WO2008109509B1 - Nucleic acid compounds for inhibiting snca gene expression and uses thereof - Google Patents

Nucleic acid compounds for inhibiting snca gene expression and uses thereof

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Publication number
WO2008109509B1
WO2008109509B1 PCT/US2008/055631 US2008055631W WO2008109509B1 WO 2008109509 B1 WO2008109509 B1 WO 2008109509B1 US 2008055631 W US2008055631 W US 2008055631W WO 2008109509 B1 WO2008109509 B1 WO 2008109509B1
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WIPO (PCT)
Prior art keywords
strand
molecule
mdrna
nucleotides
dsrna
Prior art date
Application number
PCT/US2008/055631
Other languages
French (fr)
Other versions
WO2008109509A1 (en
Inventor
Steven C Quay
James Mcswiggen
Narendra K Vaish
Mohammad Ahmadian
Original Assignee
Mdrna Inc
Steven C Quay
James Mcswiggen
Narendra K Vaish
Mohammad Ahmadian
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Mdrna Inc, Steven C Quay, James Mcswiggen, Narendra K Vaish, Mohammad Ahmadian filed Critical Mdrna Inc
Publication of WO2008109509A1 publication Critical patent/WO2008109509A1/en
Publication of WO2008109509B1 publication Critical patent/WO2008109509B1/en
Priority to US12/552,082 priority Critical patent/US20100105134A1/en
Priority to US13/327,545 priority patent/US20130011922A1/en

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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
    • C12N15/1138—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 receptors or cell surface proteins
    • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present disclosure provides meroduplex ribonucleic acid molecules (mdRNA) capable of decreasing or silencing SNCA gene expression. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to an SNCA mRNA. In addition, the meroduplex may have at least one uridine substituted with a 5-methyluridine, a nucleoside replaced with a locked nucleic acid, or optionally other modifications, and any combination thereof. Also provided are methods of decreasing expression of an SNCA gene in a cell or in a subject to treat an SNCA-related disease.

Claims

AMENDED CLAIMS received by the International Bureau on 30 September 2008 (30.09.2008)
1. A meroduplex ribonucleic acid (mdRNA) molecule that down regulates the expression of a human synuclein, alpha (non A4 component of amyloid precursor) (SNCA) mRNA, the mdRNA molecule comprising a first strand of 15 to 40 nucleotides in length that is complementary to a portion of the human SNCA mRNA as set forth in SEQ ID NOS:1158 or 1159, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double-stranded regions spaced apart by a nick or a gap.
2. The mdRNA molecule of claim 1 wherein the first strand is 15 to 25 nucleotides in length or 26 to 40 nucleotides in length.
3. The mdRNA molecule of claim 1 wherein the gap comprises from 1 to 10 unpaired nucleotides.
4. The mdRNA molecule of claim 1 wherein the mdRNA molecule comprises at least one 5-methyluridine, 2-thioribothymϊdine, or 2'-O-methyl-5- methyluridine.
5. The mdRNA molecule of claim 1 wherein the mdRNA molecule comprises at least one locked nucleic acid (LNA) molecule, deoxy nucleotide, G clamp, 2'-sugar modification, modified internucleoside linkage, or any combination thereof.
6. The mdRNA molecule of claim 1 wherein the mdRNA contains an overhang of one to four nucleotides on at least one 3 '-end that is not part of the gap or has a blunt end at one or both ends of the mdRNA.
7. An mdRNA molecule that down regulates the expression of a human SNCA πiRNA, the mdRNA molecule comprising a first strand of 15 to 40 nucleotides in length that is complementary to a portion of the human SNCA mRNA as set forth in SEQ ID NOS: 1158 or 1159, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double-stranded regions spaced apart by a nick or a gap, and wherein at least one
84 pyrimidine of the mdRNA molecule is a pyrimidine nucleosideaccording to Formula I or II:
Figure imgf000004_0001
wherein:
R1 and R2 are each independently a -H, -OH, -OCH3, -OCH2OCH2CH3, -OCH2CH2OCH3, halogen, substituted or unsubstituted C1-C]O alkyl, alkoxy, alJcoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylarninoalkyl, halσalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C2-C1O alkenyl, substituted or unsubstituted -O-aliyl, -0-CH2CH=CH2, -0-CH=CHCH3, substituted or unsubstituted C2-C1O alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH2, -NO2, -O≡, or heterocyclo group,
R3 and R4 are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an internucieoside linking group, and
R5 and Rδ are each independently O or S.
8. The mdRNA molecule of claim 7 wherein the first strand is 15 to 25 nucleotides in length or 26 to 40 nucleotides in length.
9. The mdRNA molecule of claim 7 wherein the gap comprises from 1 to 10 unpaired nucleotides.
10. The mdRNA molecule of claim 7 wherein at least one nucleoside is according to Formula I and in which R1 is methyl and R2 is -OH or -O-methyl.
11. The mdRNA molecule of claim 7 wherein at least one R2 is selected from the group consisting of 2'-0-(Ci-C5) alkyl, 2'-O-methyl, 2'-OCH2OCH2CH3, 2'-OCH2CH3OCH3, 2'-O-aIlyl, and fluoro.
85
12. The mdRN A molecule of claim 7 wherein the mdRNA molecule comprises at least one 5-methyluridine, 2-thioribothymidine, or 2'-O-methyl-5- tnethyluridine.
13. The mdRNA molecule of claim 7 wherein the mdRNA molecule comprises at least one locked nucleic acid (LNA) molecule, deoxy nucleotide, G clamp, 2'-sugar modification, modified internucleoside linkage, or any combination thereof.
14. The mdRNA molecule of claim 7 wherein contains an overhang of one to four nucleotides on at least one 3 '-end that is not a part of the gap or the dsRNA molecule has a blunt end on one or both ends of the mdRNA molecule.
15. An mdRNA molecule that down regulates the expression of a human SNCA mRNA, the mdRNA molecule comprising a first strand of 15 to 40 nucleotides in length that is complementary to a portion of the human SNCA mRNA as set forth in SEQ ID NOS :1158 or l l59, and a second strand and a third strand that is each complementary to non-overlapping regions of the first strand, wherein the second strand and third strand can anneal with the first strand to form at least two double- stranded regions spaced apart by a nick or a gap, and wherein the double-stranded regions have a combined length of about 15 base pairs to about 40 base pairs.
16. The mdRNA molecule of claim 15 wherein the first strand is 15 to 25 nucleotides in length or 26 to 40 nucleotides in length,
17. The mdRNA molecule of claim 15 wherein the gap comprises from I to 10 unpaired nucleotides.
18. The mdRNA molecule of claim 15 wherein the mdRNA molecule comprises at least one 5-methyluridine, 2-thioribo thymidine, or 2'-O-methyl-5- methyluridine,
19. The mdRNA molecule of claim 15 wherein the first strand is 19 to 23 nucleotides in length and is complementary to a human SNCA nucleic acid sequence as set forth in any one of SEQ ID NOS:1160-1417.
86
20. The mdRNA molecule of claim 15 wherein the first strand is 25 to 29 nucleotides in length, and is complementary to a human SNCA nucleic acid sequence as set forth in any one of SEQ ID NOS:1160-1417.
21. A method for reducing the expression of a human SNCA gene, comprising administering an mdRNA molecule according to any one of claims 1-20 to a cell expressing a SNCA gene, wherein the mdRNA molecule reduces the expression of the human SNCA gene in the cell.
22. The method according to claim 21 wherein the cell is a human cell.
23. Use of an mdRNA as defined in any one of the preceding claims for the manufacture of a medicament for use in the therapy of a hyperproliferative or inflammatory disease.
24. A double-stranded ribonucleic acid (dsRNA) molecule that down regulates the expression of a human synuclein, alpha (non A4 component of amyloid precursor) (SNCA) mRNA, the dsRNA molecule comprising a first strand of 26 to 40 nucleotides in length that is complementary to a portion of the human SNCA mRNA as set forth in SEQ ID NOS: 1158 or 1159, and a second strand that is complementary to the first strand, and wherein upon annealing of the first strand and the second strand the dsRNA has a 3 ' overhang and a blunt end.
25. The dsRNA molecule of claim 24 wherein the first strand is from 27 to 35 nucleotides in length.
26. The dsRNA molecule of claim 24 wherein the dsRNA molecule comprises at least one 5 -methy .uridine, 2-thioribothymidine, or 2'-O-methyl-5- methyluridine.
27. The dsRNA molecule of claim 24 wherein the dsRNA molecule comprises at least one locked nucleic acid (LNA) molecule, deoxy nucleotide, G clamp, 2-sugar modification, modified internucleoside linkage, or any combination thereof.
28. The dsRNA molecule of claim 24 wherein the 3'-overhang has from one to four nucleotides and is on the first strand.
87
29. The dsRNA molecule of claim 24 wherein the dsRNA molecule has a 5'-teπninal end comprising a hydroxy! or a phosphate.
30. A dsRNA molecule that down regulates the expression of a human SNCA mRNA, the dsRNA molecule comprising a first strand of 26 to 40 nucleotides in length that is complementary to a portion of the SNCA mRNA as set forth in SEQ ID NOS: 1158 or 1159, and a second strand that is complementary to the first strand, and wherein upon annealing of the first strand and the second strand the dsRNA has a 3' overhang and a blunt end, and wherein at least one pyrirnidine of the dsRNA molecule comprises a pyrirnidine nucleoside according to Formula I or II:
Figure imgf000007_0001
wherein:
R1 and R2 are each independently a -H, -OH, -OCH3, -OCH2OCH2CH3, -OCH2CH2OCH3, halogen, substituted or unsubstituted Ci-Ci0 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted -O-allyl, -0-CH2CH=CH2, -0-CH=CHCH3, substituted or unsubstituted C2-C10 alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, -NH2, -NO2, -C≡N, or heterocyclo group,
R3 and R4 are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an internucleoside linking group, and
R5 and R8 are each independently O or S.
31. The dsRNA molecule of claim 30 wherein the first strand is from 27 to 35 nucleotides in length.
88
32. The dsRNA molecule of claim.30 wherein at least one nucleoside is according to Formula I and in which R1 is methyl and R2 is -OH or -O-methyl.
33. The dsRNA molecule of claim 30 wherein at least one R2 is selected from the group consisting of 2'-0-(C1-C5) alkyl, 2'-O-methyL 2'-OCKbOCH2CH3, 2'-OCH2CH2OCH3, 2'-O-allyl, and 2'-fiuoro.
34. The dsRNA molecule of claim 30 wherein the dsRNA molecule comprises at least one 5-methyluridine, 2-thioribothyrnϊdine, or 2'-O-methyl-5- methyluridine,
35. The dsRNA molecule of claim 30 wherein the dsRNA molecule comprises at least one LNA, deoxy nucleotide, G clamp, 2'-sugar modification, modified intemucleoside linkage, or any combination thereof
36. The dsRNA molecule of claim 30, wherein the 3'-overhang has from one to four nucleotides and is on the first strand.
37. A method for reducing the expression of a human SNCA gene, comprising administering a dsRNA molecule according to any one of claims 24-36 to a cell expressing a SNCA gene, wherein the dsRNA molecule reduces the expression of the human SNCA gene in the cell.
38. The method according to claim 37 wherein the cell is a human cell.
39. Use of a dsRNA molecule as defined in any one of claims 24-38 for the manufacture of a medicament for use in the therapy of ahyperproliferative or inflammatory disease.
89
PCT/US2008/055631 2007-03-02 2008-03-03 Nucleic acid compounds for inhibiting snca gene expression and uses thereof WO2008109509A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US12/552,082 US20100105134A1 (en) 2007-03-02 2009-09-01 Nucleic acid compounds for inhibiting gene expression and uses thereof
US13/327,545 US20130011922A1 (en) 2007-03-02 2011-12-15 Nucleic acid compounds for inhibiting gene expression and uses thereof

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US93494007P 2007-03-02 2007-03-02
US60/934,940 2007-03-02
US93493007P 2007-03-16 2007-03-16
US60/934,930 2007-03-16
US1517107P 2007-12-19 2007-12-19
US61/015,171 2007-12-19

Related Parent Applications (1)

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Related Child Applications (3)

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PCT/US2008/055353 Continuation-In-Part WO2008109357A1 (en) 2007-03-02 2008-02-28 Nucleic acid compounds for inhibiting apob gene expression and uses thereof
US12/552,082 Continuation-In-Part US20100105134A1 (en) 2007-03-02 2009-09-01 Nucleic acid compounds for inhibiting gene expression and uses thereof
AU2009212920A Division AU2009212920A1 (en) 2007-03-02 2009-09-01 Nucleic acid compounds for inhibiting gene expression and uses thereof

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Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2076600A1 (en) 2006-10-18 2009-07-08 Nastech Pharmaceutical Company Inc. Nicked or gapped nucleic acid molecules and uses thereof
WO2009079399A2 (en) * 2007-12-14 2009-06-25 Alnylam Pharmaceuticals, Inc. Method of treating neurodegenerative disease
WO2012068405A2 (en) * 2010-11-17 2012-05-24 Isis Pharmaceuticals, Inc. Modulation of alpha synuclein expression
TWI809004B (en) 2017-11-09 2023-07-21 美商Ionis製藥公司 Compounds and methods for reducing snca expression
EP3737759A1 (en) 2018-01-12 2020-11-18 Roche Innovation Center Copenhagen A/S Alpha-synuclein antisense oligonucleotides and uses thereof

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* Cited by examiner, † Cited by third party
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EP1675948A2 (en) * 2003-10-23 2006-07-05 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED TREATMENT OF PARKINSON DISEASE USING SHORT INTERERING NUCLEIC ACID (siNA)
CA2580189C (en) * 2004-09-29 2013-05-21 Children's Memorial Hospital Sirna-mediated gene silencing of alpha synuclein
WO2007056153A2 (en) * 2005-11-04 2007-05-18 Nastech Pharmaceutical Company Inc. Peptide-dicer substrate rna conjugates as delivery vehicles for sirna
AU2007229161B2 (en) * 2006-03-23 2012-07-12 Roche Innovation Center Copenhagen A/S Small internally segmented interfering RNA
EP2076600A1 (en) * 2006-10-18 2009-07-08 Nastech Pharmaceutical Company Inc. Nicked or gapped nucleic acid molecules and uses thereof

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