WO2013173638A1 - Compositions and methods for modulating smn gene family expression - Google Patents

Compositions and methods for modulating smn gene family expression Download PDF

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Publication number
WO2013173638A1
WO2013173638A1 PCT/US2013/041440 US2013041440W WO2013173638A1 WO 2013173638 A1 WO2013173638 A1 WO 2013173638A1 US 2013041440 W US2013041440 W US 2013041440W WO 2013173638 A1 WO2013173638 A1 WO 2013173638A1
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WIPO (PCT)
Prior art keywords
single stranded
stranded oligonucleotide
nucleotide
nucleotides
oligonucleotide
Prior art date
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PCT/US2013/041440
Other languages
French (fr)
Inventor
Arthur M. Krieg
Romesh Subramanian
James Mcswiggen
Jeannie T. Lee
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Rana Therapeutics, Inc.
The General Hospital Corporation D/B/A Massachusetts General Hospital
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Publication date
Priority to US14/401,194 priority Critical patent/US10059941B2/en
Priority to SG11201407483YA priority patent/SG11201407483YA/en
Priority to AU2013262649A priority patent/AU2013262649A1/en
Priority to KR1020147035185A priority patent/KR20150030205A/en
Application filed by Rana Therapeutics, Inc., The General Hospital Corporation D/B/A Massachusetts General Hospital filed Critical Rana Therapeutics, Inc.
Priority to EP13790819.0A priority patent/EP2850186B1/en
Priority to DK13790819.0T priority patent/DK2850186T3/en
Priority to EA201492123A priority patent/EA201492123A1/en
Priority to JP2015512858A priority patent/JP2015523854A/en
Priority to CN201380037632.9A priority patent/CN104540947A/en
Priority to CA2873794A priority patent/CA2873794A1/en
Publication of WO2013173638A1 publication Critical patent/WO2013173638A1/en
Priority to IL235676A priority patent/IL235676A0/en
Priority to ZA2014/09228A priority patent/ZA201409228B/en
Priority to HK15109139.6A priority patent/HK1208700A1/en
Priority to US15/872,684 priority patent/US10837014B2/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • 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
    • C12N15/1131—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 viruses
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    • 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
    • C12N15/1131—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 viruses
    • C12N15/1132—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 viruses against retroviridae, e.g. HIV
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based compositions for treating disease.
  • SMA Spinal muscular atrophy
  • SMA type I is the most severe form and is one of the most common causes of infant mortality, with symptoms of muscle weakness and difficulty breathing occurring at birth.
  • SMA type II occurs later, with muscle weakness and other symptoms developing from ages 6 month to 2 years. Symptoms appear in SMA type III during childhood and in SMA type IV, the mildest form, during adulthood. All four types of SMA have been found to be associated with mutations in the SMN gene family, particularly SMN1.
  • SNS motor neuron
  • snRNPs small nuclear ribonucleoproteins
  • snRNPs protein-RNA complexes that bind with pre-mRNA to form a spliceosome, which then splices the pre-mRNA, most often resulting in removal of introns.
  • SMNl and SMN2 are a result of a gene duplication at 5ql3 in humans.
  • a lack of SMN activity results in widespread splicing defects, especially in spinal motor neurons, and degeneration of the spinal cord lower motor neurons.
  • aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating the expression of certain genes in cells.
  • single stranded oligonucleotides are provided that target a PRC2-associated region of a gene and thereby cause upregulation of the gene.
  • methods and related single stranded oligonucleotides that are useful for selectively inducing expression of particular splice variants of genes.
  • the methods are useful for controlling the levels in a cell of particular protein isoforms encoded by the splice variants.
  • the methods are useful for inducing expression of proteins to levels sufficient to treat disease.
  • single stranded oligonucleotides are provided that target a
  • PRC2-associated region of a SMN gene e.g., human SMNl, human SMN2
  • methods are provided for increasing expression of full-length SMN protein in a cell for purposes of treating SMA.
  • aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SMNl or SMN2 in cells.
  • single stranded oligonucleotides are provided that target a PRC2-associated region of the gene encoding SMNl or SMN2.
  • these single stranded oligonucleotides activate or enhance expression of SMNl or SMN2 by relieving or preventing PRC2 mediated repression of SMNl or SMN2.
  • the methods comprise delivering to the cell a first single stranded oligonucleotide complementary with a PRC2-associated region of an SMN gene, eg.., a PRC2-associated region of SMNl or SMN2, and a second single stranded
  • oligonucleotide complementary with a splice control sequence of a precursor mRNA of an SMN gene e.g., a precursor mRNA of SMNl or SMN2
  • a precursor mRNA of SMNl or SMN2 in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell.
  • single stranded oligonucleotides that have a region of complementarity that is complementarty with (e.g., at least 8 consecutive nucleotides of ) a PRC2-associated region of an SMN gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5.
  • the oligonucleotide has at least one of the following features: a) a sequence that is 5 'X-Y-Z, in which X is any nucleotide and in which X is at the 5 ' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine
  • the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded
  • oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has each of features a), b), c), d), and e). In certain embodiments, the oligonucleotide has the sequence 5 'X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length.
  • single stranded oligonucleotides have a sequence X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with a PRC2-associated region of an SMN gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5.
  • single stranded oligonucleotides have a sequence 5 ' -X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5.
  • Y is a sequence selected from Table 1.
  • the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 9 to 18.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087, or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087, in which the 5' end of the nucleotide sequence provided is the 5 ' end of the oligonucleotide. In some
  • the region of complementarity (e.g., the at least 8 consecutive nucleotides) is also present within the nucleotide sequence set forth as SEQ ID NO: 7 or 8.
  • a PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 9 to 14.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the oligonucleotide.
  • nucleotide sequence set forth as SEQ ID NO: 7.
  • a PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 15 to 18.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916- 3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides.
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087.
  • the oligonucleotide is up to 50 nucleotides in length.
  • the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087.
  • a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 4. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 4. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 4.
  • compounds that comprise the general formula A-B-C, wherein A is a single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mR A of the gene.
  • B comprises an oligonucleotide, peptide, low pH labile bond, or disulfide bond.
  • the splice control sequence resides in an exon of the gene.
  • the splice control sequence traverses an intron-exon junction of the gene. In some embodiments, the splice control sequence resides in an intron of the gene. In some embodiments, the splice control sequence comprises at least one hnR AP binding sequence. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
  • hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
  • the gene is SMNl or SMN2.
  • the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8 of SMNl or SMN2.
  • the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100).
  • B comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO:
  • A has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
  • the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5.
  • Y is a sequence selected from Table
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23.
  • A comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof that is at least 8 nucleotides.
  • A comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of A.
  • the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29.
  • A comprises a nucleotide sequence as set forth in any one of
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8.
  • A does not comprise three or more consecutive guanosine nucleotides.
  • A does not comprise four or more consecutive guanosine nucleotides.
  • a or C is 8 to 30 nucleotides in length. In some embodiments, A is 8 to 10 nucleotides in length and all but 1,
  • B is an oligonucleotide comprising
  • B is more susceptible to cleavage in a mammalian extract than A and C.
  • A comprises a nucleotide sequence selected from
  • GCTUTGGGAAGUAUG (SEQ ID NO: 11394), CUTUGGGAAGTATG (SEQ ID NO: 11395) and GGTACATGAGTGGCT (SEQ ID NO: 11419);
  • B comprises the nucleotide sequence TTTT or UUUU; and
  • C comprises the nucleotide sequence
  • TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO: 13091); or CTTTCATAATGCTGG (SEQ ID NO: 13092), and wherein the 3' end of A is linked to the 5' end of B, and the 3' end of B is linked to 5' end of C.
  • the single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
  • the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
  • the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, e.g., a PRC2- associated region of a nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5, in which the nucleotide sequence of the single stranded oligonucleotide comprises one or more of a nucleotide sequence selected from the group consisting of
  • At least one nucleotide of the oligonucleotide is a nucleotide analogue.
  • the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
  • At least one nucleotide of the oligonucleotide comprises a 2' O-methyl. In some embodiments, each nucleotide of the oligonucleotide comprises a 2' O- methyl. In some embodiments, the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide. In some embodiments, the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide. In some embodiments, each nucleotide of the oligonucleotide is a LNA nucleotide.
  • the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
  • the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2 '-O-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a LNA nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between between all nucleotides.
  • modified internucleotide linkages e.g., phosphorothioate internucleotide linkages or other linkages
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group. In some embodiments, the nucleotide at the 3' position of the
  • the oligonucleotide has a 3' thiophosphate.
  • the single stranded oligonucleotide has a biotin moiety or other moiety conjugated to its 5' or 3' nucleotide.
  • the single stranded oligonucleotide has cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • compositions are provided that comprise any of the oligonucleotides disclosed herein, and a carrier.
  • compositions are provided that comprise any of the oligonucleotides in a buffered solution.
  • the oligonucleotide is conjugated to the carrier.
  • the carrier is a peptide.
  • the carrier is a steroid.
  • pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
  • kits that comprise a container housing any of the compositions disclosed herein.
  • methods of increasing expression of SMNl or SMN2 in a cell involve delivering any one or more of the single stranded oligonucleotides disclosed herein into the cell.
  • delivery of the single stranded oligonucleotide into the cell results in a level of expression of SMNl or SMN2 that is greater (e.g., at least 50% greater) than a level of expression of SMNl or SMN2 in a control cell that does not comprise the single stranded oligonucleotide.
  • methods of increasing levels of SMNl or SMN2 in a subject are provided.
  • methods of treating a condition e.g., Spinal muscular atrophy
  • the methods involve
  • aspects of the invention relate to methods of increasing expression of SMN protein in a cell.
  • the method comprise delivering to the cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2- associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises exon 7 in the cell.
  • the region of complementarity with at least 8 consecutive nucleotides of a PRC2-associated region of SMN 2 has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more mismatches with a corresponding region of SMN1.
  • splice control sequence refers to a nucleotide sequence that when present in a precursor mR A influences splicing of that precursor mR A in a cell.
  • a splice control sequence includes one or more binding sites for a molecule that regulates mRNA splicing, such as a hnRNAP protein.
  • a splice control sequence comprises the sequence CAG or AAAG. In some embodiments, a splice control sequence resides in an exon (e.g., an exon of SMN1 or SMN2, such as exon 7 or exon 8). In some embodiments, a splice control sequence traverses an intron-exon junction (e.g., an intron-exon junction of SMN1 or SMN2, such as the intron 6/exon 7 junction or the intron 7/exon 8 junction). In some embodiments, a splice control sequence resides in an intron (e.g., an intron of SMN1 or SMN2, such as intron 6 or intron 7). In some embodiments, a splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof.
  • the second single stranded oligonucleotide is splice switching oligonucleotide that comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089);
  • the second single stranded oligonucleotide is 8 to 30 nucleotides in length.
  • the first single stranded oligonucleotide has a sequence 5'-X-
  • the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5.
  • Y is a sequence selected from Table 1.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof that is at least 8 nucleotides.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide.
  • the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439- 3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides.
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8.
  • the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
  • the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
  • the first single stranded oligonucleotide is 8 to 30 nucleotides in length.
  • the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
  • the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell simultaneously.
  • the cell is in a subject and the step of delivering to the cell comprises administering the first single stranded oligonucleotide and the second single stranded oligonucleotide to the subject as a co-formulation.
  • the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.
  • the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond. In some embodiments, the linker comprises an oligonucleotide, optionally wherein the oligonucleotide comprises 1 to 10 thymidines or uridines. In some embodiments, the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides. In some embodiments, the first single stranded
  • oligonucleotide is not covalently linked to the second single stranded oligonucleotide.
  • the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell separately.
  • methods for treating spinal muscular atrophy in a subject.
  • the methods comprise administering to the subject a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of SMN protein in the subject.
  • methods for treating spinal muscular atrophy in a subject that involve administering to the subject a first single stranded oligonucleotide complementary with a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of SMN protein in the subject.
  • compositions are provided that comprise a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2, and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of SMN2.
  • compositions are provided that comprise a single stranded
  • kits comprising single stranded oligonucleotides that regulate SMNl or SMN2 expression are also provided.
  • compositions are provided that comprise any of the oligonucleotides or compounds disclosed herein, and a carrier.
  • compositions are provided that comprise any of the oligonucleotides or compounds in a buffered solution.
  • the oligonucleotide is conjugated to the carrier.
  • the carrier is a peptide.
  • the carrier is a steroid.
  • pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
  • compositions that comprise a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2, and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of SMN2.
  • the splice control sequence resides in an exon of SMN2.
  • the exon is exon 7 or exon 8.
  • the splice control sequence traverses an intron-exon junction of SMN2.
  • the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction.
  • the splice control sequence resides in an intron of SMN2.
  • the intron is intron 6 or intron 7 (SEQ ID NO: 13101).
  • the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof.
  • the splice control sequence comprises at least one hnRNAP binding sequence.
  • the second single stranded oligonucleotide comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO: 13091); and
  • the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
  • the PRC2-associated region of SMN2 is a PRC2-associated region within SEQ ID NO: 1,2, 4 or 5.
  • Y is a sequence selected from Table 1.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof that is at least 8 nucleotides.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide.
  • the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides.
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8.
  • the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides. In some embodiments, the first and/or second single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
  • the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.
  • the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond.
  • the linker comprises an oligonucleotide, optionally wherein the
  • oligonucleotide comprises 1 to 10 thymidines or uridines.
  • the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides.
  • the first single stranded oligonucleotide is not covalently linked to the second single stranded oligonucleotide.
  • the composition further comprises a carrier.
  • the carrier is a
  • Further aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of SMNl or SMN2.
  • methods are provided for selecting a set of oligonucleotides that is enriched in candidates (e.g., compared with a random selection of oligonucleotides) for activating or enhancing expression of SMNl or SMN2.
  • the methods may be used to establish sets of clinical candidates that are enriched in oligonucleotides that activate or enhance expression of SMNl or SMN2.
  • Such libraries may be utilized, for example, to identify lead oligonucleotides for developing therapeutics to treat SMNl or SMN2.
  • oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and/or efficacy of the single stranded oligonucleotides for activating expression of SMNl or SMN2.
  • kits are provided that comprise a container housing any of the compositions disclosed herein.
  • kits are provided that comprise a first container housing first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene; and a second container housing a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • the splice control sequence resides in an exon of the gene.
  • the splice control sequence traverses an intron-exon junction of the gene. In some embodiments, the splice control sequence resides in an intron of the gene. In some embodiments, the splice control sequence comprises at least one hnRNAP binding sequence. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
  • hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
  • the gene is SMN1 or SMN2.
  • the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8.
  • the splice control sequence comprises the sequence:
  • the second single stranded oligonucleotide comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089);
  • the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
  • the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5.
  • Y is a sequence selected from Table 1.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof that is at least 8 nucleotides.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5 ' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide.
  • the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
  • the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29.
  • the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides.
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8.
  • FIG. 1 provides a schematic of SMN 1 and SMN2 mRNA processing
  • FIG. 2 provides a table outlining genotypes and patent information, including SMA classification, of cell lines tested in Example 2. Baseline SMN protein levels in the cell lines are also depicted.
  • FIG. 3 depicts results of RT-PCR assays showing effects on SMN mRNA expression of oligonucleotides directed against a PRC2-associated region of SMN2 (oligos 1-52 and 59-
  • FIG. 4 depicts results of RT-PCR assays showing effects on SMN mRNA expression of oligonucleotides directed against a PRC2-associated region of SMN2 (oligos 1-52 and 59-
  • FIG. 5 shows that splice switching oligonucleotides (oligoes 53-58) increase expression of full length SMN2. Results are based on a gel separation analysis of PCR products obtained following a Ddel restriction digest. Two cell lines were tested, 3813 and
  • Oligo 84 which targets a PRC2-associated region of SMN2, did not exhibit an increase in full length SMN2 expression when delivered alone to cells.
  • FIG. 6 provides results of an SMN ELISA (Enzo) showing that certain
  • oligonucleotides directed against a PRC2-associated region of SMN2 alone do not significantly increase SMN2 protein 24h post-transfection in certain SMA patient fibroblasts
  • FIG. 7 provides results of an SMN ELISA showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to Lipofectamine treated cells - dashed line).
  • FIG. 8 provides results of an SMN ELISA showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 54) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to Lipofectamine treated cells - dashed line).
  • FIG. 9 provides results of an RT-PCR assay showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to negative control oligo and Lipofectamine treated cells).
  • oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to negative control oligo and Lipofectamine treated cells).
  • oligonucleotides (DL design) were tested.
  • Table 2 Oligonucleotide sequences made for testing in the lab.
  • RQ column 2
  • RQ SE column 3
  • Table 2 shows the activity of the oligo relative to a control well (usually carrier alone) and the standard error or the triplicate replicates of the experiment, [oligo] is shown in nanomolar for in vitro experiments and in milligrams per kilogram of body weight for in vivo experiments.
  • Table 4 Oligonucleotide sequences made for testing human cells obtained from subjects with Spinal Muscular Atrophy.
  • the table shows the sequence of the modified nucleotides, where InaX represents an LNA nucleotide with 3' phosphorothioate linkage, omeX is a 2'-0-methyl nucleotide, dX is a deoxy nucleotide.
  • An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage.
  • the "-Sup" at the end of the sequence marks the fact that the 3 ' end lacks either a phosphate or thiophosphate on the 3' linkage.
  • the Formatted Sequence column shows the sequence of the oligonucleotide, including modified nucleotides, for the oligonucleotides tested in Table 2, 5, 6 and 7.
  • Appendix A contains Table 5, which shows RT-PCR data from
  • Appendix B contains Table 6, which shows RT-PCR data from
  • combination treatments e.g., two oligonucleotides, an oligonucleotide and a drug.
  • Appendix C contains Table 7, which shows ELISA data from
  • SEQID sequence identifier of base sequence of oligonucleotide used
  • Oligo Name name of oligonucleotide
  • Avg RQ average relative quantification of RT-PCR based expression levels of target gene(s);
  • Avg RQ SE standard error of mean of relative quantification of RT-PCR based expression level
  • %SMN over lipo only control refers to the ratio of SMN protein levels (ng/mg total protein) when compared to Lipofectamine2000 (transfection reagent) treated cells converted into %
  • %SMN CVV refers to coefficient of variation
  • Exp # Experiment reference number
  • Target target gene
  • [oligo] concentration of oligonucleotide used in nM unless otherwise indicated
  • Cell Line cell line used
  • Assay Type assay used
  • Time(hr) time of assay following treatment
  • 2 nd Drug name of second oligonugonu
  • SMA Spinal muscular atrophy
  • MIMs 253300, 253550, and 253400 types I, II, and III
  • type I generally understood as being the most severe.
  • Loss of function of the SMNl gene is responsible for SMA. Humans have an extra SMN gene copy, called SMN2. Both SMN genes reside within a segmental duplication on Chromosome 5ql3 as inverted repeats.
  • SMNl and SMN2 are almost identical. In some cases, SMNl and SMN2 differ by 11 nucleotide substitutions, including seven in intron 6, two in intron 7, one in coding exon 7, and one in non-coding exon 8.
  • the substitution in exon 7 involves a translationally silent C to T transition compared with SMNl, that results in alternative splicing because the substitution disrupts recognition of the upstream 3' splice site, in which exon 7 is frequently skipped during precursor mR A splicing. Consequently, SMN2 encodes primarily the exon 7-skipped protein isoform (SMNA7), which is unstable, mislocalized, and only partially functional.
  • SMNA7 exon 7-skipped protein isoform
  • Methods and related single stranded oligonucleotides that are useful for selectively inducing expression of particular splice variants of SMNl or SMN2 are provided herein.
  • the methods are useful for controlling the levels in a cell of particular SMN protein isoforms encoded by the splice variants.
  • the methods are useful for inducing expression of SMN proteins to levels sufficient to treat SMA.
  • methods are provided for increasing expression of full-length SMN protein in a cell for purposes of treating SMA.
  • the methods comprise delivering to the cell a first single stranded oligonucleotide complementary with a PRC2- associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell.
  • PRC2 Polycomb repressive complex 2
  • Polycomb repressive complex 2 (PRC2) is a histone methyltransferase and a known epigenetic regulator involved in silencing of genomic regions through methylation of histone H3.
  • PRC2 interacts with long noncoding RNAs (IncRNAs), such as RepA, Xist, and Tsix, to catalyze
  • PRC2 contains four subunits, Eed, Suzl2, RbAp48, and Ezh2. Aspects of the invention relate to the recognition that single stranded oligonucleotides that bind to PRC2-associated regions of RNAs (e.g., IncRNAs) that are expressed from within a genomic region that encompasses or that is in functional proximity to the SMNl or SMN2 gene can induce or enhance expression of SMNl or SMN2. In some embodiments, this upregulation is believed to result from inhibition of PRC2 mediated repression of SMNl or SMN2.
  • RNAs e.g., IncRNAs
  • PRC2-associated region refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2.
  • a PRC2-associated region may be present in a RNA (e.g., a long non- coding RNA (IncRNA)) that interacts with a PRC2.
  • a PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2. In some cases, the PRC2- associated region is equivalently referred to as a PRC2-interacting region.
  • a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region.
  • a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
  • a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
  • a PRC2- associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an R A-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
  • the PRC2-associated region may be referred to as a "peak.”
  • a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g. , of 40 to 60 nucleotides) that interacts with
  • a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2.
  • a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 9 to 29. In some embodiments, a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 24 to 29.
  • single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region in a genomic region that encompasses or that is in proximity to the SMN1 or SMN2 gene. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 9 to 29.
  • single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 9 to 29 combined with up to 2kb, up to 5kb, or up to lOkb of flanking sequences from a corresponding genomic region to which these SEQ IDs map (e.g., in a human genome).
  • single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID NOS: 30 to 13087.
  • single stranded oligonucleotides have a sequence as set forth in Table 2.
  • a PRC2 associated region of SMN1 or SMN2 against which a single stranded oligonucleotide is complementary is selected from SEQ ID NOS: 24-29.
  • a single stranded oligonucleotide that is complementary with a PRC2 associated region of SMNl or SMN2 comprises a sequence selected from SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916- 3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087.
  • a single stranded oligonucleotide that is complementary with a PRC2 associated region of SMNl or SMN2 comprises a sequence selected from 11395, 11394, 10169,
  • these oligonucleotides are able to interfere with the binding of and function of PRC2, by preventing recruitment of PRC2 to a specific chromosomal locus.
  • a single administration of single stranded oligonucleotides designed to specifically bind a PRC2-associated region IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin. After displacement, the full complement of PRC2 is not recovered for up to 24 hours.
  • IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.
  • Methods of modulating gene expression are provided, in some embodiments, that may be carried out in vitro, ex vivo, or in vivo. It is understood that any reference to uses of compounds throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition ⁇ e.g., Spinal muscular atrophy) associated with decreased levels or activity of SMNl or SMN2. Thus, as one nonlimiting example, this aspect of the invention includes use of such single stranded oligonucleotides in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of SMNl or SMN2.
  • methods are provided for selecting a candidate oligonucleotide for activating expression of SMNl or SMN2.
  • the methods generally involve selecting as a candidate oligonucleotide, a single stranded oligonucleotide comprising a nucleotide sequence that is complementary to a PRC2-associated region ⁇ e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 9 to 29).
  • sets of oligonucleotides may be selected that are enriched ⁇ e.g., compared with a random selection of oligonucleotides) in oligonucleotides that activate expression of SMNl or SMN2.
  • single Stranded Oligonucleotides for Modulating Expression of SMNl or SMN2 are provided for modulating expression of SMNl or SMN2 in a cell.
  • expression of SMNl or SMN2 is upregulated or increased.
  • single stranded oligonucleotides complementary to these PRC2- associated regions inhibit the interaction of PRC2 with long RNA transcripts such that gene expression is upregulated or increased.
  • oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts, resulting in reduced methylation of histone H3 and reduced gene inactivation, such that gene expression is upregulated or increased.
  • this interaction may be disrupted or inhibited due to a change in the structure of the long RNA that prevents or reduces binding to PRC2.
  • the oligonucleotide may be selected using any of the methods disclosed herein for selecting a candidate oligonucleotide for activating expression of SMNl or SMN2.
  • the single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 8.
  • oligonucleotide may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of the PRC2-associated region.
  • the PRC2-associated region may map to a position in a chromosome between 50 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 50 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 25 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 25 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 12 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 12 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 5 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 5 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene.
  • the genomic position of the selected PRC2-associated region relative to the SMNl or SMN2 gene may vary.
  • the PRC2-associated region may be upstream of the 5 ' end of the SMNl or SMN2 gene.
  • the PRC2-associated region may be downstream of the 3 ' end of the SMNl or SMN2 gene.
  • the PRC2-associated region may be within an intron of the SMNl or SMN2 gene.
  • the PRC2-associated region may be within an exon of the SMNl or SMN2 gene.
  • the PRC2-associated region may traverse an intron-exon junction, a 5 '-UTR- exon junction or a 3 '-UTR-exon junction of the SMNl or SMN2 gene.
  • the single stranded oligonucleotide may comprise a sequence having the formula X- Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of varying length.
  • X is the 5 ' nucleotide of the oligonucleotide.
  • the oligonucleotide when X is anchored at the 5 ' end of the oligonucleotide, the oligonucleotide does not have any nucleotides or nucleotide analogs linked 5 ' to X.
  • the single stranded oligonucleotide has a sequence 5 'X-Y-Z and is 8-50 nucleotides in length.
  • the Y sequence may be a nucleotide sequence of 6 nucleotides in length set forth in Table 1.
  • the single stranded oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides).
  • guanosine nucleotide stretches e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides.
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than SMNl or SMN2.
  • an oligonucleotide may be designed to ensure that it does not have a sequence that maps to any other known PRC2-associated region, particularly PRC2-associated regions that are functionally related to any other known gene (e.g., any other known protein coding gene). In either case, the oligonucleotide is expected to have a reduced likelihood of having off-target effects.
  • the threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the single stranded oligonucleotide may have a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops.
  • oligonucleotides that are complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops e.g., at least two single stranded loops
  • have a greater likelihood of being active e.g., of being capable of activating or enhancing expression of a target gene
  • the secondary structure may comprise a double stranded stem between the at least two single stranded loops. Accordingly, the region of
  • complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2-associated region that encodes at least a portion of at least two of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of the double stranded stem.
  • a PRC2-associated region (e.g., of an lncRNA) is identified (e.g., using RIP- Seq methodology or information derived therefrom).
  • the predicted secondary structure RNA (e.g., lncRNA) containing the PRC2-associated region is determined using RNA secondary structure prediction algorithms, e.g., RNAfold, mfold.
  • oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g., at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.
  • the single stranded oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content.
  • the single stranded oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
  • the sequence of the PRC2-associated region to which the single stranded oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
  • the single stranded oligonucleotide may be complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homo log of SMN1 or SMN2.
  • the single stranded oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the SMN1 or SMN2 gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of SMN1 or SMN2.
  • the single stranded oligonucleotide may be complementary to a sequence as set forth in SEQ ID NO: 1, 2, 4, or 5, which is a human genomic region encompassing or in proximity to the SMN1 or SMN2 gene, and also be complementary to a sequence as set forth in SEQ ID NO:7 or 8, which is a mouse genomic region encompassing or in proximity to the mouse homolog of the SMN1 or SMN2 gene.
  • Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease.
  • the region of complementarity of the single stranded oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or 50 consecutive nucleotides of a PRC2-associated region.
  • the region of complementarity is complementary with at least 8 consecutive nucleotides of a PRC2-associated region.
  • the sequence of the single stranded oligonucleotide is based on an RNA sequence that binds to PRC2, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
  • RNA sequence that binds to PRC2
  • portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
  • Complementary refers to the capacity for precise pairing between two nucleotides.
  • nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of PRC2-associated region
  • the single stranded nucleotide and PRC2-associated region are considered to be complementary to each other at that position.
  • the single stranded nucleotide and PRC2-associated region are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases.
  • “complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the single stranded nucleotide and PRC2-associated region.
  • a base at one position of a single stranded nucleotide is capable of hydrogen bonding with a base at the corresponding position of a PRC2-associated region, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • the single stranded oligonucleotide may be at least 80% complementary to
  • the single stranded oligonucleotide may contain 1 , 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region. In some embodiments the single stranded oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable.
  • a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target molecule (e.g., IncRNA) interferes with the normal function of the target (e.g., IncRNA) to cause a loss of activity (e.g., inhibiting PRC2-associated repression with consequent up-regulation of gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the target molecule e.g., IncRNA
  • the single stranded oligonucleotide is 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
  • the PRC2-associated region occurs on the same DNA strand as a gene sequence (sense). In some embodiments, the PRC2-associated region occurs on the opposite DNA strand as a gene sequence (anti-sense). Oligonucleotides complementary to a PRC2-associated region can bind either sense or anti-sense sequences.
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine -type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
  • GC content of the single stranded oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
  • the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome) as a single contiguous transcript (e.g., a non-spliced RNA).
  • a genome e.g., a human genome
  • a single contiguous transcript e.g., a non-spliced RNA
  • the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome), in which the distance in the genome between the 5 'end of the coding region of the RNA and the 3 ' end of the coding region of the RNA is less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7 kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.
  • a genome e.g., a human genome
  • single stranded oligonucleotides disclosed herein may increase expression of mRNA corresponding to the gene by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. It has also been found that increased mRNA expression has been shown to correlate to increased protein expression.
  • the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to the PRC2 binding RNA that is transcribed from the same strand as a protein coding reference gene.
  • the oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding sense strand of a reference gene (refGene).
  • the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to a PRC2 binding RNA that transcribed from the opposite strand (the antisense strand) of a protein coding reference gene.
  • the oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding antisense strand of a reference gene.
  • oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • the oligonucleotides can exhibit one or more of the following properties: do not induce substantial cleavage or degradation of the target RNA; do not cause
  • RNAse H pathway do not activate RNAse H pathway; do not activate RISC; do not recruit any Argonaute family protein; are not cleaved by Dicer; do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; may have improved endosomal exit; do interfere with interaction of IncRNA with PRC2, preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
  • PRC2 preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
  • Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target (e.g. , are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).
  • aspects of the invention provide strategies for targeting SMN1 or SMN2 precursor mRNA to affect splicing to minimize exon skipping. Accordingly, aspects of the invention provide therapeutic compounds useful for the treatment of SMA.
  • oligonucleotides referred to herein as "splice switching oligonucleotides” are provided that modulate SMN2 splicing.
  • Methods and related compositions, compounds, and kits are provided, in some embodiments, that are useful for increasing expression of full-length.
  • the methods generally involve delivering to a cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2- associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises (or includes) exon 7 in the cell. Any of the single stranded oligonucleotides that are complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMNl or SMN2 may be used.
  • single stranded oligonucleotides that are complementary with a splice control sequence may alternatively be referred herein, as splice switching oligonucleotides.
  • Splice switching oligonucleotides typically comprise a sequence complementary to a splice control sequence (e.g., a intronic splicing silencer sequence) of a precursor mRNA, and are capable of binding to and affecting processing of the precursor mRNA.
  • Splice switching oligonucleotides may be complementary with a region of an exon, a region of an intron or an intron/exon junction.
  • the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof. In some embodiments, the splice control sequence comprises at least one hnRNAP binding sequence. In some embodiments, splice switching oligonucleotides that target SMNl or SMN2 function based on the premise that there is a competition between the 3' splice sites of exons 7 and 8 for pairing with the 5' splice site of exon 6, so impairing the recognition of the 3' splice site of exon 8 favors exon 7 inclusion.
  • splice switching oligonucleotides are provided that promote SMN2 exon 7 inclusion and full-length SMN protein expression, in which the oligonucleotides are complementary to the intron 7/exon 8 junction.
  • splice switching oligonucleotide are composed of a segment complementary to an exon of SMNl or SMN2 (e.g., exon 7).
  • splice switching oligonucleotides comprise a tail (e.g., a non-complementary tail) consisting of RNA sequences with binding motifs recognized by a serine/arginine-rich (SR) protein.
  • SR serine/arginine-rich
  • splice switching oligonucleotides are complementary (at least partially) with an intronic splicing silencer (ISS).
  • the ISS is in intron 6 or intron 7 of SMNl or SMN2.
  • splice switching oligonucleotides comprise an antisense moiety complementary to a target exon or intron (e.g., of SMNl or SMN2) and a minimal RS domain peptide similar to the splicing activation domain of SR proteins.
  • the splice switching oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In one embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
  • any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g. , a cleavable linker.
  • a linker e.g. , a cleavable linker.
  • compounds are provided that comprise a single stranded oligonucleotide complementary with a PRC2-associated region of a gene that is linked via a linker to a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • compounds are provided that have the general formula A-B-C, in which A is a single stranded oligonucleotide complementary with a PRC2- associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • linker B comprises an oligonucleotide, peptide, low pH labile bond, or disulfide bond.
  • the compounds is orientated as 5'-A-B-C-3'. In some embodiments, the compound is orientated as 3'-A-B-C-5'.
  • the 3' end of A is linked to the 5' end of B, and the 3' end of B is linked to 5' end of C.
  • the 5' end of A is linked to the 3 ' end of B, and the 5 ' end of B is linked to 3 ' end of C.
  • the 5 ' end of A is linked to the 5' end of B, and/or the 3' end of B is linked to the 3' end of C.
  • the 3 ' end of A is linked to the 3' end of B, and/or the 5' end of B is linked to the 5' end of C.
  • linker generally refers to a chemical moiety that is capable of covalently linking two or more oligonucleotides.
  • at least one bond comprised or contained within the linker is capable of being cleaved (e.g., in a biological context, such as in a mammalian extract, such as an endosomal extract), such that at least two
  • oligonucleotides are no longer covalently linked to one another after bond cleavage.
  • a provided linker may include a region that is non- cleavable, as long as the linker also comprises at least one bond that is cleavable.
  • the linker comprises a polypeptide that is more susceptible to cleavage by an endopeptidase in the mammalian extract than the oligonucleotides.
  • the endopeptidase may be a trypsin, chymotrypsin, elastase, thermolysin, pepsin, or
  • the endopeptidase may be a cathepsin B, cathepsin D, cathepsin L, cathepsin C, papain, cathepsin S or endosomal acidic insulinase.
  • the linker comprise a peptide having an amino acid sequence selected from: ALAL, APISFFELG, FL, GFN, R/KXX, GRWHTVGLRWE, YL, GF, and FF, in which X is any amino acid.
  • the linker comprises the formula -(CH 2 ) compassionS-S(CH 2 ) m -, wherein n and m are independently integers from 0 to 10.
  • the linker may comprise an oligonucleotide that is more susceptible to cleavage by an endonuclease in the mammalian extract than the oligonucleotides.
  • the linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines.
  • the linker may have a nucleotide sequence comprising
  • the linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines linked through phosphodiester internucleotide linkages.
  • the linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines linked through phosphorothioate
  • At least one linker is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more sensitive to enzymatic cleavage in the presence of a mammalian extract than at least two oligonucleotides. It should be appreciated that different linkers can be designed to be cleaved at different rates and/or by different enzymes in compounds comprising two or more linkers. Similarly different linkers can be designed to be sensitive to cleavage in different tissues, cells or subcellular compartments in compounds comprising two or more linkers.
  • linkers are stable (e.g., more stable than the oligonucleotides they link together) in plasma, blood or serum which are richer in exonucleases, and less stable in the intracellular environments which are relatively rich in endonucleases.
  • a linker is considered “non-cleavable” if the linker's half-life is at least 24, or 28, 32, 36, 48, 72, 96 hours or longer under the conditions described here, such as in liver homogenates.
  • a linker is considered “cleavable” if the half-life of the linker is at most 10, or 8, 6, 5 hours or shorter.
  • the linker is a nuclease-cleavable oligonucleotide linker.
  • the nuclease-cleavable linker contains one or more phosphodiester bonds in the oligonucleotide backbone.
  • the linker may contain a single
  • phosphodiester bridge or 2, 3, 4, 5, 6, 7 or more phosphodiester linkages for example as a string of 1-10 deoxynucleotides, e.g., dT, or ribonucleotides, e.g., rU, in the case of RNA linkers.
  • the cleavable linker contains one or more phosphodiester linkages.
  • the cleavable linker may consist of phosphorothioate linkages only.
  • phosphorothioate-linked deoxynucleotides which in some embodiments are cleaved relatively slowly by nucleases (thus termed
  • noncleavable phosphorothioate-linked rU undergoes relatively rapid cleavage by ribonucleases and therefore is considered cleavable herein in some embodiments. It is also possible to combine dN and rN into the linker region, which are connected by phosphodiester or phosphorothioate linkages. In other embodiments, the linker can also contain chemically modified nucleotides, which are still cleavable by nucleases, such as, e.g., 2'-0-modified analogs. In particular, 2'-0-methyl or 2'-fluoro nucleotides can be combined with each other or with dN or rN nucleotides.
  • a linker is a part of the compound that is usually not complementary to a target, although it could be. This is because the linker is generally cleaved prior to action of the oligonucleotides on the target, and therefore, the linker identity with respect to a target is inconsequential. Accordingly, in some embodiments, a linker is an (oligo)nucleotide linker that is not complementary to any of the targets against which the oligonucleotides are designed.
  • the cleavable linker is an oligonucleotide linker that contains a continuous stretch of deliberately introduced Rp phosphorothioate stereoisomers (e.g., 4, 5, 6, 7 or longer stretches).
  • the Rp stereoisoform unlike Sp isoform, is known to be susceptible to nuclease cleavage (Krieg et al, 2003, Oligonucleotides, 13:491-499).
  • Such a linker would not include a racemic mix of PS linkaged oligonucleotides since the mixed linkages are relatively stable and are not likely to contain long stretches of the Rp stereoisomers, and therefore, considered “non-cleavable" herein.
