EP1495041A1 - Inhibition de l'expression genique de g72 et de la d-amino acide oxydase (daao) induite par l'interference d'arn au moyen d'un acide nucleique interferant court (nasi) - Google Patents

Inhibition de l'expression genique de g72 et de la d-amino acide oxydase (daao) induite par l'interference d'arn au moyen d'un acide nucleique interferant court (nasi)

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Publication number
EP1495041A1
EP1495041A1 EP03742736A EP03742736A EP1495041A1 EP 1495041 A1 EP1495041 A1 EP 1495041A1 EP 03742736 A EP03742736 A EP 03742736A EP 03742736 A EP03742736 A EP 03742736A EP 1495041 A1 EP1495041 A1 EP 1495041A1
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Prior art keywords
sina
nucleotides
sina molecule
molecule
rna
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EP03742736A
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German (de)
English (en)
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EP1495041A4 (fr
Inventor
James Mcswiggen
Leonid Beigelman
Peter Haeberli
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Sirna Therapeutics Inc
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Sirna Therapeutics Inc
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Publication of EP1495041A1 publication Critical patent/EP1495041A1/fr
Publication of EP1495041A4 publication Critical patent/EP1495041A4/fr
Withdrawn legal-status Critical Current

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Definitions

  • a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a G72 and or DAAO gene and thereby mediate silencing of G72 and/or DAAO gene expression, for example, wherein the siNA mediates regulation of G72 and/or DAAO gene expression by cellular processes that modulate the chromatin structure of the G72 and/or DAAO gene and prevent transcription of the G72 and/or DAAO gene.
  • an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5 '-end of the sense strand, the antisense strand, or both strands.
  • an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands.
  • an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
  • any modified nucleotides present in the siNA molecules of the invention preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides.
  • the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).
  • phenotypic change is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA).
  • a nucleic acid molecule of the invention e.g., siNA
  • the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.
  • modulate is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • inhibitor it is meant that the activity of a gene expression product or level of RNAs or equivalent RNAs encoding one or more gene products is reduced below that observed in the absence of the nucleic acid molecule of the invention.
  • inhibition with a siNA molecule preferably is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response.
  • inhibition of gene expression with the siNA molecule of the instant invention is greater in the presence of the siNA molecule than in its absence.
  • amino is meant 2'-NH 2 or 2'-0- NH 2 , which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al, U.S. Pat. No. 5,672,695 and Matulic- Adamic et al, U.S. Pat. No. 6,248,878, which are both inco ⁇ orated by reference in their entireties.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types.
  • ASGPr asialoglycoprotein receptor
  • ASOR asialoorosomucoid
  • the folate receptor is overexpressed in many cancer cells.
  • siNA transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • viral DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.
  • a cleavable linker for example, a succinyl-based linker.
  • the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5'-O-DMT group while the complementary strand comprises a terminal 5'-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group.
  • Figure 2 provides an example of MALDI-TOV mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex.
  • the same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands.
  • CGE capillary gel electrophoresis
  • Ion exchange HPLC analysis of the same siNA contract only shows a single peak.
  • Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.
  • the ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
  • siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein.
  • the sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above.
  • the siNA molecules can be chemically synthesized using methods described herein.
  • Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence.
  • siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al, US Patent Nos.
  • the support is then washed and any unreacted 5 '-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • the trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage.
  • the 5 '-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.
  • RNA target for siNA mediated RNAi cleavage wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the G72 and/or DAAO RNA target, for example by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.
  • Example 7 Nucleic acid inhibition of G72 and/or DAAO target RNA in vivo siNA molecules targeted to the human G72 and/or DAAO RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the G72 and/or DAAO RNA are given in Table II and III.
  • hNT cells can be used in cell culture experiments to assess the efficacy of nucleic acid molecules of the invention.
  • hNT cells treated with nucleic acid molecules of the invention e.g., siNA
  • G72 and/or DAAO RNA would be expected to have decreased G72 and/or DAAO expression capacity following stimulation with pro-inflammatory cytokines compared to matched control nucleic acid molecules having a scrambled or inactive sequence.
  • the transgenic mice have a small thymus at necropsy, as well as reduced thymus to body weight ratio as compared to wild-type mice.
  • the homozygous mice display a stimulus processing deficit similar to that observed in schizophrenic patients.
  • Such transgenic mice are useful as models for schizophrenia and for identifying nucleic acid molecules of the invention that modulate G72 and/or DAAO gene expression and gene function toward therapeutic use in treating schizophrenia.
  • Tandem synthesis utilizes double coupling of linker molecule

