EP1831367A2 - Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus - Google Patents

Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus

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Publication number
EP1831367A2
EP1831367A2 EP05816225A EP05816225A EP1831367A2 EP 1831367 A2 EP1831367 A2 EP 1831367A2 EP 05816225 A EP05816225 A EP 05816225A EP 05816225 A EP05816225 A EP 05816225A EP 1831367 A2 EP1831367 A2 EP 1831367A2
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EP
European Patent Office
Prior art keywords
agr2
sirna
cells
dna construct
mucus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05816225A
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German (de)
English (en)
Inventor
Lutz Zeitlmann
Johannes Grosse
Klaus Dembowsky
Andreas Popp
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Ingenium Pharmaceuticals GmbH
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Ingenium Pharmaceuticals GmbH
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Publication of EP1831367A2 publication Critical patent/EP1831367A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the epithelial mucosal layer is a physical and chemical barrier important in protecting the animal body from dryness, harmful exogenous substances and pathogens.
  • Mucus forms a gel layer covering the epithelial surface, acting as a semi-permeable barrier between the epithelium and the exterior environment. Mucus serves many functions, including protection against shear stress and chemical damage, and, especially in the respiratory tree, trapping and elimination of particulate matter and microorganisms.
  • the mucus layer on top of the intestinal epithelium is the barrier between the host's internal milieu and gut bacteria.
  • Secretion of mucus occurs by exocytosis of secretory granules (Verdugo, 1991). Constitutive or basal secretion occurs at low levels and is essentially unregulated and continuous. Stimulated secretion corresponds to regulated exocytosis of granules in response to extracellular stimuli such as hormones, neuropeptides and inflammatory mediators (Jackson, 2001; Laboisse et al., 1996). This pathway provides the ability to dramatically increase mucus secretion.
  • Mucins are high molecular mass, highly glycosylated macromolecules that are the major components of mucus secretions. Many epithelial cells express mucins. For example, mucins are secreted from the apical surface of specialized columnar epithelial cells referred to as goblet cells
  • Goblet cells are distributed among other cells in the epithelium of many organs, especially in the intestinal and respiratory tracts. In areas like the conjunctiva, their numbers are rather small compared to other cell types, whereas in tissues such as the colon, they are much more abundant. Goblet cells have a characteristic morphology, based on membrane-bound secretory granules, which contain mucus (Specian and Oliver, 1991). The goblet cells' function is the secretion of mucins and other products, including protease resistant peptides - like the trefoil peptide family, which protect epithelium from injury and promote repair through restitution of epithelial cells (Podolsky, 2000).
  • Mucins have the ability to hydrate and form a viscous gel, producing a protective scaffold overlaying epithelial surfaces.
  • Mucins consist of a polypeptide core (apomucin) covered almost entirely by O-linked carbohydrate chains, which may constitute up to 80% of the total molecular weight. All known mucin genes are characterized by tandem and irregular repeat sequences rich in codons for threonine and serine, e.g., TT and SS, the potential sites of attachment of oligosaccharide chains.
  • mucin proteins are secreted into the endoplasmatic reticulum (ER).
  • the ER serves as cellular compartment for further posttranslational modification, e.g., protein folding.
  • the O-glycosylation by glycosyltransferase occurs in the Golgi.
  • Mucin2 mucin5ac, mucin5b and mucin ⁇ , all of them exhibiting tissue- and cell specific expression, belong to the class of secreted mucins.
  • Mucin2 is mainly expressed in intestinal and in colonic goblet cells.
  • the mucin2 protein is more than 5,100 amino acids in its commonest allelic form.
  • the mucin2 product is polymerized end to end through disulfide bridges to form large secreted polymeric gel- forming mucins (Allen et al., 1998).
  • Mucin5ac is primarily expressed in tracheobronchial goblet cells and in gastric surface epithelial cells.
  • Mucin5b is expressed in tracheobronchial, salivary and esophageal mucous glands, pancreatobiliary and endocervical epithelial cells.
  • Mucin5b is composed of 14.9% protein, 78.1% carbohydrate, and 7% sulfate.
  • Mucin ⁇ is expressed in gastric and duodenal mucous glands and in pancreatobiliary and endocervical epithelial cells.
  • WO2004/056858 discloses reduced amounts of mucin2 mRNA in colon of mice carrying a point mutation in the Agr2 gene (see Example 24 therein).
  • the mutated Agr2 protein carries a charged glutamic acid (E) in position 137 instead of a non-polar valin (V) in the wild type (non mutated) protein.
  • Mutated mice display a reduction in pre-mucin storing granules in the goblet cells, a reduced mucus secretion, and secondary inflammatory infiltrations in the intestinal mucosal epithelium and submucosa.
  • Agr2 transcript is detected in mucus secreting tissues, including tissues of the digestive/gastrointestinal tract, in particular salivary gland, esophagus, stomach, small intestine, large intestine, rectum; and tissue of the respiratory tract, in particular nose epithelium, trachea and lung (see Figures 6 to 8 therein).
  • Agr2 protein expression is detected in colon goblet cells of wild type mice (see Figure 10 therein).
  • the Agr2 protein is disclosed to be involved in goblet cell differentiation, particularly terminal differentiation and/or goblet cell mucus production or secretion and/or mucus composition.
  • Mucus hyperproduction / hypersecretion is observed in diseases such as asthma, allergy, COPD and cystic fibrosis.
  • Disease-associated cytokines, bacterial products, proteinases or oxidants are inducers of goblet cell hyperplasia.