  • a linker comprises a stretch of 4, 5, 6, 7 or more phosphorothioated nucleotides, wherein the stretch does not contain a substantial amount or any of the Sp stereoisoform. The amount could be considered substantial if it exceeds 10% on a per-mole basis.
  • the linker is a non-nucleotide linker, for example, a single phosphodiester bridge.
  • the linker can be designed so as to undergo a chemical or enzymatic cleavage reaction.
  • Chemical reactions involve, for example, cleavage in acidic environments (e.g., endosomes), reductive cleavage (e.g., cytosolic cleavage) or oxidative cleavage (e.g., in liver microsomes).
  • the cleavage reaction can also be initiated by a rearrangement reaction.
  • Enzymatic reactions can include reactions mediated by nucleases, peptidases, proteases, phosphatases, oxidases, reductases, etc.
  • a linker can be pH-sensitive, cathepsin- sensitive, or predominantly cleaved in endosomes and/or cytosol.
  • the linker comprises a peptide. In certain embodiments, the linker comprises a peptide which includes a sequence that is cleavable by an endopeptidase. In addition to the cleavable peptide sequence, the linker may comprise additional amino acid residues and/or non-peptide chemical moieties, such as an alkyl chain. In certain
  • the linker comprises Ala-Leu-Ala-Leu, which is a substrate for cathepsin B. See, for example, the maleimidocaproyl-Arg-Arg-Ala-Leu-Ala-Leu linkers described in
  • a cathepsin B- cleavable linker is cleaved in tumor cells.
  • the linker comprises Ala- Pro-Ile-Ser-Phe-Phe-Glu-Leu-Gly, which is a substrate for cathepsins D, L, and B (see, for example, Fischer et al, Chembiochem 2006, 7, 1428-1434).
  • a cathepsin-cleavable linker is cleaved in HeLA cells.
  • the linker comprises Phe-Lys, which is a substrate for cathepsin B.
  • the linker comprises Phe-Lys-p-aminobenzoic acid (PABA). See, e.g., the maleimidocaproyl-Phe-Lys-PABA linker described in Walker et al, Bioorg. Med. Chem. Lett. 2002, 12, 217-219.
  • the linker comprises Gly-Phe-2- naphthylamide, which is a substrate for cathepsin C (see, for example, Berg et al. Biochem. J. 1994, 300, 229-235).
  • a cathepsin C-cleavable linker is cleaved in hepatocytes.
  • the linker comprises a cathepsin S cleavage site.
  • the linker comprises Gly-Arg-Trp-His-Thr-Val-Gly-Leu- Arg-Trp-Glu, Gly-Arg-Trp-Pro-Pro-Met-Gly-Leu-Pro-Trp-Glu, or Gly-Arg-Trp-His-Pro- Met-Gly-Ala-Pro-Trp-Glu, for example, as described in Lutzner et al, J. Biol. Chem. 2008, 283, 36185-36194.
  • a cathepsin S-cleavable linker is cleaved in antigen presenting cells.
  • the linker comprises a papain cleavage site.
  • Papain typically cleaves a peptide having the sequence -R/K-X-X (see Chapman et al, Annu. Rev. Physiol 1997, 59, 63-88).
  • a papain-cleavable linker is cleaved in endosomes.
  • the linker comprises an endosomal acidic insulinase cleavage site.
  • the linker comprises Tyr-Leu, Gly-Phe, or Phe-Phe (see, e.g., Authier et al, FEBS Lett. 1996, 389, 55-60).
  • an endosomal acidic insulinase-cleavable linker is cleaved in hepatic cells.
  • the linker is pH sensitive. In certain embodiments, the linker comprises a low pH-labile bond.
  • a low-pH labile bond is a bond that is selectively broken under acidic conditions (pH ⁇ 7). Such bonds may also be termed endosomally labile bonds, because cell endosomes and lysosomes have a pH less than 7.
  • the linker comprises an amine, an imine, an ester, a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, a polyphosphazene, an acetal, a vinyl ether, a hydrazone, an azidomethyl-methylmaleic anhydride, a thiopropionate, a masked endosomo lytic agent or a citraconyl group.
  • the linker comprises a low pH-labile bond selected from the following: ketals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and a ketone; acetals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and an aldehyde; imines or iminiums that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form an amine and an aldehyde or a ketone; silicon-oxygen-carbon linkages that are labile under acidic condition; silicon-nitrogne (silazane) linkages; silicon-carbon linkages (e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamates (amide bonds synthesized from maleic anhydride derivatives and amines); ortho esters; hydrazones; activate
  • the linker comprises a masked endosomolytic agent.
  • Endosomolytic polymers are polymers that, in response to a change in pH, are able to cause disruption or lysis of an endosome or provide for escape of a normally membrane- impermeable compound, such as a polynucleotide or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome.
  • a normally membrane- impermeable compound such as a polynucleotide or protein
  • fusogenic compounds including fusogenic peptides. Fusogenic peptides can facilitate endosomal release of agents such as oligomeric compounds to the cytoplasm. See, for example, US Patent Application Publication Nos. 20040198687, 20080281041,
  • the linker can also be designed to undergo an organ/ tissue-specific cleavage. For example, for certain targets, which are expressed in multiple tissues, only the knock-down in liver may be desirable, as knock-down in other organs may lead to undesired side effects.
  • linkers susceptible to liver-specific enzymes such as pyrrolase (TPO) and glucose-6- phosphatase (G-6-Pase), can be engineered, so as to limit the antisense effect to the liver mainly.
  • linkers not susceptible to liver enzymes but susceptible to kidney- specific enzymes can be engineered, so that the antisense effect is limited to the kidneys mainly.
  • intestine-specific peptidases cleaving Phe-Ala and Leu- Ala could be considered for orally administered multimeric oligonucleotides.
  • an enzyme recognition site into the linker, which is recognized by an enzyme over-expressed in tumors, such as plasmin (e.g., PHEA-D-Val-Leu- Lys recognition site), tumor-specific knock-down should be feasible.
  • the linker can also contain a targeting signal, such as N-acetyl galactosamine for liver targeting, or folate, vitamin A or RGD-peptide in the case of tumor or activated macrophage targeting.
  • a targeting signal such as N-acetyl galactosamine for liver targeting, or folate, vitamin A or RGD-peptide in the case of tumor or activated macrophage targeting.
  • the cleavable linker is organ- or tissue-specific, for example, liver-specific, kidney-specific, intestine-specific, etc.
  • the oligonucleotides can be linked through any part of the individual oligonucleotide, e.g., via the phosphate, the sugar (e.g., ribose, deoxyribose), or the nucleobase.
  • the linker when linking two oligonucleotides together, can be attached e.g.
  • the linker when linking two oligonucleotides together, can attach internal residues of each oligonucleotides, e.g., via a modified nucleobase.
  • the linker can attach internal residues of each oligonucleotides, e.g., via a modified nucleobase.
  • OLIGONUCLEOTIDE COMPOUNDS the contents of which relating to linkers and related chemistries are incorporated herein by referenced in its entirety.
  • the target selection methods may generally involve steps for selecting single stranded oligonucleotides having any of the structural and functional characteristics disclosed herein.
  • the methods involve one or more steps aimed at identifying oligonucleotides that target a PRC2-associated region that is functionally related to SMNl or SMN2, for example a PRC2-associated region of a IncRNA that regulates expression of SMNl or SMN2 by facilitating ⁇ e.g., in a czs-regulatory manner) the recruitment of PRC2 to the SMNl or SMN2 gene.
  • Such oligonucleotides are expected to be candidates for activating expression of SMNl or SMN2 because of their ability to hybridize with the PRC2-associated region of a nucleic acid ⁇ e.g., a IncRNA).
  • this hybridization event is understood to disrupt interaction of PRC2 with the nucleic acid ⁇ e.g., a IncRNA) and as a result disrupt recruitment of PRC2 and its associated co-repressors ⁇ e.g., chromatin remodeling factors) to the SMNl or SMN2 gene locus.
  • Methods of selecting a candidate oligonucleotide may involve selecting a PRC2- associated region ⁇ e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 9 to 29) that maps to a chromosomal position encompassing or in proximity to the SMNl or SMN2 gene ⁇ e.g., a chromosomal position having a sequence as set forth in any one of SEQ ID NOS: 1 to 8).
  • the PRC2-associated region may map to the strand of the chromosome comprising the sense strand of the SMNl or SMN2 gene, in which case the candidate oligonucleotide is complementary to the sense strand of the SMNl or SMN2 gene (i.e., is antisense to the SMNl or SMN2 gene).
  • the PRC2-associated region may map to the strand of the first chromosome comprising the antisense strand of the SMNl or SMN2 gene, in which case the oligonucleotide is complementary to the antisense strand (the template strand) of the SMNl or SMN2 gene (i.e., is sense to the SMNl or SMN2 gene).
  • Methods for selecting a set of candidate oligonucleotides that is enriched in oligonucleotides that activate expression of SMNl or SMN2 may involve selecting one or more PRC2-associated regions that map to a chromosomal position that encompasses or that is in proximity to the SMNl or SMN2 gene and selecting a set of oligonucleotides, in which each oligonucleotide in the set comprises a nucleotide sequence that is complementary with the one or more PRC2-associated regions.
  • a set of oligonucleotides that is enriched in oligonucleotides that activate expression of refers to a set of oligonucleotides that has a greater number of oligonucleotides that activate expression of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of a target gene (e.g., SMN1 or SMN2) compared with a random selection of
  • oligonucleotides of the same physicochemical properties e.g. , the same GC content, T m , length etc.
  • design and/or synthesis of a single stranded oligonucleotide involves design and/or synthesis of a sequence that is complementary to a nucleic acid or PRC2- associated region described by such sequence information
  • the skilled person is readily able to determine the complementary sequence, e.g., through understanding of Watson Crick base pairing rules which form part of the common general knowledge in the field.
  • design and/or synthesis of a single stranded oligonucleotide involves manufacture of an oligonucleotide from starting materials by techniques known to those of skill in the art, where the synthesis may be based on a sequence of a PRC2- associated region, or portion thereof.
  • Methods of design and/or synthesis of a single stranded oligonucleotide may involve one or more of the steps of:
  • Single stranded oligonucleotides so designed and/or synthesized may be useful in method of modulating gene expression as described herein.
  • oligonucleotides of the invention are synthesized chemically.
  • Oligonucleotides used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques.
  • Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5 ' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2 * -0-dimethylaminoethyl (2 * -0-DMAOE), 2 * -0-dimethylaminopropyl (2 * -0-DMAP), 2 * -0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0 ⁇ N-methylacetamido (2'-0 ⁇ NMA).
  • a 2'-modified nucleotide e.g., a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminoprop
  • the nucleic acid sequence can include at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification.
  • the nucleic acids are "locked,” i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
  • any of the modified chemistries or formats of single stranded oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • the method may further comprise the steps of amplifying the synthesized single stranded oligonucleotide, and/or purifying the single stranded
  • oligonucleotide (or amplified single stranded oligonucleotide), and/or sequencing the single stranded oligonucleotide so obtained.
  • the process of preparing a single stranded oligonucleotide may be a process that is for use in the manufacture of a pharmaceutical composition or medicament for use in the treatment of disease, optionally wherein the treatment involves modulating expression of a gene associated with a PRC2-associated region.
  • a PRC2-associated region may be, or have been, identified, or obtained, by a method that involves identifying RNA that binds to PRC2.
  • Such methods may involve the following steps: providing a sample containing nuclear ribonucleic acids, contacting the sample with an agent that binds specifically to PRC2 or a subunit thereof, allowing complexes to form between the agent and protein in the sample, partitioning the complexes, synthesizing nucleic acid that is complementary to nucleic acid present in the complexes.
  • single stranded oligonucleotide is based on a PRC2-associated region, or a portion of such a sequence, it may be based on information about that sequence, e.g., sequence information available in written or electronic form, which may include sequence information contained in publicly available scientific publications or sequence databases.
  • Nucleotide Analogues are examples of nucleotide Analogues.
  • the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide.
  • the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • LNA locked nucleic acid
  • cEt constrained ethyl
  • ENA ethylene bridged nucleic acid
  • the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
  • the oligonucleotide may have one or more 2' O-methyl nucleotides.
  • the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
  • the single stranded oligonucleotide has one or more nucleotide analogues.
  • the single stranded oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
  • the single stranded oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
  • the oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15 , 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
  • the oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides).
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
  • the oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides.
  • the oligonucleotide may have a 5 ' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide).
  • the oligonucleotide may have a 5 ' nucleotide that is a deoxyribonucleotide.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5 ' and 3 ' ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides fianked by 1 , 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5 ' and 3 ' ends of the deoxyribonucleotides.
  • the 3 ' position of the oligonucleotide may have a 3 ' hydroxyl group.
  • the 3 ' position of the oligonucleotide may have a 3 ' thiophosphate.
  • the oligonucleotide may be conjugated with a label.
  • the oligonucleotide may be conjugated with a label.
  • oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5 ' or 3 ' end.
  • the single stranded oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the
  • modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the single stranded oligonucleotides are chimeric
  • oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving R A:DNA or RNA:R A hybrids. Chimeric single stranded oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
  • oligonucleosides and/or oligonucleotide mimetics as described above.
  • Such compounds have also been referred to in the art as hybrids or gapmers.
  • Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
  • the single stranded oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with
  • phosphorothioate backbones and those with heteroatom backbones particularly CH 2 -NH-O- CH 2 , CH, ⁇ N(CH 3 ) ⁇ 0 ⁇ CH 2 (known as a methylene(methylimino) or MMI backbone, CH 2 - O-N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3*-5* to 5*-3* or 2*-5* to 5*-2*; see US patent nos. 3,687,808; 4,469,863;
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol, 2002, 243, 209-214; Nasevicius et al, Nat. Genet., 2000, 26, 216- 220; Lacerra et al, Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g. , as described in Iverson, Curr. Opin. Mol. Ther., 3 :235-238, 2001 ; and Wang et al, J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al, J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones;
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al, Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • ENAs ethylene-bridged nucleic acids
  • Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids.
  • LNAs examples include compounds of the following general formula.
  • -CH 2 -0-, -CH 2 -S-, -CH 2 -N(H)-, -CH 2 -N(R)-, -CH 2 -CH 2 - or -CH 2 -CH- (if part of a double bond), -CH CH-, where R is selected from hydrogen and Ci_4-alkyl; Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
  • the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
  • the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, -0-P(O) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, o- PO(OCH 3 )-0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )- 0-, -0-P(0) 2 -NR H -, -NR H -P(0) 2 -0-, -NR H -CO
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci_4-alkyl.
  • Amino-LNA can be in both beta-D and alpha-L-configuration.
  • Oxy-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail herein.
  • One or more substituted sugar moieties can also be included, e.g. , one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group;
  • a preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'- methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F). Similar
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Single stranded oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Single stranded oligonucleotides can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substi
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al, Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi, et al, eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the single stranded oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more single stranded oligonucleotides, of the same or different types, can be conjugated to each other; or single stranded
  • oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether,
  • single stranded oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide.
  • the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
  • additional molecules e.g. a biotin moiety or a fluorophor
  • the single stranded oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
  • the single stranded oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-0- methyl nucleotides.
  • the single stranded oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a
  • the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3 ' ends of the deoxyribonucleotides.
  • the nucleotide at the 3' position of the oligonucleotide has a 3 ' hydroxyl group or a 3' thiophosphate.
  • the single stranded oligonucleotide comprises
  • the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
  • the single stranded oligonucleotide can have any combination of modifications as described herein.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • XXXXXXx in which "X” denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or R A nucleotide unit.
  • X denotes a nucleotide analogue
  • X denotes an optional nucleotide analogue
  • x denotes a DNA or R A nucleotide unit.
  • methods for increasing expression of SMN protein in a cell.
  • the methods involve delivering to the cell a first single stranded oligonucleotide complementary with a PRC2-associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell.
  • the first and second single stranded oligonucleotides may be delivered together or separately.
  • the first and second single stranded oligonucleotides may be linked together, or unlinked, i.e., separate.
  • methods for treating spinal muscular atrophy in a subject.
  • the methods involve administering to a subject a first single stranded oligonucleotide complementary with a PRC2-associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of full length SMN protein in the subject to levels sufficient to improve one or more conditions associated with SMA.
  • the first and second single stranded oligonucleotides may be administered together or separately.
  • the first and second single stranded oligonucleotides may be linked together, or unlinked, i.e., separate.
  • the first single stranded oligonucleotide may be administered within 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours or more of administration of the second single stranded oligonucleotide.
  • the first single stranded oligonucleotide may be administered before or after the second single stranded oligonucleotide.
  • the oligonucleotides may be administered once or on multiple occasions depending on the needs of the subject and/or judgment of the treating physician. In some cases, the oligonucleotides may be administered in cycles.
  • the administration cycles may vary; for example, the administration cycle may be 2 nd oligo - 1 st oligo - 2 nd oligo - 1 st oligo and so on; or 1 st oligo-2 nd oligo- 1 st oligo-2 nd oligo, and so on; or 1 st oligo - 2 nd oligo - 2 nd oligo -1 st oligo- 1 st oligo - 2 nd oligo - 2 nd oligo -1 st oligo, and so on.
  • the skilled artisan will be capable of selecting administration cycles and intervals between each administration that are appropriate for treating a particular subject.
  • the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which SMNl or SMN2 levels are reduced) for research purposes (e.g., to study the function of the gene in the cell).
  • the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which SMNl or SMN2 levels are reduced) for gene or epigenetic therapy.
  • the cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of SMNl or SMN2.
  • methods for modulating gene expression in a cell comprise delivering a single stranded oligonucleotide as described herein.
  • delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level of expression of gene in a control cell to which the single stranded
  • oligonucleotide has not been delivered.
  • delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 50%> greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered.
  • methods comprise administering to a subject (e.g. a human) a composition comprising a single stranded oligonucleotide as described herein to increase protein levels in the subject.
  • a subject e.g. a human
  • the increase in protein levels is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering.
  • the methods include introducing into the cell a single stranded oligonucleotide that is sufficiently complementary to a PRC2-associated region (e.g., of a long non-coding R A) that maps to a genomic position encompassing or in proximity to the SMNl or SMN2 gene.
  • a PRC2-associated region e.g., of a long non-coding R A
  • a condition e.g. , Spinal muscular atrophy
  • SMNl or SMN2 a condition associated with decreased levels of expression of SMNl or SMN2 in a subject
  • the method comprising administering a single stranded oligonucleotide as described herein.
  • a subject can include a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse.
  • a subject is a human.
  • Single stranded oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, including humans.
  • Single stranded oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
  • an animal preferably a human, suspected of having Spinal muscular atrophy is treated by administering single stranded oligonucleotide in accordance with this invention.
  • the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a single stranded oligonucleotide as described herein.
  • oligonucleotides described herein can be formulated for administration to a subject for treating a condition ⁇ e.g., Spinal muscular atrophy) associated with decreased levels of SMN protein. It should be understood that the formulations, compositions and methods can be practiced with any of the oligonucleotides disclosed herein. In some embodiments, formulations are provided that comprise a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • formulations comprise a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene that is linked via a linker with a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene is linked with a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene, and in other embodiments, the single stranded oligonucleotides are not linked.
  • Single stranded oligonucleotides that are not linked may be administered to a subject or delivered to a cell simultaneously (e.g., within the same composition) or separately.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient ⁇ e.g. , an oligonucleotide or compound of the invention
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g. tumor regression.
  • compositions of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • a formulated single stranded oligonucleotide composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water).
  • the single stranded oligonucleotide is in an aqueous phase, e.g., in a solution that includes water.
  • the aqueous phase or the crystalline compositions can, e.g. , be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • the single stranded oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • a single stranded oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g., a protein that complexes with single stranded oligonucleotide.
  • another agent e.g., another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g., a protein that complexes with single stranded oligonucleotide.
  • Still other agents include chelators, e.g., EDTA (e.g., to
  • RNAse inhibitors e.g., a broad specificity
  • RNAse inhibitor such as RNAsin
  • the other agent used in combination with the single stranded oligonucleotide is an agent that also regulates SMN expression.
  • the other agent is a growth hormone, a histone deacetylase inhibitor, a hydroxycarbamide (hydroxyurea), a natural polyphenol compound (e.g., resveratrol, curcumin), prolactin, or salbutamol.
  • histone deacetylase inhibitors examples include aliphatic compounds (e.g., butyrates (e.g., sodium butyrate and sodium phenylbutyrate) and valproic acid), benzamides (e.g., M344), and hydroxamic acids (e.g., CBHA, SBHA, Entinostat (MS-275)) Panobinostat (LBH-589), Trichostatin A, Vorinostat (SAHA)),
  • aliphatic compounds e.g., butyrates (e.g., sodium butyrate and sodium phenylbutyrate) and valproic acid
  • benzamides e.g., M344
  • hydroxamic acids e.g., CBHA, SBHA, Entinostat (MS-275)
  • Panobinostat LH-589
  • Trichostatin A Vorinostat (SAHA)
  • the single stranded oligonucleotide preparation includes another single stranded oligonucleotide, e.g. , a second single stranded oligonucleotide that modulates expression and/or mR A processing of a second gene or a second single stranded
  • the single stranded oligonucleotide preparation includes at least a second therapeutic agent ⁇ e.g., an agent other than an oligonucleotide).
  • a composition that includes a single stranded oligonucleotide can be delivered to a subject by a variety of routes.
  • routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular.
  • therapeutically effective amount is the amount of oligonucleotide present in the composition that is needed to provide the desired level of SMNl or SMN2 expression in the subject to be treated to give the anticipated physiological response.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxico logical effects to the subject.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of single stranded oligonucleotide and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
  • the route and site of administration may be chosen to enhance targeting.
  • intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the single stranded oligonucleotide in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the single stranded oligonucleotide and mechanically introducing the oligonucleotide.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
  • the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • the most common topical delivery is to the skin.
  • the term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum.
  • Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition.
  • Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
  • the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin.
  • Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
  • transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
  • iontophoresis transfer of ionic solutes through biological membranes under the influence of an electric field
  • phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
  • optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
  • oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
  • GI gastrointestinal
  • compositions can be targeted to a surface of the oral cavity, e.g. , to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
  • the sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
  • a pharmaceutical composition of single stranded oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant.
  • the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration.
  • parental administration involves administration directly to the site of disease (e.g. injection into a tumor).
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • any of the single stranded oligonucleotides described herein can be administered to ocular tissue.
  • the compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid.
  • ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers.
  • Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly( vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
  • the single stranded oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably single stranded oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A single stranded oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • the term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli.
  • the powder is said to be "respirable.”
  • the average particle size is less than about 10 ⁇ in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ⁇ m and most preferably less than about 5.0 ⁇ m.
  • the particle size distribution is between about 0.1 ⁇ m and about 5 ⁇ m in diameter, particularly about 0.3 ⁇ m to about 5 ⁇ m.
  • dry means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w.
  • a dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
  • HSA human serum albumin
  • Suitable H adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • Pulmonary administration of a micellar single stranded oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
  • propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
  • Exemplary devices include devices which are introduced into the vasculature, e.g. , devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
  • Other devices include non-vascular devices, e.g., devices implanted in the
  • the device can release a therapeutic substance in addition to a single stranded oligonucleotide, e.g., a device can release insulin.
  • unit doses or measured doses of a composition that includes single stranded oligonucleotide are dispensed by an implanted device.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • Tissue e.g., cells or organs can be treated with a single stranded oligonucleotide, ex vivo and then administered or implanted in a subject.
  • the tissue can be autologous, allogeneic, or xenogeneic tissue.
  • tissue can be treated to reduce graft v. host disease .
  • the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue.
  • tissue e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.
  • the single stranded oligonucleotide treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body.
  • the porous barrier is formed from alginate.
  • a contraceptive device is coated with or contains a single stranded oligonucleotide. Exemplary devices include condoms, diaphragms, IUD
  • the invention features a method of administering a single stranded oligonucleotide ⁇ e.g., as a compound or as a component of a composition) to a subject ⁇ e.g., a human subject).
  • the methods involve administering a compound (e.g., a compound of the general formula A-B-C, as disclosed herein, or an single stranded oligonucleotide,) in a unit dose to a subject.
  • the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight.
  • the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the SMN1 or SMN2.
  • the unit dose for example, can be administered by injection ⁇ e.g., intravenous or intramuscular), an inhaled dose, or a topical application.
  • the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency ⁇ e.g., not a regular frequency). For example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g. , once an hour, two hours, four hours, eight hours, twelve hours, etc.
  • a subject is administered an initial dose and one or more maintenance doses of a single stranded oligonucleotide.
  • the maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg ofbody weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day.
  • the maintenance doses may be administered no more than once every 1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semipermanent stent ⁇ e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semipermanent stent ⁇ e.g., intravenous, intraperitoneal, intracisternal or intracapsular
  • reservoir may be advisable.
  • the pharmaceutical composition includes a plurality of single stranded oligonucleotide species.
  • the pharmaceutical composition comprises a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene (e.g., SMN1 or SMN2), and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of a gene (e.g., SMN1 or SMN2).
  • the pharmaceutical composition includes a compound comprising the general formula A-B-C, in which A is a single stranded oligonucleotide complementary with a PRC2-associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • the single stranded oligonucleotide species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence ⁇ e.g., a PRC2-associated region).
  • the plurality of single stranded oligonucleotide species is specific for different PRC2-associated regions.
  • the single stranded oligonucleotide is allele specific. In some cases, a patient is treated with a single stranded oligonucleotide in conjunction with other therapeutic modalities.
  • the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
  • the concentration of the single stranded oligonucleotide composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.
  • concentration or amount of single stranded oligonucleotide administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary.
  • nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
  • treatment of a subject with a therapeutically effective amount of a single stranded oligonucleotide can include a single treatment or, preferably, can include a series of treatments.
  • the effective dosage of a single stranded oligonucleotide used for treatment may increase or decrease over the course of a particular treatment.
  • the subject can be monitored after administering a single stranded oligonucleotide composition. Based on information from the monitoring, an additional amount of the single stranded
  • oligonucleotide composition can be administered.
  • Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of SMN1 or SMN2 expression levels in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing
  • the animal models include transgenic animals that express a human SMN1 or SMN2.
  • the composition for testing includes a single stranded oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between SMN1 or SMN2 in the animal model and the SMN1 or SMN2 in a human.
  • the administration of the single stranded oligonucleotide composition is parenteral, e.g.
  • intravenous ⁇ e.g., as a bolus or as a diffusible infusion
  • intradermal intraperitoneal
  • intramuscular intrathecal
  • intraventricular intracranial
  • subcutaneous transmucosal
  • buccal sublingual
  • endoscopic rectal
  • oral vaginal
  • topical pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • kits comprising a container housing a composition comprising a single stranded oligonucleotide.
  • the kits comprise a container housing a single stranded oligonucleotide complementary with of a PRC2-associated region of a gene; and a second container housing a single stranded oligonucleotide complementary to a splice control sequence of a precursor mR A of the gene.
  • kits comprise a container housing a single stranded oligonucleotide complementary with of a PRC2-associated region of a gene and a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
  • the composition is a pharmaceutical composition comprising a single stranded oligonucleotide and a pharmaceutically acceptable carrier.
  • the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for single stranded oligonucleotides, and at least another for a carrier compound.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • Example 1 Oligonucleotides targeting PRC2-associated regions that upregulate SMN1.
  • MA TERIALS AND METHODS Real Time PCR
  • Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatoma Hepal-6 cells, and human renal proximal tubule epithelial cells (RPTEC) were cultured using conditions known in the art (see, e.g. Current Protocols in Cell Biology).
  • Other cell lines tested were neuronal cell lines (SK-N-AS, U-87) and SMN patient fibroblasts. Details of the cell lines used in the experiments described herein are provided in Table 8.
  • Oligonucleotides were designed within PRC2-interacting regions in order to upregulate SMNl .
  • the sequence and structure of each oligonucleotide is shown in Table 2.
  • the following table provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 2.
  • Oligonucleotides were designed as candidates for upregulating SMNl expression. A total of 52 single stranded oligonucleotides were designed to be complementary to a PRC2- interacting region within a sequence as set forth in SEQ ID NO: 1, 2, 4, or 5.
  • Oligonucleotides were tested in at least duplicate. The sequence and structural features of the oligonucleotides are set forth in Table 2. Briefly, cells were transfected in vitro with the oligonucleotides as described above. SMNl expression in cells following treatment was evaluated by qRT-PCR. Oligonucleotides that upregulated SMNl expression were identified. Further details are outlined in Table 2.
  • Table 5 shows further results from experiments in which oligonucleotides were transfected into cells at a particular concentration [oligo] and 48 or 72h later RNA was prepared and qRTPCR assays carried out to determine mRNA levels of full length (FL) or delta7 SMN.
  • oligos were administered gymnotically to cells at ⁇ and R A harvested 9 days post treatment.
  • the cell lines tested were neuronal cell lines (SK-N- AS, U-87) and SMN patient fibroblasts.
  • Table 6 shows results from experiments in which oligonucleotides were transfected into cells in combination with either one or two more oligos or small molecule compounds at a particular concentration ([oligo], [2nd], [3rd]) and 48 or 72h later RNA was prepared and qRTPCR assays carried out to determine mRNA levels of full length (FL) or delta7 SMN.
  • the cell lines tested were SMN patient fibroblasts.
  • Table 7 shows results from experiments in which oligonucleotides were transfected into cells in combination with either one or two more oligos or as dimers or by gymnotic treatment at a particular concentration ([oligo], [2nd], [3rd]) and 24, 48, 72 or 216h later cell lysates were prepared and ELISA assays carried out to determine SMN protein levels.
  • the cell lines tested were SMN patient fibroblasts.
  • GGGGGU GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGU UA, GGGU UG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,
  • GGUACU GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GGUCGG, GGUCGU, GGUCUC, GGUCU U, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGU UAA, GGU UAG, GGU UAU, GGUUCA, GGU UCC, GGU UCG, GGU UGC, GGU UUC, GGUU UU, GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA, GUAAUC, GUAAUG, GUAAUU,
  • GUAGGU GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAU UG, G UAU UU, GUCAAA, GUCAAG, GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, G UCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCU U, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG,
  • GUGCAU GUGCCC
  • GUGCCG GUGCGA
  • GUGCGG GUGCGU
  • GUGCUA GUGCUC
  • GUGCUG GUGCAU
  • GUGGAG GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGU UU, GU UAAA, GU UAAC, GUUAAG, GU UACA, GU UACC, GUUACG, GU UACU, GU UAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG,
  • GU UAUU GUUCAA, GUUCAC, GUUCAG, GU UCCA, GUUCCG, GUUCGA, GU UCGC, GU UCGG, GU UCGU, GUUCUA, GUUCUG, GUUGAA, GU UGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GU UGGC, GU UGGU, GU UGUC, GUUGUG, GUUGU U, GUU UAA, GU UAC, GU UUAG, GUU UAU, GU UUCA, GUUUCC, GU UUCU, GU UUGA, GUU UGC, GUU UGG, GUU UGU, GU UUUA, GU UU UC, GU UU UU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU,
  • Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
  • SMN1 1.9782541 1.3529511 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21170U20 -14 54 56
  • SeqID range 30 to 8329, 13088-13094 SeqIDs w/o G Runs: 30-142, 156-560, 575-780, 794-912, 926-1013, 1027-1078, 1092-1286, 1300-1335, 1349- 1385, 1399-1453, 1460-1527, 1548-1555, 1571-1653, 1675-1691, 1706-1802, 1816-1883, 1897-2009, 2023-2141, 2165-2289, 2303-2320, 2334-2447, 2461-2494, 2508-2526, 2540- 2545, 2571-2635, 2651-2670, 2689-2763, 2772-2814, 2828-2854, 2868-3030, 3044-3256, 3270-3360, 3374-3400, 3414-3722, 3737, 3759-3783, 3797-3970, 3986-4059, 4073-4153, 4175-4240, 4255-4415, 4438-4441, 4456-4472, 4484-
  • SeqID range 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, 13062-13087
  • Example 2 Selective upregulation of exon 7 containing SMN2 transcripts using oligonucleotides targeting PRC2-interacting regions that upregulate SMN2 and splice- switching oligonucleotides.
  • Oligonucleotides targeting PRC2-interacting regions (IncR A peaks) in the SMN1/2 gene loci were designed. These oligos were synthesized with various DNA base
  • SSO Splice switching oligos
  • SMA fibroblast cell lines and one lymphoblast cell line were obtained from the Coriell Institute (FIG. 2).
  • the cells were either transfected with the oligos using Lipofectamine 2000 (Fibroblasts) or by electroporation or unassisted delivery (lymphoblast) to ascertain effects of the oligonucleotides on SMNl/2 mRNA and protein expression. All experiments were carried out as biological triplicates.
  • transfections were performed using Lipofectamie2000 per manufacturer's instructions with oligos at either ⁇ or 30nM.
  • SMN ELISA to determine SMN protein was carried out per manufacturer's instructions (SMN ELISA kit #ADI-900-209, Enzo Life Sciences). Briefly, SMN fibroblasts were cultures at 40,000 cells/well of a 24-well tissue culture coated plate on day 1. Cells were transfected with the oligos using Lipofectamine2000 on day 2 and cell lysates prepared at 24 and 48 hours post-treatment. ELISA was carried out per manufacturer's instructions.
  • Splice switching assay (Ddel assay) - SMN2-derived transcripts contain a unique Ddel restriction element introduced because of a nucleotide polymorphism not present in SMNl and are differentiated from SMNl -derived transcripts because of the faster migration of the SMN2 products.
  • Ddel assay Splice switching assay
  • SMA fibroblasts were treated with oligonucleotides targeting PRC2-interacting regions with or without SSO at 30nM each as described before.
  • RT-PCR was carried out with an SMN exon 5 forward primer and an exon 8 reverse primer to generate cDNAs that were then digested with Ddel.
  • the SMNl transcript if present, migrates at a slower rate than the Ddel- digested SMN2 transcript and is seen as the first band from the top of the gel.
  • the second band from the top indicates full length SMN2 (accurately spliced form) and the third band indicates the incorrectly spliced SMN2delta7. (FIG. 5)
  • the SMN1 gene is often mutated in such a way that it is unable to correctly code the SMN protein - due to either a deletion encompassing at least a portion of exon 7 or to other mutations.
  • SMA patients generally retain at least one copy of the SMN2 gene (with many having 2 or more copies) that still expresses small amounts of SMN protein.
  • the SMN2 gene has a C to T mutation (compared with SMN1) in exon 7 that alters splicing of its precursor mRNA such that exon 7 is spliced out at a high frequency. Consequently, only about 10% of the normal levels of full length SMN protein are produced from SMN2. (See FIG. 1)
  • SMA fibroblast cell lines and one lymphoblast cell line were obtained from the Coriell Institute (FIG. 2).
  • Cells were transfected with oligonucleotides (oligos 1-52 and 59- 101) directed against a PRC2-associated region of SMN2 and RT-PCR assays were conducted to evaluate effects on SMN mRNA expression. (See FIGS. 3 and 4 for results in cell lines 3814 and 3813).
  • oligonucleotides oligos 1-52 and 59- 101
  • RT-PCR assays were conducted to evaluate effects on SMN mRNA expression.
  • oligonucleotides (oligos 53-58) directed at a splice control sequence in intron 7 of SMN2 and RT-PCR assays were conducted to evaluate effects on SMN mRNA expression (See FIGS. 3 and 4 for results in cell lines 3814 and 3813).
  • Splice switching oligonucleotides (oligos 53- 58) were found to increase expression of full length SMN2 based on a gel separation analysis of PCR products obtained following a Ddel restriction digest; whereas certain
  • SMN ELISA (Enzo) assays were conducted and revealed that certain oligonucleotides directed against a PRC2-associated region of SMN2 alone did not significantly increase full length SMN protein 24h post-transfection in certain SMA patient fibroblasts.
  • FIG. 6 the same SMN ELISA assays showed that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increase full length SMN protein 24h post-transfection in SMA patient fibroblasts above that observed with splice switching oligonucleotides alone.
  • RT-PCR assays were conducted and showed that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increased SMN2 protein 24h post-transfection in SMA patient fibroblasts.
  • oligo 53 or 54 a splice switching oligonucleotide
  • FIG. 9. These experiments were conducted modified oligonucleotides with either alternating LNA and 2'OMe nucleotides or alternating DNA and LNA nucleotides.
  • Example 2 show that certain oligos targeting PRC2 associated regions of SMN2 induce SMN RNA expression (e.g.., of the SMNA7 transcript) SMA patient-derived fibroblasts.
  • SMN RNA expression e.g.., of the SMNA7 transcript
  • results show that, in some embodiments, splice- switching oligos may not induce SMN RNA expression, but rather shift SMN RNA splicing to the full-length transcript.
  • combination of splice switching oligos with PRC2-associated region targeting oligos substantially increases full length SMN protein in cells from SMA patients.
  • Table 4 Oligonucleotide sequences made for testing human cells obtained from subjects with Spinal Muscular Atrophy (See Table 3 for structural features of formatted sequence).

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of SMNl or SMN2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of SMNl or SMN2 that comprises exon 7. Methods for modulating expression of SMNl or SMN2 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of SMNl or SMN2.

Description

COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY
EXPRESSION CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/785,529, entitled "COMPOSITIONS AND METHODS FOR
MODULATING SMN GENE FAMILY EXPRESSION", filed March 14, 2013; U.S.
Provisional Application No. 61/719,394, entitled "COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION", filed October 27, 2012; and U.S. Provisional Application No. 61/647,858, entitled "COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION", filed May 16, 2012, the contents of each of which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION
The invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based compositions for treating disease.
