EP1831367A2 - Methods and agents useful in treating conditions characterized by mucus hyperproduction/hypersecretion - Google Patents

Abstract

The present invention inter alia relates to gene silencing of the Agr2 gene by short interfering RNAs. Moreover, in view of the involvement of Agr2 in mucus production, the invention also relates to pharmaceutical compositions useful as medicaments in methods for the prevention, amelioration or treatment of medical conditions associated with mucus hyperproduction / hypersecretion, particularly in the respiratory tract. Novel siRNAs which are useful in the above methods, and novel pharmaceutical compositions are likewise provided. The invention further relates to screening methods for additional siRNAs capable of silencing the expression of the Agr2 gene.

Description

Methods and Agents Useful in Treating Conditions Characterized by Mucus Hyperproduction / Hypersecretion

BACKGROUND OF THE INVENTION

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

(Verdugo, 1990; WO 2004/056858).

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. After translation, 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.

At least 13 different mucins are known which are subdivided into secreted and membrane-bound mucins (see for Review in Dekker et al., 2002 and Moniaux et al., 2001). Mucin2, mucin5ac, mucin5b and mucinό, 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. Mucinό is expressed in gastric and duodenal mucous glands and in pancreatobiliary and endocervical epithelial cells. Secreted mucin gene expression patterns in mouse are very similar to those described in humans (Audie at al., 1993, Vandenhaute et al., 1997, Bartmann et al., 1998). 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. Within a broad spectrum of different murine and human tissue mRNAs analyzed, 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). In one Example, 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.

Known therapeutic targets linked to the pathophysiology of airway mucus hypersecretion / hyperproduction are epidermal growth factor receptor (EGFR) tyrosine kinase, calcium-activated chloride channels (CLCA), and the apoptosis inhihitor Bcl-2. Clinical trials with blockers of these targets are ongoing.

Other strategies of modulating mucus production have been proposed, e.g., by LTB4 antagonists (WO 02/55065), EGF receptor antagonists (WO 02/05842), polycationic peptides (US 6,245,320), KGF (WO 94/23032) (Farrell et al., 2002) and KGF-2 (WO 99/41282).

Despite the various strategies employed in the art as described above, there is clearly a need for novel methods and agents useful for the treatment of respiratory diseases associated with mucus hyperproduction / hypersecretion, such as asthma, allergic recations of the respiratory system, COPD and cystic fibrosis.

SUMMARY OF THE INVENTION

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.

It has been found that certain short interfering RNAs (siRNAs) that are complementary to specific 19 nucleotide long sequences of mRNA encoding human Agr2 protein are suitable for effectively suppressing Agr2 gene expression. Accordingly, in a first aspect 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.

Preferably, 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.

Pharmaceutical compositions comprising such siRNA molecules and a pharmaceutically acceptable carrier are also encompassed by the present invention.

In another embodiment, the invention 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.

In yet another embodiment, 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.

In accordance with the present invention, 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.

Further embodiments of the invention relate to the use of the 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. Thus, 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. Accordingly, 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. The siRNA molecules, the DNA constructs capable of expressing said siRNAs, as well as the pharmaceutical compositions containing said siRNAs of the instant invention may be administered to a patient using well known delivery methods as described in more detail infra. For example, administration of siRNAs of the present invention may be accomplished by a method known as gene therapy. It will thus be appreciated that 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. The above embodiments and yet further embodiments of the present invention will be explained in more detail below.

DESCRIPTION OF THE FIGURES

Figure 1 shows Agr2 protein co-localization with protein disulphide isomerase (PDI) in the endoplasmatic reticulum (ER) of CaCo2 cells. PDI is a known marker for ER localization. CaCo2 cells, which endogenously express Agr2 and PDI, were stained with anti

Agr2 antiserum plus a fluorescent labeled second antibody, or with anti-PDI antibody plus a fluorescent labeled second antibody, respectively. Cellular localization of Agr2 (see Figure IB) and PDI

(see Figure IA) was performed with fluorescence microscopic techniques.

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, whereas 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.

Figure 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. In 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. These data provide evidence for a function of Agr2 in goblet cell differentiation and glycoprotein synthesis.

Figure 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.).

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and utilities of the present invention will be apparent from the following detailed description.

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).

