WO2011120023A1 - Composés d'acide nucléique pour inhiber l'expression de gène de survivine et utilisations de ceux-ci - Google Patents

Composés d'acide nucléique pour inhiber l'expression de gène de survivine et utilisations de ceux-ci Download PDF

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Publication number
WO2011120023A1
WO2011120023A1 PCT/US2011/030100 US2011030100W WO2011120023A1 WO 2011120023 A1 WO2011120023 A1 WO 2011120023A1 US 2011030100 W US2011030100 W US 2011030100W WO 2011120023 A1 WO2011120023 A1 WO 2011120023A1
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WIPO (PCT)
Prior art keywords
nucleic acid
rna
seq
antisense strand
disclosure
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PCT/US2011/030100
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English (en)
Inventor
Michael V. Templin
Michael E. Houston
Shaguna Seth
Kathy L. Fosnaugh
Narendra K. Vaish
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Marina Biotech, Inc.
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Publication of WO2011120023A1 publication Critical patent/WO2011120023A1/fr

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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
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • 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
    • C12N2310/533—Physical structure partially self-complementary or closed having a mismatch or nick in at least one of the strands

Definitions

  • the instant disclosure provides for a method for reducing the expression of a human BIRC5 gene, comprising administering a nucleic acid as disclosed herein to a cell expressing a BIRC5 gene, wherein the nucleic acid reduces expression of the BIRC5 gene in the cell.
  • the cell is a human cell.
  • nucleobase analog refers to a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring that is capable of forming Watson-Crick hydrogen bonds with a complementary nucleobase or nucleobase analog.
  • Dicer length or “Dicer length RNA complex” means a nucleic acid molecule have 25 or more base pairs, generally, from 25 to 40 base pairs.
  • nucleic acid molecules of the instant disclosure can be used to treat diseases or conditions discussed herein.
  • the dsRNA molecules can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment.
  • trifluoromethoxy refers to -OCF 3 .
  • delivery efficiency ratio refers to the ratio of the total mass of the carrier compounds in the composition or formulation to the total mass of nucleic acids in the composition or formulation.
  • BIRC5 Baculoviral IAP repeat-containing protein 5 gene
  • API4, IAP4, EPR-1 baculoviral IAP repeat-containing protein 5 gene
  • BIRC5 is an antiapoptotic protein which inhibits capsase activation (e.g., Caspase 3 and 7) and plays a role in negatively regulating apoptosis.
  • BIRC5 is thought to play a role in regulating mitosis, and expressed in most human tumors and fetal tissue, but absent in terminally differentiated cells.
  • BIRC5 and related disorders are described at the Online Mendelian Inheritance in Man database (OMIM Accession No. 603352).
  • the complete mRNA sequence of human BIRC5 has Genbank accession number NM_001012270 (SEQ ID NO: l).
  • reference to an BIRC5 mRNA or RNA sequence or sense strand means an BIRC5 RNA as set forth in SEQ ID NO: l , SEQ ID NO:2 and SEQ ID NO:3 as well as isoforms, variants, and homologs having at least 80% or more identity with the human BIRC5 mRNA sequence as set forth in SEQ ID NO: l , SEQ ID NO:2 and SEQ ID NO:3.
  • the nucleic acid further comprises that one or both of the last two positions of the 3 '-end of the sense strand are occupied by the same or different hydroxymethyi substituted nucleomonomer.
  • the nucleic acid further comprises a nucleotide analogue selected from the group consisting of 2'-0-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, PNA monomers, HNA monomers, ANA monomers, FANA monomers, CeNA monomers, ENA monomers, DNA monomers, and INA monomers.
  • a nucleotide analogue selected from the group consisting of 2'-0-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, PNA monomers, HNA monomers, ANA monomers, FANA monomers, CeNA monomers, ENA monomers, DNA monomers, and INA monomers.
  • the instant disclosure provides for the use of a nucleic acid as disclosed herein for the manufacture of a medicament for use in the therapy of cancer.
  • the positions of the sense strand may be described as follows where X represents a nucleomonomer (nucleoside or hydroxymethyl substituted
  • nucleomonomer XI occupies position 1
  • X2 occupies position 2.
  • the last two nucleomonomers of the 3'-end of the antisense strand and the last two nucleomonomers of the 3' -end of the sense strand are hydroxymethyl substituted nucleomonomers.
  • all nucleomonomers of the passenger strand of an siRNA complex of the disclosure are hydroxymethyl substituted nucleomonomers.
  • the RNA complex is single stranded and has no double stranded region.
  • the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ,25 ,26, 27, 28, 29, 30, 31, 32, 33, 34 ,35 ,36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 1, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleomonomers in length.
  • the antisense strand is 18, 19, 20, 21 , 22, 23, 24 or 25 nucleomonomers in length.
  • a conjugate molecule is attached at both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of a dsRNA molecule, or any combination thereof.
  • a conjugate molecule of this disclosure comprises a molecule that facilitates delivery of a dsRNA or analog thereof into a biological system, such as a cell.
  • a person of skill in the art can screen dsRNA of this disclosure having various conjugates to determine whether the dsRNA-conjugate possesses improved properties (e.g., pharmacokinetic profiles, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art.
  • a DILA2 amino acid compound may be admixed with a compound that releases a small molecule such as ethanol to assists in delivering an agent to a cell.
  • DELA2 amino acid compounds include (C l Oacyl)-His-Asp-NH-(C l Oalkyl),
