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`US 20130109091Al
`
`c19) United States
`c12) Patent Application Publication
`Baker et al.
`
`c10) Pub. No.: US 2013/0109091 Al
`May 2, 2013
`(43) Pub. Date:
`
`(54) COMPOSITIONS AND METHODS FOR
`MODULATION OF SMN2 SPLICING
`
`(60) Provisional application No. 60/693,542, filed on Jun.
`23, 2005.
`
`(71) Applicants:Isis Pharmaceuticals, Inc., Carlsbad,
`CA (US); Cold Spring Harbor
`Laboratory, Cold Spring Harbor, CA
`(US)
`
`(72)
`
`Inventors: Brenda F. Baker, Carlsbad, CA (US);
`Adrian R. Krainer, Huntington Station,
`NY (US); Yimin Hua, Jericho, NY (US)
`
`(73) Assignees: Cold Spring Harbor Laboratory, Cold
`Spring Harbor, CA (US); Isis
`Pharmaceuticals, Inc., Carlsbad, CA
`(US)
`(21) Appl. No.: 13/720,474
`Dec. 19, 2012
`Filed:
`(22)
`Related U.S. Application Data
`
`(63)
`
`Continuation of application No. 11/993,609, filed on
`May 6, 2010, now Pat. No. 8,361,977, filed as appli(cid:173)
`cation No. PCT/US06/24469 on Jun. 23, 2006.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12N 151113
`(52) U.S. Cl.
`CPC .................................... C12N 151113 (2013.01)
`USPC .......................................... 435/375; 536/24.5
`
`(2006.01)
`
`(57)
`
`ABSTRACT
`
`Disclosed herein are compounds, compositions and methods
`for modulating splicing of SMN2 mRNA in a cell, tissue or
`animal. Also provided are uses of disclosed compounds and
`compositions in the manufacture of a medicament for treat(cid:173)
`ment of diseases and disorders, including spinal muscular
`atrophy.
`
`

`

`US 2013/0109091 Al
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`May 2, 2013
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`1
`
`COMPOSITIONS AND METHODS FOR
`MODULATION OF SMN2 SPLICING
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is a continuation of U.S. patent
`application Ser. No. 11/993,609, filed May 6, 2010, which is
`the US National Phase application under 35 U.S.C. 371 of
`PCT Application Number PCT/US2006/024469, filed Jun.
`23, 2006, which claims priority to U.S. Provisional Patent
`Application Ser. No. 60/693,542, filed Jun. 23, 2005, each of
`which is incorporated by reference herein in its entirety.
`
`INCORPORATION OF SEQUENCE LISTING
`
`[0002] The present application is being filed along with a
`Sequence Listing in electronic format. The Sequence Listing
`is provided as a file entitled CORE0058USC1SEQ.txt, cre(cid:173)
`ated on Oct. 25, 2012 which is 24 Kb in size. The information
`in the electronic format of the sequence listing is incorporated
`herein by reference in its entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Newly synthesized eukaryotic mRNA molecules,
`also known as primary transcripts or pre-mRNA, made in the
`nucleus, are processed before or during transport to the cyto(cid:173)
`plasm for translation. Processing of the pre-mRNAs includes
`addition ofa 5' methylated cap and an approximately 200-250
`base poly(A) tail to the 3' end of the transcript.
`[0004] The next step in mRNA processing is splicing of the
`pre-mRNA, which occurs in the maturation of 90-95% of
`mammalian mRNAs. Intrans ( or intervening sequences) are
`regions of a primary transcript ( or the DNA encoding it) that
`are not included in the coding sequence of the mature mRNA.
`Exons are regions of a primary transcript that remain in the
`mature mRNA when it reaches the cytoplasm. The exons are
`spliced together to form the mature mRNA sequence. Splice
`junctions are also referred to as splice sites with the 5' side of
`the junction often called the "5' splice site," or "splice donor
`site" and the 3' side the "3' splice site" or "splice acceptor
`site." In splicing, the 3' end of an upstream exon is joined to
`the 5' end of the downstream exon. Thus the unspliced RNA
`( or pre-mRNA) has an exon/intronjunction at the 5' end ofan
`intron and an intron/exon junction at the 3' end of an intron.