BACKGROUND OF THE INVENTION
Spinal muscular atrophy (SMA) is a group of hereditary diseases that causes muscle damage leading to impaired muscle function, difficulty breathing, frequent respiratory infection, and eventually death. There are four types of SMA that are classified based on the onset and severity of the disease. SMA type I is the most severe form and is one of the most common causes of infant mortality, with symptoms of muscle weakness and difficulty breathing occurring at birth. SMA type II occurs later, with muscle weakness and other symptoms developing from ages 6 month to 2 years. Symptoms appear in SMA type III during childhood and in SMA type IV, the mildest form, during adulthood. All four types of SMA have been found to be associated with mutations in the SMN gene family, particularly SMN1.
Survival of motor neuron (SMN) is a protein involved in transcriptional splicing through its involvement in assembly of small nuclear ribonucleoproteins (snRNPs). snRNPs are protein-RNA complexes that bind with pre-mRNA to form a spliceosome, which then splices the pre-mRNA, most often resulting in removal of introns. Three genes encode SMN or a variant of SMN, including SMN1 (survival of motor neuron 1, telomeric), SMN2 (survival of motor neuron 2, centromeric), and SMNP (survival of motor neuron 1, telomeric pseudogene). SMNl and SMN2 are a result of a gene duplication at 5ql3 in humans. A lack of SMN activity results in widespread splicing defects, especially in spinal motor neurons, and degeneration of the spinal cord lower motor neurons.
SUMMARY OF THE INVENTION
Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating the expression of certain genes in cells. In some embodiments, single stranded oligonucleotides are provided that target a PRC2-associated region of a gene and thereby cause upregulation of the gene. Also provided herein are methods and related single stranded oligonucleotides that are useful for selectively inducing expression of particular splice variants of genes. In some embodiments, the methods are useful for controlling the levels in a cell of particular protein isoforms encoded by the splice variants. In some cases, the methods are useful for inducing expression of proteins to levels sufficient to treat disease.
In some embodiments, single stranded oligonucleotides are provided that target a
PRC2-associated region of a SMN gene (e.g., human SMNl, human SMN2) and thereby cause upregulation of the gene. For example, according to some aspects of the invention methods are provided for increasing expression of full-length SMN protein in a cell for purposes of treating SMA. Accordingly, aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating SMNl or SMN2 in cells. In some embodiments, single stranded oligonucleotides are provided that target a PRC2-associated region of the gene encoding SMNl or SMN2. In some embodiments, these single stranded oligonucleotides activate or enhance expression of SMNl or SMN2 by relieving or preventing PRC2 mediated repression of SMNl or SMN2.
In some embodiments, the methods comprise delivering to the cell a first single stranded oligonucleotide complementary with a PRC2-associated region of an SMN gene, eg.., a PRC2-associated region of SMNl or SMN2, and a second single stranded
oligonucleotide complementary with a splice control sequence of a precursor mRNA of an SMN gene, e.g., a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell.
According to some aspects of the invention single stranded oligonucleotides are provided that have a region of complementarity that is complementarty with (e.g., at least 8 consecutive nucleotides of ) a PRC2-associated region of an SMN gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5. In some embodiments, the oligonucleotide has at least one of the following features: a) a sequence that is 5 'X-Y-Z, in which X is any nucleotide and in which X is at the 5 ' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine
nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and e) a sequence that has greater than 60% G-C content. In some embodiments, the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded
oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has each of features a), b), c), d), and e). In certain embodiments, the oligonucleotide has the sequence 5 'X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length.
According to some aspects of the invention, single stranded oligonucleotides are provided that have a sequence X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with a PRC2-associated region of an SMN gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5. In some aspects of the invention, single stranded oligonucleotides are provided that have a sequence 5 ' -X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 9 to 18.
In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087, or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087, in which the 5' end of the nucleotide sequence provided is the 5 ' end of the oligonucleotide. In some
embodiments, the region of complementarity (e.g., the at least 8 consecutive nucleotides) is also present within the nucleotide sequence set forth as SEQ ID NO: 7 or 8.
In some embodiments, a PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 9 to 14. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to
13094 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the oligonucleotide. In some embodiments, the at least 8
consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
In some embodiments, a PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 15 to 18. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916- 3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087. In some embodiments, the oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087.
In some embodiments, a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 4. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 4. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 4.
According to some aspects of the invention, compounds are provided that comprise the general formula A-B-C, wherein A is a single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mR A of the gene. In some embodiments, B comprises an oligonucleotide, peptide, low pH labile bond, or disulfide bond. In some embodiments, the splice control sequence resides in an exon of the gene. In some embodiments, the splice control sequence traverses an intron-exon junction of the gene. In some embodiments, the splice control sequence resides in an intron of the gene. In some embodiments, the splice control sequence comprises at least one hnR AP binding sequence. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
In some embodiments, the gene is SMNl or SMN2. In some embodiments, the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8 of SMNl or SMN2. In some embodiments, the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100). In some embodiments, B comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO:
13090); ACTTTCATAATGCTG (SEQ ID NO: 13090); and CTTTCATAATGCTGG (SEQ ID NO: 13092). In some embodiments, A has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length. In some embodiments, the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In some embodiments, Y is a sequence selected from Table
1. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23. In some embodiments, A comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof that is at least 8 nucleotides. In some embodiments, A comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of A. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29.
In some embodiments, A comprises a nucleotide sequence as set forth in any one of
SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, A does not comprise three or more consecutive guanosine nucleotides. In some embodiments, A does not comprise four or more consecutive guanosine nucleotides. In some embodiments, A or C is 8 to 30 nucleotides in length. In some embodiments, A is 8 to 10 nucleotides in length and all but 1,
2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides. In some embodiments, B is an oligonucleotide comprising
1 to 10 thymidines or uridines. In some embodiments, B is more susceptible to cleavage in a mammalian extract than A and C.
In some embodiments, A comprises a nucleotide sequence selected from
GCTUTGGGAAGUAUG (SEQ ID NO: 11394), CUTUGGGAAGTATG (SEQ ID NO: 11395) and GGTACATGAGTGGCT (SEQ ID NO: 11419); B comprises the nucleotide sequence TTTT or UUUU; and C comprises the nucleotide sequence
TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO: 13091); or CTTTCATAATGCTGG (SEQ ID NO: 13092), and wherein the 3' end of A is linked to the 5' end of B, and the 3' end of B is linked to 5' end of C.
In some embodiments, the single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, e.g., a PRC2- associated region of a nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5, in which the nucleotide sequence of the single stranded oligonucleotide comprises one or more of a nucleotide sequence selected from the group consisting of
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx, (X)xxxXxX and (X)xxxxXX,
(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,
(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and
(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, wherein "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit.
In some embodiments, at least one nucleotide of the oligonucleotide is a nucleotide analogue. In some embodiments, the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
In some embodiments, at least one nucleotide of the oligonucleotide comprises a 2' O-methyl. In some embodiments, each nucleotide of the oligonucleotide comprises a 2' O- methyl. In some embodiments, the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide. In some embodiments, the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide. In some embodiments, each nucleotide of the oligonucleotide is a LNA nucleotide.
In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2 '-O-methyl nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a LNA nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages or other linkages) between between all nucleotides.
In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group. In some embodiments, the nucleotide at the 3' position of the
oligonucleotide has a 3' thiophosphate. In some embodiments, the single stranded oligonucleotide has a biotin moiety or other moiety conjugated to its 5' or 3' nucleotide. In some embodiments, the single stranded oligonucleotide has cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
According to some aspects of the invention compositions are provided that comprise any of the oligonucleotides disclosed herein, and a carrier. In some embodiments, compositions are provided that comprise any of the oligonucleotides in a buffered solution. In some embodiments, the oligonucleotide is conjugated to the carrier. In some embodiments, the carrier is a peptide. In some embodiments, the carrier is a steroid. According to some aspects of the invention pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
According to other aspects of the invention, kits are provided that comprise a container housing any of the compositions disclosed herein.
According to some aspects of the invention, methods of increasing expression of SMNl or SMN2 in a cell are provided. In some embodiments, the methods involve delivering any one or more of the single stranded oligonucleotides disclosed herein into the cell. In some embodiments, delivery of the single stranded oligonucleotide into the cell results in a level of expression of SMNl or SMN2 that is greater (e.g., at least 50% greater) than a level of expression of SMNl or SMN2 in a control cell that does not comprise the single stranded oligonucleotide.
According to some aspects of the invention, methods of increasing levels of SMNl or SMN2 in a subject are provided. According to some aspects of the invention, methods of treating a condition (e.g., Spinal muscular atrophy) associated with decreased levels of SMNl or SMN2 in a subject are provided. In some embodiments, the methods involve
administering any one or more of the single stranded oligonucleotides disclosed herein to the subject.
Aspects of the invention relate to methods of increasing expression of SMN protein in a cell. In some embodiments, the method comprise delivering to the cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2- associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises exon 7 in the cell. In some embodiments, the region of complementarity with at least 8 consecutive nucleotides of a PRC2-associated region of SMN 2 has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more mismatches with a corresponding region of SMN1. As used herein the term, "splice control sequence" refers to a nucleotide sequence that when present in a precursor mR A influences splicing of that precursor mR A in a cell. In some embodiments, a splice control sequence includes one or more binding sites for a molecule that regulates mRNA splicing, such as a hnRNAP protein. In some embodiments, a splice control sequence comprises the sequence CAG or AAAG. In some embodiments, a splice control sequence resides in an exon (e.g., an exon of SMN1 or SMN2, such as exon 7 or exon 8). In some embodiments, a splice control sequence traverses an intron-exon junction (e.g., an intron-exon junction of SMN1 or SMN2, such as the intron 6/exon 7 junction or the intron 7/exon 8 junction). In some embodiments, a splice control sequence resides in an intron (e.g., an intron of SMN1 or SMN2, such as intron 6 or intron 7). In some embodiments, a splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof.
In some embodiments, the second single stranded oligonucleotide is splice switching oligonucleotide that comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089);
CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO:
13091); and CTTTCATAATGCTGG (SEQ ID NO: 13092). In some embodiments, the second single stranded oligonucleotide is 8 to 30 nucleotides in length.
In some embodiments, the first single stranded oligonucleotide has a sequence 5'-X-
Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length. In some embodiments, the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7.
In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439- 3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides. In some embodiments, the first single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
In some embodiments, the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell simultaneously. In some embodiments, the cell is in a subject and the step of delivering to the cell comprises administering the first single stranded oligonucleotide and the second single stranded oligonucleotide to the subject as a co-formulation. In some embodiments, the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker. In some embodiments, the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond. In some embodiments, the linker comprises an oligonucleotide, optionally wherein the oligonucleotide comprises 1 to 10 thymidines or uridines. In some embodiments, the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides. In some embodiments, the first single stranded
oligonucleotide is not covalently linked to the second single stranded oligonucleotide. In some embodiments, the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell separately.
According to some aspects of the invention, methods are provided for treating spinal muscular atrophy in a subject. The methods, in some embodiments, comprise administering to the subject a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of SMN protein in the subject.
According to some aspects of the invention methods are provided for treating spinal muscular atrophy in a subject that involve administering to the subject a first single stranded oligonucleotide complementary with a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of SMN protein in the subject.
Related compositions are also provided. In some embodiments, compositions are provided that comprise a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2, and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of SMN2. In some embodiments, compositions are provided that comprise a single stranded
oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene, linked via a linker to a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. Related kits comprising single stranded oligonucleotides that regulate SMNl or SMN2 expression are also provided.
According to some aspects of the invention compositions are provided that comprise any of the oligonucleotides or compounds disclosed herein, and a carrier. In some embodiments, compositions are provided that comprise any of the oligonucleotides or compounds in a buffered solution. In some embodiments, the oligonucleotide is conjugated to the carrier. In some embodiments, the carrier is a peptide. In some embodiments, the carrier is a steroid. According to some aspects of the invention pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
According to some aspects of the invention, compositions are provided that comprise a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2, and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of SMN2. In some embodiments, the splice control sequence resides in an exon of SMN2. In some
embodiments, the exon is exon 7 or exon 8. In some embodiments, the splice control sequence traverses an intron-exon junction of SMN2. In some embodiments, the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction. In some
embodiments, the splice control sequence resides in an intron of SMN2. In some
embodiments, the intron is intron 6 or intron 7 (SEQ ID NO: 13101). In some embodiments, the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof. In some embodiments, the splice control sequence comprises at least one hnRNAP binding sequence. In some embodiments, the second single stranded oligonucleotide comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO: 13091); and
CTTTCATAATGCTGG (SEQ ID NO: 13092). In some embodiments, the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length. In some
embodiments, the PRC2-associated region of SMN2 is a PRC2-associated region within SEQ ID NO: 1,2, 4 or 5. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides. In some embodiments, the first and/or second single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides. In some embodiments, the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker. In some embodiments, the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond. In some
embodiments, the linker comprises an oligonucleotide, optionally wherein the
oligonucleotide comprises 1 to 10 thymidines or uridines. In some embodiments, the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides. In some embodiments, the first single stranded oligonucleotide is not covalently linked to the second single stranded oligonucleotide. In some embodiments, the composition further comprises a carrier. In some embodiments, the carrier is a
pharmaceutically acceptable carrier.
Further aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of SMNl or SMN2. In some embodiments, methods are provided for selecting a set of oligonucleotides that is enriched in candidates (e.g., compared with a random selection of oligonucleotides) for activating or enhancing expression of SMNl or SMN2. Accordingly, the methods may be used to establish sets of clinical candidates that are enriched in oligonucleotides that activate or enhance expression of SMNl or SMN2. Such libraries may be utilized, for example, to identify lead oligonucleotides for developing therapeutics to treat SMNl or SMN2. Furthermore, in some embodiments, oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and/or efficacy of the single stranded oligonucleotides for activating expression of SMNl or SMN2.
According to other aspects of the invention, kits are provided that comprise a container housing any of the compositions disclosed herein. According to other aspects of the invention, kits are provided that comprise a first container housing first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene; and a second container housing a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. In some embodiments, the splice control sequence resides in an exon of the gene. In some
embodiments, the splice control sequence traverses an intron-exon junction of the gene. In some embodiments, the splice control sequence resides in an intron of the gene. In some embodiments, the splice control sequence comprises at least one hnRNAP binding sequence. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell. In some embodiments, hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell. In some embodiments, the gene is SMN1 or SMN2. In some embodiments, the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8. In some embodiments, the splice control sequence comprises the sequence:
CAGCAUUAUGAAAG (SEQ ID NO: 13100). In some embodiments, the second single stranded oligonucleotide comprises a sequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO: 13089);
CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO: XX); and CTTTCATAATGCTGG (SEQ ID NO: 13091). In some embodiments, the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length. In some embodiments, the PRC2-associated region of an SMN2 gene is a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 9 to 23. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5 ' end of the nucleotide sequence provided is the 5' end of the first single stranded oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 7. In some embodiments, the PRC2-associated region is a sequence set forth in any one of SEQ ID NOS: 24 to 29. In some embodiments, the first single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 8. BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 provides a schematic of SMN 1 and SMN2 mRNA processing
FIG. 2 provides a table outlining genotypes and patent information, including SMA classification, of cell lines tested in Example 2. Baseline SMN protein levels in the cell lines are also depicted.
FIG. 3 depicts results of RT-PCR assays showing effects on SMN mRNA expression of oligonucleotides directed against a PRC2-associated region of SMN2 (oligos 1-52 and 59-
101) and splice switching oligonucleotides (oligos 53-58) (PCR primers directed against exon
1 of SMN1/2.) in cell line 3814.
FIG. 4 depicts results of RT-PCR assays showing effects on SMN mRNA expression of oligonucleotides directed against a PRC2-associated region of SMN2 (oligos 1-52 and 59-
101) and splice switching oligonucleotides (oligos 53-58) (PCR primers directed against exon
1 of SMN1/2.) in cell line 3813.
FIG. 5 shows that splice switching oligonucleotides (oligoes 53-58) increase expression of full length SMN2. Results are based on a gel separation analysis of PCR products obtained following a Ddel restriction digest. Two cell lines were tested, 3813 and
9677. Oligo 84, which targets a PRC2-associated region of SMN2, did not exhibit an increase in full length SMN2 expression when delivered alone to cells.
FIG. 6 provides results of an SMN ELISA (Enzo) showing that certain
oligonucleotides directed against a PRC2-associated region of SMN2 alone do not significantly increase SMN2 protein 24h post-transfection in certain SMA patient fibroblasts
(compared to Lipofectamine treated cells - dashed line). FIG. 7 provides results of an SMN ELISA showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to Lipofectamine treated cells - dashed line).
FIG. 8 provides results of an SMN ELISA showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 54) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to Lipofectamine treated cells - dashed line).
FIG. 9 provides results of an RT-PCR assay showing that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increase SMN2 protein 24h post-transfection in SMA patient fibroblasts (compared to negative control oligo and Lipofectamine treated cells). LNA/2'OMe alternating oligonucleotide (LM design) and DNA/LNA alternating
oligonucleotides (DL design) were tested.
BRIEF DESCRIPTION OF TABLES
Table 1: Hexamers that are not seed sequences of human miRNAs
Table 2: Oligonucleotide sequences made for testing in the lab. RQ (column 2) and RQ SE (column 3) shows the activity of the oligo relative to a control well (usually carrier alone) and the standard error or the triplicate replicates of the experiment, [oligo] is shown in nanomolar for in vitro experiments and in milligrams per kilogram of body weight for in vivo experiments.
Table 3: A listing of oligonucleotide modifications
Table 4: Oligonucleotide sequences made for testing human cells obtained from subjects with Spinal Muscular Atrophy. The table shows the sequence of the modified nucleotides, where InaX represents an LNA nucleotide with 3' phosphorothioate linkage, omeX is a 2'-0-methyl nucleotide, dX is a deoxy nucleotide. An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage. The "-Sup" at the end of the sequence marks the fact that the 3 ' end lacks either a phosphate or thiophosphate on the 3' linkage. The Formatted Sequence column shows the sequence of the oligonucleotide, including modified nucleotides, for the oligonucleotides tested in Table 2, 5, 6 and 7.
Table 8: Cell lines BRIEF DESCRIPTION OF THE APPENDICES
• Appendix A; Appendix A contains Table 5, which shows RT-PCR data from
testing of different oligonucleotides.
• Appendix B; Appendix B contains Table 6, which shows RT-PCR data from
testing of different combination treatments (e.g., two oligonucleotides, an oligonucleotide and a drug).
• Appendix C; Appendix C contains Table 7, which shows ELISA data from
testing of different oligonucleotides
Note the following column information for Tables 5-7 in Appendices A-C, respectively. SEQID: sequence identifier of base sequence of oligonucleotide used; Oligo Name: name of oligonucleotide; Avg RQ: average relative quantification of RT-PCR based expression levels of target gene(s); Avg RQ SE: standard error of mean of relative quantification of RT-PCR based expression level; "%SMN over lipo only control" refers to the ratio of SMN protein levels (ng/mg total protein) when compared to Lipofectamine2000 (transfection reagent) treated cells converted into %; "%SMN CVV" refers to coefficient of variation; Exp #: Experiment reference number; Target: target gene; [oligo] : concentration of oligonucleotide used in nM unless otherwise indicated; Cell Line: cell line used; Assay Type: assay used; Time(hr): time of assay following treatment; 2nd Drug: name of second oligonucleotide (identified by Oligo Name) or drug used in combination experiment; [2nd]: concentration of second oligonucleotide or drug; Units: units of concentration; 3rd Drug: name of third oligonucleotide (identified by Oligo Name) or drug used in combination experiment; [3rd]: concentration of third oligonucleotide or drug; Notes: comments regarding experiment. Oligo Names correspond to those in Tables 2 and 4. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
Spinal muscular atrophy (SMA), the most common genetic cause of infant mortality, is an autosomal recessive neuromuscular disease characterized by progressive loss of a-motor neurons in the anterior horns of the spinal cord, leading to limb and trunk paralysis and atrophy of voluntary muscles. Based on the severity and age of onset, SMA is clinically subdivided into types I, II, and III (MIMs 253300, 253550, and 253400), with type I generally understood as being the most severe. Loss of function of the SMNl gene is responsible for SMA. Humans have an extra SMN gene copy, called SMN2. Both SMN genes reside within a segmental duplication on Chromosome 5ql3 as inverted repeats. SMNl and SMN2 are almost identical. In some cases, SMNl and SMN2 differ by 11 nucleotide substitutions, including seven in intron 6, two in intron 7, one in coding exon 7, and one in non-coding exon 8. The substitution in exon 7 involves a translationally silent C to T transition compared with SMNl, that results in alternative splicing because the substitution disrupts recognition of the upstream 3' splice site, in which exon 7 is frequently skipped during precursor mR A splicing. Consequently, SMN2 encodes primarily the exon 7-skipped protein isoform (SMNA7), which is unstable, mislocalized, and only partially functional.
Methods and related single stranded oligonucleotides that are useful for selectively inducing expression of particular splice variants of SMNl or SMN2 are provided herein. The methods are useful for controlling the levels in a cell of particular SMN protein isoforms encoded by the splice variants. In some cases, the methods are useful for inducing expression of SMN proteins to levels sufficient to treat SMA. For example, according to some aspects of the invention methods are provided for increasing expression of full-length SMN protein in a cell for purposes of treating SMA. In some embodiments, the methods comprise delivering to the cell a first single stranded oligonucleotide complementary with a PRC2- associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell. Further aspects of the invention are described in detailed herein. Polycomb repressive complex 2 (PRC2) -inter acting RNAs
Aspects of the invention provided herein relate to the discovery of polycomb repressive complex 2 (PRC2)-interacting RNAs. Polycomb repressive complex 2 (PRC2) is a histone methyltransferase and a known epigenetic regulator involved in silencing of genomic regions through methylation of histone H3. Among other functions, PRC2 interacts with long noncoding RNAs (IncRNAs), such as RepA, Xist, and Tsix, to catalyze
trimethylation of histone H3-lysine27. PRC2 contains four subunits, Eed, Suzl2, RbAp48, and Ezh2. Aspects of the invention relate to the recognition that single stranded oligonucleotides that bind to PRC2-associated regions of RNAs (e.g., IncRNAs) that are expressed from within a genomic region that encompasses or that is in functional proximity to the SMNl or SMN2 gene can induce or enhance expression of SMNl or SMN2. In some embodiments, this upregulation is believed to result from inhibition of PRC2 mediated repression of SMNl or SMN2.
As used herein, the term "PRC2-associated region" refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2. A PRC2-associated region may be present in a RNA (e.g., a long non- coding RNA (IncRNA)) that interacts with a PRC2. A PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2. In some cases, the PRC2- associated region is equivalently referred to as a PRC2-interacting region.
In some embodiments, a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.
In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g., RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
In some embodiments, a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2- associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an R A-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region. In such embodiments, the PRC2-associated region may be referred to as a "peak."
In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g. , of 40 to 60 nucleotides) that interacts with
PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g., of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 9 to 29. In some embodiments, a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 24 to 29.
In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region in a genomic region that encompasses or that is in proximity to the SMN1 or SMN2 gene. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 9 to 29. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 9 to 29 combined with up to 2kb, up to 5kb, or up to lOkb of flanking sequences from a corresponding genomic region to which these SEQ IDs map (e.g., in a human genome). In some embodiments, single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID NOS: 30 to 13087. In some embodiments, single stranded oligonucleotides have a sequence as set forth in Table 2. In some embodiments, a PRC2 associated region of SMN1 or SMN2 against which a single stranded oligonucleotide is complementary is selected from SEQ ID NOS: 24-29. In some embodiments, a single stranded oligonucleotide that is complementary with a PRC2 associated region of SMNl or SMN2 comprises a sequence selected from SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916- 3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087. In some embodiments, a single stranded oligonucleotide that is complementary with a PRC2 associated region of SMNl or SMN2 comprises a sequence selected from 11395, 11394, 10169, and 10170.
Without being bound by a theory of invention, these oligonucleotides are able to interfere with the binding of and function of PRC2, by preventing recruitment of PRC2 to a specific chromosomal locus. For example, a single administration of single stranded oligonucleotides designed to specifically bind a PRC2-associated region IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin. After displacement, the full complement of PRC2 is not recovered for up to 24 hours. Further, IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.
Methods of modulating gene expression are provided, in some embodiments, that may be carried out in vitro, ex vivo, or in vivo. It is understood that any reference to uses of compounds throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition {e.g., Spinal muscular atrophy) associated with decreased levels or activity of SMNl or SMN2. Thus, as one nonlimiting example, this aspect of the invention includes use of such single stranded oligonucleotides in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of SMNl or SMN2.
In further aspects of the invention, methods are provided for selecting a candidate oligonucleotide for activating expression of SMNl or SMN2. The methods generally involve selecting as a candidate oligonucleotide, a single stranded oligonucleotide comprising a nucleotide sequence that is complementary to a PRC2-associated region {e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 9 to 29). In some embodiments, sets of oligonucleotides may be selected that are enriched {e.g., compared with a random selection of oligonucleotides) in oligonucleotides that activate expression of SMNl or SMN2. Single Stranded Oligonucleotides for Modulating Expression of SMNl or SMN2 In one aspect of the invention, single stranded oligonucleotides complementary to the PRC2-associated regions are provided for modulating expression of SMNl or SMN2 in a cell. In some embodiments, expression of SMNl or SMN2 is upregulated or increased. In some embodiments, single stranded oligonucleotides complementary to these PRC2- associated regions inhibit the interaction of PRC2 with long RNA transcripts such that gene expression is upregulated or increased. In some embodiments, single stranded
oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts, resulting in reduced methylation of histone H3 and reduced gene inactivation, such that gene expression is upregulated or increased. In some
embodiments, this interaction may be disrupted or inhibited due to a change in the structure of the long RNA that prevents or reduces binding to PRC2. The oligonucleotide may be selected using any of the methods disclosed herein for selecting a candidate oligonucleotide for activating expression of SMNl or SMN2.
The single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 8. The region of complementarity of the single stranded
oligonucleotide may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of the PRC2-associated region.
It should be appreciated that due the high homology between SMNl and SMN2, single stranded oligonucleotides that are complementary with a PRC2-associated region of SMNl are often also complementary with a corresponding PRC2-associated region of SMN2.
The PRC2-associated region may map to a position in a chromosome between 50 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 50 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene. The PRC2-associated region may map to a position in a chromosome between 25 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 25 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene. The PRC2-associated region may map to a position in a chromosome between 12 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 12 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene. The PRC2-associated region may map to a position in a chromosome between 5 kilobases upstream of a 5 '-end of the SMNl or SMN2 gene and 5 kilobases downstream of a 3 '-end of the SMNl or SMN2 gene. The genomic position of the selected PRC2-associated region relative to the SMNl or SMN2 gene may vary. For example, the PRC2-associated region may be upstream of the 5 ' end of the SMNl or SMN2 gene. The PRC2-associated region may be downstream of the 3 ' end of the SMNl or SMN2 gene. The PRC2-associated region may be within an intron of the SMNl or SMN2 gene. The PRC2-associated region may be within an exon of the SMNl or SMN2 gene. The PRC2-associated region may traverse an intron-exon junction, a 5 '-UTR- exon junction or a 3 '-UTR-exon junction of the SMNl or SMN2 gene.
The single stranded oligonucleotide may comprise a sequence having the formula X- Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of varying length. In some embodiments X is the 5 ' nucleotide of the oligonucleotide. In some embodiments, when X is anchored at the 5 ' end of the oligonucleotide, the oligonucleotide does not have any nucleotides or nucleotide analogs linked 5 ' to X. In some embodiments, other compounds such as peptides or sterols may be linked at the 5 ' end in this embodiment as long as they are not nucleotides or nucleotide analogs. In some embodiments, the single stranded oligonucleotide has a sequence 5 'X-Y-Z and is 8-50 nucleotides in length.
Oligonucleotides that have these sequence characteristics are predicted to avoid the miRNA pathway. Therefore, in some embodiments, oligonucleotides having these sequence characteristics are unlikely to have an unintended consequence of functioning in a cell as a miRNA molecule. The Y sequence may be a nucleotide sequence of 6 nucleotides in length set forth in Table 1.
The single stranded oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
The single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene. For example, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than SMNl or SMN2. In a similar embodiment, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to any other known PRC2-associated region, particularly PRC2-associated regions that are functionally related to any other known gene (e.g., any other known protein coding gene). In either case, the oligonucleotide is expected to have a reduced likelihood of having off-target effects. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
The single stranded oligonucleotide may have a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops. In has been discovered that, in some embodiments, oligonucleotides that are complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops (e.g., at least two single stranded loops) have a greater likelihood of being active (e.g., of being capable of activating or enhancing expression of a target gene) than a randomly selected
oligonucleotide. In some cases, the secondary structure may comprise a double stranded stem between the at least two single stranded loops. Accordingly, the region of
complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2-associated region that encodes at least a portion of at least two of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of the double stranded stem. In some embodiments, a PRC2-associated region (e.g., of an lncRNA) is identified (e.g., using RIP- Seq methodology or information derived therefrom). In some embodiments, the predicted secondary structure RNA (e.g., lncRNA) containing the PRC2-associated region is determined using RNA secondary structure prediction algorithms, e.g., RNAfold, mfold. In some embodiments, oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g., at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.
The single stranded oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content. The single stranded oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments in which the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides. In some embodiments, the sequence of the PRC2-associated region to which the single stranded oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
The single stranded oligonucleotide may be complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homo log of SMN1 or SMN2. The single stranded oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the SMN1 or SMN2 gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of SMN1 or SMN2. For example, the single stranded oligonucleotide may be complementary to a sequence as set forth in SEQ ID NO: 1, 2, 4, or 5, which is a human genomic region encompassing or in proximity to the SMN1 or SMN2 gene, and also be complementary to a sequence as set forth in SEQ ID NO:7 or 8, which is a mouse genomic region encompassing or in proximity to the mouse homolog of the SMN1 or SMN2 gene. Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease.
In some embodiments, the region of complementarity of the single stranded oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or 50 consecutive nucleotides of a PRC2-associated region. In some embodiments, the region of complementarity is complementary with at least 8 consecutive nucleotides of a PRC2-associated region. In some embodiments the sequence of the single stranded oligonucleotide is based on an RNA sequence that binds to PRC2, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases. Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of PRC2-associated region, then the single stranded nucleotide and PRC2-associated region are considered to be complementary to each other at that position. The single stranded nucleotide and PRC2-associated region are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus, "complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the single stranded nucleotide and PRC2-associated region. For example, if a base at one position of a single stranded nucleotide is capable of hydrogen bonding with a base at the corresponding position of a PRC2-associated region, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
The single stranded oligonucleotide may be at least 80% complementary to
(optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of a PRC2-associated region. In some embodiments the single stranded oligonucleotide may contain 1 , 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region. In some embodiments the single stranded oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable. In some
embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target molecule (e.g., IncRNA) interferes with the normal function of the target (e.g., IncRNA) to cause a loss of activity (e.g., inhibiting PRC2-associated repression with consequent up-regulation of gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. In some embodiments, the single stranded oligonucleotide is 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
In some embodiments, the PRC2-associated region occurs on the same DNA strand as a gene sequence (sense). In some embodiments, the PRC2-associated region occurs on the opposite DNA strand as a gene sequence (anti-sense). Oligonucleotides complementary to a PRC2-associated region can bind either sense or anti-sense sequences. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine -type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa. In some embodiments, GC content of the single stranded oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome) as a single contiguous transcript (e.g., a non-spliced RNA). In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome), in which the distance in the genome between the 5 'end of the coding region of the RNA and the 3 ' end of the coding region of the RNA is less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7 kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.
It is to be understood that any oligonucleotide provided herein can be excluded.
In some embodiments, single stranded oligonucleotides disclosed herein may increase expression of mRNA corresponding to the gene by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. It has also been found that increased mRNA expression has been shown to correlate to increased protein expression.
In some or any of the embodiments of the oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to the PRC2 binding RNA that is transcribed from the same strand as a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding sense strand of a reference gene (refGene).
In some or any of the embodiments of oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to a PRC2 binding RNA that transcribed from the opposite strand (the antisense strand) of a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding antisense strand of a reference gene.
The oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof. In addition, the oligonucleotides can exhibit one or more of the following properties: do not induce substantial cleavage or degradation of the target RNA; do not cause
substantially complete cleavage or degradation of the target RNA; do not activate the RNAse H pathway; do not activate RISC; do not recruit any Argonaute family protein; are not cleaved by Dicer; do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; may have improved endosomal exit; do interfere with interaction of IncRNA with PRC2, preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target (e.g. , are complementary to the underlying genomic DNA sequence from which the RNA is transcribed). Splice Switching Oligonucleotides
Aspects of the invention provide strategies for targeting SMN1 or SMN2 precursor mRNA to affect splicing to minimize exon skipping. Accordingly, aspects of the invention provide therapeutic compounds useful for the treatment of SMA. In some embodiments, oligonucleotides, referred to herein as "splice switching oligonucleotides" are provided that modulate SMN2 splicing. Methods and related compositions, compounds, and kits are provided, in some embodiments, that are useful for increasing expression of full-length.
SMN protein in a cell. The methods generally involve delivering to a cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2- associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises (or includes) exon 7 in the cell. Any of the single stranded oligonucleotides that are complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMNl or SMN2 may be used. It should be appreciated that single stranded oligonucleotides that are complementary with a splice control sequence may alternatively be referred herein, as splice switching oligonucleotides. Splice switching oligonucleotides typically comprise a sequence complementary to a splice control sequence (e.g., a intronic splicing silencer sequence) of a precursor mRNA, and are capable of binding to and affecting processing of the precursor mRNA. Splice switching oligonucleotides may be complementary with a region of an exon, a region of an intron or an intron/exon junction. In some embodiments, the splice control sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or a portion thereof. In some embodiments, the splice control sequence comprises at least one hnRNAP binding sequence. In some embodiments, splice switching oligonucleotides that target SMNl or SMN2 function based on the premise that there is a competition between the 3' splice sites of exons 7 and 8 for pairing with the 5' splice site of exon 6, so impairing the recognition of the 3' splice site of exon 8 favors exon 7 inclusion. In some embodiments, splice switching oligonucleotides are provided that promote SMN2 exon 7 inclusion and full-length SMN protein expression, in which the oligonucleotides are complementary to the intron 7/exon 8 junction. In some embodiments, splice switching oligonucleotide are composed of a segment complementary to an exon of SMNl or SMN2 (e.g., exon 7). In some embodiments, splice switching oligonucleotides comprise a tail (e.g., a non-complementary tail) consisting of RNA sequences with binding motifs recognized by a serine/arginine-rich (SR) protein. In some embodiments, splice switching oligonucleotides are complementary (at least partially) with an intronic splicing silencer (ISS). In some embodiments, the ISS is in intron 6 or intron 7 of SMNl or SMN2. In some embodiments, splice switching oligonucleotides comprise an antisense moiety complementary to a target exon or intron (e.g., of SMNl or SMN2) and a minimal RS domain peptide similar to the splicing activation domain of SR proteins. In some embodiments, the splice switching oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In one embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
Linkers
Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g. , a cleavable linker. Accordingly, in some embodiments, compounds are provided that comprise a single stranded oligonucleotide complementary with a PRC2-associated region of a gene that is linked via a linker to a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. In some embodiments, compounds are provided that have the general formula A-B-C, in which A is a single stranded oligonucleotide complementary with a PRC2- associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. In some embodiments, linker B comprises an oligonucleotide, peptide, low pH labile bond, or disulfide bond. In some embodiments, the compounds is orientated as 5'-A-B-C-3'. In some embodiments, the compound is orientated as 3'-A-B-C-5'. In some embodiments, where B is an oligonucleotide, the 3' end of A is linked to the 5' end of B, and the 3' end of B is linked to 5' end of C. In some embodiments, where B is an oligonucleotide, the 5' end of A is linked to the 3 ' end of B, and the 5 ' end of B is linked to 3 ' end of C. In some
embodiments, where B is an oligonucleotide, the 5 ' end of A is linked to the 5' end of B, and/or the 3' end of B is linked to the 3' end of C. In some embodiments, where B is an oligonucleotide, the 3 ' end of A is linked to the 3' end of B, and/or the 5' end of B is linked to the 5' end of C.
The term "linker" generally refers to a chemical moiety that is capable of covalently linking two or more oligonucleotides. In some embodiments, at least one bond comprised or contained within the linker is capable of being cleaved (e.g., in a biological context, such as in a mammalian extract, such as an endosomal extract), such that at least two
oligonucleotides are no longer covalently linked to one another after bond cleavage. It will be appreciated that, in some embodiments, a provided linker may include a region that is non- cleavable, as long as the linker also comprises at least one bond that is cleavable.
In some embodiments, the linker comprises a polypeptide that is more susceptible to cleavage by an endopeptidase in the mammalian extract than the oligonucleotides. The endopeptidase may be a trypsin, chymotrypsin, elastase, thermolysin, pepsin, or
endopeptidase V8. The endopeptidase may be a cathepsin B, cathepsin D, cathepsin L, cathepsin C, papain, cathepsin S or endosomal acidic insulinase. For example, the linker comprise a peptide having an amino acid sequence selected from: ALAL, APISFFELG, FL, GFN, R/KXX, GRWHTVGLRWE, YL, GF, and FF, in which X is any amino acid.
In some embodiments, the linker comprises the formula -(CH2)„S-S(CH2)m-, wherein n and m are independently integers from 0 to 10.
In some embodiments, the linker may comprise an oligonucleotide that is more susceptible to cleavage by an endonuclease in the mammalian extract than the oligonucleotides. The linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines. The linker may have a nucleotide sequence comprising
deoxyribonucleotides linked through phosphodiester internucleotide linkages. The linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines linked through phosphodiester internucleotide linkages. The linker may have a nucleotide sequence comprising from 1 to 10 thymidines or uridines linked through phosphorothioate
internucleotide linkages.