  • cationic lipids examples include l ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l ,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA), l ,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane (DLinDMA), and l ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane
  • This invention encompasses methods for treating a disease including cancer, bladder cancer, liver cancer, liver disease, hypercholesterolemia, an inflammatory disease, a metabolic disease, inflammation, arthritis, rheumatoid arthritis, encephalitis, bone fracture, heart disease, viral disease, hepatitis, and influenza.
  • a range of novel emulsion compositions are provided in this disclosure including novel compositions and uses of oils, emulsifiers, DILA2 amino acid compounds and lipid components with interfering-RNA agents.
  • this disclosure includes compositions containing a nucleic acid molecule, such as a double-stranded RNA (dsRNA), a short interfering RNA (siRNA), or a short hairpin RNA (shRNA), admixed or complexed with a DILA2 amino acid compound, and a polymeric lipid to form a composition that enhances intracellular delivery of the nucleic acid molecule.
  • a delivery composition of this disclosure may contain a dsRNA and one, two, or more DILA2 amino acid compounds, which may be cationic or non- cationic.
  • a delivery composition may contain a dsRNA, DILA2 amino acid compounds, and one or more polymeric lipids.
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of a desired compound in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co., A.R. Gennaro edit., 1985, hereby incorporated by reference herein.
  • pharmaceutical compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof.
  • Exemplary preservatives include sodium benzoate, sorbic acid, chlorobutanol, and esters of p-hydroxybenzoic acid.
  • a dsRNA of this disclosure can include a conjugate member on one or more of the terminal nucleotides of a dsRNA.
  • the conjugate member can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide.
  • Exemplary peptide conjugate members for use within these aspects of this disclosure include peptides PN27, PN28, PN29, PN58, PN61 , PN73, PN158, PN159, PN173, PN182, PN183, PN202, PN204, PN250, PN361, PN365, PN404, PN453, PN509, and PN963, described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application Nos. 60/822,896 and 60/939,578; and PCT Application PC17US2007/075744, which are all incorporated herein by reference.
  • a dsRNA or analog thereof of this disclosure may be conjugated to the polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the dsRNA as compared to delivery resulting from contacting the target cells with a naked dsRNA.
  • the mixture, complex or conjugate comprising a dsRNA and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as LipofectineTM.
  • a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
  • test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo-treated or other suitable control subjects.
  • conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • antimetabolites e.g. , aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine
  • taxanes e.g., paclitaxel, docetaxel
  • anthracyclines e.g. , doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin
  • bleomycin mytomycin, actinomycin, hydroxyurea
  • topoisomerase inhibitors e.g. , camptothecin, topotecan, irinotecan, etoposide *teniposide
  • monoclonal antibodies e.g.
  • RNA complexes having motif 10 have one blunt-ended and a 25 base pair duplex region with two non-nucleotide hydroxymethyl nucleomonomers attached to 5 '-end of the antisense strand (or at positions 26 and 27 in the antisense strand counting from the 5 '-end of the antisense strand; the hydroxymethyl nucleomonomers occupy the last two positions of that strand counting from the 5 '-end of the strand ), and one non-nucleotide hydroxymethyl nucleomonomer attached to 3'-end of the sense strand (or at position 25 in the sense strand counting from the 5'-end of the sense strand; the hydroxymethyl nucleomonomer occupies the last position of that strand counting from the 5 '-end of the strand).
  • a scrambled sequence of a Survivin siRNA (Srv-scr; DX10103) served as the negative control RNA complex (shown below). Knockdown activity in transfected and untransfected cells was normalized to the negative control Srv-scr RNA complex and presented as a normalized value of the Srv-scr RNA complex negative control (i.e., no Survivin knockdown was represented as 0%, thus a higher percentage indicates greater knockdown).
  • KU-7 cells were transfected as described above.
  • the mRNA expression levels were measured with qRT-PCR, and protein levels were measured by ELISA.
  • Tumor size for each mouse was determined by quantifying bioluminescence and directly correlating the degree of bioluminescity with tumor size as described above. The tumor bioluminescence results are shown in Table 6, below, and in Figs. 4a and 4b.

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Abstract

La présente invention concerne des complexes d'acide nucléique bicaténaire ayant un ou plusieurs nucléomonomère(s) substitué(s) par hydroxyméthyle et où un brin est complémentaire d'un ARNm BIRC5. Les complexes d'acide nucléique de la présente invention peuvent être utiles pour des applications thérapeutiques, des applications diagnostiques ou des applications de recherche. Les complexes nucléiques comprennent des complexes d'ARN interférents courts (ARNsi) capables de moduler l'expression génique comprenant un brin antisens et un brin passager continu ou discontinu (« brin sens »). De plus, un ou plusieurs nucléomonomère(s) substitué(s) par hydroxyméthyle de la présente invention peuvent être positionnés à l'extrémité 3', à l'extrémité 5', à la fois à l'extrémité 3' et l'extrémité 5', et/ou dans une région bicaténaire d'un complexe d'acide nucléique. La présente invention concerne en outre des procédés de diminution de l'expression d'un gène BIRC5 dans une cellule ou chez un sujet pour traiter une maladie associée à BIRC5.
PCT/US2011/030100 2010-03-26 2011-03-25 Composés d'acide nucléique pour inhiber l'expression de gène de survivine et utilisations de ceux-ci WO2011120023A1 (fr)

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