`After the intron is removed, the exons are contiguous at what
`is sometimes referred to as the exon/ exon junction or bound(cid:173)
`ary in the mature mRNA. Cryptic splice sites are those which
`are less often used but may be used when the usual splice site
`is blocked or unavailable. Alternative splicing, defined as the
`splicing together of different combinations of exons, often
`results in multiple mRNA transcripts from a single gene.
`[0005] Up to 50% of human genetic diseases resulting from
`a point mutation are caused by aberrant splicing. Such point
`mutations can either disrupt a current splice site or create a
`new splice site, resulting in mRNA transcripts comprised of a
`different combination of exons or with deletions in exons.
`Point mutations also can result in activation of a cryptic splice
`site or disrupt regulatory cis elements (i.e. splicing enhancers
`or silencers) (Cartegni et al., Nat. Rev. Genet., 2002, 3, 285-
`298; Drawczak et al., Hum. Genet., 1992, 90, 41-54).
`[0006] Antisense oligonucleotides have been used to target
`mutations that lead to aberrant splicing in several genetic
`diseases in order to redirect splicing to give a desired splice
`product (Kole, Acta Biochimica Polonica, 1997, 44, 231-
`
`238). Such diseases include ~-thalassemia (Dominski and
`Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677;
`Sierakowska et al., Nucleosides & Nucleotides, 1997, 16,
`1173-1182; Sierakowska et al., Proc. Natl. Acad. Sci. USA,
`1996, 93, 12840-44; Lacerra et al., Proc. Natl. Acad. Sci.
`USA, 2000, 97, 9591-9596); dystrophinKobe (Takeshima et
`al., J. Clin. Invest., 1995, 95, 515-520); Duchenne muscular
`dystrophy (Dunckley et al. Nucleosides & Nucleotides, 1997,
`16, 1665-1668; Dunckley et al. Human Mal. Genetics, 1998,
`5, 1083-90); osteogenesis imperfecta (Wang and Marini, J.
`Clin Invest., 1996, 97, 448-454); and cystic fibrosis (Fried(cid:173)
`man et al., J. Biol. Chem., 1999, 274, 36193-36199).
`[0007] Antisense compounds have also been used to alter
`the ratio of the long and short forms ofBcl-x pre-mRNA (U.S.
`Pat. No. 6,172,216; U.S. Pat. No. 6,214,986; Taylor et al.,
`Nat. Biotechnol. 1999, 17, 1097-1100) orto force skipping of
`specific exons containing premature termination codons
`(Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338). U.S.
`Pat. No. 5,627,274 and WO 94/26887 disclose compositions
`and methods for combating aberrant splicing in a pre-mRNA
`molecule containing a mutation using antisense oligonucle(cid:173)
`otides which do not activate RNAse H.
`[0008] Proximal spinal muscular atrophy (SMA) is a
`genetic, neurodegenerative disorder characterized by the loss
`of spinal motor neurons. SMA is an autosomal recessive
`disease of early onset and is currently the leading cause of
`death among infants. The severity of SMA varies among
`patients and has thus been classified into three types. Type I
`SMA is the most severe form with onset at birth or within 6
`months and typically results in death within 2 years. Children
`with type I SMA are unable to sit or walk. Type II SMA is the
`intermediate form and patients are able to sit, but cannot stand
`or walk. Patients with type III SMA, a chronic form of the
`disease, typically develop SMA after 18 months of age (Lefe(cid:173)
`bvre et al., Hum. Mo!. Genet., 1998, 7, 1531-1536).
`[0009] SMA is caused by the loss of both copies of survival
`of motor neuron 1 (SMNl ), a protein that is part of a multi(cid:173)
`protein complex thought to be involved in snRNP biogenesis
`and recycling. A nearly identical gene, SMN2, exists in a
`duplicated region on chromosome 5q13. Although SMNl
`and SMN2 have the potential to code for the same protein,
`SMN2 contains a translationally silent mutation at position
`+6 of exon 7, which results in inefficient inclusion of exon 7
`in SMN2 transcripts. Thus, the predominant form ofSMN2 is
`a truncated version, lacking exon 7, which is unstable and
`inactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-
`384).
