US20110313016A1 - Treatment of Intestinal Conditions - Google Patents

Treatment of Intestinal Conditions Download PDF

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US20110313016A1
US20110313016A1 US11/908,696 US90869606A US2011313016A1 US 20110313016 A1 US20110313016 A1 US 20110313016A1 US 90869606 A US90869606 A US 90869606A US 2011313016 A1 US2011313016 A1 US 2011313016A1
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sina
sirna
seq
sina molecule
disorder
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Ana I. Jiménez
Irene Gascön
Maria Concepción Jiménez
José P. Román
Angela Sesto
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Sylentis SA
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    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
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Definitions

  • the present invention relates to methods and compositions for the treatment of intestinal pathologies by means of intrarectal administration of the RNAi technology.
  • the compositions of the invention comprise short interfering nucleic acid molecules (siNA) and related compounds including, but not limited to, small-interfering RNAs (siRNA).
  • compositions of the invention can be used for the treatment of intestinal pathologies including: hyperproliferative diseases, in particular, colorectal cancer; autoimmune and inflammatory bowel diseases (IBD), in particular Crohn's disease; colitis, in particular ulcerative colitis; irritable bowel syndrome; infectious diseases of the intestine, such as pseudomembranous colitis, amebiasis or intestinal tuberculosis; colonic polyps; diverticular disease; constipation; intestinal obstruction; malabsorption syndromes; rectal diseases and diarrhoea.
  • IBD autoimmune and inflammatory bowel diseases
  • colitis in particular ulcerative colitis
  • irritable bowel syndrome infectious diseases of the intestine, such as pseudomembranous colitis, amebiasis or intestinal tuberculosis; colonic polyps; diverticular disease; constipation; intestinal obstruction; malabsorption syndromes; rectal diseases and diarrhoea.
  • intestinal conditions caused by increased levels of interleukin-12 (IL-12), a cytokine involved in type 1 helper T (Th1) cells immune response are to be treated by this approach: for example, autoimmune diseases and IBD.
  • IL-12 interleukin-12
  • Th1 type 1 helper T
  • Compositions and methods comprising siRNA and related compounds targeting IL12-p40 subunit and/or IL12-p35 subunit are provided for the treatment of diseases associated with over-expression of IL-12, in particular Crohn's disease.
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • RNAi The process of RNAi begins when the enzyme DICER encounters dsRNA and chops it into pieces called small-interfering RNAs or siRNA.
  • This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these siRNAs and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (see Bosher & Labouesse, 2000; and Akashi et al., 2001).
  • RNAi for gene knockdown
  • mammalian cells have developed various protective mechanisms against viral infections that could impede the use of this approach. Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Gil & Esteban, 2000).
  • dsRNA was reported to specifically inhibit three genes in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000).
  • Research at Ribopharma AG demonstrated the functionality of RNAi in mammalian cells, using short (20-24 base pairs) dsRNAs to switch off genes in human cells without initiating the acute-phase response. Similar experiments carried out by other research groups confirmed these results (Elbashir et al., 2001; Caplen et al., 2001).
  • RNAs short hairpin RNAs
  • siRNAs small RNAs
  • stRNAs small temporally regulated RNAs
  • RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene functions.
  • RNAi employing short dsRNA oligonucleotides will yield an understanding of the function of genes being only partially sequenced.
  • RNAs microRNAs
  • antiagomirs a class of specially engineered compounds that can effectively silence the action of microRNAs (miRNAs), non-coding pieces of RNA that regulate gene expression.
  • Interleukin-12 (1L-12) is a heterodimeric 70 kDa glycoprotein (IL12-p70) consisting of a 40 kDa subunit (designated IL12-p40) and a 35 kDa subunit (designated IL12p35) linked by disulfide bonds that are essential for the biological activity of IL-12.
  • IL-12 is a key cytokine that regulates cell-mediated immune responses and type 1 helper T (Th1) cells inflammatory reaction (Gately et al., 1998; Trinchieri, 1998).
  • Th1 type 1 helper T
  • the ability of IL-12 to strongly promote the development of Th1 cells makes it an ideal target for the treatment of Th1 cell-mediated diseases, such as autoimmune diseases and inflammatory bowel disease (IBD).