In some embodiments, at least one linker is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more sensitive to enzymatic cleavage in the presence of a mammalian extract than at least two oligonucleotides. It should be appreciated that different linkers can be designed to be cleaved at different rates and/or by different enzymes in compounds comprising two or more linkers. Similarly different linkers can be designed to be sensitive to cleavage in different tissues, cells or subcellular compartments in compounds comprising two or more linkers. This can advantageously permit compounds to have oligonucleotides that are released from compounds at different rates, by different enzymes, or in different tissues, cells or subcellular compartments thereby controlling release of the monomeric oligonucleotides to a desired in vivo location or at a desired time following administration.
In certain embodiments, linkers are stable (e.g., more stable than the oligonucleotides they link together) in plasma, blood or serum which are richer in exonucleases, and less stable in the intracellular environments which are relatively rich in endonucleases. In some embodiments, a linker is considered "non-cleavable" if the linker's half-life is at least 24, or 28, 32, 36, 48, 72, 96 hours or longer under the conditions described here, such as in liver homogenates. Conversely, in some embodiments, a linker is considered "cleavable" if the half-life of the linker is at most 10, or 8, 6, 5 hours or shorter.
In some embodiments, the linker is a nuclease-cleavable oligonucleotide linker. In some embodiments, the nuclease-cleavable linker contains one or more phosphodiester bonds in the oligonucleotide backbone. For example, the linker may contain a single
phosphodiester bridge or 2, 3, 4, 5, 6, 7 or more phosphodiester linkages, for example as a string of 1-10 deoxynucleotides, e.g., dT, or ribonucleotides, e.g., rU, in the case of RNA linkers. In the case of dT or other DNA nucleotides dN in the linker, in certain embodiments the cleavable linker contains one or more phosphodiester linkages. In other embodiments, in the case of rU or other RNA nucleotides rN, the cleavable linker may consist of phosphorothioate linkages only. In contrast to phosphorothioate-linked deoxynucleotides, which in some embodiments are cleaved relatively slowly by nucleases (thus termed
"noncleavable"), phosphorothioate-linked rU undergoes relatively rapid cleavage by ribonucleases and therefore is considered cleavable herein in some embodiments. It is also possible to combine dN and rN into the linker region, which are connected by phosphodiester or phosphorothioate linkages. In other embodiments, the linker can also contain chemically modified nucleotides, which are still cleavable by nucleases, such as, e.g., 2'-0-modified analogs. In particular, 2'-0-methyl or 2'-fluoro nucleotides can be combined with each other or with dN or rN nucleotides. Generally, in the case of nucleotide linkers, the linker is a part of the compound that is usually not complementary to a target, although it could be. This is because the linker is generally cleaved prior to action of the oligonucleotides on the target, and therefore, the linker identity with respect to a target is inconsequential. Accordingly, in some embodiments, a linker is an (oligo)nucleotide linker that is not complementary to any of the targets against which the oligonucleotides are designed.
In some embodiments, the cleavable linker is an oligonucleotide linker that contains a continuous stretch of deliberately introduced Rp phosphorothioate stereoisomers (e.g., 4, 5, 6, 7 or longer stretches). The Rp stereoisoform, unlike Sp isoform, is known to be susceptible to nuclease cleavage (Krieg et al, 2003, Oligonucleotides, 13:491-499). Such a linker would not include a racemic mix of PS linkaged oligonucleotides since the mixed linkages are relatively stable and are not likely to contain long stretches of the Rp stereoisomers, and therefore, considered "non-cleavable" herein. Thus, in some embodiments, a linker comprises a stretch of 4, 5, 6, 7 or more phosphorothioated nucleotides, wherein the stretch does not contain a substantial amount or any of the Sp stereoisoform. The amount could be considered substantial if it exceeds 10% on a per-mole basis.
In some embodiments, the linker is a non-nucleotide linker, for example, a single phosphodiester bridge. Another example of such cleavable linkers is a chemical group comprising a disulfide bond, for example, -(CH2)„S-S(CH2)m-, wherein n and m are integers from 0 to 10. In illustrative embodiments, n=m=6. Additional examples of non-nucleotide linkers are described below.
The linker can be designed so as to undergo a chemical or enzymatic cleavage reaction. Chemical reactions involve, for example, cleavage in acidic environments (e.g., endosomes), reductive cleavage (e.g., cytosolic cleavage) or oxidative cleavage (e.g., in liver microsomes). The cleavage reaction can also be initiated by a rearrangement reaction.
Enzymatic reactions can include reactions mediated by nucleases, peptidases, proteases, phosphatases, oxidases, reductases, etc. For example, a linker can be pH-sensitive, cathepsin- sensitive, or predominantly cleaved in endosomes and/or cytosol.
In some embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises a peptide which includes a sequence that is cleavable by an endopeptidase. In addition to the cleavable peptide sequence, the linker may comprise additional amino acid residues and/or non-peptide chemical moieties, such as an alkyl chain. In certain
embodiments, the linker comprises Ala-Leu-Ala-Leu, which is a substrate for cathepsin B. See, for example, the maleimidocaproyl-Arg-Arg-Ala-Leu-Ala-Leu linkers described in
Schmid et al, Bioconjugate Chem 2007, 18, 702-716. In certain embodiments, a cathepsin B- cleavable linker is cleaved in tumor cells. In certain embodiments, the linker comprises Ala- Pro-Ile-Ser-Phe-Phe-Glu-Leu-Gly, which is a substrate for cathepsins D, L, and B (see, for example, Fischer et al, Chembiochem 2006, 7, 1428-1434). In certain embodiments, a cathepsin-cleavable linker is cleaved in HeLA cells. In some embodiments, the linker comprises Phe-Lys, which is a substrate for cathepsin B. For example, in certain
embodiments, the linker comprises Phe-Lys-p-aminobenzoic acid (PABA). See, e.g., the maleimidocaproyl-Phe-Lys-PABA linker described in Walker et al, Bioorg. Med. Chem. Lett. 2002, 12, 217-219. In certain embodiments, the linker comprises Gly-Phe-2- naphthylamide, which is a substrate for cathepsin C (see, for example, Berg et al. Biochem. J. 1994, 300, 229-235). In certain embodiments, a cathepsin C-cleavable linker is cleaved in hepatocytes. In some embodiments, the linker comprises a cathepsin S cleavage site. For example, in some embodiments, the linker comprises Gly-Arg-Trp-His-Thr-Val-Gly-Leu- Arg-Trp-Glu, Gly-Arg-Trp-Pro-Pro-Met-Gly-Leu-Pro-Trp-Glu, or Gly-Arg-Trp-His-Pro- Met-Gly-Ala-Pro-Trp-Glu, for example, as described in Lutzner et al, J. Biol. Chem. 2008, 283, 36185-36194. In certain embodiments, a cathepsin S-cleavable linker is cleaved in antigen presenting cells. In some embodiments, the linker comprises a papain cleavage site. Papain typically cleaves a peptide having the sequence -R/K-X-X (see Chapman et al, Annu. Rev. Physiol 1997, 59, 63-88). In certain embodiments, a papain-cleavable linker is cleaved in endosomes. In some embodiments, the linker comprises an endosomal acidic insulinase cleavage site. For example, in some embodiments, the linker comprises Tyr-Leu, Gly-Phe, or Phe-Phe (see, e.g., Authier et al, FEBS Lett. 1996, 389, 55-60). In certain embodiments, an endosomal acidic insulinase-cleavable linker is cleaved in hepatic cells.
In some embodiments, the linker is pH sensitive. In certain embodiments, the linker comprises a low pH-labile bond. As used herein, a low-pH labile bond is a bond that is selectively broken under acidic conditions (pH < 7). Such bonds may also be termed endosomally labile bonds, because cell endosomes and lysosomes have a pH less than 7. For example, in certain embodiments, the linker comprises an amine, an imine, an ester, a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, a polyphosphazene, an acetal, a vinyl ether, a hydrazone, an azidomethyl-methylmaleic anhydride, a thiopropionate, a masked endosomo lytic agent or a citraconyl group.
In certain embodiments, the linker comprises a low pH-labile bond selected from the following: ketals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and a ketone; acetals that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form a diol and an aldehyde; imines or iminiums that are labile in acidic environments (e.g., pH less than 7, greater than about 4) to form an amine and an aldehyde or a ketone; silicon-oxygen-carbon linkages that are labile under acidic condition; silicon-nitrogne (silazane) linkages; silicon-carbon linkages (e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamates (amide bonds synthesized from maleic anhydride derivatives and amines); ortho esters; hydrazones; activated carboxylic acid derivatives (e.g., esters, amides) designed to undergo acid catalyzed hydrolysis); or vinyl ethers. Further examples may be found in International Patent Appln. Pub. No. WO 2008/022309, entitled POLYCONJUGATES FOR IN VIVO DELIVERY OF POLYNUCLEOTIDES, the contents of which are incorporated herein by reference.
In some embodiments, the linker comprises a masked endosomolytic agent.
Endosomolytic polymers are polymers that, in response to a change in pH, are able to cause disruption or lysis of an endosome or provide for escape of a normally membrane- impermeable compound, such as a polynucleotide or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome. A subset of endosomolytic compounds is fusogenic compounds, including fusogenic peptides. Fusogenic peptides can facilitate endosomal release of agents such as oligomeric compounds to the cytoplasm. See, for example, US Patent Application Publication Nos. 20040198687, 20080281041,
20080152661, and 20090023890, which are incorporated herein by reference. The linker can also be designed to undergo an organ/ tissue-specific cleavage. For example, for certain targets, which are expressed in multiple tissues, only the knock-down in liver may be desirable, as knock-down in other organs may lead to undesired side effects. Thus, linkers susceptible to liver-specific enzymes, such as pyrrolase (TPO) and glucose-6- phosphatase (G-6-Pase), can be engineered, so as to limit the antisense effect to the liver mainly. Alternatively, linkers not susceptible to liver enzymes but susceptible to kidney- specific enzymes, such as gamma-glutamyltranspeptidase, can be engineered, so that the antisense effect is limited to the kidneys mainly. Analogously, intestine-specific peptidases cleaving Phe-Ala and Leu- Ala could be considered for orally administered multimeric oligonucleotides. Similarly, by placing an enzyme recognition site into the linker, which is recognized by an enzyme over-expressed in tumors, such as plasmin (e.g., PHEA-D-Val-Leu- Lys recognition site), tumor-specific knock-down should be feasible. By selecting the right enzyme recognition site in the linker, specific cleavage and knock-down should be achievable in many organs. In addition, the linker can also contain a targeting signal, such as N-acetyl galactosamine for liver targeting, or folate, vitamin A or RGD-peptide in the case of tumor or activated macrophage targeting. Accordingly, in some embodiments, the cleavable linker is organ- or tissue-specific, for example, liver-specific, kidney-specific, intestine-specific, etc.
The oligonucleotides can be linked through any part of the individual oligonucleotide, e.g., via the phosphate, the sugar (e.g., ribose, deoxyribose), or the nucleobase. In certain embodiments, when linking two oligonucleotides together, the linker can be attached e.g. to the 5 '-end of the first oligonucleotide and the 3 '-end of the second nucleotide, to the 5 '-end of the first oligonucleotide and the 5 'end of the second nucleotide, to the 3 '-end of the first oligonucleotide and the 3 '-end of the second nucleotide. In other embodiments, when linking two oligonucleotides together, the linker can attach internal residues of each oligonucleotides, e.g., via a modified nucleobase. One of ordinary skill in the art will understand that many such permutations are available for multimers. Further examples of appropriate linkers as well as methods for producing compounds having such linkers are disclosed in International Patent Application Number, PCT/US2012/05535, entitled MULTIMERIC
OLIGONUCLEOTIDE COMPOUNDS the contents of which relating to linkers and related chemistries are incorporated herein by referenced in its entirety. Methods for Selecting Candidate Oligonucleotides for Activating Expression of SMNl or SMN2
Methods are provided herein for selecting a candidate oligonucleotide for activating or enhancing expression of SMNl or SMN2. The target selection methods may generally involve steps for selecting single stranded oligonucleotides having any of the structural and functional characteristics disclosed herein. Typically, the methods involve one or more steps aimed at identifying oligonucleotides that target a PRC2-associated region that is functionally related to SMNl or SMN2, for example a PRC2-associated region of a IncRNA that regulates expression of SMNl or SMN2 by facilitating {e.g., in a czs-regulatory manner) the recruitment of PRC2 to the SMNl or SMN2 gene. Such oligonucleotides are expected to be candidates for activating expression of SMNl or SMN2 because of their ability to hybridize with the PRC2-associated region of a nucleic acid {e.g., a IncRNA). In some embodiments, this hybridization event is understood to disrupt interaction of PRC2 with the nucleic acid {e.g., a IncRNA) and as a result disrupt recruitment of PRC2 and its associated co-repressors {e.g., chromatin remodeling factors) to the SMNl or SMN2 gene locus.
Methods of selecting a candidate oligonucleotide may involve selecting a PRC2- associated region {e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 9 to 29) that maps to a chromosomal position encompassing or in proximity to the SMNl or SMN2 gene {e.g., a chromosomal position having a sequence as set forth in any one of SEQ ID NOS: 1 to 8). The PRC2-associated region may map to the strand of the chromosome comprising the sense strand of the SMNl or SMN2 gene, in which case the candidate oligonucleotide is complementary to the sense strand of the SMNl or SMN2 gene (i.e., is antisense to the SMNl or SMN2 gene). Alternatively, the PRC2-associated region may map to the strand of the first chromosome comprising the antisense strand of the SMNl or SMN2 gene, in which case the oligonucleotide is complementary to the antisense strand (the template strand) of the SMNl or SMN2 gene (i.e., is sense to the SMNl or SMN2 gene).
Methods for selecting a set of candidate oligonucleotides that is enriched in oligonucleotides that activate expression of SMNl or SMN2 may involve selecting one or more PRC2-associated regions that map to a chromosomal position that encompasses or that is in proximity to the SMNl or SMN2 gene and selecting a set of oligonucleotides, in which each oligonucleotide in the set comprises a nucleotide sequence that is complementary with the one or more PRC2-associated regions. As used herein, the phrase, "a set of oligonucleotides that is enriched in oligonucleotides that activate expression of refers to a set of oligonucleotides that has a greater number of oligonucleotides that activate expression of a target gene (e.g., SMN1 or SMN2) compared with a random selection of
oligonucleotides of the same physicochemical properties (e.g. , the same GC content, Tm, length etc.) as the enriched set.
Where the design and/or synthesis of a single stranded oligonucleotide involves design and/or synthesis of a sequence that is complementary to a nucleic acid or PRC2- associated region described by such sequence information, the skilled person is readily able to determine the complementary sequence, e.g., through understanding of Watson Crick base pairing rules which form part of the common general knowledge in the field.
In some embodiments design and/or synthesis of a single stranded oligonucleotide involves manufacture of an oligonucleotide from starting materials by techniques known to those of skill in the art, where the synthesis may be based on a sequence of a PRC2- associated region, or portion thereof.
Methods of design and/or synthesis of a single stranded oligonucleotide may involve one or more of the steps of:
Identifying and/or selecting PRC2-associated region;
Designing a nucleic acid sequence having a desired degree of sequence identity or complementarity to a PRC2-associated region or a portion thereof;
Synthesizing a single stranded oligonucleotide to the designed sequence;
Purifying the synthesized single stranded oligonucleotide; and
Optionally mixing the synthesized single stranded oligonucleotide with at least one pharmaceutically acceptable diluent, carrier or excipient to form a pharmaceutical composition or medicament.
Single stranded oligonucleotides so designed and/or synthesized may be useful in method of modulating gene expression as described herein.
Preferably, single stranded oligonucleotides of the invention are synthesized chemically. Oligonucleotides used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques.
Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5 ' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2*-0-dimethylaminoethyl (2*-0-DMAOE), 2*-0-dimethylaminopropyl (2*-0-DMAP), 2*-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0~N-methylacetamido (2'-0~NMA). As another example, the nucleic acid sequence can include at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
It is understood that any of the modified chemistries or formats of single stranded oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
In some embodiments, the method may further comprise the steps of amplifying the synthesized single stranded oligonucleotide, and/or purifying the single stranded
oligonucleotide (or amplified single stranded oligonucleotide), and/or sequencing the single stranded oligonucleotide so obtained.
As such, the process of preparing a single stranded oligonucleotide may be a process that is for use in the manufacture of a pharmaceutical composition or medicament for use in the treatment of disease, optionally wherein the treatment involves modulating expression of a gene associated with a PRC2-associated region.
In the methods described above a PRC2-associated region may be, or have been, identified, or obtained, by a method that involves identifying RNA that binds to PRC2.
Such methods may involve the following steps: providing a sample containing nuclear ribonucleic acids, contacting the sample with an agent that binds specifically to PRC2 or a subunit thereof, allowing complexes to form between the agent and protein in the sample, partitioning the complexes, synthesizing nucleic acid that is complementary to nucleic acid present in the complexes.
Where the single stranded oligonucleotide is based on a PRC2-associated region, or a portion of such a sequence, it may be based on information about that sequence, e.g., sequence information available in written or electronic form, which may include sequence information contained in publicly available scientific publications or sequence databases. Nucleotide Analogues
In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide. In some
embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide may have one or more 2' O-methyl nucleotides. The oligonucleotide may consist entirely of 2' O-methyl nucleotides.
Often the single stranded oligonucleotide has one or more nucleotide analogues. For example, the single stranded oligonucleotide may have at least one nucleotide analogue that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue. The single stranded oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
The oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15 , 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
The oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues. Optionally, the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. The oligonucleotide may consist entirely of bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues. The oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides. The oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides. The oligonucleotide may have a 5 ' nucleotide that is a bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). The oligonucleotide may have a 5 ' nucleotide that is a deoxyribonucleotide.
The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5 ' and 3 ' ends of the deoxyribonucleotides. The oligonucleotide may comprise
deoxyribonucleotides fianked by 1 , 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5 ' and 3 ' ends of the deoxyribonucleotides. The 3 ' position of the oligonucleotide may have a 3 ' hydroxyl group. The 3 ' position of the oligonucleotide may have a 3 ' thiophosphate.
The oligonucleotide may be conjugated with a label. For example, the
oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5 ' or 3 ' end.
Preferably the single stranded oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the
modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, the single stranded oligonucleotides are chimeric
oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving R A:DNA or RNA:R A hybrids. Chimeric single stranded oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.
In some embodiments, the single stranded oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.
A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with
phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-O- CH2, CH,~N(CH3)~0~CH2 (known as a methylene(methylimino) or MMI backbone, CH2 - O-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al, Science 1991 , 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3*-5* to 5*-3* or 2*-5* to 5*-2*; see US patent nos. 3,687,808; 4,469,863;
4,476,301 ; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321 , 131 ; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541 ,306; 5,550, 1 1 1 ; 5,563, 253; 5,571 ,799; 5,587,361 ; and 5,625,050.
Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol, 2002, 243, 209-214; Nasevicius et al, Nat. Genet., 2000, 26, 216- 220; Lacerra et al, Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g. , as described in Iverson, Curr. Opin. Mol. Ther., 3 :235-238, 2001 ; and Wang et al, J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al, J. Am. Chem. Soc, 2000, 122, 8595-8602.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5,166,315; 5, 185,444; 5,214, 134; 5,216, 141 ; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541 ,307; 5,561 ,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al, Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
Other preferred modifications include ethylene-bridged nucleic acids (ENAs) (e.g., International Patent Publication No. WO 2005/042777, Morita et al, Nucleic Acid Res., Suppl 1 :241-242, 2001 ; Surono et al., Hum. Gene Ther., 15 :749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al, Nucleic Acids Symp. Ser (Oxf), 49: 171- 172, 2005; the disclosures of which are incorporated herein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids.
Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
Figure imgf000047_0001
where X and Y are independently selected among the groups -0-,
-S-, -N(H)-, N(R)-, -CH2- or -CH- (if part of a double bond),
-CH2-0-, -CH2-S-, -CH2-N(H)-, -CH2-N(R)-, -CH2-CH2- or -CH2-CH- (if part of a double bond), -CH=CH-, where R is selected from hydrogen and Ci_4-alkyl; Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
Preferably, the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
Figure imgf000047_0002
wherein Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci_4-alkyl. In some embodiments, the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
In some embodiments, the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O)2-O-, -0-P(0,S)-0-, -0-P(S)2-O-, -S-P(0)2-0-, -S-P(0,S)-0-, -S-P(S)2-0-, -0-P(O)2-S-, -0-P(0,S)-S-, -S-P(0)2-S-, -0-PO(RH)-0-, o- PO(OCH3)-0-, -0-PO(NRH)-0-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BH3)-0-, -0-PO(NHRH)- 0-, -0-P(0)2-NRH-, -NRH-P(0)2-0-, -NRH-CO-0-, where RH is selected from hydrogen and Ci_4-alkyl.
Specifically preferred LNA units are shown in scheme 2:
Figure imgf000048_0001
^-D-oxy-LJJA
Figure imgf000048_0002
P-D-aroino-LNA
Scheme 2
The term "thio-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L-configuration.
The term "amino-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Ci_4-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.
The term "oxy-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH2-0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
The term "ena-LNA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B).
LNAs are described in additional detail herein.
One or more substituted sugar moieties can also be included, e.g. , one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar
modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Single stranded oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g., Kornberg, "DNA Replication," W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)). A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and may be used as base substitutions.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be
incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
Single stranded oligonucleotides can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.
Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al, Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, et al, eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
In some embodiments, the single stranded oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. For example, one or more single stranded oligonucleotides, of the same or different types, can be conjugated to each other; or single stranded
oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2- di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.
These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
In some embodiments, single stranded oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate. It should be appreciated that additional molecules (e.g. a biotin moiety or a fluorophor) can be conjugated to the 5 ' or 3' end of the single stranded oligonucleotide. In some embodiments, the single stranded oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
In some embodiments, the single stranded oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3 ' ends of the deoxyribonucleotides. In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3 ' hydroxyl group or a 3' thiophosphate.
In some embodiments, the single stranded oligonucleotide comprises
phosphorothioate internucleotide linkages. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
It should be appreciated that the single stranded oligonucleotide can have any combination of modifications as described herein.
The oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx,
(X)xxxXxX and (X)xxxxXX,
(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,
(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and
(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and
XXXXXXx, in which "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or R A nucleotide unit. Each of the above listed patterns may appear one or more times within an oligonucleotide, alone or in combination with any of the other disclosed modification patterns.
Methods for Modulating Gene Expression
In some embodiments, methods are provided for increasing expression of SMN protein in a cell. The methods, in some embodiments, involve delivering to the cell a first single stranded oligonucleotide complementary with a PRC2-associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of a mature mRNA of SMNl or SMN2 that comprises (or includes) exon 7 in the cell. The first and second single stranded oligonucleotides may be delivered together or separately. The first and second single stranded oligonucleotides may be linked together, or unlinked, i.e., separate.
In some embodiments, methods are provided for treating spinal muscular atrophy in a subject. The methods, in some embodiments, involve administering to a subject a first single stranded oligonucleotide complementary with a PRC2-associated region of SMNl or SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMNl or SMN2, in amounts sufficient to increase expression of full length SMN protein in the subject to levels sufficient to improve one or more conditions associated with SMA. The first and second single stranded oligonucleotides may be administered together or separately. The first and second single stranded oligonucleotides may be linked together, or unlinked, i.e., separate. The first single stranded oligonucleotide may be administered within 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours or more of administration of the second single stranded oligonucleotide. The first single stranded oligonucleotide may be administered before or after the second single stranded oligonucleotide. The oligonucleotides may be administered once or on multiple occasions depending on the needs of the subject and/or judgment of the treating physician. In some cases, the oligonucleotides may be administered in cycles. The administration cycles may vary; for example, the administration cycle may be 2nd oligo - 1st oligo - 2nd oligo - 1st oligo and so on; or 1st oligo-2nd oligo- 1st oligo-2nd oligo, and so on; or 1st oligo - 2nd oligo - 2nd oligo -1st oligo- 1st oligo - 2nd oligo - 2nd oligo -1st oligo, and so on. The skilled artisan will be capable of selecting administration cycles and intervals between each administration that are appropriate for treating a particular subject.
In one aspect, the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which SMNl or SMN2 levels are reduced) for research purposes (e.g., to study the function of the gene in the cell). In another aspect, the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which SMNl or SMN2 levels are reduced) for gene or epigenetic therapy. The cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of SMNl or SMN2. In some embodiments, methods for modulating gene expression in a cell comprise delivering a single stranded oligonucleotide as described herein. In some embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level of expression of gene in a control cell to which the single stranded
oligonucleotide has not been delivered. In certain embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 50%> greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered.
In another aspect of the invention, methods comprise administering to a subject (e.g. a human) a composition comprising a single stranded oligonucleotide as described herein to increase protein levels in the subject. In some embodiments, the increase in protein levels is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering.
As another example, to increase expression of SMNl or SMN2 in a cell, the methods include introducing into the cell a single stranded oligonucleotide that is sufficiently complementary to a PRC2-associated region (e.g., of a long non-coding R A) that maps to a genomic position encompassing or in proximity to the SMNl or SMN2 gene.
In another aspect of the invention provides methods of treating a condition (e.g. , Spinal muscular atrophy) associated with decreased levels of expression of SMNl or SMN2 in a subject, the method comprising administering a single stranded oligonucleotide as described herein.
A subject can include a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, a subject is a human. Single stranded oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Single stranded oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
For therapeutics, an animal, preferably a human, suspected of having Spinal muscular atrophy is treated by administering single stranded oligonucleotide in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a single stranded oligonucleotide as described herein.
Formulation, Delivery, And Dosing
The oligonucleotides described herein can be formulated for administration to a subject for treating a condition {e.g., Spinal muscular atrophy) associated with decreased levels of SMN protein. It should be understood that the formulations, compositions and methods can be practiced with any of the oligonucleotides disclosed herein. In some embodiments, formulations are provided that comprise a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. In some embodiments, formulations are provided that comprise a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene that is linked via a linker with a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. Thus, it should be appreciated that in some embodiments, a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene is linked with a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene, and in other embodiments, the single stranded oligonucleotides are not linked. Single stranded oligonucleotides that are not linked may be administered to a subject or delivered to a cell simultaneously (e.g., within the same composition) or separately.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient {e.g. , an oligonucleotide or compound of the invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g. tumor regression.
Pharmaceutical formulations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
A formulated single stranded oligonucleotide composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the single stranded oligonucleotide is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can, e.g. , be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the single stranded oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.
In some embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
A single stranded oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g., a protein that complexes with single stranded oligonucleotide. Still other agents include chelators, e.g., EDTA (e.g., to
2_|_
remove divalent cations such as Mg ), salts, RNAse inhibitors (e.g., a broad specificity
RNAse inhibitor such as RNAsin) and so forth. In some embodiments, the other agent used in combination with the single stranded oligonucleotide is an agent that also regulates SMN expression. In some embodiments, the other agent is a growth hormone, a histone deacetylase inhibitor, a hydroxycarbamide (hydroxyurea), a natural polyphenol compound (e.g., resveratrol, curcumin), prolactin, or salbutamol. Examples of histone deacetylase inhibitors that may be used include aliphatic compounds (e.g., butyrates (e.g., sodium butyrate and sodium phenylbutyrate) and valproic acid), benzamides (e.g., M344), and hydroxamic acids (e.g., CBHA, SBHA, Entinostat (MS-275)) Panobinostat (LBH-589), Trichostatin A, Vorinostat (SAHA)),
In one embodiment, the single stranded oligonucleotide preparation includes another single stranded oligonucleotide, e.g. , a second single stranded oligonucleotide that modulates expression and/or mR A processing of a second gene or a second single stranded
oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different single stranded oligonucleotide species. Such single stranded oligonucleotides can mediated gene expression with respect to a similar number of different genes. In one embodiment, the single stranded oligonucleotide preparation includes at least a second therapeutic agent {e.g., an agent other than an oligonucleotide).
Route of Delivery
A composition that includes a single stranded oligonucleotide can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular. The term "therapeutically effective amount" is the amount of oligonucleotide present in the composition that is needed to provide the desired level of SMNl or SMN2 expression in the subject to be treated to give the anticipated physiological response. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term
"pharmaceutically acceptable carrier" means that the carrier can be administered to a subject with no significant adverse toxico logical effects to the subject.
The single stranded oligonucleotide molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of single stranded oligonucleotide and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the single stranded oligonucleotide in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the single stranded oligonucleotide and mechanically introducing the oligonucleotide.
Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
(inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy. In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea), and optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
Both the oral and nasal membranes offer advantages over other routes of
administration. For example, oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g. , to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
A pharmaceutical composition of single stranded oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration. In some embodiments, parental administration involves administration directly to the site of disease (e.g. injection into a tumor).
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Any of the single stranded oligonucleotides described herein can be administered to ocular tissue. For example, the compositions can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly( vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The single stranded oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably single stranded oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A single stranded oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 μιη in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μ m and most preferably less than about 5.0 μ m. Usually the particle size distribution is between about 0.1 μ m and about 5 μ m in diameter, particularly about 0.3 μ m to about 5 μ m.
The term "dry" means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. Suitable H adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Pulmonary administration of a micellar single stranded oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
Exemplary devices include devices which are introduced into the vasculature, e.g. , devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
Other devices include non- vascular devices, e.g., devices implanted in the
peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a single stranded oligonucleotide, e.g., a device can release insulin.
In one embodiment, unit doses or measured doses of a composition that includes single stranded oligonucleotide are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.
Tissue, e.g., cells or organs can be treated with a single stranded oligonucleotide, ex vivo and then administered or implanted in a subject. The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease . In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation. Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies. In some implementations, the single stranded oligonucleotide treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate. In one embodiment, a contraceptive device is coated with or contains a single stranded oligonucleotide. Exemplary devices include condoms, diaphragms, IUD
(implantable uterine devices, sponges, vaginal sheaths, and birth control devices.
Dosage
In one aspect, the invention features a method of administering a single stranded oligonucleotide {e.g., as a compound or as a component of a composition) to a subject {e.g., a human subject). In some embodimetns, the methods involve administering a compound (e.g., a compound of the general formula A-B-C, as disclosed herein, or an single stranded oligonucleotide,) in a unit dose to a subject. In one embodiment, the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the SMN1 or SMN2. The unit dose, for example, can be administered by injection {e.g., intravenous or intramuscular), an inhaled dose, or a topical application.
In some embodiments, the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency {e.g., not a regular frequency). For example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g. , once an hour, two hours, four hours, eight hours, twelve hours, etc.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a single stranded oligonucleotide. The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg ofbody weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day. The maintenance doses may be administered no more than once every 1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In some embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semipermanent stent {e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
In some embodiments, the pharmaceutical composition includes a plurality of single stranded oligonucleotide species. In some embodiments, the pharmaceutical composition comprises a first single stranded oligonucleotide complementary with a PRC2-associated region of a gene (e.g., SMN1 or SMN2), and a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of a gene (e.g., SMN1 or SMN2). In some embodiments, the pharmaceutical composition includes a compound comprising the general formula A-B-C, in which A is a single stranded oligonucleotide complementary with a PRC2-associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
In another embodiment, the single stranded oligonucleotide species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence {e.g., a PRC2-associated region). In another embodiment, the plurality of single stranded oligonucleotide species is specific for different PRC2-associated regions. In another embodiment, the single stranded oligonucleotide is allele specific. In some cases, a patient is treated with a single stranded oligonucleotide in conjunction with other therapeutic modalities.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
The concentration of the single stranded oligonucleotide composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of single stranded oligonucleotide administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a single stranded oligonucleotide can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a single stranded oligonucleotide used for treatment may increase or decrease over the course of a particular treatment. For example, the subject can be monitored after administering a single stranded oligonucleotide composition. Based on information from the monitoring, an additional amount of the single stranded
oligonucleotide composition can be administered.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of SMN1 or SMN2 expression levels in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing
methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human SMN1 or SMN2. In another embodiment, the composition for testing includes a single stranded oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between SMN1 or SMN2 in the animal model and the SMN1 or SMN2 in a human. In one embodiment, the administration of the single stranded oligonucleotide composition is parenteral, e.g. intravenous {e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
Kits
In certain aspects of the invention, kits are provided, comprising a container housing a composition comprising a single stranded oligonucleotide. In some embodiments, the kits comprise a container housing a single stranded oligonucleotide complementary with of a PRC2-associated region of a gene; and a second container housing a single stranded oligonucleotide complementary to a splice control sequence of a precursor mR A of the gene. In some embodiments, the kits comprise a container housing a single stranded oligonucleotide complementary with of a PRC2-associated region of a gene and a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene. In some embodiments, the composition is a pharmaceutical composition comprising a single stranded oligonucleotide and a pharmaceutically acceptable carrier. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for single stranded oligonucleotides, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: Oligonucleotides targeting PRC2-associated regions that upregulate SMN1. MA TERIALS AND METHODS: Real Time PCR
RNA was harvested from the cells using Promega SV 96 Total RNA Isolation system or Trizol omitting the DNAse step. In separate pilot experiments, 50 ng of RNA was determined to be sufficient template for the reverse transcriptase reaction. RNA harvested from cells was normalized so that 50ng of RNA was input to each reverse transcription reaction. For the few samples that were too dilute to reach this limit, the maximum input volume was added. Reverse transcriptase reaction was performed using the Superscript II kit and real time PCR performed on cDNA samples using icycler SYBR green chemistry (Biorad). A baseline level of mRNA expression for each target gene was determined through quantitative PCR as outlined above. Baseline levels were also determined for mRNA of various housekeeping genes which are constitutively expressed. A "control" housekeeping gene with approximately the same level of baseline expression as the target gene was chosen for comparison purposes.
Protein expression (ELISA)
ELISA to determine SMN protein was carried out per manufacturer's instructions (SMN ELISA kit #ADI-900-209, Enzo Life Sciences). Cell Culture
Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatoma Hepal-6 cells, and human renal proximal tubule epithelial cells (RPTEC) were cultured using conditions known in the art (see, e.g. Current Protocols in Cell Biology). Other cell lines tested were neuronal cell lines (SK-N-AS, U-87) and SMN patient fibroblasts. Details of the cell lines used in the experiments described herein are provided in Table 8.
Table 8. Cell lines
Culture
Cell line Source Species Gender Cell Type Tissue Status Conditions
proximal Clonetics™ tubule EGM™ epithelial BulletKit™ (CC-
RPTEC Lonza human N/A cells kidney primary 3190)
hepatocyte immortalize Eagle's MEM +
Hep3B ATCC human M s liver d 10% FBS
immortalize
SK-N-AS ATCC human F neuroblast brain d DMEM + 10% FBS gliobastom immortalize Eagle's MEM +
U-87 ATCC human M a brain d 10% FBS
Coriell immortalize
GM03813 Institute human F fibroblast skin d MEM + 10% FBS
Coriell immortalize
GM03814 Institute human M fibroblast skin d MEM + 10% FBS
Coriell immortalize
GM09677 Institute human M fibroblast skin d MEM + 10% FBS
Coriell immortalize
GM00232 Institute human M fibroblast skin d MEM + 10% FBS
Coriell immortalize
GM03815 Institute human M fibroblast skin d MEM + 10% FBS
Coriell immortalize
GM22592 Institute human M fibroblast skin d MEM + 10% FBS
B-
Coriell lymphocyt immortalize
GM 10684 Institute human F e blood d MEM + 10% FBS
GM00321 Coriell immortalize
(normal) Institute human F fibroblast skin d MEM + 10% FBS Oligonucleotide design
Oligonucleotides were designed within PRC2-interacting regions in order to upregulate SMNl . The sequence and structure of each oligonucleotide is shown in Table 2. The following table provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 2.
In vitro transfection of cells with oligonucleotides
Cells were seeded into each well of 24-well plates at a density of 25,000 cells per 500uL and transfections were performed with Lipofectamine and the single stranded oligonucleotides. Control wells contained Lipofectamine alone. At 48 hours post- transfection, approximately 200 uL of cell culture supernatants were stored at -80 C for ELISA. At 48 hours post-transfection, RNA was harvested from the cells and quantitative PCR was carried out as outlined above. The percent induction of target mRNA expression by each oligonucleotide was determined by normalizing mRNA levels in the presence of the oligonucleotide to the mRNA levels in the presence of control (Lipofectamine alone). This was compared side-by-side with the increase in mRNA expression of the "control" housekeeping gene.
RESULTS:
In vitro delivery of single stranded oligonucleotides upregulated SMNl expression
Oligonucleotides were designed as candidates for upregulating SMNl expression. A total of 52 single stranded oligonucleotides were designed to be complementary to a PRC2- interacting region within a sequence as set forth in SEQ ID NO: 1, 2, 4, or 5.
Oligonucleotides were tested in at least duplicate. The sequence and structural features of the oligonucleotides are set forth in Table 2. Briefly, cells were transfected in vitro with the oligonucleotides as described above. SMNl expression in cells following treatment was evaluated by qRT-PCR. Oligonucleotides that upregulated SMNl expression were identified. Further details are outlined in Table 2.
Table 5 shows further results from experiments in which oligonucleotides were transfected into cells at a particular concentration [oligo] and 48 or 72h later RNA was prepared and qRTPCR assays carried out to determine mRNA levels of full length (FL) or delta7 SMN. In other cases, oligos were administered gymnotically to cells at ΙΟμΜ and R A harvested 9 days post treatment. The cell lines tested were neuronal cell lines (SK-N- AS, U-87) and SMN patient fibroblasts.
Table 6 shows results from experiments in which oligonucleotides were transfected into cells in combination with either one or two more oligos or small molecule compounds at a particular concentration ([oligo], [2nd], [3rd]) and 48 or 72h later RNA was prepared and qRTPCR assays carried out to determine mRNA levels of full length (FL) or delta7 SMN. The cell lines tested were SMN patient fibroblasts.