`[0010] Chimeric peptide nucleic acid molecules designed
`to modulate splicing of SMN2 have been described (WO
`02/38738; Cartegni and Krainer, Nat. Struct. Biol., 2003, 10,
`120-125).
`[0011] Antisense technology is an effective means for
`modulating the expression of one or more specific gene prod(cid:173)
`ucts, including alternative splice products, and is uniquely
`useful in a number of therapeutic, diagnostic, and research
`applications. The principle behind antisense technology is
`that an antisense compound, which hybridizes to a target
`nucleic acid, modulates gene expression activities such as
`transcription, splicing or translation through one of a number
`of antisense mechanisms. The sequence specificity of anti(cid:173)
`sense compounds makes them extremely attractive as tools
`for target validation and gene functionalization, as well as
`therapeutics to selectively modulate the expression of genes
`involved in disease.
`
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`[0012] Disclosed herein are antisense compounds useful
`for modulating gene expression and associated pathways via
`antisense mechanisms, which may include antisense mecha(cid:173)
`nisms based on target occupancy. Provided herein are anti(cid:173)
`sense compounds targeting SMN2 for use in modulation of
`SMN2 splicing. One having skill in the art, once armed with
`this disclosure will be able, without undue experimentation,
`to identify, prepare and exploit antisense compounds for these
`uses.
`
`SUMMARY OF THE INVENTION
`
`[0013] The present invention is directed to antisense com(cid:173)
`pounds targeted to and hybridizable with a nucleic acid mol(cid:173)
`ecule encoding SMN2. Provided are antisense compounds
`targeted to intron, 6, exon 7 or intron 7 of SMN2 which
`modulate splicing of SMN2 pre-mRNAs. In one embodi(cid:173)
`ment, modulation of splicing results in an increase in exon 7
`inclusion. In another embodiment, modulation of splicing
`results in a decrease in exon 7 inclusion. Contemplated and
`provided herein are antisense compounds 12 to 20 nucle(cid:173)
`otides in length targeted to intron 6, exon 7 or intron 7 of
`SMN2, wherein the compounds comprise 2'-O-methoxyethyl
`sugar modifications.
`[0014]
`In one aspect of the invention, the antisense com(cid:173)
`pounds are targeted to cis splicing regulatory elements. Regu(cid:173)
`latory elements include exonic splicing enhancers, exonic
`splicing silencers, intronic splicing enhancers and intronic
`splicing silencers. Exonic and intronic splicing silencers are
`preferred targets.
`[0015]
`In one embodiment, the antisense compounds com(cid:173)
`prise at least an 8-nucleobase portion of one of the exemplary
`compounds provided herein.
`[0016] Also provided are methods for modulating splicing
`of SMN2 mRNA in a cell, tissue or organ using one or more
`of the compounds of the invention. In one embodiment,
`modulation of splicing is exon inclusion. In another embodi(cid:173)
`ment, modulation of splicing is exon skipping. In one aspect,
`the compound is targeted to an intronic splicing silencer
`element. In another aspect, the compound is targeted to an
`exonic splicing silencer element.
`[0017] Further provided are antisense compounds 10 to 50,
`12 to 30 or 12 to 20 nucleotides in length targeted to intron 6,
`exon 7 or intron 7 of SMN2 comprising 2'-O-methoxyethyl
`sugar modifications for use in therapy. Also provided are
`pharmaceutical compositions comprising one or more of the
`compounds of the invention. Use of an antisense oligonucle(cid:173)
`otide provided herein for the preparation of a medicament for
`modulating splicing of an SMN2 pre-mRNA is also provided.