  • Goblet cell hyperplasia and associated mucus hypersecretion are clinical and pathophysiological features of asthma and COPD. Especially in asthma, airway mucus hypersecretion contributes to morbidity and mortality.
  • EGFR epidermal growth factor receptor
  • CLCA calcium-activated chloride channels
  • LTB4 antagonists WO 02/55065
  • EGF receptor antagonists WO 02/05842
  • polycationic peptides US 6,245,320
  • KGF WO 94/23032
  • KGF-2 WO 99/41282
  • the invention described herein demonstrates for the first time that Agr2 protein directly interacts with mucins, the major components of mucus.
  • the invention therefore offers novel opportunities for the treatment of respiratory diseases where a reduction of mucus hyperproduction / hypersecretion is desirable, by suppressing the expression of the Agr2 gene, thereby preventing the interaction of Agr2 with mucins.
  • the invention relates to specific short interfering RNAs comprising a double stranded nucleotide sequence wherein one strand is complementary to an at least 19 to 25 nucleotide long segment of specific regions of an mRNA encoding an Agr2 protein that are capable of silencing or suppressing the expression of the Agr2 gene.
  • the siRNAs of the present invention are designed so as to efficiently suppress Agr2 gene expression, by gene silencing.
  • said Agr2 gene is a vertebrate Agr2 gene, in particular a mammalian Agr2 gene, more particularly an Agr2 gene selected from the group consisting of the Agr2 gene of human, mouse, rat, rabbit, hamster, dog, cat, sheep, and horse, most preferably a human Agr2 gene.
  • compositions comprising such siRNA molecules and a pharmaceutically acceptable carrier are also encompassed by the present invention.
  • the invention in another embodiment, relates to a method of preventing, treating, or ameliorating a medical condition associated with mucus hyperproduction / hypersecretion in a mammal, e.g., in a human subject, particularly a medical condition affecting the respiratory system, for example asthma, allergic reactions of the respiratory system, COPD (chronic obstructive pulmonary disease), and cystic fibrosis.
  • the method comprises administering to said mammal or said human subject a pharmaceutical composition comprising the siRNA molecules of the invention that are capable of silencing or suppressing the expression of Agr2.
  • the invention relates to a method of identifying siRNAs capable of silencing or suppressing the expression of the Agr2 gene, and thus, identifying siRNAs useful for preventing, treating, or ameliorating any of the above-mentioned medical conditions.
  • Said methods comprise assaying the ability of a test siRNA to suppress Agr2 gene expression.
  • the siRNAs as described herein can be used for the preparation of a pharmaceutical composition for preventing, treating, or ameliorating any of the above-mentioned medical conditions.
  • DNA constructs such as vectors, and/or host cells as described herein and their use in any of the methods described herein for prevention, amelioration, or treatment of the aforementioned medical conditions.
  • the use described above when applied to an animal such as a mammal (e.g., a human) has significant medicinal value.
  • another aspect of the invention is related to the use of the siRNAs, the DNA constructs capable of expressing said siRNAs, as well as the pharmaceutical compositions containing said siRNAs as described herein as a medicament.
  • the medicament may be used for suppressing or silencing the expression of the Agr2 gene.
  • the medical composition may be used to prevent, to ameliorate, or to treat a medical condition or disease associated with mucus hyperproduction / hypersecretion, particularly a medical condition affecting the respiratory system, such as asthma, allergic reactions of the respiratory system, chronic obstructive pulmonary disease (COPD), and cystic fibrosis.
  • COPD chronic obstructive pulmonary disease
  • siRNA molecules may be administered to a patient using well known delivery methods as described in more detail infra.
  • administration of siRNAs of the present invention may be accomplished by a method known as gene therapy.
  • a method of gene therapy for preventing, treating, or ameliorating any of the above-mentioned medical conditions is another aspect of the present invention.
  • Other preferred ways of administering the siRNAs of the present invention are governed by the need to reach the mucus producing cells of the respiratory system and will be apparent to those skilled in the art. Accordingly, it will be appreciated that intratracheal, transmucosal, or intranasal administration is particularly preferred for the siRNAs of the present invention.
  • intratracheal, transmucosal, or intranasal administration is particularly preferred for the siRNAs of the present invention.
  • FIG 1 shows Agr2 protein co-localization with protein disulphide isomerase (PDI) in the endoplasmatic reticulum (ER) of CaCo2 cells.
  • PDI protein disulphide isomerase
  • ER endoplasmatic reticulum
  • Agr2 antiserum plus a fluorescent labeled second antibody or with anti-PDI antibody plus a fluorescent labeled second antibody, respectively.
  • Figure 2 shows results of a yeast-two hybrid experiment, indicating protein- protein interaction between murine Agr2 and murine mucin2 (Agr2 x mucin2 fragment).
  • Agr2 protein was expressed as a fusion protein carrying the DNA binding domain of GaW
  • murine mucin2 was expressed as a fusion protein carrying the Gal4 transcriptional activator domain. Binding of the two fusions products was monitored based on the transcriptional activation of the reporter gene His3.
  • Only yeast cells carrying the fusion products of Agr2 and mucin2 in a protein-protein interaction state are capable of growing on a yeast medium lacking histidine (interaction). The strength of protein-protein interaction correlates with the size of yeast colonies grown and is indicated as a score value (+). Positive and negative controls provided by the kit were included in the assay.
  • FIG 3 shows data of a tracheal ovalbumin challenge assay applied to wild type (WT) mice and Agr2 mutant (Agr2-/-) mice.
