EP1778841A2 - Arn interférant spécifique vis-à-vis de la gastrine - Google Patents

Arn interférant spécifique vis-à-vis de la gastrine

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Publication number
EP1778841A2
EP1778841A2 EP05775595A EP05775595A EP1778841A2 EP 1778841 A2 EP1778841 A2 EP 1778841A2 EP 05775595 A EP05775595 A EP 05775595A EP 05775595 A EP05775595 A EP 05775595A EP 1778841 A2 EP1778841 A2 EP 1778841A2
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EP
European Patent Office
Prior art keywords
gastrin
rnai
gene
ribonucleic acid
gastrin gene
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EP05775595A
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German (de)
English (en)
Inventor
Susan A. Watson
Anna Grabowska
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Receptor Biologix Inc
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Receptor Biologix Inc
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Publication date
Application filed by Receptor Biologix Inc filed Critical Receptor Biologix Inc
Publication of EP1778841A2 publication Critical patent/EP1778841A2/fr
Withdrawn legal-status Critical Current

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/595—Gastrins; Cholecystokinins [CCK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to short ribonucleic acid polymers that inhibit expression of the gene encoding the hormone, gastrin, hi particular, the invention relates to inhibition of gastrin gene expression by short ribonucleic acid polymer molecules targeting gastrin messenger RNA (rnENA) to potentiate degradation and, in some cases, block translation so that expression is diminished or completely prevented.
  • rnENA gastrin messenger RNA
  • RNAi interfering RNA
  • small interfering RNAs small interfering RNAs
  • microRNAs small interfering RNAs
  • siRNAs are double-stranded RNA molecules generally consisting of paired ribonucleotide chains of about 22 nucleotides in length. Ruvkin, G. (2001) Glimpses of a Tiny RNA World. Science 294: 797-799. 27-mers have recently shown to be effective, for some targets. (Kim et al (2005) Nature Biotechnology_2:222).
  • siRNAs were first identified in plants as molecules responsible for the phenomenon of co-suppression, now known to be a manifestation of post-transcriptional gene silencing. siRNAs have also been found in the fungus, Neurospora, shown to be responsible for the phenomenon of gene "quelling” and RNA interference in the fruit fly, Drosophila. For a review, see Calpen & Mousses (2003) JV.7. Acad. ScI 1002: 56-62.
  • RNAse Ill-like enzyme having so-called "Dicer” activity was confirmed in each of these instances of gene silencing by siRNA. Dicer enzymatically cleaved double-stranded RNA yielding siRNAs with component RNA strands of less than 30 base pairs.
  • the siRNAs in turn were shown to act at a ribonucleic acid-protein complex called the RNA-induced silencing complex, or RISC, mediating specific targeting of mRNAs carrying the target sequence. Targeting is achieved by alignment of the mRNA with the antisense strand component of the siRNA in the RISC. Subsequently, the target mRNA is cleaved at a site within the target sequence, rendering it susceptible to rapid degradation by other cellular ribonucleases. See Denli and Harmon (2003) RNAi: An ever growing puzzle. Trends Biochem. Sci. 28: 196.
  • MicroRNAs are a second class of RNAi molecules distinct from siRNAs, hundreds of which have been identified in a diverse group of organisms from fruit flies, to worms and from plants to humans. See for example, Ambros, V. (2003) MicroRNA pathways in flies and worms. Cell 113: 673-676.
  • MicroRNAs are 21-23 nucleotide single- stranded RNA molecules that are processed from larger double-stranded molecules, usually in the form of stem-loop structures.
  • the double-stranded miRNA precursors are transcribed from non-protein-coding genes, forming self-complementary fold-back structures processed by "Dicer" enzyme.
  • MicroRNAs are critical for normal cellular differentiation and for development of multicellular structures. See Carrington and Ambros (2003) Role of microRNAs in Plant and Animal Development. Science 301:336-338.
  • miRNAs are endogenous regulatory molecules that bind a target mRNA by hybridizing at an imperfectly matched sequence, preventing its translation. This gene-specific blockage of protein/peptide synthesis is in contrast to the degradation of the targeted niRNAs caused by siRNA molecules. This latter function appears to be dependent on a perfect or near-perfect matching hybridizing sequence of the anti-sense strand of the siRNA and the target sequence of the mRNA.
  • MicroRNAs by contrast are imperfectly matched to the target mRNA, hybridizing with the mRNA sequence in short stretches, without providing a sufficient length of double-stranded sequence for RNA degradation by RISC.
  • siRNAs targeted to different sites in the ICAM-I mRNA have been demonstrated over a wide range of concentrations, and siRNAs have shown to be effective at nanomolar concentrations, a thousand-fold lower than the minimum concentration necessary for inhibition by antisense RNAs: Kretschmer-Kazemi & Sczakiel (2003) The Activity of siRNA in Mammalian Cells is Related to Structural Target Accessibility: A Comparison with Antisense Nucleotides, Nucl. Acids Res. 3_i(15): 4417-4424.
  • NPM-ALK kinase protein Long protein half-life and persistence of function even after complete ablation of de . novo gene expression has been shown for the tumor-specific marker, NPM-ALK kinase protein. This protein is constitutively activated in anaplastic large cell lymphomas (ALCL), triggering malignant transformation.
  • ACL anaplastic large cell lymphomas
  • RNA molecules have been shown to knock-down gene expression of the cancer related proteins, clusterin, IGFBP-2, IGBFBP-5, Mitf, and B-raf (See WO 2004/018676); growth factors such as VEGF, and other genes whose expression stimulates growth of tumors.
  • growth factors such as VEGF
  • VEGF growth factors
  • other genes whose expression stimulates growth of tumors. See for example, Yin et al. SiRNA agents inhibit oncogene expression and attenuate human tumor cell growth (2003) J. Exp. Therapeut. Oncol. 3_: 194- 204.
  • HeLa cells, lung adenocarcinoma cells, and melanoma cells were successfully transfected with siRNA-loaded cationic lipid complexes.