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Abstract

La présente invention concerne des méthodes et des réactifs qui sont utiles pour moduler l'expression génique de G72 et/ou de la D-amino acide oxydase (DAAO), dans diverses applications, y compris dans des applications de thérapie, de diagnostic, de validation de cible et de découverte du génome. De manière plus spécifique, cette invention se rapporte à des petites molécules d'acide nucléique, telles que des petites molécules d'acide nucléique interférant (siNA), des petites molécules d'ARN interférant (siARN), des molécules d'ARN double brin, des molécules de micro-ARN et des petites molécules d'ARN en épingle à cheveux capables d'induire l'interférence d'ARN s'opposant à l'expression génique et/ou à l'activité de G72 et/ou de la D-amino acide oxydase (DAAO). Les molécules siNA sont utiles dans le traitement de la schizophrénie et ou de tout autre condition qui réagit à la modulation de l'expression ou de l'activité de G72 et/ou de DAAO.
EP03742736A 2002-02-20 2003-02-13 Inhibition de l'expression genique de g72 et de la d-amino acide oxydase (daao) induite par l'interference d'arn au moyen d'un acide nucleique interferant court (nasi) Withdrawn EP1495041A4 (fr)

Applications Claiming Priority (17)

Application Number Priority Date Filing Date Title
US35858002P 2002-02-20 2002-02-20
US358580P 2002-02-20
US36312402P 2002-03-11 2002-03-11
US363124P 2002-03-11
US38678202P 2002-06-06 2002-06-06
US386782P 2002-06-06
US40678402P 2002-08-29 2002-08-29
US406784P 2002-08-29
US40837802P 2002-09-05 2002-09-05
US408378P 2002-09-05
US40929302P 2002-09-09 2002-09-09
US409293P 2002-09-09
US43110502P 2002-12-05 2002-12-05
US431105P 2002-12-05
US44012903P 2003-01-15 2003-01-15
US440129P 2003-01-15
PCT/US2003/004397 WO2003070743A1 (fr) 2002-02-20 2003-02-13 Inhibition de l'expression genique de g72 et de la d-amino acide oxydase (daao) induite par l'interference d'arn au moyen d'un acide nucleique interferant court (nasi)

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EP1495041A1 true EP1495041A1 (fr) 2005-01-12
EP1495041A4 EP1495041A4 (fr) 2006-02-01

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Families Citing this family (6)

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US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
CN103140582A (zh) 2010-08-24 2013-06-05 默沙东公司 含有内部非核酸间隔子的单链RNAi试剂
WO2012058210A1 (fr) 2010-10-29 2012-05-03 Merck Sharp & Dohme Corp. INHIBITION FACILITÉE PAR L'INTERFÉRENCE D'ARN DE L'EXPRESSION D'UN GÈNE AU MOYEN D'ACIDES NUCLÉIQUES INTERFÉRENTS COURTS (siNA)
JP2014520811A (ja) 2011-06-29 2014-08-25 ザ トラスティース オブ コロンビア ユニバーシティ イン ザ シティ オブ ニューヨーク 統合失調症の易罹病性及び認知機能障害と関連付けられるニューロン結合の阻害剤
CA2965485A1 (fr) * 2014-10-21 2016-04-28 Martin R. Schiller Procedes et compositions permettant le depistage de fonction moleculaire comprenant des mini-motifs chimeriques

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WO2000058510A2 (fr) * 1999-03-30 2000-10-05 Genset Genes, proteines, et marqueurs bialleliques associes a la schizophrenie
WO2001009118A2 (fr) * 1999-07-29 2001-02-08 Patrick T Prendergast Composes dithiolthione pour le traitement de troubles neurologiques et pour renforcer la memoire
WO2002044321A2 (fr) * 2000-12-01 2002-06-06 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Petites molecules d'arn mediant l'interference arn
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WO2001009118A2 (fr) * 1999-07-29 2001-02-08 Patrick T Prendergast Composes dithiolthione pour le traitement de troubles neurologiques et pour renforcer la memoire
WO2002044321A2 (fr) * 2000-12-01 2002-06-06 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Petites molecules d'arn mediant l'interference arn
WO2002066672A2 (fr) * 2001-01-16 2002-08-29 Genset S.A. Traitement de troubles du systeme nerveux central au moyen d'antagonistes de la d-amino acide oxydase et de la d-aspartate oxydase

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CHUMAKOV I ET AL: "Genetic and physiological data implicating in the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 99, no. 21, 15 October 2002 (2002-10-15), pages 13675-13680, XP002967443 ISSN: 0027-8424 -& DATABASE NCBI 16 October 2002 (2002-10-16), XP002342651 Database accession no. AY138546 -& DATABASE NCBI 16 October 2002 (2002-10-16), XP002342652 Database accession no. AY138547 *
ELBASHIR S M ET AL: "Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 20, no. 23, 3 December 2001 (2001-12-03), pages 6877-6888, XP002225998 ISSN: 0261-4189 *
See also references of WO03070743A1 *

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