  • Tracheal ovalbumin challenge induces goblet cell differentiation from tracheal epithelial cells and glycoprotein synthesis in the goblet cells of WT mice, as seen in Figure 3B.
  • Such mice are well known as a murine asthma model.
  • Control mice do not display such phenotypic alterations in the trachea after saline installation, as shown in Figure 3A.
  • Agr2-/- mice lacking normal Agr2 function, differentiated goblet cell are not visible, and no synthesized glycoproteins are detectable after ovalbumin instillation, as shown in Figure 3C.
  • FIG. 4 pSilencer constructs for expressing double stranded oligonucleotides, which target Agr2 coding sequences were transiently transfected into mammalian LS174T cells. These cells express Agr2. Transfection efficiency was 50%, as measured with controle transfections (data not shown). cDNA from freshly isolated RNA of transfected cells was used for quantitative PCR analysis, indicating the silencing effect of the expressed oligonucleotides on Agr2 gene expression.
  • Targeting Agr2 coding sequences shl or sh4 leads to a 78% reduction and 98% reduction of Agr2 mRNA, respectively, whereas targeting of Agr2 coding sequence sh5 has almost no gene silencing effect. Targeting with a random sequence, exhibiting no homology to Agr2 gene sequences has no gene silencing effect (neg. Ctrl.).
  • the goblet cells referred to herein are cells, which are specialized with respect to mucus secretion via granules, in particular in the gastrointestinal tract (GI), or in the respiratory tract (examples in this regard are goblet cells of the nose epithelium, of the trachea, of the bronchius, and of the submucosal glands of the trachea).
  • GI gastrointestinal tract
  • the respiratory tract examples in this regard are goblet cells of the nose epithelium, of the trachea, of the bronchius, and of the submucosal glands of the trachea).
  • differentiation refers to all steps of cellular differentiation of a goblet cell from early differentiation to late differentiation and to terminal differentiation, i.e., to the mature mucus secrecting goblet cell.
  • terminal differentiation of goblet cells means the last differentiation step to the mature goblet cell.
  • mucus secreting cell refers to cells which are specialized to mucus secretion without prior storage of the mucus in granules, e.g., submucosal cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue.
  • antisense strand refers to a polynucleotide that is substantially or 100% complementary, to a target nucleic acid of interest, such as, for example, a protein coding or a non-coding nucleic acid sequence.
  • An antisense strand may be comprised of a polynucleotide that is RNA, DNA or chimeric RNA/DNA.
  • an antisense strand may be complementary, in whole or in part, to a protein coding or a non-coding sequence, for example, an RNA sequence that is not mRNA (e.g., tRNA, rRNA and hnRNA) or a sequence of DNA that is a protein coding or a non-coding sequence.
  • Complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand.
  • Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity. Substantial complementarity refers to 78% or greater complementarity. In determining complementarity, overhang regions are excluded.
  • duplex region refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between polynucleotide strands that are complementary or substantially complementary.
  • a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region” consists of 19 base pairs.
  • the remaining base pairs may, for example, exist as 5'- and 3'- overhangs.
  • 100% complementarity is not required; substantial complementarity is allowable within a duplex region.
  • Substantial complementarity refers to 78% or greater complementarity.
  • a mismatch in a duplex region consisting of 19 base pairs i. e. , 18 base pairs and one mismatch
  • results in 94.7% complementarity thus rendering the duplex region substantially complementary.
  • three mismatches in a duplex region consisting of 19 base pairs i.e., 16 base pairs and three mismatches
  • result in 84.2% complementarity again rendering the duplex region substantially complementary.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology at the nucleotide level or amino acid level, respectively.
  • Homologous nucleotide sequences can include those sequences coding for isoforms of Agr2 polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • “Orthologues” or “orthologous nucleic acid or amino acid sequences” refer to genes/proteins in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percent amino acid/nucleic acid identity or “% amino acid/nucleic acid identity” refer to the percentage of sequence identity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be readily determined electronically, e.g., by using the MEGALIGN program (DNASTAR,
  • the MEGALIGN program can create alignments between two or more sequences according to different methods, one of them being the clustal method. See, e.g., Higgins and Sharp (Higgins and Sharp, 1988).
  • the clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity.
  • a particularly preferred method of determining amino acid identity between two protein sequences for the purposes of the present invention is using the "Blast 2 sequences" (bl2seq) algorithm described by Tatusova et al. (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247- 250).
  • This method produces an alignment of two given sequences using the "BLAST” engine. On-line access of "blasting two sequences” can be gained via the NCBI server at http://www.ncbi.nlm.mh.gov/blastM2seqM2.h1ml.
  • the standalone executable for blasting two sequences can be retrieved from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/blast/executables).
  • the settings of the program blastp used to determine the number and percentage of identical or similar amino acids between two proteins were the following:
  • a reference to percent amino acid sequence identity means in a preferred embodiment percent identity as determined in accordance with the blastp program using the above settings.
  • a reference to percent nucleic acid sequence identity preferably means percent identity as determined in accordance with the blastn program using the following settings:
  • an Agr2 protein in the present invention may be, for example, a corresponding homologue or orthologue of the human Agr2 protein according to SEQ ID NO:1. It may also be a variant of the human Agr2 protein according to SEQ ID NO:1, or of said orthologue, allelic or otherwise, wherein certain amino acids or partial amino acid sequences have been replaced, added, or deleted.
  • RNA interference refers to the process by which a polynucleotide or double stranded polynucleotide comprising at least one ribonucleotide unit exerts an effect on a biological process.