  • RNAs have been used in studies of a wide variety of cellular functions targeted for therapeutic intervention, including p53, TNF-alpha, bcl-2, and caspases in cell cycle regulation and apoptosis, growth factors and their regulation, protein kinases and signaling factors, RNA stability, DNA repair and tumor reversion. See for instance, Lu et al. (2003) siRNA-mediated anti-tumorigenesis for drug target validation and therapeutics, Curr. Opin. MoI. Therapeutics 5(3): 225-234; and Zender & Kubicka (2004) SiRNA based strategies for inhibition of apoptotic pathways in v/vo-analytical and therapeutic implications, Apoptosis 9: 51-54.
  • siRNA was delivered into specific organs in vivo
  • expression of enhanced green fluorescent protein (eGFP) from a recombinant vector injected into the striatal region of mouse brain has been shown to be decreased by eGFP targeted siRNA.
  • eGFP targeted siRNA an siRNA targeted to beta-glucuronidase in mouse liver was shown to cause a significant reduction in expression of the targeted gene.
  • the present invention provides a gastrin gene-specific interfering ribonucleic acid (RNAi) molecule comprising a ribonucleotide chain of up to about 90 nucleotides in length including a targeting sequence of about 19 to 21 nucleotides, alternatively about 19 to 24, alternatively of about 19 to 27, alternatively of about 19 to 29 nucleotides of sufficient complementarity to a stretch of the coding sequence of the gastrin gene to bind gastrin mRNA (mRNA).
  • RNAi gastrin gene-specific interfering ribonucleic acid
  • the invention also provides a double-stranded small interfering RNA (siRNA) molecule that inhibits expression of the gastrin hormone gene by RNA interference.
  • siRNA small interfering RNA
  • the siRNA of the invention specifically interferes with gastrin gene expression and targets gastrin mRNA by a targeting sequence corresponding to the anti-sense strand sequence complementary to a short stretch of from about 19 to about 21 nucleotides, alternatively about 19 to about 24 nucleotides, alternatively about 19 to about 27 nucleotides of the gastrin gene.
  • the invention provides a gastrin gene-specific micro-ribonucleic acid (miRNA) molecule comprising a ribonucleotide chain of up to about 90 nucleotides in length including a targeting sequence of about 19 to about 21 nucleotides, alternatively about 19 to about 24 nucleotides, which can be up to about 27 nucleotides of that is partially complementary to a stretch of the coding sequence of the gastrin gene and binds gastrin mRNA (mRNA).
  • miRNA micro-ribonucleic acid
  • the invention further provides a pharmaceutical composition that includes a gastrin gene-specific interfering ribonucleic acid (RNAi) molecule including a ribonucleotide chain of up to about 90 nucleotides in length including a sequence of about 19-21 nucleotides, alternatively about 19-24 nucleotides, alternatively about 19-27 nucleotides, alternatively about 19-29 nucleotides of sufficient complementarity to a stretch of the coding sequence of the gastrin gene to bind gastrin mRNA (mRNA), and a physiologically acceptable carrier.
  • RNAi gastrin gene-specific interfering ribonucleic acid
  • the invention provides a method of treating a patient suffering from a gastrin-promoted disease or condition in a patient, wherein the method includes administering to the patient an effective amount of the gastrin gene-specific interfering ribonucleic acid (RNAi) molecule including a ribonucleotide chain of up to about 90 nucleotides in length including a sequence of about 19-21 nucleotides, alternatively about 19-24 nucleotides, alternatively about 19-27 nucleotides, alternatively about 19-29 nucleotides of sufficient complementarity to a stretch of the coding sequence of the gastrin gene to bind gastrin mRNA (mRNA).
  • RNAi gastrin gene-specific interfering ribonucleic acid
  • the present invention also provides a method of reducing gastrin gene expression in a cell, the method includes treating the cell with a gastrin-specific interfering ribonucleic acid (RNAi) molecule including a ribonucleotide chain of up to about 90 nucleotides in length including a sequence of about 19-21 nucleotides, alternatively about 19-24 nucleotides, alternatively about 19-27 nucleotides, alternatively about 19-29 nucleotides of sufficient complementarity to a stretch of the coding sequence of the gastrin gene to bind gastrin mRNA (mRNA).
  • RNAi gastrin-specific interfering ribonucleic acid
  • RNAi gastrin-specific interfering ribonucleic acid
  • RNAi gastrin-specific interfering ribonucleic acid
  • mRNA gastrin mRNA
  • the invention provides a host cell carrying a nucleic acid vector expressing a gastrin-specific interfering ribonucleic acid (RNAi) molecule including a ribonucleotide chain of up to about 90 nucleotides in length including a sequence of about 19-21 nucleotides, alternatively about 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotides of sufficient complementarity to a stretch of the coding sequence of the gastrin gene to bind gastrin mRNA (mRNA).
  • RNAi gastrin- specific interfering ribonucleic acid
  • EGF Gene expression in PAN-I cells on day 2 after transfection with gastrin (tg8) or control (scrtg ⁇ ) siRNA and 24hrs treatment with or without lO ⁇ g/ml EGF. Data are expressed relative to the housekeeping gene, HPRT. Error bars indicate the 95% confidence intervals. Significant differences (p ⁇ 0.001) are indicated by *.
  • FIG 10 Inhibition of GFP-tagged gastrin protein expression by gastrin siRNA.
  • HCTl 16 cells were transfected with a plasmid expressing GFP-tagged gastrin together with the control scrtg8 siRNA (a) or gastrin tg8 siRNA (b). The percentage of positive cells is indicated. There was a significant difference between the two treatment (p ⁇ 0.0001)
  • FIG. 11 Inhibition of growth by gastrin siRNA. Growth of PAN-I, C170HM2, HCTl 16, and MGLVAl cells in serum-free medium (a) with or (b) without the addition of lO ⁇ g/ml EGF following transfection with gastrin (tg8) or control (scrtg ⁇ ) siRNAs. Growth of tg8- transfected cells is shown as a percentage of scrtg ⁇ -treated cells. Significant differences
  • FIG. 13 Interference with the OE 19 autocrine gastrin pathway using anti-gastrin (Tg8) siRNA.