Table 7 shows results from experiments in which oligonucleotides were transfected into cells in combination with either one or two more oligos or as dimers or by gymnotic treatment at a particular concentration ([oligo], [2nd], [3rd]) and 24, 48, 72 or 216h later cell lysates were prepared and ELISA assays carried out to determine SMN protein levels. The cell lines tested were SMN patient fibroblasts.
Tables
Table 1: Non-Seed hexamer sequences.
AAAAAA, AAAAAG, AAAACA, AAAAGA, AAAAGC, AAAAGG, AAAAUA, AAACAA, AAACAC, AAACAG, AAACAU, AAACCC, AAACCU, AAACGA, AAACGC, AAACGU, AAACUA, AAACUC, AAACUU, AAAGAU, AAAGCC, AAAGGA, AAAGGG, AAAGUC, AAAUAC, AAAUAU, AAAUCG, AAAUCU, AAAUGC, AAAUGU, AAAUUA, AAAUUG, AACAAC, AACAAG, AACAAU, AACACA, AACACG, AACAGA, AACAGC, AACAGG, AACAUC, AACAUG, AACCAA, AACCAC, AACCAG, AACCAU, AACCCC, AACCCG, AACCGA, AACCGC, AACCGG, AACCUA, AACCUU, AACGAA, AACGAC, AACGAG, AACGAU, AACGCU, AACGGG, AACGGU, AACGUA, AACGUC, AACGUG, AACGUU, AACUAU, AACUCA, AACUCC, AACUCG, AACUGA, AACUGC, AACUGU, AACU UA, AACUUC, AACU UG, AACUUU, AAGAAA, AAGAAG, AAGAAU, AAGACG, AAGAGA, AAGAGC, AAGAGG, AAGAGU, AAGAUU, AAGCAA, AAGCAC, AAGCAG, AAGCAU, AAGCCA, AAGCCC, AAGCCG, AAGCCU, AAGCGA, AAGCGG, AAGCGU, AAGCUA, AAGGAA, AAGGAC, AAGGCU, AAGGGC, AAGGGU, AAGGUU, AAGUAA, AAGUAC, AAGUAU, AAGUCC, AAGUCG, AAGUGA, AAGUGG, AAGUUA, AAGU UU, AAUAAA, AAUAAC, AAUAAG, AAUAAU, AAUACA, AAUACC, AAUACG, AAUAGA, AAUAGC, AAUAGG, AAUAGU, AAUAUC, AAUAU U, AAUCAA, AAUCAU, AAUCCA, AAUCCC, AAUCCG, AAUCGA, AAUCGC, AAUCGU, AAUCUA, AAUCUG, AAUCUU, AAUGAA, AAUGAC, AAUGAG, AAUGAU, AAUGCG, AAUGCU, AAUGGA, AAUGGU, AAUGUA, AAUGUC, AAUGUG, AAUUAA, AAUUAC, AAUUAG, AAU UCC, AAU UCG, AAUUGA, AAUUGG, AAU UGU, AAUUUC, AAU UUG, ACAAAA, ACAAAC, ACAAAG, ACAAAU, ACAACC, ACAACG, ACAACU, ACAAGA, ACAAGC, ACAAGU, ACAAUC, ACAAUG, ACAAUU, ACACAG, ACACCA, ACACCC, ACACCG, ACACCU, ACACGA, ACACGC, ACACGU, ACACUC, ACACUG, ACACUU, ACAGAA, ACAGAC, ACAGCC, ACAGCG, ACAGCU, ACAGGG, ACAGUC, ACAGUG, ACAGU U, ACAUAA, ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAU UG, ACAU UU, ACCAAA, ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU, ACCAUA, ACCAUG, ACCAU U, ACCCAA, ACCCAC, ACCCCA, ACCCCG, ACCCGA, ACCCGC, ACCCUA, ACCCUC, ACCCUU, ACCGAA, ACCGAC, ACCGAU, ACCGCA, ACCGCC, ACCGCG, ACCGCU, ACCGGA, ACCGGC, ACCGGU, ACCGUA, ACCGUC, ACCGUG, ACCGUU, ACCUAA, ACCUAC, ACCUAG, ACCUAU, ACCUCA, ACCUCC, ACCUCG, ACCUCU, ACCUGA, ACCUGC, ACCUGU, ACCUUA, ACCUUC, ACCU U U, ACGAAA, ACGAAC, ACGAAG, ACGAAU, ACGACA, ACGACC, ACGACG, ACGACU, ACGAGA, ACGAGC, ACGAGG, ACGAGU, ACGAUA, ACGAUC, ACGAUG, ACGAUU, ACGCAA, ACGCAG, ACGCAU, ACGCCC, ACGCCG, ACGCCU, ACGCGA, ACGCGG, ACGCGU, ACGCUA, ACGCUG, ACGCUU, ACGGAA, ACGGAC, ACGGAG, ACGGAU, ACGGCC, ACGGCG, ACGGCU, ACGGGC, ACGGGG, ACGGGU, ACGGUA, ACGGUC, ACGGUG, ACGGUU, ACGUAA, ACGUAC, ACGUAU, ACGUCC, ACGUCG, ACGUCU, ACGUGA, ACGUGC, ACGUGG, ACGUGU, ACGUUA, ACGU UC, ACGUUG, ACGUU U, AC U AAA, ACUAAG, ACUAAU, ACUACA, ACUACC, ACUACG, ACUACU, ACUAGG, ACUAUC, ACUAUG, ACUAUU, ACUCAU, ACUCCC, ACUCCG, ACUCCU, ACUCGA, ACUCGC, ACUCGG, ACUCUC, ACUCUU, ACUGAG, ACUGAU, ACUGCC, ACUGCG, ACUGCU, ACUGGG, ACUGGU, ACUGUC, ACUUAA, ACU UAC, ACUUAU, ACU UCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACU UGC, ACU UGU, ACUUUA, ACU UUC, ACUU UG, AGAAAA, AGAAAC, AGAAAG, AGAACC, AGAACG, AGAACU, AGAAGC, AGAAGU, AGAAUA, AGAAUC, AGAAUG, AGAAU U, AGACAA, AGACAC, AGACAU, AGACCA, AGACCC, AGACCG, AGACCU, AGACGA, AGACGC, AGACGU, AGACUA, AGACUC, AGACUU, AGAGAC, AGAGAG, AGAGAU, AGAGCC, AGAGCG, AGAGCU, AGAGGC, AGAGGG, AGAGGU, AGAGUA, AGAGUU, AGAUAC, AGAUAG, AGAUAU, AGAUCC, AGAUCG, AGAUCU, AGAUGA, AGAUGC, AGAUGG, AGAU UA, AGAU UC, AGAUUG, AGAUU U, AGCAAC, AGCACA, AGCACG, AGCACU, AGCAGA, AGCAUA, AGCAUC, AGCAUG, AGCCAA, AGCCAU, AGCCCA, AGCCGA, AGCCGC, AGCCGG, AGCCGU, AGCCUA, AGCCUC, AGCGAA, AGCGAG, AGCGAU, AGCGCA, AGCGCC, AGCGCG, AGCGCU, AGCGGA, AGCGGC, AGCGGU, AGCGUA, AGCGUC, AGCGUG, AGCGUU, AGCUAA, AGCUAC, AGCUAG, AGCUAU, AGCUCA, AGCUCC, AGCUCG, AGCUCU, AGCUGA, AGCUGG, AGCUGU, AGCU UC, AGCUU U, AGGAAU, AGGACC, AGGACG, AGGAGA, AGGAGU, AGGAUA, AGGCAA, AGGCAU, AGGCCG, AGGCGA, AGGCGC, AGGCGG, AGGCUA, AGGCUC, AGGCUU, AGGGAC, AGGGAU, AGGGGA, AGGGGU, AGGGUA, AGGGUG, AGGUAA, AGGUAC, AGGUCA, AGGUCC, AGGUCU, AGGUGA, AGGUGC, AGGUGG, AGGUGU, AGGU UC,
AGGUUG, AGUAAA, AGUAAG, AGUAAU, AGUACA, AGUACG, AGUAGC, AGUAGG, AGUAUA, AGUAUC, AGUAUG, AGUAUU, AGUCAA, AGUCAC, AGUCAG, AGUCAU, AGUCCA, AGUCCG, AGUCCU, AGUCGA, AGUCGC, AGUCGG, AGUCGU, AGUCUA, AGUCUC, AGUCUG, AGUCU U, AGUGAA, AGUGAC, AGUGCG, AGUGGG, AGUGUC, AGU UAA, AGU UAC, AGUUAG, AGUUCC, AGUUCG, AGUUGA, AGUUGC,
AGUUGU, AGUUUA, AGUUUC, AGUU UG, AGU UU U, AUAAAC, AUAAAU, AUAACA, AUAACC, AUAACG, AUAACU, AUAAGA, AUAAGC, AUAAGG, AUAAGU, AUAAUC, AUAAUG, AUAAUU, AUACAC, AUACAG, AUACAU, AUACCA, AUACCC, AUACCG, AUACGA, AUACGC, AUACGG, AUACGU, AUACUA, AUACUC, AUACUG, AUACUU, AUAGAA, AUAGAC, AUAGAU, AUAGCA, AUAGCG, AUAGCU, AUAGGA, AUAGGU, AUAGUA, AUAGUC, AUAGUG, AUAGUU, AUAUAC, AUAUAG, AUAUCC, AUAUCG, AUAUCU, AUAUGA, AUAUGC, AUAUGG, AUAUGU, AUAUUC, AUAU UG, AUAUU U, AUCAAA, AUCAAC, AUCAAG, AUCAAU, AUCACA, AUCACC, AUCACG, AUCAGC, AUCAGG, AUCCAA, AUCCAU, AUCCCC, AUCCCG, AUCCGA, AUCCGC, AUCCGG, AUCCUA, AUCCUC, AUCCUG, AUCGAA, AUCGAC, AUCGAG, AUCGAU, AUCGCA, AUCGCC, AUCGCG, AUCGCU, AUCGGC, AUCGGG, AUCGGU, AUCGUC, AUCGUG, AUCGU U, AUCUAA, AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU, AUCU UG, AUCUU U, AUGAAA, AUGAAC, AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG, AUGAGU, AUGAUA, AUGAUC, AUGAU U, AUGCAA, AUGCAG, AUGCCA, AUGCCC, AUGCCG, AUGCGA, AUGCGG, AUGCGU, AUGCUC, AUGCUU, AUGGAC, AUGGCC, AUGGGA, AUGGGC, AUGGGU, AUGGUC, AUGGUG, AUGUAC, AUGUAU, AUGUCA, AUGUCC, AUGUCG, AUGUGU, AUGUUA, AUGUUC, AU UAAA, AUUAAC, AU UAAG, AU UAAU, AU UACA, AUUACC, AUUACG, AUUACU, AU UAGA, AUUAGC, AUUAGG, AU UAGU, AU UAUA, AUUAUC, AUUAUG, AUUCAC, AUUCCA, AU UCCG, AU UCCU, AUUCGA, AUUCGC, AUUCGG, AU UCGU, AUUCUA, AUUCUC, AUUCU U, AUUGAA, AUUGAC, AUUGAU, AU UGCC, AUUGCG, AUUGCU, AUUGGA, AUUGGC,
AUUGGG, AU UGGU, AUUGUA, AUUGUC, AUUGUG, AU UGU U, AU UUAA, AU UUAG, AU UUAU, AUU UCC, AUU UCG, AUU UCU, AU UUGA, AU UUGC, AU UUGU, AU UUUA, AU UU UC, AUU UUG, AUU UU U, CAAAAG, CAAACA, CAAACC, CAAACG, CAAACU, CAAAGA, CAAAGG, CAAAUA, CAAAUU, CAACAC, CAACAU, CAACCA, CAACCC, CAACCG, CAACGA, CAACGC, CAACGG, CAACGU, CAACUA, CAACUC, CAACUG, CAACUU, CAAGAA, CAAGAC, CAAGAU, CAAGCA, CAAGCC, CAAGCG, CAAGCU, CAAGGA, CAAGGG, CAAGUC, CAAGUG, CAAGU U, CAAUAA, CAAUAC, CAAUAG, CAAUCC, CAAUCG, CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU, CAAU UC, CAAU UG, CAAU UU, CACAAU, CACACA, CACACG, CACACU, CACAGA, CACAGC, CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC, CACCAU, CACCCA, CACCCC, CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCU U, CACGAA, CACGAC, CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG, CACGG U, CACGUA, CACGUC, CACGUG, CACGU U, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG, CACUGA, CACUGC, CACUGG, CACUUA, CACU UC, CACU UU, CAGAAA, CAGAAG, CAGAAU, CAGACC, CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC, CAGCGG, CAGCGU, CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC, CAGGUU, CAGUAC, CAGUCG, CAGU UG, CAUAAA, CAUAAC, CAUAAG, CAUAAU, CAUACA, CAUACC, CAUACG, CAUACU, CAUAGA, CAUAGG, CAUAGU, CAUAUA, CAUAUC, CAUAUG, CAUCAA, CAUCAC, CAUCAG, CAUCAU, CAUCCA, CAUCCC, CAUCCG, CAUCGA, CAUCGC, CAUCGG, CAUCGU, CAUCUA, CAUCUC, CAUCUG, CAUCUU, CAUGAA, CAUGAC, CAUGAG, CAUGAU, CAUGCA, CAUGCC, CAUGCG, CAUGCU, CAUGGC, CAUGGG, CAUGGU, CAUGUA, CAUGUC, CAUGUU, CAU UAA, CAUUAC, CAUUAG, CAUUCA, CAU UCC, CAU UCG, CAU UCU, CAU UGA, CAU UGG, CAUU UC, CAU UUG, CAUU UU, CCAAAA, CCAAAC, CCAAAG, CCAAAU, CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG, CCAAU U, CCACAA, CCACAC, CCACAG, CCACAU, CCACCA, CCACCC, CCACCG, CCACCU, CCACGA, CCACGC, CCACGG, CCACGU, CCACUA, CCACUC, CCACUU, CCAGAA, CCAGAC, CCAGAG, CCAGCC, CCAGGU, CCAGUC, CCAGUU, CCAUAA, CCAUAC, CCAUAG, CCAUAU, CCAUCA, CCAUCC, CCAUCU, CCAUGA, CCAUGC, CCAUGG, CCAUUC, CCAUUG, CCAUU U, CCCAAC, CCCAAG, CCCAAU, CCCACA, CCCAGA, CCCAGC, CCCAGU, CCCAUA, CCCAUC, CCCAUG, CCCAUU, CCCCAA, CCCCAG, CCCCAU, CCCCCC, CCCCCG, CCCCCU, CCCCGA, CCCCGC, CCCCGU, CCCCUA, CCCCUC, CCCGAA, CCCGAC, CCCGAU, CCCGCA, CCCGCU, CCCGGA, CCCGGC, CCCGUA, CCCGUG, CCCGU U, CCCUAA, CCCUAG, CCCUCA, CCCUCU, CCCUGC, CCCUUA, CCCU UC, CCCUU U, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC, CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAU U, CCGCAA, CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA, CCGCGC, CCGCGG, CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCUU, CCGGAA, CCGGAU, CCGGCA, CCGGCC, CCGGCG, CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA, CCGGUC, CCGGUG, CCGUAA, CCGUAG, CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA, CCGUGU, CCGUUA, CCGUUC, CCGU UG, CCGUU U, CCUAAC, CCUAAG, CCUAAU, CCUACA, CCUACC, CCUACG, CCUACU, CCUAGA, CCUAGC, CCUAGG, CCUAGU, CCUAUA, CCUAUC, CCUAUG, CCUAUU, CCUCAA, CCUCAC, CCUCAG, CCUCAU, CCUCCA, CCUCCC, CCUCCG, CCUCGA, CCUCGC, CCUCGG, CCUCGU, CCUCUA, CCUCUG, CCUGAC, CCUGAU, CCUGCA, CCUGGG, CCUGGU, CCUGU U, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA, CCUUGU, CCU UUA, CCUU UC, CCU UU U, CGAAAA, CGAAAC, CGAAAG, CGAAAU, CGAACA, CGAACC, CGAACG, CGAACU, CGAAGA, CGAAGC, CGAAGG, CGAAGU, CGAAUA, CGAAUC, CGAAUG, CGAAUU, CGACAA, CGACAC, CGACAU, CGACCA, CGACCU, CGACGA, CGACGC, CGACGG, CGACGU, CGACUA, CGACUG, CGACU U, CGAGAA, CGAGAC, CGAGAG, CGAGAU, CGAGCA, CGAGCC, CGAGCG, CGAGCU, CGAGGC, CGAGGG, CGAGGU, CGAGUA, CGAGUC, CGAGUG, CGAG UU, CGAUAA, CGAUAC, CGAUAG, CGAUAU, CGAUCA, CGAUCC, CGAUCG, CGAUCU, CGAUGA, CGAUGC, CGAUGG, CGAUGU, CGAUUA, CGAU UC, CGAUUG, CGAUU U, CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG, CGCAGA, CGCAGC, CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAU U, CGCCAA, CGCCAC, CGCCAG, CGCCAU, CGCCCA, CGCCCC, CGCCCG, CGCCGA, CGCCGC, CGCCGG, CGCCGU, CGCCUA, CGCCUG, CGCCUU, CGCGAA, CGCGAC, CGCGAG, CGCGAU, CGCGCA, CGCGCC, CGCGCG, CGCGCU, CGCGGA, CGCGGC, CGCGGG, CGCGGU, CGCGUA, CGCGUC, CGCGUG, CGCGU U, CGCUAA, CGCUAC, CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA, CGCUGC, CGCUGG, CGCUGU, CGCUUA, CGCU UC, CGCU UG, CGGAAA, CGGAAC, CGGAAG, CGGACA, CGGACC, CGGACG, CGGACU, CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAU U, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC, CGGCCG, CGGCGC, CGGCGG, CGGCGU, CGGCUA, CGGCUC, CGGCUG, CGGCU U, CGGGAA, CGGGAC, CGGGAG, CGGGAU, CGGGCA, CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG, CGG UAA, CGGUAC, CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU, CGG UUA, CGGU UC, CGGUUG, CGGUUU, CGUAAA, CGUAAC, CGUAAG, CGUAAU, CGUACA, CGUACG, CGUACU, CGUAGA, CGUAGC, CGUAGG, CGUAGU, CGUAUA, CGUAUC, CGUAUG, CGUAUU, CGUCAA, CGUCAC, CGUCAG, CGUCAU, CGUCCA, CGUCCC, CGUCCG, CGUCCU, CGUCGA, CGUCGG, CGUCGU, CGUCUA, CGUCUC, CGUCUG, CGUCU U, CGUGAA, CGUGAC, CGUGAG, CGUGAU, CGUGCC, CGUGCG, CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG, CGUUAA, CGUUAC, CGU UAG,
CGU UAU, CGUUCA, CGUUCC, CGUUCG, CGU UCU, CGUUGA, CGUUGC, CGU UGU, CGU UUA, CGUUUC, CGU U UU, CUAAAA, CUAAAC, CUAAAU, CUAACA, CUAACC, CUAACG, CUAACU, CUAAGA, CUAAGC, CUAAGU, CUAAUA, CUAAUC, CUAAUG, CUACAC, CUACAU, CUACCA, CUACCC, CUACCG, CUACCU, CUACGA, CUACGC, CUACGG, CUACGU, CUACUA, CUACUC, CUACUG, CUAGAA, CUAGAG, CUAGAU, CUAGCA, CUAGCC, CUAGCG, CUAGCU, CUAGGA, CUAGGG, CUAGGU, CUAGUG, CUAGUU, CUAUAA, CUAUAG, CUAUAU, CUAUCA, CUAUCC, CUAUCG, CUAUCU, CUAUGA, CUAUGC, CUAUGG, CUAUGU, CUAUUA, CUAUUG, CUCAAC, CUCAAG, CUCAAU, CUCACC, CUCACG, CUCAGC, CUCAUA, CUCAUC, CUCAUG, CUCAU U, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG, CUCCUA, CUCCUC, CUCCUU, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC, CUCGCG, CUCGGG, CUCGGU, CUCGUA, CUCGUC, CUCGUG, CUCGU U, CUCUAA, CUCUAC, CUCUAU, CUCUCA, CUCUCC, CUCUCU, CUCUGC, CUCUGU, CUCUUA, CUCU UG, CUGAAG, CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC, CUGCCG, CUGCCU, CUGCGA, CUGCUA, CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC, CUGUCA, CUGUCC, CUGUCG, CUGUGG, CUGUGU, CUGUUA, CUGUU U, CUUAAC, CUUAAG, CU UAAU, CU UACC, CUUACG, CUUAGA, CU UAGC, CU UAGG, CUUAGU, CUUAUA, CU UAUC, CU UAUG, CUUAUU, CU UCAG, CU UCAU, CUUCCA, CUUCCC, CU UCCG, CU UCCU, CUUCGA, CUUCGC, CU UCGG, CUUCGU, CU UCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CU UGCU, CUUGGC, CUUGGU, CU UGUU, CUU UAC, CU UUAG, CU UUAU, CUU UCA, CUU UCG, CU UUCU, CUUUGA, CU UUGC, CU UUGU, CU U UUA, CUUU UC, CUU UUG, CUU UUU, GAAAAA, GAAAAG, GAAAAU, GAAACC, GAAACG, GAAAGA, GAAAGC, GAAAGU, GAAAUA, GAAAUC, GAAAUG, GAAAU U, GAACAA, GAACAC, GAACAG, GAACAU, GAACCA, GAACCC, GAACCG, GAACCU, GAACGA, GAACGC, GAACGG, GAACGU, GAACUA, GAACUG, GAACUU, GAAGAC, GAAGAG, GAAGCA, GAAGCG, GAAGCU, GAAGUC, GAAUAA, GAAUAC, GAAUAG, GAAUAU, GAAUCC, GAAUCG, GAAUCU, GAAUGA, GAAUGC, GAAUGU, GAAU UA, GAAU UC, GAAUU U, GACAAA, GACAAG, GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA, GACCAC, GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC, GACCU U, GACGAA, GACGAC, GACGAG, GACGAU, GACGCA, GACGCC, GACGCG, GACGCU, GACGGA, GACGGC, GACGGG, GACGGU, GACGUA, GACGUC, GACGUG, GACG UU, GACUAA, GACUAC, GACUAG, GACUAU, GACUCA, GACUCC, GACUCG, GACUGG, GACUGU, GACUUA, GACUUG, GACUU U, GAGAAU, GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA, GAGCAU, GAGCCA, GAGCGA, GAGCGG, GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA, GAGUAG, GAGUCC, GAGUUC, GAGU UU,
GAUAAA, GAUAAC, GAUAAG, GAUAAU, GAUACA, GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC, GAUAGG, GAUAGU, GAUAUA, GAUCAA, GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC, GAUCGG, GAUCGU, GAUCUA, GAUCUG, GAUCU U, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC, GAUGCG, GAUGCU, GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGU U, GAUUAA, GAUUAC, GAUUAG, GAUUAU, GAUUCA, GAUUCG, GAU UCU, GAUUGA, GAUUGC, GAU UUA, GAU UUC,
GAUU UG, GAUUU U, GCAAAC, GCAAAG, GCAAAU, GCAACA, GCAACC, GCAAGC, GCAAGU, GCAAUA, GCAAUC, GCAAUG, GCAAUU, GCACAA, GCACAC, GCACAG, GCACCC, GCACCG, GCACCU, GCACGA, GCACGC, GCACGU, GCACUA, GCACUC, GCACUG, GCACUU, GCAGAU, GCAGCC, GCAGCG, GCAGGC, GCAGUA, GCAGUC, GCAGUG, GCAGUU, GCAUAA, GCAUAG, GCAUAU, GCAUCG, GCAUCU, GCAUGA, GCAUGC, GCAUGG, GCAUGU, GCAU UA, GCAU UC, GCAUUG, GCAUU U, GCCAAA, GCCAAC, GCCAAU, GCCACA, GCCACC, GCCACG, GCCAGA, GCCAGU, GCCAUA, GCCAUC, GCCAUG, GCCAUU, GCCCAA, GCCCAC, GCCCAG, GCCCCG, GCCCGA, GCCCGG, GCCCGU, GCCGAA, GCCGAC, GCCGAG, GCCGAU, GCCGCA, GCCGCU, GCCGGA, GCCGGC, GCCGGG, GCCGGU, GCCGUA, GCCGUC, GCCGUG, GCCGU U, GCCUAA, GCCUAU, GCCUCA, GCCUCC, GCCUCG, GCCUGA, GCCU UA, GCCU UU, GCGAAA, GCGAAC, GCGAAG, GCGAAU, GCGACC, GCGACG, GCGACU, GCGAGA, GCGAGC, GCGAGG, GCGAGU, GCGAUA, GCGAUC, GCGAUG, GCGAUU, GCGCAA, GCGCAC, GCGCAG, GCGCAU, GCGCCA, GCGCCC, GCGCCU, GCGCGA, GCGCGU, GCGCUA, GCGCUC, GCGCUG, GCGCUU, GCGGAA, GCGGAC, GCGGAU, GCGGCA, GCGGCC, GCGGCU, GCGGGA, GCGGUA, GCGGUC, GCGGUU, GCGUAA, GCGUAC, GCGUAG, GCGUAU, GCG UCA, GCGUCC, GCGUCG, GCGUCU, GCGUGA, GCGUGC, GCGUGG, GCGUGU, GCGU UA, GCGUUC, GCG UUG, GCGUU U, GCUAAA, GCUAAC, GCUAAG, GCUAAU, GCUACC, GCUACG, GCUACU, GCUAGA, GCUAGG, GCUAGU, GCUAUA, GCUAUC, GCUAUU, GCUCAA, GCUCAC, GCUCAG, GCUCAU, GCUCCA, GCUCCC, GCUCCG, GCUCGA, GCUCGC, GCUCGU, GCUCUA, GCUCUC, GCUCU U, GCUGAA, GCUGAC, GCUGAU, GCUGCA, GCUGCC, GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU, GCU UCA, GCUUCG, GCU UGA, GCUUGG, GCUUGU, GCUU UA, GCU UUG, GGAAAG, GGAACA, GGAACC, GGAACG, GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAU U, GGACAA, GGACAC, GGACAG, GGACAU, GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACU U, GGAGAC, GGAGCA, GGAGCG, GGAGGG, GGAGUA, GGAUAA, GGAUAC, GGAUCA, GGAUCC, GGAUCG, GGAUCU, GGAUGC, GGAUUA, GGAUUG, GGCAAU, GGCACA, GGCACU, GGCAGA, GGCAUA, GGCAUC, GGCCAC, GGCCAG, GGCCCC, GGCCGA, GGCCGC, GGCCGU, GGCCUA, GGCCUG, GGCCU U, GGCGAA, GGCGAG, GGCGAU, GGCGCA, GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG, GGCGU U, GGCUAA, GGCUAC, GGCUAG, GGCUAU, GGCUCC, GGCUCG, GGCUGA, GGCU UA, GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU, GGGAUA, GGGAU U, GGGCAA, GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG,
GGGGGU, GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGU UA, GGGU UG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,
GGUACU, GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GGUCGG, GGUCGU, GGUCUC, GGUCU U, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGU UAA, GGU UAG, GGU UAU, GGUUCA, GGU UCC, GGU UCG, GGU UGC, GGU UUC, GGUU UU, GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA, GUAAUC, GUAAUG, GUAAUU, GUACAA, GUACAC, GUACAG, GUACAU, GUACCA, GUACCC, GUACCG, GUACCU, GUACGA, GUACGC, GUACGG, GUACGU, GUACUA, GUACUC, GUACUG, GUACUU, GUAGAA, GUAGAC, GUAGCA, GUAGCC, GUAGCG, GUAGCU, GUAGGA, GUAGGC, GUAGGG,
GUAGGU, GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAU UG, G UAU UU, GUCAAA, GUCAAG, GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, G UCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCU U, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGU U, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCU UC, GUCU UU, GUGAAA, GUGAAC, GUGAAG, GUGACC, GUGACG, GUGAGA, GUGAGC, GUGAGU, GUGAUC, GUGAUG, GUGAUU, GUGCAC,
GUGCAU, GUGCCC, GUGCCG, GUGCGA, GUGCGG, GUGCGU, GUGCUA, GUGCUC, GUGCUG,
GUGGAG, GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGU UU, GU UAAA, GU UAAC, GUUAAG, GU UACA, GU UACC, GUUACG, GU UACU, GU UAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG,
GU UAUU, GUUCAA, GUUCAC, GUUCAG, GU UCCA, GUUCCG, GUUCGA, GU UCGC, GU UCGG, GU UCGU, GU UCUA, GUUCUG, GUUGAA, GU UGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GU UGGC, GU UGGU, GU UGUC, GUUGUG, GUUGU U, GUU UAA, GU U UAC, GU UUAG, GUU UAU, GU UUCA, GUUUCC, GU UUCU, GU UUGA, GUU UGC, GUU UGG, GUU UGU, GU UUUA, GU UU UC, GU UU UU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA, UAAAGG, UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAU U, U A AC A A, UAACAC, UAACAG, UAACCA, UAACCC, UAACCG, UAACCU, UAACGA, UAACGC, UAACGG, UAACGU, UAACUA, UAACUG, UAACUU, UAAGAG, UAAGAU, UAAGCA, UAAGCC, UAAGCG, UAAGCU, UAAGGA, UAAGGC, UAAGGG, UAAGGU, UAAGUA, UAAGUC, UAAGUG, UAAGUU, UAAUAA, UAAUCA, UAAUCC, UAAUCG, UAAUCU, UAAUGA, UAAUGG, UAAUGU, UAAUUA, UAAU UC, UAAUUG, UACAAC, UACAAG, UACAAU, UACACC, UACACG, UACACU, UACAGA, UACAGC, UACAUA, UACAUC, UACAU U, UACCAA, UACCAC, UACCAG, UACCAU, UACCCC, UACCCG, UACCCU, UACCGA, UACCGC, UACCGG, UACCGU, UACCUA, UACCUG, UACGAA, UACGAC, UACGAG, UACGAU, UACGCA, UACGCC, UACGCG, UACGCU, UACGGC, UACGGG, UACGGU, UACGUA, UACGUC, UACGUG, UACGUU, UACUAA, UACUAC, UACUAG, UACUAU, UACUCA, UACUCC, UACUCG, UACUCU, UACUGA, UACUGC, UACUGG, UACU UA, UACUUG, UACU UU, UAGAAA, UAGAAG, UAGAAU, UAGACA, UAGACG, UAGAGA, UAGAGC, UAGAGU, UAGAUA, UAGAUC, UAGAUG, UAGCAU, UAGCCC, UAGCCG, UAGCCU, UAGCGA, UAGCGC, UAGCGU, UAGCUA, UAGCUC, UAGCUG, UAGGAA, UAGGAU, UAGGCG, UAGGCU, UAGGGU, UAGGUC, UAGGUG, UAGGUU, UAGUAA, UAGUAC, UAGUAG, UAGUAU, UAGUCA, UAGUCG, UAGUGU, UAGUUA, UAGU UC, UAGU UG, UAGUU U, UAUAAC, UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU, UAUAUA, UAUAUC, UAUAUG, UAUAU U, UAUCAA, UAUCAC, UAUCAU, UAUCCA, UAUCCC, UAUCCG, UAUCCU, UAUCGA, UAUCGC, UAUCGG, UAUCGU, UAUCUA, UAUCUC, UAUCUG, UAUCUU, UAUGAA, UAUGAC, UAUGAG,
UAUGAU, UAUGCA, UAUGCG, UAUGCU, UAUGGA, UAUGGC, UAUGUC, UAUGUG, UAUGU U, UAU UAG, UAUUCA, UAU UCC, UAUUCG, UAUUCU, UAUUGA, UAUUGG, UAUU UA, UAU UUC, UAU UUG, UAUUU U, UCAAAA, UCAAAC, UCAAAG, UCAACC, UCAACU, UCAAGA, UCAAGC, UCAAUA, UCAAUC, UCAAUG, UCAAUU, UCACCC, UCACCG, UCACCU, UCACGA, UCACGC, UCACGG, UCACGU, UCACUA, UCACUC, UCACUU, UCAGAA, UCAGAC, UCAGAG, UCAGCG, UCAGCU, UCAGGA, UCAGGC, UCAGGU, UCAGUC, UCAGU U, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU, UCAUUA, UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC, UCCAGG, UCCAUA, UCCAUC, UCCAU U, UCCCAA, UCCCAG, UCCCAU, UCCCCC, UCCCCG, UCCCCU, UCCCGA, UCCCGC, UCCCGG, UCCCGU, UCCCUA, UCCCUC, UCCGAA, UCCGAC, UCCGAG, UCCGAU, UCCGCA, UCCGCC, UCCGGA, UCCGGC, UCCGGU, UCCGUA, UCCGUC, UCCGUG, UCCUAA, UCCUCA, UCCUCG, UCCUCU, UCCUGC, UCCUGU, UCCU UA, UCCU UC, UCCUU U, UCGAAA, UCGAAC, UCGAAG, UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU, UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC, UCGAUG, UCGAU U, UCGCAA, UCGCAC, UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU, UCGCGA, UCGCGC, UCGCGU, UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA, UCGGCU, UCGGGG, UCGGGU, UCGGUC, UCGGUG, UCGGU U, UCGUAA, UCGUAC, UCGUAG, UCGUAU, UCGUCA, UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGUUC, UCGUUG, UCGUUU, UCUAAC, UCUAAG, UCUAAU, UCUACA, UCUACC, UCUACG, UCUACU, UCUAGC, UCUAGG, UCUAGU, UCUAUA, UCUAUC, UCUAUG, UCUAUU, UCUCAG, UCUCAU, UCUCCG, UCUCGC, UCUCGG, UCUCGU, UCUCUC, UCUGAA, UCUGAU, UCUGCA, UCUGCG, UCUGCU, UCUGGC, UCUGGU, UCUGUC, UCUGUG, UCUGUU, UCUUAA, UCUUAC, UCUUAG, UCUUAU, UCUUCA, UCUUCC, UCUUCG, UCUUCU, UCUUGC, UCUUGG, UCUUGU, UCUUUA, UCUUUC, UCUUUG, UCUUUU, UGAAAA, UGAAAC, UGAACA, UGAACC, UGAAGG, UGAAUC, UGAAUG, UGACAA, UGACAC, UGACAG, UGACCA, UGACCC, UGACCG, UGACGA, UGACGC, UGACGG, UGACGU, UGACUA, UGACUC, UGACUU, UGAGAG, UGAGAU, UGAGCA, UGAGCC, UGAGCU, UGAGGC, UGAGGU, UGAGUA, UGAGUU, UGAUAC, UGAUAG, UGAUAU, UGAUCA, UGAUCG, UGAUCU, UGAUGA, UGAUGC, UGAUGG, UGAUGU, UGAUUA,
UGAUUC, UGAUUG, UGAUUU, UGCAAC, UGCAAG, UGCACA, UGCACG, UGCAGG, UGCAGU, UGCAUC, UGCCCA, UGCCCC, UGCCCG, UGCCGA, UGCCGC, UGCCGG, UGCCGU, UGCCUA, UGCCUC, UGCCUG, UGCCUU, UGCGAA, UGCGAC, UGCGAU, UGCGCC, UGCGCG, UGCGCU, UGCGGC, UGCGGG, UGCGGU, UGCGUA, UGCGUC, UGCGUG, UGCGUU, UGCUAC, UGCUAU, UGCUCC, UGCUCG, UGCUGC, UGCUGG, UGCUGU, UGCUUA, UGCUUU, UGGAAC, UGGAAG, UGGAGC, UGGAUC, UGGAUU, UGGCAA,
UGGCAC, UGGCAG, UGGCCG, UGGCCU, UGGCGA, UGGCGC, UGGCGU, UGGCUA, UGGCUC, UGGCUU, UGGGAA, UGGGCA, UGGGCC, UGGGGC, UGGGUC, UGGUAA, UGGUAG, UGGUAU, UGGUCC, UGGUCG, UGGUCU, UGGUGA, UGGUGC, UGGUGG, UGGUGU, UGGUUA, UGGUUG, UGUAAA, UGUAAC, UGUAAG, UGUACC, UGUACG, UGUACU, UGUAGA, UGUAGC, UGUAGU, UGUAUC, UGUAUU, UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UGUCCG, UGUCGA, UGUCGC, UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU, UGUGCA, UGUGGU, UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC, UGUUCG, UGUUGG, UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC, UUAAAG, UUAAAU, UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG, UUAAUU, UUACAA, UUACAC, UUACAG, UUACAU, UUACCA, UUACCC, UUACCG, UUACCU, UUACGA, UUACGC, UUACGG, UUACGU, UUACUA, UUACUC, UUACUG, UUACUU, UUAGAA, UUAGAC, UUAGCC, UUAGCG, UUAGCU, UUAGGC, UUAGGU, UUAGUA, UUAGUC, UUAGUU, UUAUAA, UUAUAC, UUAUAG, UUAUAU, UUAUCC, UUAUCG, UUAUCU, UUAUGA, UUAUGG, UUAUGU, UUAUUA, UUAUUC, UUAUUG, UUAUUU, UUCAAC, UUCAAU, UUCACA, UUCACC, UUCACG, UUCACU, UUCAGC, UUCAGG, UUCAGU, UUCAUA, UUCAUC, UUCAUG, UUCAUU, UUCCAA, UUCCCA, UUCCCG, UUCCGA, UUCCGU, UUCCUU, UUCGAA, UUCGAC, UUCGAG, UUCGAU, UUCGCA, UUCGCC, UUCGCG, UUCGCU, UUCGGA, UUCGGC, UUCGGG, UUCGGU, UUCGUA, UUCGUC, UUCGUG, UUCGUU, UUCUAC, UUCUAG, UUCUCA, UUCUCG,
UUCUGG, UUCUUA, UUCUUU, UUGAAA, UUGAAC, UUGAAG, UUGAAU, UUGACC, UUGACG, UUGACU, UUGAGA, UUGAGC, UUGAGU, UUGAUA, UUGAUC, UUGAUG, UUGAUU, UUGCAA, UUGCAC, UUGCAG, UUGCAU, UUGCCC, UUGCCG, UUGCGA, UUGCGC, UUGCGG, UUGCGU, UUGCUA, UUGCUC, UUGCUG, UUGCUU, UUGGAA, UUGGAG, UUGGCC, UUGGCG, UUGGCU, UUGGGC, UUGGGU, UUGGUA, UUGGUG, UUGUAA, UUGUAC, UUGUCA, UUGUCG, UUGUCU, UUGUGC, UUGUGG, UUGUUA, UUGUUG, UUGUUU, UUUAAA, UUUAAC, UUUAAG, UUUAAU, UUUACA, UUUACC, UUUACG, UUUACU, UUUAGA, UUUAGC, UUUAGG, UUUAGU, UUUAUA, UUUAUC, UUUAUG, UUUAUU, UUUCAU, UUUCCA, UUUCCG, UUUCCU, UUUCGA, UUUCGC, UUUCGG, UUUCGU, UUUCUA, UUUCUC, UUUCUG, UUUCUU, UUUGAA, UUUGAC, UUUGAG, UUUGAU, UUUGCC, UUUGCU, UUUGGA, UUUGGC, UUUGGG, UUUGGU, UUUGUA, UUUGUC, UUUGUU, UUUUAA, UUUUAG, UUUUAU, UUUUCC, UUUUCG, UUUUCU, UUUUGA, UUUUGC, UUUUGG, UUUUGU, UUUUUA, UUUUUC, UUUUUU
Table 2: Oligonucleotide sequences made for testing
Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 0.8126719 0.1352513 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21157U20 -01 52 51
SMN1 0.8570321 0.0273187 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21157U20 -01 01 37
SMN1 0.1679989 0.1679986 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21157U20 -01 15 72
SMN1 1.0481253 0.0393027 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21157U20 -01 02 84
SMN1 1.3817042 0.0532905 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21157U20 -01 07 65
SMN1 0.9798692 0.0205152 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21157U20 -01 47 27
SMN1 0.7600003 0.0429932 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21158U20 -02 18 12
SMN1 0.9871384 0.0681879 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21158U20 -02 47 98
SMN1 2.2524945 1.8031906 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21158U20 -02 26 69
SMN1 1.1143879 0.0267332 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21158U20 -02 73 51
SMN1 1.3464192 0.0276412 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21158U20 -02 9 81
SMN1 1.1536970 0.0249999 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21158U20 -02 83 91
SMN1 1.9072297 0.5259392 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21159U20 -03 5 96
SMN1 1.1327582 0.0946401 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21159U20 -03 64 77
SMN1 0.2961917 0.1732823 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21159U20 -03 4 09
SMN1 1.4881793 0.1727195 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21159U20 -03 5 07
SMN1 1.2993282 0.0598252 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21159U20 -03 6 28
SMN1 1.5115678 0.0541781 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21159U20 -03 14 75
SMN1 1.0483065 0.2439345 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21160U20 -04 17 43
SMN1 1.3224072 0.1000223 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21160U20 -04 67 92
SMN1 0.1331700 0.0328243 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21160U20 -04 13 91
SMN1 1.2895501 0.3301959 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21160U20 -04 63 87 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 1.2802254 0.0625779 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21160U20 -04 92 72
SMN1 1.4884827 0.0446412 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21160U20 -04 95 87
SMN1 0.8767475 0.0873925 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21161U20 -05 27 04
SMN1 1.1671203 0.0698140 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21161U20 -05 45 91
SMN1 0.0883178 0.0398870 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21161U20 -05 63 14
SMN1 1.3100532 0.2342313 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21161U20 -05 56 48
SMN1 1.0386996 0.0564213 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21161U20 -05 43 62
SMN1 0.8591447 0.0399700 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21161U20 -05 51 15
SMN1 0.7046598 0.0872441 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21162U20 -06 91 19
SMN1 1.1119400 0.0885713 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21162U20 -06 6 77
SMN1 0.5768596 0.2461865 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21162U20 -06 2 41
SMN1 1.4194188 0.4324471 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21162U20 -06 84 22
SMN1 1.1462517 0.0518915 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21162U20 -06 04 41
SMN1 1.0306823 0.0130708 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21162U20 -06 17 35
SMN1 0.6820857 0.0848853 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21163U20 -07 32 51
SMN1 0.9758535 0.0341785 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21163U20 -07 52 42
SMN1 1.0132523 0.1185407 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21163U20 -07 14 59
SMN1 1.0393819 0.0598153 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21163U20 -07 02 87
SMN1 1.1569496 0.1073854 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21163U20 -07 05 05
SMN1 1.2395039 0.1346038 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21163U20 -07 54 44
SMN1 0.9487148 0.1427082 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21164U20 -08 88 31
SMN1 1.3120804 0.0584649 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21164U20 -08 45 93
SMN1 0.2165300 0.1774005 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21164U20 -08 07 55
SMN1 2.0821517 0.8151842 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21164U20 -08 81 52
SMN1 1.0100906 0.2005887 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21164U20 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 1.2239476 0.2953072 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21164U20 -08 67 43
SMN1 0.7751906 0.0986951 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21165U20 -09 3 18
SMN1 1.6857316 0.0148840 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21165U20 -09 16 28
SMN1 0.6214067 0.2272112 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21165U20 -09 81 61
SMN1 0.8559392 0.2561083 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21165U20 -09 2 37
SMN1 0.9401860 0.1970084 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21165U20 -09 97 64
SMN1 0.8644811 0.1627392 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21165U20 -09 45 71
SMN1 0.9457309 0.0807295 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21166U20 -10 86 2
SMN1 1.5745269 0.1230626 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21166U20 -10 02 84
SMN1 0.4828222 0.1315574 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21166U20 -10 42 74
SMN1 1.2806291 0.1708842 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21166U20 -10 28 5
SMN1 1.1272546 0.1524863 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21166U20 -10 54 74
SMN1 1.0695714 0.1221067 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21166U20 -10 58 58
SMN1 0.7744369 0.0380761 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21167U20 -11 79 82
SMN1 1.5627142 0.1580430 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21167U20 -11 54 98
SMN1 0.4636559 0.2955138 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21167U20 -11 38 86
SMN1 0.9576116 0.3341375 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21167U20 -11 52 41
SMN1 1.2259738 0.2234727 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21167U20 -11 18 58
SMN1 1.0893022 0.1264142 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21167U20 -11 59 68
SMN1 0.9814294 0.0793738 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21168U20 -12 76 4
SMN1 1.5850881 0.0529191 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21168U20 -12 28 2
SMN1 0.2085860 0.1870176 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21168U20 -12 47 55
SMN1 3.2669658 2.0020743 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21168U20 -12 96 69
SMN1 1.0338137 0.2043762 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21168U20 -12 9 91
SMN1 1.1374716 0.2467919 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21168U20 -12 71 54 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 0.7496364 0.1032770 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21169U20 -13 37 03
SMN1 1.1759892 0.1223555 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21169U20 -13 63 85