  • Th1 cell-mediated diseases such as autoimmune diseases and inflammatory bowel disease (IBD).
  • IBD Crohn's disease
  • TNF- ⁇ tumour necrosis factor ⁇
  • Crohn's disease causes inflammation in the small intestine.
  • the inflammation can cause pain and can make the intestines empty frequently, resulting in diarrhoea.
  • the most common symptoms of Crohn's disease are abdominal pain and diarrhoea, although rectal bleeding, weight loss and fever may also occur. Bleeding may be serious and persistent, leading to anaemia. Children with Crohn's disease may suffer delayed development and stunted growth.
  • mesalamine a substance that helps control inflammation.
  • Sulfasalazine is the most commonly used of these drugs.
  • Patients who do not benefit from it or who cannot tolerate it may be put on other mesalamine-containing drugs, generally known as 5-ASA agents, such as Asacol, Dipentum or Pentasa.
  • 5-ASA agents such as Asacol, Dipentum or Pentasa.
  • Possible side effects of mesalamine preparations include nausea, vomiting, diarrhoea and headache. Some patients take corticosteroids to control inflammation.
  • These drugs are the most effective for active Crohn's disease, but they can cause serious side effects, including greater susceptibility to infection. Drugs that suppress the immune system are also used to treat Crohn's disease.
  • azathioprine 6-mercaptopurine and a related drug, azathioprine.
  • Immunosuppressive agents work by blocking the immune reaction that contributes to inflammation. These drugs may cause side effects like nausea, vomiting, and diarrhoea and may lower a person's resistance to infection. Surgery to remove part of the intestine can help Crohn's disease but cannot cure it. Due to the side effects and the lack of effectiveness of the current treatments for Crohn's disease, researchers continue to look for more effective treatments.
  • Inhibiting the action of IL-12 has been shown to suppress development and clinical progression of disease in a multitude of experimental models of autoimmunity and chronic inflammation (Caspi, 1998). These models include experimental autoimmune encephalomyelitis (EAE), experimental autoimmune uveitis (EAU), collagen-induced arthritis (CIA), autoimmune nephritis, insulin-dependent diabetes mellitus (IDDM) and different models for IBD (Vandenbroeck et al., 2004). In these models, the role of endogenous IL-12 has been addressed by using IL-12p40 knockout mice or by administering anti-IL-12 antibodies.
  • EAE experimental autoimmune encephalomyelitis
  • EAU experimental autoimmune uveitis
  • CIA collagen-induced arthritis
  • IDDM insulin-dependent diabetes mellitus
  • IBD insulin-dependent diabetes mellitus
  • mice with trinitrobenzene sulfonate-induced colitis have a Th1-mediated gut inflammation characterized by greatly increased production of IL-12, interferon- ⁇ and tumour necrosis factor ⁇ (TNF- ⁇ ).
  • administration of a monoclonal antibody against IL-12 can result in the resolution of established colitis and, if given at the time of induction of colitis, can prevent inflammation (Neurath et al., 1995).
  • Anti-interleukin-12 can also prevent and treat the spontaneous colitis seen in models of Th1-mediated inflammation such as mice that over-express the human CD3e gene and mice deficient in interleukin-10 (Davidson et al., 1998; Simpson et al., 1998).
  • siRNA targeting of IL-12 expression has already been used to obtain modified dendritic cells (DC) that might be used in a variety of therapeutic in vitro, ex vivo and in vivo methods to modulate T cell activity, and thus have use in therapeutic approaches for the treatment of immune disorders in a mammalian subject (WO 03/104455; Hill et al., 2003).
  • siRNA targeting of IL-12 expression in mature DC has revealed a critical role for IL-12 in natural killer cell interferon ⁇ (IFN- ⁇ ) secretion promoted by mature DC (Borg et al., 2004).
  • IFN- ⁇ natural killer cell interferon ⁇
  • IL-12 p35 inhibitors including siRNA have surprisingly demonstrated to block differentiation of preadipocytes to adipocytes and triglyceride accumulation in adipocytes (WO 03/104495).