  • Figure 14 HB-EGF gene expression in STl 6, AGS or MGLVAl cells treated with the gastrin siRNA (target 8) or control siRNA (scrambled target 8)
  • FIG. 15 HB-EGF gene expression in STl 6, AGS or MGLVAl cells treated with the control siRNA alone (scrambled target 8); the control siRNA (scrambled target) and
  • H.pylori strain 60190 or gastrin siRNA (target 8) and H.pylori strain 60190.
  • Figure 16 HB-EGF ectodomain shedding in response to H. pylori strain 60190 in the presence or absence of the gastrin siRNA (target 8) or the control siRNA (scrambled target
  • FIG 17 XIAP gene expression in AGS cells treated with the gastrin siRNA (target 8) or the control siRNA (scrambled target 8)
  • Figure 18 XIAP gene expression in AGS cells treated with the gastrin siRNA (target 8) or the control siRNA (scrambled target 8) and then exposed to H. pylori strain 60190 for 24hrs
  • Figure 19 XIAP protein expression in AGS cells treated with H. pylori strain 60190 in the presence or absence of the gastrin siRNA (target 8) or the control siRNA (scrambled target
  • FIG. 21 Vector (pSilencer 2.1-U6 hygro, Ambion) used to express gastrin and control siRNAs as small hairpin RNAs. Insert structure and resulting small hairpin RNA are also illustrated. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides an interfering RNA (RNAi) molecule that includes a ribonucleotide chain of no more than about 90 nucleotides, wherein a stretch of about 19 to 21 nucleotides, alternatively 19 to about 24 nucleotides, alternatively about 19 to 27 nucleotides, alternatively about 19-29 nucleotides is sufficiently complementary to a sequence within the sense-strand of the gastrin gene such that the sequence of about 19-21 nucleotides, alternatively about 19-24 nucleotides, alternatively about 19-27 nucleotides, alternatively about 19-29 nucleotides binds gastrin mRNA.
  • RNAi interfering RNA
  • This stretch of about 19 to 21 nucleotides, alternatively about 19 to about 24 nucleotides, alternatively about 19-27 nucleotides, alternatively aboutl9-29 nucleotides is a gastrin gene targeting sequence that directs the RNAi molecule to bind gastrin mRNA.
  • the gastrin gene targeting sequence may be complementary to any sequence of about about 19 to 21 nucleotides, alternatively about 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotides within the sequence transcribed from the gastrin gene.
  • the gastrin gene targeting sequence is between about 20 to about 21 nucleotides long. More preferably, the gastrin gene targeting sequence is 21 or 22 nucleotides long. More preferably, the gastrin gene targeting sequence is up to 29 nucleotides long.
  • the gastrin gene-specific interfering RNA targeting sequences of the invention preferably have no more than about 80 percent complementarity with any other gene, especially any other human gene, i.e. when aligned base for base, considering for example, a gastrin targeting sequence of 20 nucleotides in length, no more than about 16 of nucleotides are complementary to a any other gene, especially any other human gene. More preferably, no more than 70 percent of the nucleotides of the gastrin gene-specific interfering RNA targeting sequences are complementary to any other gene, especially any other human gene.
  • the targeting sequence preferably has a GC content of from about 30 and about 60 percent, has no more than about four consecutive adenosine bases.
  • the gastrin-specific interfering RNA (RNAi) molecules of the present invention comprises a chain of ribonucleotides, i.e. a 3 '-5' phosphodiester linked pentose sugar- phosphate backbone, preferably, each sugar being substituted at the 1 '-position with a nucleotide base.
  • the ribonucleotide chain may include one or more linkages which are not phosphate diester linkages, such as for instance linkages containing a sulfur atom, a nitrogen atom or a carbon atom to prevent in vivo nuclease cleavage and degradation.
  • the nucleotide base is adenine, guanine, cytidine or uracil.
  • the sugar may be any pentose sugar, but is preferably D-ribose.
  • the ribonucleotide chain of the RNAi molecules of the present invention can include one or more modified nucleotide bases, preferably a modified purine base or a modified pyrimidine base.
  • the modified purine base may be any modified purine base, for example a hypoxanthine (inosine in the ribonucleotide chain), or a modified purine base, for instance a methylated adenine, a substituted guanine base, or the like.
  • the modified pyrimidine base may be any modified pyrimidine base, such as a modified cytidine or uracil, such as 5-methyl cytosine or the like.
  • the RNAi molecule of the invention includes a gastrin gene targeting sequence no more than about 19- 21 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotides that is perfectly complementary to a sequence within the sense-strand of the gastrin gene.
  • every one of the ribonucleotide bases of the gastrin gene targeting sequence of this embodiment of the gastrin-specific RNAi is paired in a GC or a CG pair, or in an AU or an UA base pair with the complementary base in the gastrin mRNA.
  • RNAi molecule of the present invention one or more mismatches in the approximately 19-21 nucleotides, alternatively 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotide gastrin gene targeting sequence are tolerated provided that the sequence within the gastrin gene targeting sequence that is perfectly complementary to the sense-strand of the gastrin gene is sufficient to form a stable double stranded RNA with gastrin mRNA.
  • melting temperature profile is routine and well within the ordinary skill in the art. See for example, Sambrook & Russell (2000) Molecular Cloning, A Laboratory Manual. 3d edn.
  • the gastrin gene targeting sequence of the RNAi of the invention may include one, two, three or four mismatched bases may be included in the gastrin gene targeting sequence, leaving about 18 to about 21, about 17 to about 24, or about 15 to about 27 nucleotides, or about 16 to about 29 nucleotides perfectly matched with the sense-strand sequence.