SMN1 0.1614991 0.0793562 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21169U20 -13 59 87
SMN1 1.2877635 0.0903067 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21169U20 -13 91 17
SMN1 1.3368516 0.1778149 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21169U20 -13 75
SMN1 1.0377722 0.0394045 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21169U20 -13 91 07
SMN1 0.7716351 0.0860419 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21170U20 -14 77 59
SMN1 1.4670485 0.0731138 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21170U20 -14 48 84
SMN1 1.9782541 1.3529511 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21170U20 -14 54 56
SMN1 1.3119909 0.0731216 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21170U20 -14 37 34
SMN1 1.1289277 0.1471627 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21170U20 -14 7 01
SMN1 0.8557951 0.0177971 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21170U20 -14 21 81
SMN1 0.8914919 0.0398220 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21171U20 -15 64 32
SMN1 1.5734403 0.1174530 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21171U20 -15 42 17
SMN1 0.3660431 0.1171620 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21171U20 -15 04 19
SMN1 1.7382173 0.5201481 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21171U20 -15 94 55
SMN1 1.3832013 0.1018307 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21171U20 -15 37 76
SMN1 1.6194950 0.0383649 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21171U20 -15 52 89
SMN1 0.7372188 0.0380675 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21172U20 -16 1 83
SMN1 1.4416161 0.0598239 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21172U20 -16 96 44
SMN1 0.5100566 0.2865226 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21172U20 -16 05 59
SMN1 1.3819142 0.2478802 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21172U20 -16 14 29
SMN1 1.3100735 0.0263470 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21172U20 -16 73 93
SMN1 1.4181326 0.0823717 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21172U20 -16 46 08
SMN1 1.2190656 0.2819876 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21173U20 -17 51 74 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 1.2748191 0.1795272 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21173U20 -17 95 93
SMN1 0.4167392 0.0660662 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21173U20 -17 22 42
SMN1 3.3318430 0.9701748 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21173U20 -17 17 73
SMN1 1.2608565 0.0385657 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21173U20 -17 22 99
SMN1 1.6090453 0.1048743 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21173U20 -17 11 4
SMN1 0.8684419 0.0881846 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21174U20 -18 41 98
SMN1 1.2216635 0.0644455 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21174U20 -18 74 39
SMN1 10.284551 3.9293108 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21174U20 -18 67 32
SMN1 1.8009207 0.4255904 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21174U20 -18 64 5
SMN1 1.2617526 0.0698171 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21174U20 -18 02 43
SMN1 1.5927007 0.1992809 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21174U20 -18 96 16
SMN1 0.7055124 0.0649667 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21175U20 -19 52 5
SMN1 1.4343330 0.0759369 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21175U20 -19 9 65
SMN1 0.5389321 0.3092735 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21175U20 -19 56 94
SMN1 1.1737463 0.1794157 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21175U20 -19 7 46
SMN1 1.1861414 0.0367290 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21175U20 -19 71 63
SMN1 1.8347753 0.1557617 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21175U20 -19 68 23
SMN1 0.8263034 0.0629982 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21176U20 -20 53 54
SMN1 1.5057866 0.1706979 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21176U20 -20 89 84
SMN1 0.0624499 0.0490695 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21176U20 -20 2 71
SMN1 1.5414808 0.4611586 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21176U20 -20 55 69
SMN1 1.0899856 0.0437505 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21176U20 -20 92 68
SMN1 1.4153137 0.1465027 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21176U20 -20 5 26
SMN1 0.8655664 0.2094550 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21177U20 -21 53 26
SMN1 1.4666887 0.1162677 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21177U20 -21 87 64 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 0.3882335 0.1396808 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21177U20 -21 14 69
SMN1 1.3662694 0.2394205 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21177U20 -21 47 57
SMN1 1.3545548 0.0131754 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21177U20 -21 41 63
SMN1 2.0269683 0.2782790 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21177U20 -21 82 2
SMN1 0.6399348 0.0116798 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21178U20 -22 51 91
SMN1 1.2425939 0.0284051 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21178U20 -22 23 9
SMN1 0.2298579 0.1281012 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21178U20 -22 22 82
SMN1 1.4997222 0.5687885 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21178U20 -22 55 39
SMN1 1.2347837 0.0171194 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21178U20 -22 64 32
SMN1 1.5096955 0.1757641 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21178U20 -22 91 56
SMN1 0.7480318 0.0837324 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21179U20 -23 45 79
SMN1 1.3391097 0.0708771 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21179U20 -23 3 43
SMN1 0.3841433 0.1472373 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21179U20 -23 84 5
SMN1 2.6201956 0.3421018 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21179U20 -23 11 26
SMN1 1.4736638 0.0537626 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21179U20 -23 66 05
SMN1 1.9208004 0.1273368 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21179U20 -23 18 42
SMN1 0.9074366 0.2420168 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21180U20 -24 01 1
SMN1 1.2837936 0.1586617 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21180U20 -24 9 09
SMN1 0.9631002 0.1178024 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21180U20 -24 08 22
SMN1 0.9947532 0.2684156 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21180U20 -24 99 48
SMN1 0.9654403 0.0326462 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21180U20 -24 48 95
SMN1 1.1405661 0.1016312 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21180U20 -24 71 1
SMN1 0.9088548 0.0760260 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21181U20 -25 08 35
SMN1 1.2261850 0.0444227 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21181U20 -25 41 05
SMN1 1.0550823 0.3267680 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21181U20 -25 01 36 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 0.9691850 0.2264848 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21181U20 -25 38 66
SMN1 1.0097463 0.1221207 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21181U20 -25 6 37
SMN1 1.0813036 0.1018273 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21181U20 -25 39 03
SMN1 0.8764440 0.0704579 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21182U20 -26 72 09
SMN1 1.6324348 0.0615123 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21182U20 -26 88 57
SMN1 0.0715933 0.0715928 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21182U20 -26 19 84
SMN1 1.9992025 0.4203876 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21182U20 -26 16 69
SMN1 0.9741075 0.0668636 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21182U20 -26 84 61
SMN1 1.0302278 0.0961050 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21182U20 -26 91 98
SMN1 0.8343657 0.1081028 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21157U15 -27 03 71
SMN1 1.5899542 0.0931016 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21157U15 -27 19 53
SMN1 0.7471867 0.0078077 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21157U15 -27 14 01
SMN1 1.0490687 0.0926451 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21157U15 -27 44 93
SMN1 1.0583436 0.2089315 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21158U15 -28 94 76
SMN1 1.4023484 0.1019507 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21158U15 -28 14 71
SMN1 1.1502243 0.0800777 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21158U15 -28 16 07
SMN1 1.2198283 0.0317827 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21158U15 -28 96 62
SMN1 0.7122685 0.0775728 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21159U15 -29 87 38
SMN1 1.1453055 0.0445753 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21159U15 -29 52 89
SMN1 0.9373938 0.0157007 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21159U15 -29 65 83
SMN1 1.2085219 0.0840218 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21159U15 -29 62 99
SMN1 0.8695041 0.1476827 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21160U15 -30 09 79
SMN1 1.1669957 0.1289005 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21160U15 -30 09 31
SMN1 1.0695334 0.0422583 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21160U15 -30 23 92
SMN1 1.0046189 0.0682455 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21160U15 -30 99 37 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 1.2236852 0.1552583 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21161U15 -31 97 66
SMN1 0.9365695 0.0833678 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21161U15 -31 75 99
SMN1 1.0329784 0.0231205 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21162U15 -32 69 7
SMN1 1.0530458 0.0301583 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21162U15 -32 21 89
SMN1 1.0463614 0.0389718 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21163U15 -33 07 09
SMN1 1.2333022 0.0632553 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21163U15 -33 32 41
SMN1 1.0798767 0.0985940 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21164U15 -34 51 2
SMN1 1.2710261 0.0670194 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21164U15 -34 83 76
SMN1 0.8614640 0.0240959 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21165U15 -35 08 12
SMN1 0.8369663 0.0541596 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21165U15 -35 92 19
SMN1 1.2663632 0.0469636 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21166U15 -36 4 81
SMN1 1.3262571 0.0396746 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21166U15 -36 17 49
SMN1 1.2326900 0.0434762 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21167U15 -37 86 52
SMN1 1.1446329 0.0584333 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21167U15 -37 87 53
SMN1 0.8432418 0.0338080 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21168U15 -38 63 43
SMN1 0.9381803 0.0113762 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21168U15 -38 3 17
SMN1 0.6637462 0.0455270 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21169U15 -39 49 14
SMN1 0.8917645 0.0193953 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21169U15 -39 51 27
SMN1 0.8881386 0.0814018 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21170U15 -40 53 04
SMN1 0.8716028 0.0653729 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21170U15 -40 99 36
SMN1 0.8824661 0.0310167 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21171U15 -41 48 49
SMN1 1.0936947 0.0259965 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21171U15 -41 65 02
SMN1 0.9568608 0.0435583 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21172U15 -42 36 82
SMN1 1.1517559 0.0676621 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21172U15 -42 99 07
SMN1 1.3419197 0.0808077 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21173U15 -43 82 6 Oligo RQ RQ SE Gene Expt Type Cell [Oligo] Assay Coordinates_g Name Name Line/Tissue Type
SMN1 1.6929198 0.0846691 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21173U15 -43 15 98
SMN1 0 0 SMN1 NA NA 0 NA SMN1:21174U15 -44
SMN1 1.8072368 0.1141094 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21175U15 -45 97 8
SMN1 1.3777737 0.1085400 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21175U15 -45 03 58
SMN1 1.5456495 0.0640068 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21176U15 -46 38 14
SMN1 1.3542915 0.0384989 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21176U15 -46 04 44
SMN1 2.7115983 0.2600434 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21177U15 -47 61 46
SMN1 1.9866747 0.1194366 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21177U15 -47 86 75
SMN1 1.4823421 0.0630363 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21178U15 -48 95 43
SMN1 2.5973506 0.1454398 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21178U15 -48 28 01
SMN1 1.5344939 0.1106883 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21179U15 -49 05 65
SMN1 2.2233407 0.1487029 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21179U15 -49 84 92
SMN1 0.8974213 0.0342549 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21180U15 -50 96 31
SMN1 1.1323627 0.0785230 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21180U15 -50 81 03
SMN1 1.1579213 0.0442563 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21181U15 -51 68 19
SMN1 1.1776046 0.0380603 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21181U15 -51 65 53
SMN1 0.9735483 0.0514615 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21182U15 -52 53 83
SMN1 1.0683556 0.0608511 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21182U15 -52 42 46
Table 3: Oligonucleotide Modifications
Symbol Feature Description
bio 5' biotin
dAs DNA w/3' thiophosphate
dCs DNA w/3' thiophosphate
dGs DNA w/3' thiophosphate Symbol Feature Description
dTs DNA w/3' thiophosphate dG DNA
enaAs ENA w/3' thiophosphate enaCs ENA w/3' thiophosphate enaGs ENA w/3' thiophosphate enaTs ENA w/3' thiophosphate fluAs 2'-fluoro w/3' thiophosphate fluCs 2'-fluoro w/3' thiophosphate fluGs 2'-fluoro w/3' thiophosphate fluUs 2'-fluoro w/3' thiophosphate
InaAs LNA w/3' thiophosphate
InaCs LNA w/3' thiophosphate
InaGs LNA w/3' thiophosphate
InaTs LNA w/3' thiophosphate omeAs 2'-OMe w/3' thiophosphate omeCs 2'-OMe w/3' thiophosphate omeGs 2'-OMe w/3' thiophosphate omeTs 2'-OMe w/3' thiophosphate
InaAs-Sup LNA w/3' thiophosphate at 3' terminus
InaCs-Sup LNA w/3' thiophosphate at 3' terminus
InaGs-Sup LNA w/3' thiophosphate at 3' terminus
InaTs-Sup LNA w/3' thiophosphate at 3' terminus
InaA-Sup LNA w/3' OH at 3' terminus
InaC-Sup LNA w/3' OH at 3' terminus
InaG-Sup LNA w/3' OH at 3' terminus
InaT-Sup LNA w/3' OH at 3' terminus omeA-Sup 2'-OMe w/3' OH at 3' terminus omeC-Sup 2'-OMe w/3' OH at 3' terminus omeG-Sup 2'-OMe w/3' OH at 3' terminus omeU-Sup 2'-OMe w/3' OH at 3' terminus dAs-Sup DNA w/3' thiophosphate at 3' terminus dCs-Sup DNA w/3' thiophosphate at 3' terminus dGs-Sup DNA w/3' thiophosphate at 3' terminus dTs- Sup DNA w/3' thiophosphate at 3' terminus dA-Sup DNA w/3' OH at 3' terminus dC-Sup DNA w/3' OH at 3' terminus dG-Sup DNA w/3' OH at 3' terminus dT-Sup DNA w/3' OH at 3' terminus BRIEF DESCRIPTION OF SEQUENCE LISTING
SeqID Chrom Gene Chrom Start Chrom End Strand Name
1 chr5 SMNl 70208768 70260838 + human SMNl
2 chr5 SMN2 69333350 69385422 + human SMN2
3 chr9 SMNP 20319406 20344375 + human SMNP
4 chr5 SMNl 70208768 70260838 - human SMNl_revComp
5 chr5 SMN2 69333350 69385422 - human SMN2_revComp
6 chr9 SMNP 20319406 20344375 - human SMNP_revComp
7 chrl3 Smnl 100881160 100919653 + mouse Smnl
8 chrl3 Smnl 100881160 100919653 - mouse Smnl_revComp
9 chr5 SMNl 70240095 70240127 + S48-193240
9 chr5 SMN2 69364672 69364704 + S48-193240
10 chr5 SMNl 70214393 70214822 + S48-441814
10 chr5 SMN2 69338976 69339405 + S48-441814
11 chr5 SMNl 70214064 70214108 + S48-441815
11 chr5 SMN2 69338647 69338691 + S48-441815
12 chr5 SMNl 70214276 70214317 + S48-473289
12 chr5 SMN2 69338859 69338900 + S48-473289
13 chr5 SMNl 70214445 70214472 + S48-473290
13 chr5 SMN2 69339028 69339055 + S48-473290
14 chr5 SMNl 70238095 70242127 + S48-193240 +2K
15 chr5 SMN2 69362672 69366704 + S48-193240 +2K
16 chr5 SMNl 70212393 70216822 + S48-441814 +2K
17 chr5 SMN2 69336976 69341405 + S48-441814 +2K
18 chr5 SMNl 70212064 70216108 + S48-441815 +2K
19 chr5 SMN2 69336647 69340691 + S48-441815 +2K
20 chr5 SMNl 70212276 70216317 + S48-473289 +2K
21 chr5 SMN2 69336859 69340900 + S48-473289 +2K
22 chr5 SMNl 70212445 70216472 + S48-473290 +2K
23 chr5 SMN2 69337028 69341055 + S48-473290 +2K
24 chr5 SMNl 70240510 70240551 - S48-193241
24 chr5 SMN2 69365087 69365128 - S48-193241
25 chr5 SMNl 70241924 70241968 - S48-193242
25 chr5 SMN2 69366499 69366543 - S48-193242
26 chr5 SMNl 70238510 70242551 - S48-193241 +2K
27 chr5 SMN2 69363087 69367128 - S48-193241 +2K
28 chr5 SMNl 70239924 70243968 - S48-193242 +2K
29 chr5 SMN2 69364499 69368543 - S48-193242 +2K
13100 chr5 SMNl 70247831 70247845 + Splice control sequence
13100 chr5 SMN2 69372411 69372425 + Splice control sequence 13101 chr5 SMN2 69372402 69372845 + Intron 7
Single Strand Oligonucleotides (Antisense Strand of Target Gene)
SeqID range: 30 to 8329, 13088-13094 SeqIDs w/o G Runs: 30-142, 156-560, 575-780, 794-912, 926-1013, 1027-1078, 1092-1286, 1300-1335, 1349- 1385, 1399-1453, 1460-1527, 1548-1555, 1571-1653, 1675-1691, 1706-1802, 1816-1883, 1897-2009, 2023-2141, 2165-2289, 2303-2320, 2334-2447, 2461-2494, 2508-2526, 2540- 2545, 2571-2635, 2651-2670, 2689-2763, 2772-2814, 2828-2854, 2868-3030, 3044-3256, 3270-3360, 3374-3400, 3414-3722, 3737, 3759-3783, 3797-3970, 3986-4059, 4073-4153, 4175-4240, 4255-4415, 4438-4441, 4456-4472, 4484-4505, 4513-4516, 4531-4546, 4560- 4650, 4664-4751, 4766-4918, 4932-5035, 5049-5064, 5091-5189, 5203-5448, 5459-5503, 5508-5520, 5535-5654, 5668-5863, 5877-6016, 6025-6029, 6054-6063, 6078-6215, 6229- 6701, 6715-6729, 6744-6869, 6883-6945, 6959-6968, 6982-7085, 7099-7173, 7195-7247, 7255-7268, 7273-7309, 7320-7335, 7349-7442, 7456-7465, 7479-7727, 7740-7951, 7977- 8208, 8223-8255, 8257-8296, 8304-8312, 8319-8329, 13093-13094
SeqIDs w/o miR Seeds:
30-32, 34-39, 45-62, 64-72, 77-142, 145-151, 153, 157-184, 186-202, 205-246, 249, 251-260, 263, 266-320, 322, 326-328, 331, 333-341, 343-344, 346-394, 396-445, 447-541, 543-562, 564-596, 599-605, 607-646, 648-673, 677-701, 703-735, 737-772, 774-781, 785, 787-793, 795-809, 812, 814-815, 819-820, 822-827, 833-834, 836, 838-850, 852-876, 879, 883-886, 889-890, 892, 894, 897, 899, 901-906, 909, 911, 919, 921-935, 940, 942-1012, 1016-1063, 1065-1067, 1069-1095, 1097-1147, 1149-1166, 1168-1190, 1193-1214, 1217, 1227-1237, 1240, 1244, 1246-1251, 1258-1281, 1283-1312, 1314-1333, 1335, 1337-1356, 1358-1364, 1367, 1369-1381, 1384, 1388-1389, 1392-1404, 1406-1417, 1421-1441, 1443, 1445, 1447- 1460, 1462-1492, 1494-1500, 1502, 1506-1512, 1514-1531, 1533, 1535-1539, 1543-1558, 1561-1562, 1564-1601, 1603-1614, 1616-1633, 1635-1646, 1648-1656, 1658, 1661-1675, 1678-1716, 1718-1740, 1742-1750, 1752-1785, 1788-1795, 1798, 1804-1871, 1873-1884, 1892-1973, 1976-1992, 1998-2032, 2034-2053, 2055-2077, 2079-2116, 2118-2135, 2138, 2142, 2149, 2151-2153, 2155-2162, 2165-2174, 2178, 2181-2254, 2256-2268, 2271-2293, 2296-2298, 2301-2312, 2314-2323, 2325-2427, 2429-2438, 2441, 2445, 2450-2476, 2478- 2489, 2492-2494, 2497-2513, 2515, 2521-2526, 2529-2545, 2547-2571, 2573-2610, 2613- 2620, 2622-2639, 2641, 2644, 2651-2743, 2745, 2747-2755, 2760-2775, 2777-2825, 2828- 2841, 2844-2861, 2864-2888, 2894-2954, 2956-2988, 2991-3006, 3008-3043, 3046, 3048- 3239, 3241-3253, 3256-3268, 3270-3273, 3276-3320, 3322-3355, 3357-3404, 3406-3428, 3430-3488, 3490-3491, 3493-3522, 3524-3552, 3554-3569, 3571-3650, 3653-3670, 3672- 3688, 3690-3717, 3719-3724, 3727-3736, 3743, 3746, 3749, 3751-3821, 3823-3842, 3844, 3846, 3848, 3851, 3855, 3858, 3861-3863, 3866-3881, 3883, 3887-3907, 3909-3917, 3919- 3924, 3926-3942, 3944-3952, 3956-3970, 3976-3988, 3991-3998, 4001-4008, 4010-4022, 4026, 4028-4039, 4041-4048, 4051-4059, 4063-4070, 4072-4073, 4075, 4078, 4080-4104, 4106-4124, 4126, 4129-4140, 4143-4153, 4156-4162, 4166-4171, 4174-4196, 4201-4241, 4244-4252, 4254-4285, 4289-4318, 4320-4324, 4327, 4330-4331, 4333-4335, 4337-4351, 4353-4414, 4417, 4419.4425, 4427-4433, 4435, 4438-4446, 4449-4450, 4452-4462, 4470- 4472, 4474-4510, 4512-4517, 4520-4546, 4548, 4551-4590, 4592-4619, 4623-4696, 4699, 4701, 4703-4752, 4755-4877, 4879-4949, 4951-5007, 5010, 5013-5046, 5049-5059, 5061- 5063, 5066, 5069-5078, 5080-5119, 5121-5131, 5134-5168, 5171-5189, 5191-5228, 5230- 5341, 5343-5405, 5407-5426, 5429-5437, 5439-5476, 5478-5491, 5494-5516, 5518-5556, 5558-5572, 5574-5647, 5649, 5652-5653, 5657-5729, 5731-5742, 5744-5769, 5771-5780, 5782-5866, 5868, 5870-5876, 5879-5881, 5884-5899, 5902-5951, 5954-5993, 6000-6006, 6009-6016, 6019-6033, 6035-6065, 6067-6073, 6080-6165, 6168-6249, 6252-6257, 6261- 6282, 6284-6289, 6291-6301, 6305-6367, 6369-6378, 6380-6398, 6401-6412, 6415, 6417- 6447, 6449-6456, 6458-6500, 6502-6563, 6565-6589, 6591-6612, 6616-6660, 6663-6702, 6704-6748, 6753, 6758-6763, 6765, 6768-6807, 6810, 6812-6872, 6874-6877, 6879-6913, 6915-6916, 6919, 6922, 6924-6926, 6928, 6930-6936, 6940-6959, 6962, 6964-6990, 6992, 6996, 6998-6999, 7004-7038, 7042-7085, 7087, 7089, 7092-7134, 7136-7140, 7142-7143, 7146-7150, 7152-7157, 7159-7171, 7175, 7177-7178, 7180-7196, 7198-7220, 7223, 7231- 7237, 7239, 7242-7246, 7250-7273, 7275-7308, 7310-7312, 7314-7317, 7319-7330, 7332- 7400, 7402-7437, 7439, 7441-7466, 7470-7491, 7493, 7495, 7497, 7499, 7502-7614, 7622- 7628, 7631-7646, 7649-7651, 7655-7657, 7661-7672, 7676, 7679-7721, 7723-7800, 7802- 7803, 7805-7906, 7908, 7910-7939, 7943-7953, 7956-7964, 7966-7981, 7983, 7985-7999, 8002, 8004-8034, 8036-8046, 8048-8080, 8084-8094, 8096-8112, 8114-8115, 8117-8139, 8141-8143, 8146-8148, 8150-8187, 8190-8216, 8218-8229, 8232-8238, 8240-8250, 8253- 8255, 8257-8275, 8278-8296, 8299-8304, 8306-8329, 13093-13094
Single Strand Oligonucleotides (Sense Strand of Target Gene)
SeqID range: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, 13062-13087
SeqID s w/o G Runs:
1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2545, 2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-8495, 8520-8560, 8574-8837, 8857-8882, 8907-8964, 8978-9298, 9312-9382, 9394-9640, 9656- 9753, 9767-9974, 9988-10261, 10275-10276, 10290-10301, 10315-10434, 10448-10613, 10623-10641, 10644-10676, 10678-10704, 10714-10802, 10822-11161, 11175-11192, 11207-11386, 11400-11730, 11744-11745, 11759-11852, 11857-11900, 11914-11984, 11999-12011, 12026-12153, 12163-12175, 12178-12195, 12198-12212, 12216-12536, 12547-12564, 12575-12664, 12674-12758, 12772-12797, 12800-12840, 12854-13061, 13062-13069
SeqIDs w/o miR Seeds:
1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2489, 2542-2545, 2656-2657, 2833-2835, 3439-3440, 3916-3917, 4470-4472, 4821, 5429, 5537, 6061, 7327, 8330-8334, 8336-8345, 8347, 8351-8373, 8375-8390, 8392-8399, 8401-8413, 8415-8455, 8457-8493, 8495, 8497-8502, 8510-8517, 8520, 8525, 8527-8634, 8637-8653, 8655-8671, 8673-8718, 8721-8822, 8824-8825, 8827-8842, 8849-8879, 8881-8892, 8894-8902, 8905-8906, 8914- 8927, 8929, 8931, 8935, 8937-8975, 8980-8992, 8994, 8996-8997, 8999-9001, 9003, 9005- 9086, 9089-9124, 9126-9286, 9288-9307, 9310-9359, 9362-9420, 9425-9427, 9429-9432, 9434, 9436-9437, 9439-9461, 9464-9483, 9486, 9488-9498, 9500-9511, 9513, 9515-9650, 9653-9667, 9669, 9671-9723, 9725-9869, 9871-9872, 9874-9879, 9881-9889, 9891-9973, 9975-10077, 10080-10097, 10099, 10101-10127, 10129-10166, 10168-10170, 10172-10184, 10186-10230, 10232-10237, 10239-10260, 10262-10272, 10274-10342, 10344-10400, 10402-10423, 10426-10441, 10445-10556, 10560, 10562-10580, 10582-10606, 10609- 10647, 10650, 10652-10704, 10706, 10710-10713, 10716-10731, 10733-10824, 10826- 10842, 10844-10903, 10906-10907, 10909-11101 , 11104, 11106-11134, 11137-11138, 11145-11161, 11164-11173, 11175-11181, 11184, 11186-11203, 11207-11212, 11214- 11239, 11243-11259, 11261-11347, 11351-11397, 11399-11740, 11742-11747, 11749- 11790, 11792-11817, 11821-11852, 11854-11904, 11908-11944, 11946-11959, 11961, 11964-11984, 11986-12007, 12009-12022, 12024-12092, 12095-12119, 12121-12133, 12135-12144, 12146-12157, 12159-12225, 12227-12231, 12233-12300, 12302-12329, 12332-12333, 12335-12382, 12385-12411, 12414-12416, 12418-12444, 12446-12455, 12457-12461, 12465, 12468-12474, 12476-12499, 12501-12536, 12538-12544, 12547- 12553, 12559-12610, 12612-12626, 12628-12631 , 12633-12637, 12640-12645, 12648- 12657, 12659-12671, 12673-12679, 12683-12710, 12712, 12714-12747, 12750, 12752- 12766, 12769, 12771-12806, 12808-12826, 12828-12829, 12831, 12833-12846, 12848- 12849, 12854-12931, 12933-12946, 12948-13061 , 13064, 13066-13068, 13071-13072, 13075, 13077-13081, 13083, 13085, 13087
Example 2: Selective upregulation of exon 7 containing SMN2 transcripts using oligonucleotides targeting PRC2-interacting regions that upregulate SMN2 and splice- switching oligonucleotides.
Oligo design:
Oligonucleotides targeting PRC2-interacting regions (IncR A peaks) in the SMN1/2 gene loci were designed. These oligos were synthesized with various DNA base
modifications, modification placements, inter-nucleoside bonds and inter-oligo linkers (oligos 1-52 and 59-101) as outlined in Table 4.
Splice switching oligos (SSO) were designed based on sequences of SMN2. Various modifications of such SSOs in length and chemistry were prepared (oligos 53-58).
Universal negative control oligos (oligo 232 and 293) were also designed using on bioinformatic analysis.
Methods:
Cell Culture:
Six SMA fibroblast cell lines and one lymphoblast cell line were obtained from the Coriell Institute (FIG. 2). The cells were either transfected with the oligos using Lipofectamine 2000 (Fibroblasts) or by electroporation or unassisted delivery (lymphoblast) to ascertain effects of the oligonucleotides on SMNl/2 mRNA and protein expression. All experiments were carried out as biological triplicates.
mRNA and protein expression:
mRNA expression -
1. On day 1 , SMA fibroblasts were seeded into each well of 96-well plates at a density of 5,000 cells per lOOuL.
2. On day 2, transfections were performed using Lipofectamie2000 per manufacturer's instructions with oligos at either ΙΟηΜ or 30nM.
3. 48 hours post-transfection, Ambion Cells-to-CT kit was used to directly obtain qRT-PCR results from the cells per manufacturer's instructions.
4. Quantitative PCR evaluation was completed using Taqman FAST qPCR on StepOne Plus, and change in mRNA expression was quantified using the delta delta Ct method by normalizing SMN expression to a housekeeper gene (B2M).
Protein expression (ELISA) -
ELISA to determine SMN protein was carried out per manufacturer's instructions (SMN ELISA kit #ADI-900-209, Enzo Life Sciences). Briefly, SMN fibroblasts were cultures at 40,000 cells/well of a 24-well tissue culture coated plate on day 1. Cells were transfected with the oligos using Lipofectamine2000 on day 2 and cell lysates prepared at 24 and 48 hours post-treatment. ELISA was carried out per manufacturer's instructions.
Subsequently fold induction of SMN protein was determined by normalizing SMN protein levels induced by oligonucleotides to the SMN protein levels induced by Lipofectamine treatment alone.
Splice switching assay (Ddel assay) - SMN2-derived transcripts contain a unique Ddel restriction element introduced because of a nucleotide polymorphism not present in SMNl and are differentiated from SMNl -derived transcripts because of the faster migration of the SMN2 products. Briefly, SMA fibroblasts were treated with oligonucleotides targeting PRC2-interacting regions with or without SSO at 30nM each as described before. RT-PCR was carried out with an SMN exon 5 forward primer and an exon 8 reverse primer to generate cDNAs that were then digested with Ddel. The SMNl transcript, if present, migrates at a slower rate than the Ddel- digested SMN2 transcript and is seen as the first band from the top of the gel. The second band from the top indicates full length SMN2 (accurately spliced form) and the third band indicates the incorrectly spliced SMN2delta7. (FIG. 5)
Results
In Spinal Muscular Atrophy patients, the SMN1 gene is often mutated in such a way that it is unable to correctly code the SMN protein - due to either a deletion encompassing at least a portion of exon 7 or to other mutations. SMA patients, however, generally retain at least one copy of the SMN2 gene (with many having 2 or more copies) that still expresses small amounts of SMN protein. The SMN2 gene has a C to T mutation (compared with SMN1) in exon 7 that alters splicing of its precursor mRNA such that exon 7 is spliced out at a high frequency. Consequently, only about 10% of the normal levels of full length SMN protein are produced from SMN2. (See FIG. 1)
Six SMA fibroblast cell lines and one lymphoblast cell line were obtained from the Coriell Institute (FIG. 2). Cells were transfected with oligonucleotides (oligos 1-52 and 59- 101) directed against a PRC2-associated region of SMN2 and RT-PCR assays were conducted to evaluate effects on SMN mRNA expression. (See FIGS. 3 and 4 for results in cell lines 3814 and 3813). In separate experiments, cells were transfected with
oligonucleotides (oligos 53-58) directed at a splice control sequence in intron 7 of SMN2 and RT-PCR assays were conducted to evaluate effects on SMN mRNA expression (See FIGS. 3 and 4 for results in cell lines 3814 and 3813). Splice switching oligonucleotides (oligos 53- 58) were found to increase expression of full length SMN2 based on a gel separation analysis of PCR products obtained following a Ddel restriction digest; whereas certain
oligonucleotides directed against a PRC2-associated region of SMN2 did not. (FIG. 5)
SMN ELISA (Enzo) assays were conducted and revealed that certain oligonucleotides directed against a PRC2-associated region of SMN2 alone did not significantly increase full length SMN protein 24h post-transfection in certain SMA patient fibroblasts. (FIG. 6) However, the same SMN ELISA assays showed that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increase full length SMN protein 24h post-transfection in SMA patient fibroblasts above that observed with splice switching oligonucleotides alone. (FIGS. 7 and 8). RT-PCR assays were conducted and showed that oligonucleotides directed against a PRC2-associated region of SMN2 in combination with a splice switching oligonucleotide (oligo 53 or 54) significantly increased SMN2 protein 24h post-transfection in SMA patient fibroblasts. (FIG 9.) These experiments were conducted modified oligonucleotides with either alternating LNA and 2'OMe nucleotides or alternating DNA and LNA nucleotides.
In summary, the results of Example 2 show that certain oligos targeting PRC2 associated regions of SMN2 induce SMN RNA expression (e.g.., of the SMNA7 transcript) SMA patient-derived fibroblasts. The results also show that, in some embodiments, splice- switching oligos may not induce SMN RNA expression, but rather shift SMN RNA splicing to the full-length transcript. Finally, the results show that combination of splice switching oligos with PRC2-associated region targeting oligos substantially increases full length SMN protein in cells from SMA patients.
Table 4: Oligonucleotide sequences made for testing human cells obtained from subjects with Spinal Muscular Atrophy (See Table 3 for structural features of formatted sequence).
Oligo Base Formatted Sequence SeqID
Name Sequence
SM N 1- ATTCTCTTGA omeAs;omeUs;omeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;om 13062
01 m03 TGATGCTGAT eAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs
;omeU-Sup
SM N 1- TTCTCTTGAT omeUs;omeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;om 13063
02 m03 GATGCTGAT eUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs
G ;omeG-Sup
SM N 1- TCTCTTGATG omeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;om 13064
03 m03 ATGCTGATGC eGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs
;omeC-Sup
SM N 1- CTCTTGATGA omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;om 13065
04 m03 TGCTGATGCT eAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs
;omeU-Sup
SM N 1- TCTTGATGAT omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;om 13066
05 m03 GCTGATGCTT eUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs
;omeU-Sup
SM N 1- CTTGATGATG omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;om 13067
06 m03 CTGATGCTTT eGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs
;omeU-Sup
SM N 1- TTGATGATGC omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;om 13068
07 m03 TGATGCTTTG eCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs
;omeG-Sup
SM N 1- TGATGATGCT omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;om 13069
08 m03 GATGCTTTGG eUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs
;omeG-Sup
SM N 1- GATGATGCT omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;om 13070
09 m03 GATGCTTTGG eGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs
G ;omeG-Sup
SM N 1- ATGATGCTGA omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;om 13071
10 m03 TGCTTTGGGA eAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs ;omeA-Sup
SM N l- TGATGCTGAT omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;om 13072
11 m03 GCTTTGGGA eUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs
A ;omeA-Sup
SM N 1- GATGCTGAT omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;om 13073
12 m03 GCTTTGGGA eGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs
AG ;omeG-Sup
SM N 1- ATGCTGATGC omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;om 13074
13 m03 TTTGGGAAGT eCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs
;omeU-Sup
SM N 1- TGCTGATGCT omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;om 13075
14 m03 TTGGGAAGT eUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs
A ;omeA-Sup
SM N 1- GCTGATGCTT omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;om 13076
15 m03 TGGGAAGTA eUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs
T ;omeU-Sup
SM N 1- CTGATGCTTT omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;om 13077
16 m03 GGGAAGTAT eUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs
G ;omeG-Sup
SM N 1- TGATGCTTTG omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;om 13078
17 m03 GGAAGTATG eGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs
T ;omeU-Sup
SM N 1- GATGCTTTGG omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;om 13079
18 m03 GAAGTATGTT eGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs
;omeU-Sup
SM N 1- ATGCTTTGGG omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;om 13080
19 m03 AAGTATGTTA eGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs
;omeA-Sup
SM N 1- TGCTTTGGGA omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;om 13081 0 m03 AGTATGTTAA eAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs
;omeA-Sup
SM N 1- GCTTTGGGA omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;om 13082 1 m03 AGTATGTTAA eAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs
T ;omeU-Sup
SM N 1- CTTTGGGAA omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;om 13083 2 m03 GTATGTTAAT eGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs
T ;omeU-Sup
SM N 1- TTTGGGAAGT omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;om 13084 3 m03 ATGTTAATTT eUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs
;omeU-Sup
SM N 1- TTGGGAAGT omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;om 13085 4 m03 ATGTTAATTT eAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs
C ;omeC-Sup
SM N 1- TGGGAAGTA omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;om 13086 5 m03 TGTTAATTTC eUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs;omeCs
A ;omeA-Sup
SM N 1- GGGAAGTAT omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;om 13087 6 m03 GTTAATTTCA eGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs;omeCs;omeAs
T ;omeU-Sup
SM N 1- ATTCTCTTGA lnaAs;omeUs;lnaTs;omeCs;lnaTs;omeCs;lnaTs;omeUs;lnaGs;omeAs;lnaT 11374 7 mOl TGATG s;omeGs;lnaAs;omeUs;lnaG-Sup
SM N 1- TTCTCTTGAT lnaTs;omeUs;lnaCs;omeUs;lnaCs;omeUs;lnaTs;omeGs;lnaAs;omeUs;lna 11375 8 mOl GATGC Gs;omeAs;lnaTs;omeGs;lnaC-Sup
SM N 1- TCTCTTGATG lnaTs;omeCs;lnaTs;omeCs;lnaTs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaA 11376 9 mOl ATGCT s;omeUs;lnaGs;omeCs;lnaT-Sup
SM N 1- CTCTTGATGA lnaCs;omeUs;lnaCs;omeUs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeAs;lnaT 11377 0 mOl TGCTG s;omeGs;lnaCs;omeUs;lnaG-Sup
SM N 1- TCTTGATGAT lnaTs;omeCs;lnaTs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaAs;omeUs;lnaG 11378 1 mOl GCTGA s;omeCs;lnaTs;omeGs;lnaA-Sup
SM N 1- CTTGATGATG lnaCs;omeUs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaC 11379 2 mOl CTGAT s;omeUs;lnaGs;omeAs;lnaT-Sup
SM N 1- TTGATGATGC lnaTs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaT 11380 3 mOl TGATG s;omeGs;lnaAs;omeUs;lnaG-Sup
SM N 1- TGATG ATGCT lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lna 11381 4 mOl GATGC Gs;omeAs;lnaTs;omeGs;lnaC-Sup
SM N 1- GATG ATGCT lnaGs;omeAs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeGs;lnaA 11382 5 mOl GATGCT s;omeUs;lnaGs;omeCs;lnaT-Sup
SM N 1- ATGATGCTGA lnaAs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaGs;omeAs;lna 11383 6 mOl TGCTT Ts;omeGs;lnaCs;omeUs;lnaT-Sup
SM N 1- TGATG CTGAT lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeGs;lnaAs;omeUs;lna 11384 7 mOl GCTTT Gs;omeCs;lnaTs;omeUs;lnaT-Sup
SM N 1- GATGCTGAT lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaC 11385 8 mOl GCTTTG s;omeUs;lnaTs;omeUs;lnaG-Sup
SM N 1- ATGCTGATGC lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaT 11386 9 mOl TTTGG s;omeUs;lnaTs;omeGs;lnaG-Sup
SM N 1- TGCTGATGCT lnaTs;omeGs;lnaCs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaT 11387 0 mOl TTGGG s;omeUs;lnaGs;omeGs;lnaG-Sup
SM N 1- GCTGATGCTT lnaGs;omeCs;lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeUs;lnaT 11388 1 mOl TGGGA s;omeGs;lnaGs;omeGs;lnaA-Sup
SM N 1- CTGATGCTTT lnaCs;omeUs;lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaTs;omeUs;lna 11389 2 mOl GGGAA Gs;omeGs;lnaGs;omeAs;lnaA-Sup
SM N 1- TGATG CTTTG lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeUs;lnaTs;omeGs;lnaG 11390 3 mOl GGAAG s;omeGs;lnaAs;omeAs;lnaG-Sup
SM N 1- GATGCTTTGG lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaTs;omeUs;lnaGs;omeGs;lna 11391 4 mOl GAAGT Gs;omeAs;lnaAs;omeGs;lnaT-Sup
SM N 1- ATG CTTTG GG lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeUs;lnaTs;omeGs;lnaGs;omeGs;lna 11392 5 mOl AAGTA As;omeAs;lnaGs;omeUs;lnaA-Sup
SM N 1- TGCTTTGGGA lnaTs;omeGs;lnaCs;omeUs;lnaTs;omeUs;lnaGs;omeGs;lnaGs;omeAs;lna 11393 6 mOl AGTAT As;omeGs;lnaTs;omeAs;lnaT-Sup
SM N 1- GCTTTGGGA dGs;lnaCs;dTs;lnaTs;dTs;lnaGs;dGs;lnaGs;dAs;lnaAs;dGs;lnaTs;dAs;lnaTs; 11394 7 m02 AGTATG dG-Sup
SM N 1- GCTTTGGGA lnaGs;omeCs;lnaTs;omeUs;lnaTs;omeGs;lnaGs;omeGs;lnaAs;omeAs;lna 11394 7 mOl AGTATG Gs;omeUs;lnaAs;omeUs;lnaG-Sup
SM N 1- CTTTGGGAA dCs;lnaTs;dTs;lnaTs;dGs;lnaGs;dGs;lnaAs;dAs;lnaGs;dTs;lnaAs;dTs;lnaGs; 11395 8 m05 GTATGT dT-Sup
SM N 1- CTTTGGGAA lnaCs;omeUs;lnaTs;omeUs;lnaGs;omeGs;lnaGs;omeAs;lnaAs;omeGs;lna 11395 8 mOl GTATGT Ts;omeAs;lnaTs;omeGs;lnaT-Sup
SM N 1- TTTGGGAAGT lnaTs;omeUs;lnaTs;omeGs;lnaGs;omeGs;lnaAs;omeAs;lnaGs;omeUs;lna 11396 9 mOl ATGTT As;omeUs;lnaGs;omeUs;lnaT-Sup
SM N 1- TTGGGAAGT lnaTs;omeUs;lnaGs;omeGs;lnaGs;omeAs;lnaAs;omeGs;lnaTs;omeAs;lnaT 11397 0 mOl ATGTTA s;omeGs;lnaTs;omeUs;lnaA-Sup
SM N 1- TGGGAAGTA lnaTs;omeGs;lnaGs;omeGs;lnaAs;omeAs;lnaGs;omeUs;lnaAs;omeUs;lna 11398 1 mOl TGTTAA Gs;omeUs;lnaTs;omeAs;lnaA-Sup
SM N 1- GGGAAGTAT lnaGs;omeGs;lnaGs;omeAs;lnaAs;omeGs;lnaTs;omeAs;lnaTs;omeGs;lnaT 11399 2 mOl GTTAAT s;omeUs;lnaAs;omeAs;lnaT-Sup
SM N 1- TCACTTTCAT dTs;lnaCs;dAs;lnaCs;dTs;lnaTs;dTs;lnaCs;dAs;lnaTs;dAs;lnaAs;dTs;lnaGs; 13088 3 m02 AATGCTGG dCs;lnaTs;dGs;lnaG-Sup
SM N 1- TCACTTTCAT lnaTs;dCs;lnaAs;dCs;lnaTs;dTs;lnaTs;dCs;lnaAs;dTs;lnaAs;dAs;lnaTs;dGs;l 13088 3 ml2 AATGCTGG naCs;dTs;lnaGs;dG-Sup
SM N 1- TCACTTTCAT lnaTs;omeCs;lnaAs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaA 13088 4 mOl AATGCTGG s;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaGs;omeG-Sup
SM N 1- TCACTTTCAT omeUs;omeCs;omeAs;omeCs;omeUs;omeUs;omeUs;omeCs;omeAs;ome 13088 3 m03 AATGCTGG Us;omeAs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeG-Sup
SM N 1- TCACTTTCAT lnaTs;omeCs;lnaAs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaA 13089 5 mOl AATGC s;omeAs;lnaTs;omeGs;lnaC-Sup
SM N 1- CACTTTCATA lnaCs;omeAs;lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeAs;lnaTs;omeAs;lnaA 13090 6 mOl ATGCT s;omeUs;lnaGs;omeCs;lnaT-Sup
SM N 1- ACTTTCATAA dAs;lnaCs;dTs;lnaTs;dTs;lnaCs;dAs;lnaTs;dAs;lnaAs;dTs;lnaGs;dCs;lnaTs; 13091 7 m02 TGCTG dG-Sup
SM N 1- ACTTTCATAA lnaAs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeAs;lnaT 13091 7 mOl TGCTG s;omeGs;lnaCs;omeUs;lnaG-Sup
SM N 1- CTTTCATAAT lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeAs;lnaTs;omeAs;lnaAs;omeUs;lnaG 13092 8 mOl GCTGG s;omeCs;lnaTs;omeGs;lnaG-Sup
SM N 1- AGACCAGTTT lnaAs;omeGs;lnaAs;omeCs;lnaCs;omeAs;lnaGs;omeUs;lnaTs;omeUs;lnaT 3650 9 mOl TACCT s;omeAs;lnaCs;omeCs;lnaT-Sup
SM N 1- CCTAGCTACT lnaCs;omeCs;lnaTs;omeAs;lnaGs;omeCs;lnaTs;omeAs;lnaCs;omeUs;lnaT 13093 0 mOl TTGAA s;omeUs;lnaGs;omeAs;lnaA-Sup
SM N 1- TCCTAGCTAC lnaTs;omeCs;lnaCs;omeUs;lnaAs;omeGs;lnaCs;omeUs;lnaAs;omeCs;lnaT 13094 1 mOl TTTGA s;omeUs;lnaTs;omeGs;lnaA-Sup
SM N 1- GAAATATTCC lnaGs;omeAs;lnaAs;omeAs;lnaTs;omeAs;lnaTs;omeUs;lnaCs;omeCs;lnaT 10065 2 mOl TTATA s;omeUs;lnaAs;omeUs;lnaA-Sup
SM N 1- AAATATTCCT lnaAs;omeAs;lnaAs;omeUs;lnaAs;omeUs;lnaTs;omeCs;lnaCs;omeUs;lnaT 10066 3 mOl TATAG s;omeAs;lnaTs;omeAs;lnaG-Sup
SM N 1- AATATTCCTT lnaAs;omeAs;lnaTs;omeAs;lnaTs;omeUs;lnaCs;omeCs;lnaTs;omeUs;lnaA 10067 4 mOl ATAGC s;omeUs;lnaAs;omeGs;lnaC-Sup
SM N 1- ATATTCCTTA lnaAs;omeUs;lnaAs;omeUs;lnaTs;omeCs;lnaCs;omeUs;lnaTs;omeAs;lnaT 10068 5 mOl TAGCC s;omeAs;lnaGs;omeCs;lnaC-Sup
SM N 1- TATTCCTTAT lnaTs;omeAs;lnaTs;omeUs;lnaCs;omeCs;lnaTs;omeUs;lnaAs;omeUs;lnaA 10069 6 mOl AGCCA s;omeGs;lnaCs;omeCs;lnaA-Sup
SM N 1- ATTCCTTATA lnaAs;omeUs;lnaTs;omeCs;lnaCs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaG 10070 7 mOl GCCAG s;omeCs;lnaCs;omeAs;lnaG-Sup
SM N 1- TTCCTTATAG lnaTs;omeUs;lnaCs;omeCs;lnaTs;omeUs;lnaAs;omeUs;lnaAs;omeGs;lnaC 10071 8 mOl CCAGG s;omeCs;lnaAs;omeGs;lnaG-Sup
SM N 1- TCCTT ATAGC lnaTs;omeCs;lnaCs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaGs;omeCs;lnaC 10072 9 mOl CAGGT s;omeAs;lnaGs;omeGs;lnaT-Sup
SM N 1- CCTTATAGCC lnaCs;omeCs;lnaTs;omeUs;lnaAs;omeUs;lnaAs;omeGs;lnaCs;omeCs;lnaA 10073 0 mOl AGGTC s;omeGs;lnaGs;omeUs;lnaC-Sup
SM N 1- CTTATAGCCA lnaCs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaGs;omeCs;lnaCs;omeAs;lnaG 10074 1 mOl GGTCT s;omeGs;lnaTs;omeCs;lnaT-Sup
SM N 1- TTATAGCCAG lnaTs;omeUs;lnaAs;omeUs;lnaAs;omeGs;lnaCs;omeCs;lnaAs;omeGs;lna 10075 2 mOl GTCTA Gs;omeUs;lnaCs;omeUs;lnaA-Sup
SM N 1- GCCAGGTCTA lnaGs;omeCs;lnaCs;omeAs;lnaGs;omeGs;lnaTs;omeCs;lnaTs;omeAs;lnaA 10080 3 mOl AAATT s;omeAs;lnaAs;omeUs;lnaT-Sup
SM N 1- CCAGGTCTAA lnaCs;omeCs;lnaAs;omeGs;lnaGs;omeUs;lnaCs;omeUs;lnaAs;omeAs;lna 10081 4 mOl AATTC As;omeAs;lnaTs;omeUs;lnaC-Sup
SM N 1- CAGGTCTAAA lnaCs;omeAs;lnaGs;omeGs;lnaTs;omeCs;lnaTs;omeAs;lnaAs;omeAs;lnaA 10082 75 mOl ATTCA s;omeUs;lnaTs;omeCs;lnaA-Sup
SM N 1- GGTCTAAAAT lnaGs;omeGs;lnaTs;omeCs;lnaTs;omeAs;lnaAs;omeAs;lnaAs;omeUs;lnaT 10084 76 mOl TCAAT s;omeCs;lnaAs;omeAs;lnaT-Sup
SM N 1- CTAAAATTCA lnaCs;omeUs;lnaAs;omeAs;lnaAs;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaA 10087 77 mOl ATGGC s;omeUs;lnaGs;omeGs;lnaC-Sup
SM N 1- CTAAAATTCA omeCs;omeUs;omeAs;omeAs;omeAs;omeAs;omeUs;omeUs;omeCs;ome 10087 77 m03 ATGGC As;omeAs;omeUs;omeGs;omeGs;omeC-Sup
SM N 1- GGACCACCA lnaGs;omeGs;lnaAs;omeCs;lnaCs;omeAs;lnaCs;omeCs;lnaAs;omeGs;lnaT 10168 78 mOl GTAAGT s;omeAs;lnaAs;omeGs;lnaT-Sup
SM N 1- GACCACCAGT dGs;lnaAs;dCs;lnaCs;dAs;lnaCs;dCs;lnaAs;dGs;lnaTs;dAs;lnaAs;dGs;lnaTs; 10169 79 m02 AAGTA dA-Sup
SM N 1- GACCACCAGT lnaGs;omeAs;lnaCs;omeCs;lnaAs;omeCs;lnaCs;omeAs;lnaGs;omeUs;lnaA 10169 79 mOl AAGTA s;omeAs;lnaGs;omeUs;lnaA-Sup
SM N 1- ACCACCAGTA dAs;lnaCs;dCs;lnaAs;dCs;lnaCs;dAs;lnaGs;dTs;lnaAs;dAs;lnaGs;dTs;lnaAs; 10170 80 m02 AGTAA dA-Sup
SM N 1- ACCACCAGTA lnaAs;omeCs;lnaCs;omeAs;lnaCs;omeCs;lnaAs;omeGs;lnaTs;omeAs;lnaA 10170 80 mOl AGTAA s;omeGs;lnaTs;omeAs;lnaA-Sup
SM N 1- TTCTGTTACC lnaTs;omeUs;lnaCs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaCs;omeCs;lnaC 10337 81 mOl CAGAT s;omeAs;lnaGs;omeAs;lnaT-Sup
SM N 1- TCTGTTACCC lnaTs;omeCs;lnaTs;omeGs;lnaTs;omeUs;lnaAs;omeCs;lnaCs;omeCs;lnaA 10338 82 mOl AGATG s;omeGs;lnaAs;omeUs;lnaG-Sup
SM N 1- CTGTTACCCA lnaCs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaCs;omeCs;lnaCs;omeAs;lnaG 10339 83 mOl GATGC s;omeAs;lnaTs;omeGs;lnaC-Sup
SM N 1- 1 1 1 1 I AG I A dTs;lnaTs;dTs;lnaTs;dTs;lnaAs;dGs;lnaGs;dTs;lnaAs;dTs;lnaTs;dAs;lnaAs; 10763 84 m02 TTAAC dC-Sup
SM N 1- 1 1 1 1 I AG I A lnaTs;omeUs;lnaTs;omeUs;lnaTs;omeAs;lnaGs;omeGs;lnaTs;omeAs;lnaT 10763 84 mOl TTAAC s;omeUs;lnaAs;omeAs;lnaC-Sup
SM N 1- 1 1 1 I AG I A I lnaTs;omeUs;lnaTs;omeUs;lnaAs;omeGs;lnaGs;omeUs;lnaAs;omeUs;lna 10764 85 mOl TAACA Ts;omeAs;lnaAs;omeCs;lnaA-Sup
SM N 1- CATAGCTTCA lnaCs;omeAs;lnaTs;omeAs;lnaGs;omeCs;lnaTs;omeUs;lnaCs;omeAs;lnaT 10949 86 mOl TAGTG s;omeAs;lnaGs;omeUs;lnaG-Sup
SM N 1- TAGCTTCATA lnaTs;omeAs;lnaGs;omeCs;lnaTs;omeUs;lnaCs;omeAs;lnaTs;omeAs;lnaG 10951 87 mOl GTGGA s;omeUs;lnaGs;omeGs;lnaA-Sup
SM N 1- AGCTTCATAG lnaAs;omeGs;lnaCs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeGs;lnaT 10952 88 mOl TGGAA s;omeGs;lnaGs;omeAs;lnaA-Sup
SM N 1- GCTTCATAGT lnaGs;omeCs;lnaTs;omeUs;lnaCs;omeAs;lnaTs;omeAs;lnaGs;omeUs;lnaG 10953 89 mOl GGAAC s;omeGs;lnaAs;omeAs;lnaC-Sup
SM N 1- CTTCATAGTG lnaCs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeGs;lnaTs;omeGs;lnaG 10954 90 mOl GAACA s;omeAs;lnaAs;omeCs;lnaA-Sup
SM N 1- TCATGGTACA lnaTs;omeCs;lnaAs;omeUs;lnaGs;omeGs;lnaTs;omeAs;lnaCs;omeAs;lnaT 11415 91 mOl TGAGT s;omeGs;lnaAs;omeGs;lnaT-Sup
SM N 1- TGGTACATGA lnaTs;omeGs;lnaGs;omeUs;lnaAs;omeCs;lnaAs;omeUs;lnaGs;omeAs;lna 11418 92 mOl GTGGC Gs;omeUs;lnaGs;omeGs;lnaC-Sup
SM N 1- GGTACATGA dGs;lnaGs;dTs;lnaAs;dCs;lnaAs;dTs;lnaGs;dAs;lnaGs;dTs;lnaGs;dGs;lnaCs 11419 93 m02 GTGGCT ;dT-Sup
SM N 1- GGTACATGA lnaGs;omeGs;lnaTs;omeAs;lnaCs;omeAs;lnaTs;omeGs;lnaAs;omeGs;lnaT 11419 93 mOl GTGGCT s;omeGs;lnaGs;omeCs;lnaT-Sup
SM N 1- TACATGAGTG lnaTs;omeAs;lnaCs;omeAs;lnaTs;omeGs;lnaAs;omeGs;lnaTs;omeGs;lnaG 11421 94 mOl GCTAT s;omeCs;lnaTs;omeAs;lnaT-Sup
SM N 1- ACATGAGTG lnaAs;omeCs;lnaAs;omeUs;lnaGs;omeAs;lnaGs;omeUs;lnaGs;omeGs;lna 11422 95 mOl GCTATC Cs;omeUs;lnaAs;omeUs;lnaC-Sup
SM N 1- CATGAGTGG lnaCs;omeAs;lnaTs;omeGs;lnaAs;omeGs;lnaTs;omeGs;lnaGs;omeCs;lnaT 11423 96 mOl CTATCA s;omeAs;lnaTs;omeCs;lnaA-Sup
SM N 1- CTGGCTATTA lnaCs;omeUs;lnaGs;omeGs;lnaCs;omeUs;lnaAs;omeUs;lnaTs;omeAs;lnaT 11440
97 mOl TATGG s;omeAs;lnaTs;omeGs;lnaG-Sup
SM N 1- TGGCTATTAT lnaTs;omeGs;lnaGs;omeCs;lnaTs;omeAs;lnaTs;omeUs;lnaAs;omeUs;lnaA 11441
98 mOl ATGGT s;omeUs;lnaGs;omeGs;lnaT-Sup
SM N 1- GGCTATTATA lnaGs;omeGs;lnaCs;omeUs;lnaAs;omeUs;lnaTs;omeAs;lnaTs;omeAs;lnaT 11442
99 mOl TGGTA s;omeGs;lnaGs;omeUs;lnaA-Sup
SM N 1- GCTATTATAT lnaGs;omeCs;lnaTs;omeAs;lnaTs;omeUs;lnaAs;omeUs;lnaAs;omeUs;lna 11443
100 GGTAA Gs;omeGs;lnaTs;omeAs;lnaA-Sup
mOl
SM N 1- GTATCATCTG lnaGs;omeUs;lnaAs;omeUs;lnaCs;omeAs;lnaTs;omeCs;lnaTs;omeGs;lnaT 12369
101 TGTGT s;omeGs;lnaTs;omeGs;lnaT-Sup
mOl
SM N 1- GCTTTGGGA lnaGs;omeCs;lnaTs;omeUs;lnaTs;omeGs;lnaGs;omeGs;lnaAs;omeAs;lna 13097
102 AGTATG 1 1 1 1 Gs;omeUs;lnaAs;omeUs;lnaG;dT;dT;dT;dT;lnaTs;omeCs;lnaAs;omeCs;lna mOl TCACTTTCAT Ts;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeAs;lnaTs;omeGs;lnaCs;o
AATGCTGG meUs;lnaGs;omeG-Sup
SM N 1- CTTTGGGAA lnaCs;omeUs;lnaTs;omeUs;lnaGs;omeGs;lnaGs;omeAs;lnaAs;omeGs;lna 13102
103 GTATG 1 1 1 1 1 Ts;omeAs;lnaTs;omeGs;lnaT;dT;dT;dT;dT;lnaTs;omeCs;lnaAs;omeCs;lnaT mOl TCACTTTCAT s;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeAs;lnaTs;omeGs;lnaCs;o
AATGCTGG meUs;lnaGs;omeG-Sup
SM N 1- GGTACATGA lnaGs;omeGs;lnaTs;omeAs;lnaCs;omeAs;lnaTs;omeGs;lnaAs;omeGs;lnaT 13099
104 GTGGC I 1 1 1 1 s;omeGs;lnaGs;omeCs;lnaT;dT;dT;dT;dT;lnaTs;omeCs;lnaAs;omeCs;lnaTs mOl TCACTTTCAT ;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeAs;lnaTs;omeGs;lnaCs;om
AATGCTGG eUs;lnaGs;omeG-Sup
SM N 1- TGATGCTGAT lnaTs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaTs;omeGs;lnaAs;omeUs;lna 13103
105 GC I 1 1 1 1 1 I C I Gs;omeCs;lnaTs;omeUs;lnaT;dT;dT;dT;dT;lnaCs;omeUs;lnaAs;omeAs;lna mOl AAAATTCAAT As;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaC- GGC Sup
SM N 1- CTAAAATTCA lnaCs;omeUs;lnaAs;omeAs;lnaAs;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaA 13104
106 ATGGC I I M C s;omeUs;lnaGs;omeGs;lnaC;dT;dT;dT;dT;lnaCs;omeUs;lnaAs;omeAs;lnaA mOl TAAAATTCAA s;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaC- TGGC Sup
SM N 1- CTGTTACCCA lnaCs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaCs;omeCs;lnaCs;omeAs;lnaG 13105
107 GATGC 1 1 1 1 C s;omeAs;lnaTs;omeGs;lnaC;dT;dT;dT;dT;lnaCs;omeUs;lnaAs;omeAs;lnaA mOl TAAAATTCAA s;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaC- TGGC Sup
SM N 1- CTTCATAGTG lnaCs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeGs;lnaTs;omeGs;lnaG 13106
108 GAACA 1 1 1 1 C s;omeAs;lnaAs;omeCs;lnaA;dT;dT;dT;dT;lnaCs;omeUs;lnaAs;omeAs;lnaA mOl TAAAATTCAA s;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaC- TGGC Sup
SM N 1- TCACTTTCAT lnaTs;omeCs;lnaAs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaA 13107
109 AATGCTGGTT s;omeAs;lnaTs;omeGs;lnaCs;omeUs;lnaGs;omeG;dT;dT;dT;dT;lnaTs;ome mOl TTTCACTTTC Cs;lnaAs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaAs;omeUs;lnaAs;omeAs;ln
ATAATGCTGG aTs;omeGs;lnaCs;omeUs;lnaGs;omeG-Sup
SM N l- AAATTCAATG lnaAs;omeAs;lnaAs;omeUs;lnaTs;omeCs;lnaAs;omeAs;lnaTs;omeGs;lnaG 10090
110 GCCCA s;omeCs;lnaCs;omeCs;lnaA-Sup
mOl
SM N 1- AATTCAATGG lnaAs;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lna 10091
111 CCCAC Cs;omeCs;lnaCs;omeAs;lnaC-Sup
mOl
SM N 1- ATTCAATGGC lnaAs;omeUs;lnaTs;omeCs;lnaAs;omeAs;lnaTs;omeGs;lnaGs;omeCs;lnaC 10092
112 CCACC s;omeCs;lnaAs;omeCs;lnaC-Sup mOl
SM N 1- TTCAATGGCC lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaCs;omeCs;lnaC 10093 113 CACCA s;omeAs;lnaCs;omeCs;lnaA-Sup
mOl
SM N 1- TCAATGGCCC lnaTs;omeCs;lnaAs;omeAs;lnaTs;omeGs;lnaGs;omeCs;lnaCs;omeCs;lnaA 10094 114 ACCAC s;omeCs;lnaCs;omeAs;lnaC-Sup
mOl
SM N 1- CAATGGCCCA lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeGs;lnaCs;omeCs;lnaCs;omeAs;lnaC 10095 115 CCACC s;omeCs;lnaAs;omeCs;lnaC-Sup
mOl
SM N 1- AATGGCCCAC lnaAs;omeAs;lnaTs;omeGs;lnaGs;omeCs;lnaCs;omeCs;lnaAs;omeCs;lnaC 10096 116 CACCG s;omeAs;lnaCs;omeCs;lnaG-Sup
mOl
SM N 1- ATGGCCCACC lnaAs;omeUs;lnaGs;omeGs;lnaCs;omeCs;lnaCs;omeAs;lnaCs;omeCs;lnaA 10097 117 ACCGC s;omeCs;lnaCs;omeGs;lnaC-Sup
mOl
SM N 1- AATGCCTTTC lnaAs;omeAs;lnaTs;omeGs;lnaCs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaTs 10330 118 TGTTA ;omeGs;lnaTs;omeUs;lnaA-Sup
mOl
SM N 1- ATGCCTTTCT lnaAs;omeUs;lnaGs;omeCs;lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeUs;lna 10331 119 GTTAC Gs;omeUs;lnaTs;omeAs;lnaC-Sup
mOl
SM N 1- TGCCTTTCTG lnaTs;omeGs;lnaCs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaTs;omeGs;lnaTs 10332 120 TTACC ;omeUs;lnaAs;omeCs;lnaC-Sup
mOl
SM N 1- GCCTTTCTGT lnaGs;omeCs;lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeUs;lnaGs;omeUs;lnaT 10333 121 TACCC s;omeAs;lnaCs;omeCs;lnaC-Sup
mOl
SM N 1- CCTTTCTGTT lnaCs;omeCs;lnaTs;omeUs;lnaTs;omeCs;lnaTs;omeGs;lnaTs;omeUs;lnaA 10334 122 ACCCA s;omeCs;lnaCs;omeCs;lnaA-Sup
mOl
SM N 1- CTTTCTGTTA lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaC 10335 123 CCCAG s;omeCs;lnaCs;omeAs;lnaG-Sup
mOl
SM N 1- TTTCTGTTAC lnaTs;omeUs;lnaTs;omeCs;lnaTs;omeGs;lnaTs;omeUs;lnaAs;omeCs;lnaC 10336 124 CCAGA s;omeCs;lnaAs;omeGs;lnaA-Sup
mOl
SM N 1- TGTTACCCAG lnaTs;omeGs;lnaTs;omeUs;lnaAs;omeCs;lnaCs;omeCs;lnaAs;omeGs;lnaA 10340 125 ATGCA s;omeUs;lnaGs;omeCs;lnaA-Sup
mOl
SM N 1- GTTACCCAGA lnaGs;omeUs;lnaTs;omeAs;lnaCs;omeCs;lnaCs;omeAs;lnaGs;omeAs;lnaT 10341 126 TGCAG s;omeGs;lnaCs;omeAs;lnaG-Sup
mOl
SM N 1- TTACCCAGAT lnaTs;omeUs;lnaAs;omeCs;lnaCs;omeCs;lnaAs;omeGs;lnaAs;omeUs;lnaG 10342 127 GCAGT s;omeCs;lnaAs;omeGs;lnaT-Sup
mOl
SM N 1- ACCCAGATGC lnaAs;omeCs;lnaCs;omeCs;lnaAs;omeGs;lnaAs;omeUs;lnaGs;omeCs;lnaA 10344 128 AGTGC s;omeGs;lnaTs;omeGs;lnaC-Sup
mOl
SM N 1- CCCAGATGCA lnaCs;omeCs;lnaCs;omeAs;lnaGs;omeAs;lnaTs;omeGs;lnaCs;omeAs;lnaG 10345 129 GTGCT s;omeUs;lnaGs;omeCs;lnaT-Sup
mOl SM N l- CCAGATGCA lnaLs;omeLs;lnaAs;omeGs;lnaAs;omeUs;lnaGs;omeLs;lnaAs;omeGs;lnaT 10346 130 GTGCTC s;omeGs;lnaLs;omeUs;lnaL-Sup
mOl
SM N 1- CAGATGCAGT lnaLs;omeAs;lnaGs;omeAs;lnaTs;omeGs;lnaLs;omeAs;lnaGs;omeUs;lna 10347 131 GCTCT Gs;omeLs;lnaTs;omeLs;lnaT-Sup
mOl
SM N 1- AGATGCAGT lnaAs;omeGs;lnaAs;omeUs;lnaGs;omeLs;lnaAs;omeGs;lnaTs;omeGs;lna 10348 132 GCTCTT Ls;omeUs;lnaLs;omeUs;lnaT-Sup
mOl
SM N 1- 1 1 1 1 AC 1 LA 1 lnaTs;omeUs;lnaTs;omeUs;lnaAs;omeLs;lnaTs;omeLs;lnaAs;omeUs;lnaA 10942 133 AG TT s;omeGs;lnaLs;omeUs;lnaT-Sup
mOl
SM N 1- TTTALTLATA lnaTs;omeUs;lnaTs;omeAs;lnaLs;omeUs;lnaLs;omeAs;lnaTs;omeAs;lnaG 10943 134 GLTTL s;omeLs;lnaTs;omeUs;lnaL-Sup
mOl
SM N 1- TTALTLATAG lnaTs;omeUs;lnaAs;omeLs;lnaTs;omeLs;lnaAs;omeUs;lnaAs;omeGs;lnaL 10944 135 LTTLA s;omeUs;lnaTs;omeLs;lnaA-Sup
mOl
SM N 1- TALTLATAGL lnaTs;omeAs;lnaLs;omeUs;lnaLs;omeAs;lnaTs;omeAs;lnaGs;omeLs;lnaT 10945 136 TTLAT s;omeUs;lnaLs;omeAs;lnaT-Sup
mOl
SM N 1- ALTLATAGLT lnaAs;omeLs;lnaTs;omeLs;lnaAs;omeUs;lnaAs;omeGs;lnaLs;omeUs;lnaT 10946 137 TLATA s;omeLs;lnaAs;omeUs;lnaA-Sup
mOl
SM N 1- LTLATAGLTT lnaLs;omeUs;lnaLs;omeAs;lnaTs;omeAs;lnaGs;omeLs;lnaTs;omeUs;lnaL 10947 138 LATAG s;omeAs;lnaTs;omeAs;lnaG-Sup
mOl
SM N 1- TLATAGLTTL lnaTs;omeLs;lnaAs;omeUs;lnaAs;omeGs;lnaLs;omeUs;lnaTs;omeLs;lnaA 10948 139 ATAGT s;omeUs;lnaAs;omeGs;lnaT-Sup
mOl
SM N 1- ATA GLTT AT lnaAs;omeUs;lnaAs;omeGs;lnaLs;omeUs;lnaTs;omeLs;lnaAs;omeUs;lna 10950 140 AGTGG As;omeGs;lnaTs;omeGs;lnaG-Sup
mOl
SM N 1- TTLATAGTGG lnaTs;omeUs;lnaLs;omeAs;lnaTs;omeAs;lnaGs;omeUs;lnaGs;omeGs;lna 10955 141 AALAG As;omeAs;lnaLs;omeAs;lnaG-Sup
mOl
SM N 1- TLATAGTGGA lnaTs;omeLs;lnaAs;omeUs;lnaAs;omeGs;lnaTs;omeGs;lnaGs;omeAs;lnaA 10956 142 ALAGA s;omeLs;lnaAs;omeGs;lnaA-Sup
mOl
SM N 1- LATAGTGGA lnaLs;omeAs;lnaTs;omeAs;lnaGs;omeUs;lnaGs;omeGs;lnaAs;omeAs;lnaL 10957 143 ALAGAT s;omeAs;lnaGs;omeAs;lnaT-Sup
mOl
SM N 1- ATAGTGGAA lnaAs;omeUs;lnaAs;omeGs;lnaTs;omeGs;lnaGs;omeAs;lnaAs;omeLs;lna 10958 144 LAGATA As;omeGs;lnaAs;omeUs;lnaA-Sup
mOl
SM N 1- TAGTGGAAL lnaTs;omeAs;lnaGs;omeUs;lnaGs;omeGs;lnaAs;omeAs;lnaLs;omeAs;lna 10959 145 AGATAL Gs;omeAs;lnaTs;omeAs;lnaL-Sup
mOl
SM N 1- AGTGGAALA lnaAs;omeGs;lnaTs;omeGs;lnaGs;omeAs;lnaAs;omeLs;lnaAs;omeGs;lna 10960 146 GATALA As;omeUs;lnaAs;omeLs;lnaA-Sup
mOl
SM N 1- GTGGAALAG lnaGs;omeUs;lnaGs;omeGs;lnaAs;omeAs;lnaLs;omeAs;lnaGs;omeAs;lna 10961 147 ATALAT Ts;omeAs;lnaLs;omeAs;lnaT-Sup mOl
SM N 1- TGGAACAGA lnaTs;omeGs;lnaGs;omeAs;lnaAs;omeCs;lnaAs;omeGs;lnaAs;omeUs;lna 10962 148 TACATA As;omeCs;lnaAs;omeUs;lnaA-Sup
mOl
SM N 1- TGTCCAGATT lnaTs;omeGs;lnaTs;omeCs;lnaCs;omeAs;lnaGs;omeAs;lnaTs;omeUs;lnaC 11367 149 CTCTT s;omeUs;lnaCs;omeUs;lnaT-Sup
mOl
SM N 1- GTCCAGATTC lnaGs;omeUs;lnaCs;omeCs;lnaAs;omeGs;lnaAs;omeUs;lnaTs;omeCs;lnaT 11368 150 TCTTG s;omeCs;lnaTs;omeUs;lnaG-Sup
mOl
SM N 1- TCCAGATTCT lnaTs;omeCs;lnaCs;omeAs;lnaGs;omeAs;lnaTs;omeUs;lnaCs;omeUs;lnaC 11369 151 CTTGA s;omeUs;lnaTs;omeGs;lnaA-Sup
mOl
SM N 1- CCAGATTCTC lnaCs;omeCs;lnaAs;omeGs;lnaAs;omeUs;lnaTs;omeCs;lnaTs;omeCs;lnaT 11370 152 TTGAT s;omeUs;lnaGs;omeAs;lnaT-Sup
mOl
SM N 1- CAGATTCTCT lnaCs;omeAs;lnaGs;omeAs;lnaTs;omeUs;lnaCs;omeUs;lnaCs;omeUs;lnaT 11371 153 TGATG s;omeGs;lnaAs;omeUs;lnaG-Sup
mOl
SM N 1- AGATTCTCTT lnaAs;omeGs;lnaAs;omeUs;lnaTs;omeCs;lnaTs;omeCs;lnaTs;omeUs;lnaG 11372 154 GATGA s;omeAs;lnaTs;omeGs;lnaA-Sup
mOl
SM N 1- GATTCTCTTG lnaGs;omeAs;lnaTs;omeUs;lnaCs;omeUs;lnaCs;omeUs;lnaTs;omeGs;lnaA 11373 155 ATGAT s;omeUs;lnaGs;omeAs;lnaT-Sup
mOl
SM N 1- GGAAGTATG lnaGs;omeGs;lnaAs;omeAs;lnaGs;omeUs;lnaAs;omeUs;lnaGs;omeUs;lna 11400 156 TTAATT Ts;omeAs;lnaAs;omeUs;lnaT-Sup
mOl
SM N 1- GAAGTATGTT lnaGs;omeAs;lnaAs;omeGs;lnaTs;omeAs;lnaTs;omeGs;lnaTs;omeUs;lnaA 11401 157 AATTT s;omeAs;lnaTs;omeUs;lnaT-Sup
mOl
SM N 1- AAGTATGTTA lnaAs;omeAs;lnaGs;omeUs;lnaAs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lna 11402 158 ATTTC As;omeUs;lnaTs;omeUs;lnaC-Sup
mOl
SM N 1- AGTATGTTAA lnaAs;omeGs;lnaTs;omeAs;lnaTs;omeGs;lnaTs;omeUs;lnaAs;omeAs;lnaT 11403 159 TTTCA s;omeUs;lnaTs;omeCs;lnaA-Sup
mOl
SM N 1- GTATGTTAAT lnaGs;omeUs;lnaAs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaAs;omeUs;lna 11404 160 TTCAT Ts;omeUs;lnaCs;omeAs;lnaT-Sup
mOl
SM N 1- TATG TTAATT lnaTs;omeAs;lnaTs;omeGs;lnaTs;omeUs;lnaAs;omeAs;lnaTs;omeUs;lnaT 11405 161 TCATG s;omeCs;lnaAs;omeUs;lnaG-Sup
mOl
SM N 1- ATGTTAATTT lnaAs;omeUs;lnaGs;omeUs;lnaTs;omeAs;lnaAs;omeUs;lnaTs;omeUs;lna 11406 162 CATGG Cs;omeAs;lnaTs;omeGs;lnaG-Sup
mOl
SM N 1- TGAAATATTC lnaTs;omeGs;lnaAs;omeAs;lnaAs;omeUs;lnaAs;omeUs;lnaTs;omeCs;lnaC 10064 163 CTTAT s;omeUs;lnaTs;omeAs;lnaT-Sup
mOl
SM N 1- TATAGCCAGG lnaTs;omeAs;lnaTs;omeAs;lnaGs;omeCs;lnaCs;omeAs;lnaGs;omeGs;lnaT 10076 164 TCTAA s;omeCs;lnaTs;omeAs;lnaA-Sup
mOl SM N l- ATAGCCAGGT lnaAs;omeUs;lnaAs;omeGs;lnaCs;omeCs;lnaAs;omeGs;lnaGs;omeUs;lna 10077 165 CTAAA Cs;omeUs;lnaAs;omeAs;lnaA-Sup
mOl
SM N 1- AGGTCTAAAA lnaAs;omeGs;lnaGs;omeUs;lnaCs;omeUs;lnaAs;omeAs;lnaAs;omeAs;lna 10083 166 TTCAA Ts;omeUs;lnaCs;omeAs;lnaA-Sup
mOl
SM N 1- GTCTAAAATT lnaGs;omeUs;lnaCs;omeUs;lnaAs;omeAs;lnaAs;omeAs;lnaTs;omeUs;lna 10085 167 CAATG Cs;omeAs;lnaAs;omeUs;lnaG-Sup
mOl
SM N 1- TCTAAAATTC lnaTs;omeCs;lnaTs;omeAs;lnaAs;omeAs;lnaAs;omeUs;lnaTs;omeCs;lnaA 10086 168 AATGG s;omeAs;lnaTs;omeGs;lnaG-Sup
mOl
SM N 1- TAAAATTCAA lnaTs;omeAs;lnaAs;omeAs;lnaAs;omeUs;lnaTs;omeCs;lnaAs;omeAs;lnaT 10088 169 TGGCC s;omeGs;lnaGs;omeCs;lnaC-Sup
mOl
SM N 1- AAAATTCAAT lnaAs;omeAs;lnaAs;omeAs;lnaTs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaG 10089 170 GGCCC s;omeGs;lnaCs;omeCs;lnaC-Sup
mOl
unc-232 CTACGCGTCG lnaCs;dTs;lnaAs;dCs;lnaGs;dCs;lnaGs;dTs;lnaCs;dGs;lnaAs;dCs;lnaGs;dGs; 13095 ml2 ACGGT InaT-Sup
unc-232 CTACGCGTCG lnaCs;omeUs;lnaAs;omeCs;lnaGs;omeCs;lnaGs;omeUs;lnaCs;omeGs;lna 13095 mOl ACGGT As;omeCs;lnaGs;omeGs;lnaT-Sup
unc-293 CCGATTCGCG lnaCs;dCs;lnaGs;dAs;lnaTs;dTs;lnaCs;dGs;lnaCs;dGs;lnaCs;dGs;lnaTs;dAs; 13096 ml2 CGTAA InaA-Sup
unc-293 CCGATTCGCG lnaCs;omeCs;lnaGs;omeAs;lnaTs;omeUs;lnaCs;omeGs;lnaCs;omeGs;lnaC 13096 mOl CGTAA s;omeGs;lnaTs;omeAs;lnaA-Sup
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. Appendix A: Table 5
Figure imgf000107_0001
Appendix A: Table 5
Figure imgf000108_0001
Appendix A: Table 5
Figure imgf000109_0001
Appendix A: Table 5
Figure imgf000110_0001
Appendix A: Table 5
Figure imgf000111_0001
Appendix A: Table 5
Figure imgf000112_0001
Appendix A: Table 5
Figure imgf000113_0001
Appendix A: Table 5
Figure imgf000114_0001
Appendix A: Table 5
Figure imgf000115_0001
Appendix A: Table 5
Figure imgf000116_0001
Appendix A: Table 5
Figure imgf000117_0001
Appendix A: Table 5
Figure imgf000118_0001
Appendix A: Table 5
Figure imgf000119_0001
Appendix A: Table 5
Figure imgf000120_0001
Appendix A: Table 5
Figure imgf000121_0001
Appendix A: Table 5
Figure imgf000122_0001
Appendix A: Table 5
Figure imgf000123_0001
Appendix A: Table 5
Figure imgf000124_0001
Appendix A: Table 5
Figure imgf000125_0001
Appendix A: Table 5
Figure imgf000126_0001
Appendix A: Table 5
Figure imgf000127_0001
Appendix A: Table 5
Figure imgf000128_0001
Appendix A: Table 5
Figure imgf000129_0001
Appendix A: Table 5
Figure imgf000130_0001
Appendix A: Table 5
Figure imgf000131_0001
Appendix A: Table 5
Figure imgf000132_0001
Appendix A: Table 5
Figure imgf000133_0001
Appendix A: Table 5
Figure imgf000134_0001
Appendix A: Table 5
Figure imgf000135_0001
Appendix A: Table 5
Figure imgf000136_0001
Appendix A: Table 5
Figure imgf000137_0001
Appendix A: Table 5
Figure imgf000138_0001
Appendix A: Table 5
Figure imgf000139_0001
Appendix A: Table 5
Figure imgf000140_0001
Appendix A: Table 5
Figure imgf000141_0001
Appendix A: Table 5
Figure imgf000142_0001
Appendix A: Table 5
Figure imgf000143_0001
Appendix A: Table 5
Figure imgf000144_0001
Appendix A: Table 5
Figure imgf000145_0001
Appendix A: Table 5
Figure imgf000146_0001
Appendix A: Table 5
Figure imgf000147_0001
Appendix A: Table 5
Figure imgf000148_0001
Appendix A: Table 5
Figure imgf000149_0001
Appendix A: Table 5
Figure imgf000150_0001
Appendix A: Table 5
Figure imgf000151_0001
Appendix A: Table 5
Figure imgf000152_0001
Appendix A: Table 5
Figure imgf000153_0001
Appendix A: Table 5
Figure imgf000154_0001
Appendix A: Table 5
Figure imgf000155_0001
Appendix A: Table 5
Figure imgf000156_0001
Appendix A: Table 5
Figure imgf000157_0001
Appendix A: Table 5
Figure imgf000158_0001
Appendix A: Table 5
Figure imgf000159_0001
Appendix A: Table 5
Figure imgf000160_0001
Appendix A: Table 5
Figure imgf000161_0001
Appendix A: Table 5
Figure imgf000162_0001
Appendix A: Table 5
Figure imgf000163_0001
Appendix A: Table 5
Figure imgf000164_0001
Appendix A: Table 5
Figure imgf000165_0001
Appendix A: Table 5
Figure imgf000166_0001
Appendix A: Table 5
Figure imgf000167_0001
Appendix A: Table 5
Figure imgf000168_0001
Appendix A: Table 5
Figure imgf000169_0001
Appendix A: Table 5
Figure imgf000170_0001
Appendix A: Table 5
Figure imgf000171_0001