  • the process includes but is not limited to gene silencing by degrading mRNA, interactions with tRNA, rRNA, hnRNA, cDNA and genomic DNA, as well as methylation of DNA and ancillary proteins.
  • siRNA and the term “short interfering RNA” refer to a double stranded nucleic acid that is capable of performing RNA interference and that is usually between 18 and 30 base pairs in length (i.e., a duplex region of between 18 and 30 base pairs).
  • short interfering RNAs according to the present invention have a duplex region of between 18 and 25 base pairs. It is particularly preferred that the siRNAs have a duplex region of between 19 and 21 base pairs.
  • siRNA and “short interfering RNA” may be understood to include nucleic acids that also contain moieties other than ribonucleotide moieties, including, but not limited to, modified nucleotides, modified internucleotide linkages, non-nucleotides, deoxynucleotides and analogs of the aforementioned nucleotides.
  • siRNAs can be duplexes, and can also comprise short hairpin RNAs, RNAs with loops as long as, for example, 4 to 23 or more nucleotides, RNAs with stem loop bulges, micro-RNAs, and short temporal RNAs.
  • RNAs having loops or hairpin loops can include structures where the loops are connected to the stem by linkers such as flexible linkers.
  • Flexible linkers can be comprised of a wide variety of chemical structures, as long as they are of sufficient length and materials to enable effective intramolecular hybridization of the stem elements.
  • gene silencing or “silencing” refers to the reduction in transcription, translation or expression or activity of a nucleic acid, as measured by transcription level, mRNA level, enzymatic activity, methylation state, chromatin state or configuration, or other measure of its activity or state in a cell or biological system.
  • Gene silencing refers to the reduction or amelioration of activity known to be associated with a nucleic acid sequence, such as its ability to function as a regulatory sequence, its ability to be transcribed, its ability to be translated and result in expression of a protein, regardless of the mechanism whereby such silencing occurs.
  • siRNA a decreased amount of mRNA when compared to the amount of mRNA encoding the Agr2 protein found in the absence of said siRNA, i.e., found in the presence of a control siRNA (scrambled siRNA without homology to
  • Agr2 gene expression can be attenuated by RNA interference, i.e., siRNA mediated suppression of Agr2 gene expression, where expression products of an Agr2 gene are targeted by specific double stranded Agr2-derived siRNA nucleotide sequences that are complementary to an at least a 19 to 25 nt long segment of the Agr2 gene transcript.
  • RNA interference i.e., siRNA mediated suppression of Agr2 gene expression
  • said siRNAs comprise a double stranded nucleotide sequence wherein one strand is substantially or perfectly complementary to an at least 19, 20, 21, 22, 23, 24, or 25 nucleotide long segment of an mRNA encoding the human Agr2 protein according to SEQ ID NO:1; or a homologue or orthologue thereof having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity compared to the human Agr2 protein according to SEQ ID NO:1.
  • said segment of an mRNA encoding the Agr2 protein as defined above may represent or comprise non-coding sequence.
  • said segment may represent or comprise coding sequence of said mRNA.
  • the above-mentioned segment may also include sequences from the 5' untranslated (UT) region. Alternatively or in addition, it may include sequences corresponding to the open reading frame (ORF). Again alternatively or in addition, it may include sequences from the 3 ' untranslated (UT) region.
  • the antisense strand is substantially complementary to said segment of an mRNA encoding the human Agr2 protein. More preferably, the antisense strand is perfectly, i.e., 100% complementary to said segment of an mRNA encoding the human Agr2 protein.
  • the sense strand is preferably substantially complementary to the region of the antisense strand with which it forms a duplex (excluding overhangs, if present). More preferably, the sense strand is 100% complementary to the region of the antisense strand with which it forms a duplex.
  • the invention provides siRNAs, wherein one strand is complementary to a 19 nucleotide long segment of an mRNA encoding said Agr2 protein, and wherein said segment encodes the shl region (coding position +93 to +111) of the human Agr2 gene according to SEQ ID NO:3, or the sh4 region (coding position +203 to +221) of the human Agr2 gene according to SEQ ID NO:4, respectively.
  • siRNAs described herein including these particularly preferred siRNAs targeting the shl and sh4 regions of the human Agr2 gene are useful in any method described in the present invention, including methods of gene therapy, methods for treating medical conditions associated with mucus hyperproduction / hypersecretion, as well as methods for the preparation of any pharmaceutical compositions and medicaments set forth herein.
  • the amount of Agr2 mRNA found in cells that are contacted with the siRNA molecules of the present invention is preferably reduced by at least 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100%.
  • the reduction of Agr2 gene expression is preferably measured by quantitative PCR, e.g., Light CyclerTM PCR (PerkinElmer Inc.).
  • the decrease in mRNA expression is determined by comparing Agr2 mRNA levels in cells co-transfected with the siRNA molecules of the present invention to Agr2 mRNA levels in cells cotransfected with non-complementary short RNA molecules which are not able to suppress expression of the Agr2 gene in the same assay, preferably side-by-side and under the same assay conditions, therefore resulting in relative values, it will be appreciated that the skilled person will be readily able to determine the above percentages for reduced mRNA levels in the in vitro assays contemplated in connection with the present invention.
  • DNA constructs that are capable of directing the expression of the siRNAs of the present invention.
  • such DNA constructs are vectors.
  • Particularly preferred vectors are viral vectors.
  • Other suitable vectors are known in the art, or are described in more detail herein below.
  • the preferred DNA constructs according to the present invention are capable of transiently expressing the siRNAs contemplated in the instant invention.