  • the RNAi molecule of the present invention may include no more than four bases in the approximately 19-21 nucleotides, alternatively 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotide gastrin gene targeting sequence that are mismatched with the targeted sequence from within gastrin gene.
  • the maximum permissible number of mismatches between the gastrin gene-specific targeting sequence of the RNAi of the invention and the gastrin gene that still allows hybridization can be determined by routine methods in the known to those of skill in the art. See for example Urakawa et al. (2003) Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays. J Appl. Envir. Microbiol. 69(5): 2848-2856.
  • the ribonucleotide chain of no more than about 90 nucleotides of the gastrin gene-specific RNAi molecule of the invention includes a second nucleotide sequence of between about 19 and about 21 nucleotides, alternatively about 19 and about 24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotides having sufficient complementarity to bind the approximately 19 to approximately 21 nucleotides, alternatively 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotide gastrin gene targeting sequence.
  • this complementarity is complete sequence complementarity such that every base of the approximately 19 to approximately 21 nucleotides, alternatively 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotide gastrin gene targeting sequence is paired with the second nucleotide sequence in a GC or an AU base pair.
  • the second nucleotide sequence may include one, two, three or more bases mismatched with the opposing base in the gastrin gene targeting sequence in the double stranded gastrin gene-specific interfering ribonucleic acid molecule.
  • the second nucleotide sequence includes no more than about 9 mismatched bases in the gastrin gene targeting sequence. More preferably, no more than about 6 mismatched bases and most preferably, no more than about 3 mismatched bases are included in the gastrin gene targeting sequence.
  • gastrin-specific interfering RNA of the invention may be chemically synthesized in vitro or expressed from a recombinant clone and annealed to form the double-stranded RNAi molecule.
  • the gastrin-specific interfering RNA of the invention may be synthesized or expressed as a single strand with the complementary sequences connected by a loop sequence. The complementary sequences can then be folded back and annealed under appropriate conditions to form a single molecular siRNA species known as a double- stranded "hairpin siRNA" molecule.
  • the two complementary strands of the small interfering RNA can be linked by an oligonucleotide linker, or by a non-nucleotide linker.
  • Suitable non-nucleotide linkers for instance short inorganic polymer linkers, peptide linkers, such as polyglycine or polylysine linkers and the like are well known in the art and are readily adapted to in vitro RNAi synthesis by chemical and or enzymatic methods.
  • the gastrin-specific interfering RNA of the invention comprises two separate ribonucleotide chains forming a double-stranded RNAi molecule.
  • the ribonucleotide chain that includes the gastrin anti-sense targeting sequence and the second ribonucleotide chain are fully complementary along the full length of the approximately 19-21 nucleotides, alternatively 19-24 nucleotides, alternatively 19-27 nucleotides, alternatively 19-29 nucleotide gastrin gene-specific targeting sequence.
  • the double-stranded RNAi molecule can also include an unpaired "overhang" ribonucleotide sequences at one or more of the four ribonucleotide chain ends.
  • the overhang at any particular end of the ribonucleotide chain can be of any length such that the total number of ribonucleotides in the molecule does not exceed about 90 in all.
  • the gastrin-specific double-stranded RNAi molecule of the invention includes an overhang sequence of ribonucleotides at the 3' end of one or both of the ribonucleotide chains or alternatively comprises a blunt-end sequence.
  • the gastrin-specific double-stranded RNAi molecule of the invention includes a overhang of one, two or three ribonucleotides at the 3' end of one or both of the ribonucleotide chains.
  • the gastrin-specific double- stranded RNAi molecule of the invention includes an overhang of two bases at the 3' end of both of the ribonucleotide chains and no overhang at the 5' ends of the chains.
  • Embodiments of the double-stranded RNAi molecule of the invention that include unpaired ribonucleotide overhangs at all four ends of the two chains are also contemplated within the scope of the present invention.
  • Such unpaired ribonucleotide overhangs form "ragged ends" of the RNAi molecule which may be cleaved by ribonucleases in vivo to form the active RNAi species.
  • the gastrin-specific RNAi of the invention can be expressed as a single stranded ribonucleotide structure or as a hairpin ribonucleotide structure from an inducible recombinant gene.
  • the recombinant gene can be expressed from a DNA vector such as a DNA plasmid, or a DNA virus vector, or alternatively, the recombinant gene can be expressed from an RNA viral vector such as a retrovirus vector or the like.
  • the knock-down of target gene expression can then be achieved by the single step of inducing the expression of the recombinant RNAi gene. This targeted-knock-down can be reversed by shutting down the expression of the RNAi and thereby permitting the gastrin gene to be expressed normally.
  • RNA interference with gene expression is manifested at the level of protein synthesis as a reduction in the expression of the protein or peptide product of the target gene.
  • This down-regulation of gene expression of the target gene by an RNAi molecule exhibits stringent sequence specificity and is referred to as "knock-down" of the expression of the target gene, in contrast to the "down-regulation” mediated by anti-sense RNA molecules and the "knock-out” seen after deletion of the target gene.
  • the knock-down of gastrin gene expression may be partial inhibition of expression of gastrin gene products, or may be complete ablation of expression of gastrin gene products, referred to as "silencing" of the gastrin gene.
  • Gastrin-specific gene products include all gastrin-specific peptide translation products, for instance, gastrin mRNA transcripts and the peptide products of translation of gastrin mRNA.
  • the peptide products of gastrin mRNA translation include for instance, preprogastrin (the primary translation product including a leader sequence for transmembrane transport of the peptide); progastrin (the gastrin precursor pro-peptide which is produced from preprogastrin by cleavage of the leader sequence and is matured to one or more of the gastrin hormone forms by specific proteolytic cleavage); the gastrin hormone forms: gastrin 17, and gastrin 34 named according to the number of amino acids in the peptide chains; and glycine-extended gastrin 17 and glycine-extended gastrin 34, having a glycine residue extension to the C-terminal of gastrin 17 or gastrin 34, respectively; modified forms such as sulfated forms of each of the above gastrin hormone forms;
  • gastrin hormone forms exhibit differing biological activities. For instance, G17-Gly and G34-Gly are implicated in stimulation of growth and proliferation of gastrin-promoted neoplasms through activating a receptor that specifically binds the glycine-extended C-terminal sequences of these gastrin hormone forms.