  • siRNA targeting IL-12p40 has successfully been delivered by means of liposome encapsulation to murine peritoneal cavity to modulate the local and systemic inflammatory response after endotoxin challenge (Flynn et al., 2004).
  • siRNA targeting IL-12p40 has successfully been delivered by means of liposome encapsulation to murine peritoneal cavity to modulate the local and systemic inflammatory response after endotoxin challenge (Flynn et al., 2004).
  • intrarectal administration of siRNA for the downregulation of IL-12 nor of any other gene involved in intestinal pathologies.
  • compositions of the invention comprise short interfering nucleic acid molecules (siNA) and related compounds including, but not limited to, siRNA.
  • siNA short interfering nucleic acid molecules
  • compositions of the invention can be used in the preparation of a medicament for the treatment of intestinal pathologies including: hyperproliferative diseases, in particular, colorectal cancer; autoimmune and inflammatory bowel diseases (1BD), in particular Crohn's disease; colitis, in particular ulcerative colitis; irritable bowel syndrome; infectious diseases of the intestine, such as pseudomembranous colitis, amebiasis or intestinal tuberculosis; colonic polyps; diverticular disease; constipation; intestinal obstruction; malabsorption syndromes; rectal diseases and diarrhoea.
  • hyperproliferative diseases in particular, colorectal cancer
  • autoimmune and inflammatory bowel diseases (1BD) in particular Crohn's disease
  • colitis in particular ulcerative colitis
  • irritable bowel syndrome infectious diseases of the intestine, such as pseudomembranous colitis, amebiasis or intestinal tuberculosis; colonic polyps; diverticular disease; constipation; intestinal obstruction; malabsorption syndromes
  • siNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • antagomirs short hairpin RNA molecules capable of mediating RNA interference.
  • shRNA short hairpin RNA
  • the methods of the invention comprise the administration to a patient in the need thereof of an effective amount of one or more siNA of the invention for the treatment of an intestinal condition.
  • the methods of the invention comprise intrarectal administration of the therapeutic siNA.
  • the present invention relates to siNA or similar chemically synthesized entities that are directed at interfering with the mRNA expression of either the p35 or the p40 subunits of the cytokine IL-12, and that ultimately modulate the amount of protein produced.
  • Compositions and methods comprising above-mentioned siRNA and related compounds are intended for the treatment of diseases associated with over-expression of IL-12, such as autoimmune diseases and inflammatory bowel diseases (IBD), in particular, Crohn's disease.
  • diseases associated with over-expression of IL-12 such as autoimmune diseases and inflammatory bowel diseases (IBD), in particular, Crohn's disease.
  • IBD inflammatory bowel diseases
  • FIG. 1 Oligonucleotide sequences for siRNA molecules targeting IL-12 p35 and p40 subunits encompassed by the present invention.
  • the SEQ ID Nos given in the Figure refer to the sense (5′->3′) strand; typically sIRNA will be administered as dsRNA, so will include both the sense strand and its complement.
  • FIG. 2 Effect of siRNA on IL-12 p35 subunit expression in an in vitro system.
  • siRNA treatment reduces the levels of IL-12 p35 gene transcript.
  • RNA was prepared from SW480 cells treated with siRNAs for different times. The samples were analyzed by RT-PCR using specific primers. The values show the mean expression levels of different transcripts normalized to 18S as housekeeping gene.
  • FIG. 3 Effect of siRNA on IL-12 p40 subunit expression in an in vitro system.
  • A siRNA treatment reduces the levels of IL-12 p40 gene transcript in human cells.
  • RNA was prepared from SW480 cells treated with siRNA SEQ ID 67 and SEQ ID 79 at different times, at dose treatment of 200 nM. The values show the mean expression levels of different transcripts normalized to 18S as housekeeping gene. The values represent the mean of the percentage of the normalized mRNA levels upon siRNA interference over the control gene expression and their medium standard deviations (SEM).
  • B siRNA treatment reduces the levels of IL-12 p40 gene transcript in murine cells.
  • RNA was prepared from C2C12 cells treated with siRNA SEQ ID 86 and SEQ ID 87 at different times, at dose treatment of 100 nM.