The term "differentiation" as used herein in connection with goblet cells 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. Thus, terminal differentiation of goblet cells means the last differentiation step to the mature goblet cell.

The term "mucus secreting cell" as used herein 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.

The term "antisense strand" as used herein, 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. For example, 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. The term "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.

The term "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. For example, 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. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to 78% or greater complementarity. For example, 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. Accordingly, 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.

The following definitions apply to any reference to nucleic acid or amino acid sequence identity described herein. The term "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. The phrases "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,

Inc., Madison Wis.). 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 (bl2seq) can be retrieved from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/blast/executables). Preferrably, 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:

Program: blastp

Matrix: BLOSUM62

Open gap penalty: 11

Extension gap penalty: 1

Gap x dropoff: 50

Expect: 10.0

Word size: 3

Low-complexity filter: on

For the purposes of the present specification, 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. Similarly, a reference to percent nucleic acid sequence identity preferably means percent identity as determined in accordance with the blastn program using the following settings:

Program: blastn

Open gap penalty: 5 Extension gap penalty: 2 Mismatch penalty: -3

Match reward: 1

Expect: 10.0

Word size: 11

Low-complexity filter: on

In view of the above, it will be understood that whenever it is referred to an Agr2 protein in the present invention, said protein 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.

The phrase "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. The term "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). Preferably, 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.

Additionally, the terms "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. The term "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.

The term "silencing" or "suppressing Agr2 gene expression" as used herein refers to 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 sequences), as measured by quantitative PCR.

As demonstrated by the present invention, 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. In a preferred embodiment of this aspect of the present invention, 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. It will be understood by those skilled in the art, that said segment of an mRNA encoding the Agr2 protein as defined above may represent or comprise non-coding sequence. Alternatively or in addition, 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.

In a preferred embodiment of this aspect of the present invention, 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.

In a particularly preferred embodiment, 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.

It will be appreciated that the 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 Cycler™ PCR (PerkinElmer Inc.). Since 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.

Another aspect of the present invention is the provision of DNA constructs that are capable of directing the expression of the siRNAs of the present invention. In a preferred embodiment of this aspect of the 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. In preferred embodiments, the preferred DNA constructs according to the present invention are capable of transiently expressing the siRNAs contemplated in the instant invention. It will thus be appreciated that the use of 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. Cells transfected with a siRNA expression vector 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.

In a specific embodiment of the above aspect of the instant 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. One example of a vector system is the GeneSuppressor™ 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. In yet another aspect 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. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety (Tuschl et al., 1999), (Sharp, 1999). When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format. The most efficient silencing is generally observed with siRNA 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.

In one embodiment, the nucleotides in the 3' overhang are ribonucleotides. In an alternative embodiment, 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.

In general, 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. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER. 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. Alternatively, 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. See Elbashir et al. 2001 EMBO J. 20(23):6877-88 (Elbashir et al., 2001b). Hence, consideration should be taken to accommodate SNPs, polymorphisms, allelic variants or species-specific variations when targeting a desired gene. A complete Agr2 siRNA experiment must include a suitable negative control. 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. In addition, 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. Alternatively, if 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.

In a typical experiment, for one well of a 24-well plate, approximately 0.84 μg 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.

Depending on the abundance and the half life (or turnover) of the targeted Agr2 polynucleotide in a cell, 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. Two days after transfection, total 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.

In another embodiment of the present invention, 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.

It will be appreciated that 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.

The 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. In such diagnostic methods of 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.

As used herein, "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. The use of such media and agents for pharmaceutically active substances is well known in the art. 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. Examples of 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); 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.

Pharmaceutical 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.

For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^™ (BASF, Parsippany, NJ, U.S.A.) or phosphate buffered saline (PBS). In all cases, 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. In many cases, it will be preferable to include 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.

For instance, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, 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.

Injectable 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. Additionally, solid forms suitable for dissolving in liquid prior to injection can be formulated. Injectable compositions are preferably aqueous isotonic solutions 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, 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. For solid compositions, 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. In some embodiments, 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. In some embodiments, 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. In other embodiments, large unilamellar vesicles (LUVs) are composed of a mixture of cationic and anionic lipids (see, e.g., Hafez IM, Ansell S, Cullis PR, 2000).

The 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.

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. Furthermore, 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.