  • Complete knock-down of gastrin gene expression can be achieved by administering an effective amount of a gastrin-specific interfering RNA of the invention selected for the ability to cause complete blockage of gastrin hormone synthesis, and which may be accompanied by targeted degradation of gastrin mRNA as explained below.
  • the siRNAs of the present invention are useful as therapeutic agents for inhibition of gastrin-promoted tumor growth and proliferation.
  • Complete knock-down of gastrin gene expression may be necessary for gastrin-promoted tumors and other conditions stimulated by an autocrine mechanism, where the diseased tissue itself produces gastrin that promotes its own growth and proliferation.
  • complete knock-down of gastrin gene expression by the gastrin-specific RNAi may be targeted to a different tissue that produces gastrin in a paracrine mechanism.
  • partial knock-down of the gastrin mRNA can be achieved by administering an effective amount of a gastrin-specific interfering RNA of the invention that leaves residual gastrin gene expression, due to administration of a dose of RNAi of the invention that results in under titration of the gastrin mRNA, or is targeted to a sequence that is ineffective in completely blocking gastrin gene expression.
  • Partial knock-down of the gastrin gene expression may be advantageous in the treatment of gastrin-promoted diseases and conditions such as Zollinger-Ellison syndrome due to overproduction of gastrin.
  • Reduction in the level of gastrin production to normal levels in such diseases and conditions can be achieved by knock-down of gastrin expression by administration of an effective amount of the gastrin-specific interfering RNA according to the methods of the present invention and as exemplified below.
  • An effective amount of a gastrin-specific RNAi molecule according to the present invention is that amount which when administered to a patient suffering from a gastrin- promoted disease or condition causes a knock-down of gastrin gene expression.
  • the knock ⁇ down of gastrin gene expression may occur only at the translational level, i.e. blocking or reducing synthesis of gastrin peptide products.
  • the knock-down of gastrin gene expression may include targeted degradation of gastrin mRNA, as well as blocking or reducing its translation.
  • the knock-down of gastrin gene expression may be a complete knock-down in which all, or substantially all gastrin gene expression is prevented.
  • the knock-down of gastrin gene expression caused by administration of a gastrin-specific RNAi molecule according to the present invention can be a partial knock-down wherein gastrin gene expression is reduced but not completely prevented.
  • the partial knock-down of gastrin gene expression can be a reduction of about 25 percent in the level of gastrin peptide products of the gastrin gene.
  • the partial knock-down of gastrin gene expression is a reduction of about 50 percent in the level of gastrin peptide products of the gastrin gene. More preferably, the partial knock-down of gastrin gene expression is a reduction of about 75 percent in the level of gastrin peptide products of the gastrin gene.
  • the partial knock ⁇ down of gastrin gene expression is a reduction of about 85 percent in the level of gastrin peptide products of the gastrin gene.
  • the level of gastrin mRNA may be reduced by administration of a gastrin-specific RNAi molecule of the present invention.
  • the level of gastrin mRNA can be reduced by about 25 percent, preferably by about 50 percent, more preferably by about 75 percent, and most preferably by about 85 percent upon administration of the gastrin-specific interfering RNA of the present invention.
  • administration of a gastrin-specific interfering RNA of the present invention causes complete, or substantially complete (i.e. about 90-100 percent) degradation of gastrin mRNA.
  • Combinations of gastrin-specific interfering RNAi molecules targeted to different gastrin sense-strand sequences can be administered to a patient suffering from a gastrin- promoted disease or condition in order to improve therapeutic efficacy by augmenting the knock-down of gastrin gene expression achieved with each of the RNAi molecules alone.
  • the invention provides a method of using a gastrin-specific RNAi molecule in combination with a chemotherapeutic agent to knock-down expression of gastrin for the treatment of gastrin-promoted gastrointestinal tumors in concert with chemotherapy.
  • the chemotherapeutic agents suitable for use in combination with the gastrin-specific RNAi of the present invention may be any chemotherapeutic agent, such as for instance gemcitabine, camptothecin, doxorubicin, 5-fluorouracil (5FU), docetaxel, paclitaxel, vinblastine, etoposide (VP- 16), oxaloplatin, carboplatin, cisplatin (CDDP), a kinase inhibitor (e.g. erlotinib, Iressa®), an angiogenesis inhibitor (e.g. Bevacizumab), and an EGF receptor inhibitor (e.g. cetuximab).
  • chemotherapeutic agent such as for instance gemcitabine, camptothec
  • the chemotherapeutic agent may be administered at any stage of a gastrin-specific RNAi therapy regimen according to the methods of the present invention, including continuously or intermitently during the gastrin-specific RNAi therapy regimen.
  • Methods treatment of gastrin-promoted tumors of the present invention include methods wherein a gastrin-specific RNAi molecule is administered directly at the site of the tumor.
  • the gastrin-specific RNAi molecule can administered at a distant site.
  • the treatment can be administered in conjunction with a chemotherapy regimen.
  • Gastrin-promoted diseases or conditions amenable to treatment with a gastrin- specific RNAi of the present invention include all gastrin-promoted tumors (both GI and non-GI), gastric-esophageal reflux disease (GERD), premalignatn conditions like for example Barrett's esophagus, atrophic gastritis, gastric ulceration, duodenal ulceration, hypergastrinemia, such as in Zollinger-Ellison syndrome, pernicious anemia and Helicobacter pylori infection.
  • GSD gastric-esophageal reflux disease
  • premalignatn conditions like for example Barrett's esophagus, atrophic gastritis, gastric ulceration, duodenal ulceration, hypergastrinemia, such as in Zollinger-Ellison syndrome, pernicious anemia and Helicobacter pylori infection.