  • a DNA construct as described herein in a method of treating a human subject suffering from a medical condition associated with mucus hyperproduction / hypersecretion, said method comprising delivering said DNA construct to at least some of the cells of said human subject, preferably the subject's goblet cells or or submucosal cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue, is also encompassed within the present invention.
  • siRNA vectors appear to have an advantage over synthetic siRNAs where long term knock-down of expression is desired.
  • siRNA expression vectors of the present invention may experience steady, long-term mRNA inhibition, hi contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division.
  • the long-term gene silencing ability of siRNA expression vectors of the present invention is suitable for applications in gene therapy. It will be understood by those skilled in the art that DNA constructs which are capable of being stably integrated into the genome of cells transfected with said DNA constructs are thus particularly suitable for gene therapy methods. Accordingly, such DNA constructs represent particularly preferred embodiments of the present invention.
  • siRNAs are transcribed intracellularly by cloning the Agr2 gene templates into a vector containing, e.g., a RNA pol III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA Hl.
  • a vector system is the GeneSuppressorTM RNA Interference kit (commercially available from Imgenex).
  • the U6 and Hl promoters are members of the type III class of Pol III promoters.
  • the +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for Hl promoters is adenosine.
  • the termination signal for these promoters is defined by five consecutive thymidines.
  • the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoters, therefore they are ideally suited for the expression of around 21 -nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.
  • Prokaryotic and eukaryotic host cells transformed with the above siRNAs or DNA constructs are likewise within the scope of the present invention.
  • methods are provided that are suitable for identifying novel siRNAs capable of suppressing Agr2 gene expression.
  • Short interfering RNA capable of Agr2 gene expression silencing can be obtained using an Agr2 polynucleotide sequence, for example, by processing the Agr2 ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded Agr2 RNA or by chemical synthesis of nucleotide sequences homologous to a Agr2 sequence.
  • RNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3' overhang.
  • the sequence of the 2-nt 3' overhang may provide an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases.
  • the nucleotides in the 3' overhang are ribonucleotides.
  • the nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-deoxynucleotides in the 3' overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.
  • siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER.
  • DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex.
  • siRNAs/protein complex siRNP
  • RISC RNA-induced silencing complex
  • RISC uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.
  • An Agr2 mRNA region to be targeted by siRNA is generally selected from a desired Agr2 sequence beginning 50 to 100 nt downstream of the start codon.
  • 5' or 3' UTRs and regions nearby the start codon can be used but are generally avoided, as these may be richer in regulatory protein binding sites.
  • UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex.
  • An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only one gene is targeted. Specificity of target recognition by siRNA duplexes indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation.
  • Negative control siRNA should have the same nucleotide composition as the Agr2 siRNA, but lack significant sequence homology to the genome. Typically, one would scramble the nucleotide sequence of the Agr2 siRNA and do a homology search to ascertain it lacks homology to any other gene.
  • control transfection with vectors not including an insert are performed to determine whether vector sequences itself (i.e., without an insert) are capable of silencing Agr2 gene expression.
  • a targeted Agr2 region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N 19) residues (e.g., AA(Nl 9)TT).
  • a desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21).
  • the sequence of the Agr2 sense siRNA corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3' end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the Agr2 polynucleotide.
  • the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
  • Symmetric 3' overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs (see, Elbashir, Lendeckel and Tuschl (2001), Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely) (Elbashir et al., 2001a).
  • the modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as it is usually the antisense siRNA strand that guides target recognition.
  • the Agr2 target mRNA does not contain a suitable
  • AA(N21) sequence one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5' (N19)TT, as it is believed that the sequence of the 3'-most nucleotide of the antisense siRNA does not contribute to specificity. Unlike antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. See Harborth et al. (2001) J. Cell Science 114: 4557- 4565, incorporated herein by reference in its entirety (Harborth et al., 2001).
  • Transfection of Agr2 siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially available from Invitrogen).
  • An assay for Agr2 gene silencing is generally performed approximately 2 days after transfection. No Agr2 gene silencing is observed in the absence of transfection reagent, allowing for a comparative analysis of the wild type and silenced Agr2 phenotypes.
  • siRNA duplex In a typical experiment, for one well of a 24-well plate, approximately 0.84 ⁇ g of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful Agr2 silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used.
  • Preferred cells are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human.
  • Particularly preferred cells useful in the methods described herein are mammalian cells such as CaCo2, LS174T or HT-29 cells. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.
  • a knock-down phenotype may become apparent after 1 to 3 days, or even later. Depletion of the Agr2 polynucleotide may also be observed by immunofluorescence or Western blotting. If the Agr2 polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the Agr2 protein is observed, it may be desirable to analyze whether the target mRNA was effectively destroyed by the transfected siRNA duplex.
  • RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs.
  • RT-PCR of a non- targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable Agr2 protein may exist in the cell. Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfections are required, cells are split 2 to 3 days after transfection. The cells may be re-transfected immediately after splitting.
  • two independent Agr2 siRNA duplexes can be used to knock-down a target Agr2 gene. This helps to control for specificity of the silencing effect, hi addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different Agr2 siRNA duplexes. Availability of siRNA- associating proteins is believed to be more limiting than target mRNA accessibility.
  • a therapeutic method according to the present invention contemplates administering an Agr2-specific siRNA construct as therapy to reduce mucus hyperproduction / hypersecretion, particularly in the respiratory system, by suppressing Agr2 gene expression .
