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WO2009115834A2 - Inhibition hormonale - Google Patents

Inhibition hormonale Download PDF

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WO2009115834A2
WO2009115834A2 PCT/GB2009/050237 GB2009050237W WO2009115834A2 WO 2009115834 A2 WO2009115834 A2 WO 2009115834A2 GB 2009050237 W GB2009050237 W GB 2009050237W WO 2009115834 A2 WO2009115834 A2 WO 2009115834A2
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seq
sirna
rna sequence
sense strand
rna
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WO2009115834A3 (fr
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John Newell-Price
Alia Munir
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University of Sheffield
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University of Sheffield
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • 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 nucleic acids [NA]

Definitions

  • the present invention relates to siRNA's capable of mediating target-specific inhibition of proopiomelancortin.
  • Cushing's disease is a devastating condition caused primarily by a pituitary tumour expressing excess proopiomelancortin (POMC) that is cleaved to adrenocorticotropin (ACTH) that in turn drives the adrenals to synthesise and secrete excess Cortisol (Newell-Price et al (2006) Lancet 367:1605-1617). Modern series show that if left untreated Cushing's disease has a five-fold excess mortality (Lindholm J et al (2001 ) . The Journal of clinical endocrinology and metabolism 86:1 17-123). In rare instances Cushing's disease may result from the ectopic production of ATCH from locations other than the pituitary. Ectopic sites of ATCH production can arise from many different tissues including medullary carcinoma of the thyroid, oat cell carcinoma of the lung, thymoma, islet cell tumors, and pheochromocytoma.
  • tumours have high proliferative activity and low expression of p27, and mis-expression of Brg1 (Bilodeau S et a/ (2006) Genes Dev. Oct
  • Corticotrope tumours express pro-opiomelanocortin gene (POMC) whose product is cleaved to corticotrophin (ACTH).
  • POMC pro-opiomelanocortin gene
  • Patients who are not cured or relapse can receive external beam radiation (in certain cases sterotactic radiosurgery maybe appropriate) and/or medical therapy with steroidogenic inhibitors or neuromodulators of ACTH.
  • Metyrapone, ketoconazole, etomidate and aminoglutethimide inhibit steroid biosynthesis.
  • Valproic acid has also been used in patients with Cushing's and Nelson's syndrome, which may enhance GABA inhibition of hypothalamic CRH release, however the efficacy is unproven in Cushing's disease.
  • Cyproheptadine may suppress ACTH by acting at hypothalamo- pituitary level but further studies are required. Very recent studies are embarking on testing the ability of mifeprostone to block the action of Cortisol in the body.
  • Spampinato et al used an AtT20 model and treated cells with an oligonucleotide complementary to a region of ⁇ -endorphin mRNA. This markedly reduced the levels of ACTH and ⁇ -endorphin. In addition they performed an in vivo infusion of the oligonucleotide in rats via an implanted cannula in to the hypothalamic arcuate nucleus and noted a reduction in the ACTH and ⁇ -endorphin immunopositive neurons. Grooming behavior was reduced (Spampinato et al (1994) Proc Natl Acad Sci U S A, 91 , 8072-6).
  • Woloschak et al treated human ACTH-secreting pituitary adenoma cells cultured from two uncommon ACTH-secreting macroadenomas.
  • the tumour tissue was enzymatically dispersed and cultured for four days. They were then treated for 18 hours with antisense POMC oligomer or control, and one set of tumour cells was treated with dexamethasone. They showed a reduction in POMC mRNA levels and ACTH levels by 76% and 62% in the two tumours and 58% and 48% relative to controls (Woloschak et al (1994) J Endocrinol Invest, 17, 817-9).
  • the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to an RNA sequence encoding POMC or a portion thereof, and said antisense strand is complementary to said sense strand.
  • siRNA short interfering RNA
  • said sense strand is at least 80% identical to an RNA sequence selected from: i) RNA target sequence of siRNA 1 (SEQ ID NO:1 ); ii) RNA target sequence of siRNA 2 (SEQ ID NO:2); iii) RNA target sequence of siRNA 3 (SEQ ID NO:3).
  • sense strand and antisense strand each comprise about 21 nucleotides.
  • siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.
  • siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon.
  • said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:4; ii) an RNA sequence of SEQ ID NO:5; and iii) an RNA sequence of SEQ ID NO:6.
  • said sense strand comprises an RNA sequence of SEQ ID NO:4 and said antisense strand comprises an RNA sequence of SEQ ID NO:7.
  • said sense strand comprises an RNA sequence of SEQ ID NO:5 and said antisense strand comprises an RNA sequence of SEQ ID NO:8.
  • said sense strand comprises an RNA sequence of SEQ ID NO:6 and said antisense strand comprises an RNA sequence of SEQ ID NO:9.
  • said sense strand consists of an RNA sequence of SEQ ID NO:4 and said antisense strand consists of an RNA sequence of SEQ ID NO:7.
  • said sense strand consists of an RNA sequence of SEQ ID NO:5 and said antisense strand consists of an RNA sequence of SEQ ID NO:8.
  • said sense strand consists of an RNA sequence of SEQ ID NO:6 and said antisense strand consists of an RNA sequence of SEQ ID NO:9.
  • the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to a DNA sequence encoding pituitary promoter region of POMC or a portion thereof and said antisense strand is complementary to said sense strand.
  • siRNA short interfering RNA
  • said sense strand is at least 80% identical to an DNA sequence selected from: i) DNA target sequence of ProPOMCI (SEQ ID NO:10); ii) DNA target sequence of ProPOMC2 (SEQ ID NO:1 1 ); iii) DNA target sequence of ProPOMC3 (SEQ ID NO:12); iv) DNA target sequence of ProPOMC4 (SEQ ID NO:13); v) DNA target sequence of ProPOMC ⁇ (SEQ ID NO:14); vi) DNA target sequence of ProPOMC ⁇ (SEQ ID NO:15).
  • sense strand and antisense strand each comprise about 25 nucleotides.
  • siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.
  • said siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon.
  • said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:16; ii) an RNA sequence of SEQ ID NO:17; iii) an RNA sequence of SEQ ID NO:18; iv) an RNA sequence of SEQ ID NO:19; v) an RNA sequence of SEQ ID NO:20; and vi) an RNA sequence of SEQ ID NO:21.
  • said sense strand comprises an RNA sequence of SEQ ID NO:16 and said antisense strand comprises an RNA sequence of SEQ ID NO:22.
  • sense strand comprises an RNA sequence of SEQ ID NO:17 and said antisense strand comprises an RNA sequence of SEQ ID NO:23.
  • said sense strand comprises an RNA sequence of SEQ ID NO:18 and said antisense strand comprises an RNA sequence of SEQ ID NO:24.