If desired, 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.

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. When provided in tablet or capsule form, the dosage per unit may be varied according to well known techniques. For example, 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. In addition, 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.

It will be understood that all of the aforementioned methods may be useful in the present invention. In a preferred embodiment of this aspect of the invention, a method is provided 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. In some embodiments of this aspect of the invention, the expression of the siRNAs is transient. In other preferred embodiments, the DNA construct is capable of being stably integrated into the genome of the target cells to be treated.

It will be understood that all pharmaceutical 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. In a preferred embodiment of this aspect of the invention, 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.

Examples

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 is a known marker for ER localization. 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. Blocking was performed with 2% bovine serum albumine (BSA)/PBS for 30min at room temperature. 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. A21069, Molecular Probes, USA). Nucleus staining was performed with Hoechst33342 (cat. no. H3570, Molecular Probes, USA). Fluorescence staining was monitored with fluorescence microscopic technique, showing co-localization of Agr2 (Agr2, Figure IB) and PDI (PDI (ER marker), Figure IA) in the endoplasmatic reticulum of CaCo2 cells

Example 2: Yeast Two-Hybrid Assay A yeast-two hybrid experiment was performed, using a

Matchmaker Gal4 Two-hybrid-sytem 3 (cat. no. PT3247-1, Clontech, USA). Protein-protein interaction between murine Agr2 and murine mucin2 (Agr2 x mucin2 fragment) was observed in this experiment (shown in Figure 2).

Expressed Agr2 protein was fused to the DNA binding domain of Gal4, whereas expressed murine mucin2 was fused to 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 score value (+). Positive and negative controls were included.

In detail, 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.

Example 3: Tracheal Ovalbumin Challenge in Wild Type and in Agr2-/- Mice

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

3A. hi Agr2-/- mice, lacking normal Agr2 function, neither differentiated goblet cell are visable nor synthesized glycoproteins are detectable after 21 days of ovalbumin instillation, as shown in Figure 3C. These data provide evidence for

Agr2 function in goblet cell differentiation and glycoprotein synthesis. Mucins, in particular mucin5ac, are major components of such glycoproteins. Ovalbumin instillation (125μgram ovalbumine; 50μl 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.

Example 4 Production of RNAs

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 μM) 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). To monitor annealing, 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)).

Example 5: Lysate Preparation

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.

Subsequently, Agr2 mRNAs are added and the incubation is continued for an additional 60 min. The molar ratio of dsRNA and mRNA is about 200:1. The

Agr2 mRNA is radiolabeled using techniques known in the art and its stability is monitored by gel electrophoresis.

In a parallel experiment made under the same conditions, the dsRNA is internally radiolabeled with α-³²P-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.

Example 6: 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).

These single strand RNAs (20 μM) 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.

Example 7: CeU Culture

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 (1-3x10⁵ cells/ml) and transferred into 24-well plates (500 μl/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. It is observed that base-paired 21-23 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific mRNA degradation in lysates and in cell culture. In this experiment, different 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 In this experiment, double stranded oligonucleotides of 61 base pairs in length, representing templates cloned into the vector system pSilencer™ 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). These 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 pSilencer™ 2.1-U6 neo (AMBION Cat# 5764), following the manufacturer's instruction manual for Cat. #5764, 5770.

The templates are complementary to human Agr2 nucleotide sequences described in SEQ ID NO:3 (5^Λ-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 Cycler™ quantitative PCR analysis (PerkinElmer Inc.), as shown in Figure 4.

Control transfections indicate a 50% transfection efficiency. Consequently, a maximum of 50% reduction in Agr2 mRNA amount would have been achievable in quantitative PCR under these experimental conditions, see Figure 4. The % reduction values shown in Figure 4 represent % reduction observed for all cells (second row) and an effective % reduction (calculated based on an assumed transfection efficiency of 50%), see last row.

The results indicate an effective reduction of 78% (averaged from two independent transient transfection experiments) of Agr2 mRNA by targeting coding positions +93 to +111 (referred to as the shl region of human Agr2) and a

98% reduction (averaged from two independent experiments) of Agr2 mRNA by targeting coding positions +203 to +221 (sh4 region of human Agr2). Average values of reduction are calculated from independent transfections. Reduction of Agr2 mRNA by targeting target sequence sh5 is close to values obtained for control transfections (neg. Ctrl.), as indicated in Figure 4. Negative controls comprise transfection with vector constructs carrying a scrambled RNA without homology to the human Agr2 gene sequences.