`In one aspect, modulation of splicing results in an increase in
`exon 7 inclusion. Use of an antisense oligonucleotide pro(cid:173)
`vided herein for the preparation of a medicament for the
`treatment of spinal muscular atrophy is further provided.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0018] Antisense technology is an effective means for
`modulating the expression of one or more specific gene prod(cid:173)
`ucts and is uniquely useful in a number of therapeutic, diag(cid:173)
`nostic, and research applications. Provided herein are anti(cid:173)
`sense compounds useful for modulating gene expression via
`antisense mechanisms of action, including antisense mecha(cid:173)
`nisms based on target occupancy. In one aspect, the antisense
`compounds provided herein modulate splicing of a target
`gene. Such modulation includes promoting or inhibiting exon
`
`inclusion. Further provided herein are antisense compounds
`targeted to cis splicing regulatory elements present in pre(cid:173)
`mRNA molecules, including exonic splicing enhancers,
`exonic splicing silencers, intronic splicing enhancers and
`intronic splicing silencers. Disruption of cis splicing regula(cid:173)
`tory elements is thought to alter splice site selection, which
`may lead to an alteration in the composition of splice prod(cid:173)
`ucts.
`[0019] Processing of eukaryotic pre-mRNAs is a complex
`process that requires a multitude of signals and protein factors
`to achieve appropriate mRNA splicing. Exon definition by the
`spliceosome requires more than the canonical splicing signals
`which define intron-exon boundaries. One such additional
`signal is provided by cis-acting regulatory enhancer and
`silencer sequences. Exonic splicing enhancers (ESE), exonic
`splicing silencers (ESS), intronic splicing enhancers (ISE)
`and intron splicing silencers (ISS) have been identified which
`either repress or enhance usage of splice donor sites or splice
`acceptor sites, depending on their site and mode of action
`(Yeo eta!. 2004,Proc. Natl.Acad. Sci. U.S.A. 101(44):15700-
`15705). Binding of specific proteins (trans factors) to these
`regulatory sequences directs the splicing process, either pro(cid:173)
`moting or inhibiting usage of particular splice sites and thus
`modulating the ratio of splicing products (Scamborova et al.
`2004, Mal. Cell. Biol. 24(5):1855-1869; Hovhannisyan and
`Carstens, 2005, Mal. Cell. Biol. 25(1):250-263; Minovitsky
`et al. 2005, Nucleic Acids Res. 33(2):714-724). Little is
`known about the trans factors that interact with intronic splic(cid:173)
`ing elements; however, several studies have provided infor(cid:173)
`mation on exonic splicing elements. For example, ESEs are
`known to be involved in both alternative and constitutive
`splicing by acting as binding sites for members of the SR
`protein family. SR proteins bind to splicing elements via their
`RNA-binding domain and promote splicing by recruiting
`spliceosomal components with protein-protein interactions
`mediated by their RS domain, which is comprised of several
`Arg-Ser dipeptides (Cartegni and Krainer, 2003, Nat. Struct.
`Biol. 10(2):120-125; Wang et al. 2005, Nucleic Acids Res.
`33(16):5053-5062). ESEs have been found to be enriched in
`regions of exons that are close to splice sites, particularly 80
`to 120 bases from the ends of splice acceptor sites (Wu et al.
`2005, Genomics 86:329-336). Consensus sequences have
`been determined for four members of the SR protein family,
`SF2/ASF, SC35, SRp40 and SRp55 (Cartegni et al. 2003,
`Nucleic Acids Res. 31(13):3568-3571).
`[0020] Although the trans factors that bind intronic splicing
`regulatory elements have not been extensively studied, SR
`proteins and heterogeneous ribonucleoproteins (hnRNPs)
`have both been suggested to interact with these elements (Yeo
`et al. 2004, Proc. Natl. Acad. Sci. U.S.A. 101(44):15700-
`15705). Two intronic splicing enhancer elements (IS Es) have
`been identified in SMN2, one in intron 6 and the other in
`intron 7 (Miyajima et al. 2002, J. Biol. Chem. 22:23271-
`23277). Gel shift assays using the ISE in intron 7 showed
`formation of RNA-protein complexes, which suggests these
`trans proteins may be important for regulation of splicing
`(Miyaso et al. 2003, J. Biol. Chem. 278(18):15825-15831).