  • said sense strand comprises an RNA sequence of SEQ ID NO:19 and said antisense strand comprises an RNA sequence of SEQ ID NO:25.
  • said sense strand comprises an RNA sequence of SEQ ID NO:20 and said antisense strand comprises an RNA sequence of SEQ ID NO:26.
  • said sense strand comprises an RNA sequence of SEQ ID NO:21 and said antisense strand comprises an RNA sequence of SEQ ID NO:27.
  • said sense strand consists of an RNA sequence of SEQ ID NO:16 and said antisense strand consists of an RNA sequence of SEQ ID NO:22.
  • said sense strand consists of an RNA sequence of SEQ ID NO:17 and said antisense strand consists of an RNA sequence of SEQ ID NO:23.
  • said sense strand consists of an RNA sequence of SEQ ID NO:18 and said antisense strand consists of an RNA sequence of SEQ ID NO:24.
  • said sense strand consists of an RNA sequence of SEQ ID NO:19 and said antisense strand consists of an RNA sequence of SEQ ID NO:25.
  • said sense strand consists of an RNA sequence of SEQ ID NO:20 and said antisense strand consists of an RNA sequence of SEQ ID NO:26.
  • said sense strand consists of an RNA sequence of SEQ ID NO:21 and said antisense strand consists of an RNA sequence of SEQ ID NO:27.
  • the invention provides a short interfering RNA (siRNA) comprising a sense strand and an antisense strand which each independently comprise from about 19 to about 25 nucleotides, wherein said sense strand is at least 80% identical to an RNA sequence encoding Tpit or a portion thereof, and said antisense strand is complementary to said sense strand.
  • siRNA short interfering RNA
  • said sense strand is at least 80% identical to an RNA sequence selected from: i) RNA target sequence of TpitsiRNA 1 (SEQ ID NO:28); or ii) RNA target sequence of TpitsiRNA 2 (SEQ ID NO:29).
  • said sense strand and antisense strand each comprise about 21 nucleotides.
  • said siRNA comprises a nucleotide overhang at the 3' end, the 5' end or both the 3' and 5' ends of said siRNA.
  • said siRNA comprises at least one nucleotide wherein a methylene bridge connects a 2'- oxygen of a ribose to a 4'carbon
  • said sense strand of said siRNA comprises an RNA sequence selected from: i) an RNA sequence of SEQ ID NO:30; and ii) an RNA sequence of SEQ ID NO:32.
  • said sense strand comprises an RNA sequence of SEQ ID NO:30 and said antisense strand comprises an RNA sequence of SEQ ID NO:31.
  • said sense strand comprises an RNA sequence of SEQ ID NO:32 and said antisense strand comprises an RNA sequence of SEQ ID NO:33.
  • said sense strand consists of an RNA sequence of SEQ ID NO:30 and said antisense strand consists of an RNA sequence of SEQ ID NO:31 .
  • said sense strand consists of an RNA sequence of SEQ ID NO:32 and said antisense strand consists of an RNA sequence of SEQ ID NO:33.
  • a therapeutic composition comprising an siRNA according to the invention and a pharmaceutically acceptable carrier.
  • an siRNA according to the invention for use as a medicament.
  • an siRNA according to the invention for treating a disorders associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid.
  • said disorder is Cushing's disease.
  • the invention provides use of an siRNA according to the invention to down-regulate the expression or activity of proopiomelancortin (POMC).
  • POMC proopiomelancortin
  • the invention provides methods of treating a subject having a disease or disorder associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid by administering an siRNA of the invention to the subject.
  • a method of treating a disorder associated with the expression or activity of a POMC nucleic acid or polypeptide encoded by the POMC nucleic acid in a subject in need thereof comprising administering to the subject an effective amount of an siRNA in accordance with the invention.
  • said disorder is Cushing's disease.
  • a method of down-regulating the expression or activity of proopiomelancortin (POMC) in a subject in need thereof comprising administering to the subject an effective amount of an siRNA in accordance with the invention.
  • POMC proopiomelancortin
  • Figure 1 is a schematic representation of the cellular RNAi response
  • Figure 2 is a schematic representation of the polycystronic products of the POMC gene
  • Figure 3 is a schematic representation of mouse PO/WC with positions of siRNAs aligned
  • Figure 4a is a graphical representation of siRNA silencing of pome mRNA with siRNA 1 :. siRNAI silences pome mRNA by 86% at 24 hours;
  • Figure 4b is a graphical representation of siRNA silencing of pome mRNA with siRNA 2:. siRNA2 silences pome mRNA by 89% at 24 hours;
  • Figure 4c is a graphical representation of siRNA silencing of pome mRNA with siRNA 3:.
  • siRNA3 silences pome mRNA by 95% at 24 hours;
  • Figure 4d is a graphical representation of the stability of beta-actin mRNA at 48 hours after treatment with siRNA3: beta-actin expression remains stable in keeping with pomc- specific effect of siRNA3;
  • Figure 5a is a graphical representation of ACTH levels upon siRNA-3 treatment - effect at 48, 120 and 240 hours respectively;
  • Figure 5b is a graphical representation of siRNA silencing of pome mRNA with siRNA 3, demonstrating significant silencing of pome mRNA over 48, 120 and 240 hours, respectively;
  • Figure 6a is a graphical representation of the knockdown of pome mRNA following a single transfection with siRNA targeting the pome promoter showing ⁇ 95% reduction of expression of pome at 96 hours p ⁇ 0.0001
  • Figure 6b is a graphical representation of the knockdown of pome mRNA following a single transfection with siRNA targeting the pome promoter showing recovery of expression to 50% by 120 hours;
  • Figure 7a and b provide a graphical representation of methylation analysis of the promoter of pome.
  • the number of rows is the number of clones sequenced
  • Figure 7a illustrates lack of methylation-induction of pome after AtT20 cells are treated with 6 siRNAs targeted to the pome promoter region
  • figure 7b illustrates methylation of the pome promoter in non-expressing tissue (3T3 cells);
  • Figure 8 is a schematic representation of the regulatory effect of Tpit on the expression of POMC
  • Figure 9 illustrates a BLAST alignment of mouse Tpit with human TPIT.
  • the target site for TpitsiRNA 1 is highlighted in dark grey (top of diagram).
  • the target site for TpitsiRNA 2 is highlighted in light grey.(bottom of diagram).
  • the numbers correspond to bases in mouse Tpit and human TPITmRUA;
  • Figure 10a is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 1 - treated samples 24 hours post-transfection.
  • Figure 10b is a graphical representation of the Relative quantities of Tpit mRNA in TpitsiRNA 2- treated samples 24 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 1 1 a is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 1 - treated samples 24 hours post-transfection.
  • Figure 1 1 b is a graphical representation of the Relative quantities of POMC mRNA in TpitsiRNA 2- treated samples 24 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 12 is a graphical representation of the relative quantities of beta Actin mRNA in samples 24 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 13a is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 1 - treated samples 72 hours post-transfection.