  • the Agr2 ribopolynucleotide is obtained and processed into siRNA fragments as described herein.
  • the Agr2- derived siRNAs of the instant invention are administered to cells or tissues using known nucleic acid transfection techniques, as described herein.
  • An Agr2-derived siRNA specific for an Agr2 gene will decrease or knockdown Agr2 transcription products, which will lead to reduced Agr2 polypeptide production, thus ultimately resulting in reduced mucus production in the cells or tissues.
  • the siRNAs of the present invention that are to be administered to a subject in need of such treatment by any of the methods described herein must preferably affect Agr2 gene expression in the cells that produce mucus components. Accordingly, the siRNAs of the present invention will preferably suppress expression of the Agr2 gene in mucus producing cells of the respiratory system, such as goblet cells and submucosal cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue.
  • mucus producing cells of the respiratory system such as goblet cells and submucosal cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue.
  • siRNAs of the present invention are also useful in diagnostic methods, e.g., whether a subject suffering from a medical condition associated with mucus hyperproduction / hypersecretion is amenable to therapeutic treatment according to the present invention.
  • expression levels are detected using the assays as described herein or as generally known in the art, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like.
  • a subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state.
  • These cells or tissues are treated by administering Agr2-specific siRNAs to the cells or tissues by methods described for the transfection of nucleic acids into a cell or tissue, and a change in Agr2 polypeptide or polynucleotide expression is observed in the subject sample relative to the control sample, using the assays described.
  • This Agr2 gene knockdown approach provides a rapid method for determination of a Agr2-phenotype in the treated subject sample.
  • the Agr2-phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.
  • the invention also includes pharmaceutical compositions containing the siRNAs or the DNA constructs as described herein.
  • the compositions are preferably suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed.), Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes large unilamellar vesicles (LUVs) and other non-aqueous vehicles such as fixed oils may also be used.
  • LUVs large unilamellar vesicles
  • Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid
  • EDTA EDTA
  • buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ, U.S.A.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
  • the compositions of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the siRNAs or DNA constructs of the present invention are dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • Injectable compositions are preferably aqueous isotonic solutions or suspensions.
  • compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers, hi addition, they may also contain other therapeutically valuable substances.
  • adjuvants such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers, hi addition, they may also contain other therapeutically valuable substances.
  • the compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
  • Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow- release or sustained-released system, which assures that a constant level of dosage is maintained, according to US Pat. No. 3,710,795, incorporated herein by reference.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above, may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in US Pat. No. 5,262,564.
  • large unilamellar vesicles are composed of a mixture of cationic and anionic lipids (see, e.g., Hafez IM, Ansell S, Cullis PR, 2000).
  • siRNAs or DNA constructs of the present invention are preferably administered intratracheal, e.g., by inhalation. Particularly preferred for intratracheal delivery are aerosols containing the siRNAs or DNA constructs of the present invention together with suitable additives known by those skilled in the art. Alternatively, the siRNAs or DNA constructs of the present invention may be administered to the target cells or tissues in intranasal form, e.g., via aerosols or via topical use of suitable intranasal vehicles.
  • Liposomal formulations containing the siRNAs or DNA constructs of the present invention are particularly preferred for intratracheal delivery. Such formulations may conveniently be formulated into aerosol formulations suitable for inhalation or intranasal delivery (see, for example, WO 99/34837). In addition, formulations containing large unilamellar vesicles (LUVs) composed of a mixture of cationic and anionic lipids are likewise particularly suitable for these applications.
  • LUVs large unilamellar vesicles
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl- methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydro gels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydro gels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Oral dosages of the present invention when used for the indicated effects, may be preferably provided in any form commonly used for oral dosage such as, for example, in scored tablets, time released capsules, liquid filled capsule, gels, powder or liquid forms.
  • the dosage per unit may be varied according to well known techniques.
  • individual dosages may contain 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient. It is well known that daily dosage of a medication, such as a medication of this invention, may involve between one to ten or even more individual tables per day.
  • the compounds comprised in the pharmaceutical compositions of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • a further aspect of the present invention relates to a method of gene therapy using the siRNAs or DNA constructs provided by the present invention.
  • Gene therapy is known in the art. This term has been used to describe a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. Such methods include, for example, the delivery of a gene, antisense RNA, an siRNA molecule, an aptamer, a cytotoxic agent, etc., by a vector to a mammalian cell, preferably a human cell either in vivo or ex vivo. Most work has focused on the use of viral vectors to transform these cells. This focus has resulted from the ability of some viruses, to infect cells and have their genetic material integrated into the host cell with high efficiency.
  • Viruses useful for this approach include retroviruses, adenoviruses, pox viruses (including vaccinia), herpes virus, etc.
  • various non-viral vectors such as ligand-DNA-conjugates have been used.
  • Transient expression of transgenes has been developed also by the use of non-integrative viral vectors with low replicative efficiency. It should be noted that in order to be useful, gene therapy does not need to be completely efficacious.
  • a method that encompasses delivering to cells in a human subject suffering from a condition associated with mucus hyperproduction / hypersecretion, particularly cells in the respiratory tract, the DNA constructs of the present invention.
  • Target cells of the respiratory tract of the human subject to be treated include goblet cells and/or other mucus secreting cells, such as submucosal cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue.
  • AGR2-specific siRNA viral vectors capable of directing expression of said siRNA are particularly preferred DNA constructs for the gene therapy applications of the instant invention.
  • the expression of the siRNAs is transient.
  • the DNA construct is capable of being stably integrated into the genome of the target cells to be treated.