It will be appreciated by those of skill in the art that the above Examples are intended to describe ways for the deduction of suitable Agr2 siRNA sequences (target sequences) besides the target sequences shl and sh4, as well as the use of such siRNAs for in vitro suppression of Agr2 gene expression.

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 .

This invention has been described in detail including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements thereon without departing from the spirit and scope of the invention as set forth in the claims. All references, patents, patent applications and Genbank references recited in this patent application are hereby incorporated by reference in their entirety.

REFERENCES

Aberger,F., Weidinger,G., Grunz,H., and Richter,K. (1998). Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2. Mech. Dev. 72, 115-130. Ahlstedt,S. and Enander,I. (1987). Immune regulation of goblet cell development. Int. Arch. Allergy Appl. Immunol. 82, 357-360.

Allen et al., (1998) Allen, A.; Hutton, D. A.; Pearson, J. P.: The MUC2 gene product: a human intestinal mucin. Int. J. Biochem. Cell Biol. 30: 797-801, 1998. Audie et al., (1993): Expression of Human Genes in Respiratory,

Digestive, and Reproductive Tracts Ascertained by In Situ Hybridization, J. Histochem. Cytochem 41(10):1479-85.

Bartmann et al., (1998) The MUC6 secretory mucin gene is expressed in a wide varitey of epithelial tissues. J. Pathol. 1998 Dec. 186(4): 398- 405.

Bhat,S.P. (2001). The ocular lens epithelium. Biosci. Rep. 21, 537- 563.

Brennan,M., Davison,P.F., and Paulus,H. (1985). Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin Gl fragments. Science 229, 81-83.

Brittan,M. and Wright,N.A. (2002). Gastrointestinal stem cells. J. Pathol. 197, 492-509.

Caron,P.C, Laird,W., Co,M.S., Avdalovic,N.M., Queen,C, and Scheinberg,D.A. (1992). Engineered humanized dimeric forms of IgG are more effective antibodies. J. Exp. Med. 176, 1191 - 1195.

Corfield,A.P., Carroll,D., Myerscough,N., and Probert,C.S. (2001). Mucins in the gastrointestinal tract in health and disease. Front Biosci. 6:D1321- 57., D1321-D1357. Cote,R.J., Morrissey,D.M., Houghton,A.N., Beattie,E.J., Jr., Oettgen,H.F., and Old,L.J. (1983). Generation of human monoclonal antibodies reactive with cellular antigens. Proc. Natl. Acad. Sci. U. S. A 80, 2026-2030.

Daniels,J.T., Dart,J.K., Tuft,SJ., and Khaw,P.T. (2001). Corneal stem cells in review. Wound. Repair Regen. 9, 483-494.

Dekker,J., RossenJ.W., Buller,H.A., and Einerhand,A.W. (2002). The MUC family: an obituary. Trends Biochem. Sci. 27, 126-131.

Deplancke,B. and Gaskins,H.R. (2001). Microbial modulation of innate defense: goblet cells and the intestinal mucus layer. Am. J. Clin. Nutr. 73, 1131S-1141S.

Einerhand,A.W., Renes,I.B., Makkink,M.K., van der,S.M., Buller,H.A., and Dekker,J. (2002). Role of mucins in inflammatory bowel disease: important lessons from experimental models. Eur. J. Gastroenterol. Hepatol. 14,

757-765. Elbashir,S.M., Lendeckel,W., and Tuschl,T. (2001a). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188- 200.

Elbashir,S.M., Martinez,J., Patkaniowska,A., Lendeckel,W., and Tuschl,T. (2001b). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877-6888.

Emura,M. (2002). Stem cells of the respiratory tract. Paediatr. Respir. Rev. 3, 36-40.

Fahy,J.V. (2001). Remodeling of the airway epithelium in asthma. Am. J. Respir. Crit Care Med. 164, S46-S51. Farrell,C.L., Rex,K.L., Chen,J.N., Bready,J.V., DiPalma,C.R.,

Kaufrnan,S.A., Rattan,A., Scully,S., and Lacey,D.L. (2002). The effects of keratinocyte growth factor in preclinical models of mucositis. Cell Prolif. 35 Suppl 1:78-85., 78-85.