  • Figure 13b is a graphical representation of the relative quantities of Tpit mRNA in TpitsiRNA 2- treated samples 72 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 14a is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 1 - treated samples 72 hours post-transfection.
  • Figure 14b is a graphical representation of the relative quantities of POMC mRNA in TpitsiRNA 2- treated samples 72 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Lane 1 TpitsiRNA 1
  • Lane 2 TpitsiRNA 2
  • Figure 16a is a graphical representation of the relative levels of ACTH in TpitsiRNA 1 - treated samples 24 hours post-transfection.
  • Figure 16b is a graphical representation of the relative levels of ACTH in TpitsiRNA 2- treated samples 24 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 17a is a graphical representation of the relative levels of ACTH in TpitsiRNA 1 - treated samples 72 hours post-transfection.
  • Figure 17b is a graphical representation of the relative levels of ACTH in TpitsiRNA 2- treated samples 72 hours post-transfection.
  • Neg siRNA Negative control siRNA;
  • Figure 18 is a Light microscope image of live primary human corticotroph tumour cells (x 20);
  • Figure 19 is a graphical representation of the knock down of ACTH levels with siRNA 2 74%, siRNA 3 27%, TpitsiRNA 2 14 % when compared with Negative siRNA control at 24 hours;
  • Figure 20 is a graphical representation of the knock down of ACTH levels with siRNA 2 68%, siRNA 23%, TpitsiRNA 2 23 % when compared with Negative siRNA control at 48 hours;
  • Figure 21 is a graphical representation of ACTH levels 5 days post siRNA 3 treatment in mice with tumours less than or equal to 5mm;
  • Figure 22 is a graphical representation of ACTH levels 5 days post siRNA 2 treatment in mice with tumours less than or equal to 5mm;
  • Figure 23 is a graphical representation of tumour growth 5 days post siRNA 3 treatment.
  • RNA Interference RNA Interference
  • siRNA's short interfering RNA molecules
  • RNA interference is a process for sequence specific control of gene expression at a transcriptional, post-transcriptional and translational level using short interfering RNA molecules (Fire et al (1998) Nature, 391 , 806-1 1 ). It has been found that dsRNA mixture is at least ten fold more potent as a silencing trigger than sense or anti-sense alone (Guo and Kemphues, (1996) Nature, 382, 455-8).
  • RNA-induced silencing complex RISC is a high molecular weight multienzyme protein complex that endonulceolytically cleaves the target mRNA's before they are translated to protein. This complex was first isolated by Hannon's lab, with a species of small RNA about 25 nuleotides in length (Hammond et al (2000) Nature, 404, 293-6). It was these RNAs that were termed small interfering RNAs (siRNAs) and were shown to be required for degradation of the mRNA.
  • siRNAs small interfering RNAs
  • Dicer The introduction of dsRNA into a cell, stimulates the activity of an enzyme responsible for processing the dsRNA to siRNAs (Bernstein et al (2001 ) Nature, 409, 363-6).
  • This enzyme from the Ribonuclease III family of enzymes is referred to as Dicer.
  • the Dicer enzyme possesses dual dsRNA catalytic domains: PAZ and helicase. Dicer is highly conserved with homologues in nearly every species.
  • siRNAs produced by the enzymatic reaction of Dicer are double stranded, and typically between about 20 to 25, more typically 21 to 23, nucleotides in length with 5'-phosphate and 3'-hydroxyl termini. They comprise about 19 to 22, more typically about 19, central complementary paired bases and have short 3'-nucleotide overhangs, typically 3'- dinucleotide overhangs.
  • silencing occurs upon recognition of dsRNA by Dicer, which converts the dsRNA in to 21 -25nt RNAs.
  • siRNAs small interfering RNAs
  • RISC small interfering RNAs
  • mRNA of interest the guide strand which is anti-sense to the target mRNA
  • AGO Argonaute
  • RISC mediates several different modes of silencing. Sequence specific degradation of mRNA targets, termed slicing, is one method of post-transcriptional gene silencing. The target RNA is cleaved at the position opposite the phosphate between the 10 th and 1 1 th base from the 5 prime end of the guide. Read through is prevented and mRNA degraded.
  • the alternative method is one of translational repression used by microRNA (endogenous non coding RNAs processed in to stem loops called pre-imRNAs; important in development, oncogenesis and immunity).
  • the third method is transcriptional gene silencing (TGS) initially observed in plants.
  • the 5 prime nucleotide of the siRNA is unpaired and the base and phosphate interact with residues of the middle domain via base-stacking and ionic interactions. Consistent with the seed locator of nucleotides 2-8 in the guide RNA, Argonaute substrate specificity is determined by the sequence of the bound guide RNA.
  • One strand of the siRNA is incorporated in to RISC this is mediated by Dicer and dependent on thermodynamic differences between the 2 ends of the double stranded siRNA. The passenger strand undergoes destruction requiring an active slicing Argonaute.
  • the loading of the guide RNA strand is also reliant on the presence of a 5 prime phosphate for entry in to the RISC loading complex (RLC), and this enhances binding of the guide to Argonaute.
  • RLC RISC loading complex
  • the complex once loaded is capable of multiple rounds of target binding and cleavage (Tolia and Joshua-Tor, (2007) Nat Chem Biol, 3, 36-43).
  • RNA relates to a polymer comprising at least one ribonucleotide monomer.
  • RNA relates to an isolated double stranded RNA molecule, comprising a sense strand and an antisense strand, wherein each strand is from about 19 to 25 nucleotides in length, more particularly from about 19 to 23 nucleotides in length, more particularly 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • at least one strand has a 3' overhang of from about 1 to 5 nucleotides, more preferably a 3' overhang of 2 nucleotides.
  • at least one strand has a 5' overhang of from about 1 to 5 nucleotides, more preferably a 3' overhang of 2 nucleotides.
  • least one strand has a 3' overhang and a 5' overhang of from about 1 to 5 nucleotides, more preferably a 3' overhang and a 5' overhang of 2 nucleotides.
  • the sense and antisense strands of an siRNA may comprise two single stranded RNA molecules.
  • the siRNA may comprise a single RNA molecule in which two sections are complimentary and base pair by hydrogen bonding to give a "hair pin” conformation.
  • sense strand or " sense RNA strand” relates to a nucleic acid sequence which identical to a target mRNA sequence.
  • antisense strand or “antisense RNA strand” relates to a nucleic acid molecule which is complementary to a sense RNA strand, i.e. may anneal to a sense strand by complementary base-pairing.
  • complementary refers to the ability of a first nucleic acid molecule, i.e. an RNA, to hydrogen bond to with another nucleic acid, either via conventional nucleotide complementarity, i.e. Watson-Crick base pairing or by an alternative non traditional pairing.