`[0021] The role of SMN2 in diseases such as spinal mus(cid:173)
`cular atrophy (SMA) makes it an important therapeutic target.
`SMA is a genetic disorder characterized by degeneration of
`spinal motor neurons. SMA is caused by the loss of both
`functional copies of SMNl. However, SMN2 has the poten(cid:173)
`tial to code for the same protein as SMNl and thus overcome
`the genetic defect of SMA patients. SMN2 contains a trans-
`
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`US 2013/0109091 Al
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`3
`
`lationally silent mutation (C-T) at position +6 of exon 7
`(nucleotide 66 of SEQ ID NO: 1), which results in inefficient
`inclusion of exon 7 in SMN2 transcripts. Therefore, the pre(cid:173)
`dominant form of SMN2, one which lacks exon 7, is unstable
`and inactive. Thus, therapeutic compounds capable of modu(cid:173)
`lating SMN2 splicing such that the percentage of SMN2
`transcripts containing exon 7 is increased, would be useful for
`the treatment of SMA.
`
`Overview
`
`[0022] Disclosed herein are oligomeric compounds,
`including antisense oligonucleotides and other antisense
`compounds for use in modulating the expression of nucleic
`acid molecules encoding SMN2. This is accomplished by
`providing oligomeric compounds which hybridize with one
`or more target nucleic acid molecules encoding SMN2. As
`used herein, the terms "target nucleic acid" and "nucleic acid
`molecule encoding SMN2" have been used for convenience
`to encompass DNA encoding SMN2, RNA (including pre(cid:173)
`mRNA and mRNA or portions thereof) transcribed from such
`DNA, and also cDNA derived from such RNA.
`[0023] Provided herein are anti sense compounds for use in
`modulation of SMN2 pre-mRNA splicing. In one embodi(cid:173)
`ment, the disclosed antisense compounds are targeted to exon
`7 of SMN2 such that SMN mRNA splicing is modulated. In
`another embodiment, the anti sense compounds are targeted to
`intron 6 of SMN2. In another embodiment, the antisense
`compounds are targeted to intron 7 of SMN2. Modulation of
`splicing may result in exon 7 inclusion or exon 7 skipping.
`[0024] Also provided are antisense compounds targeted to
`cis regulatory elements. In one embodiment, the regulatory
`element is in an exon. In another embodiment, the regulatory
`element is an in intron.
`
`Modulation of Splicing
`
`[0025] As used herein, modulation of splicing refers to
`altering the processing of a pre-mRNA transcript such that the
`spliced mRNA molecule contains either a different combina(cid:173)
`tion of exons as a result of exon skipping or exon inclusion, a
`deletion in one or more exons, or additional sequence not
`normally found in the spliced mRNA ( e.g., intron sequence).
`In the context of the present invention, modulation of splicing
`refers to altering splicing of SMN2 pre-mRNA to achieve
`exon skipping or exon inclusion. In one embodiment, exon
`skipping results in an SMN2 mRNA transcript lacking exon 7
`and exon inclusion results in an SMN2 mRNA transcript
`containing exon 7.
`[0026] As used herein, alternative splicing is defined as the
`splicing together of different combinations of exons, which
`may result in multiple mRNA transcripts from a single gene.
`In the context of the present invention, an SMN2 mRNA
`transcript containing exon 7 and an SMN2 mRNA transcript
`lacking exon 7 are two products of alternative splicing.