  • Gastrin-promoted tumors treatable with a gastrin-specific RNAi of the present invention include colonic adenomas, pan-intraepithelial neoplasias, esophageal tumors, gastric neoplasias, intestinal tumors, pancreatic tumors, small cell lung cancers, medullary thyroid carcinomas, hepatic tumors, pulmonary tumors, ovarian tumors, glioblastomas, astrocytomas, tumors of brain origin such as glioblastomas or astrocytomas and tumors of neuroendocrine origin.
  • the gastrin-specific RNAi molecule according to the present invention is preferably administered to a patient in a pharmaceutical composition.
  • the gastrin-specific RNAi molecule can be administered as a bolus or continuously, either systemically or locally at the site of the tissue that responds to gastrin.
  • a patient is an animal or a human suffering from a gastrin-promoted disease or condition.
  • the disease can also be any disease where the gastrin gene is upregulated by growth factors, environmental stimuli or transcription factors.
  • compositions useful in the practice of the methods of the present invention include a gastrin-specific RNAi molecule and a physiologically compatible carrier or excipient.
  • Suitable carriers or excipients include any physiologically compatible buffer, such as, for instance phosphate buffered saline (PBS).
  • compositions useful can further have a peptide covalently linked to the RNAi molecule.
  • these could be peptides such as TAT which enable uptake into cells (Cell. 1988 Dec 23;55(6):1189-93). These peptides are used for increasing transport into cells e.g. liposomes (PNAS 2001 98:8786-8781). Exemplary, penetratin and transportan have also been tried for siRNAs (Muratovska 2004 FEBS Letters 558:63-68).
  • peptides could be used that would allow targeting to particular cell-types e.g. gastrin peptide to get the siRNAs into gastrin receptor-expressing cells.
  • the peptides could be either directly linked to the siRNA or attached to the carrier (polymer/liposome etc).
  • the pharmaceutical compositions useful in the practice of the methods of the present invention include a nucleic acid vector expressing a gastrin-specific RNAi molecule according to the present invention and a physiologically compatible carrier or excipient.
  • the pharmaceutical compositions of the present invention include a recombinant vector expressing a gastrin-specific RNAi of the present invention as a single stranded ribonucleotide structure or as a hairpin ribonucleotide structure from an inducible recombinant gene, and a physiologically compatible carrier or excipient. Incorporation by reference: The texts of each of the patents and other publications cited in this specification are hereby specifically incorporated by reference in their entireties.
  • siRNA synthesis An in vitro system for siRNA synthesis (marketed by Ambion Ltd., Huntingdon, Cambridgeshire, UK) was used, in which the two strands of the siRNA are synthesized using T7 polymerase, annealed together to form a double stranded structure and purified by glass fiber filter binding and elution. The concentration of the double stranded nucleotide was calculated by measurement of the absorbance at 260nm.
  • a fluorescent siRNA was synthesised by modification of the above method, involving addition of fluorescein- 12-uridine-5' -triphosphate during single strand synthesis. This required optimization of the ratio of fluorescent to non-fluorescent nucleotides and use of an increased overall concentration of ribonucleotide triphosphates (rNTPs). The higher concentration of rNTPs gave a higher efficiency of siRNA synthesis which may also be applicable to synthesis of non-fluorescent siRNAs.
  • rNTPs ribonucleotide triphosphates
  • siRNAs were synthesized commercially (Dharmacon, Lafayette, CO, USA). The two strands were synthesized separately using standard RNA oligonucleotide chemistry, converted to the 2'-hydroxyl form and annealed. The duplex was PAGE - purified and desalted by standard methods well known in the art.
  • Panl - a human pancreatic adenocarcinoma cell-line.
  • HCTl 16 a poorly differentiated human colon adenocarcinoma cell-line (ECACC, ref.no. 91091005)
  • MGLVAl a gastric adenocarcinoma cell-line, an ascitic variant of the gastric cell line MKN45G (Watson et al, 1990. Int.J.Cancer, 45, 90-4)
  • OEl 9 an esophageal adenocarcinoma cell line.
  • AGS - human gastric adenocarcinoma cell line ECACC, Wiltshire UK
  • STl 6 human gastric adenocarcinoma cell line
  • the fluorescent siRNA was used to check the efficiency of transfection. Almost 100% of the cells were fluorescent when viewed through a fluorescent microscope 24hrs after transfection. The distribution of fluorescence in the cells transfected with fluorescently labeled siRNA, tg5, was examined using confocal microscopy. Fluorescence was associated with vesicle-like structures in the cytoplasm.
  • gastrin siRNA transfections of cell-lines was undertaken. For several of these experiments gastrin gene expression was measured using real-time PCR. Gastrin gene expression in cells treated with gastrin siRNAs was calculated relative to cells treated with the scrambled tg8 siRNA (scrtg ⁇ ) and the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT) or relative to the housekeeping gene alone using the"2 " ⁇ Ct method” and the "T ACt method” respectively.
  • scrtg ⁇ scrambled tg8 siRNA
  • HPRT hypoxanthine-guanine phosphoribosyl transferase
  • Ct is the "threshhold cycle" where the relative copy number is seen to breakthrough into a detectable exponential phase of amplification.
  • the amount of target for a sample (q), normalized to the endogenous reference and relative to a calibrator (cb) is given by
  • ⁇ C t,q is the difference in threshold cycles for targets T and q.
  • ⁇ C t ,cb is the difference in threshold cycles for targets t and calibrator, cb.
  • This latter method compensates for differences between the reference control sample and the test samples for factors such as precise cell numbers, and efficiency of RNA and cDNA synthesis ( ⁇ Q, q ).
  • the method also provides a measure of gene expression in the test sample relative to the control-treated sample ( ⁇ Ct, C b)-
  • ⁇ Q Q, ⁇ - 0,Cb (i-e. the difference in threshold cycles for the target and the housekeeping genes). This method is used to illustrate differences in absolute expression levels, for example between different cell lines.
  • siPort Amine (Ambion, UK) transfection reagent were used to transfect Panl cells, referred to in the figures as Al, A2 and A4, corresponding to 1 ⁇ l, 2 ⁇ l and 4 ⁇ l of the siRNA reagent per transfection.
  • Gastrin gene expression monitored by PCR using the 2 " ⁇ Ct method is shown in Figure 1.
  • siRNAs were tested, including six different targets within gastrin and two control siRNAs targeting scrambled (scr) versions of two of the gastrin siRNAs.
  • the targets were chosen for the following properties: a) preceded by the dinucleotide AA in the gene sequence; b) avoiding the extreme 5' or 3' ends of the gene; c) low GC content (approximately 30-60%) d) absence of long stretches of A's; and e) lack of significant homology with other genes.
  • Targets tg5 and tg7 were initially investigated as the best potential targets since target 7 had the lowest GC content of the targets in the 3 ! end of the gene whilst target 5 targeted an independent region of the gene compared with target 7, but avoided the extreme 5' end of the gene. Approximately 85% knock-down of gene expression was achieved with tg5 whilst tg7 gave more variable and generally lower knock-down. The remaining targets were then assessed for knock-down of gastrin gene expression. Target tgl is located towards the 5' end of the gene, which has generally been found to be less successful in other siRNA gene silencing systems. Targets 8, 9 and 10 are immediately adjacent to tg7 with only a three nucleotide shift between each target sequence.
  • siRNAs tgl, tg5, tg7, tg8, tg9 and tglO, and the scrtg5 control siRNA were synthesized using the in vitro method described above and tested on Pan-1 cells at a range of concentrations (undiluted, 1:3 and 1:10 dilutions, corresponding to final concentrations of approximately 2OnM 5 6nM and 2nM respectively). Transfections were carried out using siPort Amine (Ambion). RNA was extracted using RNABee (Ambion), cDNA sythesised using Superscript II (Invitrogen, Carlsbad, California) and random hexamers as primers.
  • HPRT hypoxanthine-guanine phosphotransferase
  • the most effective targets were tg8 and tg9, providing 95% knock-down of gastrin gene expression.
  • the siRNA, tgl gave the least knock-down of gastrin gene expression.
  • Knock-down by the scrambled tg5 siRNA was lower (about 30%). For each of the siRNAs, the knock-down effect decreased with decreasing concentrations of the transfected siRNA.
  • RNA structure of gastrin mRNA was predicted using the RNA2 software available at rhttp://www. genebee.msu.su/services/rna2 reduced.html).
  • siRNAs tg7, tg8, tg9, and tglO appear to target the same loop structure within the gastrin mRNA.
  • tg5 targets a different structure in the precursor RNA from that targeted by tg7, tg8, tg9, and tglO.
  • hybridization of the antisense strands of tg7, tg8, tg9, or tglO would involve opening up similar structures within the RNA to allow targeting by the RISC, believed to be the complex responsible for mediation of gene silencing.
  • Example 7 Effect of gastrin siRNAs on a range of cell-lines
  • Panl cells were transfected with 4OnM (undiluted) or 4nM (1:10) siRNAs. Gene expression was monitored at days 1, 4, 7 and 11. The data are shown in Figure 7. The experiment was repeated with HCTl 16 cells and gene expression measured at days 1, 3, 6 and 9. These data are shown in Figure 8.
  • FIG. 9 shows gastrin gene expression measured by real-time PCR in siRNA-transfected PAN-1 cells in the presence or absence of lO ⁇ g/ml EGF. The gastrin siRNA inhibited the induction of gastrin gene expression by EGF.
  • Pan-1 cells were harvested using trypsin/EDTA on day 3 following transfection with 4OnM tg8 or scrtg ⁇ siRNA. The transfection was carried out in duplicate for each siRNA. After washing, the cells were fixed using 4% formalin and stored at 4 0 C. The cells were used to make cytospins, permeabilised using Triton XlOO and stained with a polyclonal rabbit antibody raised against amino acids 6-14 of progastrin. Binding of the primary antibody was detected using a fluorescent secondary antibody. Staining was detected using fluorescent microscopy.
  • Example 12 Inhibition of GFP-tagged gastrin protein expression by gastrin siRNA
  • the effect of siRNA transfection on gastrin protein expression was also analysed using HCTl 16 cells transfected with a GFP-tagged gastrin gene.
  • the complete gastrin coding sequence was amplified by PCR and cloned upstream of the GFP coding sequence in the plasmid pHRGFP-C (Stratagene) under control of a CMV promoter using the BamHl and Xhol restriction sites.
  • a complete Kozak sequence was incorporated into the forward primer, involving modification of the 4th nucleotide of the gastrin coding sequence from C to G, indicated in the primer sequence by underlining.
  • the primers used were as follows: Forward: CGCGGATCCGCCGCCGCCATGGAGCGACTGTGTGTG (SEQ ID NO 10) Reverse: CCGCCGCTCGAGGCCGAAGTCCATCCATC (SEQ ID NO 11)
  • the unmodified pHRGFP-C plasmid was used as a vector control. Since it lacks a Kozak sequence, no GFP expression was expected.
  • Dual transfection of plasmids and siRNAs was carried out by preparing the siRNA transfection mix as above but in half the volume, and pre-incubating 500ng of plasmid with l ⁇ l Lipofectamine (InVitrogen) in 25 ⁇ l serum-free medium. The two transfection reagents were then mixed immediately before addition to the cells. 24 hours following transfection, GFP expression was measured by flow cytometry.
  • MTT assays were carried out on cells transfected with the siRNAs. This assay is based upon the reduction of the colorless tetrazolium salt 3, [4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the deeply colored red dye product in the mitochondria of metabolically active cells. During initial experiments, growth effects were not apparent immediately following transfection and day 5 was selected as the optimal time-point for the assay.