  • the percentage complementarity is a measure of the number of contiguous nucleic acid residues in a first nucleic acid molecule that are capable of hydrogen bonding with a second nucleic acid molecule.
  • RNA target refers to the mRNA sequence or DNA sequence to which the sense strand of an siRNA is identical.
  • RNA target is a contiguous stretch of between 19 and 25 nucleotides of an mRNA.
  • RNA target is a contiguous stretch of between 19 and 25 nucleotides of an DNA.
  • the target mRNA encodes POMC (Proopiomelanocortin) or a portion thereof.
  • the target mRNA comprises a transcribed POMC gene or a portion thereof.
  • the target mRNA may also include a mutant, homologue or alternative splice form of POMC mRNA.
  • the POMC gene is expressed in the anterior and intermediate lobes of the pituitary gland (in corticotrope cells), the hypothalamus (3000 neurones in the arcuate nucleus, dorsomedial hypothalamus and the brainstem) and the skin (in melanocytes) (Chretien et al., (1979) Can J Biochem, 57, 1 1 1 1 -21 ).
  • the primary product of the POMC gene is a 285 amino acid precursor peptide that can undergo differential processing to yield at least 8 peptides dependent upon the location of stimulus i.e. POMC is polycistronic, as shown in Figure 2.
  • POMC is processed by the convertases PC1/3 and generates pro-adrenocorticotrophin and ⁇ -lipotrophin.
  • Pro-ACTH in turn generates a joining peptide (JP-not shown on the figure), N-terminal pro-opiocortin (NPOC-not shown on the figure) and ACTH (1 -39).
  • This ACTH is cleaved to generate ACTH 1 -17 and corticotrophin like intermediate lobe peptide by PC2.
  • a number of post translational modifications result in ACTH 1 -17 which is cleaved to ⁇ -melanocyte stimulating hormone (MSH).
  • N-POC is processed in to v- MSH in humans, but not in rodents.
  • ⁇ -lipotrophin is processed to ⁇ -MSH.
  • ⁇ -lipotrophin also generates lipotrophin and endorphin which in turn are cleaved to ⁇ -MSH , y- endorphin and ⁇ -endorphin (Pritchard LE and White A (2007) Endocrinology. 2007 Sep;148(9):4201 -7).
  • siRNA's directed to the exonic coding sequence of POMC are capable of mediating long duration post-transcriptional silencing of the POMC gene.
  • the POMC gene is a mammalian POMC gene. More preferably, the POMC gene is a human POMC gene (Chang AC et al (1980) Proc Natl Acad Sci U S A, 77,
  • the POMC gene is a feline, canine, equine or proboscine POMC gene.
  • the term "gene” refers to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences, such as promoter regions and introns.
  • the POMC gene is assigned to chromosome 2p23.3 and originally isolated in 1980 (Chang et al (1980) Proc Natl Acad Sci U S A, 77, 4890-4). In mouse the POMC gene is located on chromosome 12 and was sequenced in 1982 (Uhler and Herbert, (1983) J Biol Chem, 258, 257-61 ).
  • the target mRNA is specific to POMC. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 1 , 2 or 3 of table 1 below.
  • the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:1 , 2 or 3.
  • siRNA's directed to the POMC promoter region are capable of mediating transcriptional silencing of the POMC gene.
  • the target DNA to which the siRNA's of the present application are directed are located at the POMC promoter region. More preferable, the target mRNA is directed to the transcription binding sites of the factors: Nurr 77, Ptx 1 , Tpit and NeuroDI (NCBI accession (NM_008895), Lamolet B ef a/ (2001 ) Cell. Mar 23;104(6):849-59, Poulin G ef a/ (1997) MoI Cell Biol. Nov;17(1 1 ):6673-82, Philips A et al (1997) MoI Cell Biol. Oct;17(10):5946-51 ,Lamonerie T ef a/ (1996) Genes Dev. May 15;10(10):1284-95).
  • the factors include Nurr 77, Ptx 1 , Tpit and NeuroDI (NCBI accession (NM_008895), Lamolet B ef a/ (2001 ) Cell. Mar 23;104(6):849-59, Poulin
  • the target DNA is specific to a POMC promoter region. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 10, 1 1 , 12, 13, 14 or 15 of table 2 below. Preferably, the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:10, 1 1 , 12, 13, 14 or 15.
  • the target mRNA encodes Tpit or a portion thereof.
  • the target mRNA comprises a transcribed Tpit gene or a portion thereof.
  • the target mRNA may also include a mutant, homologue or alternative splice form of Tpit mRNA.
  • the Tpit gene is a pituitary restricted transcription factor, present only in POMC expressing cells and is a partner of Pitxi on the POMC promoter (Lamolet B et al (2001 ) Cell, 104:849-859). Tpit mutations have been identified in patients with ATCH deficiency, suggesting a role for Tpit in POMC regulation (Pulichino AM et al (2003) Genes Dev 17:71 1 -716). Mutations in the human and mouse Tpit genes are known to cause a neonatal onset form of ACTH deficiency (Vallette-Kasic S et al (2005) J Clin Endocrinol Metab 90:1323-1331 ).
  • Tpit plays a crucial role in corticotroph development and in the corticotrope-specific expression of POMC, and interacts with other factors to drive POMC expression (Fig 8).
  • the Tpit gene is a mammalian Tpit gene. More preferably, the Tpit gene is a human Tpit gene (NCBI accession no. NG_008244). Alternatively, the Tpit gene is a feline, canine, equine or proboscine Tpit gene.
  • the target mRNA is specific to Tpit. More preferably the target mRNA comprises a nucleic acid sequence as illustrated in SEQ ID NO: 28 or 29 of table 5 below.
  • the target mRNA is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA of SEQ ID NO:28 or 29.
  • the sense strand of the siRNA must be sufficiently complementary to the target mRNA to mediate RNAi.
  • the sense strand is at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the target mRNA.
  • the percentage identity of the siRNA to the target mRNA is calculated over double stranded portion of the siRNA.
  • the percentage identity of the siRNA to the target mRNA may be calculated including any 3' or 5' overhang. The percentage identity between two sequences may be determined using alignment algorithms known in the art, see for example the Needleman algorithm (Needleman et al. (1970) J. MoI. Biol.
  • the sense strand of the siRNA may contain insertions, deletions or point mutations when compared to the target mRNA sequence.
  • the double stranded portion of the sense strand of the siRNA is identical to the target mRNA.
  • the siRNA sense strand comprises a nucleotide sequence which is at least at least 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, 99 or 100% identical to the nucleotide sequence as represented in SEQ ID NO:4, 5, 6, as illustrated in table 3 below or SEQ ID NO:16, 17, 18, 19, 20 and 21 as illustrated in table 4 below, or SEQ ID NO:30 and 32.