  • compositions and formulations described herein may principally be used in the gene therapy methods of the present invention.
  • Particularly preferred formulations for delivery of the DNA constructs of the present invention to the target cells of a human subject to be treated include the liposome or unilamellar vesicle formulations as described herein above.
  • the DNA constructs of the invention are administered intratracheal, intranasal or transmucosal, e.g. by inhalation.
  • Other suitable formulations and modes of administration will be apparent to the skilled person based on the teaching disclosed in the present invention.
  • Example 1 Co-Localization of Agr2 and PDI in the Endoplasmatic Reticulum of CaCo2 CeUs Agr2 protein co-localizes with protein disulphide isomerase (PDI) in the endoplasmatic reticulum (ER) of CaCo2 cells.
  • PDI protein disulphide isomerase
  • ER endoplasmatic reticulum
  • CaCo2 cells which endogenously express Agr2 and PDI, were grown in Dulbecco's modified eagle medium (DMEM) + 10% fetal calf serum (FCS) on glass slides. Cells were fixed and permeabilized by treatment with methanol/aceton for 20min at -20°C.
  • DMEM Dulbecco's modified eagle medium
  • FCS fetal calf serum
  • Blocking was performed with 2% bovine serum albumine (BSA)/PBS for 30min at room temperature.
  • BSA bovine serum albumine
  • Grown cells were split for parallel assays of protein detection: PDI protein was detected by immunhistochemistry, incubating cells for 1 hour at room temperature with a mouse anti-PDI antibody (cat. no. P71720, BecktonDickinson, USA), followed by a 30min incubation with a second antibody, goat anti-mouse Alexa (cat. no. Al 1017, Molecular Probes, USA).
  • Agr2 protein was detected by incubating cells for 1 hour with a rabbit anti Agr2 antiserum, followed by a 30min incubation with a second antibody, goat anti mouse Alexa (cat. no.
  • Example 2 Yeast Two-Hybrid Assay A yeast-two hybrid experiment was performed, using a
  • nucleotide sequence encoding amino acid positions 21 to 175 of murine Agr2 and a nucleotide sequence including positions 248 to 478 of the murine mucin2 mRNA, encoding 77 amino acids of murine mucin 2 were cloned into the provided vectors, as described above and according to the instructions in the user manual. Transformation was done into yeast strain AH 109, according to the user manual. Fresh transformed yeast cells were plated on medium lacking histidine. Yeast transformed with Agr2 and mucin2 expression vectors grew at medium lacking histidine, indicating Agr2 — mucin2 protein- protein interaction. Positive and negative controls were provided within the kit.
  • a tracheal ovalbumin challenge assay was performed at wild type (WT) mice and at Agr2 mutant (Agr2-/-) mice.
  • Tracheal ovalbumin challenge performed by intratracheal ovalbumine instillation over a period of 21 days, induces goblet cell differentiation from tracheal epithelial cells and glycoprotein synthesis in the goblet cells of wt mice, as seen in Figure 3B.
  • Such mice are well known as murine asthma model.
  • Control mice do not display such phenotypic alterations in the trachea after 21 days of saline installation, as shown in Figure
  • Mucins in particular mucin5ac, are major components of such glycoproteins.
  • Ovalbumin instillation (125 ⁇ gram ovalbumine; 50 ⁇ l final volume) was performed for 5 times at anesthetized mice within a period of 21 days. Mice were sacrificed at day 22 and histologically analyzed. Thin sections of trachea from treated and untreated mice were stained with rhodamine labeled wheat germ agglutinin (WGA) (cat. no. RL- 1022, Vector Laboratories, USA), binding to glycoproteins, including mucin5ac and mucin5b.
  • WGA rhodamine labeled wheat germ agglutinin
  • Sense RNA (ssRNA) and antisense RNA (asRNA) of Agr2 are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each.
  • the thus produced ssRNA and asRNA (0.5 ⁇ M) dissolved in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCl are heated to 95°C for 1 min, then cooled and annealed at room temperature for 12 to 16 h. The RNAs are precipitated and resuspended in lysis buffer (see below).
  • RNAs are electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide (Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989)).
  • Untreated rabbit reticulocyte lysate (Ambion, Inc.) is assembled according to the manufacturer's protocol. DsRNA as prepared in Example 4 is incubated with the lysate at 30°C for 10 min prior to the addition of mRNAs.
  • the molar ratio of dsRNA and mRNA is about 200:1.
  • Agr2 mRNA is radiolabeled using techniques known in the art and its stability is monitored by gel electrophoresis.
  • the dsRNA is internally radiolabeled with ⁇ - 32 P-ATP. Reactions are stopped by the addition of 2x proteinase K buffer and deproteinized as described previously (Tuschl, Zamore, Lehmann, Bartel, and Sharp 1999b). Products are analyzed by electrophoresis in 15% or 18% polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels for radioactivity, the natural production of 10 to 25 nt RNAs originating from the double stranded RNA can be determined.
  • the bands of dsRNA having about 21-23 bp, are eluted.
  • the efficacy of these 21-23 mers for suppressing Agr2 transcription is assayed in vitro using the same rabbit reticulocyte assay described above using 50 nM of the double stranded 21-23 mer RNA for each assay.
  • the sequence of these 21-23 mers is then determined using standard nucleic acid sequencing techniques.
  • RNA Preparation 21 nt RNAs based on the sequence determined in Example 2 above are chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel-purified (Elbashir, Lendeckel, and Tuschl 2001b), followed by Sep-Pak Cl 8 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, Ng, Pieken, Benseler, and Eckstein 1993).