`
`Compounds
`
`[0027] The term "oligomeric compound" refers to a poly(cid:173)
`meric structure capable ofhybridizing to a region of a nucleic
`acid molecule. This term includes oligonucleotides, oligo(cid:173)
`nucleosides, oligonucleotide
`analogs, oligonucleotide
`mimetics and chimeric combinations of these. An "antisense
`compound" or "anti sense oligomeric compound" refers to an
`oligomeric compound that is at least partially complementary
`to the region of a nucleic acid molecule to which it hybridizes
`
`and which modulates its expression. Consequently, while all
`antisense compounds can be said to be oligomeric com(cid:173)
`pounds, not all oligomeric compounds are antisense com(cid:173)
`pounds. An "antisense oligonucleotide" is an antisense com(cid:173)
`pound that is a nucleic acid-based oligomer. An antisense
`oligonucleotide can be chemically modified. Nonlimiting
`examples of oligomeric compounds include primers, probes,
`antisense compounds, antisense oligonucleotides, external
`guide sequence (EGS) oligonucleotides, alternate splicers,
`and siRNAs. As such, these compounds can be introduced in
`the form of single-stranded, double-stranded, circular,
`branched or hairpins and can contain structural elements such
`as internal or terminal bulges or loops. Oligomeric double(cid:173)
`stranded compounds can be two strands hybridized to form
`double-stranded compounds or a single strand with sufficient
`self complementarity to allow for hybridization and forma(cid:173)
`tion of a fully or partially double-stranded compound.
`[0028] The oligomeric compounds in accordance with this
`invention may comprise a complementary oligomeric com(cid:173)
`pound from about 10 to about 50 nucleobases (i.e. from about
`10 to about 50 linked nucleosides ). One having ordinary skill
`in the art will appreciate that this embodies antisense com(cid:173)
`pounds of 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 nucleobases.
`In one embodiment, the antisense compounds of the
`[0029]
`invention are 12 to 30 nucleobases. One having ordinary skill
`in the art will appreciate that this embodies antisense com(cid:173)
`pounds of 12, 13,14,15,16, 17,18,19,20,21,22,23,24,25,
`26, 27, 28, 29 or 30 nucleobases.
`In one embodiment, the antisense compounds of the
`[0030]
`invention are 12 to 20 nucleobases. One having ordinary skill
`in the art will appreciate that this embodies antisense com(cid:173)
`pounds of 12, 13, 14, 15, 16, 17, 18, 19or20nucleobases.
`In one embodiment, the antisense compounds of the
`[0031]
`invention have antisense portions of 20 nucleobases.
`In one embodiment, the antisense compounds of the
`[0032]
`invention have antisense portions of 18 nucleobases.
`In one embodiment, the antisense compounds of the
`[0033]
`invention have antisense portions of 15 nucleobases.
`In one embodiment, the antisense compounds of the
`[0034]
`invention have antisense portions of 12 nucleobases.
`[0035] Anti sense compounds 10-50 nucleobases in length
`comprising a stretch of at least eight (8) consecutive nucleo(cid:173)
`bases selected from within the illustrative antisense com(cid:173)
`pounds are considered to be suitable antisense compounds as
`well.
`[0036] Compounds of the invention include oligonucle(cid:173)
`otide sequences that comprise at least the 8 consecutive
`nucleobases from the 5'-terminus of one of the illustrative
`antisense compounds (the remaining nucleobases being a
`consecutive stretch of nucleobases continuing upstream of
`the 5'-terminus of the antisense compound until the oligo(cid:173)
`nucleotide contains about 10 to about 50 nucleobases ). Other
`compounds are represented by oligonucleotide sequences
`that comprise at least the 8 consecutive nucleobases from the
`3'-terminus of one of the illustrative antisense compounds
`(the remaining nucleobases being a consecutive stretch of
`nucleobases continuing downstream of the 3'-terminus of the
`antisense compound and continuing until the oligonucleotide
`contains about 10 to about 50 nucleobases). It is also under(cid:173)
`stood that compounds may be represented by oligonucleotide
`sequences that comprise at least 8 consecutive nucleobases
`from an internal portion of the sequence of an illustrative
`
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`compound, and may extend in either or both directions until
`the oligonucleotide contains about 10 to about 50 nucleo(cid:173)
`bases. The compounds described herein are specifically
`hybridizable to the target nucleic acid.
`[0037] One having skill in the art armed with the antisense
`compounds illustrated herein will be able, without undue
`experimentation, to identify further antisense compounds.