  • the siRNA sense strand has the nucleotide sequence as represented in SEQ ID N0:4, 5, 6, 16, 17, 18, 19, 20, 21 , 30 and 32.
  • siRNA 1 (SEQ ID NO:4 and SEQ ID NO:7): rat and mouse homology siRNA 2 (SEQ ID N0:5 and 8): rat, mouse and man homology siRNA 3 (SEQ ID N0:6 and 9): rat, mouse and man homology
  • POMCPRO1 (SEQ ID NO:16 and 22) rat and mouse homology: Nurr 77 P0MCPR02 (SEQ ID N0:17 and 23) mouse only: Neuro D1 P0MCPR03 (SEQ ID N0:18 and 24) mouse only: Ptx 1 and Tpit P0MCPR04 (SEQ ID N0:19 and 25) mouse only P0MCPR05 (SEQ ID NO:20 and 26) mouse only P0MCPR06 (SEQ ID N0:21 and 27) mouse only: Nurr 77
  • the siRNA sense strand is identical to an exonic region of the POMC gene and and comprises SEQ ID N0:4 and the antisense strand comprises SEQ ID N0:7.
  • the sense strand comprises SEQ ID N0:5 and the antisense strand comprises SEQ ID N0:8.
  • the sense strand comprises SEQ ID N0:6 and the antisense strand comprises SEQ ID N0:9.
  • the siRNA sense strand is complementary to the POMC promoter region and comprises SEQ ID N0:16 and the antisense strand comprises SEQ ID NO:22.
  • the sense strand comprises SEQ ID N0:17 and the antisense strand comprises SEQ ID NO:23.
  • the sense strand comprises SEQ ID N0:18 and the antisense strand comprises SEQ ID NO:24.
  • the sense strand comprises SEQ ID N0:19 and the antisense strand comprises SEQ ID NO:25.
  • the sense strand comprises SEQ ID NO:20 and the antisense strand comprises SEQ ID NO:26.
  • the sense strand comprises SEQ ID N0:21 and the antisense strand comprises SEQ ID NO:27.
  • the siRNA sense strand is complementary to the Nurr 77 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:21 and the antisense strand comprises SEQ ID NO:27.
  • the siRNA strand is complementary to the Nurr 77 transcription binding site and the siRNA sense strand comprises SEQ ID N0:16 and the antisense strand comprises SEQ ID NO:22.
  • the siRNA sense strand is complementary to the Neuro D1 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:17 and the antisense strand comprises SEQ ID NO:23.
  • the siRNA sense strand is complementary to the Ptx 1 transcription binding site, more preferably the siRNA sense strand comprises SEQ ID N0:18 and the antisense strand comprises SEQ ID NO:24.
  • the siRNA sense strand is complementary to an exonic region of the Tpit gene and comprises SEQ ID NO:30 and the antisense strand comprises SEQ ID N0:31 .
  • the sense strand comprises SEQ ID NO:32 and the antisense strand comprises SEQ ID NO:33.
  • Figure 3 illustrates the mouse POMC gene region with positions of each of the POMC exonic siRNA's and each of the POMC promoter siRNAs of the invention aligned.
  • Modification of the siRNA's of the invention may be used to improve nuclease resistance and cellular uptake of the siRNA molecules.
  • Modification may comprise modification of the phosphate backbone.
  • the siRNA may include phosphate backbone modifications, such as phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications (Harborth J et al. Antisense Nucleic Acid Drug Dev. 2003;13:83-105), the introduction of 2'-deoxy-2'-fluorouridine (Braasch D. A et al, RNA interference in mammalian cells by chemically-modified RNA. Biochemistry.
  • LNA Locked Nucleic Acid
  • the siRNA's of the invention may also include modifications of the sugar group (Choung S et al (2006) Biochem Biophys Res Commun. Apr 14;342(3):919-27.
  • the siRNA's may comprise combinations of the aforementioned modifications.
  • siRNA's Methods for the synthesis of siRNA's are well known in the art (Birmingham, A. et al (2006) Nat Methods, 3, 199-204 and Jackson, A et al, (2006) Rna, 12, 1 197-205).
  • Systems for both transient and permanent expression of siRNA have been developed which may be incorporated into the expression vectors, such as Ad vector
  • the invention provides an expression vector comprising a promoter and a polynucleotide sequence that encodes an siRNA of the invention.
  • the polynucleotide encodes a nucleic acid sequence of any one of SEQ ID NO's: 4 to 9, or SEQ ID NO's: 16 to 26.
  • the vector is a viral vector.
  • the vector is a non-viral vector.
  • siRNA's of the invention may be used to inhibit, the expression or activity of POMC gene or polypeptide encoded by the POMC gene, i.e. ACTH or ⁇ -lipotropin.
  • the terms “inhibit”, “down-regulate” and “reduce” refer to an alteration of the level of gene expression or the level of RNA molecules encoding POMC, or one or more protein subunits thereof, such that the aforementioned expression, level, or activity of the POMC nucleic acid or polypeptide is than less that observed in the absence of the modulation.
  • the inhibition can be performed in vitro (e.g., by culturing the cell with the siRNA) or, alternatively, in vivo (e.g, by administering the siRNA to a subject).
  • In vitro inhibition provides a method of reducing POMC activity or expression in a cell, comprising contact a cell with an siRNA of the invention.
  • the cell is contacted under conditions wherein modulation of siRNA mediated inhibition of POMC may occur.
  • said contact comprises introducing the siRNA into the cell.
  • the cells are corticotrope cells, neurones in the arcuate nucleus, dorsomedial hypothalamus and the brainstem, or melanocytes.
  • the cell is isolated from a cancerous tissue. More preferably the cell is isolated from pituitary adenoma, such as a corticotrope tumour.
  • the cell is isolated from an ACTH-producing tumour, such as small cell lung cancer, a thymoma, a pancreatic islet cell tumour, or a medullary carcinoma of the thyroid or an adrenal cancer, such as an adrenocortical carcinoma, or adrenal adenomas, including phaeochromocytomas.
  • the cells are mammalian cells. More preferably the cells are human.
  • In vitro inhibition methods including cell culture, may be carried out in any suitable vessel.
  • the vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate.
  • “Vessel” is construed as any means suitable to contain a cell culture.
  • In vivo inhibition provides methods of treating a subject having a disease or disorder, or at risk of having a disease or disorder, associated the expression or activity of a POMC nucleic acid or polypeptide derived there from, for example a disease or disorder associated with the activity of ACTH. Accordingly, the invention provides both prophylactic and therapeutic methods.
  • the siRNA's disclosed herein may be used alone or in combination with one another in prophylactic and therapeutic methods.
  • ACTH Diseases and disorders associated with the activity of ACTH include diseases and disorders associated with the expression of Cortisol.
