the present invention may be formulated in any of many possible dosage forms such as, but not limited to, tablets, capsules, gel caps, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, nonaqueous or mixed media. Aqueous suspensions may additionally contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran. The suspension can also contain stabilizers.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams, and formulations containing liposomes. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients.

Emulsions are usually heterogeneous systems of one liquid dispersed in another in the form of droplets that normally exceed 0.1 Bales; The emulsions are the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, the oil phase or itself as an independent phase. Microemulsions are included as one embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in US Patent No. 6,287,860.

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 that have a membrane formed by a lipophilic material and an aqueous interior that contains the composition to be administered. 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 trap DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA to cells.

Liposomes also include sterically stabilized liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids produce liposomes with improved circulating lives over liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-formed lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.

Liposomes and their uses are further described in US Patent No. 6,287,860.

The formulations and pharmaceutical compositions of the present invention may also comprise surfactants. The use of surfactants in drugs, formulations, and emulsions is well known in the art. Surfactants and their uses are further described in US Pat. No.

6.287.860.

In one embodiment, the present invention employs various penetration enhancers to effect efficient delivery of nucleic acids, particularly oligonucleotides. In addition to helping in the dissemination of

ES 2 664 591 T3 non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad categories, ie surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in US Pat. No. 6,287,860.

One skilled in the art will recognize that formulations are routinely designed according to their intended use, ie, their route of administration.

Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical administration agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include negative neutrals (eg, dioleoyl-phosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DmPc, distearoylphosphatidylcholine) (eg, dimyristoylphosphatidylglycerol DMPG) and cationics (eg, dioleoMAOT-DMPG) and cationics (eg, dioleoMAOT-D-phosphatidyl-phosphatidyl-phosphatidylcholine).

For topical or other administration, the oligonucleotides of the invention can be encapsulated within liposomes or can form complexes with them, in particular with cationic liposomes. Alternatively, the oligonucleotides can be complexed with lipids, in particular with cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in US Patent No. 6,287,860.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets, or minicills. Thickeners, flavors, diluents, emulsifiers, dispersing aids, or binders may be desirable. Preferred oral formulations are those in which the 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 US Patent No. 6,287,860. Combinations of penetration enhancers are also preferred, 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. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligonucleotides of the invention can be administered orally, in granular form that includes spray-dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in US Patent No. 6,287,860.

Compositions and formulations for parenteral, intrathecal, or intraventricular administration may comprise 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 vehicles or excipients.

Certain embodiments of the invention provide pharmaceutical compositions that contain 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, maphosfamide, ifosforoetilin, cytosin, bisureycin arabomycin, cytosin, bisureycin arabomycin, bushycin, cytosine arabicin. 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-azacitidine, hydroxyurea, deoxycoformycin, 5-phosphorous-ureacyclooF (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol (DES). When used with the compounds of the disclosure, such chemotherapeutic agents can be used individually (eg, 5-FU and oligonucleotide), sequentially (eg, 5-FU and oligonucleotide over a period of time followed by MTX and oligonucleotide), or in combination with one or more of the other such chemotherapeutic agents (eg, 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including, but not limited to, non-steroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including, but not limited to, ribivirine, vidarabine, acyclovir, and ganciclovir, can also be combined in compositions of the invention. Combinations of antisense and other compounds

ES 2 664 591 T3 non-antisense drugs are also within the scope of the present invention. Two or more compounds can be used in combination together or sequentially.

In another related embodiment, the compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, directed to a first nucleic acid and one or more additional antisense compounds directed to a second target nucleic acid. For example, the first target can be a particular antisense sequence of filaggrin (FLG), and the second target can be a region of another nucleotide sequence. Alternatively, the compositions of the invention may contain two or more antisense compounds that target different regions of the same filaggrin target nucleic acid (FLG). Numerous examples of antisense compounds are illustrated herein and others may be selected from suitable compounds known in the art. Two or more compounds can be used in combination together or sequentially.

Dosage:

The formulation of therapeutic compositions and their subsequent administration (dosing) are believed to be within the skill of those skilled in the art. The dosage depends on the severity and sensitivity of the pathology to be treated, the treatment cycle lasting from several days to several months, or until a cure is effected or a reduction of the pathology is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the patient's body. Experts can easily determine optimal dosages, dosing methodologies, and repeat rates. Optimal dosages can vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on the EC50's that have been found to be effective in in vitro and in vivo animal models. In general, the dosage is 0.01 pg to 100 g per kg of body weight, and it can be administered once or more daily, weekly, monthly or annually, or even once every 2 to 20 years. Those skilled in the art can easily estimate repeat rates for dosing based on measured residence times and drug concentrations in body fluids or tissues. After successful treatment, it may be desired that the patient receive maintenance therapy to prevent recurrence of the pathology, in which the oligonucleotide is administered in maintenance doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more daily, to once every 20 years.

In embodiments, a patient is treated with a drug dosage that is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about less 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 of weight bodily. Certain injected doses of antisense oligonucleotides are described, e.g. eg, in the US patent. Pat. No. 7,563,884, Antisense modulation of PTP1B 8 expression.

EXAMPLES

The following non-limiting examples serve to illustrate selected embodiments of the invention.

Example 1: Design of antisense oligonucleotides specific for a nucleic acid molecule antisense to the Filaggrin gene (FLG) and / or a sense strand of the FLG polynucleotide.

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

Selection of the appropriate oligonucleotides is facilitated using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare obtained nucleic acid sequences, 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 show an appropriate degree of identity between species. In the case of genes that have not been sequenced. Southern blots are performed to

ES 2 664 591 T3 allow a determination of the degree of identity between genes of 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 a rough measure of identity. These procedures allow the selection of oligonucleotides that show a high degree of complementarity with target nucleic acid sequences in a subject to be monitored and a lower degree of complementarity with 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.

An antisense compound is specifically hybridizable when the 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 nonspecific binding of the compound. antisense to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo tests or therapeutic treatment, and under conditions under which tests are performed in the case of in vitro tests

The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays, as is known in the art. For example, the properties of the oligonucleotides described herein can be obtained by determining the binding intensity between the target natural antisense and a potential drug molecule using the melting curve assay.

The intensity of binding between the target natural antisense and a possible drug molecule (Molecule) can be estimated using any of the established methods of measuring the intensity of intermolecular interactions, eg, a melting curve assay.

The melting curve test 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 intensity of interaction between the two molecules.

A melting curve assay can be performed using a cDNA copy of the actual natural antisense RNA molecule or a nucleotide of DNA or synthetic RNA corresponding to the binding site of the molecule. Multiple kits are available that contain all the reagents necessary to perform this assay (eg, MeltDoctor kit from Applied Biosystems Inc.). These kits include a suitable buffer solution containing one of the double-stranded DNA-binding (dcDNA) dyes (such as aBi HRM, SYBR Green, SYTO dyes, etc.). The properties of dsDNA dyes are such that they almost do not fluoresce in free form, but are highly fluorescent when bound to dsDNA.

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

Melting peaks are constructed by plotting the negative derivative of fluorescence with respect to temperature (-d (Fluorescence) / dT) on the y-axis) versus temperature (x-axis) using appropriate software (e.g., LightTyper (Roche) or SDS Dissociation Curve, ABI). The data is analyzed to identify the temperature of the rapid transition of the dsDNA complex to single-stranded molecules. This temperature is called Tm and it is directly proportional to the intensity of the interaction between the two molecules. Typically, the Tm will exceed 40 ° C.

Example 2: Modulation of polynucleotides of the FLG gene

Treatment of HepG2 cells with antisense oligonucleotides

ATCC HepG2 cells (cat # HB-8065) ​​were grown in culture medium (MEM / EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV) + 10% FBS ( Mediatech cat no. MT35-011-CV) + penicillin / streptomycin (Mediatech cat no. MT30-002-CI)) at 37 ° C and 5% CO2. On the day of the experiment,

ES 2 664 591 T3 medium in the 6-well plates was changed to fresh culture medium. All antisense oligonucleotides were diluted to the concentration of 20 µΜ. Two μΙ of this solution were incubated with 400 μΙ of Opti-MEM medium (Gibco, cat # 31985-070) and 4 μI of Lipofectamine 2000 (Invitrogen, cat # 11668039) at room temperature for 20 min and applied to each well of the 6-well plates with HEPG2 cells. A similar mix including 2 µl of water was used in place of the oligonucleotide solution for mock vector transfected controls. After 3-18 h incubation at 37 ° C and 5% CO2, the medium was changed to fresh culture medium 48 h after the addition of antisense oligonucleotides, the medium was removed and RNA was extracted from the cells using Promega SV Total RNA Isolation System (Cat # Z3105) or Qiagen RNeasy Total RNA Isolation Kit (Cat # 74181) following the manufacturer's instructions. 600 ng of RNA was added to the reverse transcription reaction performed using the Thermo Scientific Verso cDNA Kit (Cat # AB1453B) or the High Capacity cDNA Reverse Transcription Kit (Cat # 4368813) as described in manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using the ABI Taqman Gene Expression Mix (cat # 4369510) and ABI-designed primers / probes (Taqman Gene Expression Assay from Applied Biosystems; Hs00856927 gl from Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) using the Mx4000 thermal cycler (Stratagene). The fold change in gene expression after antisense oligonucleotide treatment was calculated based on the difference in normalized dCt values ​​against 18S between the treated and mock-transfected samples. Results: Real-time PCR results show that FLG1 mRNA levels in HepG2 cells are significantly increased with two of the oligos designed for FLG1 antisense BGAK056431 (Fig 1). Treatment of 518A2 cells with antisense oligonucleotides

518A2 cells obtained from the Albert Einstein-Montefiore Cancer Center, NY were cultured in culture medium (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 ° C and 5% CO2. One day before the experiment, the cells were re-seeded at the density of 1.5 x 105 / ml in 6-well plates and incubated at 37 ° C and 5% CO2. On the day of the experiment, the medium in the 6-well plates was changed to fresh culture medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. Two μΙ of this solution was incubated with 400 μΙ of Opti-MEM medium (Gibco, cat no. 31985-070) and 4 μΙ of Lipofectamine 2000 (invitrogen, no. cat 11668019) at room temperature for 20 min and applied to each well of the 6-well plates with 518A2 cells. A similar mix including 2 µl of water was used in place of the oligonucleotide solution for mock vector transfected controls. After 3-18 h of incubation at 37 ° C and 5% CO2, the medium was changed to fresh culture medium 48 h after the addition of antisense oligonucleotides, the medium was removed and RNA was extracted from the cells using Promega SV Total RNA Isolation System (Cat # Z3105) or Qiagen RNeasy Total RNA Isolation Kit (Cat # 74181) following the manufacturer's instructions. 600 ng of RNA was added to the reverse transcription reaction performed using the Thermo Scientific Verso cDNA Kit (Cat # AB1453B) or the High Capacity cDNA Reverse Transcription Kit (Cat # 4368813) as described in manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using the ABI Taqman Gene Expression Mix (cat # 4369510) and ABI-designed primers / probes (Taqman Gene Expression Assay from Applied Biosystems: Hs00856927 gl from Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) using the StepOne Plus Real Time PCR Machine (Applied Biosystems). The fold change in gene expression after antisense oligonucleotide treatment was calculated based on the difference in normalized dCt values ​​against 18S between the treated and mock-transfected samples. Results: the real-time PCR results show that the FLG mRNA levels in 518A2 cells are significantly increased with two of the oligos designed for FLG1 antisense AK056431 (Fig 2). Another set of real-time PCR results show that FLG mRNA levels in 518A2 cells are significantly increased with two of the oligos designed for FLG1 antisense AK056431 (Fig 3). Example 3: Modulation of the expression and activity of the FLG gene

Treatment of 518A2 cells with small compounds

518A2 cells were grown in culture medium ((Mediatech cat # 10-013-CV) + 5% FBS (Mediatech cat # MT35-011-CV) + penicillin / streptomycin (Mediatech cat # MT30-002-C1)] at 37 ° C with 5% CO2. One day before the experiment, the cells were re-seeded at the density of approximately 1x10^TO4 / ml (or a dilution of approximately 1/5 from 90% confluence) in 6-well plates and incubated at 37 ° C and 5% CO2 overnight. On the day of the experiment, the medium in the 6-well plates was changed to 2 ml of medium.