  • SEQ ID 86 which is homologous to human SEQ ID 67, targets the mouse IL-12 p40 subunit. Further targeting the mouse IL-12 p40 subunit, SEQ ID 87 is the siRNA with the best score in mouse, and has no homologous siRNA duplex in human.
  • siNA molecules SEQ ID 86 and SEQ ID 87 are as described below, with 2 thymidine nucleotide 3′ overhangs. The values represent the mean of the percentage of the normalized mRNA levels compared to 18S upon siRNA interference over the control gene expression and their medium standard deviations (SEM).
  • FIG. 4 siRNA treatment reduces the levels of GFP gene transcript in small intestine.
  • the collected tissue in OCT was analyzed by microscopy and measured by photoshop program.
  • Data show single dose siRNA treatment (mice 2-3) and repeated dose treatment (mice 4-5).
  • the values show the expression levels of 25 representative images per mouse referred to control untreated mouse. Standard deviation of the data is represented.
  • FIG. 5 siRNA treatment reduces the levels of GFP gene transcript in small intestine.
  • the tissue collected in RNA later was analyzed by RT-PCR. Data show single dose siRNA treatment (mice 2-3) and repeated dose treatment (mice 4-5). Standard deviation is represented.
  • FIG. 6 siRNA treatment reduces the levels of GFP gene transcript in large intestine.
  • the tissue collected in OCT was analyzed by microscopy and measured by photoshop program.
  • Data show single dose siRNA treatment (mice 2-3) and repeated dose treatment (mice 4-5).
  • the values show the expression levels of 25 representative images per mouse referred to control untreated mouse. Standard deviation of the data is represented.
  • FIG. 7 siRNA treatment reduces the levels of GFP gene transcript in large intestine.
  • FIG. 8 Data of samples collected in OCT medium.
  • FIG. 9 Data of samples collected in RNA later.
  • the present invention relates to methods and compositions for the treatment of intestinal pathologies by means of intrarectal administration of the RNAi technology.
  • the compositions of the invention comprise short interfering nucleic acid molecules (siNA) that modulate the expression of target genes associated with conditions of the intestinal wall.
  • siNA short interfering nucleic acid molecules
  • the methods of the invention comprise the administration to a patient in need thereof of an effective amount of one or more siNA of the invention.
  • a gene is “targeted” by siNA according to the invention when, for example, the siNA selectively decrease or inhibit the expression of the gene.
  • siNA target a gene when the siNA hybridize under stringent conditions to the gene transcript.
  • siNA can be tested either in vitro or in vivo for the ability to target a gene.
  • a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the sequence of the siNA of the invention.
  • the siNA is siRNA.
  • the short fragment of target gene sequence is a fragment of the target gene mRNA.
  • the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include: 1) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5′ or 3′ end of the native mRNA molecule; 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%; 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT); 4) a sequence from the target gene mRNA that is accessible in the mRNA; and 5) a sequence from the target gene mRNA that is unique to the target gene.
  • the sequence fragment from the target gene mRNA may meet one or more of the above-mentioned identified criteria.
  • the siRNA has a G/C content below 60% and/or lacks repetitive sequences.
  • the gene of interest is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides.
  • This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNA.
  • the output of this analysis is a score of possible siRNA oligonucleotides.
  • the highest scores are used to design double stranded RNA oligonucleotides (typically 21 bp long, although other lengths are also possible) that are typically made by chemical synthesis.
  • Candidate oligonucleotides can further be filtered for interspecies sequence conservation in order to facilitate the transition from animal to human clinical studies.
  • siNA molecules may be used to target homologous regions.
  • WO2005/045037 describes the design of siNA molecules to target such homologous sequences, for example by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences.
  • non-canonical base pairs for example, mismatches and/or wobble bases
  • non-canonical base pairs can be used to generate siNA molecules that target more than one gene sequence.
  • non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing targets that share sequence homology.
  • siNAs of the invention are designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between homologous genes.
  • a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target different genes.
  • Sequence identity may be calculated by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90%, 95%, or 99% sequence identity between the siNA and the portion of the target gene is preferred.