Appendix A: Table 5
Figure imgf000172_0001
Appendix A: Table 5
Figure imgf000173_0001
Appendix A: Table 5
Figure imgf000174_0001
Appendix A: Table 5
Figure imgf000175_0001
Appendix A: Table 5
Figure imgf000176_0001
Appendix A: Table 5
Figure imgf000177_0001
Appendix A: Table 5
Figure imgf000178_0001
Appendix A: Table 5
Figure imgf000179_0001
Appendix A: Table 5
Figure imgf000180_0001
Appendix A: Table 5
Figure imgf000181_0001
Appendix A: Table 5
Figure imgf000182_0001
Appendix A: Table 5
Figure imgf000183_0001
Appendix A: Table 5
Figure imgf000184_0001
Appendix A: Table 5
Figure imgf000185_0001
Appendix A: Table 5
Figure imgf000186_0001
Appendix A: Table 5
Figure imgf000187_0001
Appendix A: Table 5
Figure imgf000188_0001
Appendix A: Table 5
Figure imgf000189_0001
Appendix A: Table 5
Figure imgf000190_0001
Appendix A: Table 5
Figure imgf000191_0001
Appendix A: Table 5
Figure imgf000192_0001
Appendix A: Table 5
Figure imgf000193_0001
Appendix A: Table 5
Figure imgf000194_0001
Appendix A: Table 5
Figure imgf000195_0001
Appendix A: Table 5
Figure imgf000196_0001
Appendix A: Table 5
Figure imgf000197_0001
Appendix A: Table 5
Figure imgf000198_0001
Appendix A: Table 5
Figure imgf000199_0001
Appendix A: Table 5
Figure imgf000200_0001
Appendix A: Table 5
Figure imgf000201_0001
Appendix A: Table 5
Figure imgf000202_0001
Appendix A: Table 5
Figure imgf000203_0001
Appendix A: Table 5
Figure imgf000204_0001
Appendix A: Table 5
Figure imgf000205_0001
Appendix A: Table 5
Figure imgf000206_0001
Appendix A: Table 5
Figure imgf000207_0001
Appendix A: Table 5
Figure imgf000208_0001
Appendix A: Table 5
Figure imgf000209_0001
Appendix A: Table 5
Figure imgf000210_0001
Appendix A: Table 5
Figure imgf000211_0001
Appendix A: Table 5
Figure imgf000212_0001
Appendix A: Table 5
Figure imgf000213_0001
Appendix A: Table 5
Figure imgf000214_0001
Appendix A: Table 5
Figure imgf000215_0001
Appendix A: Table 5
Figure imgf000216_0001
Appendix A: Table 5
Figure imgf000217_0001
Appendix A: Table 5
Figure imgf000218_0001
Appendix A: Table 5
Figure imgf000219_0001
Appendix A: Table 5
Figure imgf000220_0001
Appendix A: Table 5
Figure imgf000221_0001
Appendix A: Table 5
Figure imgf000222_0001
Appendix A: Table 5
Figure imgf000223_0001
Appendix A: Table 5
Figure imgf000224_0001
Appendix A: Table 5
Figure imgf000225_0001
Appendix A: Table 5
Figure imgf000226_0001
Appendix A: Table 5
Figure imgf000227_0001
Appendix A: Table 5
Figure imgf000228_0001
Appendix A: Table 5
Figure imgf000229_0001
Appendix A: Table 5
Figure imgf000230_0001
Appendix A: Table 5
Figure imgf000231_0001
Appendix A: Table 5
Figure imgf000232_0001
Appendix A: Table 5
Figure imgf000233_0001
Appendix A: Table 5
Figure imgf000234_0001
Appendix A: Table 5
Figure imgf000235_0001
Appendix A: Table 5
Figure imgf000236_0001
Appendix A: Table 5
Figure imgf000237_0001
Appendix A: Table 5
Figure imgf000238_0001
Appendix A: Table 5
Figure imgf000239_0001
Appendix A: Table 5
Figure imgf000240_0001
Appendix A: Table 5
Figure imgf000241_0001
Appendix A: Table 5
Figure imgf000242_0001
Appendix A: Table 5
Figure imgf000243_0001
Appendix A: Table 5
Figure imgf000244_0001
Appendix A: Table 5
Figure imgf000245_0001
Appendix A: Table 5
Figure imgf000246_0001
Appendix A: Table 5
Figure imgf000247_0001
Appendix A: Table 5
Figure imgf000248_0001
Appendix A: Table 5
Figure imgf000249_0001
Appendix A: Table 5
Figure imgf000250_0001
Appendix A: Table 5
Figure imgf000251_0001
Appendix A: Table 5
Figure imgf000252_0001
Appendix A: Table 5
Figure imgf000253_0001
Appendix A: Table 5
Figure imgf000254_0001
Appendix A: Table 5
Figure imgf000255_0001
Appendix A: Table 5
Figure imgf000256_0001
Appendix A: Table 5
Figure imgf000257_0001
Appendix A: Table 5
Figure imgf000258_0001
Appendix A: Table 5
Figure imgf000259_0001
Appendix A: Table 5
Figure imgf000260_0001
Appendix A: Table 5
Figure imgf000261_0001
Appendix A: Table 5
Figure imgf000262_0001
Appendix A: Table 5
Figure imgf000263_0001
Appendix A: Table 5
Figure imgf000264_0001
Appendix A: Table 5
Figure imgf000265_0001
Appendix A: Table 5
Figure imgf000266_0001
Appendix A: Table 5
Figure imgf000267_0001
Appendix A: Table 5
Figure imgf000268_0001
Appendix A: Table 5
Figure imgf000269_0001
Appendix A: Table 5
Figure imgf000270_0001
Appendix A: Table 5
Figure imgf000271_0001
Appendix A: Table 5
Figure imgf000272_0001
Appendix A: Table 5
Figure imgf000273_0001
Appendix A: Table 5
Figure imgf000274_0001
Appendix A: Table 5
Figure imgf000275_0001
Appendix A: Table 5
Figure imgf000276_0001
Appendix A: Table 5
Figure imgf000277_0001
Appendix A: Table 5
Figure imgf000278_0001
Appendix A: Table 5
Figure imgf000279_0001
Appendix A: Table 5
Figure imgf000280_0001
Appendix A: Table 5
Figure imgf000281_0001
Appendix A: Table 5
Figure imgf000282_0001
Appendix A: Table 5
Figure imgf000283_0001
Appendix A: Table 5
Figure imgf000284_0001
Appendix A: Table 5
Figure imgf000285_0001
Appendix A: Table 5
Figure imgf000286_0001
Appendix A: Table 5
Figure imgf000287_0001
Appendix A: Table 5
Figure imgf000288_0001
Appendix B: Table 6
Figure imgf000289_0001
Appendix B: Table 6
Figure imgf000290_0001
Appendix B: Table 6
Figure imgf000291_0001
Appendix B: Table 6
Figure imgf000292_0001
Appendix B: Table 6
Figure imgf000293_0001
Appendix B: Table 6
Figure imgf000294_0001
Appendix B: Table 6
Figure imgf000295_0001
Appendix B: Table 6
Figure imgf000296_0001
Appendix B: Table 6
Figure imgf000297_0001
Appendix B: Table 6
Figure imgf000298_0001
Appendix B: Table 6
Figure imgf000299_0001
Appendix B: Table 6
Figure imgf000300_0001
Appendix B: Table 6
Figure imgf000301_0001
Appendix B: Table 6
Figure imgf000302_0001
Appendix B: Table 6
Figure imgf000303_0001
Appendix B: Table 6
Figure imgf000304_0001
Appendix B: Table 6
Figure imgf000305_0001
Appendix B: Table 6
Figure imgf000306_0001
Appendix B: Table 6
Figure imgf000307_0001
Appendix B: Table 6
Figure imgf000308_0001
Appendix B: Table 6
Figure imgf000309_0001
Appendix B: Table 6
Figure imgf000310_0001
Append] ixC:Table7
Figure imgf000311_0001
Appendix C: Table 7
Figure imgf000312_0001
Appendix C: Table 7
Figure imgf000313_0001
Appendix C: Table 7
Figure imgf000314_0001
Appendix C: Table 7
Figure imgf000315_0001
Appendix C: Table 7
Figure imgf000316_0001
Appendix C: Table 7
Figure imgf000317_0001
Appendix C: Table 7
Figure imgf000318_0001
Appendix C: Table 7
Figure imgf000319_0001
Appendix C: Table 7
Figure imgf000320_0001
Appendix C: Table 7
Figure imgf000321_0001
Appendix C: Table 7
Figure imgf000322_0001
Appendix C: Table 7
Figure imgf000323_0001
Appendix C: Table 7
Figure imgf000324_0001
Appendix C: Table 7
Figure imgf000325_0001
Appendix C: Table 7
Figure imgf000326_0001
Appendix C: Table 7
Figure imgf000327_0001
Appendix C: Table 7
Figure imgf000328_0001
Appendix C: Table 7
Figure imgf000329_0001
Appendix C: Table 7
Figure imgf000330_0001
Appendix C: Table 7
Figure imgf000331_0001
Appendix C: Table 7
Figure imgf000332_0001
Appendix C: Table 7
Figure imgf000333_0001
Appendix C: Table 7
Figure imgf000334_0001
Appendix C: Table 7
Figure imgf000335_0001
Appendix C: Table 7
Figure imgf000336_0001
Appendix C: Table 7
Figure imgf000337_0001
Appendix C: Table 7
Figure imgf000338_0001
Appendix C: Table 7
Figure imgf000339_0001
Appendix C: Table 7
Figure imgf000340_0001
Appendix C: Table 7
Figure imgf000341_0001
Appendix C: Table 7
Figure imgf000342_0001
Appendix C: Table 7
Figure imgf000343_0001
Appendix C: Table 7
Figure imgf000344_0001
Appendix C: Table 7
Figure imgf000345_0001
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Figure imgf000346_0001
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Figure imgf000347_0001
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Figure imgf000348_0001
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Figure imgf000349_0001
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Figure imgf000350_0001
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Figure imgf000351_0001
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Figure imgf000355_0001
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Figure imgf000360_0001
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Figure imgf000361_0001
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Figure imgf000364_0001
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Figure imgf000365_0001
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Figure imgf000366_0001
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Figure imgf000367_0001
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Figure imgf000368_0001
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Figure imgf000381_0001
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Figure imgf000382_0001
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Figure imgf000383_0001
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Figure imgf000384_0001
Append] ixC:Table7
Figure imgf000385_0001
Append] ixC:Table7
Figure imgf000386_0001
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Figure imgf000387_0001
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Figure imgf000388_0001
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Figure imgf000389_0001
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Figure imgf000395_0001
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Figure imgf000397_0001
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Figure imgf000398_0001
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Figure imgf000399_0001
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Figure imgf000400_0001

Claims

CLAIMS What is claimed is:
1. A single stranded oligonucleotide having a sequence 5 '-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length, wherein the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene.
2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
3. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
4. The single stranded oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide is 8 to 30 nucleotides in length.
5. The single stranded oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
6. The single stranded oligonucleotide of any one of claims 1 to 5, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.
7. The single stranded oligonucleotide of claim 7, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
8. The single stranded oligonucleotide of any one of claims 1 to 7, wherein at least one nucleotide of the oligonucleotide comprises a 2' O-methyl.
9. The single stranded oligonucleotide of any one of claims 1 to 9, wherein each nucleotide of the oligonucleotide comprises a 2' O-methyl.
10. The single stranded oligonucleotide of any one of claims 1 to 9, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
11. The single strand oligonucleotide of claim 10, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
12. The single stranded oligonucleotide of any one of claims 1 to 6, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.
13. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro- deoxyribonucleotides.
14. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides.
15. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
16. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.
17. The single stranded oligonucleotide of any one of claims 13 to 16, wherein the 5' nucleotide of the oligonucleotide is a deoxyribonucleotide.
18. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides.
19. The single stranded oligonucleotide of claim 19, wherein the 5' nucleotide of the oligonucleotide is a LNA nucleotide.
20. The single stranded oligonucleotide of any one of claims 1 to 8, wherein the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one
LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
21. The single stranded oligonucleotide of any one of claims 1 to 20, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.
22. The single stranded oligonucleotide of claim 21, further comprising phosphorothioate internucleotide linkages between all nucleotides.
23. The single stranded oligonucleotide of any one of claims 1 to 22, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group.
24. The single stranded oligonucleotide of any one of claims 1 to 22, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' thiophosphate.
25. The single stranded oligonucleotide of any one of claims 1 to 24, further comprising a biotin moiety conjugated to the 5' nucleotide.
26. A single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an
SMN gene, wherein the oligonucleotide has at least one of: a) a sequence that is 5'X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5 ' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microR A, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine
nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that are between 50 kilobases upstream of a 5 '- end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and/or e) a sequence that has greater than 60% G-C content.
27. The single stranded oligonucleotide of claim 26, wherein the oligonucleotide has the sequence 5'X-Y-Z and wherein the oligonucleotide is 8-50 nucleotides in length.
28. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 27 and a carrier.
29. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 27 in a buffered solution.
30. A composition of claim 29, wherein the oligonucleotide is conjugated to the carrier.
31. The composition of claim 30, wherein the carrier is a peptide.
32. The composition of claim 30, wherein the carrier is a steroid.
33. A pharmaceutical composition comprising a composition of any one of claims 28 to 32 and a pharmaceutically acceptable carrier.
34. A kit comprising a container housing the composition of any one of claims 28 to 33.
35. A method of increasing expression of SMNl or SMN2 in a cell, the method comprising delivering the single stranded oligonucleotide of any one of claims 1 to 28 into the cell.
36. The method of claim 35, wherein delivery of the single stranded
oligonucleotide into the cell results in a level of expression of SMNl or SMN2 that is at least 50% greater than a level of expression of SMNl or SMN2 in a control cell that does not comprise the single stranded oligonucleotide.
37. A method increasing levels of SMNl or SMN2 in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 27 to the subject.
38. A method of treating a condition associated with decreased levels of SMNl or SMN2 in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 27 to the subject.
39. The method of claim 38, wherein the condition is Spinal muscular atrophy.
40. A method of increasing expression of SMN protein in a cell, the method comprising: delivering to the cell a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mR A of SMN2, in amounts sufficient to increase expression of a mature mRNA of SMN2 that comprises exon 7 in the cell.
41. The method of claim 40, wherein the splice control sequence resides in an exon of SMN2.
42. The method of claim 41, wherein the exon is exon 7 or exon 8.
43. The method of claim 40, wherein the splice control sequence traverses an intron-exon junction of SMN2.
44. The method of claim 43, wherein the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction.
45. The method of claim 40, wherein the splice control sequence resides in an intron of SMN2.
46. The method of claim 45, wherein the intron is intron 6 or intron 7.
47. The method of any one of claims 40 to 46, wherein the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
48. The method of any one of claims 40 to 47, wherein the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
49. The method of any one of claims 40 to 47, wherein the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
50. The method of any one of claims 40 to 49, wherein the first single stranded oligonucleotide is 8 to 30 nucleotides in length.
51. The method of any one of claims 40 to 50, wherein the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
52. The method of any one of claims 40 to 51 , wherein the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell simultaneously.
53. The method of any one of claims 40 to 52, wherein the cell is in a subject and the step of delivering to the cell comprises administering the first single stranded oligonucleotide and the second single stranded oligonucleotide to the subject as a co- formulation.
54. The method of any one of claims 40 to 53, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.
55. The method of claim 54, wherein the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond.
56. The method of claim 54 or 55, wherein the linker comprises an
oligonucleotide, optionally wherein the oligonucleotide comprises 1 to 10 thymidines or uridines.
57. The method of any one of claims 54 to 56, wherein the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides.
58. The method of any one of claims 40 to 53, wherein the first single stranded oligonucleotide is not covalently linked to the second single stranded oligonucleotide.
59. The method of any one of claims 40 to 51 and 58, wherein the first single stranded oligonucleotide and the second single stranded oligonucleotide are delivered to the cell separately.
60. The method of any one of claims 40 to 59, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a nucleotide analogue.
61. The method of claim 60, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
62. The method of any one of claims 40 to 61, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide comprises a 2' O-methyl.
63. The method of any one of claims 40 to 62, wherein each nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide comprises a 2' O- methyl.
64. The method of any one of claims 40 to 62, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide comprises at least one
ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
65. The method of claim 64, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
66. The method of any one of claims 40 to 60, wherein each nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a LNA nucleotide.
67. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and 2'-f uoro-deoxyribonucleotides.
68. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and 2 '-O-methyl nucleotides.
69. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
70. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.
71. The method of any one of claims 67 to 70, wherein the 5 ' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a deoxyribonucleotide.
72. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides.
73. The method of claim 72, wherein the 5 ' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a LNA nucleotide.
74. The method of any one of claims 40 to 60, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5 ' and 3 ' ends of the deoxyribonucleotides.
75. The method of any one of claims 40 to 74, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide further comprises phosphorothioate internucleotide linkages between at least two nucleotides.
76. The method of claim 75, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide further comprises phosphorothioate internucleotide linkages between all nucleotides.
77. The method of any one of claims 40 to 76, wherein the nucleotide at the 3 ' position of the first single stranded oligonucleotide or second single stranded oligonucleotide has a 3' hydroxyl group.
78. The method of any one of claims 40 to 76, wherein the nucleotide at the 3 ' position of the first single stranded oligonucleotide or second single stranded oligonucleotide has a 3' thiophosphate.
79. The method of any one of claims 40 to 78, a biotin moiety is conjugated to the 5 ' or 3 ' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide.
80. A method of treating spinal muscular atrophy in a subject, the method comprising: administering to the subject a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2 and a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2, in amounts sufficient to increase expression of SMN protein in the subject.
81. A composition comprising:
a first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of SMN2, and a second single stranded
oligonucleotide complementary to a splice control sequence of a precursor mRNA of SMN2.
82. The composition of claim 81, wherein the splice control sequence resides in an exon of SMN2.
83. The composition of claim 82, wherein the exon is exon 7 or exon 8.
84. The composition of claim 81, wherein the splice control sequence traverses an intron-exon junction of SMN2.
85. The composition of claim 84, wherein the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction.
86. The composition of claim 81, wherein the splice control sequence resides in an intron of SMN2.
87. The composition of claim 86, wherein the intron is intron 6 or intron 7.
88. The composition of any one of claims 81 to 87, wherein the splice control sequence comprises at least one hnRNAP binding sequence.
89. The composition of any one of claims 81 to 88, wherein the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
90. The composition of any one of claims 81 to 89, wherein the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
91. The composition of any one of claims 81 to 90, wherein the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
92. The composition of any one of claims 81 to 91, wherein the first single stranded oligonucleotide is 8 to 30 nucleotides in length.
93. The composition of any one of claims 81 to 92, wherein the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
94. The composition of any one of claims 81 to 93, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.
95. The composition of claim 94, wherein the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond.
96. The composition of claim 94 or 95, wherein the linker comprises an oligonucleotide, optionally wherein the oligonucleotide comprises 1 to 10 thymidines or uridines.
97. The composition of any one of claims 94 to 96, wherein the linker is more susceptible to cleavage in a mammalian extract than the first and second single stranded oligonucleotides.
98. The composition of any one of claims 81 to 93, wherein the first single stranded oligonucleotide is not covalently linked to the second single stranded
oligonucleotide.
99. The composition of any one of claims 81 to 98 further comprising a carrier.
100. The composition of claim 99, wherein the carrier is a pharmaceutically acceptable carrier.
101. The composition of any one of claims 81 to 100, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded
oligonucleotide is a nucleotide analogue.
102. The composition of claim 101, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
103. The composition of any one of claims 81 to 101, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded
oligonucleotide comprises a 2' O-methyl.
104. The composition of any one of claims 81 to 103, wherein each nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide comprises a 2' O-methyl.
105. The composition of any one of claims 81 to 103, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
106. The composition of claim 105, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
107. The composition of any one of claims 81 to 101, wherein each nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a LNA nucleotide.
108. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
109. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and 2 '-O-methyl nucleotides.
110. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
111. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.
112. The composition of any one of claims 108 to 111, wherein the 5' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a deoxyribonucleotide.
113. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise alternating LNA nucleotides and 2'-0-methyl nucleotides.
114. The composition of claim 113, wherein the 5' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a LNA nucleotide.
115. The composition of any one of claims 81 to 101, wherein the nucleotides of the first single stranded oligonucleotide or second single stranded oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5 ' and 3 ' ends of the deoxyribonucleotides.
116. The composition of any one of claims 81 to 115, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide further comprises phosphorothioate internucleotide linkages between at least two nucleotides.
117. The composition of claim 115 or 116, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide further comprises phosphorothioate internucleotide linkages between all nucleotides.
118. The composition of any one of claims 81 to 117, wherein the nucleotide at the 3 ' position of the first single stranded oligonucleotide or second single stranded
oligonucleotide has a 3' hydroxyl group.
119. The composition of any one of claims 81 to 118, wherein the nucleotide at the 3 ' position of the first single stranded oligonucleotide or second single stranded
oligonucleotide has a 3' thiophosphate.
120.. The composition of any one of claims 81 to 119, further comprising a biotin moiety conjugated to the 5 ' or 3 ' nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide.
121. A compound comprising the general formula A-B-C, wherein A is a single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene, B is a linker, and C is a single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
122. The compound of claim 121, wherein B comprises an oligonucleotide, peptide, low pH labile bond, or disulfide bond.
123. The compound of claim 121 or 122, wherein the splice control sequence resides in an exon of the gene.
124. The compound of claim 121 or 122, wherein the splice control sequence traverses an intron-exon junction of the gene.
125. The compound of claim 121 or 122, wherein the splice control sequence resides in an intron of the gene.
126. The compound of any one of claims 121 to 125, wherein the splice control sequence comprises at least one hnRNAP binding sequence.
127. The compound of any one of claims 121 to 126, wherein hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
128. The compound of any one of claims 121 to 126, wherein hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
129. The compound of any one of claims 121 to 128, wherein the gene is SMN2.
130. The compound of claim 129, wherein the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8.
131. The compound of any one of claims 121 to 130, wherein A has a sequence 5'- X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
132. The compound of any one of claims 121 to 131, wherein A does not comprise three or more consecutive guanosine nucleotides.
133. The compound of any one of claims 121 to 132, wherein A does not comprise four or more consecutive guanosine nucleotides.
134. The compound of any one of claims 121 to 133, wherein A is 8 to 30 nucleotides in length.
135. The compound of any one of claims 121 to 134, wherein A is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
136. The compound of any one of claims 121 to 135, wherein the B is an oligonucleotide comprising 1 to 10 thymidines or uridines.
137. The compound of any one of claims 121 to 136, wherein B is more susceptible to cleavage in a mammalian extract than A and C.
138. The compound of any one of claims 121 to 137, wherein at least one nucleotide of the compound is a nucleotide analogue.
139. The compound of any one of claims 121 to 138, wherein at least one nucleotide of the compound comprises a 2' O-methyl.
140. The compound of any one of claims 121 to 139, wherein each nucleotide of the compound comprises a 2' O-methyl.
141. The compound of any one of claims 121 to 140, wherein the compound comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
142.. The compound of claim 141, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
143. A kit comprising :
a first container housing first single stranded oligonucleotide complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a gene; and
a second container housing a second single stranded oligonucleotide complementary to a splice control sequence of a precursor mRNA of the gene.
144. The kit of claim 143, wherein the splice control sequence resides in an exon of the gene.
145. The kit of claim 143, wherein the splice control sequence traverses an intron- exon junction of the gene.
146. The kit of claim 145, wherein the splice control sequence resides in an intron of the gene.
147. The kit of any one of claims 143 to 146, wherein the splice control sequence comprises at least one hnR AP binding sequence.
148. The kit of any one of claims 143 to 147, wherein hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in inclusion of a particular exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
149. The kit of any one of claims 143 to 149, wherein hybridization of an oligonucleotide having the sequence of C with the splice control sequence of the precursor mRNA in a cell results in exclusion of a particular intron or exon in a mature mRNA that arises from processing of the precursor mRNA in the cell.
150. The kit of any one of claims 143 to 149, wherein the gene is SMN2.
151. The kit of claim 150, wherein the splice control sequence resides in intron 6, intron 7, exon 7, exon 8 or at the junction of intron 7 and exon 8.
152. The kit of any one of claims 143 to 151, wherein the first single stranded oligonucleotide has a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length.
153. The kit of any one of claims 143 to 152, wherein the first single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
154. The kit of any one of claims 143 to 153, wherein the first single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
155. The kit of any one of claims 143 to 154, wherein the first single stranded oligonucleotide is 8 to 30 nucleotides in length.
156. The kit of any one of claims 143 to 155, wherein the first single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
157. The kit of any one of claims 143 to 156, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide is a nucleotide analogue.
158. The kit of any one of claims 143 to 157, wherein at least one nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide comprises a 2' O-methyl.
159. The kit of any one of claims 143 to 158, wherein each nucleotide of the first single stranded oligonucleotide or second single stranded oligonucleotide comprises a 2' O- methyl.
160. The kit of any one of claims 143 to 159, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide comprises at least one
ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
161. The kit of claim 160, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
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