  • the invention provides a method of treatment of a subject, preferably a mammal, more preferably a human, comprising administering to said subject an siRNA according to the invention.
  • the invention also provides the use of an siRNA according to the invention for the treatment of diseases or disorders associated with the expression or activity of a POMC nucleic acid or an ACTH polypeptide.
  • the invention also provides siRNA's according to the invention for the treatment of diseases or disorders associated with the expression or activity of a POMC nucleic acid or an ACTH polypeptide.
  • a POMC nucleic acid or an ACTH polypeptide derived therefrom include, but are not limited to, Cushing's disease, Cushing's syndrome and impaired immune response due to hypersecretion of corticosteroid.
  • siRNA's which inhibit POMC may be used to exert an effect upon the hypothalamus-pituitary-adrenal axis for initiation of pre-term labour, and for congenital adrenal hyperplasia where untreated there are high levels of pituitary POMC expression and high circulating levels of ACTH. If delivered to the CNS antagonism of POMC will be of benefit for the treatment of anorexic syndromes, including those associated with cancer cachexia and anorexia nervosa.
  • the siRNA's of the present invention may also be used to treat congenital adrenal hyperplasia, a condition in which an enzyme defect inherited in the steroid synthetic pathway in the adrenal gland results in lower levels of circulating Cortisol and hence causes ACTH levels to rise.
  • the higher levels of ACTH drive the adrenal to make pre- cursor steroids that cause hypertension, premature puberty, hirsutism, and ultimately short stature in the child, and infertility in the adult.
  • Treatment is with glucocorticoids, but be effective the levels of ACTH have to be inhibited and the side effects of existing treatments resemble Cushing's syndrome.
  • the siRNAs of the present invention could be used to lower ACTH levels to control the androgenic and mineralocorticoid effects associated with high levels of ACTH, which would allow the condition to be treated with lower doses of glucocorticoid.
  • the siRNA's of the present invention could be used alone or in combination with glucocorticoid in the treatment of congenital adrenal hyperplasia, for example the siRNA's may be administered, or prepared for administration, in a combined dosing regimen with with glucocorticoid.
  • the siRNA's of the present invention may also be used to treat small cell lung cancer (SCLC).
  • SCLC small cell lung cancer
  • the POMC gene is activated giving rise to an mRNA product that is similar in length to that seen in the pituitary, and it is likely that Cushing's syndrome is frequently not recognized.
  • the CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression (Newell-Price J et al. (2001 ) MoI Endocrinol 15(2):338-48).
  • the peptide products from POMC cleavage may confer a cellular survival advantage in an autocrine or paracrine fashion.
  • Beta-endorphin and neurotensin stimulate in vitro clonal growth of human SCLC cells (Davis TP et al (1989) Eur J Pharmacol 161 (2-3):283-5. Accordingly administration of any of the siRNA's of the present invention would be of therapeutic benefit.
  • compositions suitable for administration.
  • Such compositions typically comprise the siRNA and a pharmaceutically acceptable carrier.
  • pharmaceutically- acceptable carrier refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the targeted gene has limited tissue expression, thereby reducing the possibility of any off-target inhibition.
  • the restricted expression of the Tpit gene also makes it an attractive target for siRNA. This specificity is advantageous for an siRNA therapy.
  • the pituitary is outside the blood brain barrier and has a dense blood supply, providing an accessible administration route.
  • cholesterol conjugation with the 3' end of the sense strand by means of a pyrrolidine linker has prolonged in vivo half life to at least 95 minutes(Soutschek et al (2004) Nature, 432, 173-8).
  • Liposomal encapsulation may also be used, for example encapsulation by iontophoresis.
  • the stable nucleic acid lipid particle (SNALP) stabilized the siRNA used to target HBV RNA in a mouse model of HBV replication (Morrissey et al (2005) Nat Biotechnol, 23, 1002-7.).
  • This consists of a lipid bilayer containing a mixture of cationic and fusogenic lipid that enables the cellular uptake and endosomal release of the particle's nucleic acid. It is also coated with a diffusible polyethylene glycol lipid conjugate to neutralize the exterior and stabiles the particle. This also prevents rapid systemic clearance.
  • siRNA coding sequence may be linked to the c-terminus of the heavy chain Fab fragment in order to deliver siRNA's to specific cell targets (Song et al (2005) Nat Biotechnol, 23, 709-17).
  • siRNA's may be delivered utilising chemically modified liposomes conjugated to monocloncal antibodies (Kumar et al (2007) Nature, 448, 39- 43).
  • RVG-Arg-siRNA rabies virus in a glycoprotein envelope fused with a peptide conjugated to the siRNA
  • RVG-Arg-siRNA may be used as a delivery construct (Cantin and Rossi (2007) Nature, 448, 33-4).
  • Viral vectors such as retroviruses, may also be used as a delivery method (Ralph et al (2005) Nat Med, 1 1 , 429-33) as may adenovirus recombinant derivatives.
  • the invention provides a method of administering to a subject an siRNA according to the invention comprising contacting the subject with an siRNA, under conditions suitable for administration, i.e. in the presence of a delivery agent such as a lipid, liposome, phospholipid or liposome.
  • a delivery agent such as a lipid, liposome, phospholipid or liposome.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be topical, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal, intranasal, intracerebral or epidural and compositions of the invention are prepared accordingly.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is the amount of a composition that alone, or together with further doses, produces the desired response.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by measuring the physiological effects of the composition, such as decrease of disease symptoms etc.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • siRNA's can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. ScL USA 91 :3054-3057).
  • siRNAs were designed siRNAs to avoid homology to any other areas of the genome, and to regions of exact sequence homology in the mouse, rat and human genomes, as assessed by NCBI BLAST. Thus the sequences were the same for the coding sequence of human POMC and mouse pome. Both POMC exonic and promoter sequences were targeted. Chemically synthesised pre-annealed lyophilised siRNAs (Ambion, Applied Biosystems, Warrington UK, and Dharmacon, Perbio, Northumberland, UK) were reconstituted in filtered de-ionised distilled water prior to transfection. The Nucleotide RNAs were chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • siRNA's used are illustrated in table 3 and table 4.
  • AtT20v D16 16 cells were obtained from the European Collection of Cell Cultures. Cells were maintained in DMEM + glutamax, 10% fetal calf serum, and 1 % penicillin and streptomycin, and cultured at 37° in 5% carbon dioxide.
  • Transfections were optimized using fluorescently labelled siRNAs with a FITC fluorophor (Block-iTTM Fluorescent oligos) and Lipofectamine 2000 (both Invitrogen, Loughborough, Leicestershire, UK).
  • FITC fluorophor Bosset-iTTM Fluorescent oligos
  • Lipofectamine 2000 both Invitrogen, Loughborough, Leicestershire, UK.
  • reagents were prepared in antibiotic free OptiMEM I reduced serum media (Invitrogen, Fisher LTD Loughborough, Leicestershire, UK) as recommended by the manufacturer. Following transfection culture was maintained in antibiotic-free conditions. Fluorescent activated cell sorting (FACS) analysis and confocal microscopy were used to assess transfection efficiency. Trypan blue and cell counting were used to confirm cell viability following transfection, and found to be the same for all treatments. Transfection efficiency was >95% (data not shown).
  • the RNeasy kit (Qiagen, Crawley, Uk) was used in accordance with the manufacturers instructions. RNA was treated twice with DNAse 1 (DNA free protocol, Ambion, Warrington, UK) prior to quantification in order to eliminate any contamination with genomic DNA. Quantification of RNA was performed using Ribogreen® chemistry (Invitrogen, Paisly, UK) and plate reader settings at excitation 485 nm and emission 525 nm according to manufacturers instructions.
  • the RETRO-script® kit (Ambion) was used to make cDNA in accordance with manufacturers instructions. 2 ⁇ g of input RNA was used to make cDNA using random decamers as first strand primers (RETRO-script® kit Ambion, Applied Biosystems, Warrington UK) in accordance with manufacturers instructions.
  • RT-qPCR was carried out in MicroAmp® (Alpha Labs, Eastleigh, Hampshire, UK) Optical 96 well plates sealed with optically clear caps. Components of the reaction were made up to a final volume of 25 ⁇ l_ using the SYBR Green Core Reagent Kit and The Stratagene M x 3000 real time thermocycler (Stratagene, Europe, Amsterdam, The Netherlands). Standard curve efficiencies of 95-1 10% with Rsq values of > 0.98 were generated. cDNA for the standard curve. Standards were generated by serially diluting the stock cDNA in ddH 2 O. The same standards were included on every plate and amplified in triplicate.
  • Total cDNA input amounts for the standards ranged from 100ng to 0.41 ng in five 1 in 3 dilutions. These dilutions were found to empirically to produce optimum standard curves with all samples amplifying within the standards.
  • a negative control PCR reaction with nuclease free water was added in place of the sample cDNA, as was a no RT control from the same RT step.
  • Primers for amplification of converted DNA of POMC promoter were as follows: MBSPOMC FORWARD 5'CGG AAT TCC GTT TTT GTT TAG TTT TAA GTG GAG3' MBSPOMC REVERSE 5'GCT CTA GAC AAA ACT AAA ACA CCC TTA CCT ATC 3'.
  • the PCR product was cloned into the TOPO TA vector (Invitrogen, Loughborough, Leicestershire, UK ) and sequenced with T7 primers, using an ABI Big Dye Terminator kit and resolved on an ABI 3100 platform (both ABI, Epsom, UK).
  • siRNAs targeted to the exons 2 and 3 of POMC/pomc, regions of ( Figure 2). Sites were selected with homology to mouse, rat and man, but with no homology to other areas of the genomes. Three siRNAs, named siRNA-1 to 3, were used. Transfection efficiency as assessed by FACS analysis was >95%, (data not shown). A final concentration of 3OnM of siRNA was used in all transfections.
  • Beta-actin mRNA was found to be stable ( Figure 4d). Since siRNA-3 had the most potent effect on pome expression a longer time course was explored.
  • siRNAs directed to the promoter region of POMC exhibit potent knock down of POMC expression and significantly lower levels of secreted ACTH .
  • siRNAs targeting the promoter could induce knockdown of POMC
  • 6 siRNAs to the pituitary promoter region of POMC We specifically targeted regions including response elements for the following positively-acting transcription factors, nurr 77, neuro D1 , ptx1 and tpit, since DNA methylation may induce interference of transcription factor binding (Newell-Price J et al (2000) Trends Endocrinol Metab 1 1 :142-8).
  • 3OnM After transfection of all six siRNAs at a final combined concentration of 3OnM, cells and media were harvested at 96 and 120 hours. At 96 hours POMC expression was reduced by 96 % (p ⁇ 0.001 , Figure 6a).
  • siRNAs directed to the promoter region of POMC do not induce promoter methylation in AtT20 Cells
  • siRNAs would be particularly useful for treatment of man since in the majority of patients with Cushing's disease control of hormonal hypersecretion is a clinical priority, particularly for those 40% of patients in whom no tumour is visible on MRI (Newell-Price J et al (2006) Lancet 367:1605-17, Newell-Price J et a/ (1998) Endocr Rev 19:647-72).
  • siRNA-3 (with the greatest activity against exonic sequences) maintained potent knockdown for up to 10 days. It is predicted that repeated dosing would be even more effective.
  • RNA interference The clinical potential of RNA interference is enormous, and has clear applicability to the field of endocrinology where highly-tissue restricted hypersecretion is a common pathology.
  • TpitRNAs were prepared as described in section 1 .1 -1.4 above using the following primers: Table 7:
  • siRNA's used are illustrated in table 6.
  • Target sites for TpitsiRNAs on mouse and human Tpit are illustrated in figure 9.
  • the two test TpitsiRNAs and a negative control siRNA were each transfected into AtT20 cells at a concentration of 30 nM in duplicate. Cells were incubated for 24 hours post transfection. A 62.0% knockdown in Tpit mRNA levels was observed in TpitsiRNA 1 samples relative to No siRNA samples (p ⁇ 0.001 ) as illustrated in figure 10a. The knockdown in Tpit mRNA levels in TpitsiRNA 2 samples relative to No siRNA samples was 75.4% (p ⁇ 0.001 ) as illustrated in figure 10b.
  • TpitsiRNA or control solution were transfected with a TpitsiRNA or control solution. Complexes contained Lipofectamine 2000 and either TpitsiRNA 1 , TpitsiRNA 2, a Negative control siRNA or no siRNA. Fresh media was changed 24 hours post- transfection, and cells were cultured for a total of 72 hours post-transfection. Cells were washed, dissociated, pelleted, then resuspended in PBS to wash. Cells were then pelleted again, resuspended in PBS and counted. In the previous Western blots, 5 x 10 6 cells from a T75 cells had been lysed in 125 ⁇ l of lysis buffer.
  • TpitsiRNA 1 or TpitsiRNA 2 was caused by the siRNAs or due to lower levels of total protein in these samples, the membrane shown in Figure 15a was re-probed with an anti-alpha Tubulin antibody solution.
  • the results, pictured in Figure 15b, show that the bands produced from samples treated with TpitsiRNA 1 or TpitsiRNA 2 were as strong as those from samples treated with a control solution. This indicates that TpitsiRNA 1 and TpitsiRNA 2 caused a reduction in Tpit protein.
  • ACTH levels in Negative control siRNA samples were 8.3 % higher than in
  • TpitsiRNA 1 reduced Tpit mRNA levels by 62.0 % (p ⁇ 0.001 ) and POMC mRNA levels by 33.7%.