  • RNAs (20 ⁇ M) are incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C, followed by 1 h at 37°C.
  • annealing buffer 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate
  • Cell cultures that regularly express Agr2, including, but not limited to CaCo2 cells, HT-29 cells, and LS174T cells, are propagated using standard conditions. 24 hours before transfection, at approx. 80% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3x10 5 cells/ml) and transferred into 24-well plates (500 ⁇ l/well). Transfection is performed using a commercially available lypofection kit and Agr2 expression is monitored using standard techniques with a positive and a negative control. As positive control, cells are used that naturally express Agr2, such as the cell lines mentioned above, while as negative control, cells are used that do not express Agr2.
  • siRNAs base-paired 21-23 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific mRNA degradation in lysates and in cell culture.
  • concentrations of siRNAs are used.
  • An efficient concentration for suppression in vitro in mammalian cell culture is generally found to be between 25 nM and 100 nM final concentration for the siRNAs. This indicates that siRNAs are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene targeting experiments.
  • Example 8 Construction of siRNAs Capable of Silencing Agr2 Gene Expression
  • double stranded oligonucleotides of 61 base pairs in length representing templates cloned into the vector system pSilencerTM 2.1-U6 neo (Ambion Inc.) and targeting the particular human Agr2 nucleotide sequences described in SEQ ID NO's: 3, 4, and 5 as described herein are customer synthesized and purified (Qiagen, Hilden, Germany).
  • oligonucleotides comprise a first Agr2 nucleotide to be transcribed (guanidine), a loop sequence of 9 bases, a sequence which is reverse complementary to a target sequence, six thymidine residues (serving as transcription stop signal for the RNA Polymerase III), a sequence motif GGAA, as recommended by AMBION Inc., and sequences for generating the required restriction enzyme cloning sites BamHI and HindIIL Reverse complementary oligonucleotides are annealed and double stranded oligonucleotides are subsequently cloned into pSilencerTM 2.1-U6 neo (AMBION Cat# 5764), following the manufacturer's instruction manual for Cat. #5764, 5770.
  • pSilencerTM 2.1-U6 neo AMBION Cat# 5764
  • the templates are complementary to human Agr2 nucleotide sequences described in SEQ ID NO:3 (5 ⁇ -GGACACAAAGGACTCTCGA; coding position +93 to +111; target sequence shl), SEQ ID NO:4 (5'- GCAACAAACCCTTGATGAT; coding position +203 to +221; target sequence sh4), or SEQ ID NO:5 (5'- GCTTTAAAGAAAGTGTTTG; coding position +256 to +274; target sequence sh5).
  • Double transient transfections of pSilencer constructs for targeting the Agr2 target sequences shl, sh4, or sh5 are performed into CaCo2 cells or LS174T cells, respectively. Transfected LS174T cells are used for Light CyclerTM quantitative PCR analysis (PerkinElmer Inc.), as shown in Figure 4.
  • Negative controls comprise transfection with vector constructs carrying a scrambled RNA without homology to the human Agr2 gene sequences.
  • In vivo suppression may be performed using the same siRNAs using in vivo transfection techniques well-known in the art, including the gene therapy transfection techniques described herein, as well as other techniques that will be apparent to those skilled in the art in view of the teaching of the present invention .
  • RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188- 200.
  • Emura,M. (2002). Stem cells of the respiratory tract. Paediatr. Respir. Rev. 3, 36-40.
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Abstract

Cette invention concerne notamment l'extinction génique du gène Agr2 par des ARN interférents courts (ARNic). En outre, étant donné l'implication du gène Agr2 dans la production de mucus production, cette invention concerne également des compositions pharmaceutiques utiles comme médicaments dans des procédés de prévention, d'amélioration ou de traitement d'états médicaux associés à l'hyperproduction/hypersécrétion de mucus, en particulier dans les voies respiratoires. De nouveaux ARNic utiles dans ces procédés et de nouvelles compositions pharmaceutiques sont également décrites. Cette invention concerne en outre des procédés de criblage d'ARNic additionnels capables d'éteindre l'expression du gène Agr2.
EP05816225A 2004-12-09 2005-12-08 Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus Withdrawn EP1831367A2 (fr)

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US63458704P 2004-12-09 2004-12-09
PCT/EP2005/056610 WO2006061418A2 (fr) 2004-12-09 2005-12-08 Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus

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WO2012116357A2 (fr) 2011-02-25 2012-08-30 The Board Of Trustees Of The Leland Utilisation d'agr3 pour traitement du cancer
WO2021142245A1 (fr) * 2020-01-10 2021-07-15 Translate Bio, Inc. Composés, compositions pharmaceutiques et méthodes pour moduler l'expression de la muc5b dans des cellules et des tissus pulmonaires

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AU6762298A (en) * 1997-03-19 1998-10-12 Zymogenetics Inc. Secreted polypeptides with homology to xenopus cement gland proteins
IL154129A0 (en) * 2000-07-28 2003-07-31 Compugen Inc Oligonucleotide library for detecting rna transcripts and splice variants that populate a transcriptome
GB0222787D0 (en) * 2002-10-02 2002-11-06 Univ Liverpool Metastasis inducing compounds
US20050186574A9 (en) * 2002-12-23 2005-08-25 Johannes Grosse Methods and agents for diagnosis and prevention, amelioration or treatment of goblet cell-related disorders

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Title
See references of WO2006061418A2 *

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WO2006061418A2 (fr) 2006-06-15

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