ES 2 664 591 T3 from fresh culture. Small compounds were diluted in DMSO to a concentration of 1000 uM. On the day of the experiment, this solution was diluted 1: 100 in fresh growth medium. Pure DMSO was diluted in medium at the same ratio (1: 100) to treat vehicle control samples. To dispense one well, 200 µΙ of the compound or pure DMSO solution was added directly to the well of a 6-well plate. The final concentration of compounds was 1 uM. The dosage volume was adjusted if a different concentration of compound was desired. After dosing the plates were incubated overnight at 37 ° C and 5% CO2. 24 h after the addition of small compounds, the medium was replaced with fresh culture medium and the dosing was repeated as described above. 24 h after the second dosing, RNA was extracted from cells using Promega's Sv Total RNA Isolation System (cat # Z3105) following the manufacturer's instructions. 600 ng of total RNA was added to the reverse transcription reaction performed using the Applied Biosystems High Throughput cDNA 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 ABI designed primers / probes (eg Assay ID # Hs00856927_gl for FLG). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) using the StepOne thermal cycler (ABI). The assay for 18S used to normalize mRNA levels was manufactured by ABI (cat # 4319413E). The fold change in gene expression after treatment with small compounds was calculated based on the difference in normalized 18S dCt values ​​between mock-transfected and treated samples.

Results: real-time PCR results show a fold change in filaggrin mRNA expression in 518A2 cells treated with small molecules (Fig. 4)

Treatment of primary keratinocytes with small compounds

Primary keratinocytes (from Promocell) were grown in growth medium (Keratinocyte Growth Media, Promocell cat # C-20011) at 37 ° C with 5% CO2. One day before the experiment, the cells were re-seeded at the density of approximately 5x10^TO4 / ml (or approximately 1/3 dilution from 90% confluence) in 24-well collagen coated plates (Beckton Dickinson BioCoat plates cat # 35 6408) and incubated at 37 ° C and the 5% CO2 overnight. On the day of the experiment, the medium in the 24-well plates was changed to 1 ml of fresh culture medium. Small compounds were diluted in DMSO to a concentration of 1000 uM. On the day of the experiment, this solution was diluted 1: 100 in fresh growth medium. Pure DMSO was diluted in the media at the same ratio (1: 100) to treat vehicle control samples. To dispense one well, 100 µl of the compound or neat DMSO solution was added directly to the well of a 24-well plate. The final concentration of compounds was 1 uM. The dosage volume was adjusted if a different concentration of compound was desired. After dosing, the plates were incubated overnight at 37 ° C and 5% CO2, 24 h after the addition of small compounds, the medium was replaced with fresh culture medium and dosing was repeated as described above. 24 h after the second dosing, RNA was extracted from cells using the Promega SV Total RNA Isolation System (cat # Z3105) following the manufacturer's instructions. 600 ng of total DNA was added to the reverse transcription reaction performed using the Applied Biosystems High Throughput cDNA 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's Taqman Gene Expression Mix (cat # 4369510) and ABI-designed primers / probes (e.g., assay no. ID # Hs0856927_gl for FLG). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles (95 ° C for 15 seconds, 60 ° C for 1 min) using the StepOne thermal cycler (ABI). The assay for 18S used to normalize mRNA levels was manufactured by AB1 (cat # 4319413E). The fold change in gene expression after treatment with small compounds was calculated based on the difference in normalized 18S dCt values ​​between mock-transfected and treated samples. Results: Real-time PCR results show a fold change in Filaggrin mRNA expression in primary keratinocytes treated with small molecules (Fig. 4). Another set of real-time PCR results shows that the levels of natural antisense transcripts of the FLG gene in primary keratinocytes decrease significantly after treatment with small molecules of Bupropion, Bendipine and Topiramate (Fig. 5).

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 after reading and understanding this specification and the accompanying drawings. Furthermore, although a particular feature of the disclosure has been described with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations, as it may be desired and advantageous for any given or particular application. .