  • TpitsiRNA 2 reduced Tpit mRNA levels by 75.4% (p ⁇ 0.001 ) and POMC mRNA levels by 54.3 %.
  • Expression of the housekeeping gene beta Actin was constant across all treatment groups.
  • TpitsiRNA 1 reduced Tpit mRNA levels by 39.8% (p ⁇ 0.001 ) and POMC mRNA levels by 55.2% (p ⁇ 0.001 ).
  • TpitsiRNA 2 reduced Tpit mRNA levels by 62.3% and POMC mRNA levels by 71.67%.
  • tumour specimen was placed on a glass petri dish and washed twice with HBSS. Large blood clots were carefully dissected out and tissue chopped finely with a sterile scalpel. The specimen was then placed in complete media and centrifuged for 5 minutes at 2000rpm. Supernatant was removed and washing process repeated twice. This was followed by enzyme digestion with 5 mis of dispase (Gibco)
  • the specimen was placed in a sterile container with a magnetic stirrer and placed in the 37 ⁇ incubator to enzymatically digest the tissue. Depending on the amount of tissue this was for 30-60 minutes.
  • To check for cell dispersal a small amount was viewed on the haemocytometer, if adequate 5mls of complete media was added, passed through a 19 gauge needle and filtered through sterile gauze. The sample was then centrifuged at 2000rpm for 5 minutes. The supernatant was removed and a further wash step with 5 mis media performed. The cell pellet was then vortexed with 1 ml of media and the cells counted. Generally 12 x10 4 cells were plated in a 24 well plate. In general the cells remained in suspension (see figure 18).
  • Tumour cells were maintained in MEM plus 1% L-glutamine, 1% penicillin, streptomycin and 0.1 % fungizone, and 10 % FCS were added to complete the media. Cells were cultured in a 37 0 C incubator with 5% carbon dioxide.
  • siRNA 2, siRNA3 and TpitsiRNA2 The fluorescently labeled siRNAs (siRNA 2, siRNA3 and TpitsiRNA2), scrambled siRNA with no known homology to any gene sequence was used as controls (Block-iT Fluorescent oligos, Invitrogen, UK) and siRNA's of no known homology, with a FITC fluorophor, were individually transfected in to human pituitary cells with Lipofectamine 2000. Reagents were prepared in OptiMEM I reduced serum media (Invitrogen) as recommended by manufacturers. Controls of no transfection reagent and no flurorescent oligo were included. 500 ⁇ L of the complete transfection reagent was added to each well and plates gently rocked back and forth to distribute the complexes through the well evenly. The plate was then protected from light using an aluminium foil wrapping (to avoid degradation of the fluorophor) and returned to the incubator for analysis at 24 hours.
  • the encapsulated fluorescent siRNA labelled with Cy3
  • ACTH levels were measured using a chemoluminescent immunometric assay (Immunolite 2000).
  • siRNAs 2,3 and TpitsiRNA2 were transfected using lipofectamine and compared to the siRNA controls (scrambled siRNA controls and siRNA's of no known homology) at a final concentration of 30 nM. The data shown here is up to 48 hours. As the cells were predominantly in suspension, plated cells were spun to remove media then re-seeded in fresh media.
  • BALB/cANnCRL-Foxn1 nu female mice with AtT20 cells inoculated subcutaneously to form ACTH-secreting tumours (Leung C et al (1982) Pathol Anat 396:303-312; Paez- Pereda M et al (2001 ) 108:1 123-1 131 ; Heaney A et al (2002) Nature Med 8:1281 -1287). Mice were maintained at Charles River Laboratories, Kent , UK ender an handled on a defined protocol and MTA.
  • BALB/cAn NCrI- Foxni nu Female were transferred to the isolator at 5-6w/o for 1 week acclimatisation. At 6-7w.o all animals underwent subcutaneous inoculation of murine
  • AtT20 cells (2,000,000cells in 20OuI vol per animal) in to the flank. Animals were observed daily for 3 weeks to monitor how the tumour development was progressing.
  • Grp 1 Scrambled siRNA (fluorescent) control plus invivofectamine Grp 2. LNA alone (control) Grp 3. Invivofectamine alone (control) Grp 4. siRNA 2 plus invivofectamine Grp 5. siRNA 3 plus invivofectamine Grp 6. TpitsiRNA2 plus invivofectamine
  • ACTH levels were measured on the mouse plasma. ACTH levels were measured using a chemoluminescent immunometric assay (Immunolite 2000), in duplicate. Due to poor sample volume limiting data points to only few mice in the pre- siRNA dose group analysis was only performed on the samples obtained at euthanisation. Statistical analysis was performed on data from the tumours less than 10mm against the scrambled siRNA + in vivo fectamine control group. Graphpad prism version 5 was used for statistical analysis and to generate graphical data.

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Abstract

L’invention concerne des petits ARNi. Lesdits petits ARNi sont appropriés pour le traitement de maladies ou de troubles associés avec l’expression ou l’activité d’un acide nucléique POMC ou d’un polypeptide codé par l’acide nucléique POMC.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DROUIN J ET AL: "Of old and new diseases: genetics of pituitary ACTH excess (Cushing) and deficiency." CLINICAL GENETICS SEP 2007, vol. 72, no. 3, September 2007 (2007-09), pages 175-182, XP002544264 ISSN: 0009-9163 *
MUNIR A ET AL: "RNA interference as a therapy for a model of Cushing's disease" ENDOCRINE ABSTRACTS, [Online] vol. 13, no. P248, 2007, XP002534682 ISSN: 1479-6848 Retrieved from the Internet: URL:http://www.endocrine-abstracts.org/ea/0013/ea0013p248.htm> [retrieved on 2009-06-30] *
SLOMINSKI ANDRZEJ ET AL: "CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH." AMERICAN JOURNAL OF PHYSIOLOGY. ENDOCRINOLOGY AND METABOLISM APR 2005, vol. 288, no. 4, April 2005 (2005-04), pages E701-E706, XP002534683 ISSN: 0193-1849 *
TATENO TORU ET AL: "Differential gene expression in ACTH -secreting and non-functioning pituitary tumors." EUROPEAN JOURNAL OF ENDOCRINOLOGY / EUROPEAN FEDERATION OF ENDOCRINE SOCIETIES DEC 2007, vol. 157, no. 6, December 2007 (2007-12), pages 717-724, XP002534684 ISSN: 1479-683X *
VILA GREISA ET AL: "Sonic hedgehog regulates CRH signal transduction in the adult pituitary." THE FASEB JOURNAL : OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY FEB 2005, vol. 19, no. 2, February 2005 (2005-02), pages 281-283, XP002534685 ISSN: 1530-6860 *

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