Fishwild,D.M., O'Donnell,S.L., Bengoechea,T., Hudson,D.V., Harding,F., Bernhard,S.L., Jones,D., Kay,R.M., Higgins,K.M., Schramm,S.R., and Lonberg,N. (1996a). High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat. Biotechnol. 14, 845-851.

Forstner,J.F. (1978). Intestinal mucins in health and disease. Digestion 17, 234-263. Foster,C.S., Dodson,A., Karavana,V., Smith,P.H., and Ke,Y.

(2002). Prostatic stem cells. J. Pathol. 197, 551-565.

Gruber,M., Schodin,B.A., Wilson,E.R., and Kranz,D.M. (1994). Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli. J. Immunol. 152, 5368-5374. HarborthJ., Elbashir,S.M., Bechert,K., Tuschl,T., and Weber,K.

(2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557-4565.

Higgins,D.G. and Sharp,P.M. (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237-244. Higgins,DG and Sharp,PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr;5(2): 151-3.

Holliger,P., Prospero,T., and Winter,G. (1993). "Diabodies": small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. U. S. A 90, 6444-6448. Hoogenboom,H.R. and Winter,G. (1992). By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J. MoI. Biol 227, 381-388.

Hopp,T.P. and Woods,K.R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. U. S. A 78, 3824- 3828.

Huse,W.D., Sastry,L., Iverson,S.A., Kang,A.S., Alting-Mees,M., Burton,D.R., Benkovic,S.J., and Lerner,R.A. (1989). Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275-1281. Jackson,A.D. (2001). Airway goblet-cell mucus secretion. Trends Pharmacol. Sci. 22, 39-45.

Jass,J.R. and Walsh,M.D. (2001). Altered mucin expression in the gastrointestinal tract: a review. J. Cell MoI. Med. 5, 327-351. Jones,P.T., Dear,P.H., Foote,J., Neuberger,M.S., and Winter,G.

(1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522-525.

Katz,J.P., Perreault,N., Goldstein,B.G., Lee,C.S., Labosky,P.A., Yang, V. W., and Kaestner,K.H. (2002). The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129, 2619-2628.

Kohler,G. and Milstein,C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497.

Komiya,T., Tanigawa,Y., and Hirohashi,S. (1999). Cloning of the gene gob-4, which is expressed in intestinal goblet cells in mice. Biochim. Biophys. Acta 1444, 434-438.

Kostelny,S.A., Cole,M.S., and Tso,J.Y. (1992). Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 148, 1547- 1553.

Kozbor,D., Tripputi,P., Roder,J.C, and Croce,C.M. (1984). A human hybrid myeloma for production of human monoclonal antibodies. J. Immunol. 133, 3001-3005.

Kyte,J. and Doolittle,R.F. (1982a). A simple method for displaying the hydropathic character of a protein. J. MoI. Biol 157, 105-132.

Laboisse,C, Jarry,A., Branka,J.E., Merlin,D., Bou-Hanna,C, and Vallette,G. (1996). Recent aspects of the regulation of intestinal mucus secretion. Proc. Nutr. Soc. 55, 259-264.

Lonberg,N., Taylor,L.D., Harding,F.A., Trounstine,M., Higgins,K.M., Schramm,S.R., Kuo,C.C, Mashayekh,R., Wymore,K., McCabe,J.G., and . (1994). Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368, 856-859. Lonberg,N. and Huszar,D. (1995). Human antibodies from transgenic mice. Int. Rev. Immunol. 13, 65-93.

Maestrelli,P., Saetta,M., Mapρ,C.E., and Fabbri,L.M. (2001). Remodeling in response to infection and injury. Airway inflammation and hypersecretion of mucus in smoking subjects with chronic obstructive pulmonary disease. Am. J. Respir. Crit Care Med. 164, S76-S80.

Marks,J.D., Griffiths,A.D., Malmqvist,M., Clackson,T.P., Bye,J.M., and Winter,G. (1992). By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N. Y. ) 10, 779-783. Marks, J.D., Hoogenboom,H.R., Bonnert,T.P., McCafferty,J.,

Griffiths, A.D., and Winter,G. (1991a). By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. MoI. Biol 222, 581-597.