  • the complementarity between the siNA and native RNA molecule may be defined functionally by hybridisation as well as functionally by its ability to decrease or inhibit the expression of a target gene. The ability of a siNA to affect gene expression can be determined empirically either in vivo or in vitro.
  • Preferred siNA molecules of the invention are double stranded.
  • double stranded siNA molecules comprise blunt ends.
  • double stranded siNA molecules comprise overhanging nucleotides (e.g., 1-5 nucleotide overhangs, preferably 2 nucleotide overhangs).
  • the overhanging nucleotides are 3′ overhangs.
  • the overhanging nucleotides are 5′ overhangs. Any type of nucleotide can be a part of the overhang.
  • the overhanging nucleotide or nucleotides are ribonucleic acids.
  • the overhanging nucleotide or nucleotides are deoxyribonucleic acids. In a preferred embodiment, the overhanging nucleotide or nucleotides are thymidine nucleotides. In another embodiment, the overhanging nucleotide or nucleotides are modified or non-classical nucleotides. The overhanging nucleotide or nucleotides may have non-classical internucleotide bonds (e.g., other than phosphodiester bond).
  • siNA can be synthesized by any method known in the art.
  • RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • siRNA can be obtained from commercial RNA oligo synthesis suppliers, including, but not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK), Qiagen (Germany), Ambion (USA) and Invitrogen (Scotland).
  • siNA molecules of the invention can be expressed in cells by transfecting the cells with vectors containing the reverse complement siNA sequence under the control of a promoter. Once expressed, the siNA can be isolated from the cell using techniques well known in the art.
  • RNA oligo 50 ⁇ M solution 30 ⁇ l of each RNA oligo 50 ⁇ M solution are to be combined in 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate. The solution is then incubated for 1 minute at 90° C., centrifuged for 15 seconds, and incubated for 1 hour at 37° C.
  • siRNA is a short hairpin RNA (shRNA); the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non-nucleotide linker).
  • a linker region e.g., a nucleotide linker or a non-nucleotide linker.
  • the siNAs of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siNA. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules (see International Publications WO03/070744 and WO2005/045037 for an overview of types of modifications).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides (especially on the sense strand of a double stranded siRNA), 2′-deoxy-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation (see generally GB2406568).
  • modifications can be used to enhance the stability of the siRNA or to increase targeting efficiency.
  • Modifications include chemical cross linking between the two complementary strands of an siRNA, chemical modification of a 3′ or 5′ terminus of a strand of an siRNA, sugar modifications, nucleobase modifications and/or backbone modifications, 2′-fluoro modified ribonucleotides and 2′-deoxy ribonucleotides (see generally International Publication WO2004/029212).
  • modifications can be used to increase or decrease affinity for the complementary nucleotides in the target mRNA and/or in the complementary siNA strand (see generally International Publication WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deza, 7-alkyl, or 7-alkenyl purine.
  • the siNA when the siNA is a double-stranded siRNA, the 3′-terminal nucleotide overhanging nucleotides are replaced by deoxyribonucleotides (see generally Elbashir et al., 2001).
  • the cells used for the experiments were human SW480 cells and murine muscle cells C2C12. After incubation of the cells with the corresponding siRNA duplexes, the levels of p35 and p40 expression were analyzed.
  • siRNA knockdown to specific phenotypes in cultured cells, it is necessary to demonstrate the decrease of the targeted protein or at least to demonstrate the reduction of the targeted mRNA.
  • mRNA levels of the target gene can be quantitated by real time PCR(RT-PCR). Further, the protein levels can be determined in a variety of ways well known in the art, such as Western blot analysis with specific antibodies to the different target allow direct monitoring of the reduction of targeted protein.
  • siRNA are introduced into cells by means of any transfection technique well known in the art.
  • a single transfection of siRNA duplex can be performed, for instance, by using a cationic lipid, such as Lipofectamine 2000 Reagent (Invitrogen), followed by an assay of silencing efficiency 24, 48 and 72 hours after transfection.