  • Panl, HCTl 16, MGLVAl or C170HM2 cells were transfected with 0.5 ⁇ g of the tg8 or scrtg8 siRNAs, and treated with MTT for 4hrs on days 5 post-transfection. Six replicates were performed for each siRNA and reduction of MTT was measured photometrically. Growth was measured on a minimum of 3 separate occasions for each cell-line and, whilst there was some variation between experiments, in all 4 cell-lines there was reduced growth in the tg8 compared with the scrtg ⁇ -treated cells.
  • gastrin is known to be anti-apoptotic as well as acting as a growth factor
  • the effect of the siRNAs on apoptosis in the 4 cell-lines was also investigated.
  • the degree of apoptosis in gastrin siRNA-treated cells was examined using an assay that involves treating cells with a fluorescent caspase 3-specific inhibitor (BioCarta).
  • Caspase 3 is a key mediator in the initiation of the apoptotic cascade.
  • the rationale behind this assay is that cells in which the caspase is activated are marked by binding of the fluorescent inhibitor, but prevented from continuing through the apoptotic pathway.
  • Panl, HCTl 16, MGLVAl or C170HM2 cells were transfected with 0.5 ⁇ g of the tg8 or scrtg ⁇ siRNAs. On day 4 following transfection they were harvested using EDTA and treated with the FAM-DEVD- FMK reagent at the specified concentration. After 1 hour they were washed and fixed in 0.4% formalin, then analysed by flow cytometry. The percentage of positive cells was calculated relative to untreated cells. The data is shown in Figure 12. There was increased apoptosis in the cells treated with the gastrin siRNA compared with the control siRNA. In the absence of EGF the difference was significant in the Panl and C170HM2 cells. The effect was enhanced in the presence of EGF, with a significant difference seen additionally in the HCTl 16 cells. Significance was not reached in the MGLVAl cells under either condition.
  • Example 15 Reduced PKB phosphorylation in cells transfected with the gastrin siRNA
  • H.pylori infection results in a pronounced hypergastrinaemia and upregulation of other human growth factors, including HB-EGF, that may contribute to the neoplastic progression associated with this infection (Parsonnet et al (1991) Helicobacter pylori infection and the risk of gastric carcinoma N. Engl. J. Med. 325: 1127 -1131).
  • HB-EGF expression is upregulated by gastrin
  • the effect of the gastrin siRNA on HB-EGF expression was investigated in three gastric cancer cell lines, MGLVAl, ST16 and AGS. Following transfection with the gastrin siRNA or control siRNA, HB-EGF expression was measured by real-time PCR. The results are shown in Figure 14. A significant decrease in HB-EGF gene expression was seen in all three cell-lines in the gastrin siRNA-treated cells compared with cells treated with the control siRNA.
  • Example 17 Effect of gastrin siRNA on XIAP expression
  • the X-linked inhibitor of apoptosis protein is a member of the inhibitor of apoptosis protein (IAP) family and a potent inhibitor of caspase-3, -6, -7 and 9 . Since XIAP expression is raised in a number of cancers and is upregulated by phospho-Akt, the effect of the gastrin siRNA on XIAP expression in the gastric cancer cell line, AGS, was investigated. Following transfection with the gastrin (target 8) and control siRNA (scrambled tg8), XIAP expression was measured by real time PCR. The data are shown in Figure 17.
  • the 27-mer and tg8 gastrin siRNAs and the control siRNA (scrtg ⁇ ) were used to transfect C170HM2 cells at a range of concentrations between O.lnM and 4OnM. Gene expression was monitored on day 1 by real time PCR. The data are shown in Figure 20.
  • the 27-mer gastrin siRNA was as effective at knocking down gastrin gene expression as the tg8 siRNA.
  • Oligonucleotides were designed to encode tg5, tg7, tg8 and scrtg ⁇ hairpin RNAs, with BamHl and HindIII restriction sites to allow insertion into vector, pSilencer 2.1-U6 (Ambion Inc., Huntingdon, Cambridgeshire, UK). Their sequences are given in Table 2.
  • Each of the hairpin loops was cloned into the vector pSilencer 2.1 U6 obtained from Ambion (Huntingdon, Cambridgeshire, UK). An EcoRI/Hindlll digest was used to visualize the insert.
  • the structure of the gastrin siRNA stem-loop structure can be expressed from the recombinant pSilencer 2.1 U6 vector clone from Ambion.

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Abstract

L'invention concerne des molécules d'acide ribonucléique interférant spécifique vis-à-vis de la gastrine (ARNi) qui exercent une régulation négative sur le gène de l'hormone gastrine. Les molécules d'ARNi de l'invention sont des molécules d'ARN double brin qui peuvent comprendre des bases modifiées et des liaisons qui ne sont pas des liaisons de type phosphate entre les bases. Les brins complémentaires des molécules d'ARNi de l'invention peuvent être liés par une chaîne de nucléotides ou par des groupes de liaison qui ne sont pas des nucléotides. Les molécules d'ARNi de l'invention peuvent être incorporées dans une formulation pharmaceutique utile dans un procédé servant à traiter des maladies ou affections médiées par la gastrine dont des tumeurs médiées par la gastrine, la maladie du reflux gastro-oesophagien (GERD), le syndrome de Zollinger-Ellison, l'hypergastrinémie, l'anémie pernicieuse, l'ulcère gastrique, l'ulcère duodénal et l'infection par H. pylori.
EP05775595A 2004-08-11 2005-08-10 Arn interférant spécifique vis-à-vis de la gastrine Withdrawn EP1778841A2 (fr)

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EP1608984A2 (fr) 2003-03-28 2005-12-28 Aphton Corporation Dosages immunologiques pour la detection de l'hormone gastrine
US8889640B1 (en) * 2008-06-20 2014-11-18 Jill P. Smith Composition and method for the treatment of gastrin mediated cancers
EP3624841A4 (fr) 2017-06-15 2021-01-27 Cancer Advances, Inc., Compositions et procédés pour induire des immunités humorales et cellulaires contre des tumeurs et un cancer
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