Melton,L. (2002). Does mucus hypersecretion matter in airway disease? Lancet 359, 1924. Milstein,C. and Cuello,A.C. (1983). Hybrid hybridomas and their use in immunohistochemistry. Nature 305, 537-540.

Moniaux et al., (2001) Mucin (MUC) Gene Expression in Human Pancreatic Adenocarcinoma and Chronic Pancreatitis: A Potential Role of MUC4 as a Tumor Marker of Diagnostic Significance. Clin. Cancer Res. 7(12):4033-40 Morrison,S.L. (1994). Immunology. Success in specification.

Nature 368, 812-813.

Munson,P.J. and Rodbard,D. (1980). Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 220-239. Nadel,J.A. (2001). Role of epidermal growth factor receptor activation in regulating mucin synthesis. Respir. Res. 2, 85-89.

Neuberger,M. (1996a). Generating high-avidity human Mabs in mice. Nat. Biotechnol. 14, 826. Nielsen,H., EngelbrechtJ., Brunak,S., and von Heijne,G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6.

Otto,W.R. (2002). Lung epithelial stem cells. J. Pathol. 197, 527- 535.

Podolsky,D.K. (2000). Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J. Gastroenterol. 35 Suppl 12:69-74., 69- 74.

Puchelle,E., Bajolet,O., and Abely,M. (2002). Airway mucus in cystic fibrosis. Paediatr. Respir. Rev. 3, 115.

Riechmann,L., Clark,M., Waldmann,H., and Winter,G. (1988). Reshaping human antibodies for therapy. Nature 332 , 323-327.

Schnoelzer, M., Alewood, P., Jones, A., Alewood, D., Kent, S.B. (1992). I situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40(3-4): 180- 193.

Schreiber,J., Bohnsteen,B., and Rosahl,W. (2002). Influence of mucolytic therapy on respiratory mechanics in patients with chronic obstructive pulmonary disease. Eur. J. Med. Res. 7, 98-102. Shalaby,M.R., Shepard,H.M., Presta,L., Rodrigues,M.L.,

Beverley,P.C, Feldmann,M., and Carter,P. (1992). Development of humanized bispecifϊc antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J. Exp. Med. 175, 217-225.

Shopes,B- (1992). A genetically engineered human IgG mutant with enhanced cytolytic activity. J. Immunol. 148 , 2918-2922.

Skerra, A. (2001). 'Anticalins': a new class of engineered ligand- binding proteins with antibody-like properties. J. Biotechnol. 74(4):257-275

Slomiany,B.L. and Slomiany,A. (2002). Disruption in gastric mucin synthesis by Helicobacter pylori lipopolysaccharide involves ERK and p38 mitogen-activated protein kinase participation. Biochem. Biophys. Res. Commun. 294, 220-224.

Specian,R.D. and Oliver,M.G. (1991). Functional biology of intestinal goblet cells. Am. J. Physiol 260, C 183 -C 193. Stappenbeck,T.S. and Gordon,J.I. (2000). Racl mutations produce aberrant epithelial differentiation in the developing and adult mouse small intestine. Development 127, 2629-2642.

Stevenson,G.T., Pindar,A., and Slade,CJ. (1989). A chimeric antibody with dual Fc regions (bisFabFc) prepared by manipulations at the IgG hinge. Anticancer Drug Des 3, 219-230.

Suresh,M.R., Cuello,A.C, and Milstein,C. (1986). Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays. Proc. Natl. Acad. Sci. U. S. A 83, 7989-7993.

Tatusova,T.A. and Madden, T.L. (1999). Blast 2 sequences - a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174, 247-250.

Thompson,D.A. and Weigel,R.J. (1998). hAG-2, the human homologue of the Xenopus laevis cement gland gene XAG-2, is coexpressed with estrogen receptor in breast cancer cell lines. Biochem. Biophys. Res. Commun. 251, 111-116.

Traunecker,A., Lanzavecchia,A., and Karjalainen,K. (1991). Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. EMBO J. 10, 3655-3659.

Tuschl,T., Zamore,P.D., Lehmann,R., Bartel,D.P., and Sharp,P.A. (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191-3197.