  • a cationic lipid such as Lipofectamine 2000 Reagent (Invitrogen)
  • a typical transfection protocol can be performed as follows: for one well of a E-well plate, we transfect using 100 nM for murine C2C12 cells or 200 nM for human SW480 cells as final concentration of siRNA. Following Lipofectamine 2000 Reagent protocol, the day before transfection, we seed 2-4 ⁇ 10 5 cells per well in 3 ml of an appropriate growth medium, containing DMEM, 10% serum, antibiotics and glutamine, and incubate cells under normal growth conditions (37° C. and 5% CO 2 ). On the day of transfection, cells have to be at 30-50% confluence.
  • 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 are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful silencing. Good transfection is a non-trivial issue and needs to be carefully examined for each new cell line to be used.
  • Transfection efficiency may be tested transfecting reporter genes, for example a CMV-driven EGFP-expression plasmid (e.g. from Clontech) or a B-Gal expression plasmid, and then assessed by phase contrast and/or fluorescence microscopy the next day.
  • a knock-down phenotype may become apparent after 1 to 3 days, or even later.
  • depletion of the protein may be observed by immunofluorescence or Western blotting.
  • RNA fractions extracted from cells are pre-treated with DNase I and used for reverse transcription using a random primer.
  • PCR-amplified with a specific primer pair covering at least one exon-exon junction is used as 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 protein may exist in the cell.
  • RT-PCR amplification can be used to test in a more precise way the mRNA decrease or disappearance.
  • RT-PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly.
  • RT-PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle, in a light cycler apparatus. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template.
  • the PCR conditions were an initial step of 30 s at 95° C., followed by 40 cycles of 5 s at 95° C., 10 s at 62° C. and 15 s at 72° C.
  • Quantification of 18S mRNA was used as a housekeeping gene as a control for data normalization. Relative gene expression comparisons work best when the gene expression of the chosen endogenous/internal control is more abundant and remains constant, in proportion to total RNA, among the samples.
  • RNA target By using an invariant endogenous control as an active reference, quantitation of an mRNA target can be normalised for differences in the amount of total RNA added to each reaction.
  • the amplification curves obtained with the light cycler were analyzed in combination with the control kit DNA, which targets in vitro transcribed beta-globin DNA template, according to the manufacturer protocol.
  • a melting curve analysis was performed. The resulting melting curves allow discrimination between primer-dimers and specific PCR product.
  • GFP C57BL/6-TG mice This transgenic mouse line was bought from “The Jackson Laboratory”. Transgenic mice have been used because homozygous mice for this transgene die within the first two weeks following birth.
  • the transgenic mouse line with an “enhanced” GFP (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer makes all of the tissues, with the exception of erythrocytes and hair, appear green under excitation light.
  • This strain was generated in C57BL/6 mice.
  • the strain cDNA encoding enhanced green fluorescent protein (EGFP) was adjoined to the chicken beta actin promoter and cytomegalovirus enhancer.
  • a bovine globin polyadenylation signal was also included in the construct.
  • the EcoR1 sites included in the PCR primers were used to introduce the amplified EGFP cDNA into a pCAGGS expression vector containing the chicken beta-actin promoter and cytomegalovirus enhancer, beta-actin intron and bovine globin poly-adenylation signal.
  • the entire insert with the promoter and coding sequence was excised with Bam-HI and SalI and gel-purified.
  • siRNA duplex used for intrarectal injection in mice was purchased from Dharmacon.
  • Dharmacon Research Inc (Lafayette, Colo.) have developed a new generation of modified siRNA for in vivo use as a therapeutic, named siSTABLEv2.
  • Dharmacon's siSTABLEv2 siRNA have demonstrated an enhanced stability in serum with respect to that of non-modified siRNA.
  • Conventional siRNA are typically degraded within minutes in serum-containing environments, making in vivo use of siRNA problematic.
  • the siSTABLEv2 modification dramatically extends the siRNA stability in serum as described in Dharmacon's web page (http://www.dharmacon.com/docs/siSTABLE %20v2%20Flier.pdf).
  • the siRNA used to downregulate EGFP mRNA expression targeted the following sequence in EGFP mRNA: 5′-GGC UAC GUC CAG GAG CGC ACC-3′ (SEQ ID No 88).
  • the sense strand of the siRNA duplex was 5′-P GGC UAC GUC CAG CGC ACC-3′ (SEQ ID No 89) and the antisense strand was 5′-P U GCG CUC CUG GAC GUA GCC UU-3′ (SEQ ID No 90). This sequence is distributed by Dharmacon as pre-synthesized control siRNA green fluorescent protein duplex.
  • mice Males, 8 weeks old mice were used. The animals were kept in cages with free access to food and water until one day before the experimental protocol.
  • intrarectal therapeutic silencing mice were fasted for one day prior to the treatment.
  • the drugs are typically administered by injecting a small volume (120 ⁇ L) in the rectum.
  • Control mouse is treated with the vehicle alone.
  • animals were sacrificed two days after the first injection by cervical dislocation.
  • the protocol for the siRNA application in mouse is as follows. For each experimental administration, 60 ⁇ l siRNA duplexes were premixed with 60 ⁇ l of NaCl (1.8% w/v) up to physiological levels. In all cases animals were sacrificed two days after the first injection.
  • mice 2 and 3 were treated intrarectally with one dose of 250 ⁇ g (19 nanomols) of the siRNA vs GFP, while mice 4 and 5 were treated with two doses of 125 ⁇ g of siRNA during two consecutive days.
  • OCT blocks were storaged at ⁇ 80° C. until data processing.
  • OCT blocks were cut in slices of 12 ⁇ m by a cryostat (Leica CM 1850) at ⁇ 20° C.
  • the collected slices were analyzed on a fluorescence microscope (Olympus BX51) coupled to a digital camera (DP70), using a filter of 488 nm.
  • the sensitive conditions (ISO200), resolution image size (2040 ⁇ 1536) and time exposure (1 second) were set up for all the samples in order to be compared between them.
  • Green fluorescence was measured as an index of GFP expression by an Adobe Photoshop program (version 8.0).
  • the present invention may comprise the administration of one or more species of siNA molecule simultaneously. These species may be selected to target one or more target genes.
  • a single type of siNA is administered in the therapeutic methods of the invention.
  • a siNA of the invention is administered in combination with another siNA of the invention and/or with one or more other non-siNA therapeutic agents useful in the treatment, prevention or management of a disease condition of the intestine wall.
  • the term “in combination with” is not limited to the administration of therapeutic agents at exactly the same time, but rather it is meant that the siNAs of the invention and the other agent are administered to a patient in a sequence and within a time interval such that the benefit of the combination is greater than the benefit if they were administered otherwise.
  • each therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic effect.
  • Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route.
  • siNAs of the invention may be formulated into pharmaceutical compositions by any of the conventional techniques known in the art (see for example, Alfonso, G. et al., 1995, in: The Science and Practice of Pharmacy, Mack Publishing, Easton Pa., 19th ed.).
  • Formulations comprising one or more siNAs for use in the methods of the invention may be in numerous forms, and may depend on the various factors specific for each patient (e.g., the type and severity of disorder, type of siNA administered, age, body weight, response, and the past medical history of the patient), the number and type of siNAs in the formulation, the form of the composition (e.g., in liquid, semi-liquid or solid form), the therapeutic regime (e.g.
  • the therapeutic agent is administered over time as a slow infusion, a single bolus, once daily, several times a day or once every few days), and/or the route of administration (e.g., topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, or sublingual means).
  • route of administration e.g., topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, or sublingual means.
  • siNA molecules of the invention and formulations or compositions thereof may be administered directly or topically as is generally known in the art.
  • a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject.
  • Carriers and diluents and their salts can be present in pharmaceutically acceptable formulations.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins poly (lactic-co-glycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors.
  • nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine PEI-PEG-GAL
  • PEI-PEG-triGAL polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine
  • a siNA molecule of the invention may be complexed with membrane disruptive agents and/or a cationic lipid or helper lipid molecule.
  • Delivery systems which may be used with the invention include, for example, aqueous and non aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
  • the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
  • a pharmaceutical formulation of the invention is in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art.
  • preservatives, stabilizers, dyes and flavouring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.
  • compositions of the invention can be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles.
  • Formulations can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • colouring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavouring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • compositions of the invention can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavouring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • a sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parentally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug.
  • suppositories e.g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules of the invention can be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • siNA molecules of the invention can be expressed within cells from eukaryotic promoters.
  • Recombinant vectors capable of expressing the siNA molecules can be delivered and persist in target cells.
  • vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary.
  • the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
  • GenBank Accession numbers corresponding to IL-12 p35 (Interleukin 12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35) and p40 (Interleukin 12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) subunits are NM 000882 and NM 002187, respectively.
  • siNA molecules directed to target IL-12 p35 and p40 subunits were obtained.
  • the output of this analysis was a score of possible siNA oligonucleotides, the highest scores being used to design double stranded RNA oligonucleotides (typically 19 bp long) that were typically made by chemical synthesis.
  • siNA compositions of the invention are any of SEQ ID NOS:1-81 of FIG. 1 ; typically administered as a duplex of the sense strand and the antisense strand.
  • the invention also encompasses siNA that are 40 nucleotides or less and comprise a nucleotide sequence of any of SEQ ID NOS:1-81.
  • the siNA is 21-30 nucleotides long and comprises any one of SEQ ID NOS:1-81 of FIG. 1 . All siNA molecules used in the experiments described below were designed to have a 2 thymidine nucleotide 3′ overhang.
  • siRNA contained within FIG. 1 has been analysed in cell cultures. SiRNA with the best characteristics were selected to be tested and were applied to proper cell cultures, such as SW480. The effect of siRNA over the target gene was analyzed by RT-PCR according to the manufacturer's protocol. The gene target transcript levels were normalized using 18S as housekeeping gene. Some of the different siRNA that were tested and their different efficacies in the interference of the target gene are included in the FIG. 2 . These results correspond to SEQ ID 8 and SEQ ID 17 of FIG. 1 in SW480 cells expressing p35.
  • the values represent the mean of the percentage of the normalized mRNA levels upon siRNA interference over the control gene expression and their medium standard deviations (SEM).
  • SEM medium standard deviations
  • siRNA contained within FIG. 1 has been analyzed.
  • the siRNA with the best characteristics designed as described before, were tested in human and murine cells.
  • the p40 transcript level was analyzed by RT-PCR and normalized using 18S as housekeeping gene. These results correspond to SEQ ID 67, SEQ ID 79 in SW480 cells expressing p40 ( FIG. 3A ); and SEQ ID 86 and SEQ ID 87 in C2C12 cells expressing p40 ( FIG. 3B ).
  • siNA molecules SEQ ID 86 and SEQ ID 87 are as described in the figure, with 2 thymidine nucleotide 3′ overhangs.
  • siRNA corresponding to SEQ ID 67 in SW480, up to 65% compared to control cells.
  • siRNA corresponding to SEQ ID 86 decreased the gene expression to 61% at 48 h compared to the control. It is important to note that SEQ ID 67 and SEQ ID 86 correspond to homologous regions of human and mouse IL-12 p40 gene, respectively.
  • the siRNA application is made in order to determine the proper siRNA delivery in the intestine.
  • small intestine samples were collected in OCT medium and analyzed as previously described. Since the goal is to determine the downregulation of GFP gene transcript, levels of fluorescence were measured following siRNA application. No secondary effects were observed in the animals during the experimental protocols.
  • the first group of work (animals 2 and 3) was treated with a single dose of 250 ⁇ g of siRNA and sacrificed 48 h later.
  • the results indicate a significant decrease of fluorescence when compared with the control mouse.
  • the siRNA 250 ⁇ g
  • the siRNA is administered in two doses of 125 ⁇ g and analyzed 48 h after the first injection
  • the decrease of GFP expression was similar to that after a single application.
  • the results are shown in FIG. 4 . For each experimental condition an average of the data is represented.
  • the administered dose of 250 ⁇ g of siRNA in one or two applications was enough and sufficient to downregulate the level of GFP mRNA in small intestine, confirming the delivery of the siRNA in small intestine by intrarectal administration.
  • the level of downregulation compared to the control is higher when the analysis is done by RT-PCR, this being due to the higher sensitivity of the technique.
  • FIGS. 8 and 9 Data of samples collected in OCT medium and in RNA later are summarized in FIGS. 8 and 9 respectively.

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