`
`Hybridization
`
`[0038] As used herein, "hybridization" means the pairing
`of complementary strands of antisense compounds to their
`target sequence. While not limited to a particular mechanism,
`the most common mechanism of pairing involves hydrogen
`bonding, which may be Watson-Crick, Hoogsteen or reversed
`Hoogsteen hydrogen bonding, between complementary
`nucleoside or nucleotide bases (nucleobases). For example,
`the natural base adenine is complementary to the natural
`nucleobases thymidine and uracil which pair through the
`formation of hydrogen bonds. The natural base guanine is
`complementary to the natural bases cytosine and 5-methyl
`cytosine. Hybridization can occur under varying circum(cid:173)
`stances.
`[0039] An antisense compound is specifically hybridizable
`when there is a sufficient degree of complementarity to avoid
`non-specific binding of the antisense compound to non-target
`nucleic acid sequences under conditions in which specific
`binding is desired, i.e., under physiological conditions in the
`case of in vivo assays or therapeutic treatment, and under
`conditions in which assays are performed in the case of in
`vitro assays.
`[0040] As used herein, "stringent hybridization conditions"
`or "stringent conditions" refers to conditions under which an
`antisense compound will hybridize to its target sequence, but
`to a minimal number of other sequences. Stringent conditions
`are sequence-dependent and will be different in different
`circumstances, and "stringent conditions" under which anti(cid:173)
`sense compounds hybridize to a target sequence are deter(cid:173)
`mined by the nature and composition of the antisense com(cid:173)
`pounds and the assays in which they are being investigated.
`
`Complementarity
`
`[0041]
`"Complementarity," as used herein, refers to the
`capacity for precise pairing between two nucleobases on
`either two oligomeric compound strands or an antisense com(cid:173)
`pound with its target nucleic acid. For example, if a nucleo(cid:173)
`base at a certain position of an anti sense compound is capable
`ofhydrogen bonding with a nucleobase at a certain position of
`a target nucleic acid, then the position of hydrogen bonding
`between the oligonucleotide and the target nucleic acid is
`considered to be a complementary position.
`[0042]
`"Complementarity" can also be viewed in the con(cid:173)
`text of an anti sense compound and its target, rather than in a
`base by base manner. The anti sense compound and the further
`DNA or RNA are complementary to each other when a suf(cid:173)
`ficient number of complementary positions in each molecule
`are occupied by nucleobases which can hydrogen bond with
`each other. Thus, "specifically hybridizable" and "comple(cid:173)
`mentary" are terms which are used to indicate a sufficient
`degree of precise pairing or complementarity over a sufficient
`number ofnucleobases such that stable and specific binding
`occurs between the antisense compound and a target nucleic
`acid. One skilled in the art recognizes that the inclusion of
`mismatches is possible without eliminating the activity of the
`
`antisense compound. The invention is therefore directed to
`those anti sense compounds that may contain up to about 20%
`nucleotides that disrupt base pairing of the antisense com(cid:173)
`pound to the target. Preferably the compounds contain no
`more than about 15%, more preferably not more than about
`10%, most preferably not more than 5% or no mismatches.
`The remaining nucleotides do not disrupt hybridization ( e.g.,
`universal bases).
`[0043]
`It is understood in the art that incorporation of
`nucleotide affinity modifications may allow for a greater
`number of mismatches compared to an unmodified com(cid:173)
`pound. Similarly, certain oligonucleotide sequences may be
`more tolerant to mismatches than other oligonucleotide
`sequences. One of the skill in the art is capable of determining
`an appropriate number of mismatches between oligonucle(cid:173)
`otides, or between an oligonucleotide and a target nucleic
`acid, such as by determining melting temperature.
`
`Identity
`
`[0044] Antisense compounds, or a portion thereof, may
`have a defined percent identity to a SEQ ID NO, or a com(cid:173)
`pound having a specific Isis number. As used herein, a
`sequence is identical to the sequence disclosed herein if it has
`the same nucleobase pairing ability. For example, a RNA
`which contains uracil in place of thymidine in the disclosed
`sequences of the instant invention would be considered iden(cid:173)
`tical as they both pair with adenine. This identity may be over
`the entire length of the oligomeric compound, or in a portion
`of the antisense compound (e.g., nucleobases 1-20 of a
`27-mer may be compared to a 20-mer to determine percent
`identity of the oligomeric compound to the SEQ ID NO.) It is
`understood by those skilled in the art that an antisense com(cid:173)
`pound need not have an identical sequence to those described
`herein to function similarly to the antisense compound
`described herein. Shortened versions of anti sense compound
`taught herein, or non-identical versions of the antisense com(cid:173)
`pound taught herein fall within the scope of the invention.
`Non-identical versions are those wherein each base does not
`have the same pairing activity as the antisense compounds
`disclosed herein. Bases do not have the same pairing activity
`by being shorter or having at least one abasic site. Alterna(cid:173)
`tively, a non-identical version can include at least one base
`replaced with a different base with different pairing activity
`( e.g., G can be replaced by C, A, or T). Percent identity is
`calculated according to the number of bases that have identi(cid:173)
`cal base pairing corresponding to the SEQ ID NO or antisense
`compound to which it is being compared. The non-identical
`bases may be adjacent to each other, dispersed through out the
`oligonucleotide, or both.
`[0045] For example, a 16-mer having the same sequence as
`nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer.
`Alternatively, a 20-mer containing four nucleobases not iden(cid:173)
`tical to the 20-mer is also 80% identical to the 20-mer. A
`14-mer having the same sequence as nucleobases 1-14 of an
`18-mer is 78% identical to the 18-mer. Such calculations are
`well within the ability of those skilled in the art.
`[0046] The percent identity is based on the percent of
`nucleobases in the original sequence present in a portion of
`the modified sequence. Therefore, a 30 nucleobase antisense
`compound comprising the full sequence of the complement
`ofa 20 nucleobase active target segment would have a portion
`of 100% identity with the complement of the 20 nucleobase
`active target segment, while further comprising an additional
`10 nucleobase portion. In the context of the invention, the
`
`

`

`US 2013/0109091 Al
`
`May 2, 2013
`
`5
`
`complement of an active target segment may constitute a
`single portion. In a preferred embodiment, the oligonucle(cid:173)
`otides of the instant invention are at least about 80%, more
`preferably at least about 85%, even more preferably at least
`about 90%, most preferably at least 95% identical to at least
`a portion of the complement of the active target segments
`presented herein.
`It is well known by those skilled in the art that it is
`[0047]
`possible to increase or decrease the length of an antisense
`compound and/or introduce mismatch bases without elimi(cid:173)
`nating activity. For example, in Woolf et al. (Proc. Natl. Acad.
`Sci. USA 89:7305-7309, 1992, incorporated herein by refer(cid:173)
`ence), a series of ASOs 13-25 nucleobases in length were
`tested for their ability to induce cleavage of a target RNA.
`ASOs 25 nucleobases in length with 8 or 11 mismatch bases
`near the ends oftheASOs were able to direct specific cleavage
`of the target mRNA, albeit to a lesser extent than the ASOs
`that contained no mismatches. Similarly, target specific
`cleavage was achieved using a 13 nucleobase ASOs, includ(cid:173)
`ing those with 1 or 3 mismatches. Maher and Dolnick (Nuc.
`Acid. Res. 16:3341-3358, 1988, incorporated herein by ref(cid:173)
`erence) tested a series of tandem 14 nucleobase ASOs, and a
`28 and42 nucleobaseASOs comprised of the sequence of two
`or three of the tandem ASOs, respectively, for their ability to
`arrest translation of human DHFR in a rabbit reticulocyte
`assay. Each of the three 14 nucleobaseASOs alone were able
`to inhibit translation, albeit at a more modest level than the 28
`or 42 nucleobase ASOs. It is understood that antisense com(cid:173)
`pounds of the instant invention can vary in length and percent
`complementarity to the target provided that they maintain the
`desired activity. Methods to determine desired activity are
`disclosed herein and well known to those skilled in the art.
`
`Target Nucleic Acids
`
`[0048] As used here