Tutt,A., Stevenson,G.T., and Glennie,MJ. (1991). Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. J. Immunol. 147, 60-69. van Den Brink,G.R., de Santa,B.P., and Roberts,D.J. (2001). Development. Epithelial cell differentiation—a Mather of choice. Science 294, 2115-2116.

Vandenhaute et al., (1997) Mucin gene expression in biliary epithelial cells. J. Hepatology 27(6): 1057-66.

Velcich,A., Yang,W., Heyer,J., Fragale,A., Nicholas,C, Viani,S., Kucherlapati,R., Lipkin,M., Yang,K., and Augenlicht,L. (2002). Colorectal cancer in mice genetically deficient in the mucin Muc2. Science 295, 1726-1729.

Verdugo,P. (1990). Goblet cells secretion and mucogenesis. Annu. Rev. Physiol 52:157-76., 157-176.

Verdugo,P. (1991). Mucin exocytosis. Am. Rev. Respir. Dis. 144, S33-S37.

Verhoeyen,M., Milstein,C, and Winter,G. (1988a). Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534-1536. Vitetta,E.S., Krolick,K.A., Miyama-Inaba,M., Cushley,W., and

Uhr,J.W. (1983). Immunotoxins: a new approach to cancer therapy. Science 219, 644-650.

Voynow,J. (2002). What does mucin have to do with lung disease? Paediatr. Respir. Rev. 3, 98. Watanabe,H. (2002). Significance of mucin on the ocular surface.

Cornea 21, Sl 7-S22.

Weiss, G. A., Lowman, H.B. (2001). Anticalins versus antibodies: made-to-order binding proteins for small molecules. Chem.Biol. 7(8), Rl 77-184

Wolff,E.A., Schreiber,G.J., Cosand,W.L., and Raff,H.V. (1993). Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. Cancer Res. 53, 2560-2565.

Yang,Q., Bermingham,N.A., Finegold,MJ., and Zoghbi,H.Y. (2001). Requirement of Mathl for secretory cell lineage commitment in the mouse intestine. Science 294, 2155-2158.

Claims

WE CLAIM:

1. A short interfering RNA (siRNA) comprising 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

(a) the human Agr2 protein according to SEQ ID NO: 1 ; or

(b) 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

2. The siRNA of claim 1, 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 a protein according to any one of claims 1 to 2, said segment representing or comprising a non-coding segment of said mRNA.

3. The siRNA of claims 1 or 2. 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 a protein according to any one of claims 1 to 2, said segment representing or comprising a coding segment of said mRNA.

4. The siRNA of any one of claims 1 to 3, wherein said segment includes sequences from the 5' untranslated (UT) region, the open reading frame (ORF), or the 3' UT region of said mRNA.

5. The siRNA of any one of claims 1 to 4, wherein one strand is substantially or perfectly complementary to a 19 nucleotide long segment of an mRNA encoding a protein according to claim 1, said segment encoding the shl region of the human Agr2 gene according to SEQ ID NO:3, or the sh4 region of the human Agr2 gene according to SEQ ID NO:4, respectively.

6. The siRNA of any one of claims 1 to 5, wherein said siRNA is capable of silencing or suppressing the expression of the Agr2 gene.

7. The siRNA of any one of claims 1 to 6, wherein the siRNA is capable of reducing the amount of mRNA encoding the Agr2 protein as defined in claim 1 by at least 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% when compared to the amount of mRNA encoding the Agr2 protein found in the absence of said siRNA, as measured by quantitative PCR.

8. A DNA construct capable of directing expression of the siRNA according to any one of claims 1 to 7.

9. The DNA construct according to claim 8, wherein the DNA construct is a viral vector.

10. The DNA construct according to claims 8 or 9, wherein said expression is transient.

11. The DNA construct according to any one of claims 8 to 10, wherein the DNA construct is capable of being stably integrated into the genome of cells transfected with said DNA construct.

12. A host cell transformed with an siRNA according to any one of claims 1 to 7, or the DNA construct according to any one of claims 8 to 11.

13. The host cell according to claim 12, wherein said host cell is a eukaryotic cell.

14. The host cell according to claim 12, wherein said host cell is a prokaryotic cell.

15. A pharmaceutical composition comprising an siRNA according to any one of claims 1 to 7 or the DNA construct according to any one of claims 8 to 11, and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15, wherein the composition is in the form of a liposome delivery system.

17. The pharmaceutical composition of claim 16, wherein the liposomes are selected from small unilamellar vesicles, large unilamellar vesicles, preferably large unilamellar vesicles composed of a mixture of cationic and anionic lipids, and multilamellar vesicles. 18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the composition is suitable for delivery as an aerosol.

19. The pharmaceutical composition according to any of claims 15 to 18, wherein the pharmaceutical composition is suitable for intratracheal, intranasal or transmucosal administration.

20. The siRNA according to any one of claims 1 to 7, the DNA construct according to any one of claims 8 to 11, or the pharmaceutical composition according to any one of claims 15 to 19 for use as a medicament.

21. A method of identifying siRNA sequences capable of silencing or suppressing the expression of the Agr2 gene, the method comprising a) transfecting cells that express Agr2 with a sample dsRNA; b) determining whether the amount of Agr2 mRNA is reduced in the presence of the transiently expressed sample dsRNA.

22. The method according to claim 21, wherein the expression of Agr2 is determined via quantitative PCR analysis using Agr2 gene-specific primers.

23. A method of identifying an siRNA useful in the prevention, amelioration, or treatment of a medical condition associated with mucus hyperproduction / hypersecretion in the respiratory tract, the method comprising a) culturing mammalian cells expressing Agr2 protein in the presence or absence of a candidate siRNA; and b) determining whether the presence of the siRNA results in a decrease in the production by the cells of mucus and/or one or more particular mucus constituents;

24. The method according to any one of claims 21 to 23, wherein said mammalian cells are CaCo2, LS174T or HT29 cells.

25. The method according to claim 24, wherein the mucus constituent is mucin2, mucin5 ac, mucin5 b, or a trefoil peptide. 26. An siRNA identified or identifiable by a method according to any one of claims 21 to

25.

27. Use of an siRNA according to claim 26 for the prevention, amelioration, or treatment of a medical condition associated with mucus hyperproduction / hypersecretion in the respiratory tract.

28. A method of preparing a pharmaceutical composition useful in the treatment of a medical condition associated with mucus hyperproduction / hypersecretion, said method comprising

(a) identifying by the method of any of claims 21 to 25 an siRNA capable of suppressing the expression of the human Agr2 gene; and

(b) formulating the siRNA thus identified into a pharmaceutical composition.

29. The method according to claim 28, wherein the medical condition is selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis.

30. A method of gene therapy comprising delivering to cells in a human subject suffering from a condition associated with mucus hyperproduction / hypersecretion a DNA construct as defined in any of claims 8 to 11, or the pharmaceutical composition according to any one of claims 15 to 19.

31. The method of claim 30, wherein said cells are cells of the respiratory tract of said human subject, preferably goblet cells and/or mucus secreting cells of the trachea, bronchi, nose, larynx, pharynx, and salivary glands of the tongue.

32. The method according to claims 30 or 31 , wherein the DNA construct is a viral vector.

33. The method according to any one of claims 30 to 32, wherein said DNA construct is capable of directing expression of said siRNA.

34. The method of claim 33, wherein said expression is transient. 35. The method of any one of claims 30 to 34, wherein the DNA construct is capable of being stably integrated into the genome of said cells.

36. A method of preventing, treating, or ameliorating a medical condition in a human subject associated with mucus hyperproduction / hypersecretion, said method comprising administering to said human subject the siRNA according to any one of claims 1 to 7, the DNA construct of any one of claims 8 to 11, or the pharmaceutical composition according to any one of claims 15 to 19.

37. The method according to claim 36, wherein said medical condition is selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis.

38. The method according to any one of claims 30 to 37, wherein the delivery or administration is intratracheal, intranasal, or transmucosal.

39. Use of the siRNA according to any one of claims 1 to 7, the DNA construct of any one of claims 8 to 11, or the pharmaceutical composition according to any one of claims 15 to 19 for the preparation of a pharmaceutical for preventing, treating, or ameliorating a medical condition in a human subject associated with mucus hyperproduction / hypersecretion.

40. The use according to claim 39, wherein said medical condition is restricted to the respiratory tract of a human subject.

41. The use according to claims 39 or 40, wherein said medical condition is selected from the group consisting of allergic reactions of the respiratory tract, asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis.