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(11) EP 2 790 736 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/11 (2006.01) A61K 31/7084 (2006.01) 31.01.2018 Bulletin 2018/05 A61K 48/00 (2006.01)

(21) Application number: 12858522.1 (86) International application number: PCT/US2012/069294 (22) Date of filing: 12.12.2012 (87) International publication number: WO 2013/090457 (20.06.2013 Gazette 2013/25)

(54) IN VIVO DELIVERY OF OLIGONUCLEOTIDES IN-VIVO-VERABREICHUNG VON OLIGONUCLEOTIDEN ADMINISTRATION IN VIVO D’OLIGONUCLÉOTIDES

(84) Designated Contracting States: • M. B. BAKER ET AL: "In vitro quantification of AL AT BE BG CH CY CZ DE DK EE ES FI FR GB specific microRNA using molecular beacons", GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO NUCLEIC ACIDS RESEARCH, vol. 40, no. 2, 21 PL PT RO RS SE SI SK SM TR November 2011 (2011-11-21), pages e13-e13, XP055207619, ISSN: 0305-1048, DOI: (30) Priority: 12.12.2011 US 201161630446 P 10.1093/nar/gkr1016 • Veena Vijayanathan ET AL: "Direct measurement (43) Date of publication of application: of the association constant of HER2/neu 22.10.2014 Bulletin 2014/43 antisense oligonucleotide to its target RNA sequence using a molecular beacon", Antisense (73) Proprietor: ONCOIMMUNIN, INC. & nucleic acid drug development, 1 August 2002 Gaithersburg, (2002-08-01), pages 225-233, XP055207586, Maryland 20877 (US) United States DOI: 10.1089/108729002320351548 Retrieved from the Internet: (72) Inventors: URL:http://www.ncbi.nlm.nih.gov/pubmed/122 • PACKARD, Beverly 38811 [retrieved on 2015-08-12] Potomac • LERGA ET AL: "Rapid determination of total Maryland 20877 (US) hardness in water using fluorescent molecular • KOMORIYA, Akira aptamer beacon", ANALYTICA CHIMICA ACTA, Potomac ELSEVIER, AMSTERDAM, NL, vol. 610, no. 1, 18 Maryland 20877 (US) January 2008 (2008-01-18), pages 105-111, XP022473228, ISSN: 0003-2670, DOI: (74) Representative: Lee, Nicholas John et al 10.1016/J.ACA.2008.01.031 Kilburn & Strode LLP • BEVERLY Z PACKARD ET AL: "A Method in Lacon London Enzymology for Measuring Hydrolytic Activities 84 Theobalds Road in Live Cell Environments", 1 January 2008 London WC1X 8NL (GB) (2008-01-01), FLUORESCENCE SPECTROSCOPY;[METHODS IN ENZYMOLOGY; (56) References cited: ISSN 0076-6879; VOL. 450], ELSEVIER, ACAD. WO-A2-03/106631 WO-A2-2009/045536 PRESS, NL, PAGE(S) 1 - 19, XP008174215, ISBN: US-A1- 2003 224 377 US-A1- 2005 142 581 978-0-12-374586-6 [retrieved on 2009-01-17] * the US-A1- 2009 325 168 whole document *

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 790 736 B1

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• EMMANUEL CHANG ET AL: "Novel siRNA-based • PACKARD ET AL.: ’A Method in Enzymology for molecular beacons for dual imaging and Measuring Hydrolytic Activities in Live Cell therapy", BIOTECHNOLOGY JOURNAL, vol. 2, Environments’ METHODS IN ENZYMOLOGY vol. no. 4, 1 April 2007 (2007-04-01), pages 422-425, 450, 2008, pages 1 - 19, XP008174215 XP055207373, ISSN: 1860-6768, DOI: 10.1002/biot.200600257 • HARBORTH J ET AL: "Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing", ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT, MARY ANN LIEBERT, INC., NEW YORK, US, vol. 13, no. 2, 1 April 2003 (2003-04-01), pages 83-105, XP002284355, ISSN: 1087-2906, DOI: 10.1089/108729003321629638

2 1 EP 2 790 736 B1 2

Description an infectious disease, cancer, a proliferative disease or disorder, a neurological disease or disorder, an inflam- BACKGROUND OF THE INVENTION matory disease or disorder, a disease or disorder of the immune system, a disease or disorder of the cardiovas- Field of the Invention 5 cular system, a metabolic disease or disorder, a disease or disorder of the skeletal system, and a disease or dis- [0001] This invention pertains to the field of oligonu- order of the skin or eyes. cleotide therapeutics. In particular, this invention pro- [0005] Preferred features are set out in the dependent vides improved in vivo delivery for oligonucleotides in- claims herein. The invention is based in part on the im- cluding modified oligonucleotides and oligonucleotide 10 portant discovery of the inventors that the linkage of one mimics. or a plurality of HES to single, double and multiple strand [0002] Over the past several decades the use of oligo- oligonucleotide sequences results in an increased deliv- nucleotides as therapeutic agents has been the focus of ery of the HES-oligonucleotide sequences across phys- much interest. Both blockage of the transcription of spe- iologic boundaries found in in vivo systems. cific genes and addition of oligonucleotide sequences 15 [0006] One of the toughest obstacles limiting the use coding for particular proteins have been attempted as of RNAi and antisense oligonucleotides, (PNAs) and therapies for a plethora of pathologic conditions including PMOs in gene expression altering therapy has been the cancer, infectious diseases, and neurodegenerative con- low uptake of these compounds by eukaryotic cells, ditions. Moreover, multiple chemical approaches have which with currently available delivery methodologies is been developed to address the synthetic, immunogenic, 20 compounded by the sequestration and/or degradation of and biophysical properties of potential oligonucleotide- the compounds that actually do enter the cell; the latter based drugs and drug formulations. However, despite is predominantly via endocytosis. As will be immediately some success in solution and ex vivo systems, delivery apparent to a person of skill in the art, the surprisingly of oligonucleotides across biologic barriers such as cell high efficiency with which the non-toxic HES-oligonucle- membranes and extracellular matrices present in live or- 25 otide complexes useful in the invention are delivered into ganisms as well as structural components of infectious cells through sequence independent passive diffusion agents such as cell walls has been suboptimal. Thus, and the discovery by the inventors that these oligonucle- accessibility to molecular targets inside cells and tissues otides do not co-localize with lysozomes within cells, in- in vivo has been limiting development of the oligonucle- dicate that the HES-oligonucleotide delivery vehicles otide therapeutics field. 30 have the ability to enter all intracellular spaces/compart- [0003] In recent efforts to overcome some of the limi- ments. Thus, there are essentially limitless applications tations of the delivery of DNA and RNA sequences, de- in for example, research, diagnostics and therapeutics livery vehicles composed of lipids, sugars, and proteins arenas. Disclosed herein is the in vivo delivery of HES- conjugated to or encapsulating oligonucleotide sequenc- oligonucleotide complexes containing HES and at least es of interest, e.g., liposomes and lipid nanoparticles, 35 one therapeutic oligonucleotide for the treatment or pre- cholesterol conjugates, and antibody conjugates, have vention of a disease, disorder or condition. been developed. However, none of these formulations [0007] Moreover, with the currently available delivery has enabled delivery of oligonucleotide cargoes for the methodologies the induction of innate antiviral defenses field of oligonucleotide therapeutics to reach its anticipat- inmammalian cellsto exogenousnucleic acid sequences ed role in disease treatment. Accordingly, there is a need 40 have likewise significantly limited the development and for improved in vivo delivery systems of oligonucleotide- use of therapeutic oligonucleotides. The inventors have based therapeutics. discovered that HES-oligonucleotides have low toxicity (at concentrations greater than 10 fold the determined BRIEF SUMMARY OF THE INVENTION oligonucleotide in vivo cell loading level) and in fact, have 45 surprisingly found that the chemical linkage of HES. oli- [0004] In a first aspect, the present invention provides gonucleotides does not induce the interferon response a composition for delivering a therapeutic oligonucleotide in a host subject ( i.e., mouse) compared to that observed to a subject, said composition comprising a therapeuti- with other delivery vehicles. Accordingly, disclosed here- cally effective amount of an H-type excitonic structure in is a method of limiting the interferon response to an (HES)- oligonucleotide containing a therapeutic oligonu- 50 administered exogenous nucleic acid (e.g., oligonucle- cleotide that specifically hybridizes in vivo, to a nucleic otide) in a host, comprising linking 1, 2, 3 or more oligo- acid sequence and modulates the level of a protein en- nucleotides with an HES to form an HES-oligonucleotide coded or regulated by the nucleic acid, the therapeutic complex and administering the HES-oligonucleotide oligonucleotide having at least 85% sequence comple- complex to a subject. mentarity to the nucleic acid sequence or being comple- 55 [0008] An HES-oligonucleotide complex delivery vehi- mentary to the nucleic acid sequence and including 1, 2 cle can be used as a diagnostic to identify and/or quan- or 3 base substitutions, wherein the composition is for titate the presence of a nucleic acid of interestin vivo. use in the treatment of disease or disorder selected from: An HES-oligonucleotide complex delivery vehicle can be

3 3 EP 2 790 736 B1 4 used to identify the presence of an infectious agent in a been delivered to, and have hybridized with the target host organism such as, a virus or bacterium in a mam- nucleic acid. Thus, disclosed herein is a method for mon- malian tissue. The altered fluorescence that results upon itoring and/or quantitating the delivery of a therapeutic the disruption of the HES of the complex can serve as oligonucleotide to a target nucleic acid invivo, comprising an in vivo marker for binding of one or more HES-oligo- 5 administering to a subject, a HES oligonucleotides con- nucleotide sequences in the complex to a nucleic acid taining a therapeutic oligonucleotide that specifically hy- target sequence in a cell. Thus, the complexes useful in bridizes to the target nucleic acid, and determining the the invention have both diagnostic and therapeutic-ap- level of fluorescence in a cell or tissue of the subject, plications. This approach can also be used to quantitate wherein an increased fluorescence in the cell or tissue the number of copies of an aberrant gene in a host in vivo. 10 compared to a control cell or tissue indicates that that [0009] Disclosed herein is a method for detecting an thetherapeutic oligonucleotidehas been delivered toand altered level of a nucleic acid biomarker for a disease or hybridized with the target nucleic acid. disorder in vivo comprising, administering to a subject an [0013] In some embodiments of the invention, the ther- HES-oligonucleotide containing an oligonucleotide that apeutic oligonucleotide is from about 8 nucleotides to specifically hybridizes with the nucleic acid biomarker, 15 about 750 nucleotides in length. In some embodiments, determining the level of fluorescence in the subject, and the therapeutic oligonucleotide is from about 10 nucle- comparing the level of fluorescence with that obtained otides to about 100 nucleotides in length. In some em- for a control subject that has been administered the HES- bodiments, the therapeutic oligonucleotide is single oligonucleotide, wherein an altered fluorescence com- stranded. In other embodiments, the therapeutic oligo- pared to the control indicates that the subject has an al- 20 nucleotide is double stranded, In additional embodi- tered level of the nucleic acid biomarker. This approach ments, the HES-oligonucleotide comprises 3 or more can also be used to quantitate the number of copies of fluorophores capable of forming one or more HES. In an aberrant gene of host origin in vivo. further embodiments, the therapeutic oligonucleotide is [0010] The disease or disorder may be: cancer, fibro- a member selected from: siRNA, shRNA, miRNA, a Dicer sis, a proliferative disease or disorder, a neurological dis- 25 substrate, an aptamer, a decoy and antisense. In further ease or disorder, an inflammatory disease or disorder, a embodiments, the antisense oligonucleotide is DNA or a disease or disorder of the immune system, a disease or DNA mimic. disorder of the cardiovascular system, a metabolic dis- [0014] In some embodiments, the therapeutic oligonu- ease or disorder, a disease or disorder of the skeletal cleotide in an HES-oligonucleotide for use of the inven- system, or a disease or disorder of the skin or eyes. 30 tion is an antisense oligonucleotide that specifically hy- [0011] The methods disclosed herein can be used to bridizes to an RNA. In further embodiments, the anti- identify and/or distinguish between different diseases or sense oligonucleotide is a substrate for RNAse H when disorders. The methods can likewise be used to deter- hybridized to the RNA. In particular embodiments, the mine among other things, altered nucleic acid ( e.g., DNA antisense oligonucleotide is a gapmer. and RNA) profiles that distinguish between normal and 35 [0015] In additional embodiments, the therapeutic oli- diseased (e.g., cancerous) tissue or cells, discriminate gonucleotide in an HES-oligonucleotide for use of the between different subtypes of diseased cells e.g( ., be- invention is an antisense oligonucleotide that specifically tween different cancers and subtypes of a particular can- hybridizes to an RNA, but the antisense oligonucleotide cer), to discriminate between mutations ( e.g., oncogenic is not a substrate for RNAse H when hybridized to the mutations) giving rise to or associated with different dis- 40 RNA. In some embodiments, the antisense oligonucle- ease states, and to identify tissues of origin e.g.,( in a otide is DNA or a DNA mimic. metastasized tumor). [0016] In further embodiments, the therapeutic oligo- [0012] Disclosed herein are compositions and meth- nucleotide in an HES-oligonucleotide for use of the in- ods for modulating nucleic acids and protein encoded or vention is an antisense oligonucleotide containing a se- regulated by these modulated nucleic acids. In particular, 45 quence that specifically hybridizes to: (a) a sequence disclosed are compositions and methods for modulating within 30 nucleotides of the AUG start codon of an mRNA; the levels, expression, processing or function of a mRNA, (b) nucleotides 1-10 of a miRNA; (c) a sequence in the small non-coding RNA (e.g., miRNA), a gene or a protein. 5’ untranslated region of an mRNA; (d) a sequence in Disclosed is a method of delivering an oligonucleotide to the 3’ untranslated region of an mRNA; (e) an intron/exon a cell in vivo by administering to a subject an HES- oli- 50 junction of an mRNA; (f) a sequence in a precursor-miR- gonucleotide complex containing the oligonucleotide. NA (pre-miRNA) or primary-miRNA (pri-miRNA) that The oligonucleotide may be a therapeutic oligonucle- when bound by the oligonucleotide blocks miRNA otide. Moreover, the oligonucleotides in the HES-oligo- processing; and (g) an intron/exon junction and a region nucleotides may be therapeutic oligonucleotides, and the 1 to 50 nucleobases 5’ of an intron/exon junction of an destruction or significant loss of HES that results in an 55 RNA. increased fluorescence when the therapeutic HES oligo- [0017] In another embodiment, the invention modu- nucleotides specifically hybridize with target nucleic ac- lates the level of a protein encoded or regulated by the ids indicates that the therapeutic oligonucleotides have nucleic acid through the induction of RNA interference

4 5 EP 2 790 736 B1 6

(RNAi). In some embodiments, the therapeutic oligonu- acids comprising or encoding the nucleic acid and may cleotide is siRNA, shRNA or a Dicer substrate. In further act to reduce the levels of the nucleic acid and/or interfere embodiments, the therapeutic oligonucleotide is 18-35 with its function in the cell. In particular embodiments, nucleotides in length. In some embodiments, the thera- the target nucleic acid is a small-non coding RNA, such peutic oligonucleotide is a dicer substrate and contains 5 as, a miRNA. In some embodiments, the oligonucleotide 2 nucleic complementary nucleic acid strands that are comprises a sequence substantially complementary to each 18-25 nucleotides in length and contain a 2 nucle- the target nucleic acid. otide 3’ overhang. In some embodiments, the oligonu- [0023] Disclosed herein is a method of reducing the cleotide is dsRNA or a dsRNA mimic that is processed expression of a target RNA in a subject in need of reduc- by Dicer enzymatic activity. In additional embodiments, 10 ing expression of said target RNA, comprising adminis- the therapeutic oligonucleotide is single stranded RNA tering to said subject an antisense HES-oligonucleotide or RNA mimic capable of inducing RNA interference. complex. In particular embodiments, an oligonucleotide [0018] In some embodiments, the therapeutic oligonu- in the complex is a substrate for RNAse H when bound cleotide contains one or more modified internucleoside to said target mRNA. In further embodiments, the oligo- linkages selected from: phosphorothioate, phospho-15 nucleotide is a gapmer. rodithioate, phosphoramide, 3’-methylene phosphonate, [0024] The oligonucleotide may comprise or encode O-methylphosphoroamidiate, PNA and morpholino. In the nucleic acid or increase the endogenous expression, additional embodiments, the therapeutic oligonucleotide processing or function of the nucleic acid ( e.g., by binding contains one or more modified nucleobases selected regulatory sequences in the gene encoding the nucleic from C-5 propyne and 5-methyl C. In some embodiments, 20 acid) and act to increase the level of the nucleic acid at least one nucleotide of the antisense oligonucleotide and/or increase its function in the cell. In some embodi- contains a modified sugar moiety comprising a modifica- ments, the oligonucleotide comprises a sequence sub- tion at the 2 - position, a PNA motif, or a morpholino motif. stantially the same as nucleic acids comprising or encod- In further embodiments, at least one nucleotide of the ing the nucleic acid. therapeutic oligonucleotide comprising a modified sugar 25 [0025] The invention also relates to treating a disease motif selected from: 2’OME, LNA, alpha LNA, 2’-Fluoro or disorder characterized by the overexpression of a nu- (2’F), 2’-O(CH2)2OCH3(2’-MOE) and 2’-OCH3(2’-O-me- cleic acid in a subject, comprising administering to the thyl). In some embodiments, the modified nucleoside mo- subject an HES-oligonucleotide complex containing an tif is an LNA or alpha LNA in which a methylene (--CH2- oligonucleotide which is targeted to a nucleic acid com- 30 -)n group bridges the 2’ oxygen atom and the 4’ carbon prising or encoding the nucleic acid and which acts to atom wherein n is 1 or 2. In further embodiments, the reduce the levels of the nucleic acid and/or interfere with LNA or alpha LNA contains a methyl group at the 5’ po- its function in the subject. The invention relates to treating sition. In further embodiments, each nucleoside of the a disease or disorder characterized by the overexpres- oligonucleotide comprises a modified nucleoside motifs sion of a protein in a subject, comprising administering selected from: 2’OME, LNA, alpha LNA, 2’-Fluoro (2’F), 35 to the subject an HES-oligonucleotide complex contain-

2’-O(CH2)2CH3(2’-MOE) and 2’-OCH3(2’-O-methyl). ing an oligonucleotide which is targeted to a nucleic acid [0019] The HES-oligonucleotide complexes useful in encoding the protein or decreases the endogenous ex- the invention provide a highly efficient in vivo delivery of pression, processing or function of the protein in the sub- oligonucleotides into cells, essentially have limitless ap- ject. In some embodiments, the nucleic acid is DNA, mR- plications in modulating target nucleic acid and protein 40 NA or miRNA. In additional embodiments the oligonucle- levels and activity The HES-oligonucleotide complexes otide is selected from a siRNA, shRNA, miRNA, an anti- are particularly useful in therapeutic applications. miRNA, a dicer substrate, an antisense oligonucleotide, [0020] The oligonucleotide may comprise a sequence a plasmid capable of expressing a siRNA, a miRNA, a substantially complementary to the target nucleic acid ribozyme and an antisense oligonucleotide. that specifically hybridizes to and modulates levels of the 45 [0026] In an additional embodiment, the invention also nucleic acid or interferes with its processing or function. relates to treating ( e.g., alleviating) a disease or disorder In some embodiments, the target nucleic acid is RNA, in characterized by the aberrant expression of a protein in further embodiments the RNA is mRNA or miRNA. In a subject, comprising administering to the subject an further embodiments, the oligonucleotide reduces the HES-oligonucleotide complex, containing an oligonucle- level of a target RNA by at least 10%, at least 20%, at 50 otide which specifically hybridizes to the mRNA encoding least 30%, at least 40% or at least 50% in one or more the protein and alter the splicing of the target RNA ( e.g., cells or tissues of the subject. In some embodiments, the promoting exon skipping in instances where production target nucleic acid is a DNA. or overproduction of a particular splice product is impli- [0021] Compositions and methods for modulating the cated in disease). In some embodiments, each nucleo- levels, expression, processing or function of a mRNA, 55 side of the oligonucleotide comprises at least one mod- small non-coding RNA (e.g., miRNA), a gene or a protein ified sugar moiety comprising a modification at the 2’- are disclosed herein. position. In particular embodiments, the modified oligo- [0022] The oligonucleotide may be targeted to nucleic nucleotide is a 2’ OME or 2’ allyl. In additional embodi-

5 7 EP 2 790 736 B1 8 ments, the modified oligonucleotide is LNA, alpha LNA nucleotide analogs are included that have alternate (e.g., an LNA or alpha LNA containing a steric bulk moiety backbones, comprising, for example, phospho- at the 5’ position ( e.g., a methyl group). In some embod- ramide (see, e.g., Beaucage et al. (1993) Tetrahe- iments the oligonucleotide is a PNA or phosphorodiam- dron 49(10):1925) and references therein; Letsinger idate morpholino (PMO). In some embodiments, the ol- 5 (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) igonucleotide sequence specifically hybridizes to a se- Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. quence within 30 nucleotides of the AUG start codon, a Acids Res. 14:3587; Sawai et al. (1984) Chem. Lett. sequence in the 5’ or 3’ untranslated region of a target 805; Letsinger et al. (1988) J. Am. Chem. Soc. RNA, or a sequence that alters the splicing of a target 110:4470; and Pauwels et al. (1986) Chemica mRNA. In particular the oligonucleotide specifically hy- 10 Scripta 26:1419, phosphorathioate (Mag et al. bridizes to a sequence that alters the splicing of target (1991) Nucleic Acids Res. 19:1437; and U.S. Pat. mRNA in Duchenne Muscular Dystrophy (DMD). In fur- No 5,644,048), phosphorodithioate (Briu et al (1989) ther embodiments, the altered splicing results in the J. Am. Chem. Soc. 111:2321), O-methylphosphoro- "skipping" of exon 51 in the resulting mRNA. amidiate linkages see, ( e.g., Eckstein, Oligonu- [0027] In various embodiments, the invention provides 15 cleoetides and Analogues: A Practical Approach, compositions for use in modulating a target nucleic acid Oxford University Press), and peptide nucleic acid or protein in a cell, in vivo in a subject, or ex vivo. The backbones and linkages (see, e.g., Egholm (1992) HES-oligonucleotide compositions for use of the inven- J. Am. Chem. Soc. 114:1895; Meier et al. (1992) tion have applications in for example, treating a disease Chem. Int. Ed. Engl. 31:1008:; Nielsen (1993) Nature or disorder characterized by an overexpression, under- 20 365:566; Carlsson et al. (1996) Nature 380:207). expression and/or aberrant expression of a nucleic acid Other analog nucleic acids/oligonucleotides include or protein in a subject in vivo or ex vivo. Compositions of those with positive backbones (see, e.g., Dempcy et the invention are used in a method for treating diseases al. (1995) Proc. Natl. Acad. Sci USA 92:6097); non- or disorders selected from: ionic backbones ( see, e.g., U.S. Pat. Nos. 5,386,023, 25 5,637,684, 5,602,240, 5,216,141, and 4,469,863; an infectious disease, cancer, a proliferative disease Angew. (1991) Chem. Intl, Ed. English 30:423; Let- or disorder, a neurological disease or disorder, and singer et al. (1988) J. Am. Chem. Soc. 110:4470; inflammatory disease or disorder, a disease or dis- Letsinger et al. (1994) Nucleoside & Nucleotide order of the immune system, a disease or disorder 13:1597; Chapters 2 and 3, ASC Symposium Series of the cardiovascular system, a metabolic disease 30 580, "Carbohydrate Modifications in Antisense Re- or disorder, a disease or disorder of the skeletal sys- search", Ed. Y. S. Sanghui and P. Dan Cook; Mes- tem, and a disease or disorder of the skin or eyes. maeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4:395; Jeffs et al. (1994) J. Biomolecular NMR [0028] Disclosed herein is a method for cell nuclear 34:17; Tetrahedron Lett. 37:743 (1996)), and non- reprograming. HES-oligonucleotides containing one or 35 ribose backbones, including those described in U.S. more mimics and/or inhibitor of a miRNA or a plurality of Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 miRNAs can be administered ex vivo into cells such as, and 7, ASC Symposium Series 580, Carbohydrate human and mouse somatic cells to reprogram the cells Modifications in Antisense Research, Ed. Y.S. San- to have one or more properties of induced pluripotent ghui and P. Dan Cook. Nucleic acids/oligonucle- stem cells (iPSCs) or embryonic stem (ES)-like pluripo- 40 otides containing one or more carbocyclic sugars are tent cells. The non-toxic and highly efficient HES-oligo- also included within the definition of nucleic acids/ol- nucleotide delivery system of the invention provides a igonucleotides (see, e.g., Jenkins et al. (1995), greatly increased efficiency of delivery method for repro- Chem. Soc. Rev. pp 169-176). Several nucleic ac- gramming cells compared to conventional oligonucle- id/oligonucleotide analogs are described in Rawls, otide delivery methods see, ( e.g., U.S. Publ. Nos.45 C & E News Jun. 2 1997 page 35). These modifica- 2010/0075421, US 2009/0246875, US 2009/0203141, tions of the ribose-phosphate backbone may be and US 2008/0293143). done for example, to facilitate the addition of addi- tional moieties such as labels, or to increase the sta- DEFINITIONS bility and half-life of such molecules in physiological 50 environments. Nucleic acid/oligonucleotide back- [0029] The following abbreviations are used herein: bones of oligonucleotides used in the invention range from about 5 nucleotides to about 750 nucle- The terms "nucleic acid" or "oligonucleotide" refer to otides. Preferred nucleic acid/oligonucleotide back- at least two nucleotides covalently linked together. bones used in this invention range from about 5 nu- Anucleic acid/oligonucleotide of theinvention is pref- 55 cleotides to about 500 nucleotides, and preferably erably single-stranded or double-stranded and gen- from about 10 nucleotides to about 100 nucleotides erally contains phosphodiester bonds, although in in length. As used herein, the term "about" or "ap- some cases, as outlined below, nucleic acid/oligo- proximately" when used in conjunction with a

6 9 EP 2 790 736 B1 10

number refers to any number within 0.25%, 0.5%, to a target nucleic acid. The terms "specifically hybridiz- 1%, 5% or 10% of the referenced number. es" and specifically hybridizable" are used interchange- ably herein to indicate a sufficient degree of complemen- [0030] The oligonucleotides in the HES-oligonucle- tarity such that stable and specific binding occurs be- otide complexes of the invention are polymeric structures 5 tween the oligonucleotide and the target nucleic acid ( i.e., of nucleoside and/or nucleotide monomers capable of DNA or RNA). It is understood that an oligonucleotide specifically hybridizing to at least a region of a nucleic need not be 100% complementary to its target nucleic acid target. As indicated above, HES-oligonucleotides acid sequence to be specifically hybridizable. In particu- include, but are not limited to, compounds comprising lar embodiments, an oligonucleotide is considered to be naturally occurring bases, sugars and intersugar (back- 10 specifically hybridizable when binding of the oligonucle- bone) linkages, non-naturally occurring modified mono- otide to a target nucleic acid sequence interferes with the mers, or portions thereof (e.g., oligonucleotide analogs normal function of the target nucleic acid and results in or mimetics) which function similarly to their naturally oc- a loss or altered utility or expression therefrom. In pre- curring counterpart, and combinations of these naturally ferred embodiments, there is a sufficient degree of com- occurring and non-naturally occurring monomers. As15 plementarity between the oligonucleotide and target nu- used herein, the term "modified" or "modification" in- cleic acid to avoid or minimize non-specific binding of the cludes any substitution and/or any change from a starting oligonucleotide to undesired non-target sequences un- or natural oligomeric compound, such as an oligonucle- der the conditions in which specific binding is desired otide. Modifications to oligonucleotides encompass sub- (e.g., under physiological conditions in the case of in vivo stitutions or changes to internucleoside linkages, sugar 20 assays or therapeutic treatment, and in the case ofin moieties, or base moieties, such as those described vitro assays, under conditions in which the assays are herein and those otherwise known in the art. performed). It is well within the level of skill of scientists [0031] The term "antisense" as used herein, refers to in the oligonucleotide field to routinely determine when an oligonucleotide sequence, written in the 5’ to 3’ direc- conditions are optimal for specific hybridization to a target tion, comprises the reverse complement of the corre- 25 nucleic acid with minimal non-specific hybridization sponding region of a target nucleic acid and/or that is events. Thus, in some embodiments, oligonucleotides in able to specifically hybridize to the target nucleic acid the complexes of the invention include 1, 2, or 3 base under physiological conditions. Thus, in some embodi- substitutions compared to the corresponding comple- ments, the term antisense refers to an oligonucleotide mentary sequence of a region of a target DNA or RNA that comprises the reverse complement of the corre-30 sequence to which it specifically hybridizes. In some em- sponding region of a small noncoding RNA, untranslated bodiments, the location of a non-complementary nucle- mRNA and/or genomic DNA sequence. In particular em- obase is at the 5’ end or 3’ end of an antisense oligonu- bodiments, an antisense HES-oligonucleotide in a com- cleotide. In additional embodiments, a non-complemen- plex of the invention, once hybridized to a nucleic acid tary nucleobase is located at an internal position in the target, is able to induce or trigger a reduction in target 35 oligonucleotide. When two or more non-complementary gene expression, target gene levels, or levels of the pro- nucleobases are present in an oligonucleotide, they may tein encoded by the target nucleic acid. be contiguous (i.e., linked), non-contiguous, or both. In [0032] "Complementary," as used herein, refers to the some embodiments, the oligonucleotides in the complex- capacity for pairing between a monomeric component of es of the invention have at least 85%, at least 90%, or at an oligonucleotide and a nucleotide in a targeted nucleic 40 least 95% sequence complementarity to a target region acid (e.g., DNA, mRNA, and a non-coding RNA such as, within the target nucleic acid. In other embodiments, ol- a miRNA). For example, if a nucleotide at a certain po- igonucleotides have 100% sequence complementarity sition of an oligonucleotide is capable of hydrogen bond- to a polynucleotide sequence within a target nucleic acid. ing with a nucleotide at the same position of a DNA/RNA Percent identity is calculated according to the number of molecule, then the oligonucleotide and DNA/RNA are 45 bases that are identical to the corresponding nucleic acid considered to be complementary at that position. sequence to which the oligonucleotide being compared. [0033] In the context of this application, "hybridization" This identity may be over the entire length of the oligo- means the pairing of an oligonucleotide with a comple- meric compound (i.e., oligonucleotide), or in a portion of mentary nucleic acid sequence. Such pairing typically the oligonucleotide (e.g., nucleobases 1-20 of a 27-mer involves hydrogen bonding, which may be Watson-Crick, 50 may be compared to a 20-mer to determine percent iden- Hoogsteen or reversed tity of the oligonucleotide to the oligonucleotide). Percent [0034] Hoogsteen hydrogen bonding, between com- identity between an oligonucleotide and a target nucleic plementary nucleoside or nucleotide bases (nucleobas- acid can routinely be determined using alignment pro- es) of an oligonucleotide and a target nucleic acid se- grams and BLAST programs (basic local alignment quence (e.g., wherein the oligonucleotide comprises the 55 search tools) known in the art (see, e.g., Altschul et al., reverse complementary nucleotide sequence of the cor- J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, responding region of the target nucleic acid). In particular Genome Res., 1997, 7, 649-656). embodiments, an oligonucleotide specifically hybridizes [0035] As used herein, the terms "target nucleic acid"

7 11 EP 2 790 736 B1 12 and "nucleic acid encoding a target" are used to encom- or in different oligonucleotides so long as the collective pass any nucleic acid capable of being targeted includ- HES-oligonucleotide contains one or more HES. The ing, without limitation, DNA encoding a given molecular fluorophores are optionally attached to the oligonucle- target ( i.e., a protein or polypeptide), RNA (including miR- otide via a linker, such as a flexible aliphatic chain. NA, pre-mRNA and mRNA) transcribed from such DNA, 5 [0040] An HES-oligonucleotide may contain 1, 2, 3, 4, and also cDNA derived from such RNA. Exemplary DNA or more HES. Additionally, an HES in an HES-oligonu- functions to be interfered with include replication, tran- cleotide may contain 2, 3, 4 or more of the same or dif- scription and translation. The overall effect of such inter- ferent fluorophores, See, e.g., Toptygin et al., Chem. ference with target nucleic acid function is modulation of Phys. Lett 277:430-435 (1997). In some embodiments, the expression of the target molecule. In the context of 10 an HES is formed as a consequence of fluorophore ag- the present invention, "modulation" means a quantitative gregates between HES-oligonucleotides of the invention. change, either an increase (stimulation) or a decrease In some embodiments, an HES is formed as a conse- (inhibition), for example in the expression of a gene. The quence of fluorophore aggregates between oligonucle- inhibition of gene expression through reduction in RNA otides of the invention that are singly labeled with a fluor- levels is a preferred form of modulation according to the 15 ophore capable of forming an HES. present invention. [0041] As used herein, the terms "pharmaceutically ac- [0036] A "chromophore" is a group, substructure, or ceptable," or "physiologically tolerable" and grammatical molecule that is responsible for the absorbance of light. variations thereof, as they refer to compositions, carriers, Typical chromophores each have a characteristic ab- diluentsand reagents, are usedinterchangeably and rep- sorbance spectrum. 20 resent that the materials are capable of administration to [0037] A "fluorophore" is a chromophore that absorbs or upon a subject ( e.g., a mammal such as a mouse, rat, light at a characteristic wavelength and then re-emits the rabbit, or a primate such as a human), without the pro- light most typically at a characteristic different wave- duction of therapeutically prohibitive undesirable physi- length. Fluorophores are well known to those of skill in ological effects such as nausea, dizziness, gastric upset the art and include, but are not limited to xanthenes and 25 and the like. xanthene derivatives, rhodamine and rhodamine deriv- [0042] As used herein, a "pharmaceutical composition atives, cyanines and cyanine derivatives, coumarins and comprising an antisense oligonucleotide" refers to a com- coumarin derivatives, and chelators with the lanthanide position comprising an HES-oligonucleotide complex ion series. A fluorophore is distinguished from a chromo- and a pharmaceutically acceptable diluent. By way of phore which absorbs, but does not characteristically re- 30 example, a suitable pharmaceutically acceptable diluent emit light. is phosphate-buffered saline. [0038] An "H-type excitonic structure" (HES) refers to [0043] A "stabilizing modification" or "stabilizing motif’ two or more fluorophores whose transition dipoles are means providing enhanced stability, in the presence of arranged in a parallel configuration resulting in a splitting nucleases, relative to that provided by 2’-deoxynucleo- of the excited singlet state; transitions between a ground 35 sides linked by phosphodiester internucleoside linkages. state and an upper excited state are considered allowed Thus, such modifications provide "enhanced nuclease and transitions between a ground state and lower excited stability" to oligonucleotides. Stabilizing modifications in- state forbidden. HES formation in connection with certain clude at least stabilizing nucleosides and stabilizing in- fluorophores is known in the art and the invention en- ternucleoside linkage groups. compasses the attachment of these fluorophores to oli- 40 [0044] The term "in vivo organism" refers to a contig- gonucleotides (e.g., diagnostic and therapeutic oligonu- uous living system capable of responding to stimuli such cleotides) and the use of the resulting HES-oligonucle- as reproduction, growth and development, and mainte- otides according to the methods described herein. Ex- nance of homeostasis as a stable whole. Examples in- amples of HES forming fluorophores that can be used clude mammals, plants, and microorganisms such as according to the invention are disclosed 45 bacteria, protozoa, and viruses. herein or otherwise known in the art and include, but are [0045] The term "subject" refers to any animal ( e.g., a not limited to, xanthenes and xanthene derivatives, cya- mammal), including, but not limited to humans, non-hu- nine and cyanine derivatives, coumarins and chelators man primates, rodents, and the like, which is to be the with the lanthanide ion series. recipient of a particular treatment. Typically, the terms [0039] The term"HES-oligonuclcotide" refers toa com- 50 "subject" and "patient" are used interchangeably herein plex of one or more oligonucleotide strands ( e.g,. a single in reference to a human subject. strand, double strand, triple strand or a further plurality [0046] The terms "administering" and "administration" of strands of linear or circular oligonucleotides containing as used herein, refer to adding a chemical such as an the same, complementary or distinct oligonucleotide se- oligonucleotide to a subject in vivo or ex vivo. Thus, ad- quences) that contain 2 or more fluorphores that form an 55 ministering encompasses both the addition of an HES- HES. The fluorophores of the HES-oligonucleotide may oligonucleotide directly to a subject and also contacting be attached at the 5’ and/or 3’ terminal backbone phos- cells with HES-oligonucleotide compositions and then in- phates and/or at another base within an oligonucleotide troducing the contacted cells into a subject. In one em-

8 13 EP 2 790 736 B1 14 bodiment, cells removed from a subject are contacted complex to elicit a desired response in the subject. A with an HES-oligonucleotide and the contacted cells are therapeutically effective amount is also one in which any then re-introduced to the subject. toxic or detrimental effects of the HES-oligonucleotide [0047] The term "contacting" refers to adding a chem- complex are outweighed by the therapeutically beneficial ical such as an oligonucleotide to anin vivo organism 5 effects. such as a mammal, plant, bacterium, or virus. For mam- [0050] "Therapeutic index" means the ratio of the dose mals, common routes of contacting include peroral of an HES-oligonucleotide complex which produces an (through the mouth), topical (skin), transmucosal (nasal, undesired effect to the dose which causes desired ef- buccal/sublingual, vaginal, ocular and rectal), inhalation fects. In the context of the present disclosure, an HES- (lungs), intramuscular (muscle) and intravenous (vein). 10 oligonucleotide complex exhibits an "improved therapeu- For bacteria and viruses contact may be delivery inside tic index" when activity is retained, but undesired effects a cell or tissue of a host organism. are reduced or absent. For example, an HES-oligonucle- [0048] "Treating" or "treatment" includes the adminis- otide complex having an improved therapeutic index re- tration of an HES-oligonucleotide to prevent or delay the tains the ability to inhibit miRNA activity without resulting onset of the symptoms, complications, or biochemical 15 in undesired effects such as immunostimulatory activity, indicia of a disease, condition, or disorder, alleviating the or, at least, without resulting in undesired effects to a symptoms or arresting or inhibiting further development degree that would prohibit administration of the complex. of the disease, condition, or disorder. Treatment can be [0051] As used herein a "therapeutic oligonucleotide" prophylactic (to prevent or delay the onset of the disease, refers to an oligonucleotide capable of achieving a de- or to prevent the manifestation of clinical or subclinical 20 sired therapeutic result and/or to "treat" a disease or dis- symptoms thereof) or therapeutic suppression or allevi- order in a subject or ex vivo when administered at suffi- ation of symptoms after the manifestation of the disease, cient doses. Such desirable results include for example, condition, or disorder. Treatment can be with an HES- a slowing of disease progression, an increase in survival oligonucleotide complex containing composition alone, time, and/or an improvement in one or more indicators or in combination with 1, 2, 3 or more additional thera- 25 of disease, disease progression, or disease related con- peutic agents. ditions in a subject suffering from the disease. Exemplary [0049] The term "therapeutically effective amount" re- therapeutic oligonucleotides include an siRNA, an shR- fers to an amount of an HES-oligonucleotide complex NA, a Dicer substrate ( e.g., dsRNA), an miRNA, an anti- ("therapeutic agent") or other drug effective to achieve a miRNA, an antisense, a decoy, an aptamer and a plasmid desired therapeutic result and/or to "treat" a disease or 30 capable of expressing a siRNA, a miRNA, a ribozyme, disorder in a subject. The term "therapeutically effective an antisense oligonucleotide, or a protein coding se- amount" may also refer to an amount required to produce quence. Oligonucleotides such as probes and primers a slowing of disease progression, an increase in survival that are not able to achieve a desired therapeutic result time, and/or an improvement in one or more indicators are not considered therapeutic oligonucleotides for the of disease or the progression of a disease in a subject 35 purpose of this disclosure. On average, less than 1% of suffering from the disease. For example, in the case of mRNA is a suitable target for antisense oligonucleotides. cancer, a therapeutically effective amount an HES-oligo- Numerous antisense oligonucleotides suitable for incor- nucleotide complex may: reduce angiogenesis and ne- poration to the HES-oligonucleotides of the invention are ovascularization; reduce the number of cancer cells, a described herein or otherwise known in the art. Likewise, therapeutically effective amount an HES-oligonucleotide 40 suitabletherapeutic oligonucleotides can routinelybe de- complex may reduce tumor size, inhibit ( i.e., slow or stop) signed using guidelines, algorithms and programs known cancer cell infiltration into peripheral organs, inhibit (i.e., in the art (see, e.g., Aartsma-Rus et al., Mol Ther 17(3) slow or stop) tumor metastasis, inhibit or slow tumor 548-553 (2009) and Reynolds et al., Nat. Biotech. growth or tumor incidence, stimulate immune responses 22(3):326-330 (2004), and Zhang et al., Nucleic Acids against cancer cells and/or relieve one or more symp- 45 Res. 31e72 (2003)). Suitable therapeutic oligonucle- toms associated with the cancer. In the case of an infec- otides can likewise routinely be designed using commer- tious disease, a therapeutically effective amount an HES- cially available programs e.g., ( MysiRNA-Designer, oligonucleotide complex may be associated with a re- AsiDesigner (Bioinformatics Research Center, KRIBB), duced number of the infectious agent (e.g., viral load) siRNA Target Finder (Ambion), Block-iT RNAi Designer and/or in amelioration of one or more symptoms or con- 50 (Invitrogen), Gene specific siRNA selector (The Wistar ditions associated with infection caused by the infectious Institute), siRNA Target Finder (GeneScript), siDESIGN agent. A "therapeuticallyeffective amount"also may refer Center (Dharmacon), SiRNA at Whitehead, siRNA De- to an amount effective, at dosages and for periods of time sign (IDT), D: T7 RNAi Oligo Designer (Dudek P and necessary, to achieve a desired therapeutic result. A Picard D.), sfold-software, and RNAstructure 4.5); pro- therapeutically effective amount of an HES-oligonucle- 55 grams available over the internet such as, human splicing otide complex of the invention may vary according to fac- finder software (e.g., at ".umd.be/HSF/") and Targetfind- tors such as, the disease state, age, sex, and weight of er (available at "bioit.org.cn/ao/targetfinder"); and com- the subject, and the ability of the HES-oligonucleotide mercial providers (e.g., Gene Tools, LLC). In certain in-

9 15 EP 2 790 736 B1 16 stances, an HES-oligonucleotide and a therapeutic oli- cleotide sequences capable of blocking gene transcrip- gonucleotide may be used interchangeably herein unless tion and translation in vivo. the context clearly dictates otherwise. [0054] Both biologic and chemical approaches have been used to develop delivery methods. For example, a BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES 5 biologic approach has been the construction of several viral vectors with promoter-expressed sequences while [0052] chemically-based delivery vehicles have been created by conjugation of nucleicacids with avariety of molecules FIG. 1 shows fields histograms of blood cells isolated including cholesterol, sugars, aptamers, and antibodies. from BALB/C mice three hours after an injection of 10 However, the most studied chemical in vivo delivery sys- 200 microliters of buffer (PBS) or a Dicer substrate. tem has utilized nanoparticles wherein nucleic acids are The latter contains a sequence for a gene not present encapsulated in liposomes which are vesicles composed in these mice. In Panel a, cells were isolated after a of lipid bilayers. The latter when decorated with polyeth- single ip injection of PBS or the Dicer substrate at a ylene glycol (PEG) polymer chains for enhanced stability concentration of 1.5 mg/kg. In Panel b, cells were 15 are termed SNALPs and they are sometimes further isolated after an iv injection of PBS, the Dicer sub- modified with peptide ligands on the nanoparticle surface strate at a concentration of 1.5 mg/kg, or the Dicer for targeting receptors on specific cell types. substrate at a concentration of 0.75 mg/kg. [0055] Although some success has been achieved FIG. 2 shows (left column) emission spectra and with the above approaches, the following problems have (right column) hplc chromatograms of individual20 been encountered: with viral delivery, there is a high po- complementary single fluorophore-labeled strands tential for triggering immunogenicity in the host. Addition- of RNA (top two rows) before and (bottom row) after ally, the risk of mutations or aberrant gene expression in addition to each other. The middle column of the fig- the host due to mutations in the viral sequence must be ure shows the fluorescence intensity of the sense monitored. As for the in vivo chemical delivery vehicles, strand alone (between 0 and ca. 80 sec.) followed 25 unfortunately, even with enhanced modifications for spe- by quenching upon addition of the antisense strand cificity, delivery has been shown to be lacking with re- (at ca. 80 sec.). spect to: (1) Specific uptake by target cells. Rather, cells FIG. 3 shows the fluorescence intensity of the duplex of the reticuloendothelial system nonspecifically take up formed between a labeled sense and labeled anti- nucleic acid constructs, particularly nanoparticles, by a sense strand of RNA as a function of time after ad- 30 phagocytic-like process. (2) Even when targeting of the dition of the recombinant Dicer enzyme. desired cell is successful, internalization of the probe with FIG. 4 shows fluorescence intensity of single blood or without the delivery vehicle is often into the cells’ en- cells from mice transgenic for eGFP. Histogram from docytic system with the oligonucleotide ending up in lys- control cells and superimposed on that of cells ex- osomes where the chemical environment, e.g., low pH, posed to a duplex RNA targeting eGFP. 35 can lead to (a) destruction of the nucleic acid or (b) se- questration from the targeted mRNA in the cytoplasm or DETAILED DESCRIPTION OF THE INVENTION DNA in the nucleus. [0056] In contrast to the above described delivery ve- [0053] Molecular targets for detection and treatment hicles, the present invention provides a highly efficient in of pathologic conditions such as cancer, infectious dis- 40 vitro and in vivo oligonucleotide delivery system that re- eases, and neurodegenerative disorders can be unique quires the administration of orders of magnitude of less DNA and RNA sequences. Studies in which binding be- oligonucleotide to achieve therapeutic effect than that tween such targets and probes containing complemen- required using conventional delivery technologies. The tary sequences, a process known as hybridization, have HES-oligonucleotide delivery vehicles for use of the in- been carried out with high precision and specificity; more- 45 vention are sequence independent (e.g., delivery of nu- over, these data have provided a basis for optimism for cleic acids, modified nucleic acids, PNAs, morpholinos) development of treatments not currently available. How- and exploit passive diffusion to bypass cellular endoctyic ever, such studies have largely been carried out under systems, thereby providing access to all intracellular en- nonphysiologic conditions, e.g., in solution or in perme- vironments and increasing the delivery of oligonucle- abilized or fixed cells and tissues. Unfortunately, when 50 otides (e.g., therapeutic oligonucleotides such as, siR- the same probes have been tried under physiologic con- NA, shRNA, Dicer substrates ( e.g., dsRNA), miRNA, an- ditions, due to the complementary sequences’ sizes and ti-miRNA, decoys, aptamers and antisense to for exam- charges combined with the presence of permeability bar- ple, targeted RNA in the cell cytoplasm or DNA in the riers, e.g., host cell membranes, extracellular matrices, nucleus. In particular, in preferred embodiments, the in- or cell walls, accessibility to these targets has often been 55 vention uses HES-oligonucleotide complexes compris- considerably limited resulting in reduced effectiveness. ing an oligonucleotide and 2 or more fluorophores capa- Thus, in the past decade many resources have been di- ble of forming an HES to deliver a nucleic acid sequence rected toward developing methods of delivering oligonu- of interest into the cytoplasm and/or nucleus of cells and

10 17 EP 2 790 736 B1 18 tissues of an organism in vivo. The HES-oligonucleotide when they are separated, e.g. in solution at approximate- delivery vehicle is nontoxic to cells and organisms. The ly 1 uM or less. The maximum of an HES absorbance superior sequence-independent cell membrane perme- spectrum as compared with spectra of the individual ability of delivery vehicles of the invention facilitates the fluorophores shows the maximum absorbance wave- ability of oligonucleotides contained in the HES-oligonu- 5 length to be shifted to a shorter wavelength, i.e., a blue cleotide complex to cross membranes in a receptor-in- shift. Fluorescence intensity of H-type Excitonic Struc- dependent manner and leads to increased delivery and tures or aggregates (herein "HES") exhibits an intensity targetingof theoligonucleotide tocomplementary nucleic less than those of its components. Either a blue shift in acid sequences in the cytoplasm as well as in the nucleus the absorbance spectrum or a decrease in fluorescence of live cells. HES-oligonucleotide delivery systems for 10 intensity behavior of the H-type excitonic structures or use of the invention can also be used to target nucleic aggregates can be utilized as an indicator of a signal acid sequences of bacterial or viral origin. Moreover, the reporter moiety. In preferred embodiments two or more HES-oligonucleotide delivery vehicles for use of the in- fluorophores in the HES-oligonucleotide complex in- ventionhave applications inthe delivery ofa diverse array crease or quench by at least 50%, preferably by at least of diagnostic and functional oligonucleotides to cells in 15 70%, more preferably by at least 80%, and most prefer- vivo, including but not limited to, antisense oligonucle- ably by at least 90%, 95%, or even at least 99%. Exam- otides, siRNAs, shRNAs, Dicer substrates, ribozymes, ples of fluorophores that can form H-type excitonic struc- miRNAs,anti-miRNAs, aptamers, decoys,protein coding tures include xanthenes, cyanines and coumarins. sequences, or any nucleic acid sequence in a living or- [0060] In some embodiments, the HES-oligonucle- ganism. Such living organisms include, for example,20 otide complex contains a fluorophore selected from the mammals, plants, and microorganisms such as bacteria, group consisting of: carboxyrhodamine 110, carboxyte- protozoa, and viruses. tramethylrhodamine, carboxyrhodamine-X, diethylami- [0057] Where aspects or embodiments of the invention nocoumarin and a carbocyanine dye. In further embod- are described in terms of a Markush group or other group- iments, the HES-oligonucleotide complex contains a ing of alternatives, the present invention encompasses 25 fluorophore selected from the group consisting of: Rhod- not only the entire group listed as a whole, but also each amine Green™ carboxylic acid, succinimidyl ester or hy- member of the group individually and all possible sub- drochloride; Rhodamine Green™ carboxylic acid, trif- groups of the main group, and also the main group absent luoroacetamide or succinimidyl ester; Rhodamine oneor more ofthe groupmembers. The present invention Green™-X succinimidyl ester or hydrochloride; Rhodol also envisages the explicit exclusion of one or more of 30 Green™ carboxylic acid, N,O-bis-(trifluoroacetyl) or suc- any of the group members in the claimed invention. cinimidyl ester; bis-(4-carboxypiperidinyl) sulfonerhod- [0058] The term "and/or" as used in a phrase such as amine or di(succinimidyl, ester); 5-(and-6)-carboxynaph- "A and/or B" herein is intended to include both A and B; thofluorescein, 5-(and-6)-carboxynaphthofluorescein A or B; A (alone); and B (alone). Likewise, the term succinimidyl ester; 5-carboxyrhodamine 6G hydrochlo- "and/or" as used in a phrase such as "A, B, and/or C" is 35 ride; 6-carboxyrhodamine 6G hydrochloride, 5-carbox- intended to encompass each of the following embodi- yrhodamine 6G succinimidyl ester; 6-carboxyrhodamine ments: A, B, and C; A, B, or C; A or C; A or B; B or C; A 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G and C; A and B; B and C; A (alone); B (alone); and C succinimidyl ester; 5-carboxy-2’,4’,5’,7’-tetrabromosul- (alone). fonefluorescein succinimidyl ester or bis-(diisopropyle- [0059] The fluorophores in the oligonucleotide com- 40 thyl ammonium) salt; 5-carboxytetramethylrhodamine; plexes used in the invention can be any fluorophores in 6-carboxytetramethylrhodamine; 5-(and-6)-carboxyte- the complex that are capable of forming an HES with a tramethylrhodamine; 5-carboxytetra methylrhodamine homotypic or heterotypic cognate fluorophore(s) in the succinimidyl ester; 6-carboxytetramethylrhodamine suc- complex. In some embodiments, the HES-oligonucle- cinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine otide complex comprises 2 fluorophores capable of form- 45 succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy- ing an H-type excitonic structure. In additional, embodi- X-rhodamine succinimidyl ester; 6-carboxy-X-rhodam- ments, the HES-oligonucleotide complex comprises 3, ine succinimidyl ester; 5-(and-6)-carboxy-X-rhodamine 4, 5 or more fluorophores capable of forming an H-type succinimidyl ester; 5-carboxy-X-rhodamine triethylam- excitonic structure. In further embodiments, the HES-ol- monium salt; Lissamine™ rhodamine B sulfonyl chloride; igonucleotide complex contains from about 2-20, from 50 malachite green isothiocyanate; Rhodamine Red™-X about 2-10, from about 2-6, or from about 2-4 fluoro- succinimidyl ester; 6-(tetramethylrhodamine-5-(and- phores capable of forming an H-type excitonic structure. 6)-carboxamido)hexanoic acid succinimidyl ester; te- In additional embodiments, the HES-oligonucleotide tramethylrhodamine-5-isothiocyanate; tetramethylrhod- complex comprises 3, 4, 5 or more fluorophores capable amine-6-isothiocyanate; tetramethylrhodamine-5- (and- of forming an H-type excitonic structure. Two or more 55 6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® fluorophores are said to quench each other in an HES sulfonyl chloride; Texas Red®-X STP ester or sodium when their aggregate fluorescence is detectably less salt; Texas Red®-X succinimidyl ester; Texas Red®-X than the aggregate fluorescence of the fluorophores succinimidyl ester; X-rhodamine-5-(and-6)-isothiocy-

11 19 EP 2 790 736 B1 20 anate; and the carbocyanines. the invention are sequence independent and according- [0061] In some embodiments, the HES-oligonucle- ly, the oligonucleotides contained in the HES-oligonucle- otide complex contains a hetero-HES composed of dif- otide vehicles can be any form of nucleic acid or mimetic ferent fluorophore. In particular embodiments, the hete- that is known that would be desirable to be introduced ro-HES contains a rhodamine or rhodamine derivative 5 into a cell. and a fluorescein or a fluorescein derivative or two car- [0064] Oligonucleotides in the HES-oligonucleotide bocyanines. In further embodiments, the hetero-HES vehicles can be in the form of single-stranded, double- contains a fluorescein or fluorescein derivative selected stranded, circular or hairpin oligonucleotides. In some from: 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluores- embodiments, the oligonucleotides are single-stranded cein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-car- 10 DNA, RNA, or a nucleic acid mimetic (e.g., PMO, MNO, boxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-car- PNA, or oligonucleotides containing one or more modi- boxyfluorescein; 5-carboxyfluorescein-bis-(5-car- fied nucleotides such as a 2’OME and LNA). In some boxymethoxy-2-nitrobenzyl)ether, -alanine-carboxam- embodiments, the oligonucleotides are double-stranded ide, or succinimidyl ester; 5-carboxyfluorescein succin- DNA, RNA, nucleic acid mimetic, DNA/nucleic acid mi- imidyl ester; 6-carboxyfluorescein succinimidyl ester,15 metic, DNA-RNA and RNA-nucleic acid mimetic. 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6- [0065] The inventors have surprisingly discovered that dichlorotriazinyl) aminofluorescein; 2’,7’-difluorofluores- complexes containing HES-oligonucleotides such as cein; eosin-5-isothiocyanate; erythrosin-5-isothiocy- ssDNA and dsRNA display superior sequence independ- anate; 6-(fluorescein-5-carboxamido) hexanoic acid or ent intracellular delivery that require the administration succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxami- 20 of orders of magnitude of less oligonucleotides that that do) hexanoic acid or succinimidyl ester; fluorescein-5- required by conventional oligonucleotide delivery vehi- EX succinimidyl ester; fluorescein-5-isothiocyanate; and cles. Examples of single-stranded nucleic acids con- fluorescein-6-isothiocyanate. tained in the complexes of the invention include, but are notlimited to, antisense, siRNA, shRNA, ribozymes,miR- Oligonucleotides 25 NA, antimiRNA, triplex-forming oligonucleotides and aptamers. [0062] In the context of this invention, the term "oligo- [0066] In some embodiments an oligonucleotide in an nucleotide"refers to an oligomer orpolymer of ribonucleic HES-oligonucleotide complex is single stranded DNA acid (RNA), deoxyribonucleic acid (DNA) or mimetics (ssDNA). In preferred embodiments, at least a portion of thereof. This term includes oligonucleotides composed 30 the ssDNA oligonucleotide specifically hybridizes with a of naturally-occurring nucleobases, sugars and covalent target RNA to form an oligonucleotide-RNA duplex. In internucleoside(backbone) linkages ( i.e., "unmodified ol- further preferred embodiments, the oligonucleotide-RNA igonucleotide), as well as oligomeric compounds having duplex is susceptible to an RNase cleavage mechanism non-naturally-occurring nucleobases, sugars and/or in- (e.g.,RNase H). Insome embodiments, asingle stranded ternucleoside linkages and/or analogs of DNA and/or 35 oligonucleotide in the complex comprises at least one RNA which function in a similar manner (i.e., nucleic modified backbone linkage, at least one modified sugar, acid "mimetics"). Such mimetic oligonucleotide are often and/or at least one modified nucleobase e.g., ( as de- preferred over native forms because of desirable prop- scribed herein). In some embodiments, a single stranded erties such as: enhanced affinity for nucleic acid target oligonucleotide in the complex comprises at least one and increased stability in the presence of nucleases. For 40 modified backbone linkage, at least one modified sugar, example, as used herein,and/or at least the one modified nucleobase e.g., ( as de- term "oligonucleotide" includes morpholino (MNO) scribed herein) and is capable of forming an oligonucle- wherein one or more ribose rings of the nucleotide back- otide-RNA duplex that is susceptible to an RNase cleav- bone is replaced with a morpholine ring and phosphoro- age mechanism. In particular embodiments, the single diamidate morpholino oligomers (PMOs) wherein one or 45 stranded oligonucleotide is a gapmer (i.e., as described more ribose ring of the nucleotide backbone is replaced herein or otherwise known in the art). In additional em- with a morpholine ring and the negatively charged inter- bodiments, an oligonucleotide in the HES-oligonucle- subunit linkages are replaced by uncharged phosphoro- otide complex comprises at least one modified backbone diamidate linkages. Likewise, the term oligonucleotide linkage, at least one modified sugar, and/or at least one encompasses PNAs in which one or more sugar phos- 50 modified nucleobase that decreases the sensitivity of the phate backbone of an oligonucleotide is replaced with an oligonucleotide to an RNase cleavage mechanism ( e.g., amide containing backbone. For the purposes of this as described herein). In particular embodiments, the sin- specification, and as sometimes referenced in the art, gle stranded oligonucleotide comprises at least one modified oligonucleotides that do not have a phosphorus 2’OME, LNA, MNO or PNA motif atom in their internucleoside backbone can also be con- 55 [0067] The inventors have also surprisingly discovered sidered to be oligonucleosides. Moreover the oligonucle- that HES-oligonucleotide complexes containing double otides may be refers to as oligomers stranded oligonucleotides display superior sequence in- [0063] The delivery of HES-oligonucleotide vehicles of dependent intracellular delivery of the double stranded

12 21 EP 2 790 736 B1 22 oligonucleotides (also in the nanomolar and mid-micro- ments, nucleic acid/oligonucleotides in the HES-oligonu- molar range) over conventional oligonucleotide delivery cleotide complexes of the invention range from about 5 vehicles. Examples of double-stranded DNA oligonucle- nucleotides to about 500 nucleotides, and preferably otides contained in the complexes of the invention in- from about 10 nucleotides to about 100 nucleotides in clude, but are not limited to, dsRNAi and dicer substrates 5 length. and other RNA interference reagents, and sequences [0073] In some embodiments, an oligonucleotide in the corresponding to structural genes and/or control and ter- HES-oligonucleotide complex comprises at least 8 con- mination regions. tiguous nucleobases that are complementary to a target [0068] In some embodiments, the oligonucleotide is a nucleic acid sequence. In various related embodiments, linear double-stranded RNA (dsRNA). In preferred em- 10 an oligonucleotide in the HES-oligonucleotide complex bodiments, the ds-RNA is susceptible to an RNase cleav- is from about 8 to about 100 monomeric subunits (used age mechanism (e.g., Dicer and Drosha (an RNase III interchangeably with the term "nucleotides" herein) or enzyme)). In additional embodiments, the dsRNA is able from about 8 to about 50 nucleotides in length. to be inserted into the RNA Induced Silencing Complex [0074] In additional embodiments an oligonucleotide (RISC) of a cell. In further embodiments, a RNA strand 15 in the HES-oligonucleotide complex ranges in length of the dsRNA is able to use the RISC complex to effect from about 8 to about 30 nucleotides, from about 15 to cleavage of an RNA target. about 30 nucleotides, from about 20 to about 30 nucle- [0069] In additional embodiments, the HES-oligonu- otides, from about 18 to 26 nucleotides, from about 19 cleotide complex contains a double stranded oligonucle- to 25 nucleotides, from about 20 to 25 or from about 21 otide in which one or both oligonucleotides contain at 20 to 25 nucleotides. least one modified backbone linkage, at least one mod- [0075] In further embodiments, an oligonucleotide in ified sugar, and/or at least one modified nucleobase. In the HES-oligonucleotide complex is 8, 9, 10, 11, 12, 13, preferred embodiments, the double strand oligonucle- 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, otide is susceptible to an RNase cleavage mechanism 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, (e.g., Dicer and Drosha (an RNase III enzyme). In addi- 25 44, 45, 46, 47, 48, 49 or 50 subunits (nucleotides) in tional embodiments, the double stranded oligonucleotide length. In particular embodiments, the oligonucleotides is able to be inserted into the RNA Induced Silencing are 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. Complex (RISC) of a cell. In further embodiments, an [0076] In particular embodiments, the HES-oligonucle- oligonucleotide strand of the double stranded oligonucle- otide complex contains a double strand of RNA oligonu- otide is able to use the RISC complex to effect cleavage 30 cleotides of between 21-25 nucleotides in length and of an RNA target. have 1, 2, or 3 nucleotide overhangs at either or both [0070] In further embodiments the HES-oligonucle- ends. In other embodiments, the HES-oligonucleotide otide complex contains a triple stranded oligonucleotide. complex contains a double strand of oligonucleotides in In some embodiments the oligonucleotide is a triple- which at least one of the oligonucleotide strands is a nu- stranded DNA/RNA chimeric. In some embodiments, the 35 cleic acid mimetic of between 21-25 nucleotides in length oligonucleotide complex contains at least one oligonu- and the double stranded oligonucleotide has a 1, 2, or 3 cleotide comprising at least one modified backbone link- nucleotide overhang at either or both ends. age, at least one modified sugar, and/or at least one mod- ified nucleobase. In particular embodiments, at least one Oligonucleotides containing Modifications oligonucleotide in the complex comprises at least one 40 2’OME, LNA, MNO or PNA motif. [0077] HES-oligonucleotide complexes useful in the [0071] Oligonucleotides in the HES-oligonucleotide invention preferably include oligonucleotides containing vehicles are routinely prepared linearly but can be joined one or more modified internucleoside linkages, modified or otherwise prepared to be circular and may also include sugar moieties and/or modified nucleobases. Such mod- branching.Separate oligonucleotidescan specifically hy- 45 ified oligonucleotides (i.e., mimetics) are typically pre- bridize to form double stranded compounds that can be ferred over native forms because of desirable properties blunt-ended or may include overhangs on one or both including for example, enhanced cellular uptake, en- termini. In particular embodiments, double stranded oli- hanced affinity for nucleic acid target, increased stability gonucleotides (e.g., dsRNA and double stranded oligo- in the presence of nucleases and/or increased inhibitory nucleotide in which at least one of the oligonucleotide 50 activity. strands is a nucleic acid mimetic) contained in the com- plexes of the invention are between 21-25 nucleotides in Modified Internucleoside Linkages length and have 1, 2, or 3 nucleotide overhangs at either or both ends. [0078] The term "oligonucleotide" as used herein, re- [0072] Oligonucleotides in the HES-oligonucleotide 55 fers to those oligonucleotides that retain a phosphorus complexes of the invention may be of various lengths, atom in their internucleoside backbone as well as those generally dependent upon the particular form of nucleic that do not have a phosphorus atom in their internucle- acid or mimetic and its intended use. In some embodi- osidebackbone. In some embodiments, oligonucleotides

13 23 EP 2 790 736 B1 24 in the HES-oligonucleotide complexes useful in the in- [0081] Another suitable phosphorus-containing modi- vention comprise one or more modified internucleoside fied internucleoside linkage is the N3’-P5’ phosphoroam- linkages. Modified internucleoside linkages in the oligo- idates (NPs) in which the 3’-hydroxyl group of the 2’-de- nucleotides useful in the invention may include for exam- oxyribose ring is replaced by a 3’-amino group. Oligonu- ple, any manner of internucleoside 5 cleotides containing NPs internucleoside linkages exhibit linkages known to provide enhanced nuclease stability high affinity towards complementary RNA and resistance to oligonucleotides relative to that provided by phos- to nucleases. Since phosphoroamidate do not induce phodiester internucleoside linkages. Oligonucleotides RNase H cleavage of the target RNA, oligonucleotides having modified internucleoside linkages include inter- containing these internucleoside linkages have applica- nucleoside linkages that retain a phosphorus atom as 10 tions in those instances where RNA integrity needs to be well as internucleoside linkages that do not contain phos- maintained, such as those instances in which the oligo- phorus. In some embodiments the oligonucleotides com- nucleotides modulation mRNA splicing. In some embod- prise modified internucleoside linkages that alternate be- iments, at least 2, 3, 4, 5, 10 or 15 of the internucleoside tween modified and unmodified internucleoside linkages. linkages contained in the oligonucleotide is a phospho- In some embodiments most of the internucleoside link- 15 roamidate linkage. In some embodiments, at least 1-10, ages in the oligonucleotide are modified. In further em- 1-20, 1-30 of the modified internucleoside linkages is a bodiments, every internucleoside linkage in the oligonu- phosphoroamidate linkage. In some embodiments, at cleotide is modified. least 2, 3, 4, 5, 10 or 15 of the modified internucleoside [0079] Preferred modified oligonucleotide backbones linkages is a phosphoroamidates linkage. In additional include,for example, phosphorothioates, chiralphospho- 20 embodiments, each internucleoside linkage of an anti- rothioates, phosphorodithioates, phosphodiesters, sense compound is a phosphoroamidate internucleoside phosphotriesters, aminoalkyl-phosphotriesters, methyl linkage. and other alkyl phosphonates including 3’-alkylene phos- [0082] Numerous modified internucleoside linkages phonates, 5’-alkylene phosphonates and chiral phospho- and their method of synthesis are known in the art and nates, phosphinates, phosphoramidates including 3 ’- 25 encompassed by the modifications that may be con- amino phosphoramidate and aminoalkylphosphorami- tained in the oligonucleotides of the invention. Exemplary dates, thionophosphoramidates, thiono-alkylphospho- U.S. patents that teach the preparation of phosphorus- nates, thionoalkylphosphotriesters, selenophosphates containing internucleoside linkages include, but are not and boranophosphates having normal 3’-5’ linkages, 2’- limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 5’ linked analogs of these, and those having inverted po- 30 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,194,599; larity wherein one or more internucleotide linkages is a 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage. Preferred oligonucle- 5,399,676; 5,405,939; 5,489,677; 5,453,496; 5,455,233; otides having inverted polarity comprise a single 3’ to 3’ 5,466,677; 5,476,925; 5,519,126; 5,527,899; 5,536,821; linkage at the 3’-most internucleotide linkage i.e., a single 5,541,306; 5,550,111; 5,563,253; 5,565,555; 5,602,240; inverted nucleoside residue which may be abasic (the 35 5,571,799; 5,587,361; 5,625,050; 5,646,269; 5,663,312; nucleobase is missing or has a hydroxyl group in place 5,672,697; 5,677,439; and 5,721,218. thereof). Various salts, mixed salts and free acid forms [0083] HES-oligonucleotide complexes containing ol- are also included. igonucleotides that do not include a phosphorus atom [0080] In preferred embodiments, the HES-oligonucle- are also encompassed by the invention. Examples of otide complexes useful in the invention include at least 40 such oligonucleotides include those containing back- one phosphorothioate (PS) internucleoside linkage bones formed by short chain alkyl or cycloalkyl internu- wherein one of the nonbridging oxygen atoms in the cleoside linkages, mixed heteroatom and alkyl or cy- phosphodiester bond is replaced by sulfur. Oligonucle- cloalkyl internucleoside linkages, or one or more short otidescontaining PSinternucleoside linkage form regular chain heteroatomic or heterocyclic internucleoside link- Watson-Crick base pairs, activate RNase H, carry neg- 45 ages. These modified backbones include, but are not lim- ative charges for cell delivery and display other additional ited to oligonucleotides having morpholino linkages desirable pharmacokinetic properties. In some embodi- (formed in part from the sugar portion of a nucleoside); ments the at least one modified internucleoside linkage siloxane backbones; sulfide, sulfoxide and sulfone back- is phosphorothioate. In some embodiments, at least 2, bones; formacetyl and thioformacetyl backbones; meth- 3, 4, 5, 10 or 15 of the internucleoside linkages contained 50 ylene formacetyl and thioformacetyl backbones; ri- in the oligonucleotide is a phosphorothioate linkage. In boacetyl backbones; alkene containing backbones; sul- some embodiments, at least 1-10,1-20, 1-30 of the mod- famate backbones; methyleneimino and methylenehy- ified internucleoside linkages is a phosphorothioate link- drazino backbones; sulfonate and sulfonamide back- age. In some embodiments, at least 2, 3, 4, 5, 10 or 15 bones; amide backbones; and others having mixed N, 55 of the modified internucleoside linkages is a phospho- O, S and CH2 component parts. Methods of making oli- rothioate linkage. In additional embodiments, each inter- gonucleotides containing backbones that do not include nucleoside linkage of an oligonucleotide is a phospho- a phosphorous atom are known in the art and include, rothioate internucleoside linkage. but are not limited to, those methods and compositions

14 25 EP 2 790 736 B1 26 disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; compassed by the invention include PNA analogues in- 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; cluding PNAs having modified backbones with positively 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; charged groups and/or one or more chiral constrained 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; stereogenic centers at the C2(alpha), such as a D-amino 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,646,269; 5 acid, or C5(gamma), such as an L-amino acid e.g.,( L- 5,663,312; 5,633,360; 5,677,437; 5,677,439; 5,792,608. lysine) position of one or more monomeric units of the [0084] In some embodiments, oligonucleotides useful oligonucleotide. in the invention contain one or more modified backbone [0088] The RNAse and nuclease resistant properties linkages selected from: 3’-methylene phosphonate, of PNA oligonucleotides make them particularly useful in methylene (methylimino) (also known as MMI), mor-10 regulating RNA (e.g., mRNA and miRNA) in a cell via a pholino, locked nucleic acid, and a peptide nucleic acid steric block mechanism. In some embodiments, HES- linkage. The modified backbone linkages may be uniform oligonucleotides comprise at least one PNA oligonucle- or may be alternated with other linkages, particularly otide. In some embodiments, HES-oligonucleotides phosphodiester or phosphorothioate linkages, as long as comprise at least one PNA oligonucleotide and modulate RNAse H cleavage is not supported. 15 gene expression by strand invasion of chromosomal du- [0085] In some embodiments, the HES complexes plex DNA. In a further embodiment, HES-oligonucle- contain oligonucleotides that are nucleic acid mimetics. otides contain at least one PNA oligonucleotide and alter The term mimetic as it is applied to oligonucleotides is mRNA splicing in a subject. In additional embodiments, intended to include oligonucleotides wherein the sugar HES-oligonucleotides comprise at least one PNA oligo- or both the sugar and the internucleotide linkage are re- 20 nucleotide such as, a PMO, and act as an antisense. placed with alternative groups. I [0089] Similarly, the RNAse and nuclease resistant [0086] In some embodiments, the complexes useful in properties of morpholino containing oligonucleotides the invention contain an oligonucleotide having one or make these oligonucleotides: useful in regulating RNA more morpholino linkages. The RNAse and nuclease re- (e.g., mRNA and miRNA) in a cell via a steric block mech- sistant properties of morpholinos make them particularly 25 anism. In some embodiments, HES-oligonucleotides useful in regulating transcription in a cell. Accordingly, in comprise at least one morpholino oligonucleotide such some embodiments, a complex containing a morpholino as, a PMO, and modulate gene expression by strand unit is used to modulate gene expression. In some em- invasion of chromosomal duplex DNA. In a further em- bodiments, morpholino unit is a phosphorodiamidate bodiment, HES-oligonucleotides comprise at least one morpholino. In further embodiments, all the monomeric 30 morpholino oligonucleotide such as, a PMO, and alter units of the oligonucleotide correspond to a morpholino. mRNA splicing in a subject. In additional embodiments, In further embodiments, all the monomeric units of the HES-oligonucleotides comprise at least one morpholino oligonucleotide correspond to a phosphorodiamidate oligonucleotide such as, a PMO, and act as an antisense. morpholino. In particular embodiments, each monomeric [0090] Additionally, the RNAse and nuclease resistant unit of the oligonucleotide corresponds to a phosphoro- 35 properties of bicyclic sugar-containing nucleotides make diamidate morpholino (PMO). In additional embodiments these oligonucleotides useful in regulating RNA e.g., ( a complex containing a morpholino oligonucleotide ( e.g., mRNA and miRNA) in a cell via a steric block mechanism. PMO) is used to alter mRNA splicing in a subject. In ad- In some embodiments, complexes useful in the invention ditional embodiments, a complex containing one or more contain at least one bicyclic sugar containing nucleotide. morpholino nucleobases such as a PMO, is used as an 40 In some embodiments, the bicyclic sugar containing nu- antisense agent. cleotide is a locked nucleic acid (LNA). In further embod- [0087] In additional embodiments, an oligonucleotide iments, the LNA has a 2’-hydroxyl group linked to the 3’ a complex useful in the invention is a peptide nucleic acid or 4’ carbon atom of the sugar ring. In a further embod- (PNA). PNAs are nucleic acid mimetics in which the sugar iment, the oligonucleotide comprises at least one locked 45 phosphate backbone of an oligonucleotide is replaced nucleic acid (LNA) in which a methylene (--CH2--) n group with an amide containing backbone. In particular embod- bridges the 2’ oxygen atom and the 4’ carbon atom iments, the phosphate backbone of an oligonucleotide is wherein n is 1 or 2. In some embodiments, HES-oligo- replaced with an aminoethylglycine backbone and the nucleotides comprise at least bicyclic sugar containing nucleobases are bound directly or indirectly to aza nitro- nucleotide such as an LNA, and modulate gene expres- gen atoms of the amide portion of the backbone. Numer- 50 sion by strand invasion of chromosomal duplex DNA. In ous PNAs and methods of making PNAs are known in other embodiments, HES-oligonucleotides contain at the art (see, e.g., Nielsen et al., Science, 1991, 254, least one bicyclic sugar oligonucleotide, such as an LNA, 1497-150 and U.S. Pat. Nos. 5,539,082; 5,714,331; and and alter mRNA splicing in a subject. In additional em- 5,719,262), PNA containing oligonucleotides bodiments, HES-oligonucleotides comprise at least one provide increased stability and favorable hybridization ki- 55 bicyclic sugar oligonucleotide, such as an LNA, and act netics and have a higher affinity for RNA than DNA com- as an antisense. pared to unsubstituted counterpart nucleic acids and do not activate RNAse H mediated degradation. PNAs en-

15 27 EP 2 790 736 B1 28

Modified Sugar Moieties the complexes useful in the invention comprise at least one 2-substituted sugar having: a 2’-dimethylaminoox- [0091] In some embodiments, oligonucleotide com- yethoxy (2’-O(CH2)2ON(CH3)2 group, also known as 2’- pounds useful in the invention comprise one or more nu- DMAOE) substituent group; a 2’-dimethylaminoethox- 5 cleosides having one or more modified sugar moieties yethoxy (2’-O-CH2--O--CH2--N(CH2)2, also known as 2’- which are structurally distinguishable from, yet function- O-dimethylaminoethoxyethyl or 2’-DMAEOE) substitu- ally interchangeable with, naturally occurring or synthetic ent group; or a 2’-O-methyl (2’-O-CH 3) substituent group. unmodified nucleobases. In further embodiments, the ol- In further embodiments, an oligonucleotide in a complex igonucleotide in the HES- oligonucleotide complex com- ofthe inventioncomprises at leastone 2’-substituted sug- prises a modified sugar at each nucleoside (unit). 10 ar having a 2’-fluoro (2’-F) substituent group. [0092] Examples of sugar modifications useful in the [0097] In some embodiments, oligonucleotides in the oligonucleotides useful in the invention include, but are complexes useful in the invention contain at least one not limited to, compounds comprising a sugar substituent bicyclic sugar. In specific embodiments, the oligonucle- group selected from: OH; F; O-, S-, or N-alkyl; or O-alkyl- otides have at least one locked nucleic acid (LNA) in O-alkyl, wherein the alkyl, alkenyl and alkynyl may be 15 which the 2’-hydroxyl group is linked to the 3’ or 4’ carbon substituted or unsubstituted C1 to C10 alkyl or C2 to C10 atom of the sugar ring. In a particular embodiment, the alkenyl and alkynyl. oligonucleotides comprise at least one locked nucleic ac- [0093] Representative modified sugars include carbo- id (LNA) in which a methylene (--CH2--)n group bridges cyclicor acyclic sugars, sugars having substituentgroups the 2’ oxygen atom and the 4’ carbon atom wherein n is at one or more of their 2’, 3’ or 4’ positions, sugars having 20 1 or 2. In another embodiment, the oligonucleotide con- substituents in place of one or more hydrogen atoms of tains at least one bicyclic modified nucleoside having a the sugar, and sugars having a linkage between any two bridge between the 4’ and the 2’ ribosyl ring atoms where- other atoms in the sugar. Examples of 2’-sugarsubstitu- in the bridge is selected from selected from: 4’-(CH 2)-O- ent groups useful in the oligonucleotides of the invention 2’ (LNA); 4’-(CH2)-S-2; 4’-(CH2)2-O-2’ (ENA); 4’- 25 include, but are not limited to: OH; F; O-, S-, or N-alkyl; C(CH3)2-O-2’; 4’-CH(CH3)-Q-2’; 4’-CH(CH2OCH3)-O-2’; O-, S-, or N-alkenyl; allyl, amino; azido; thio; O-allyl; 4’-CH2-N(OCH3)-2’; 4’-CH2-O--N(CH3)-2’, 4’- O(CH2)2SCH3; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, CH2-N(R)-O-2’; 4’-CH2-CH(CH3)-2’ and 4’-CH2-C(-- wherein the alkyl, alkenyl and alkynyl may be substituted CH2)-2’, wherein R is independently, H, a C1-C12 alkyl, or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and or a protecting group. Oligonucleotides in the complexes alkynyl. In particular embodiments, the oligonucleotides 30 of the invention may also have at least one of the fore- contain at least one 2’-sugar substituent group selected going sugar configurations and an additional motif such from: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, as, alpha-L-ribofuranose, beta-D-ribofuranose or alpha- O(CH2)nCH3, 2)nONH O(CH2, andL-methyleneoxy (4’-CH2--O-2’). Further LNAs useful in O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to of the oligonucleotides of the invention and their prepa- about 10. Other preferred oligonucleotides contain at 35 ration are known in the art.See, e.g., U.S. Pat. Nos. least one 2’-sugar substituent group selected from: a C 1 6,268,490, 6,670,461, 7,217,805, 7,314,923,and to C 10lower alkyl, substitutedlower alkyl,alkenyl, alkynyl, 7,399,845; WO 98/39352 and WO 99/14226; and Singh alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3, OCN, et al, Chem. Commun., 1998, 4, 455-456. Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, [0098] In some embodiments, oligonucleotides in the 40 N3, HH2, heterocycloalkyl, heterocycloalkaryl, ami- complexes useful in the invention comprise a chemically noalkylamino, polyalkylamino, substituted silyl, an RNA modified furanosyl (e.g., ribofuranose) ring moiety. Ex- cleaving group, a reporter group, an intercalator, a group amples of chemically modified ribofuranose rings in- for improving pharmacokinetic properties, or a group for clude, but are not limited to, addition of substitutent improving the pharmacodynamic properties of an oligo- groups (including 5’ and 2’ substituent groups, and par- nucleotide compound, and other substituents having45 ticularly the 2’ position, bridging of non-geminal ring at- similar properties. oms to form bicyclic nucleic acids (BNA), replacement of [0094] In particular embodiments, the oligonucleotides the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 in the complexes useful in the invention comprise at least (R--H, C1-C12 alkyl or a protecting group) and combina- one 2-substituted sugar having a 2’-methoxyethoxy (2’- tions thereof. Examples of chemically modified sugars 50 O-CH2CH2OCH3, aka 2’-MOE) substituent group. include 2’-F-5’-methyl substituted nucleoside (see e.g., [0095] In some embodiments the oligonucleotides in WO 2008/101157, for other disclosed 5’,2’-bis substitut- the complexes useful in the invention comprise at least ed nucleosides) or replacement of the ribosyl ring oxygen one 2’-modified nucleoside selected from the group: 2’- atom with S with further substitution at the 2’-position allyl (2’-CH2-CH-CH2), 2’-O-allyl (2’-O--CH2--CH--CH2), (see e.g., US20050130923) or alternatively 5’-substitu- 55 2’-aminopropoxy (2’-OCH2CH2CH2NH2), and 2’-aceta- tion of a BNA (WO 2007/134181 wherein LNA is substi- mido (2’--O-CH2C(--O)NR1R1 wherein each R1 is inde- tuted with for example, a 5’-methyl or a 5’-vinyl group). pendently, H or C1-C1 alkyl. [0099] Complexes containing oligonucleotides com- [0096] In further embodiments the oligonucleotides in prising at least one nucleotide having a similar modifica-

16 29 EP 2 790 736 B1 30 tion to those described above, at the 3’ position of the prises at least one 5’ methylcytosine or a C-5 propyne. sugar on the 3’ terminal nucleotide or in 2’-5’ linked oli- In some embodiments, each cytosine in the oligonucle- gonucleotides and the 5’ position of 5’ terminal nucleotide otide is a methylcytosine. are also encompassed by the invention. Representative [0106] Modified nucleobases are also referred to here- U.S. patents that teach the preparation of 2’-modified nu- 5 in as heterocyclic base moieties and include other syn- cleosides contained in the oligonucleotides of the inven- thetic and natural nucleobases such as xanthine, hypox- tion include, but are not limited to, U.S. Pat. Nos. anthine, 2-aminoadenine, 6-methyl and other alkyl deriv- 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; atives of adenine and guanine, 2-propyl and other alkyl 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; derivatives of adenine and guanine, 2-thiouracil, 2-thi- 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 10 othymine and 2-thiocytosine, 5-halouracil and cytosine, 5,646,265; 5,658,873; 5,670,633; 5,700,920; and 5-propynyl(--CC--CH3) uracil and cytosine and other 5,792,747. alkynyl derivatives of pyrimidine bases, 6-azo uracil, cy- [0100] In some embodiments, the oligonucleotides in tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, the complexes useful in the invention have at least one 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other heterocyclic bicyclic nucleic acid. For example, in some 15 8-substituted adenines and guanines, 5-halo particularly embodiments, the oligonucleotides have at least one 5-bromo, 5-trifluoromethyland other 5-substituted uracils ENA motif (see, e.g., WO 01/49687). and cytosines, 7-methylguanine and 7-methyladenine, [0101] In additional embodiments, the oligonucle- 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8- otides in the complexes useful in the invention have at azaadenine, 3-deazaguanine and 3-deazaadenine. least one replacement of a five-membered furanose ring 20 [0107] Heterocyclic base moieties contained in the ol- by a six-membered ring. In at least one embodiment, the igonucleotides useful in the invention may also include oligonucleotides have at least one cyclohexene nucleic those in which the purine or pyrimidine base is replaced acid (CeNAs). They form stable duplexes with comple- with other heterocycles such as, 7-deazaadenine, 7-dea- mentary DNA or RNA and protect oligonucleotides zaguanosine, 2-aminopyridine and 2-pyridone. Nucleo- against nucleolytic degradation. 25 bases that are particularly useful for increasing the bind- [0102] In some embodiments, the oligonucleotides in ing affinity of the oligonucleotides useful in the invention the complexes useful in the invention have at least one include 5-substituted pyrimidines, 6-azapyrimidines and tricyclo-DNA (tcDNA). N-2, N-6 and O-6 substituted purines, including 2 ami- [0103] In particular embodiments, the oligonucleotides nopropyladenine, 5-propynyluracil and 5-propynylcyto- in the complexes useful in the invention contain phos- 30 sine. phorothioate backbones and oligonucleosides with het- [0108] Additional modified nucleobases that are op- eroatom backbones, such as --CH2--NH--O--CH2--, -- tionally included in the oligonucleotides useful in the in- CH2--N(CH3)--O--CH2---(also known as a methylene vention, include, but are not limited to, tricyclic pyrimi- (methylimino) or MMI backbone), --CH2--O--N(CH3)-- dines such as phenoxazine cytidine (1H-pyrimido[5,4- CH2--, --CH2--N(CH3)-N(CH3)--CH2-- and --Q--35 b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine N(CH3)--CH2--CB2-, and an amide backbone ( see, e.g., (1H-pyrimido[5,4-b][1,4]-benzothiazin-2(3H)-one), G- U.S. Pat. No. 5,602,240). In additional embodiments, the clamps such as a substituted phenoxazine cytidine ( e.g., oligonucleotides in the complexes useful in the invention 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin- have a phosphorodiamidate backbone structure. In fur- 2(3H)-one), carbazole cytidine (2H-pyrimido [4,5-b]indol- ther embodiments, the oligonucleotides in the complexes 40 2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrro- useful in the invention have a phosphorodiamidate mor- lo[2,3-d]pyrimidin-2-one), or guanidinium G-clamps and pholino (i.e., PMO) backbone structure (see, e.g., U.S. analogs. Representative guanidino substituent groups Pat. No. 5,034,506). are disclosed in U.S. Pat. No. 6,593,466. Representative acetamido substituent groups are disclosed in U.S. Pat. Modified Nucleobases 45 No. 6,147,200. [0109] Numerous modified nucleobases encom- [0104] Oligonucleotides in the complexes useful in the passed by the oligonucleotides contained in the complex- invention may also contain one or more nucleobase mod- es useful in the invention and their methods of synthesis ifications which are structurally distinguishable from, yet are known in the art, and include, for example, the mod- functionally interchangeable with, naturally occurring or 50 ified nucleobases disclosed in The Concise Encyclope- synthetic unmodified nucleobases. dia Of Polymer Science And Engineering, pages [0105] The terms "unmodified" or "natural" nucleobas- 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; es as used herein, include the purine bases adenine (A) Englisch et al., Angewandte Chemie, International Edi- and guanine (G), and the pyrimidine bases thymine (T), tion, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense cytosine (C) and uracil (U). Modified nucleobases include 55 Research and Applications, pages 289-302; Crooke, S. synthetic and natural nucleobases such as, for example, T. and Lebleu, B., ed., CRC Press, 1993; and U.S. Pat. 5-methylcytosine (5-me-C). In some embodiments, an Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; oligonucleotide in a complex useful in the invention com- 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;

17 31 EP 2 790 736 B1 32

5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; use uniformly modified oligonucleotides, such as designs 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; using modified oligonucleotides that do not support 5,646,269; 5,681,941; 5,750,692; 5,830,653; 5,763,588; RNAse H activity at each nucleotide or nucleoside posi- 6,005,096;6,028,183 and 6,007,992 andU.S. Appl. Publ. tion. As used in the present invention the term "fully mod- No. 20030158403. 5 ified motif" is meant to include a contiguous sequence of sugar modified nucleosides wherein essentially each nu- Chimeric Oligonucleotides: cleoside is modified to have the same modified sugar moiety. Suitable sugar modified nucleosides for fully [0110] The oligonucleotides in the complexes useful in modified oligonucleotides of the invention include, but 10 the invention preferably contain one or more modified are not limited to, 2’-Fluoro (2’F), 2’-O(CH2)2OCH3 (2’- internucleoside linkages, modified sugar moieties and/or MOE), 2’-OCH3 (2’-O-methyl), and bicyclic sugar modi- modified nucleobases. In some embodiments, oligonu- fied nucleosides. In one aspect the 3’ and 5’-terminal cleotides are chimeric oligonucleotides ( e.g., chimeric ol- nucleosides are left unmodified. In a preferred embodi- igomeric compounds). The terms "chimeric oligonucle- ment, the modified nucleosides are either 2’-MOE, 2’-F, otides" or "chimeras" are oligonucleotides that contain at 15 2’-O-Me or a bicyclic sugar modified nucleoside. least 2 chemically distinct regions (i.e., patterns and/or [0113] Oligonucleotides used in the compositions use- orientations of motifs of chemically modified subunits ar- ful in the present invention can also be modified to have ranged along the length of the oligonucleotide) each one or more stabilizing groups. In some embodiments, made up of at least one monomer unit, i.e., a nucleotide the stabilizing groups are attached to one or both termini or nucleoside in the case of a nucleic acid based oligo- 20 of oligonucleotides to enhance properties such as, nu- nucleotide compound. Chimeric oligonucleotides have clease stability. In some embodiments, the stabilizing also been referred to as for example, hybrids e.g.( , fu- groups are cap structures. By "cap structure or terminal sions) and gapmers. Representative United States pat- cap moiety" is meant chemical modifications, which have ents that teach the preparation of such chimeric oligonu- been incorporated at either terminus of oligonucleotides cleotide structures include, but are not limited to, U.S. 25 (see for example WO 97/26270). These terminal modifi- Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; cations may serve to protect the oligonucleotides having 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; terminal nucleic acid molecules from exonuclease deg- 5,652,355; 5,652,356; and 5,700,922. radation and/or may help in the delivery and/or localiza- [0111] Chimeric antisense compounds typically con- tion of the oligonucleotide within a cell. The oligonucle- tain at least one region modified so as to confer increased 30 otide may contain the cap at the 5’-terminus (5 ’-cap), resistance to nuclease degradation, increased cellular the 3’-terminus (3 -cap), or both the 5’-terminus, and the uptake, increased binding affinity for the target nucleic 3 ’-termini. In the case of double-stranded oligonucle- acid, and/or increased inhibitory activity. By way of ex- otides, the cap may be present at either or both termini ample, gapmers are chimeric oligonucleotides compris- of either strand. Cap structures are known in the art and ing a contiguous sequence of nucleosides that is divided 35 include,for example, inverted deoxyabasic caps. Further into 3 regions, an central region (gap) flanked by two 3’ and 5 ’-stabilizing groups that can be used to cap one external regions (wings). Gapmer design typically in- or both ends of an oligonucleotide ( e.g., antisense) com- cludes a central region of about 5-10 contiguous 2’-de- pound to impart nuclease stability include those dis- oxynucleotides which serves as a substrate for RNase closed in WO 03/004602. H is typically flanked by one or two regions of 2’-modified 40 [0114] In some embodiments, the 5’-cap of an oligo- oligonucleotides that provide enhanced target RNA bind- nucleotide contained in an HES-oligonucleotide complex ing affinity, but do not support RNAse H cleavage of the useful in the invention includes a structure that is an in- target RNA molecule. Consequently, comparable results verted abasic residue (moiety), 4’,5’-methylene nucle- can often be obtained with shorter oligonucleotides hav- otide; 1-(beta-D-erythrofuranosyl) nucleotide, 4’-thio nu- ing substrate regions when chimeras are used, com-45 cleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nu- pared to for example, phosphorothioate deoxyoligonu- cleotide; L-nucleotides; alpha-nucleotides; modified cleotides hybridizing to the same target region. Other chi- base nucleotides; phosphorodithioate linkage; threo- meric oligonucleotides rely on regions conferring for ex- pentofuranosyl nucleotide; acyclic 3’,4’-seco nucleotide; ample, altered levels of binding affinity over the length of acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihy- an oligonucleotide for its target including regions of mod- 50 droxypentyl nucleotide, 3’-3’-inverted nucleotide moiety; ified nucleosides which exhibit either increased or de- 3’-3’-inverted abasic: moiety; 3’-2’-inverted nucleotide creased affinity as compared to the other regions. So moiety; 3’-2’-inverted abasic moiety; 1,4-butanediol called, "MOE-gapmers" have 2’-MOE modifications in phosphate; 3’-phosphoramidate; hexylphosphate; ami- the wings, often contain full PS backbones, and frequent- nohexyl phosphate; 3’-phosphate; 3’-phosphorothioate; ly include 5’MeC modifications on all cytosines. 55 phosphorodithioate; or bridging or non-bridging methyl- [0112] Alternatively, for those situations in which phosphonate moiety (see e.g., WO 97/26270). RNAse H activity may be undesirable, such as in the [0115] In some embodiments, the 3’-cap of an oligo- modulation of RNA processing, it may be preferable to nucleotide contained in an HES-oligonucleotide complex

18 33 EP 2 790 736 B1 34 useful in the invention includes for example a 4’,5’ -meth- Examples of functional groups that are routinely used in ylene nucleotide; 1-(bteta-D-erythrofuranosyl) nucle- bifunctional linking moieties include, but are not limited otide; 4’-thio nucleotide, carbocyclic nucleotide; 5’-ami- to, electrophiles for reacting with nucleophilic groups and no-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3- nucleophiles for reacting with electrophilic groups. In aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- 5 some embodiments, bifunctional linking moieties include aminododecyl phosphate; hydroxypropyl phosphate; amino, hydroxyl, carboxylic acid, thiol, unsaturations 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nu- (e.g., double or triple bonds), and the like. Some nonlim- cleotide; modified base nucleotide; phosphorodithioate; iting examples of bifunctional linking moieties include 8- threo-pentofuranasyl nucleotide; acyclic 3’,4-seco nucle- amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- otide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydrox-10 maleimidomethyl)cyclohexane-1-carboxylate (SMCC) ypentyl nucleotide, 5’-5’-inverted nucleotide moiety; 5’- and 6-aminohexanoic acid (AHEX or AHA). Other linking

5’-inverted abasic moiety; 5’-phosphoramidate; 5’-phos- groups include, but are not limited to, substituted C 1-C10) phorothioate; 1,4-butanedipl phosphate; 5’-amino; bridg- alkyl, substituted or unsubstituted C 2-C10 alkenyl or sub- ing and/or non-bridging 5’-phosphoramidate, phospho- stituted or unsubstituted C2-C10 alkynyl, wherein a non- rothioate and/or phosphorodithioate, bridging or non- 15 limiting list of preferred substituent groups includes hy- bridging methylphosphonate and 5’-mercapto moieties droxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thi- (see also the stabilizing groups disclosed in Beaucage ol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. et al, 1993, Tetrahedron 49: 1925). Further representative linking groups are disclosed for [0116] In some embodiments, an oligonucleotide in a example in WO 94/01550 and WO 94/01550. complex useful in the invention comprises one or more 20 [0119] Representative United States patents that cationic tails. In farther embodiments, the oligonucleotide teach the preparation of such oligonucleotide conjugates is conjugated with at least 1, 2, 3, 4 or more positively- include, but are not limited to, U.S. Pat. Nos. 4,828,979; charged amino acids such as, lysine or arginine. In spe- 4,948,882; 5,109,124; 5,118,802; 5,218,105; 5,414,077; cific embodiments, the oligonucleotide is a PNA and one 5,486,603; 5,525,465; 5,541,313; 5,545,730; 5,552 538; or more lysine or arginine residues are conjugated to the 25 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,512,439; C-terminal end of the molecule. In a further preferred 5,578,718; 4,587,044; 4,605,735; 4,667,025; 4,762,779; embodiment, the oligonucleotide is a PNA and comprises 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; from 1 to 4 lysine and/or arginine residues are conjugated 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,245,022; to each PNA linkage. 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; [0117] In one embodiment such modified oligonucle- 30 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; otides are prepared by covalently attaching conjugate 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; groups to functional groups such as hydroxyl or amino 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; groups. Useful conjugate groups include, but are not lim- 5,599,923; 5,599,928, 5,688,941 and 6,114,513, and ited to, intercalators, reporter molecules, polyamines, U.S. Publ. Nos. 2012/0095075; 2012/0101148; and polyamides, polyethylene glycols, polyethers, and35 2012/0128760. groups that enhance the pharmacodynamic or pharma- [0120] In additional related embodiments, the present cokinetic properties of the oligonucleotides. Typical con- invention includes HES-oligonucleotide complexes jugate groups include cholesterols, carbohydrates, bi- and/or pharmaceutical compositions containing HES-ol- otin, phenazine, folate, phenanthridine and anthraqui- igonucleotide complexes that further comprise one or none. Representative conjugate groups are disclosed in 40 more active agents or therapeutic agents. In one embod- WO/1993/007883 and U.S. Pat. No. 6,287,860. iment the active agent or therapeutic agent is a nucleic [0118] Conjugate groups can be attached to various acid. In various embodiments, the nucleic acid is a plas- positions of an oligonucleotide directly or via an optional mid, an immunostimulatory oligonucleotide, a siRNA, a linking group. The term linking group is intended to in- shRNA, a miRNA, an anti-mjRNA, a dicer substrate, a clude all groups amenable to attachment of a conjugate 45 decoy, an aptamer, an antisense oligonucleotide, or a group to an oligomeric compound. Linking groups are ribozyme. bivalent groups useful for attachment of chemical func- tional groups, conjugate groups, reporter groups and oth- Oligonucleotide Synthesis er groups to selective sites in a parent compound such as for example an oligomeric compound. In general a 50 [0121] Oligonucleotides and phosphoramidites can be bifunctional linking moiety comprises a hydrocarbyl moi- synthesized and/ormodified by methods well established ety having two functional groups. One of the functional in the art. Oligomerization of modified and unmodified groups is selected to bind to a parent molecule or com- nucleosides is performed according to literature proce- pound of interest and the other is selected to bind essen- dures for DNA-like compounds (Protocols for Oligonu- tially any selected group such as chemical functional55 cleotides and Analogs, Ed. Agrawal (1993), Humana group or a conjugate group. In some embodiments, the Press) and/or RNA-like compounds (Scaringe, Methods linker comprises a chain structure or an oligomer of re- (2001), 23, 206-217. Gait et al., Applications of Chemi- peating units such as ethylene glycol or amino acid units. cally synthesized RNA in RNA: Protein Interactions, Ed.

19 35 EP 2 790 736 B1 36

Smith (1998),1-36. Gallo et al., Tetrahedronof the invention are well known in the art and can be used 57:5707-5713 (2001),) synthesis as appropriate. see,( or routinely modified to prepare the HES- oligonucle- also, Current Protocols in Nucleic Acid Chemistry, Beau- otides useful in the invention. See, e.g., Connolly et al., cage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New Nucleic Acids Res. 13:4485-4502 (1985); Dreyer et al., York, N.Y., USA). Oligonucleotides are preferably chem- 5 Proc. Natl. Acad. Sci. 86:9752-9756 (1989) ; Nelson et ically synthesized using appropriately al., Nucleic Acids Res. 17:7187-7194 (1989); Sproat et protected reagents and a commercially available oligo- al., Nucleic Acids Res. 15, 6181-6196 (1987) and Zuck- nucleotide synthesizer. Suppliers of oligonucleotide syn- erman et al., Nucleic Acids Res. 15:5305-5321 (1987). thesis reagents useful in manufacturing the oligonucle- Many fluorophores normally contain suitable reactive otides of the invention include, but are not limited to, Pro- 10 sites. ligo (Hamburg, Germany), Dharmacon Research (Lafay- [0126] Alternatively, the fluorophores may be deriva- ette, CO, USA), Pierce Chemical (part of Perbio Science, tized to provide reactive sites for linkage to another mol- Rockford, IL , USA), Glen Research (Sterling, VA, USA), ecule. Fluorophores derivatized with functional groups ChemGenes (Ashland, MA, USA), and Cruachem (Glas- for coupling to a second molecule are commercially avail- gow, UK). Alternatively, oligomers may be purchased 15 able from a variety of manufacturers. The derivatization from various oligonucleotide synthesis companies such may be by a simple substitution of a group on the fluor- as, for example, Dharmaeon Research Inc., (Lafayette, ophore itself, or may be by conjugation to a linker. Colo.), Qiagen (Gemiantown, MD), Proligo and Ambion. [0127] Fluorophores are optionally attached to the 5’ [0122] In certain embodiments, the preparation of oli- and/or 3’ terminal backbone phosphates and/or other gonucleotides as disclosed herein is performed accord- 20 bases of the oligonucleotide via a linker. Various suitable ing to literature procedures for DNA: Protocols for Oligo- linkers are known to those of skill in the art and/or are nucleotides and Analogs, Agrawal, Ed., Humana Press, discussed below. In some embodiments, the linker is a 1993, and/or RNA: Scaringe, Methods, 2001, 23, flexible aliphatic linker. In additional embodiments, the 206-217; Gait et al., Applications of Chemically synthe- linker is a C1 to C30 linear or branched, saturated or sized RNA in RNA: Protein Interactions, Smith, Ed.,25 unsaturated hydrocarbon chain. In some embodiments, 1998, 1-36; Gallo et al., Tetrahedron, 2001, 57,the linker is a C2 to C6 linear or branched, saturated or 5707-5713. Additional methods for solid-phase synthesis unsaturated hydrocarbon chain. In additional embodi- may be found U.S. Patent Nos. 4,415,732; 4,458,066; ments the hydrocarbon chain linker is substituted by one 4,500,707; 4,668,777; 4,725,677; 4,973,679;and or more heteroatoms, aryls; or lower alkyls, hydroxyla- 5,132,418; and Re, 34,069. 30 lkyls or alkoxys. [0123] Irrespective of the particular protocol used, the [0128] In some embodiments, one or more fluoro- oligonucleotides used in accordance with this invention phores are incorporated into an oligonucleotide during may be conveniently and routinely made through the automated synthesis using one or more fluoropophore- well-known technique of solid phase synthesis. Equip- modified nucleosides, fluorophore and sugar/base/ ment for such synthesis is sold by several vendors in- 35 and/or linkage modified nucleosides, and/or deoxynucl- cluding, for example, Gene Forge (Redwood City, Calif.). eoside phosphoramidites. Suitable solid phase techniques, including automated [0129] In some embodiments, one or more fluoro- synthesis techniques, are described in Oligonucleotides phores are incorporated into an oligonucleotide in a post- and Analogues, a Practical Approach, F. Eckstein, Ed., synthesis labeling reaction. Appropriate post-synthesis Oxford University Press, New York, 1991. Any other40 labeling reactions are known in the art and can routinely means for such synthesis known in the art may addition- be applied or modified to synthesize the HES-oligonu- ally or alternatively be employed (including solution cleotides of the invention. In one embodiment, one or phase synthesis). more fluorophores are incorporated into an oligonucle- [0124] The synthesis and preparation of the bicyclic otide in a post-synthesis labeling reaction in which an sugar modified monomers adenine, cytosine, guanine, 45 amine- or thiol-modified nucleotide or deoxynucleotide 5-methyl-cytosine, thymine and uracil, along with their in the synthesized oligonucleotide is reacted with an oligomerization, and nucleic acid recognition properties amine- or thiol-reactive fluorophore such as, a succinim- have been described (Koshkin et al., Tetrahedron, idyl ester fluorophore. 54:3607-3630 (1998); WO 98/39352 and WO 99/14226). [0130] In further embodiments, one or more of the Other bicyclic sugar modified nucleoside analogs such 50 same fluorophores are integrated into the oligonucleotide as the 4’-CH 2--S-2’ analog have also been prepared (Ku- in a single reaction that involves contacting a reactive mar et al, Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). form of the dye with an oligonucleotide containing a de- Preparation of other bicyclic sugar analogs containing sired number of reactive groups capable of reacting with oligodeoxyribonucleotide duplexes as substrates for nu- the fluorpohore in a suitable buffer under conditions and cleic acid polymerases has also been described (WO 98- 55 for an amount of time sufficient to accomplish the inte- DK393 19980914). gration of the fluorphores into the oligonucleotide. The [0125] Techniques for linking fluorophores to oligonu- reactive groups can routinely be incorporated into the cleotides such as those used according to the methods oligonucleotide during synthesis using standard tech-

20 37 EP 2 790 736 B1 38 niques and reagents known in the art. mentalanions such as chlorine,bromine, andiodine. Pro- drugs include for example, the incorporation of additional Formulations: nucleosides at one or both ends of an oligonucleotide which are cleaved by endogenous nucleases within the [0131] The HES-oligonucleotide complexes are op- 5 body, to form the active oligonucleotide. tionally admixed with a suitable pharmaceutically accept- [0134] In some embodiments, prodrug versions of the able diluent or carrier pharmaceutically acceptable active oligonucleotides of the invention are prepared as SATE or inert substance for the preparation of pharmaceutical [(S-acetyl-2-thioethyl)phosphate] derivatives according compositions. Thus, also disclosed herein are pharma- to the methods disclosed in WO 93/24510and WO ceutical compositions that include HES-oligonucleotide 10 94/26764. complexes. Compositions and methods for the formula- [0135] In the context of the present invention, a phar- tion of pharmaceutical compositions are dependent upon maceutically acceptable diluent includes phosphate- a number of criteria, including, but not limited to, route of buffered saline (PBS). PBS is a diluent suitable for use administration, extent of disease, or dose to be adminis- in compositions to be tered. Such considerations are well understood by those 15 [0136] Pharmaceutical compositions disclosed herein skilled in the art. include, but are not limited to, solutions and formulations. [0132] Subject doses of the HES-oligonucleotides for These compositions may be generated from a variety of mucosal or local delivery typically range from about 0.1 components that include, but are not limited to, pre- ug to 50 mg per administration (e.g., in the case of exon formed liquids. skipping drugs such as AVI-4658 (morpholino) wherein 20 [0137] The pharmaceutical compositions can conven- trial doses include the administration of the drug at 30 iently be presented in unit dosage form and can be pre- mg/kg and 50 mg/kgwk IV), which depending on the ap- pared according to conventional techniques well known plication could be given daily, weekly, or monthly and in the pharmaceutical industry. Such techniques include any other amount of time there between. However, dos- the step of bringing into association the active ingredients ing may be at substantially higher or lower ranges. De- 25 with the pharmaceutical diluent(s) or carrier(s). In general termination of appropriate dosing ranges and frequency the formulations are prepared by uniformly and intimately is well within the ability of those skilled in the art. The bringing into association the active ingredients with liquid administration of a given dose can be carried out both carriers or finely divided solid carriers or both, and then, by single administration in the form of an individual dose if necessary, shaping the product. unit or else several smaller dose units. 30 [0138] The pharmaceutical compositions can be for- [0133] Pharmaceutical compositions comprising HES- mulated into any of many possible dosage forms includ- oligonucleotide complexes encompass any pharmaceu- ing, but not limited to, tablets, capsules, liquid syrups, tically acceptable salts, esters, or salts of such esters, or soft gels, suppositories, and enemas. The compositions any other oligonucleotide which, upon administration to can also be formulated as suspensions in aqueous or a subject such as a mouse, rat, rabbit or human, is ca- 35 mixed media. Aqueous suspensions can further contain pable of providing (directly or indirectly) the biologically substances which increase the viscosity of the suspen- active metabolite or residue thereof. Accordingly, for ex- sion including, for example, sodium carboxymethylcellu- ample, the disclosure is also drawn to physiologically and lose, sorbitol and/or dextran. The suspension may addi- pharmaceutically acceptable salts (i.e., salts that retain tionally contain one or more stabilizers. the desired biological activity of the parent compound 40 [0139] As used herein, the term "dose" refers to a spec- and do not impart undesired toxicological effects thereto) ified quantity of a pharmaceutical agent provided in a of HES-oligonucleotide complexes, prodrugs, physiolog- single administration. In certain embodiments, a dose ically and pharmaceutically acceptable salts of such pro- may be administered in one or more boluses, tablets, or drugs, and other bioequivalents. Suitable pharmaceuti- injections. For example, in certain embodiments, where cally acceptable salts include, but are not limited to (a) 45 subcutaneous administration is desired and the desired salts formed with cations such as sodium, potassium, dose requires a volume not easily accommodated by a ammonium, magnesium, calcium, polyamines such as single injection, then two or more injections may be used spermine and spermidine, etc.; (b) acid addition salts to achieve the desired dose. In certain embodiments, a formed with inorganic acids, for example hydrochloric ac- dose may be administered in two or more injections to id, hydrobromic acid, sulfuric acid, phosphoric acid, nitric 50 minimize injection site reaction in an individual. acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, Administration succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic [0140] Disclosed herein are pharmaceutical composi- acid, palmitic acid, alginic acid, polyglutamic acid, naph- 55 tions and formulations which include the HES-oligonu- thalenesulfonic acid, methanesulfonic acid, p-tolue- cleotide complexes useful in the invention. The invention nesulfonic acid, naphthalenedisulfonic acid, polygalac- can be practiced using any mode of administration that turonic acid, and the like; and (d) salts formed from ele- is medically acceptable, meaning any mode that produc-

21 39 EP 2 790 736 B1 40 es a therapeutic effect without causing clinically unac- [0145] In some embodiments, an HES-oligonucleotide ceptable adverse effects (i.e., where undesired effects complex can be administered to a subject via an oral are to such an extent so as to prohibit administration of route of administration. The subject may be a mammal, the HES-oligonucleotide complex). The pharmaceutical such as a mouse, a rat, a dog, a guinea pig, or a non- compositions may be administered in a number of ways 5 human primate. In some embodiments, the subject may depending upon whether local or systemic treatment is be a human subject. In certain embodiments, the subject desired and upon the area to be treated. Thus, for use may be in need of modulation of the level or expression in therapy, an effective amount of the HES-oligonucle- of one or more pri-miRNAs as discussed in more detail otide can be administered to a subject by any mode that herein. In some embodiments, compositions for admin- delivers the nucleic acid to the desired surface, e.g., mu- 10 istration to a subject cosal or systemic. Suitable routes of administration in- [0146] In the context of the present invention, a pre- clude, but are not limited, to topical oral, pulmonary, ferred meansfor delivery ofan HES-oligonucleotide com- parenteral, intranasal, intratracheal, inhalation, ocular, plex employs an infusion pump such as Medtronic Syn- vaginal, and rectal. Such formulations and their prepa- croMed® II pump. ration are well known by those skilled in the art, as are 15 [0147] The antisense oligonucleotides useful in the in- considerations for optimal dosing routes vention can be utilized for diagnostics, therapeutics, [0141] Administration may be topical (including oph- prophylaxis and as research reagents and kits. For ther- thalmic and to mucous membranes including vaginal and apeutics, a subject such as a mouse, rabbit or primate, rectal delivery), pulmonary, e.g., by inhalation or insuf- preferably a human, suspected of having a disease or flation of powders or aerosols, including by nebulizer; 20 disorder which can be treated by modulating the behavior intratracheal, intranasal, epidermal and transdermal), of a cell can be treated by administering an HES-oligo- peroral or parenteral. Parenteral administration includes nucleotide complex of the invention. intravenous, intraarterial, subcutaneous, intraperitoneal [0148] In some embodiments, the HES-oligonucle- or intramuscular injection or infusion; or intracranial, e.g., otide delivery system useful in the invention is combined intrathecal or intraventricular, administration. HES-oligo- 25 with one or more additional oligonucleotide delivery sys- nucleotides with at least one 2’-O-methoxyethyl modifi- tems to further facilitate HES-oligonucleotide complex cation, including chimeric molecules or molecules which delivery into a cell and/or targeted delivery of the oligo- may have a 2’-O-methoxyethyl modification of every nu- nucleotide, Marcromolecular delivery systems that can cleotide sugar, are believed to be particularly useful for be combined with the HES-oligonucleotide delivery sys- oral administration. Pharmaceutical compositions and 30 tem include, but are not limited to the use of dendrimers, formulations for topical administration may include biodegradable polymers. Additional, delivery systems transdermal patches, drops, suppositories, sprays, liq- that can be combined with the HES-oligonucleotide de- uids and powders. Conventional pharmaceutical carri- livery system include, but are not limited to, conjugates ers, aqueous, powder or and the like may be necessary with amino acids, sugars, or targeting nucleic acid motifs. or desirable. 35 In particular embodiments, an HES-oligonucleotide com- [0142] Compositions and formulations for oral admin- plex is conjugated with an aptamer, peptide, or antibody istration include powders or granules, suspensions or so- (or antibody fragment) that specifically hybridizes to a lutions in water, capsules, sachets or tablets. certain receptor or serum protein, which modulates the [0143] Compositions and formulations for parenteral, half-life of the complex or which facilitates the uptake of intrathecal or intraventricular administration may include 40 the complex. sterile aqueous solutions which may also contain buffers, [0149] The HES-oligonucleotide delivery system can diluents and other suitable additives and pharmaceuti- also be covalently attached to cholesterol molecules. cally acceptable carriers or excipients known in the art. [0150] The HES-oligonucleotide complexes useful in [0144] In certain embodiments, parenteral administra- the invention may be admixed, conjugated or otherwise tion is by infusion. Infusion can be chronic or continuous 45 associated with other molecules, molecule structures or or short or intermittent. In certain embodiments, infused mixtures of compounds, as for example, receptor target- pharmaceutical agents are delivered via cannulae or ed molecules, oral, rectal, topical or other formulations. catheters. In certain embodiments, infused pharmaceu- tical agents are delivered with a pump. In certain embod- Exemplary Modes of Action iments, the compounds and compositions as described 50 herein are administered parenterally. In additional em- Antisense bodiments, parenteral administration is by injection. The injection can be delivered with a syringe or a pump. In [0151] In some embodiments, an oligonucleotide in an certain embodiments, the injection is a bolus injection. HES-oligonucleotide complex is an antisense oligonu- In certain embodiments, the injection is administered di- 55 cleotide. The term "antisense oligonucleotide" or simply rectly to a tissue or organ. In additional embodiments, "antisense" is meant to include oligonucleotides corre- the parenteral administration comprises subcutaneous sponding to single strands of nucleic acids e.g.( , DNA, or intravenous administration. RNA and nucleic acid mimetics such as PNAs mor-

22 41 EP 2 790 736 B1 42 pholinos (e.g., PMOS), and compositions containing nucleotide is from about 10 to about 50 nucleotides, more modified nucleosides and/or internucleoside linkages) preferably about 15 to about 30 nucleotides. that bind to their cognate mRNA in the cells of the treated [0154] In additional embodiments, an oligonucleotide subject and modulate RNA function by for example., al- in a complex useful in the invention interferes with the tering the translocation of target RNA to the site of protein 5 transcription of a target RNA of interest. In some embod- translation, translation of protein from the target RNA, iments, the oligonucleotide interferes with transcription altering splicing of the target RNA (e.g., promoting exon of an mRNA or miRNA of interest by strand displacement. skipping) and altering catalytic activity which may be en- In other embodiments, the oligonucleotide interferes with gaged in or facilitated by the target RNA, and targeting the transcription of an mRNA by forming a stable complex the mRNA for degradation by endogenous RNase H. In 10 with a portion of a targeted gene by strand invasion or some embodiments, the antisense oligonucleotides alter triplex formation (triplex forming oligonucleotides cellular activity by hybridizing specifically with chromo- (THOs), such as those containing LNAs see, e.g., U.S. somal DNA. The term antisense oligonucleotide also en- Appl. Publ. No. 2012/0122104). In additional embodi- compasses antisense oligonucleotides that may not be ments, the HES-oligonucleotides of the exactly complementary to the desired target gene. Thus, 15 invention interfere with the transcription of a target RNA the invention can be utilized in instances where non-tar- (e.g., mRNA or miRNA) by interfering with the transcrip- get specific-activities are found with antisense, or where tion apparatus of the cell. In some embodiments, the an antisense sequence containing one or more mis- HES-oligonucleotides are designed to specifically bind a matches with the target sequence is preferred for a par- region in the 5’ end of an mRNA or the AUG start codon ticular use. The overall effect of such interference with 20 (e.g., within 30 nucleotides of the AUG start codon) and target nucleic acid function is modulation of a targeted to reduce translation. In some embodiments, the HES- protein of interest. In the context of the present invention, oligonucleotides are designed to specifically hybridize to "modulation" means either an increase (stimulation) or a an intron/exon junction in an RNA. In some embodi- decrease (inhibition) in the expression of a gene or pro- ments, the HES-oligonucleotides are designed to specif- tein in the amount, or levels, of a small non-coding RNA, 25 ically bind the 3’ untranslated target sequence in an RNA nucleic acid target, an RNA or protein associated with a (e.g., mRNA). In further embodiments, the HES-oligonu- small non-coding RNA, or a downstream target of the cleotides are designed to specifically bind nucleotides small non-coding RNA ( e.g., a mRNA representing a pro- 1-10 of a miRNA. In additional embodiments, the HES- tein-coding nucleic acid that is regulated by a small non- oligonucleotides are designed to specifically bind a se- coding RNA). Inhibition is a suitable form of modulation 30 quence in a precursor-miRNA (pre-miRNA) or primary- and small non-coding RNA is a suitable nucleic acid tar- miRNA (pri-miRNA) that when bound by the oligonucle- get. Small non-coding RNAs whose levels can be mod- otide blocks miRNA processing. ulated include miRNA and miRNA precursors. In the con- [0155] In other embodiments, the HES-oligonucle- text of the present disclosure, "modulation of function" otides target sites of critical RNA secondary structure or means an alteration in the function or activity of the small 35 act as steric blockers that cause truncation of the trans- non-coding RNA or an alteration in the function of any lated polypeptide. In some embodiments, the HES-oli- cellular component with which the small non-coding RNA gonucleotides (e.g., PNAs and PMOs) are designed to has an association or downstream effect. In one embod- interfere with intron excision, by for example, binding at iment, modulation of function is an inhibition of the activity or near a splice junction of the targeted mRNA. In some of a small non-coding RNA. 40 embodiments, the HES-oligonucleotide are designed to [0152] Antisense oligonucleotides are preferably from interferewith intron excision or toincrease the expression about 8 to about 80 contiguous linked nucleosides in of an alternative splice variant. length. In some embodiments, the antisense oligonucle- [0156] RNase H is an endogenous enzyme that spe- otides are from about 10 to about 50 nucleosides or from cifically cleaves the RNA moiety of an RNA:DNA duplex. about 13 to about 30 nucleotides. Antisense oligonucle- 45 In some embodiments, the antisense oligonucleotides otides of the invention include ribozymes, antimiRNAs, elicit RNase H activity when bound to a target nucleic external guide sequence (EGS) oligonucleotides (oli- acid. In some embodiments, the oligonucleotides are gozymes), and other short catalytic RNAs or catalytic ol- DNA or nucleic acid mimetics. HES-oligonucleotides that igonucleotides which specifically hybridize to the target elicit RNase H activity have particular advantages in for nucleic acid and modulate its expression. 50 example, harnessing endogenous ribonucleases to re- [0153] The antisense oligonucleotides in accordance duces targeted RNA. with this invention comprise from about 15 to about 30 [0157] One antisense design for eliciting RNase H ac- nucleosides in length, (i.e., from 15 to 30 linked nucleo- tivity is the gapmer motif design in which a chimeric oli- sides) or alternatively, from about 17 to about 25 nucle- gonucleotide with a central block composed of DNA, ei- osides in length. In particular embodiments, an antisense 55 ther with or without phosphorothioate modifications, and oligonucleotide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, nuclease resistant 5’ and 3’ flanking blocks, usually 2’- 19,20, 21, 22, 23,24, 25,26, 27,28, 29,or 30 nucleosides O-methyl RNA but a wide range of 2’ modifications have in length. In additional embodiments, an antisense oligo- been used (see Crooke, 2004). Other gapmer designs

23 43 EP 2 790 736 B1 44 are described herein or otherwise known in the art. or phosphorodiamidate morpholino linkage. In further [0158] In additional embodiments, antisense oligonu- embodiments, the antisense oligonucleotide contains at cleotides in the complexes of the invention are designed least one modified nucleobase. Preferably, the modified to avoid activation of RNase H in a cell. Oligonucleotides nucleobase is a C-5 propyne or 5-methyl C. that do not elicit RNase H activity have particular advan- 5 [0162] The antisense oligonucleotide compounds use- tages in for example, blocking transcriptional machinery ful in the invention can routinely be synthesized using (via a steric block mechanism) and altering splicing of techniques known in the art. the target RNA. In some embodiments, the oligonucle- otides are designed to interfere with and/or alter intron RNAi - post transcriptional gene silencing excision, by for example, binding at or near a splice junc- 10 tion of the targeted mRNA. In additional embodiments, [0163] Short double-stranded RNA molecules and the oligonucleotides are designed to increase the expres- short hairpin RNAs (shRNAs), i.e. fold-back stem-loop sion of an alternative splice variant of a message. In one structures that give rise to siRNA can induce RNA inter- preferred embodiment, the antisense oligonucleotide ference (RNAi). In some embodiments, an oligonucle- useful in the invention is a morpholino (e.g., PMO). In 15 otide in an HES-oligonucleotide complex useful in the another preferred embodiment, the antisense oligonu- invention induces RNAi. RNAi oligonucleotides in the cleotide useful in the invention is a PNA. complexes useful in the invention include, but are not [0159] In particular embodiments, the antisense oligo- limited to siRNAs, shRNAs and dsRNA DROSHA and/or nucleotide is targeted to at least a portion of a region up Dicer substrates. The siRNAs, shRNAs, and one or both to 50 nucleobases upstream of an intron/exon junction 20 strands of the dsRNAs preferably contain one or more of a target mRNA. More preferably the antisense oligo- modified internucleoside linkages, modified sugar moie- nucleotide is targeted to at least a portion of a region ties and/or modified nucleobases described herein or 20-24 or 30-50 nucleobases upstream of an intron/exon otherwise known in the art. These RNAi oligonucleotides junction of a target mRNA and which preferably does not have applications including, but not limited to, disrupting support RNAse H cleavage of the mRNA target upon 25 the expression of a gene(s) or polynucleotide(s) of inter- binding. Preferably, the antisense compound contains at est in a subject. Thus, in some embodiments, the oligo- least one modification which increases binding affinity nucleotides in the complexes useful in the invention are for the RNA target (e.g., mRNA and miRNA) and which used to specifically inhibit the expression of target nucleic increases nuclease resistance of the antisense com- acid. In some embodiments, double-stranded RNA-me- pound. 30 diated suppression of gene and/or nucleic acid expres- [0160] In one embodiment, the antisense oligonucle- sion is accomplished by administering a complex of the otide comprises at least one nucleoside having a 2’ mod- invention comprising a dsRNA DROSHA substrate, dsR- ification of its sugar moiety. In a further embodiment, the NA Dicer substrate, siRNA or shRNA to a subject and/or antisense oligonucleotide comprises at least 2, 3, 4, 5, cell. Double-stranded RNA-mediated suppression of 6, 7, 8, 9, 10, 15, or 20 nucleoside having a 2’ modification 35 gene and nucleic acid expression may be accomplished of its sugar moiety. In yet a further embodiment, every according to the invention by administering dsRNA, siR- nucleoside of the antisense oligonucleotide has a 2’ mod- NA, or shRNA into a subject. SiRNA may be double- ificationof its sugar moiety. Preferably, the2’ modification stranded RNA, or a hybrid molecule comprising both is2’-fluoro, 2’-OME, 2-methoxyethyl (2’-MOE) or a locked RNA and DNA, e.g., one RNA strand and one DNA nucleic acid (LNA). In some embodiments, the modified 40 strand. nucleoside motif is an LNA or alpha LNA in which a meth- [0164] siRNAs useful in the invention are RNA:RNA ylene (- -CH2--)n group bridges the 2’ oxygen atom and hybrid, DNA sense:RNA antisense hybrids, RNA sense: the 4’ carbon atom wherein n is 1 or 2. In further embod- DNA antisense hybrids, and DNA:DNA hybrid duplexes iments, the LNA or alpha LNA contains a methyl group normally 21-30 nucleotides long that can associate with at the 5’ position. In some embodiments, the oligonucle- 45 a cytoplasmic multi-protein complex known as RNAi-in- otide contains a 2’ modification and at least one internu- duced silencing complex (RISC). RISC loaded with siR- cleoside linkage. In particular embodiment, antisense ol- NA mediates the degradation of homologous mRNA tran- igonucleotide contains at least one phosphorothioate in- scripts. The invention includes the use of RNAi molecules ternucleoside linkage. In one embodiment, the internu- comprising any of these different types of double-strand- cleoside linkages of the oligonucleotide alternate be-50 ed molecules. In addition, it is understood that RNAi mol- tween phosphodiester and phosphorothioate backbone ecules may be used and introduced to cells in a variety linkages. In another embodiment, every internucleoside of forms. Accordingly, as used herein, RNAi molecules linkage of the oligonucleotide is a phosphorothioate link- encompass any and all molecules capable of inducing ages. an RNAi response in cells, including, but not limited to, [0161] In additional preferred embodiments, the anti- 55 double-stranded polynucleotides comprising two sepa- sense oligonucleotide in the complexes useful in the in- rate strands, i.e. a sense strand and an antisense strand, vention comprises at least one 3’-methylene phospho- e.g., small interfering RNA (siRNA); polynucleotides nate, linkage, LNA, peptide nucleic acid (PNA) linkage comprising a hairpin loop of complementary sequences,

24 45 EP 2 790 736 B1 46 which forms a double-stranded region, e.g., shRNAi mol- in approximately 75 bases of the start codon) of the target ecules, and expression vectors that express one or more RNA in order to avoid potential interference of the binding polynucleotides capable of forming a double-stranded of the siRNP endonuclease complex by proteins that bind polynucleotide alone or in combination with another poly- regulatory regions of the target RNA. nucleotide. 5 [0169] RNAi oligonucleotide targeting specific polynu- [0165] In some embodiments, dsRNA oligonucleotide cleotides can be readily prepared using or routinely mod- contained in a complex useful in the invention is double- ifying reagents and procedures known in the art. Struc- stranded and 16-30 or 18-25 nucleotides in length. In tural characteristics of effective siRNA molecules have particular embodiments, the dsRNA is 21 nucleotides in been identified. Elshabir et al. (2001) Nature length. In certain embodiments, the dsRNA 0-7 nucle- 10 411:494-498 and Elshabir et al. (2001), EMBO otide 3’ overhangs or 0-4 nucleotide 5’ overhangs. In par- 20:6877-6888. Accordingly, one of skill in the art would ticular embodiments, the dsRNA has a two nucleotide 3’ understand that a wide variety of different siRNA mole- overhang. In a further embodiment, the dsRNA contains cules may be used to target a specific gene or transcript. two complementary RNA strands of 21 nucleotides in length with two nucleotide 3’ overhangs ( i.e., contains a 15 Enzymatic nucleic acids 19 nucleotide complementary region between the sense and antisense strands). In certain embodiments, the [0170] In some embodiments, the complexes useful in overhangs are UU or dTdT 3’ overhangs. the invention comprise an enzymatic oligonucleotide. [0166] In some embodiments, an siRNA oligonucle- Two preferred features of enzymatic oligonucleotides otide in a complex useful in the invention is completely 20 used according to the invention are that they have a spe- complementary to the corresponding reverse comple- cific substrate binding site which is complementary to mentary strand of a target RNA. In other embodiments, one or more of the target gene DNA or RNA regions, and the siRNA contains 1 or 2 substitutions, deletions or in- that they have nucleotide sequences within or surround- sertions compared to the corresponding reverse comple- ing the substrate binding site which impart an RNA cleav- mentary strand of a target RNA, 25 ing activity to the oligonucleotide. In some embodiments, [0167] In additional embodiments, the complexes use- the enzymatic oligonucleotide is a ribozyme. Ribozymes ful in the invention comprise an RNAi oligonucleotide that are RNA-protein complexes having specific catalytic do- is a short hairpin RNA. shRNA is a form of hairpin RNA mains that possess endonuclease activity. Exemplary ri- containing a fold-back stem-loop structure that give rise bozyme HES-oligonucleotides of the invention are to siRNA and is thus, likewise capable of sequence-spe- 30 formed in a hammerhead, hairpin, a hepatitis delta virus, cifically reducing expression of a target gene. Short hair- group I intron or RNaseP RNA (in association with an pin RNAs are generally more stable and less susceptible RNA guide sequence) or a Neurospora VS RNA motif. to degradation in the cellular environment than siRNAs. [0171] While the enzymatic oligonucleotides in the The stem loop structure of ShRNAs can vary in stem complexes useful in the invention may contain modified length, typically from 19 to 29 nucleotides in length. In 35 nucleotides described herein or otherwise known in the certain embodiments, the complexes useful in the inven- art, it is important that such modifications do not lead to tion comprise an shRNA having a stem that is 19 to 21 conformational changes that abolish catalytic activity of or 27 to 29 nucleotides in length. In additional embodi- the enzymatic oligonucleotide. Methods of designing, ments, the shRNA has a loop size of between 4 to 30 producing, testing and optimizing enzymatic oligonucle- nucleotides in length. While complete complementarity 40 otides such as, ribozymes are known in the art and are between the portion of the stem that specifically hybrid- encompassed by the invention ( see, e.g., WO 91/03162; izes to the target mRNA (antisense strand) and the mR- WO 92/07065; WO 93/15187; WO 93/23569; WO NA is preferred, the shRNA may optionally contain mis- 94/02595, WO 94/13688; EP 92110298; and U.S. Pat. matches between the two strands of the shRNA hairpin No. 5,334,711). stem. For example, in some embodiments, the shRNA 45 includes one or several G-U pairings in the hairpin stem Aptamers and Decoys to stabilize hairpins during propagation in bacteria. [0168] In one embodiment, the nucleic acid target of [0172] In some embodiments, the HES-oligonucle- an RNAi oligonucleotide contained in a complex useful otides useful in the invention contain an aptamer and/or in the invention is selected by scanning the target RNA 50 a decoy. As used herein, aptamers refer to a single- (e.g., mRNA or miRNA) for the occurrence of AA dinu- stranded nucleic acid molecule (such as DNA or RNA) cleotide sequences. Each AA dinucleotide sequence in that assumes a specific, sequence-dependent shape combination with the 3’ adjacent approximately 19 nu- and specifically hybridizes to a target protein with high cleotides are potential siRNA target sites based off of affinity and specificity. Aptamers in the compositions use- which an RNAi oligonucleotide can routinely be de-55 ful in the invention are generally fewer than 100 nucle- signed. In some embodiments, the RNAi oligonucleotide otides, fewer than 75 nucleotides, or fewer than 50 nu- target site is not located within the 5’ and 3’ untranslated cleotides in length. The term "aptamer" as used herein, regions(UTRs) or regions nearthe start codon ( e.g.,with- encompasses mirror-image aptamer(s) (high-affinity L-

25 47 EP 2 790 736 B1 48 enantiomeric nucleic acids such as, L-ribose or L-2’-de- through translational inhibition, transcript cleavage, or oxyribose units) that confer resistance to enzymatic deg- both. RISC is also implicated in transcriptional silencing radation compared to D-oligonucleotides. Methods for in the nucleus of a wide range of eukaryotes. making and identifying aptamers are known in the art and [0178] The present invention provides, inter alia, com- can routinely be modified to identify aptamers having de- 5 positions useful for modulating small non-coding RNA sirable diagnostic and/or therapeutic properties and to activity, including miRNA activity associated with disease incorporate these aptamers into the HES-oligonucle- states. Certain compositions useful in the invention are otides useful in the invention. See, e.g., Wlotzka et al, particularly suited for use in in vivo methods due to their Proc. Natl. Acad. Sci. 99(13):8898-8902, 2002. improved delivery, potent activity and/or improved ther- [0173] As used herein, the term "decoy" refers to short 10 apeutic index. double-stranded nucleic acids (including single-stranded [0179] The invention provides compositions useful for nucleic acids designed to "fold back" on themselves) that modulating small non-coding RNAs, including miRNA. In mimic a site on a nucleic acid to which a factor, such as particular embodiments, the invention provides compo- a protein, binds. Such decoys competitively inhibit and sitions for modulating the levels, expression, processing thereby decrease the activity and/or function of the factor. 15 or function of one or a plurality of small non-coding RNAs, Methods for making and identifying decoys are known in such as miRNAs. Thus, in some embodiments, the in- the art and can routinely be modified to identify decoys vention encompasses compositions, such as pharma- having desirable diagnostic and/or therapeutic proper- ceutical compositions, comprising an HES-oligonucle- ties, and to incorporate these decoys into the HES-oli- otide complex having at least one oligonucleotide spe- gonucleotides of the invention. See, e.g., U.S. Pat. No. 20 cifically hybridizable with a small noncoding RNA, such 5,716,780 to Edwards et al. as a miRNA. [0180] In some embodiments, an oligonucleotide in an Small non-coding RNA and antagonists (e.g., miR- HES-oligonucleotidecomplex useful inthe inventionspe- NAs and anti-miRNAs cifically hybridizes with or sterically interferes with nucleic 25 acid molecules comprising or encoding one or more small [0174] There is a need for agents that regulate gene non-coding RNAs, such as, miRNAs. In particular em- expression via the mechanisms mediated by small non- bodiments, the invention provides HES-oligonucleotide coding RNAs. The present invention meets this and other complexes useful for modulating the levels, activity, or needs. function of miRNAs, including those relying on antisense [0175] As used herein, the term "small non-coding30 mechanisms and those that are independent of antisense RNA" is used to encompass, without limitation, a poly- mechanisms. nucleotide molecule ranging from 17 to 29 nucleotides [0181] As used herein, the terms. "target nucleic acid," in length. In one embodiment, a small non-coding RNA "target RNA," "target RNA transcript" or "nucleic acid tar- is a miRNA (also known as miRNAs, Mirs, miRs, mirs, get" are used to encompass any nucleic acid capable of and mature miRNAs). 35 being targeted including, without limitation, RNA. In a one [0176] MicroRNAs (miRNAs), also known as "mature" embodiment, the target nucleic acids are non-coding se- miRNA") are small (approximately 21-24 nucleotides in quences including, but not limited to, miRNAs and miRNA length), non-coding RNA molecules that have been iden- precursors. In a preferred embodiment, the target nucleic tified as key regulators of development, cell proliferation, acid is a miRNA, which may also be referred to as the apoptosis and differentiation. Examples of particular de- 40 miRNA. An oligonucleotide is "targeted to a miRNA" velopmental processes in which miRNAs participate in- when an oligonucleotide comprises a sequence substan- clude stem cell differentiation, neurogenesis, angiogen- tially, including 100% complementary to a miRNA. esis, hematopoiesis, and exocytosis (reviewed by Alva- [0182] As used herein, oligonucleotides are "substan- rez-Garcia and Miska, Development, 2005, 132,tially complementary" to for example, an RNA such as a 4653-4662). miRNA have been found to be aberrantly 45 small non-coding RNA, when they are capable of specif- expressed in disease states, i.e., specific miRNAs are ically hybridizing to the small non-coding RNA under present at higher or lower levels in a diseased cell or physiologic conditions. In some embodiments, an oligo- tissue as compared to healthy cell or tissue. nucleotide is "targeted to a miRNA" when an oligonucle- [0177] miRNAs are believed to originate from long en- otide comprises a sequence substantially, including dogenous primary miRNA transcripts (also known as pri- 50 100% complementary to at least 8 contiguous nucle- imRNAs, pri-mirs, pri-miRs or pri-pre-miRNAs) that are otides of a miRNA. In some embodiments, an oligonu- often hundredsof nucleotides in length(Lee, et al., EMBO cleotide in a complex of the invention specifically hybrid- J., 2002, 21(17), 4663-4670). One mechanism by which izes to an miRNA and ranges in length from about 8 to miRNAs regulate gene expression is through binding to about 21 nucleotides, from about 8 to about 18 nucle- the 3’-untranslated regions (3’-UTR) of specific mRNAs. 55 otides, or from about 8 to about 14 nucleotides. In addi- miRNAs nucleotide (nt) RNA molecules that become in- tional embodiments, the oligonucleotide specifically hy- corporated into the RNA-induced silencing complex bridizes to an miRNA and ranges in length from about (RISC) mediate down-regulation of gene expression 12 to about 21 nucleotides, from about 12 to about 18

26 49 EP 2 790 736 B1 50 nucleotides, or from about 12 to about 14 nucleotides. In seed region). In additional embodiments, the oligonucle- particular embodiments, the oligonucleotides are 8, 9, otide specifically hybridizes to a sequence in a precursor- 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 monomer miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that subunits (nucleotides) in length. In certain embodiments, when bound by the oligonucleotide blocks miRNA oligonucleotides, the oligonucleotides are 14, 15, 16, 17 5 processing. or 18 monomer subunits (nucleotides) in length. [0187] In some embodiments, the composition con- [0183] In particular embodiments, the oligonucleotide tains an HES-oligonucleotide complex contains an oligo- has full length complementarity to the miRNA, In other nucleotide which is targeted to nucleic acids comprising embodiments, the length complementarity between the or encoding a small non-coding RNA and which acts to oligonucleotide and the target nucleic acid as well as up 10 reduce the levels of the small non-coding RNA and/or to 3 "mismatches" between the oligonucleotide and the interfere with its function in a cell. target miRNA such that the oligonucleotide is still capable [0188] In other embodiments, the composition con- of hybridizing with the target miRNA and the function of tains an HES-oligonucleotide complex contains an oligo- the oligonucleotide is not substantially impaired. In other nucleotide which comprises or encodes the small non- embodiments, the oligonucleotide contains a truncation 15 coding RNA or increases the endogenous expression, or expansion with respect to the length of target miRNA processing or function of the small non-coding RNA ( e.g., by up to 6 nucleosides, at either the 3’ or 5’ end, or at by binding regulatory sequences in the gene encoding both the 3’ and 5’ end of the oligonucleotide. In certain the non-coding RNA) and which acts to increase the level embodiments, the oligonucleotide is truncated by 1 or 2 of the small non-coding RNA and/or increase its function nucleosides compared with the length of the target miR- 20 in a cell. NA. As a non-limiting example, if the target miRNA is 22 [0189] Oligonucleotides contained in the HES-oligonu- nucleotides in length, the oligonucleotide which has es- cleotides useful in the invention can modulate the levels, sentially full length complementarity may be 20 or 21 nu- expression or function of small non-coding RNAs by hy- cleotides in length. In a particular embodiment, the oli- bridizing to a nucleic acid comprising or encoding a small gonucleotide is truncated by 1 nucleotide on either the 25 non-coding RNA nucleic acid target resulting in alteration 3’ or 5’ end compared to the miRNA. of normal function. For example, non-limiting mecha- [0184] Disclosed herein is a method of modulating a nisms by which the oligonucleotides might decrease the small non-coding RNA comprising contacting a cell with activity (including levels, expression or function) of a an HES-oligonucleotide complex, wherein an oligonucle- small non-coding RNA include facilitating the destruction otide of the HES-oligonucleotide complex comprises a 30 of the small non-coding RNA through cleavage, seques- sequence substantially complementary to the small non- tration, steric occlusion and by hybridizing to the small coding RNA, a small non-coding RNA precursor ( e.g., a non-coding RNA and preventing it from hybridizing to, miRNA precursor), or a nucleic acid encoding the small and regulating the activity of, its normal cellular target(s). non-coding RNA. As used herein, the term "small non- [0190] Disclosed herein is a method of inhibiting the coding RNA precursor miRNA precursor" is used to en- 35 activity of a small non-coding RNA, comprising contact- compass any longer nucleic acid sequence from which ing a cell with an HES-oligonucleotide complex compris- a small (mature) non-coding RNA is derived and may ing an oligonucleotide which is targeted to nucleic acids include, without limitation, primary RNA transcripts, pri- comprising or encoding a small non-coding RNA and small non-coding RNAs, and pre-small non-coding which acts to reduce the levels of the small non-coding RNAs. For example, an "miRNA precursor" encompass- 40 RNA and/or interfere with its function in the cell. In some es any longer nucleic acid sequence from which a miRNA embodiments, the oligonucleotide comprises a se- is derived and may include, without limitation, primary quence substantially complementary nucleic acids com- RNA transcripts, pri-miRNAs, and pre-miRNAs. prising or encoding the non-coding RNA. In particular [0185] The invention may use, inter alia, compositions embodiments, the small non-coding RNA is a miRNA. such as pharmaceutical compositions, containing an45 [0191] Disclosed herein is a method of inhibiting the HES-oligonucleotide complex containing an oligonucle- activity of a small non-coding RNA, comprising adminis- otide which is targeted to nucleic acids comprising or tering to a subject an HES-oligonucleotide complex con- encoding small non-coding RNA, and which acts to mod- taining an oligonucleotide which is targeted to nucleic ulate the levels of the small non-coding RNA, or modulate acids comprising or encoding a small non-coding RNA its function. In further embodiments, the invention may 50 and which acts to reduce the levels of the small non- use a composition such as a pharmaceutical composi- coding RNA and/or interfere with its function in the sub- tion, containing an HES-oligonucleotide complex com- ject. In some embodiments, the oligonucleotide compris- prising an oligonucleotide which is targeted to a miRNA es a sequence substantially complementary nucleic ac- and which acts to modulate the levels of the miRNA, or ids comprising or encoding the non-coding RNA. In par- interfere with its processing or function. 55 ticular embodiments, the small non-coding RNA is a miR- [0186] In some embodiments, the HES-oligonucle- NA. otide complex contains an oligonucleotide that specifi- [0192] Disclosed herein is a method of increasing the cally hybridizes to nucleotides 1-10 of a miRNA ( i.e., the activity of a small non-coding RNA, comprising contact-

27 51 EP 2 790 736 B1 52 ing a cell with an HES-oligonucleotide complex contain- and which acts to reduce the levels of the small non- ing an oligonucleotide which comprises or encodes the coding RNA and/or interfere with its function in the sub- small non-coding RNA or increases the endogenous ex- ject. In some embodiments, the HES-oligonucleotide is pression, processing or function of the small non-coding an anti-miRNA (anti-miR). In additional embodiments the RNA (e.g., by binding regulatory sequences in the gene 5 anti-miRNA is double stranded. In further embodiments, encoding the non-coding RNA) and which acts to in- the HES-oligonucleotide contains an anti-miRNA that is crease the level of the small non-coding RNA and/or in- double stranded and contains oligonucleotides of 18-23 crease its function in the cell. In some embodiments, the units in length and is blunt ended or comprises one or oligonucleotide comprises a sequence substantially the more 3’ overhangs of 1, 2, or 3 nucleotides. In additional same as nucleic acids comprising or encoding the non- 10 embodiments, the HES-oligonucleotide contains a single coding RNA. In some embodiments, the oligonucleotide stranded anti-miR that is 8-25 units in length. HES-oligo- shares 100% identity with at least 15 contiguous nucle- nucleotides containing expression vectors that express otides, at least 20 contiguous nucleotides or over the full- these anti-MiRs are also encompassed by the invention. length of the small non-coding RNA sequence. In partic- In some embodiments, the oligonucleotide comprises a ular embodiments, the small non-coding RNA is a miR- 15 sequence substantially complementary to the overex- NA. pressed small-noncoding RNA. [0193] Disclosed herein is a method of increasing the [0196] In further embodiments, the invention encom- activity of a small non-coding RNA, comprising adminis- passes treating a disease or disorder characterized by tering to a subject an HES-oligonucleotide complex con- the overexpression of a miRNA in a subject, comprising taining an oligonucleotide which comprises or encodes 20 administering to the subject an HES-oligonucleotide the small non-coding RNA or increases the endogenous complex containing an oligonucleotide which is targeted expression, processing or function of the small non-cod- to nucleic acids comprising or encoding the miRNA and ing RNA, and which acts to increase the level of the small which acts to reduce the levels of the miRNA and/or in- non-coding RNA and/or increase its function in the sub- terfere with its function in the subject. In some embodi- ject. In some embodiments, the oligonucleotide compris- 25 ments, the oligonucleotide comprises a sequence sub- es a sequence substantially the same as nucleic acids stantially complementary to the overexpressed miRNA. comprising or encoding the non-coding RNA. In some embodiments, the oligonucleotide shares 100% identity a. Families of miRNAs can be characterized by nu- with at least 15 contiguous nucleotides, at least 20 con- cleotide identity at positions 2-8 of the miRNA, a re- tiguous nucleotides or over the full-length of the small 30 gion known as the seed sequence. The members of non-coding RNA sequence. In particular embodiments, a miRNA family are herein termed "related miRNAs". the small non-coding RNA is a miRNA. Each member of a miRNA family shares an identical [0194] In additional embodiments, the HES-oligonu- seed sequence that plays an essential role in miRNA cleotide comprises a sequence substantially the same targeting and function. As used herein, the term as nucleic acids comprising or encoding the small non- 35 "seed sequence" or "seed region" refers to nucle- coding RNA. In some embodiments, the HES-oligonu- otides 2 to 9 from the 5’-end of a mature miRNA cleotide is a miRNA mimic. In some embodiments the sequence. Examples of miRNA families are known miRNA mimic is double stranded. In further embodi- in the art and include, but are not limited to, the let- ments, the HES-oligonucleotide contains an miRNA 7 family (having 9 miRNAs), the miR-15 family (com- mimic that is double stranded and contains oligonucle- 40 prising miR-15a, miR-15b, miR15-16, miR-16-1, and otides of 18-23 units in length and is blunt ended or com- miR-195), and the miR-181 family (comprising miR- prises one or more 3’ overhangs of 1, 2, or 3 nucleotides. 181a, miR-181b, and miR-181c). In some embodi- In additional embodiments, the HES-oligonucleotide ments, an HES-oligonucleotide specifically hybridiz- contains a single stranded miRNA mimic that is 18-23 es to the seed region of a miRNA and interferes with units in length. HES-oligonucleotides containing expres- 45 the processing or function of the miRNA. In some sion vectors that express these miRNA mimics are also embodiments, the HES-oligonucleotide specifically encompassed by the invention. In some embodiments, hybridizes to the seed region of a miRNA and inter- the oligonucleotide shares 100% identity with at least 15 feres with the processing or function of multiple miR- contiguous nucleotides, at least 20 contiguous nucle- NAs. In further embodiments, at least 2 of the mul- otides or over the full-length of the small non-coding RNA 50 tiple miRNAs have related seed sequences or are sequence. In particular embodiments, the small non-cod- members of the miRNA superfamily. ing RNA is a miRNA. [0195] The invention also encompasses treating a dis- [0197] The association of miRNA dysfunction with dis- ease or disorder characterized by the overexpression of eases such as cancer, fibrosis, metabolic disorders and a small-noncoding RNA in a subject, comprising admin- 55 inflammatory disorders and the ability of miRNAs to in- istering to the subject an HES-oligonucleotide complex, fluence an entire network of genes involved in a common containing an oligonucleotide which is targeted to nucleic cellular process makes the selective modulation of miR- acids comprising or encoding the small non-coding RNA NAs using anti-miRNAs and miRNA mimics particularly

28 53 EP 2 790 736 B1 54 attractive disease modulating therapeutics. The inven- In some embodiments, the oligonucleotide comprises a tion also encompasses treating a disease or disorder sequence substantially complementary to the small-non- characterized by the overexpression of a protein in a sub- coding RNA, ject, comprising administering to the subject an HES-ol- [0202] The invention also encompasses treating a dis- igonucleotide complex, containing an oligonucleotide 5 ease or disorder characterized by the overexpression of which is targeted to nucleic acids comprising or encoding a protein in a subject, comprising administering to the a small non-coding RNA that influences the increased subject an HES-oligonucleotide complex, containing an production of the protein, wherein the oligonucleotide act oligonucleotide which comprises or encodes the small to reduce the levels of the small non-coding RNA and/or non-coding RNA or increases the endogenous expres- interfere with its function in the subject. In some embod- 10 sion, processing or function of the small non-coding RNA, iments, the oligonucleotide comprises a sequence sub- and which acts to increase the level of the small non- stantially complementary to the small-noncoding RNA, coding RNA and/or increase its function in the subject. [0198] The invention also encompasses treating a dis- In some embodiments, the oligonucleotide comprises a ease or disorder characterized by the overexpression of sequence substantially complementary (specifically hy- a protein in a subject, comprising administering to the 15 bridizable) to the miRNA. subject an HES-oligonucleotide complex, containing an [0203] Disclosed herein is a method of inhibiting miR- oligonucleotide which is targeted to nucleic acids com- NA activity comprising administering to subject an HES- prising or encoding a miRNA that influences the in- oligonucleotide complex having anti-miRNA activity, creased production of the protein, wherein the oligonu- such as those described herein. cleotide acts to reduce the levels of the miRNA and/or 20 [0204] In some embodiments, the HES-oligonucle- interfere with its function in the subject. In some embod- otide complex contains an oligonucleotide selected from: iments, the oligonucleotide comprises a sequence sub- a siRNA, a miRNA, a dicer substrate e.g.,( dsRNA), a stantially complementary (specifically hybridizable) to ribozyme, a decoy, an aptamer, an antisense oligonucle- the miRNA. otide and a plasmid capable of expressing an siRNA, a [0199] The invention also encompasses treating a dis- 25 miRNA, or an antisense oligonucleotide. ease or disorder characterized by the under expression [0205] In some embodiments, the oligonucleotides are of a small-noncoding RNA in a subject, comprising ad- chimeric oligonucleotides comprising an internal region ministering to the subject an HES-oligonucleotide com- containing at least. 1, at least 2, at least 3, at least 4, at plex, containing an oligonucleotide which comprises or least 5, or all 2’-F modified nucleotides and external re- encodes the small non-coding RNA or increases the en- 30 gions comprising at least one stability enhancing modi- dogenous expression, processing or function of the small fications. In one embodiment, an oligonucleotide in the non-coding RNA, and which acts to increase the level of HES-oligonucleotide complex comprises an internal re- the small non-coding RNA and/or increase its function in gion having a first 2’-modified nucleotide and external the subject. In some embodiments, the oligonucleotide regions each comprising a second 2’-modified nucle- comprisesa sequence substantially complementary spe- 35 otide. In a further embodiment, the gap region comprises cifically hybridizable)to the overexpressed small-non- one or more 2’-fluoro modifications and the wing regions coding RNA. comprise one or more 2’-methoxyethyl modifications. In [0200] In further embodiments, the invention encom- one embodiment, the oligonucleotide in the HES-oligo- passes treating a disease or disorder characterized by nucleotide complex is ISIS 393206 or ISIS 327985. the overexpression of a miRNA in a subject, comprising 40 administering to the subject an HES-oligonucleotide Therapeutic complex, containing an oligonucleotide which comprises or encodes the small non-coding RNA or increases the Diagnostics, Drug Discovery and Therapeutics endogenous expression, processing or function of the small non-coding RNA, and which acts to increase the 45 [0206] The oligonucleotides, complexes and other level of the small non-coding RNA and/or increase its compositions disclosed herein have uses that include, function in the subject. In some embodiments, the oligo- but are not limited to, research, drug discovery, kits and nucleotide comprises a sequence substantially comple- diagnostics, and therapeutics. The complexes of the in- mentary to the overexpressed miRNA. vention are particularly suited for use in in vivo methods [0201] The invention also encompasses treating a dis- 50 due to their improved oligonucleotide delivery over con- ease or disorder characterized by the overexpression of ventional delivery techniques. a protein in a subject, comprising administering to the [0207] The invention provides compositions for detect- subject an HES-oligonucleotide complex, containing an ing a nucleic acid sequence in vitro or in vivo. Thus, dis- oligonucleotide which comprises or encodes the small closed are compositions comprising an HES-oligonucle- non-coding RNA or increases the endogenous expres- 55 otide complex containing an oligonucleotide that specif- sion, processing or function of the small non-coding RNA, ically hybridizes with a target nucleic acid under physio- and which acts to increase the level of the small non- logic conditions. coding RNA and/or increase its function in the subject. [0208] In some embodiments, an HES-oligonucleotide

29 55 EP 2 790 736 B1 56 delivery vehicle useful in the invention is used to identify tored using techniques known to those skilled in the art. the presence of an infectious agent in a host organism For example, in some embodiments, fluorescence is such as a virus in a mammalian cell or a bacterium in a monitored via fluorescence endoscopy. Fluorescence mammalian tissue. In this embodiment an HES-oligonu- endoscopy can be performed using equipment such as, cleotide which is composed of an HES, serves as an in 5 the Olympus EVIS ExERA-II CLV-80 system (Olympus vivo marker of binding to a complementary sequence. Corp., Tokyo Japan) using the appropriate excitation This identification is accomplished by the detection of wavelengths and the emission filters for the administered changes in fluorescence when binding of the HES-oligo- fluorphores. Fluorescence intensities can be determined nucleotide to a complementary foreign (e.g., infectious using techniques and software known in the art such as, agent) nucleic acid sequence results in destruction or 10 the Image-J software (NIH, Bethesda, MD). significant loss of the HES and results in a loss of fluo- [0213] In the present invention, the disease or disorder rescence: quenching. Thus, the invention encompasses is: cancer, fibrosis, a proliferative disease or disorder, a determining the presence of, and/or quantitating the lev- neurological disease or disorder, an inflammatory dis- els of, a foreign nucleic acid in a host organism (subject). ease or disorder, a disease or disorder of the immune In some embodiments, this is performed in vitro. In other 15 system, a disease or disorder of the cardiovascular sys- embodiments, this is performed in vivo. tem, a metabolic disease or disorder, a disease or dis- [0209] In some embodiments, the invention provides order of the skeletal system, or a disease or disorder of for detecting the presence of an infectious agent in a the skin or eyes. In additional embodiments, the disease subject in vitro or in vivo, comprising, contacting a cell, or disorder is a disease or disorder of the kidneys, liver, tissue or subject with an HES-oligonucleotide containing 20 lymph nodes, spleen or adipose tissue. In particular em- an oligonucleotide that specifically hybridizes with the bodiments, the disease or disorder is not a disease or nucleic acid of an infectious agent, determining the level disorder of the kidneys, liver, lymph nodes, spleen or of fluorescence in the cell, tissue or subject tissue, and adipose tissue. comparing said level of fluorescence with that obtained [0214] In further embodiments, the disease or disorder for a control cell, tissue or subject not containing the in- 25 is a proliferative disorder such as, cancer. For example, fectious agent that has been contacted with the HES- the overexpression of numerous miRNA such, as mIR- oligonucleotide, wherein an increased fluorescence 10b, mIR17-92, mIR-21, mIR125b, mIR-155, mIR193a, compared to the control indicates that the cell, tissue, or mIR-205a and mIR-210, have been associated with var- subject has the infectious agent. ious forms of cancer. In some embodiments, the biomar- [0210] In additional embodiments, an HES-oligonucle- 30 ker is a miRNA selected from mIR-10b, mIR17-92, mIR- otide useful in the invention is used to identify an altered 21, mIR125b, mIR-155, mIR193a, mIR-205a, and mIR- level of a nucleic acid that is a biomarker for a disease 210, and an increased fluorescence of the cell, tissue, or or disorder. In some embodiments, the invention pro- subject relative to a control indicates that the subject has vides for detecting the presence of an altered level of a cancer or has a predisposition for cancer. nucleic acid biomarker for a disease or disorder in vitro 35 [0215] In additional embodiments, the invention is comprising, contacting a cell or tissue with an HES-oli- used to identify and/or distinguish between different dis- gonucleotide containing an oligonucleotide that specifi- eases or disorders. The invention can likewise be used cally hybridizes with the nucleic acid biomarker, deter- to determine among other things, altered nucleic acid mining the level of fluorescence in the cell or tissue and (e.g., DNA and RNA) profiles that distinguish between comparing said level of fluorescence with that obtained 40 normal and diseased e.g( ., cancerous) tissue or cells, for a control cell or tissue that has been contacted with discriminate between different subtypes of diseased cells the HES-oligonucleotide, wherein an altered fluores- (e.g., between different cancers and subtypes of a par- cence compared to the control indicates that the cell or ticular cancer), to discriminate between mutations (e.g., tissue has an altered level of the nucleic acid biomarker. oncogenic mutations) giving rise to or associated with [0211] In further embodiments, the invention provides 45 different disease states, and to identify tissues of origin for detecting an altered level of a nucleic acid biomarker (e.g., in a metastasized tumor). fora disease or disorderin vivo comprising, administering [0216] Moreover, in some embodiments, the oligonu- to a subject an HES-oligonucleotide containing an oligo- cleotides in the HES-oligonucleotides useful in the inven- nucleotide that specifically hybridizes with the nucleic ac- tion are therapeutic oligonucleotides, and the destruction id biomarker, determining the level of fluorescence in the 50 or significant loss of HES that results in an increased subject, and comparing said level of fluorescence with fluorescence when the therapeutic HES oligonucleotides that obtained for a control subject that has been admin- specifically hybridizes with target nucleic acids indicates istered the HES-oligonucleotide, wherein an altered flu- that the therapeutic oligonucleotides have been deliv- orescence compared to the control indicates that the sub- ered to, and have hybridized with the target nucleic acid. ject has an altered level of the nucleic acid biomarker. 55 Thus, in some embodiments, the invention provides for This approach can also be used to quantitate the number monitoring and/or quantitating the delivery of a therapeu- of copies of an aberrant gene of host origin in vivo. tic oligonucleotide to a target nucleic acid in vivo, com- [0212] In vitro and in vivo fluorescence can be moni- prising administering to a subject, a HES oligonucle-

30 57 EP 2 790 736 B1 58 otides containing a therapeutic oligonucleotide that spe- an HES-oligonucleotide complex to the subject, wherein cifically hybridizes to the target nucleic acid, and deter- an oligonucleotide of the complex comprises a sequence mining the level of fluorescence in a cell or tissue of the substantially complementary to the target nucleic acid subject, wherein an increased fluorescence in the cell or that specifically hybridizes to and modulates levels of the tissue compared to a control cell or tissue indicates that 5 nucleic acid or interferes with its processing or function. that the therapeutic oligonucleotide has been delivered In some embodiments, the target nucleic acid is RNA, in to and hybridized with the target nucleic acid. further embodiments the RNA is mRNA or miRNA. In [0217] The delivery vehicles useful in the invention are further embodiments, the oligonucleotide reduces the based, in part, on the surprising discovery that the linking level of a target RNA by at least 10%, at least 20%, at of one or more HES to a single or multiple strands of10 least 30%, at least 40% or at least 50% in one or more oligonucleotides significantly enhances the in vivo deliv- cells or tissues of the subject. In some embodiments, the ery of the HES-oligonucleotides inside a cell or tissue of target nucleic acid is a DNA. a live organism. Thus, the HES-oligonucleotide vehicles [0224] Disclosed herein is a method of modulating a useful in the invention have applications as therapeutic protein in a subject comprising, administering an HES- delivery vehicles for a broad range of therapeutic appli- 15 oligonucleotide complex to the subject, wherein an oli- cations as well as in conjunction with assays and thera- gonucleotide of the complex comprises a sequence sub- pies to evaluate for example, the activity and/or number stantially complementary to a nucleic acid that encodes of copies of a specific gene or RNA in vivo. the protein or influences the transcription, translation, [0218] For use in research and drug discovery, an production, processing or function of the protein. In some HES-oligonucleotide useful in the invention can be used 20 embodiments, the oligonucleotide specifically hybridizes for example, to interfere with the normal function of the to an RNA. In further embodiments the RNA is mRNA or nucleic acid molecules to which they are targeted. Ex- miRNA. In additional embodiments, the oligonucleotide pression patterns within cells, tissues, or subjects treated reduces the level of the protein or RNA by at least 10%, with one or more HES-oligonucleotides useful in the in- at least 20%, at least 30%, at least 40% or at least 50% vention are then compared to control cells, tissues or 25 in one or more cells or tissues of the subject. In some subjects not treated with the compounds and the patterns embodiments, the oligonucleotide specifically hybridizes produced are then analyzed for differential levels of nu- to a DNA. cleic acid and/or protein expression and as they pertain, [0225] In particular embodiments, the oligonucleotide for example, to disease association, signaling pathway, in the HES-oligonucleotide complex is selected from an cellular localization, expression level, size, structure or 30 siRNA, an shRNA, a miRNA, an anti-miRNA, a dicer sub- function of the genes examined. These analyses can be strate (e.g., dsRNA), an aptamer, a decoy, an antisense performed on stimulated or unstimulated cells and in the oligonucleotide, and a plasmid capable of expressing an presence or absence of other compounds that affect ex- siRNA, a miRNA, or an antisense oligonucleotide. In pression patterns. some embodiments, the oligonucleotide specifically hy- [0219] Disclosed herein are compositions and meth- 35 bridizes with an RNA or a sequence encoding an RNA. ods for modulating nucleic acids and protein encoded or In other embodiments, the oligonucleotide specifically regulated by these modulated nucleic acids. In particular, hybridizes with DNA sequence encoding an RNA or the disclosed are compositions and methods for modulating regulatory sequences thereof. the levels, expression, processing or function of a mRNA, [0226] In additional embodiments, the expression of a small non-coding RNA (e.g., miRNA), a gene or a protein. 40 nucleic acid or protein is modulated in a subject by con- [0220] Disclosed herein is a method of delivering an tacting the subject with an HES-oligonucleotide complex oligonucleotide to a cell in vivo by administering to a sub- containing an antisense oligonucleotide. In particular em- ject an HES-oligonucleotide complex containing the oli- bodiments, the antisense oligonucleotide in the HES-ol- gonucleotide. In particular embodiments, the oligonucle- igonucleotide complex is a substrate for RNAse H when otide is a therapeutic oligonucleotide. 45 bound to a target RNA. In some embodiments, the anti- [0221] Disclosed herein are compositions, such as sense oligonucleotide is a gapmer. As used herein, a pharmaceutical compositions, comprising an HES-oligo- "gapmer" refers an antisense compound having a central nucleotide complex having at least one oligonucleotide region (also referred to as a "gap" or "gap segment") hybridizable with a target nucleic acid sequence under positioned between two external flanking regions (also physiologic conditions. 50 referred to as "wings" or "wing segments"). The regions [0222] Disclosed herein is a method of delivering an are differentiated by the types of sugar moieties compris- oligonucleotide to a subject comprising administering an ing each distinct region. The types of sugar moieties that HES-oligonucleotide complex to the subject, wherein the are used to differentiate the regions of a gapmer may in complex contains a therapeutically effective amount of some embodiments include beta-D-ribonucleosides, be- an oligonucleotide sufficient to modulate a target RNA 55 ta-D-deoxyribonucleosides, 2’-modified nucleosides (e.g., mRNA and miRNA) or target gene. (such 2’-modified nucleosides may include 2’-MOE, 2’- [0223] Disclosed herein is a method of modulating a fluoro and 2’-O--CH3, among others), and bicyclic sugar target nucleic acid in a subject comprising administering modified nucleosides (such bicyclic sugar modified nu-

31 59 EP 2 790 736 B1 60 cleosides may include LNA™ or ENA™, among others). second 2’-modified nucleoside. For example, in one em- [0227] In some embodiments, each wing of a gapmer bodiment, the central region contains 2’-F modified nu- oligonucleotides comprises the same number of subu- cleotides flanked on each end by external regions each nits. In other embodiments, one wing of a gapmer oligo- having two 2’-MOE modified nucleotides (2’-MOE/2’- nucleotide comprises a different number of subunits than 5 F/2’-MOE). In particular embodiments, the gapmer is the other wing of the gapmer. In one embodiment, the ISIS 393206. In another embodiment, the central region wings of gapmer oligonucleotides have, independently, contains 2’-F modified nucleotides flanked on each end from 1 to about 5 nucleosides of which, 1, 2 3 4 or 5 of by external regions each having two 2’-MOE modified the wing nucleosides are sugar modified nucleosides. In nucleotides (2’-MOE/2’-F/2’-MOE). In particular embod- one embodiment, the central or gap region contains 8-25 10 iments, the external regions each having two LNA or al- beta-D-ribonucleosides or beta-D-deoxyribonucleosides pha LNA modified nucleotides in the wings of the gapmer. (i.e., is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, In further embodiments, the LNA or alpha LNA modified 22, 24 or 25 nucleosides in length). In a further embod- nucleotides contain one or more methyl groups in the (R) iment, the central or gap region contains 17-24 nucle- or (S) configuration at the 6’ (2’,4’-constrained-2’-O- ethyl otides (i.e., is 17, 18, 19, 20, 21, 22, 23 or 24 nucleosides 15 BNA, S-cEt) or the 5’-position (-5’-Me-LNA or -5’-Me-al- in length). In some embodiments, the gapmer oligonu- pha LNA) of LNA or alternatively contain a substituted cleotide comprises phosphodiester internucleotide link- carbon atom in place of the 2’-oxygen atom in the LNA ages, phosphorothioate internucleotide linkages, or a or alpha LNA. combination of phosphodiester and phosphorothioate in- [0229] Disclosed herein is the use of an HES-oligonu- ternucleotide linkages. In particular embodiments the 20 cleotide complex in the manufacture of a composition for central region of the gapmer oligonucleotide contains at the treatment of one or more of the conditions associated least 2, 3, 4, 5 or 10 modified nucleosides, modified in- with a miRNA or an miRNA family. ternucleoside linkages or combinations thereof. In par- [0230] According to one embodiment, the methods ticular embodiments the central region of the gapmer ol- comprise the step of administering to or contacting the igonucleotide contains at least 10 beta-D-2’-deoxy-2’- 25 subject with an effective amount of an HES-oligonucle- fluororibofuranosyl nucleosides. In some embodiments, otide of the invention sufficient to modulate the target each nucleoside in the central region of the oligonucle- gene or RNA (e.g., mRNA and miRNA) expression and otide a beta-D-2’-deoxy-2’-fluororibofuranosyl nucleo- to thereby treat one or more conditions or symptoms as- side. In one embodiment, the gapmer oligonucleotides sociated with the disease or disorder. Exemplary com- is fully complementary over the length complementarity 30 pounds effectively modulate the expression, activity or with the target RNA. In one embodiment, one or both function of the gene, mRNA or small-non-coding RNA wings of the gapmer contains at least one 2’ modified target. In preferred embodiments, the small non-coding nucleoside. In one embodiment, one or both wings of the RNA target is a miRNA, a pre-miRNA, or a polycistronic gapmer contains 1, 2 or 3 2’-MOE modified nucleosides. or monocistronic pri-miRNA. In additional embodiments, In one embodiment, one or both wings of the gapmer35 the small non-coding RNA target is a single member of contains 1, 2 or 3 2’-OCH3 modified nucleosides. In an- a miRNA family. In a further embodiment, two or more other embodiment, one or both wings of the gapmer con- members of a miRNA family are selected for modulation. tains 1, 2 or 3 LNA or alpha-LNA nucleosides. In some [0231] Also disclosed herein is a method of inhibiting embodiments, the LNA or alpha LNA in the wings of the theactivity of atarget nucleic acidin asubject, comprising gapmer contain one or more methyl groups in the (R) or 40 administering to the subject an HES-oligonucleotide (S) configuration at the 6’ (2’,4’-constrained-2’- O- ethyl complex comprising an oligonucleotide which is targeted BNA, S-cEt) or the 5’-position (-5’-Me-LNA or -5’-Me-al- to nucleic acids comprising or encoding the nucleic acid pha LNA) of LNA or alternatively contain a substituted and which acts to reduce the levels of the nucleic acid carbon atom in place of the 2’-oxygen atom in the LNA and/or interfere with its function in the cell. In particular or alpha LNA. In further embodiments, the LNA or alpha 45 embodiments, the target nucleic acid is a small-non cod- LNA in the gapmer contain a steric bulk moiety at the 5’ ing RNA, such as, a miRNA. In some embodiments, the position (e.g., a methyl group). In a further embodiment, oligonucleotide comprises a sequence substantially the gap comprises at least one 2’fluoro modified nucle- complementary to the target nucleic acid. osides. In an additional embodiment, the wings are each [0232] Disclosed herein is a method of reducing ex- 2 or 3 nucleosides in length and the gap region is 1950 pression of a target RNA in an subject in need of reducing nucleotides in length. In additional embodiments, the expression of said target RNA, comprising administering gapmer has at least one 5-methylcytosine. to said subject an antisense HES-oligonucleotide com- [0228] In another embodiment, the nucleosides of the plex. In particular embodiments, an oligonucleotide in the central region (gap) contain uniform sugar moieties that complex is a substrate for RNAse H when bound to said are different than the sugar moieties in one or both of the 55 target mRNA. In some embodiments, the oligonucleotide external wing regions. In one non-limiting example, the is a gapmer. gap is uniformly comprised of a first 2’-modified nucleo- [0233] Also disclosed herein is a method of reducing side and each of the wings is uniformly comprised of a expression of a target RNA in a subject in need of reduc-

32 61 EP 2 790 736 B1 62 ing expression of said target RNA, comprising adminis- with the corresponding miRNA and prevent the associ- tering to said subject a HES-oligonucleotide complex ationof targetmiRNA with its endogenoustarget mRNAs. containing an antisense oligonucleotide to said subject In additional embodiments, the nucleic acid is an mRNA wherein the antisense sequence specifically hybridizes and the oligonucleotide sequence is specifically hybrid- to the target RNA. In particular embodiments, the anti- 5 izable to a target region of a RNA selected from the group sense oligonucleotide in the HES-oligonucleotide com- consisting of: an intron/exon junction of a target RNA, an plex is a substrate for RNAse H when bound to a target intron/exon junction and a region 1 to 50 nucleobases 5’ RNA. In additional embodiments, the antisense oligonu- of an intron/exon junction of the target RNA. In some cleotide is a gapmer. In some embodiments, the oligo- embodiments, the target region is selected from the nucleotide is 18 to 24 nucleotides in length comprising: 10 group consisting of: a region 1 to 15 nucleobases 5’ of a gap region having greater than 11 contiguous 2’-deox- an intron/exon junction, 20 to 24 nucleobases 5’ of an yribonucleotides; and a first wing region and a second intron/exon junction, and 30 to 50 nucleobases 5’ of an wing region flanking the gap region, wherein each of said intron/exon. junction. In further embodiments, the HES- first and second wing regions independently have 1 to 8 oligonucleotide complex contains an oligonucleotide that 2’-O-(2-methoxyethyl)ribonucleotides. 15 specifically hybridizes to nucleotides 1-10 of a miRNA [0234] In another embodiment, the antisense oligonu- (i.e., the seed region) or that specifically hybridizes to a cleotide is not a substrate for RNAse H when bound to sequencein a precursor-miRNA (pre-miRNA) or primary- the target RNA (e.g., mRNA and miRNA). In some em- miRNA (pri-miRNA) that when bound by the oligonucle- bodiments, the oligonucleotide comprises at least one otide blocks miRNA processing. modified sugar moiety comprising a modification at the 20 [0235] Disclosed herein is a method of inhibiting the 2’-position. In some embodiments, each nucleoside of production of a protein, comprising administering to a the oligonucleotide comprises a modified sugar moiety subject an HES-oligonucleotide complex containing an comprising a modification at the 2’-position. In some em- oligonucleotide which is targeted to nucleic acids encod- bodiments the oligonucleotide comprises at least one ing the protein or decreases the endogenous expression, PNA motif. In further embodiments, all the monomeric 25 processing or function of the protein in the subject. In units of the oligonucleotide correspond to a PNA. In other some embodiments, the oligonucleotide comprises a se- embodiments the oligonucleotide comprises at least one quence substantially complementary to a nucleic acid morpholino motif. In some embodiments, the morpholino encoding the protein. is a phosphorodiamidate morpholino. In further embod- [0236] Disclosed herein is a method of decreasing the iments, all the monomeric units of the oligonucleotide 30 amount of a target cellular RNA or corresponding protein correspond to a morpholino. In further embodiments, all in a cell by contacting a cell expressing the target RNA the monomeric units of the oligonucleotide correspond with an HES-oligonucleotide complex having an oligonu- to a phosphorodiamidate morpholino e.g., ( PMO). In cleotide sequence that specifically hybridizes to the tar- some embodiments, the oligonucleotide sequence is get RNA, wherein the amount of the target RNA or cor- specifically hybridizable to a sequence within 30 nucle- 35 responding protein is reduced. In some embodiments, otides of the AUG start codon of the target RNA. In ad- the RNA is an mRNA or a miRNA. In additional embod- ditional embodiments, the HES-oligonucleotide se- iments the oligonucleotide is selected from a siRNA, a quence is specifically hybridizable to a sequence in the shRNA, a miRNA, a anti-miRNA, a dicer substrate ( e.g., 5’ untranslated region of the target RNA. In some em- dsRNA), a decoy, an aptamer, a decoy, an antisense bodiments, the HES-oligonucleotides are designed to 40 oligonucleotide and a plasmid capable of expressing an target the 3’ untranslated sequence in an RNA (e.g., mR- siRNA, a miRNA, a anti-miRNA, a ribozyme or an anti- NA). In further embodiments, the HES-oligonucleotides sense oligonucleotide. are designed to target the 3’ untranslated sequence in [0237] In particular embodiments, the oligonucleotide an RNA that is bound by an miRNA (i.e., the miRNA in the HES-oligonucleotide is an antisense oligonucle- 3’UTR target site in an mRNA). One such example is45 otide. In one embodiment, the antisense oligonucleotide "miR-Mask" or "target protector," which are single- is a substrate for RNAse H when bound to a target RNA. stranded 2’- O-methyl-modified (or other chemically mod- Inadditional embodiments,the antisenseoligonucleotide ified) antisense oligonucleotide fully complementary to is a gapmer. In some embodiments, the oligonucleotide predicted miRNA binding sites in the 3’-UTR of a specific is 18 to 24 nucleotides in length comprising: a gap region target mRNA, covering up the access of the miRNA to 50 having greater than 11 contiguous 2’-deoxyribonucle- its binding site on the target mRNA ( see, e.g., Choi et al otides; and a first wing region and a second wing region (2007) Science 318:271; Wang (2011) Methods Mol. Bi- flanking the gap region, wherein each of said first and ol. 676:43). In further embodiments, the HES-oligonucle- second wing regions independently have 1 to 8 2’-O-(2- otides are designed to mimic the 3’ untranslated se- methoxyethyl)ribonucleotides. In particular embodi- quence in an mRNA that is bound by an miRNA. One 55 ments, the oligonucleotide contains 12 to 30 linked nu- such example is "miRNA sponges," competitive miRNA cleosides. inhibitory transgene expressing multiple tandem binding [0238] In another embodiment, the oligonucleotide is sites for an endogenous miRNA, which stably interact not a substrate for RNAse H when bound to the target

33 63 EP 2 790 736 B1 64

RNA (e.g., mRNA and miRNA). In some embodiments, ments one or both strands of the Dicer substrate contains the oligonucleotide comprises at least one modified sug- one or more modified nucleosides, modified internucle- ar moiety comprising a modification at the 2’-position. In oside linkages, or combinations thereof. some embodiments, each nucleoside of the oligonucle- [0240] Also disclosed is a method of reducing the ex- otide comprises a modified sugar moiety comprising a 5 pression of a target RNA in a subject in need of such modification at the 2’-position. In some embodiments the reduced expression of the target RNA, comprising ad- oligonucleotide comprises at least one PNA motif. In fur- ministering to the subject an HES-oligonucleotide com- ther embodiments, all the monomeric units of the oligo- plex having an oligonucleotide sequence that specifically nucleotide correspond to a PNA. In other embodiments hybridizes to the target RNA, wherein the expression of the oligonucleotide comprises at least one morpholino 10 the target RNA in a cell or tissue of the subject is reduced. motif. In a further embodiment the oligonucleotide com- In some embodiments, the RNA is an mRNA or a miRNA. prises at least one phosphorodiamidate morpholino. In In additional embodiments the oligonucleotide is select- further embodiments, all the monomeric units of the oli- ed from a siRNA, shRNA, miRNA, an anti-miRNA, a dicer gonucleotide correspond to a morpholino. In further em- substrate, an aptamer, a decoy, an antisense oligonu- bodiments, all the monomeric units of the oligonucleotide 15 cleotide, a plasmid capable of expressing a siRNA, a correspond to a phosphorodiamidate morpholino (PMO). miRNA, a ribozyme and an antisense oligonucleotide. In some embodiments, the oligonucleotide sequence [0241] In particular embodiments, the oligonucleotide specifically hybridizes to a sequence within 30 nucle- in the HES-oligonucleotide is an antisense oligonucle- otides of the AUG start codon of the target RNA. In some otide. In one embodiment, the antisense oligonucleotide embodiments, the HES-oligonucleotides are designed to 20 is a substrate for RNAse H when bound to the target RNA target the 3’ untranslated sequence in an RNA (e.g., mR- (e.g., mRNA and miRNA). In additional embodiments, NA). In further embodiments, the HES-oligonucleotides the antisense oligonucleotide is a gapmer. In some em- are designed to target the 3’ untranslated sequence in bodiments, the oligonucleotide is 18 to 24 nucleotides in an RNA that is bound by an miRNA. In additional em- length comprising: a gap region having greater than 11 bodiments, the target RNA is mRNA and the oligonucle- 25 contiguous 2’-deoxyribonucleotides; and a first wing re- otide sequence specifically hybridizes to a target region gion and a second wing region flanking the gap region, of the mRNA selected from the group consisting of: an wherein each of said first and second wing regions inde- intron/exon junction of a target RNA, and an intron/exon pendently have 1 to 8 2’-O-(2-methoxyethyl)ribonucle- junction and a region 1 to 50 nucleobases 5’ of an in- otides. In particular embodiments, the oligonucleotide tron/exon junction of the target RNA. In some embodi- 30 contains 12 to 30 linked nucleosides. ments, the target region is selected from the group con- [0242] In another embodiment, the antisense oligonu- sisting of: a region 1 to 15 nucleobases 5’ of an intron/ex- cleotide is not a substrate for RNAse H when bound to on junction, 20 to 24 nucleobases 5’ of an intron/exon the target RNA (e.g., mRNA and miRNA). In some em- junction, and 30 to 50 nucleobases 5’ of an intron/exon bodiments, the oligonucleotide comprises at least one junction. In further embodiments, the HES-oligonucle- 35 modified sugar moiety comprising a modification at the otide complex contains an oligonucleotide that specifi- 2’-position. In some embodiments, each of the nucleo- cally hybridizes to nucleotides 1-10 of a miRNA ( i.e., the sides of the oligonucleotide comprise a modified sugar seed region) or that specifically hybridizes to a sequence moiety comprising a modification at the 2’-position. In in a precursor-miRNA (pre-miRNA) or primary-miRNA some embodiments the oligonucleotide comprises at (pri-miRNA) that when bound by the oligonucleotide40 least one PNA motif. In further embodiments, all the mon- blocks miRNA processing. omeric units of the oligonucleotide correspond to a PNA. [0239] In some embodiments, the oligonucleotide can In other embodiments the oligonucleotide contains at induce RNA interference (RNAi). In some embodiments least one morpholino motif. In some embodiments, the the oligonucleotide is siRNA, shRNA or a Dicer substrate. morpholino is a phosphorodiamidate morpholino. In fur- In some embodiments, the oligonucleotide is a siRNA 45 ther embodiments, all the monomeric units of the oligo- that is 18-35 nucleotides in length. In some embodi- nucleotide correspond to a morpholino. In further embod- ments, the oligonucleotide is an shRNA that has a stem iments, all the monomeric units of the oligonucleotide of 19 to 29 nucleotides in length and a loop size of be- correspond to a phosphorodiamidate morpholino (PMO). tween 4-30 nucleotides. In further embodiments the siR- In some embodiments, the oligonucleotide sequence NA or shRNA oligonucleotide contains one or more mod- 50 specifically hybridizes to a sequence within 30 nucle- ified nucleosides, modified internucleoside linkages, or otides of the AUG start codon of the target RNA. In ad- combinations thereof. In some embodiments, the oligo- ditional embodiments, the oligonucleotide sequence nucleotide is a Dicer substrate and contains 2 nucleic specifically hybridizes to a sequence in the 5’ untranslat- acid strands that are each 18-25 nucleotides in length ed region of the target RNA. In some embodiments, the and contain a 2 nucleotide 3’ overhang. In particular em- 55 HES-oligonucleotides are designed to target the 3’ un- bodiments, the Dicer substrate is a double stranded nu- translated sequence in an RNA (e.g., mRNA). In further cleic acid containing 21 nucleotides in length and con- embodiments, the HES-oligonucleotides are designed to tains a two nucleotide 3’ overhang. In further embodi- target the 3’ untranslated sequence in an RNA that is

34 65 EP 2 790 736 B1 66 bound by an miRNA. In additional embodiments, the tar- quence specifically hybridizes to a DNA or mRNA encod- get RNA is mRNA and the oligonucleotide sequence spe- ing the polypeptide, such that the expression of the cifically hybridizes to a target region of the target mRNA polypeptide of interest is reduced. In further embodi- selected from the group consisting of: an intron/exon ments the oligonucleotide is selected from a siRNA, shR- junction of a target RNA, and an intron/exon junction and 5 NA, miRNA, an anti-miRNA, a dicer substrate, an anti- a region 1 to 50 nucleobases 5’ of an intron/exon junction sense oligonucleotide, a plasmid capable of expressing of the target RNA. In some embodiments, the target re- a siRNA, a miRNA, a ribozyme and an antisense oligo- gion is selected from the group consisting of: a region 1 nucleotide, and wherein the oligonucleotide specifically to 15 nucleobases 5’ of an intron/exon junction, 20 to 24 hybridizes to a nucleic acid that encodes the polypeptide, nucleobases 5’ of an intron/exon junction, and 30 to 50 10 or a complement thereof, such that the expression of the nucleobases 5’ of an intron/exon junction. In further em- polypeptide is reduced. In particular embodiments, the bodiments, the HES-oligonucleotide complex contains oligonucleotide contains 12 to 30 linked nucleosides. In an oligonucleotide that specifically hybridizes to nucle- some embodiments, the complex contains a double- otides 1-10 of a miRNA i.e.,( the seed region) or that stranded RNA (dsRNA). In some embodiments, the oli- specifically hybridizes to a sequence in a precursor-miR- 15 gonucleotide comprises at least one modified oligonu- NA (pre-miRNA) or primary-miRNA (pri-miRNA) that cleotide. In further embodiments, the oligonucleotide when bound by the oligonucleotide blocks miRNA comprises at least one modified oligonucleotide motif se- processing. lected from a 2’ modification (e.g., 2’-fluoro, 2’-OME and [0243] In some embodiments, the oligonucleotide can 2’-methoxyethyl (2’-MOE)) a locked nucleic acid (LNA induce RNA interference (RNAi). In some embodiments 20 and alpha LNA), a PNA motif, and morpholino motif. the oligonucleotide is siRNA, shRNA or a Dicer substrate. [0246] In particular embodiments, the oligonucleotide In some embodiments, the oligonucleotide is a siRNA in the HES-oligonucleotide complex is antisense se- that is 18-35 nucleotides in length. In some embodi- quence and is a substrate for RNAse H when bound to ments, the oligonucleotide is an shRNA that has a stem a target RNA. In additional embodiments, the antisense of 19 to 29 nucleotides in length and a loop size of be- 25 oligonucleotide is a gapmer. In some embodiments, the tween 4-30 nucleotides. In further embodiments the siR- gapmer is an antisense oligonucleotide that is a chimeric NA or shRNA oligonucleotide contains one or more mod- oligonucleotide. In some embodiments, the chimeric ol- ified nucleosides, modified internucleoside linkages, or igonucleotide comprises a 2’-deoxynucleotide central combinations thereof. In some embodiments, the oligo- gap region positioned between 5’ and 3’ wing segments. nucleotide is a Dicer substrate and contains 2 nucleic 30 The wing segments contain nucleosides containing at acid strands that are each 18-25 nucleotides in length least one 2’- modified sugar. The wing segments are con- and contain a 2 nucleotide 3’ overhang. In particular em- tain nucleosides containing at least one 2’ sugar moiety bodiments, the Dicer substrate is a double stranded nu- selected from a 2’-O-methoxyethyl sugar moiety or a bi- cleic acid containing 21 nucleotides in length and con- cyclic nucleic acid sugar moiety. In some embodiments, tains a two nucleotide 3’ overhang. In further embodi- 35 the gap segment may be ten 2’-deoxynucleotides in ments one or both strands of the Dicer substrate contains length and each of the wing segments may be five 2’-O- one or more modified nucleosides, modified internucle- methoxyethyl nucleotides in length. The chimeric oligo- oside linkages, or combinations thereof. nucleotide may be uniformly comprised of phospho- [0244] In some embodiments, an HES-oligonucleotide rothioate internucleoside linkages. Further, each cyto- complex is administered to a subject to deliver an oligo- 40 sine of the chimeric oligonucleotide may be a 5’-methyl- nucleotide that specifically hybridizes to a target nucleic cytosine. acid (e.g., gene, mRNA or miRNA), which provides a [0247] In another embodiment, the antisense oligonu- growth advantage for a tumor cell or enhances the rep- cleotide is not a substrate for RNAse H when hybridized lication of a microorganism. In other embodiments, an to the RNA. In some embodiments, each nucleoside of HES-oligonucleotide complex is administered to deliver 45 the oligonucleotide comprises a modified sugar moiety an antisense, siRNA, shRNA, Dicer substrate or miRNA comprising a modification at the 2’-position. In some em- targeting an mRNA sequence coding for a protein ( e.g., bodiments the oligonucleotide contains at least one PNA a protein variant) which has been implicated in a disease. motif. In further embodiments, all the monomeric units of Thus, disclosed herein is an in vivo delivery system for the oligonucleotide correspond to a PNA. In other em- transporting specific nucleic acid sequences into live50 bodiments the oligonucleotide contains at least one mor- cells to for example, silence genes in organisms afflicted pholino motif. In some embodiments, the morpholino is with pathologic conditions due to aberrant gene expres- a phosphorodiamidate morpholino. In further embodi- sion. ments, all the monomeric units of the oligonucleotide cor- [0245] Disclosed herein is a method of decreasing the respond to a morpholino. In further embodiments, all the amount of a polypeptide of interest in a cell, comprising: 55 monomeric units of the oligonucleotide correspond to a contacting a cell expressing a nucleic acid that encodes phosphorodiamidate morpholino (PMO). In some em- the polypeptide, or a complement thereof, with an HES- bodiments, the oligonucleotide sequence specifically hy- oligonucleotide complex having an oligonucleotide se- bridizes to a sequence within 30 nucleotides of the AUG

35 67 EP 2 790 736 B1 68 start codonof the targetRNA. In additional embodiments, production of a protein, comprising administering to a the oligonucleotide sequence specifically hybridizes to a subject an HES-oligonucleotide complex containing an sequence in the 5’ untranslated region of the target RNA. oligonucleotide which encodes the protein or increases In some embodiments, the HES-oligonucleotides are de- the endogenous expression, processing or function of signed to target the 3’ untranslated sequence in an RNA 5 the protein in the subject. In some embodiments the ol- (e.g., mRNA). In further embodiments, the HES-oligonu- igonucleotide comprises a sequence substantially the cleotides are designed to target the 3’ untranslated se- same as nucleic acids encoding the protein. In some em- quence in an RNA that is bound by an miRNA. In addi- bodiments, the oligonucleotide shares 100% identity with tional embodiments, the oligonucleotide sequence spe- at least 15 contiguous nucleotides, at least 20 contiguous cifically hybridizes to a target region of a target mRNA 10 nucleotides or over the full-length of an endogenous nu- selected from the group consisting of an intron/exon junc- cleic acid sequence encoding the protein. tion of a target RNA, and an intron/exon junction and a [0251] Also disclosed herein is a method of treating a region 1 to 50 nucleobases 5’ of an intron/exon junction disease or disorder characterized by the overexpression of the target RNA. In some embodiments, the target re- of a nucleic acid in a subject, comprising administering gion is selected from the group consisting of: a region 1 15 to the subject an HES-oligonucleotide complex contain- to 15 nucleobases 5’ of an intron/exon junction, 20 to 24 ing an oligonucleotide which is targeted to a nucleic acid nucleobases 5’ of an intron/exon junction, and 30 to 50 comprising or encoding the nucleic acid and which acts nucleobases 5’ of an intron/exon junction. In further em- to reduce the levels of the nucleic acid and/or interfere bodiments, the HES-oligonucleotide complex contains with its function in the subject. In some embodiments, an oligonucleotide that specifically hybridizes to nucle- 20 the nucleic acid is DNA, mRNA or miRNA. In additional otides 1-10 of a miRNA i.e( ., the seed region) or that embodiments the oligonucleotide is selected from an siR- specifically hybridizes to a sequence in a precursor-miR- NA, an shRNA, a miRNA, an anti-miRNA, a dicer sub- NA (pre-miRNA) or primary-miRNA (pri-miRNA) that strate, an antisense oligonucleotide, a plasmid capable when bound by the oligonucleotide blocks miRNA of expressing an siRNA, a miRNA, a ribozyme and an processing. 25 antisense oligonucleotide. [0248] In further embodiments, the oligonucleotide can [0252] In particular embodiments, the nucleic acid is induce RNA interference (RNAi). In some embodiments RNA and the oligonucleotide in the HES-oligonucleotide the oligonucleotide is siRNA, shRNA or a Dicer substrate. is an antisense oligonucleotide. In one embodiment, the In some embodiments, the oligonucleotide is an siRNA antisense oligonucleotide is a substrate for RNAse H that is 18-35 nucleotides in length. In some embodi-30 when hybridized to the RNA. In additional embodiments, ments, the oligonucleotide is an shRNA that has a stem the antisense oligonucleotide is a gapmer. In some em- of 19 to 29 nucleotides in length and a loop size of be- bodiments, the oligonucleotide is 18 to 24 nucleotides in tween 4-30 nucleotides. In further embodiments the siR- length comprising: a gap region having greater than 11 NA or shRNA oligonucleotide contains one or more mod- contiguous 2’-deoxyribonucleotides; and a first wing re- ified nucleosides, modified internucleoside linkages, or 35 gion and a second wing region flanking the gap region, combinations thereof. In some embodiments, the oligo- wherein each of said first and second wing regions inde- nucleotide is a Dicer substrate and contains 2 nucleic pendently have 1 to 8 2’-O-(2-methoxyethyl)ribonucle- acid strands that are each 18-25 nucleotides in length otides. In particular embodiments, the oligonucleotide and contain a 2 nucleotide 3’ overhang. In particular em- contains 12 to 30 linked nucleosides. In some embodi- bodiments, the Dicer substrate is a double stranded nu- 40 ments, the oligonucleotide comprises a sequence sub- cleic acid containing 21 nucleotides in length and con- stantially complementary to the nucleic acid. tains a two nucleotide 3’ overhang. In further embodi- [0253] In another embodiment, the oligonucleotide is ments one or both strands of the Dicer substrate contains not a substrate for RNAse H when bound to the nucleic one or more modified nucleosides, modified internucle- acid. In some embodiments, each nucleoside of the oli- oside linkages, or combinations thereof 45 gonucleotide comprises a modified sugar moiety com- [0249] Disclosed herein is a method of increasing the prising a modification at the 2’-position. In some embod- activity of a nucleic acid in a subject, comprising admin- iments the oligonucleotide contains at least one PNA mo- istering to the subject an HES-oligonucleotide complex tif. In further embodiments, all the monomeric units of the containing an oligonucleotide which comprises or en- oligonucleotide correspond to a PNA. In other embodi- codes the nucleic acid or increases the endogenous ex- 50 ments the oligonucleotide contains at least one mor- pression, processing or function of the nucleic acid ( e.g., pholino motif. In some embodiments, the morpholino is by binding regulatory sequences in the gene encoding a phosphorodiamidate morpholino. In further embodi- the nucleic acid) and which acts to increase the level of ments, all the monomeric units of the oligonucleotide cor- the nucleic acid and/or increase its function in the cell. respond to a morpholino. In further embodiments, all the In some embodiments, the oligonucleotide comprises a 55 monomeric units of the oligonucleotide correspond to a sequence substantially the same as nucleic acids com- phosphorodiamidate morpholino (PMO). In some em- prising or encoding the nucleic acid. bodiments, the oligonucleotide sequence specifically hy- [0250] Disclosed herein is a method of increasing the bridizes to a sequence within 30 nucleotides of the AUG

36 69 EP 2 790 736 B1 70 start codonof the targetRNA. In additional embodiments, gonucleotide. In some embodiments, the oligonucleotide the oligonucleotide sequence specifically hybridizes to a shares 100% identity with at least 15 contiguous nucle- sequence in the 5’ untranslated region of the target RNA. otides, at least 20 contiguous nucleotides or over the full- In some embodiments, the HES-oligonucleotides are de- length of an endogenous nucleic acid sequence encod- signed to target the 3’ untranslated sequence in an RNA 5 ing the protein. (e.g., mRNA). In further embodiments, the HES-oligonu- [0256] In particular embodiments, the targeted nucleic cleotides are designed to target the 3’ untranslated se- acid is RNA and the oligonucleotide in the HES-oligonu- quence in an RNA that is bound by an miRNA. In addi- cleotide is an antisense oligonucleotide. In one embod- tional embodiments, the nucleic acid is mRNA and the iment, the antisense oligonucleotide is a substrate for oligonucleotide sequence specifically hybridizes to a tar- 10 RNAse H when hybridized to the RNA. In additional em- get region of the mRNA selected from the group consist- bodiments, the antisense oligonucleotide is a gapmer. In ing of: an intron/exon junction of a target RNA, and an some embodiments, the oligonucleotide is 18 to 24 nu- intron/exon junction and a region 1 to 50 nucleobases 5’ cleotides in length comprising: a gap region having great- of an intron/exon junction of the target RNA. In some erthan 11contiguous 2’-deoxyribonucleotides;and a first embodiments, the target region is selected from the15 wing region and a second wing region flanking the gap group consisting of: a region 1 to 15 nucleobases 5’ of region, wherein each of said first and second wing re- an intron/exon junction, 20 to 24 nucleobases 5’ of an gions independently have 1 to 8 2’-O-(2-methoxyethyl)ri- intron/exon junction, and 30 to 50 nucleobases 5’ of an bonucleotides. In particular embodiments, the oligonu- intron/exon junction. In further embodiments, the HES- cleotide contains 12 to 30 linked nucleosides. In some oligonucleotide complex contains an oligonucleotide that 20 embodiments, the oligonucleotide comprises a se- specifically hybridizes to nucleotides 1-10 of a miRNA quence substantially complementary to the nucleic acid. (i.e., the seed region) or that specifically hybridizes to a [0257] In another embodiment, the oligonucleotide is sequence in a precursor-miRNA (pre-miRNA) or primary- not a substrate for RNAse H when bound to the target miRNA (pri-miRNA) that when bound by the oligonucle- RNA (e.g., mRNA and miRNA). In some embodiments, otide blocks miRNA processing. 25 the oligonucleotide comprises at least one modified sug- [0254] In further embodiments, the oligonucleotide can ar moiety comprising a modification at the 2’-position. In induce RNA interference (RNAi). In some embodiments some embodiments, each nucleoside of the oligonucle- the oligonucleotide is siRNA, shRNA or a Dicer substrate. otide comprises a modified sugar moiety comprising a In some embodiments, the oligonucleotide is an siRNA modification at the 2’-position. In some embodiments the that is 18-35 nucleotides in length. In some embodi-30 oligonucleotide comprises at least one PNA motif In fur- ments, the oligonucleotide is an shRNA that has a stem ther embodiments, all the monomeric units of the oligo- of 19 to 29 nucleotides in length and a loop size of be- nucleotide correspond to a PNA. In other embodiments tween 4-30 nucleotides. In further embodiments the siR- the oligonucleotide comprises at least one morpholino NA or shRNA oligonucleotide contains one or more mod- motif. In some embodiments, the morpholino is a phos- ified nucleosides, modified internucleoside linkages, or 35 phorodiamidate morpholino. In further embodiments, all combinations thereof. In some embodiments, the oligo- the monomeric units of the oligonucleotide correspond nucleotide is a Dicer substrate and contains 2 nucleic to a morpholino. In further embodiments, all the mono- acid strands that are each 18-25 nucleotides in length meric units of the oligonucleotide correspond to a phos- and contain a 2 nucleotide 3’ overhang. In particular em- phorodiamidate morpholino (PMO). In some embodi- bodiments, the Dicer substrate is a double stranded nu- 40 ments, the oligonucleotide sequence specifically hybrid- cleic acid containing 21 nucleotides in length and con- izes to a sequence within 30 nucleotides of the AUG start tains a two nucleotide 3’ overhang. In further embodi- codon of the target RNA. In additional embodiments, the ments one or both strands of the Dicer substrate contains oligonucleotide sequence is specifically hybridizable to one or more modified nucleosides, modified internucle- a sequence in the 5’ untranslated region of the target oside linkages, or combinations thereof. 45 RNA. (e.g., within 30 nucleotides of the AUG start codon) [0255] Disclosed herein is a method of treating a dis- and to reduce translation. In some embodiments, the ease or disorder characterized by the overexpression of HES-oligonucleotides are designed to target the 3’ un- a protein in a subject, comprising administering to the translated sequence in an RNA (e.g., mRNA). In further subject an HES-oligonucleotide complex containing an embodiments, the HES-oligonucleotides are designed to oligonucleotide which is targeted to a nucleic acid encod- 50 target the 3’ untranslated sequence in an RNA that is ing the protein or decreases the endogenous expression, bound by an miRNA. In additional embodiments, the nu- processing or function of the protein in the subject. In cleic acid is mRNA and the oligonucleotide sequence some embodiments, the nucleic acid is DNA, mRNA or specifically hybridizes to a target region of an mRNA en- miRNA. In additional embodiments the oligonucleotide coding the protein selected from the group consisting of: is selected from an siRNA, an shRNA, miRNA, an anti- 55 an intron/exon junction of a target RNA, and an intron/ex- miRNA, a dicer substrate, an aptamer, a decoy, an anti- on junction and a region 1 to 50 nucleobases 5’ of an sense oligonucleotide, a plasmid capable of expressing intron/exon junction of the target RNA. In some embod- an siRNA, an miRNA, a ribozyme and an antisense oli- iments, the target region is selected from the group con-

37 71 EP 2 790 736 B1 72 sisting of: a region 1 to 15 nucleobases 5’ of an intron/ex- cleotide sequence specifically hybridizes to a sequence on junction, 20 to 24 nucleobases 5’ of an intron/exon in the 5’ untranslated region of the target RNA. In some junction, and 30 to 50 nucleobases 5’ of an intron/exon embodiments, the HES-oligonucleotides are designed to junction. In further embodiments, the HES-oligonucle- target the 3’ untranslated sequence in an RNA (e.g., mR- otide complex contains an oligonucleotide that specifi- 5 NA). In further embodiments, the HES-oligonucleotides cally hybridizes to nucleotides 1-10 of a miRNA ( i.e., the are designed to target the 3’ untranslated sequence in seed region) or that specifically hybridizes to a sequence an RNA that is bound by an miRNA. In additional em- in a precursor-miRNA (pre-miRNA) or primary-miRNA bodiments, oligonucleotide sequence is specifically hy- (pri-miRNA) that when bound by the oligonucleotide bridizable to a target region of an mRNA selected from blocks miRNA processing. 10 the group consisting of: an intron/exon junction of a target [0258] In further embodiments, the oligonucleotide can RNA, and an intron/exon junction and a region 1 to 50 induce RNA interference (RNAi). In some embodiments nucleobases 5’ of an intron/exon junction of the target the oligonucleotide is siRNA, shRNA or a Dicer substrate. RNA. In some embodiments, the target region is selected In some embodiments, the oligonucleotide is an siRNA from the group consisting of: a region 1 to 15 nucleobases that is 18-35 nucleotides in length. In some embodi-15 5’ of an intron/exon junction, 20 to 24 nucleobases 5’ of ments, the oligonucleotide is an shRNA that has a stem an intron/exon junction, and 30 to 50 nucleobases 5’ of of 19 to 29 nucleotides in length and a loop size of be- an intron/exon junction. tween 4-30 nucleotides. In further embodiments the siR- [0260] In particular embodiments, the disease or dis- NA or shRNA oligonucleotide contains one or more mod- order is Duchenne Muscular Dystrophy (DMD). In some ified nucleosides, modified internucleoside linkages, or 20 embodiments, the oligonucleotide specifically hybridizes combinations thereof. In some embodiments, the oligo- to mRNA sequence that promotes message splicing to nucleotide is a Dicer substrate and contains 2 nucleic "skip over" exon 44, 45, 50, 51, 52, 53 or 55 of the dys- acid strands that are each 18-25 nucleotides in length trophin gene. In particular embodiments, the oligonucle- and contain a 2 nucleotide 3’ overhang. In particular em- otide specifically hybridizes to mRNA sequence that pro- bodiments, the Dicer substrate is a double stranded nu- 25 motes message splicing to "skip over" exon 51 of the cleic acid containing 21 nucleotides in length and con- dystrophin gene. In particular embodiments, the oligonu- tains a two nucleotide 3’ overhang. In further embodi- cleotide in the HES-oligonucleotide complex is AVI-4658 ments one or both strands of the Dicer substrate contains (AVI Biopharma). In other embodiments, the oligonucle- one or more modified nucleosides, modified internucle- otide in the HES-oligonucleotide complex is competes oside linkages, or combinations thereof. 30 for dystrophin mRNA binding with AVI-4658. [0259] Disclosed herein is a method of treating (e.g., [0261] Disclosed herein is a method comprising, se- alleviating) a disease or disorder characterized by the lecting a subject who has received a diagnosis of a dis- aberrant expression of a protein in a subject, comprising ease or disorder, administering to the subject a thera- administering to the subject an HES-oligonucleotide peutically effective amount of a HES-oligonucleotide complex,containing an oligonucleotide which specifically 35 complex containing an oligonucleotide that specifically hybridizes to the mRNA encoding the protein and alter hybridizes to a nucleic acid sequence believed to be as- the splicing of the target RNA ( e.g., promoting exon skip- sociated with or to encode a protein associated with the ping). In some embodiments, each nucleoside of the ol- disease or disorder or a condition related thereto, and igonucleotide comprises at least one modified sugar moi- monitoring disease progression in the subject. ety comprising a modification at the 2’-position. In par- 40 [0262] In some embodiments, the nucleic acid is DNA, ticular embodiments, the modified oligonucleotide is a 2’ mRNA or miRNA. In additional embodiments the oligo- OME or 2’ allyl. In additional embodiments, the modified nucleotide is selected from an siRNA, an shRNA, a miR- oligonucleotide is LNA, alpha LNA ( e.g., an LNA or alpha NA, an anti-miRNA, a dicer substrate, an aptamer, a de- LNAcontaining asteric bulk moiety at the 5’position ( e.g., coy, an antisense oligonucleotide, a plasmid capable of a methyl group). In some embodiments the oligonucle- 45 expressing an siRNA, a miRNA, a ribozyme and an an- otide contains at least one PNA motif. In further embod- tisense oligonucleotide. In some embodiments, the oli- iments, all the monomeric units of the oligonucleotide gonucleotide shares 100% identity with at least 15 con- correspond to a PNA. In other embodiments the oligo- tiguous nucleotides, at least 20 contiguous nucleotides nucleotide contains at least one morpholino motif. In or over the full-length of the nucleic acid. some embodiments, the morpholino is a phosphorodi- 50 [0263] In particular embodiments, the nucleic acid is amidate morpholino.In further embodiments,all themon- RNA and the oligonucleotide in the HES-oligonucleotide omeric units of the oligonucleotide correspond to a mor- is an antisense oligonucleotide. In one embodiment, the pholino. In further embodiments, all the monomeric units antisense oligonucleotide is a substrate for RNAse H of the oligonucleotide correspond to a phosphorodiami- when hybridized to the RNA. In additional embodiments, date morpholino (PMO). In some embodiments, the oli- 55 the antisense oligonucleotide is a gapmer. In some em- gonucleotide sequence specifically hybridizes to a se- bodiments, the oligonucleotide is 18 to 24 nucleotides in quence within 30 nucleotides of the AUG start codon of length comprising: a gap region having greater than 11 the target RNA. In additional embodiments, the oligonu- contiguous 2’-deoxyribonucleotides; and a first wing re-

38 73 EP 2 790 736 B1 74 gion and a second wing region flanking the gap region, tween 4-30 nucleotides. In further embodiments the siR- wherein each of said first and second wing regions inde- NAor shRNA oligonucleotide, contains one or more mod- pendently have 1 to 8 2’-O-(2-methoxyethyl)ribonucle- ified nucleosides, modified internucleoside linkages, or otides. In particular embodiments, the oligonucleotide combinations thereof. In some embodiments, the oligo- contains 12 to 30 linked nucleosides. In some embodi- 5 nucleotide is a Dicer substrate and contains 2 nucleic ments, the oligonucleotide comprises a sequence sub- acid strands that are each 18-25 nucleotides in length stantially complementary to the nucleic acid. and contain a 2 nucleotide 3’ overhang. In particular em- [0264] In another embodiment, the oligonucleotide is bodiments, the Dicer substrate is a double stranded nu- not a substrate for RNAse H when bound to the target cleic acid containing 21 nucleotides in length and con- RNA (e.g., mRNA and miRNA). In some embodiments, 10 tains a two nucleotide 3’ overhang. In further embodi- the oligonucleotide comprises at least one modified sug- ments one or both strands of the Dicer substrate contains ar moiety comprising a modification at the 2’-position. In one or more modified nucleosides, modified internucle- some embodiments, all the nucleosides of the oligonu- oside linkages, or combinations thereof. cleotide comprise a modified sugar moiety comprising a [0266] Disclosed herein is a method of slowing disease modification at the 2’-position. In some embodiments the 15 progression in a subject suffering from a disease or dis- oligonucleotide comprises at least one PNA motif. In fur- order correlated with the overexpression of a protein ther embodiments, all the monomeric units of the oligo- comprising, administering to the subject an HES-oligo- nucleotide correspond to a PNA. In other embodiments nucleotide complex containing an oligonucleotide that the oligonucleotide comprises at least one morpholino specifically hybridizes to a DNA or mRNA encoding the motif. In some embodiments, the morpholino is a phos- 20 protein, such that the expression of the polypeptide is phorodiamidate morpholino. In additional embodiments, reduced. In additional embodiments the oligonucleotide all the monomeric units of the oligonucleotide correspond is selected from an siRNA, an shRNA, a miRNA, an anti- to a morpholino. In further embodiments all the mono- miRNA, a dicer substrate, an antisense oligonucleotide, meric units of the oligonucleotide correspond to a phos- a plasmid capable of expressing an siRNA, a miRNA, a phorodiamidate morpholino (PMO). In some embodi-25 ribozyme and an antisense oligonucleotide. In some em- ments, the oligonucleotide sequence specifically hybrid- bodiments, the oligonucleotide shares 100% identity with izes to a sequence within 30 nucleotides of the AUG start at least 15 contiguous nucleotides, at least 20 contiguous codon of the target RNA. In additional embodiments, the nucleotides or over the full-length of the DNA or mRNA oligonucleotide sequence specifically hybridizes to a se- encoding the protein. quence in the 5’ untranslated region of the target RNA. 30 [0267] In particular embodiments, the nucleic acid is In some embodiments, the HES-oligonucleotides are de- mRNA and the oligonucleotide in the HES-oligonucle- signed to target the 3’ untranslated sequence in an RNA otide is an antisense oligonucleotide. In one embodi- (e.g., mRNA). In further embodiments, the HES-oligonu- ment, the antisense oligonucleotide is a substrate for cleotides are designed to target the 3’ untranslated se- RNAse H when hybridized to the RNA. In additional em- quence in an RNA that is bound by an miRNA. In addi- 35 bodiments, the antisense oligonucleotide is a gapmer. In tional embodiments, the oligonucleotide specifically hy- some embodiments, the oligonucleotide is 18 to 24 nu- bridizes to a target region of the mRNA selected from the cleotides in length comprising: a gap region having great- group consisting of: an intron/exon junction of a target erthan 11contiguous 2’-deoxyribonucleotides;and a first RNA, and an intron/exon junction and a region 1 to 50 wing region and a second wing region flanking the gap nucleobases 5’ of an intron/exon junction of the target 40 region, wherein each of said first and second wing re- RNA. In some embodiments, the target region is selected gions independently have 1 to 8 2’-O-(2-methoxyethyl)ri- from the group consisting of: a region 1 to 15 nucleobases bonucleotides. In particular embodiments, the oligonu- 5’ of an intron/exon junction, 20 to 24 nucleobases 5’ of cleotide contains 12 to 30 linked nucleosides. In some an intron/exon junction, and 30 to 50 nucleobases 5’ of embodiments, the oligonucleotide comprises a se- an intron/exon junction. In additional embodiments, the 45 quence substantially complementary to the nucleic acid. HES-oligonucleotide complex contains an oligonucle- [0268] In another embodiment, the oligonucleotide is otide that specifically hybridizes to nucleotides 1-10 of a not a substrate for RNAse H when bound to the target miRNA (i.e., the seed region) or that specifically hybrid- RNA (e.g., mRNA and miRNA). In some embodiments, izes to a sequence in a precursor-miRNA (pre-miRNA) the oligonucleotide comprises at least one modified sug- or primary-miRNA (pri-miRNA) that when bound by the 50 ar moiety comprising a modification at the 2’-position. In oligonucleotide blocks miRNA processing. some embodiments, each nucleoside of the oligonucle- [0265] In further embodiments, the oligonucleotide can otide comprises a modified sugar moiety comprising a induce RNA interference (RNAi). In some embodiments modification at the 2’-position. In some embodiments the the oligonucleotide is siRNA, shRNA or a Dicer substrate. oligonucleotide comprises at least one PNA motif. In fur- In some embodiments, the oligonucleotide is an siRNA 55 ther embodiments, all the monomeric units of the oligo- that is 18-35 nucleotides in length. In some embodi- nucleotide correspond to a PNA. In other embodiments ments, the oligonucleotide is an shRNA that has a stem the oligonucleotide comprises at least one morpholino of 19 to 29 nucleotides in length and a loop size of be- motif. In some embodiments, the morpholino is a phos-

39 75 EP 2 790 736 B1 76 phorodiamidate morpholino. In further embodiments, all ease. In one embodiment, an oligonucleotide in the HES- the monomeric units of the oligonucleotide correspond oligonucleotide complex inhibits miR-122. Miravirsen to a morpholino. In further embodiments, all the mono- (SPC3649), an inhibitor of miR-122 developed by meric units of the oligonucleotide correspond to a phos- Santaris Pharma A/S. Mir-122 is a liver specific miRNA phorodiamidate morpholino (PMO). In some embodi- 5 that the Hepatitis C virus requires for replication as a ments, the oligonucleotide sequence specifically hybrid- critical endogenous host factor. Clinical trial data for 4- izes to a sequence within 30 nucleotides of the AUG start week Miravirsen monotherapy has shown robust dose- codon of the target RNA. In additional embodiments, the dependent anti-viral activity. Regulus Therapeutics and oligonucleotide sequence is specifically hybridizable to GlaxoSmithKline (GSK) have likewise demonstrated in a sequence in the 5’ untranslated region of the target10 a preclinical study that miR-122 is essential in the repli- RNA. In some embodiments, the HES-oligonucleotides cation of HCV and plan to advance an anti-miR-122 into are designed to target the 3’ untranslated sequence in clinical studies for the treatment of HCV infection. an RNA (e.g., mRNA). In further embodiments, the HES- [0272] In another embodiment, an oligonucleotide in oligonucleotides are designed to target the 3’ untranslat- an HES-oligonucleotide complex is an inhibitor or mimic ed sequence in an RNA that is bound by an miRNA. In 15 of an miRNA associated with fibrosis. In one embodi- additional embodiments, the nucleic acid is an mRNA ment, an oligonucleotide in the HES-oligonucleotide and the oligonucleotide sequence specifically hybridizes complex of the invention inhibits miR-21. Preclinical stud- to a target region of the mRNA selected from the group ies by Regulus Pharmaceutical and Sanofi Aventis have consisting of: an intron/exon junction of a target RNA, shown that inhibition of miR-21, which is upregulated in and an intron/exon junction and a region 1 to 50 nucle- 20 human fibrotic tissues, can improve organ function in obases 5’ of an intron/exon junction of the target RNA. multiple models of fibrosis including heart and kidney. In In some embodiments, the target region is selected from another embodiment, an oligonucleotide in the HES-oli- the group consisting of: a region 1 to 15 nucleobases 5’ gonucleotide complex of an intron/exon junction, 20 to 24 nucleobases 5’ of an corresponds to or mimics miR-29. MGN-4220, mimics or intron/exon junction, and 30 to 50 nucleobases 5’ of an 25 miRNA replacement therapy by Mirna Therapeutics, tar- intron/exon junction. gets miR-29 implicated in cardiac fibrosis. [0269] In further embodiments, the oligonucleotide can [0273] In another embodiment, an oligonucleotide in induce RNA interference (RNAi). In some embodiments an HES-oligonucleotide complex is an inhibitor or mimic the oligonucleotide is siRNA, shRNA or a Dicer substrate. of an miRNA associated with a cardiovascular disease, In some embodiments, the oligonucleotide is an siRNA 30 including, but not limited to, stroke, heart disease, athero- that is 18-35 nucleotides in length. In some embodi- sclerosis, restenosis, thrombosis, anemia, leucopenia, ments, the oligonucleotide is an shRNA that has a stem neutropenia, thrombocytopenia, granuloctopenia, pan- of 19 to 29 nucleotides in length and a loop size of be- cytoia and idiopathic thrombocytopenic purpura. In one tween 4-30 nucleotides. In further embodiments the siR- embodiment, an oligonucleotide in the HES-oligonucle- NA or shRNA oligonucleotide contains one or more mod- 35 otide complex inhibits miR-33. Regulus Pharmaceutical ified nucleosides, modified internucleoside linkages, or and AstraZeneca has shown in preclinical studies that combinations thereof. In some embodiments, the oligo- the inhibition of miR-33 reduces arterial plaque size and nucleotide is a Dicer substrate and contains 2 nucleic increase levels of HDL. In another embodiment, an oli- acid strands that are each 18-25 nucleotides in length gonucleotidein theHES-oligonucleotide complexinhibits and contain a 2 nucleotide 3’ overhang. In particular em- 40 miR-92, miR-378, miR-206 and/or the miR-143/145 fam- bodiments, the Dicer substrate is a double stranded nu- ily. MGN-6114, MGN-5804, MGN-2677, MGN-8107, de- cleic acid containing 21 nucleotides in length and con- veloped by Miragen Therapeutics, respectively targets tains a two nucleotide 3’ overhang. In further embodi- miR-92 implicated in peripheral arterial disease, miR-378 ments one or both strands of the Dicer substrate contains implicated in cardiometablolic disease, miR- 143/145 one or more modified nucleosides, modified internucle- 45 family implicated in vascular disease, and miR-206 im- oside linkages, or combinations thereof. plicated in amylotrophic lateral sclerosis. In a further em- bodiment, an oligonucleotide in the HES-oligonucleotide Therapeutic applications on miRNA-related patholo- complex inhibits the miR-208/209 family and/or the miR- gies 15/195 family. Miragen Therapeutics’s MGN-9103 and 50 MGN-1374 are miRNA inhibitors that respectively target [0270] There currently exist several distinct groups of miR-208/209 family for chronic heart failure and miR- pathological conditions that are known to be regulated 15/195 family for post-myocardial infarction remodeling. by an miRNA or a family of miRNA, which can be targeted In another embodiment, an oligonucleotide in the HES- using the HES-oligonucleotide complexes of the present oligonucleotide complex inhibits miR-126 and/or invention. 55 miR92a. miR-126 and miR-92a play central roles in the [0271] In one embodiment, an oligonucleotide in an development of an atherosclerotic plaque. HES-oligonucleotide complex is an inhibitor or mimic of [0274] In another embodiment, an oligonucleotide in one or more miRNAs associated with an infectious dis- the HES-oligonucleotide complex is an inhibitor of an

40 77 EP 2 790 736 B1 78 miRNA associated with a neurological disease or condi- 132, miR-137, miR-184. These microRNAs are among tions. In one embodiment, an oligonucleotide in the HES- those that have been reported to have various roles in oligonucleotide complex inhibits miR-206. miR-206 plays adult neurogenesis in neural stem cells (NSCs). a crucial role in ALS and in neuromuscular synapse re- [0281] In some embodiments, an oligonucleotide in the generation. 5 HES-oligonucleotide complex is an inhibitor or mimic of [0275] In another embodiment, an oligonucleotide in an miRNAs selected from: let-7a, miR-21, mir-26, miR- the HES-oligonucleotide complex is an inhibitor or mimic 125b, mir-145, miR-155, miR-191, miR-193a, miR-200 of an miRNAs associated with oncological conditions. In family, miR-205, miR-221, and miR-222. These microR- one embodiment, an oligonucleotide in the HES-oligo- NAs are among those that have been reported to function nucleotide complex inhibits miR-21. miR-21 has been 10 as diagnostic or prognostic biomarkers for various types suggested by numerous scientific publications to play an of cancers. In particular embodiment, an oligonucleotide important role in the initiation and progression of cancers in the HES-oligonucleotide complex is an inhibitor of a including liver, kidney, breast, prostate, lung and brain. miRNA selected from: miR-21, mir-26, miR-125b, miR- Anti-miR-21 in hepatocellular carcinoma (HCC) mouse 155, miR-193a, miR-200 family, miR-221, and miR-222. model has shown delayed tumor progression in a pre- 15 In particular embodiment, an oligonucleotide in the HES- clinical study by Regulus oligonucleotide complex contains the sequence of, or [0276] Pharmaceutical and Sanofi Aventis. In another mimics a miRNA selected from: let-7a, mir-145, miR-191, embodiment, an oligonucleotide in the HES-oligonucle- and miR-205. otide complex of the invention inhibits miR-10b. Preclin- [0282] In some embodiments, an oligonucleotide in the ical animal studies of anti-miR-10b by Regulus Pharma- 20 HES-oligonucleotide complex is an inhibitor of an miR- ceutical also showed therapeutic effect in GBM model. NAs selected from: miR-138, mir-182, miR-21, mir- In an additional embodiment, an oligonucleotide in the 103/107, miR-29c. These microRNAs are among those HES-oligonucleotide complex corresponds to or mimics that have been reported to have roles in arthritis, lupus, miR-34. Mimics or atherosclerosis, insulin sensitivity, and albuminuria, re- miRNA replacement therapy by Mirna Therapeutics of 25 spectively. miR-34, which is lost or expressed at reduced levels in [0283] In some embodiments, an oligonucleotide in the most solid and hematologic malignancies, showed inhi- HES-oligonucleotide complex is an inhibitor or mimic of bition of growth for various types of cancers in preclinical an miRNAs selected from: let-7, let-7-a3, lin-28, miR-1, studies of MRX34. miR-9-1, miR-15a, miR-16-1, miR-17-92 cluster, miR-21, [0277] In someembodiments, an oligonucleotidein the 30 miR-29 family, miR-34 family, miR-124, miR-127, and HES-oligonucleotide complex is an inhibitor of an miR- miR-290. These microRNAs are among those that have NAs selected from: let-7a, miR-9, miR-10b, miR-15a- been reported to be dysregulated in various types of can- miR-16-1, miR-16, miR-21, miR-24, miR-26a, miR-34a, cers due to abnormalities in genetic or epigenetic regu- miR-103-107, miR-122, miR-133, miR-181, miR-192, lations responsible for miRNA expression. In particular miR-194, miR-200. These microRNAs are among those 35 embodiment, an oligonucleotide in the HES-oligonucle- that have been reported to be associated with cancer. otide complex is an inhibitor of a miRNA selected from: [0278] In someembodiments, an oligonucleotidein the let-7-a3, lin-28, miR-17-92 cluster, and miR-21. In par- HES-oligonucleotide complex inhibits a miRNA selected ticular embodiment, an oligonucleotide in the HES-oligo- from: let-7, let-7a, let-7f, miR-1, Mir-10b, miR-15a-miR- nucleotide complex contains the sequence of, or mimics 16-1, Mir-17-5p, Mir-17-92, miR-21, Mir-23-27, miR-25, 40 a miRNA selected from: let-7, miR-1, miR-9-1, miR-15a, miR-27b, miR-29, miR-30a, Mir-31, miR-34a, miR-92-1, miR-16-1, miR-21, miR-29 family, miR-34 family, miR- miR-106a, miR-125, Mir-126, Mir-130a, Mir-132, miR- 124, miR-127, and miR-290. 133b, Mir-155, miR-206, Mir-210, Mir-221/222, miR-223, [0284] In further embodiments, an oligonucleotide in Mir-296, miR-335, Mir-373, Mir-378, miR-380-5p, Mir- the HES-oligonucleotide complex contains the sequence 424, miR-451, miR-486-5p, and Mir-520c. These micro- 45 of, or mimics an miRNA selected from: Mir-20a, Mir-34, RNAs are among those that have been reported to pro- Mir-92a, Mir-200c, Mir-217 and Mir-503. These miRNAs mote neovascularization, metastasis and/or the onset of are among those that have been reported to be antian- cancer. giogenic. [0279] In someembodiments, an oligonucleotidein the [0285] In an additional embodiment, an oligonucle- HES-oligonucleotide complex inhibits a miRNA selected 50 otide in the HES-oligonucleotide complex contains the from: miR-15 family, miR-21, miR-23, miR-24, miR-27, sequence of or mimics: miR-1, miR-2, miR-6, miR-7 or miR-29, miR-33, miR-92a, miR-145, miR-155, miR- let-7. In particular embodiments, the oligonucleotides are 199b, miR-208a/b family, miR-320, miR-328, miR-499. miR-Rx07, miR-Rx06, miR-Rxlet-7, miR-Rx01, miR- These microRNAs are among those that have been re- Rx02 or miR-Rx03. In an additional embodiment, an ol- ported to have various roles in cardiovascular functions. 55 igonucleotide in the HES-oligonucleotide complex corre- [0280] In someembodiments, an oligonucleotidein the sponds to or mimics miR-451. miR-451 has been dem- HES-oligonucleotide complex inhibits a miRNA selected onstrated to regulate erythropoiesis in vivo (Patrick et al., from: let-7b, miR-9, miR106b-25 cluster, miR-124, miR- Genes & Dev., 2010) and thus to be implicated in dis-

41 79 EP 2 790 736 B1 80 eases such as, polycythemia vera, red cell dyscrasias disclosed herein and thereby induce reprogramming of generally, or other hematopoietic malignancies. In par- the somatic cells to display one or more properties of ticular embodiments, the oligonucleotide is MGN-4893. iPSC include an inhibitor of a miRNA selected from: let- [0286] In additional embodiments, pharmaceutical 7, miR-145, as well as the family members and variants compositions comprising an antisense compound target- 5 of these miRNAs (see, e.g., Lakshmipathy et al (2010) ed to a nucleic acid of interest are used for the preparation Regenerative Medicine 5:4; Xu et al. (2009) Cell of a composition for treating a patient suffering or sus- 137:647). Disclosed herein is a method of inducing the ceptible to a disease or disorder associated with the nu- reprogramming of somatic cells comprising administer- cleic acid. ing to the cells HES- oligonucleotides containing a miR- 10 NA, miRNA mimic or miRNA inhibitor of 1, 2, 3, 4, 5 or Ex vivo deliveryof miRNAs fornuclear reprograming more of the above miRNAs. Methods for inducing the and generation of iPSCs reprogramming of somatic cells that involve the admin- istration of HES-oligonucleotides containing expression [0287] Disclosed herein is a method for cell nuclear constructs encoding an miRNA, miRNA mimic or miRNA reprograming. In some embodiments, an HES-oligonu- 15 inhibitor of 1, 2, 3, 4, 5 or more of the above miRNAs are cleotides containing one or more mimics and/or inhibitor also disclosed herein. of a miRNA or a plurality of miRNAs are administered ex [0289] Methods for inducing the reprogramming of so- vivo into cells such as, human and mouse somatic cells matic cells that involve the administration of HES-oligo- to reprogram the cells to have one or more properties of nucleotides containing expression constructs encoding inducedpluripotent stemcells (iPSCs) orembryonic stem 20 an miRNA, miRNA mimic or miRNA inhibitor of 1, 2, 3, (ES)-like pluripotent cells ( e.g., colony morphology of in- 4, 5 or more of the above miRNAs are disclosed herein. duced iPSC and embryoid body (EB), expression of stem "Expression construct" means any double-stranded DNA cell marker genes in the reprogrammed stem cell lines or double-stranded RNA designed to transcribe an RNA shown by qRT-PCR, hematoxylin and eosin staining of of interest, e.g., a construct that contains at least one teratomas derived from iPSC clones showing pluripoten- 25 promoter which is or may be operably linked to a down- cy of forming mesoderm, endoderm, and ectoderm, im- stream gene, coding region, or polynucleotide sequence munohistochemistry analysis of iPSC-derived teratoma of interest (e.g., a cDNA or genomic DNA fragment that tissues showing expression of germ layer-specific differ- encodes a polypeptide or protein, or an RNA effector entiation markers, teratoma formation upon transplanta- molecule, e.g., an antisense RNA, triplex-forming RNA, tion into SCID mouse). The non-toxic and highly efficient 30 ribozyme, an artificially selected high affinity RNA ligand HES-oligonucleotide delivery system of the invention (aptamer), a double-stranded RNA, e.g., an RNA mole- providesa greatly increasedefficiency ofdelivery method cule comprising a stem-loop or hairpin dsRNA, or a bi- for reprogramming cells compared to conventional oligo- finger or multi-finger dsRNA or a microRNA, or any RNA nucleotide delivery methods (see, e.g., U.S. Publ. Nos. of interest). An "expression construct" includes a double- 2010/0075421, US 2009/0246875, US 2009/0203141, 35 stranded DNA or RNA comprising one or more promot- and US 2008/0293143). ers, wherein one or more of the promoters is not in fact [0288] Examples of miRNAs or mimics of miRNAs that operably linked to a polynucleotide sequence to be tran- can be administered to somatic cells according to the scribed, but instead is designed for efficient insertion of methods disclosed herein and thereby induce repro- an operably-linked polynucleotide sequence to be tran- gramming of the somatic cells to display one or more40 scribed by the promoter. Transfection or transformation properties of iPSC include a miRNA or miRNA mimic of of the expression construct into a recipient cell allows the a miRNA selected from: lin-28, miR-17-92 cluster, miR- cell to express an RNA effector molecule, polypeptide, 93, miR-106b, miR-106b-25 cluster, miR-106a-363 clus- or protein encoded by the expression construct. An ex- ter, miR-181a, miR-199b, miR-200c, miR-214, miR-302, pression construct may be a genetically engineered plas- miR-367, miR-302-367 cluster, miR-369, miR-371, miR- 45 mid, virus, recombinant virus, or an artificial chromosome 372, miR-373, and miR-520, as well as the family mem- derived from, for example, a bacteriophage, adenovirus, bers and variants of these miRNAs (see, e.g., Anokye- adeno-associated virus, retrovirus, lentivirus, poxvirus, Danso et al. (2011) Cell Stem Cell 8, 376; Miyoshi et al. or herpesvirus, etc. An expression construct can be rep- (2011) Cell Stem Cell 8, 1; Subramanyam et al. (2011) licated in a living cell, or it can be made synthetically. Nature Biotechnology, 29:5; Li et al. (2011) The EMBO 50 [0290] In particular embodiment, the HES-oligonucle- Journal 30:5; Lin et al. (2011) Nucleic Acids Research otides contain or encode tandem copies of an miRNA, 39:3; Lakshmipathy et al (2010) Regenerative Medicine miRNA mimic, and or miRNA inhibitor. For example, in 5:4; Xu et al. (2009) Cell 137:647; Goff et al. (2009) PLoS some embodiments, the HES-oligonucleotide contains One 4:9; Wilson et al. (2009) Stem Cells Dev. 18:5; Chin an expression construct that encodes one or more tan- et al. (2009) Cell Stem Cell 5:1; Ren et al. (2009) Journal 55 dem copies of one or more miRNAs, miRNA mimics of Translational Medicine 7:20; Lin et al. (2008) RNA and/or miRNA inhibitors wherein the coded sequences 14:2115). Examples of inhibitors of miRNAs that can be are expressed in cis or trans from a single transcription administered to somatic cells according to the methods unit or multiple polycistronic transcription units to gener-

42 81 EP 2 790 736 B1 82 ate a plurality (e.g., 2, 3, 4, or more) of the same or dif- phy and other neurodegenerative diseases), metabolic ferent, miRNAs, miRNA mimics and/or miRNA inhibitors diseases (e.g., type II diabetes, obesity), cardiovascular within the cell ( see, e.g., Chung et al. (2006) Nucleic Ac- diseases (e.g., clotting disorders, thrombosis, coronary ids Research 34:7, U.S. Patent No. 6,471,957, and U.S. artery disease,restenosis, amyloidosis,hemophilia, ane- Publ. Nos. US 2006/0228800 and US 2011/0105593). 5 mia, hemoglobulinopathies, atherosclerosis, high cho- [0291] Somatic cells that can be reprogramed accord- lesterol, high tryglycerides), endocrine related diseases ing to the methods of the invention can be obtained from and disorders (e.g., NASH, diabetes mellitus, diabetes any source using techniques known to those of skill in insipidus, Addison’s disease, Turner syndrome, Cush- the art, including from a subject to which the reprogramed ing’s syndrome, osteoporosis,) and infectious disease. cells are optionally readministered. Examples of human 10 [0296] In additional embodiments, the disease or dis- and mouse sources of somatic cells that can be used order treated with an HES-oligonucleotide of the inven- according to the methods of the invention, include, but tion is a disease or disorder of the kidneys, liver, lymph are not limited to human foreskin fibroblasts, human der- nodes, spleen or adipose tissue. mal fibroblasts (HDFs), human adipose stromal cells [0297] Disclosed herein is a method of monitoring the (hASCs), various human cancer cell lines, mouse em- 15 delivery of a therapeutic oligonucleotide to a cell or tissue bryonic fibroblasts (MEFs), and mouse adipose stromal in a subject, comprising administering to the subject an cells (mASCs). HES-oligonucleotide complex containing a therapeutic [0292] In some embodiments, the methods disclosed oligonucleotide and monitoring the fluorescence of cells herein involve the step of inducing the somatic repro- or tissue in the subject, wherein an increased fluores- gramed cells to differentiate into a progenitor or terminal 20 cence in the cells or tissue of the subject indicates that cell lineage by administering to the cells one or more the therapeutic oligonucleotide has been delivered to the HES-oligonucleotides containing or encoding a miRNA, cells or tissue of the subject. miRNA mimic or miRNA inhibitor that drives cell lineage [0298] Disclosed herein is a method of monitoring the specification, for example, to hematopietic cells, cardio- delivery of a therapeutic oligonucleotide to a cell or tissue myocytes, hepatocytes, or neurons. 25 in a subject, comprising administering to the subject an [0293] The ability of the HES-oligonucleotides to safely HES-oligonucleotide complex containing a therapeutic and efficiently delivery cell nuclear reprogramming oligo- oligonucleotide and monitoring the fluorescence of cells nucleotides such as certain miRNAs and miRNAs into or tissue in the subject, wherein an increased fluores- somatic cell populations additionally makes the methods cence in the cells or tissue of the subject to a predeter- of the invention amenable to a large-scale high-through- 30 mined value indicates that a therapeutically effective put generation of patient-specific iPSC-like cells from amount of the oligonucleotide has been delivered to the large patient populations for therapeutic uses, that to cells or tissue of the subject. In particular embodiments, date, has been hampered by the low reprogramming ef- the predetermined value is determined by extrapolating ficiency and cell cytoxicity concerns presented by con- from corresponding changes in fluorescence associated ventional nucleic acid delivery systems. 35 with delivery of a therapeutically effective amount of the therapeutic HES-oligonucleotide to cellsin vitro or Exemplary therapeutic applications of HES-oligonu- through quantitative fluorescence modeling analysis. cleotides [0299] Disclosed herein is a method of treating a dis- ease or disorder characterized by the under expression [0294] As will be immediately apparent to a person of 40 of a nucleic acid in a subject, comprising administering skill in the art, due in part to the surprising highly efficient to the subject an HES-oligonucleotide complex contain- in vivo delivery of oligonucleotides into cells, the HES- ing an oligonucleotide which comprises or encodes the oligonucleotide complexes disclosed herein essentially nucleic acid or increases the endogenous expression, have limitless applications in modulating target nucleic processing or function of the nucleic acid ( e.g., by binding acid and protein levels and activity and are particularly 45 regulatory sequences in the gene encoding the nucleic useful in therapeutic applications. acid) and which acts to increase the level of the nucleic [0295] Diseases and disorder that may be treated with acid and/or increase its function in the cell. In some em- theHES-oligonucleotides include,a proliferative disorder bodiments, the oligonucleotide comprises a sequence (e.g., a cancer, such as hematological cancerse.g., ( substantially the same as a nucleic acid comprising or AML, CML, CLL and multiple myeloma) and solid tumors 50 encoding the nucleic acid. (e.g., melanoma, renal cancer, pancreatic cancer, pros- [0300] Disclosed herein is a method of treating a dis- tate cancer, ovarian cancer, breast cancer, NSCLC,), im- ease or disorder characterized by the underexpression mune (e.g., ulcerative colitis, Crohn’s disease, IBD, pso- of a protein in a subject, comprising administering to the riasis, asthma, autoimmune diseases such as rheuma- subject an HES-oligonucleotide complex, containing an toid arthritis, multiple sclerosis, and SLE) and inflamma- 55 oligonucleotide which encodes the protein or increases tory diseases, neurologic diseases (e.g., diabetic retin- the endogenous expression, processing or function of opathy, Duchenne’s muscular dystrophy, myotinic dys- the protein in the subject. trophy, Huntington’s disease and spinal muscular atro- [0301] The invention provides for treating cancer or

43 83 EP 2 790 736 B1 84 one or more conditions associated with cancer by admin- administered to treat multiple myeloma. Other types of istering a therapeutically effective amount of an HES- cancer and tumors that can be treated using HES-oligo- oligonucleotide to a subject in need thereof. "Cancer," nucleotides are described herein or otherwise known in "tumor," or "malignancy" are used herein as synonymous the art. terms and refer to any of a number of diseases that are 5 [0303] In particular embodiments, the HES-oligonucle- characterized by uncontrolled, abnormal proliferation of otide contains an oligonucleotide selected from: AVI- cells, the ability of affected cells to spread locally or 4557 (Cyp 3A4m; AVI Biopharma), ISIS-2372 (Survivin; through the bloodstream and lymphatic system to other ISIS); Gem-640 (XIAP; Hybridon), Atu027 (PKN3; Si- parts of the body (metastasize), as well as any of a lence Therapeutics), CEQ508 (B catenin; Marina Bio- number of known characteristic structural and/or molec- 10 tech), GEM 231 (PKA R1 α subunit; Idera), Affinitak (Apri- ular features. A "cancerous tumor" or "malignant cell" is nocarsen, ISIS 3521/LY900003; PKC-α; ISIS/Lilly); understood as a cell having specific structural properties, Aezea (OL(1)p53/EL-625; p53; Eleos Pharma); ISIS lacking differentiation and being capable of invasion and 2503 (H-ras; ISIS), EZN-2968 (Hif-1α; Enzon Pharma- metastasis, Examples of cancers that may be treated ceuticals); G4460/ LR 3001 (c-Myb;Inex/Genta); LEra- using HES-oligonucleotide complexes include solid tu- 15 fAON (c-Raf; NeoPharm), ISIS 5132 (c-Raf; ISIS), Ge- mors and hematologic cancers. Additional, examples of nasense (Oblimersen/G3139; Bc1-2; Genta); SPC2996 cancers that can be treated using HES-oligonucleotide (Bcl-2; Santaris Pharma), OGX-427 (Hsp27; ISIS/ Onco- complexes of the invention include, breast, lung, brain, Gene X), LY2181308 (Surivin; Lilly), LY2275796 bone, liver, kidney, colon, head and neck, ovarian, he- (EIF4E ; Lilly), ISIS-STAT3 Rx (STAT3; ISIS), OGX-011 matopoietic (e.g., leukemia), and prostate cancer. Fur- 20 (Custirsen; clusterin; Teva),Veglin (VEGF; VasGene ther examples of cancer that can be treated using HES- Therapeutics, AP12009 (TGF-β2; Antisense Pharma), oligonucleotide complexes include, but are not limited to, GTI-2501 (Ribonucleotide Reductase R1; Lorus Thera- carcinoma, lymphoma, myeloma, blastoma, sarcoma, peutics), Gem-220 (VEGF; Hybridon); Gem-240 (MEM2; and leukemia. More particular examples of such cancers, Hybridon), CALAA-19 (M2 subunit ribonucleotide reduct- include, but are not limited to, squamous cell cancer,25 ase; Arrowhead Research Corporation), Trabedersen small-cell lung cancer, non-small cell lung cancer, ade- (AP 12009; TGFB2; Antisense), GTI-2040 (Ribonucle- nocarcinoma of the lung, squamous carcinoma of the otide Reductase R2 Lorus Therapeutics), AEG 3515.6 lung, cancer of the peritoneum, hepatocellular cancer, (XIAP; Aegera Pharma), and MG 98 (DNA methyltrans- gastrointestinal cancer, pancreatic cancer, glioblastoma, ferase; MethylGene/ MGI Pharma/ British Biotech). In cervical cancer, ovarian cancer, liver cancer, bladder 30 particular embodiments, an oligonucleotide in an HES- cancer, hepatoma, breast cancer, colon cancer, colorec- oligonucleotide competes for target binding with one of tal cancer, endometrial or uterine carcinoma, salivary the above oligonucleotides. gland carcinoma, kidney cancer, liver cancer, prostate [0304] In additional embodiments, the HES-oligonu- cancer, vulval cancer, thyroid cancer, hepatic carcinoma cleotide contains an oligonucleotide that specifically hy- and various types of head and neck cancers. 35 bridizes to a nucleic acid sequence that modulates ap- [0302] In additional embodiments, a therapeutically ef- optosis, cell survival, angiogenesis, metastasis, aberrant fective amount of an HES-oligonucleotide is adminis- gene regulation, cell cycle, mitogenic pathways and/or tered to treat a hematologic cancer. In further embodi- growth signaling. In further embodiments, the HES-oli- ments, the, HES-oligonucleotide is administered to treat gonucleotide contains an oligonucleotide that specifically a cancer selected from: lymphoma, leukemia, myeloma, 40 hybridizes to a nucleic acid sequence that modulates the lymphoid malignancy, cancer of the spleen, and cancer expression of a protein selected from: from: EGFR, HER- of the lymph nodes. In additional embodiments, a thera- 2/neu, ErbB3, cMet, p561ck, PDGFR, VEGF, VEGFR, peutically effective amount of an HES-oligonucleotide FGF, FGFR, ANG1, ANG2, bFGF,TIE2, protein kinase complex is administered to treat a lymphoma selected C-alpha (PKC-alpha), p561ck PKA, TGF-beta, IGFIR, from: Burkitt’s lymphoma, diffuse large cell lymphoma, 45 P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGF1R, follicular lymphoma, Hodgkin’s lymphoma, mantle cell HIF1 alpha, ferritin, transferrin receptor, TMPRSS2, IRE, lymphoma, marginal zone lymphoma, mucosa-associat- HSP27, HSP70, HSP90, MITF, clusterin, PARP1C-fos, ed-lymphoid tissue B cell lymphoma, non-Hodgkin’s lym- C-myc, n-myc, C-raf, B-raf, Al, H-raf, Skp2, K-ras, N-ras, phoma, small lymphocytic lymphoma, and a T cell lym- H-ras, farensyltransferase, c-Src, Jun, Fos, Bcr-Abl, c- phoma. In additional embodiments, a therapeutically ef- 50 Kit, EphA2, PDGFB, ARF, NOX1, NF1STAT3, E6/E7, fective amount of an HES-oligonucleotide complex is ad- APC, WNT, beta catenin, GSK3b, PI3k, mTOR, Akt, ministered to treat a leukemia selected from: chronic lym- PDK-1, CDK, Mek1, ERK1, AP-1, p53, Rb, Syk, oste- phocytic leukemia, B cell leukemia (CD5+ B lym- opontin, CD44, MEK, MAPK, NF kappa beta, E cadherin, phocytes), chronic myeloid leukemia, lymphoid leuke- cyclin D, cyclin E, Bcl-2, Bax, BXL-XL, BCL-W, MCL1, mia, acute lymphoblastic leukemia, myelodysplasia, my- 55 ER,MDR, telomerase, telomerase reverse transcriptase, eloid leukemia, acute myeloid leukemia, and secondary a DNA methyltransferase, a histone deacetlyase(e.g,. leukemia. In additional embodiments, a therapeutically HDAC1 and HDAC2), an integrin, an IAP, an aurora ki- effective amount of an HES-oligonucleotide complex is nase, a metalloprotease e.g (., MMP2, MMP3 and

44 85 EP 2 790 736 B1 86

MMP9), a proteasome, and a metallothionein gene. [0308] In further embodiment, the disorder or disorder [0305] In another embodiment, the invention provides of the immune system is an autoimmune disease. Au- for treating cancer or one or more conditions associated toimmune diseases, disorders or conditions that may be with cancer by administering an HES-oligonucleotide in treated using the HES-oligonucleotide complexes of the combination with one or more therapies currently being 5 invention include, but are not limited to, autoimmune used, have been used, or are known to be useful in the hemolytic anemia, autoimmune neonatal thrombocyto- treatment of cancer or conditions associated with cancer. penia, idiopathic thrombocytopenia purpura, autoim- [0306] In some embodiments, the invention provides mune neutropenia, autoimmunocytopenia, hemolytic for treating an inflammatory or other disease or disorder anemia, antiphospholipid syndrome, dermatitis, gluten- of the immune system, or one or more conditions asso- 10 sensitive enteropathy, allergic encephalomyelitis, myo- ciated with an inflammatory or other disease or disorder carditis, relapsing polychondritis, rheumatic heart dis- of the immune system, said method comprising admin- ease, glomerulonephritis (e.g., IgA nephropathy), Multi- istering to a subject in need thereof ( i.e., having or at risk ple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendo- of having an inflammatory or other immune system dis- crinopathies, Purpura ( e.g., Henloch Scoenlein purpura), ease or disorder), a therapeutically effective amount of 15 Reiter’s Disease, Stiff-Man Syndrome, Autoimmune Pul- one or more HES-oligonucleotides As immediately ap- monary Inflammation, myocarditis, IgA glomerulonephri- parent to those skilled in the art, any type of immune or tis, densedeposit disease, rheumaticheart disease, Guil- inflammatory disease or condition resulting from or as- lain-Barre Syndrome, insulin dependent diabetes melli- sociated withan immunesystem orinflammatory disease tus, and autoimmune inflammatory eye, autoimmune thy- can be treated in accordance with the invention. In par- 20 roiditis, hypothyroidism (i.e ., Hashimoto’s thyroiditis, sys- ticular embodiments, the invention provides for treating temic lupus erythematous, discoid lupus, Goodpasture’s an immune system and/or inflammatory disease or dis- syndrome, Pemphigus, Receptor autoimmunities for ex- order, or one or more conditions associated with such an ample, (a) Graves’ Disease, (b) Myasthenia Gravis, and immune disease or disorder. (c) insulinresistance, autoimmunehemolytic anemia, au- [0307] The term "inflammatory disorders", as used25 toimmune thrombocytopenic purpura, rheumatoid arthri- herein, refers to those diseases or conditions that are tis, scleroderma with anti-collagen antibodies, mixed characterized by one or more of the signs of pain (dolor, connective tissue disease, polymyositis/dermatomyosi- from the generation of noxious substances and the stim- tis, pernicious anemia, idiopathic Addison’s disease, in- ulation of nerves), heat (calor, from vasodilatation), red- fertility, glomerulonephritis such as primary glomerulone- ness (rubor, from vasodilatation and increased blood30 phritis and IgA nephropathy, bullous pemphigoid, flow), swelling (tumor, from excessive inflow or restricted Sjogren’s syndrome, diabetes mellitus, and adrenergic outflow of fluid), and loss of function (functio laesa, which drug resistance (including adrenergic drug resistance may be partial or complete, temporary or permanent). with asthma or cystic fibrosis), chronic active hepatitis, Inflammation takes many forms and includes, but is not primary biliary cirrhosis, other endocrine gland failure, limited to, inflammation that is one or more of the follow- 35 vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticar- ing: acute, adhesive, atrophic, catarrhal, chronic, cirrhot- ia, atopic dermatitis, asthma, inflammatory myopathies, ic, diffuse, disseminated, exudative, fibrinous, fibrosing, and other inflammatory, granulomatous, degenerative, focal, granulomatous, hyperplastic, hypertrophic, inter- and atrophic disorders. In particular embodiments, the stitial, metastatic, necrotic, obliterative, parenchyma- autoimmune disease or disorder is selected from Crohn’s tous, plastic, productive, proliferous, pseudomembra- 40 disease, Systemic lupus erythematous (SLE), inflamma- nous, purulent, sclerosing, seroplastic, serous, simple, tory bowel disease, psoriasis, diabetes, ulcerative colitis, specific, subacute, suppurative, toxic, traumatic, and/or multiple sclerosis, and rheumatoid arthritis. ulcerative. Inflammatory disorders additionally include [0309] In some embodiments, the invention provides but are not limited to those affecting the blood vessels for treating an immune or cardiovascular disease com- (polyarteritis, temporarl arteritis); joints (arthritis: crystal- 45 prising administering to a subject a therapeutically effec- line, osteo-, psoriatic, reactive, rheumatoid, Reiter’s); tive amount of an HES-oligonucleotide. In particular em- gastrointestinal tract (Disease); skin (dermatitis); or mul- bodiments, the HES-oligonucleotide complex contains tiple organs and tissues (systemic lupus erythematosus). an oligonucleotide selected from: Alicaforsen (ICAM-1; The terms "fibrosis" or "fibrosing disorder," as used here- ISIS 2302), QPI-1002 (p53; Silence Thera/No- in, refers to conditions that follow acute or chronic inflam- 50 vartis/Quark), XEN701 (Isis/Xenon Pharmaceuticals), mation and are associated with the abnormal accumula- ISIS 104838 (TNF-α; ISIS/Orasense), EPI-2010 (RA- tion of cells and/or collagen and include but are not limited SON; Adenosine A1 receptor; Epigenesis/ Genta), Pla- to fibrosis of individual organs or tissues such as the zomicin (Isis/Achaogen), ALN-PCS02 (PCSK9; Alny- heart, kidney, joints, lung, or skin, and includes such dis- lam), ALN-AT3 (SERPINC1; Alnylam), ALN-HPN (TFR2; orders as idiopathic pulmonary fibrosis and cryptogenic 55 Alnylam), ALN-HPN (TMPRSS6; Alnylam), ASM8-003 fibrosing alveolitis. In particular embodiments, the inflam- (CCR3; Topigen Pharmaceuticals), ISIS CRP Rx (CRP; matory disorder is selected from the group consisting of ISIS), Kynamro™ (ISIS 301012; Apo-B100; ISIS/Gen- asthma, allergic disorders, and rheumatoid arthritis. zyme), ISIS-APOCIII Rx (ApoCIII; ISIS), ISIS-APO(a)

45 87 EP 2 790 736 B1 88

(Apo(a); ISIS); ISIS-FVII rx Factor VII; ISIS), and ISIS- condition associated with a member selected from: Eb- FXI (Factor XI; ISIS). In particular embodiments, an oli- ola, Marburg, Junin, Denge West Nile, Lassa SARS Co- gonucleotide in an HES-oligonucleotide complex com- V, Japanese encephalitis, Venezuelan equine encepha- petes with one of the above oligonucleotides for target litis, Saint Louis encephalitis, Manchupo, Yellow fever, binding. 5 and Influenza. [0310] In some embodiments, the invention provides [0314] Examples of viruses which cause viral infec- for treating an infectious disease or one or more condi- tions and conditions that can be treated with the HES- tions associated with an infectious disease, said method oligonucleotides of the invention include, but are not lim- comprising administering to a subject in need thereof ited to, infections and conditions associated with retrovi- (i.e., having or at risk of having an infectious disease), a 10 ruses (e.g., human T-cell lymphotrophic virus (HTLV) therapeutically effective amount of one or more HES- types I and II and human immunodeficiency virus (HIV)), oligonucleotides. In some embodiments the infectious herpes viruses (e.g., herpes simplex virus (HSV) types I disease is a viral infection, a bacterial infection, a fungal and II, Epstein-Barr virus, HHV6-HHV8, and cytomega- infection or a parasite infection. lovirus), arenavirus (e.g., lassa fever virus), paramyxo- [0311] In some embodiments, the invention provides 15 viruses (e.g., morbillivirus virus, human respiratory syn- for treating an infection or condition associated with a cytial virus, mumps, hMPV, and pneumoviras), adenovi- category A infectious agent or disease, by administering ruses,bunyaviruses ( e.g., hantavirus), cornaviruses, filo- to a subject in need thereof (i.e., having or at risk of having viruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C an infectious disease), a therapeutically effective amount virus (HCV), yellow fever virus, and Japanese encepha- of one or more HES- oligonucleotides of the invention. 20 litis virus), hepadnaviruses e.g., ( hepatitis B viruses In particular embodiments, the infectious agent is select- (HBV)), orthomyoviruses ( e.g., influenza viruses A, B and ed from Bacillus anthracis, Clostridium botulinum toxin, C and PIV), papovaviruses ( e.g., papillomavirues), picor- yersina pestis, variola major a filovirus (e.g., Ebola and naviruses (e.g., rhinoviruses, enteroviruses and hepatitis Marburg) and an arenavirus (e.g., Lassa and Machupo). A viruses), poxviruses, reoviruses (e.g., rotavirues), to- In particular embodiments, the condition treated accord- 25 gaviruses (e.g., rubella virus), and rhabdoviruses (e.g., ing to the invention is selected from: anthrax, botulism, rabies virus). plague, smallpox, tularemia, and a viral hemorrhagic fe- [0315] In additional embodiments, the invention pro- ver. vides for treating or alleviating conditions associated with [0312] In some embodiments, the invention provides viral respiratory infections associated with or that cause for treating an infection or condition associated with a 30 the common cold, viral pharyngitis, viral laryngitis, viral category B infectious agent or disease, by administering croup, viral bronchitis, influenza, parainfluenza viral dis- to a subject in need thereof (i.e., having or at risk of having eases ("PIV") diseases (e.g., croup, bronchiolitis, bron- an infectious disease), a therapeutically effective amount chitis, pneumonia), respiratory syncytial virus ("RSV") of one or more HES-oligonucleotides of the invention. In diseases, metapneumavirus diseases, and adenovirus particular embodiments, the infectious agent is selected 35 diseases (e.g., febrile respiratory disease, croup, bron- from: a Bacilla species, Clostridium perfringens, a Sal- chitis, and pneumonia). monella species, E. coli 0157:H7, Shigella, Burkholderia [0316] In some embodiment, the HES-oligonucleotide pseudomallei, Chyamydia psittaci, Coxiella burnetii, contains an oligonucleotide selected from: AVI-4065 Rickettsia prowazekii, a viral encephalitis alphavirus (HCV; AVI Biopharma), VRX496 (HIV; VIRxSYS corpo- (e.g., Venezuelan equine encephalitis, eastern equine 40 ration), Miravirsen (antimiR-122, Santaris),GEM 91 encephalitis, western equine encephalitis), Vibrio chol- (Trecorvirsen)/92; Gag HIV; Hybridon), Vitravene (Fomi- erae and Cryptosporidium parvum. In particular embod- virsen; CMV; ISIS/Novartis), ALN-RSV01 (RSV; Alny- iments, the condition treated according to the invention lam), AVI-6002 (Ebola; AVI Biopharma), AVI-6003 (Eb- is selected from: Brucellosis, epsilon toxin of Clostridium ola; AVI Biopharma), MBI-1121 (human papillomavirus; perfringens, food poisoning, Glanders, Melioidosis, Psit- 45 Hybridon), ARC-520 (HPV hepatitis; Arrowhead Re- tacosis, Q fever, ricin toxin poisoning, typhus fever, viral search Corporation) and AVI-6001 (Influenza/avian flu; encephalitis and dysentery. AVI Biopharma). In particular embodiments, an oligonu- [0313] In some embodiments, the invention provides cleotide in an HES-oligonucleotide competes for target for treating a viral infection or one or more conditions binding with one of the above oligonucleotides. associated with a viral infection, by administering to a 50 [0317] In an additional embodiment, the invention pro- subject in need thereof (i.e., having or at risk of having a vides for treating a viral infection or one or more condi- viral infection), a therapeutically effective amount of one tions associated with a viral infection by administering a or more HES-oligonucleotides of the invention. As imme- combination of at least 1, at least 2, at least 3, at least 4, diately apparent to those skilled in the art, any type of or at least 5 HES-oligonucleotides. In some embodi- viral infection or condition resulting from or associated 55 ments at least 2, at least 3, or at least 4 of the HES- with a viral infection ( e.g., a respiratory condition) can be oligonucleotides specifically hybridizes to the same tar- treated in accordance with the invention. In particular em- get nucleic acid. In additional embodiments, at least 2, bodiments, the viral disease or disorder is an infection or at least 3, or at least 4 or at least 5 of the HES-oligonu-

46 89 EP 2 790 736 B1 90 cleotides bind to a different target nucleic acid. ther embodiments one or more of the HES-oligonucle- [0318] In one embodiment, the invention provides for otides are PMOs or PPMOs. In additional embodiments treating a filovirus (e.g., Ebola and Marbury) infection or one or more of the HES-oligonucleotides is an antisense, one or more conditions associated with the infection by an siRNA or an shRNA. administering to a patient in need thereof, a therapeuti- 5 [0322] In one embodiment, the invention provides for cally effective amount of HES-oligonucleotides that spe- treating a SARS-associated coronavirus (SARS Co-V) cifically hybridize to at least 1, at least 2, at least 3, at infection or one or more conditions associated with the least 4, at least 5, at least 6, at least 7, at least 8, at least infection by administering to a patient in need thereof, a 9 or at least 10 RNA sequences of a filovirus. In particular therapeutically effective amount of HES- oligonucle- embodiments, the HES-oligonucleotides bind. VP35,10 otides that specifically hybridize to at least 1, at least 2, VP24 and/or RNA polymerase L. In further embodiments at least 3, at least 4, or at least 5 family SARS Co-V one or more of the HES-oligonucleotides are PMOs or nucleic acid sequences. In particular embodiments, the PPMOs. In additional embodiments one or more of the HES-oligonucleotides bind the replica se gene (orf HES-oligonucleotides is an antisense, an siRNA or an 1a/1b), orf 1b ribosomal frameshift point, 5’ untranslated shRNA. 15 region (UTR) of the transcription regulatory sequence [0319] In one embodiment, the invention provides for (TRS), 3’ UTR of the TRS sequence, spike protein-coding treating an Ebola virus infection or one or more conditions region and/or the NSP12 region. In further embodiments associated with the infection by administering to a patient one or more of the HES-oligonucleotides are PMOs or in need thereof, HES-oligonucleotides that bind to at least PPMOs. In additional embodiments one or more of the 1, at least 2, at least 3, at least 4, at least 5, at least 6, 20 HES-oligonucleotides is an antisense, an siRNA or an at least 7, at least 8, at least 9 or at least 10 Ebola RNA shRNA. sequences. In particular embodiments, the HES-oligo- [0323] In one embodiment, the invention provides for nucleotides bind VP24, VP35, and/or RNA polymerase treating an Retroviridae (e.g., HIV viruses) family viral L. In additional embodiments, the HES-oligonucleotides infection or one or more conditions associated with the bind VP24, VP30, VP35, VP40, NP, GP and/or RNA25 infection by administering to a patient in need thereof, a polymerase L. In further embodiments one or more of therapeutically effective amount of HES-oligonucle- the HES-oligonucleotides are PMOs or PPMOs. In addi- otides that specifically hybridize to at least 2, at least 3, tional embodiments one or more of the HES-oligonucle- at least 4, or at least 5 RNA sequences of a member of otides is an antisense, an siRNA or an shRNA. the family Retroviridae. In particular embodiments, the [0320] In one embodiment, the invention provides for 30 HES-oligonucleotide(s) bind the highly conserved re- treatingan Flaviviridae ( e.g.,West Nile,yellow fever, Jap- gions of the gag, pol, int, and Vpu regions. In further em- anese encephalitis, and dengue viruses) viral infection bodiments one or more of the HES-oligonucleotides are or one or more conditions associated with the infection PMOs or PPMOs. In additional embodiments one or by administering to a patient in need thereof, a therapeu- more of the HES-oligonucleotides is an antisense, an ticallyeffective amount of HES-oligonucleotides that spe- 35 siRNA or an shRNA. cifically hybridize to at least 1, at least 2, at least 3, at [0324] In another embodiment, the invention provides least 4, or at least 5 RNA sequences of a member of the for treating an influenza A ( e.g., H1N1, H3N2 and H5N1) family Flaviviridae. In particular embodiments, the HES- infection or one or more conditions associated with influ- oligonucleotides bind the highly conserved non coding enza by administering to a patient in need thereof, a ther- sequence in the 5’ or 3’ regions of the viral genome, or 40 apeutically effective amount of HES-oligonucleotides sequence corresponding to the envelope coding gene that specifically hybridize to at least 2, at least 3, at least (E). In further embodiments one or more of the HES- 4, or at least 5 influenza RNA sequences. In particular oligonucleotides are PMOs or PPMOs. In additional em- embodiments, the HES-oligonucleotides bind NP and PA bodiments one or more of the HES-oligonucleotides is nucleic acid sequence of the virus. In particular embod- an antisense, an siRNA or an shRNA. 45 iments, the HES-oligonucleotides bind an NP, M2, and/or [0321] In one embodiment, the invention provides for PB2 (e.g., targeting the AUG start codon of PA, PB1, treating an Arenavirideae (e.g., Lassa, Junin and Ma- PB2, and NP), or terminal region of NP),NS1 and/or PA chupo viruses) family viral infection or one or more con- nucleic acid sequence of the virus. In further embodi- ditions associated with the infection by administering to ments one or more of the HES-oligonucleotides are a patient in need thereof, a therapeutically effective50 PMOs or PPMOs. In additional embodiments one or amount of HES-oligonucleotides that specifically hybrid- more of the HES-oligonucleotides is an antisense, an izes to at least 1, at least 2, at least 3, at least 4, or at siRNA or an shRNA. least 5 RNA sequences of a member of the family Are- [0325] In an additional embodiment, the invention pro- navirideae. In particular embodiments, the HES-oligonu- vides for treating an alphavirus (equine encephalitis virus cleotidesbind thehighly conservednon coding sequence 55 (VEEV)) infection or one or more conditions associated in the 5’ or 3’ viral mRNAs transcript coding for the Z with an alphavirus infection by administering to a patient protein (zinc-binding protein), L protein (viral polymer- in need thereof, a therapeutically effective amount of ase), or the GPC (glycoprotein precursor) protein. In fur- HES-oligonucleotides that specifically hybridize to at

47 91 EP 2 790 736 B1 92 least 2, at least 3, at least 4, or at least 5 alphavirus RNA using the invention. In particular embodiments, the fungal sequences. In particular embodiments, the HES-oligo- infection or condition treated according to the invention nucleotides bind NP and PA nucleic acid sequence of is associated with a fungus selected from: Cryptococcus the virus. In particular embodiments, the HES-oligonu- neoformans; Blastomyces dermatitidis; Aiellomyces der- cleotides bind an nsp1, nsp4 and/or E1 RNA sequence 5 matitidis; Histoplasma capsulatum; Coccidioides immi- of the virus. In further embodiments one or more of the tis; a Candida species, including C. albicans, C. tropica- HES-oligonucleotides are PMOs or PPMOs. In additional lis, C. parapsilosis, C. guilliermondii and C. krusei, an embodiments one or more of the HES-oligonucleotides Aspergillus species, including A, fumigatus, A, flavus and is an antisense, an siRNA or an shRNA. A. niger; a Rhizopus species; a Rhizomucor species; a [0326] In some embodiments, the invention provides 10 Cunninghammella species; a Apophysomyces species, for treating a bacterial infection or one or more conditions including A. saksenaea, A. mucor and A. absidia; Spo- associated with a bacterial infection, by administering to rothrix schenckii, Paracoccidioides brasiliensis; Pseu- a subject in need thereof ( i.e., having or at risk of having dalleseheria boydii, Torulopsis glabrata; a Trichophyton a bacterial infection), a therapeutically effective amount species, a Microsporum species and a Dermatophyres of one or more HES-oligonucleotides of the invention. 15 species, or any other fungus ( e.g., yeast) known or iden- Any type of bacterial infection or condition resulting from, tified to be pathogenic. In additional embodiments, the or associated with a bacterial infection can be treated invention provides for treating a fungal infection or con- using the invention. dition associated with a fungal infection by administering [0327] In particular embodiments, the bacterial infec- to a patient in need thereof, a therapeutically effective tion or condition treated according to the methods of the 20 amount of HES-oligonucleotides that specifically hybrid- invention is associated with a member of a bacterial ge- ize to at least 1, at least 2, at least 3, at least 4, or at least nus selected from:Salmonella, Shigella, Chlamydia, 5 nucleic acid sequences of at least 1, at least 2, at least Helicobacter, Yersinia, Bordatella, Pseudomonas, Neis- 3, at least 4, or at least 5 of the above funghi. seria, Vibrio, Haemophilus, Mycoplasma, Streptomyces, [0329] In additional embodiments, the invention pro- Treponema, Coxiella, Ehrlichia, Brucella, Streptobacil- 25 vides for treating a parasite infection or one or more con- lus, Fusospirocheta, Spirillum, Ureaplasma, Spirochae- ditions associated with a parasite infection, said method ta, Mycoplasma, Actinomycetes, Borrelia, Bacteroides, comprising administering to a subject in need thereof Trichomoras, Branhamella, Pasteurella, Clostridium, (i.e., having or at risk of having a parasite infection), a Corynebacterium, Listeria, Bacillus, Erysipelothrix, Rho- therapeutically effective amount of one or more HES- dococcus, Escherichia, Klebsiella, Pseudomanas, En- 30 oligonucleotides. Any type of parasite infection or condi- terobacter, Serratia, Staphylococcus, Streptococcus, tion resulting from or associated with a parasite infection Legionella, Mycobacterium, Proteus, Campylobacter, can be treated using the invention. In particular embod- Enterococcus, Acinetobacter, Morganella, Moraxella, iments, the parasite infection or condition treated accord- Citrobacter, Rickettsia and Rochlimeαe. In further em- ing to the invention is associated with a parasite selected bodiments, the bacterial infection or condition treated ac- 35 from: a member of theApicomplexa phylum such as, cording to the invention is associated with a member of Babesia, Toxoplasma, Plasmodium, Eimeria, Isospora, a bacterial genus selected from: P. aeruginosa; E. coli, Atoxoplasma, Cystoisospora, Hammondia, Besniotia, P. cepacia, S. epidermis, E. faecalis, S. pneumonias, S Sarcocystis, Frenkelia, Haemoproteus, Leucocytozoon, aureus, N. meningitidis, S. pyogenes, Pasteurella mul- Theileria, Perkinsus or Gregarina spp.; Pneumocystis tocida, Treponema pallidum, and P. mirabilis. In some 40 carinii; a member of the Microspora phylum such as, embodiments, the bacterial infection is an intracellular Nosema, Enterocytozoon, Encephalitozoon, Septata, bacterial infection. In additional embodiments, the inven- Mrazekia, Amblyospora, Arneson, Glugea, Pleistophora tion provides for treating an bacterial infection or one or and Microsporidium spp.; and a member of the Ascet- more conditions associated with a bacterial infection by ospora phylum such as, Haplosporidium spp. In further administering to a patient in need thereof, a therapeuti- 45 embodiments, the parasite infection or condition treated cally effective amount of HES-oligonucleotides that spe- according to the invention is associated with a parasite cifically hybridize to at least 1, at least 2, at least 3, at species selected from: Plasmodium falciparum, P. vivax, least 4, or at least 5 nucleic acid sequences of at least P. ovale, P. malaria; Toxoplasma gondii; Leishmania 1, at least 2, at least 3, at least 4, or at least 5 of the mexicana, L. tropica, L. major, L. aethiopica, L. donovani, above bacteria. 50 Trypanosoma cruzi, T. brucei, Schistosoma mansoni, S. [0328] In additional embodiments, the invention pro- haematobium, S. japonium; Trichinella spiralis; vides for treating a fungal infection or one or more con- Wuchereria bancrofti; Brugia malayli; Entamoeba histo- ditions associated with a fungal infection, by administer- lytica; Enterobius vermiculoarus; Taenia solium, T. sag- ing to a subject in need thereof (i.e., having or at risk of inata, Trichomonas vaginatis, T. hominis, T. tenax; Gia- having a fungal Infection), a therapeutically effective55 rdia lamblia; Cryptosporidium parvum; Pneumocytis car- amount of one or more HES-oligonucleotides of the in- inii, Babesia bovis, B. divergens, B. microti, Isospora bel- vention. Any type of fungal infection or condition resulting li, L. hominis; Dientamoeba fragilis; Onchocerca volvu- from or associated with a fungal infection can be treated lus; Ascaris lumbricoides; Necator americanis; Ancylos-

48 93 EP 2 790 736 B1 94 toma duodenale; Strongyloides stercoralis; Capillaria apeutically effective amount of one or more HES-oligo- philippinensis; Angiostrongylus cantonensis; Hyme- nucleotides. The term "neurological condition or disor- nolepis nana; Diphyllobothrium latum; Echinococcus der" is used herein to refer to conditions that include neu- granulosus, E. multilocularis; Paragonimus westermani, rodegenerative conditions, neuronal cell or tissue injuries P. caliensis; Chlonorchis sinensis; Opisthorchis felineas, 5 characterized by dysfunction of the central or peripheral G. Viverini, Fasciola hepatica, Sarcoptes scabiei, Pedic- nervous system or by necrosis and/or apoptosis of neu- ulus humanus; Phthirlus pubis; and Dermatobia hominis, ronal cells or tissue, and neuronal cell or tissue damage as well as any other parasite known or identified to be associated with trophic factor deprivation. Examples of pathogenic. In additional embodiments, the invention neurodegenerative diseases that can be treated using provides for treating an parasite infection or one or more 10 the HES-oligonucleotide of the invention include, but are conditions associated with a parasite infection by admin- not limited to, familial and sporadic amyotrophic lateral istering to a patient in need thereof, a therapeutically ef- sclerosis (FALS and ALS, respectively), familial and spo- fective amount of HES-oligonucleotides that specifically radic Parkinson’s disease, Huntington’s disease (Hunt- hybridize to at least 1, at least 2, at least 3, at least 4, or ington’s chorea), familial and sporadic Alzheimer’s dis- at least 5 nucleic acid sequences of at least 1, at least 15 ease, Spinal Muscular Atrophy (SMA), optical neuropa- 2, at least 3, at least 4, or at least 5 of the above parasites. thies such as glaucoma or associated disease involving [0330] In another embodiment, the invention provides retinal degeneration, diabetic neuropathy, or macular de- for treating a viral infection or one or more conditions generation, hearing loss due to degeneration of inner ear associated with a viral infection by administering an HES- sensory cells or neurons, epilepsy, Bell’s palsy, fronto- oligonucleotide in combination with one or more thera- 20 temporal dementia with parkinsonism linked to chromo- pies currently being used, have been used, or are known some 17 (FTDP-17), multiple sclerosis, diffuse cerebral to be useful in the treatment of a viral infection or condi- cortical atrophy, Lewy-body dementia, Pick disease, tri- tions associated with a viral infection, including but not nucleotide repeat disease, prion disorder, and Shy-Drag- limited to, antiviral agents such as amantadine, oseltami- er syndrome. Examples of neuronal cell or tissue injuries vir, ribaviran, palivizumab, and anamivir. In certain em- 25 that can be treating using HES-oligonucleotides of the bodiments, a therapeutically effective amount of one or invention include, but are not limited to acute and non- more HES-oligonucleotides is administered in combina- acute injury found after blunt or surgical trauma (including tion with one or more antiviral agents such as, but not post-surgical cognitive dysfunction and spinal cord or limited to, amantadine, rimantadine, oseltamivir, znami- brain stem injury) and ischemic conditions restricting vir, ribaviran, RSV-IVIG (i.e., intravenous immune glob- 30 (temporarily or permanently) blood flow such as that as- ulin infusion) (RESPIGAM™), and palivizumab. sociated with global and focal cerebral ischemia (stroke); [0331] In some embodiments, the invention provides incisions or cuts for instance to cerebral tissue or spinal for treating an respiratory disease or one or more condi- cord; lesions or placques in neuronal tissues; deprivation tions associated with a respiratory disease, by adminis- oftrophic factor(s)needed for growthand survival of cells; tering to a subject in need thereof ( i.e., having or at risk 35 and exposure to neurotoxins such as chemotherapeutic of having an respiratory disease), a therapeutically ef- agents; as well as incidental to other disease states such fective amount ofone or moreHES-oligonucleotides. The as chronic metabolic diseases such as diabetes and re- term "respiratory disease" as used herein, refers to a dis- nal dysfunction. ease affecting organs involved in breathing, such as the [0333] In some embodiments, the invention is directed nose, throat, larynx, trachea, bronchi, and lungs. Respi- 40 totreating a neurological condition ordisorder comprising ratory diseases, that can be treated according to the in- administering to a subject a therapeutically effective vention include, but are not limited to, asthma, adult res- amount of an HES-oligonucleotide. In particular embod- piratory distress syndrome and allergic (extrinsic) asth- iments, the HES-oligonucleotide complex contains an ol- ma, non-allergic (intrinsic) asthma, acute severe asthma, igonucleotide selected from: AVI-4658 (Dystrophin (ex- chronic asthma, clinical asthma, nocturnal asthma, aller- 45 on-skipping); AVI Biopharma), ISIS-SMN Rx (SMN; ISIS/ gen-induced asthma, aspirin-sensitive asthma, exercise- Biogen Idec), AVI-5126 (CABG; AVI Biopharma) and induced asthma, isocapnic hyperventilation, child-onset ATL1102 (VLA-4 (CD49d); ISIS/Antisense Therapeutics asthma, adult-onset asthma, cough-variant asthma, oc- Ltd). In particular embodiments, an oligonucleotide in an cupational asthma, steroid-resistant asthma, seasonal HES-oligonucleotide complex competes with one of the asthma, seasonal allergic rhinitis, perennial allergic rhin- 50 above oligonucleotides for target binding. itis, chronic obstructive pulmonary disease, including [0334] In some embodiments, the invention is directed chronic bronchitis or emphysema, pulmonary hyperten- to treating a metabolic disorder comprising administering sion, interstitial lung fibrosis and/or airway inflammation to a subject a therapeutically effective amount of an HES- and cystic fibrosis, and hypoxia. oligonucleotide. In particular embodiments, the HES-ol- [0332] In some embodiments, the invention provides 55 igonucleotide complex contains an oligonucleotide se- for treating an neurological condition or disorder, by ad- lected from: ISIS-FGFR4 (FGFR4; ISIS), ISIS-GCCR RX ministering to a subject in need thereof ( i.e., having or at (GCC; ISIS), ISIS-GCGR RX (GCG; ISIS), ISIS- PTP1B risk of having a neurological condition or disorder), a ther- (PTP1VB; ISIS), iCo-007 (c-Raf; Isis/iCo Therapeutics

49 95 EP 2 790 736 B1 96

Inc)ISIS-DGATRX (DGAT; ISIS), PF-04523655 (DME, tem, and a disease or disorder of the skin or eyes Silence Thera/Pfizer/Quark), ISIS-TTR Rx (TTR: are encompassed by the invention. ISIS/GSK); ISIS-AAT Rx (AAT: ISIS/GSK), ALN-TTRsc (Transerythrin; Alnylam), ALN-TTR01 (Transerythrin; Al- [0338] As one of skill in the art will immediately appre- nylam), and ALN-TTR02(Transerythrin; Alnylam). In par- 5 ciate, the therapeutic and companion diagnostic uses of ticular embodiments, an oligonucleotide in an HES-oli- the HES-oligonucleotides are essentially limitless. Pro- gonucleotide complex competes with one of the above vided herein are exemplary diagnostic and therapeutic oligonucleotides for target binding. uses of the compositions of the HES-oligonucleotides. [0335] In some embodiments, the invention is directed However,the description hereinis not meantto belimiting to treating a disease comprising administering to a sub- 10 and it is envisioned that the HES-oligonucleotides have ject a therapeutically effective amount of an HES-oligo- uses in any situations where it is desirable to detect a nucleotide. In particular embodiments, the HES-oligonu- nucleic acid sequence or to modulate levels of one or cleotide complex contains an oligonucleotide selected more nucleic acids or related proteins in a cell and/or from: ATL1103-GHr Rx (GHr; ISIS/Antisense Therapeu- organism. tics Ltd), EXC 001 (CTGF; ISIS/Excaliard), and Atu111 15 (PKN3; Silence Thera). In particular embodiments, an Plurality of HES-oligonucleotides oligonucleotide in an HES-oligonucleotide complex com- petes with one of the above oligonucleotides for target [0339] In some embodiments, the pharmaceutical binding. compositions disclosed herein comprise a combination [0336] In addition to those described above, HES-oli- 20 of at least 2, at least 3, at least 4, at least 5, or at least gonucleotides have applications including but not limited 10 different HES-oligonucleotide complexes having dif- to; treating metabolic diseases or disorders (e.g., melli- ferent oligonucleotide sequences. In some embodi- tus, obesity, high cholesterol, high triglycerides), in treat- ments, the pharmaceutical compositions contain be- ing diseases and disorder of the skeletal system e.g( ., tween 2-15, 2-10, or 2-5 different HES-oligonucleotide osteoporosis and osteoarthritis), in treating diseases and 25 complexes. In some embodiments, at least 2 or at least disorders of the cardiovascular systeme.g ( ., stroke, 3 of the different oligonucleotides in the complex specif- heart disease, atherosclerosis, restenosis, thrombosis, ically hybridize to a DNA and/or mRNA corresponding to anemia, leucopenia, neutropenia, thrombocytopenia, the same polypeptide. In some embodiments, at least 2, granuloctopenia, pancytoia or idiopathic thrombocyto- at least3, at least 4, at least 5, or at least 10 of the different penic purpura); in treating diseases and disorders of the 30 oligonucleotides in the complex specifically hybridizes to kidneys (e.g., nephropathy), pancreas (e.g., pancreati- a DNA and/or mRNA corresponding to different polypep- tis), skin and eyes (e.g., conjunctivitis, retinitis, scleritis, tides. In some embodiments, the pharmaceutical com- uveitis, allergic conjuctivitis, vernal conjunctivitis, pappil- positions contain between 2-15, 2-10, or 2-5 oligonucle- lary conjunctivitis glaucoma, retinopathy, and ocular otides that specifically hybridize to different polypeptides. ischemic conditions including anterior ischemic optic35 In some embodiments, one or more of the different HES- neuropathy, age-related macular degeneration (AMD), oligonucleotides are administered to a subject concur- Ischemic Optic Neuropathy (ION), dry eye syndrome); in rently. In other embodiments, one or more of the different preventing organ transplantation rejection ( e.g., lung, liv- HES-oligonucleotides are administered to a subject sep- er, heart, pancreas, and kidney transplantation) and uses arately. in regenerative medicine ( e.g., in counteracting aging, in 40 [0340] In certain embodiments, an HES-oligonucle- promotingwound healingand stimulatingbone, collagen, otide complex is co-administered with one or more addi- tissue and organ growth and repair). tional agents. In certain embodiments, such additional [0337] Disclosed herein are compositions for use in agents are designed to treat a different disease, disorder, modulating a target nucleic acid or protein in a cell,in or condition as the HES-oligonucleotide complex. In vivo in a subject, or ex vivo. The HES-oligonucleotide 45 some embodiments, the additional agent is co-adminis- compositions have applications in for example, treating tered with the HES-oligonucleotide complex to treat an a disease or disorder characterized by an overexpres- undesired effect of the complex. In additional embodi- sion, underexpression and/or aberrant expression of a ments, the additional agent is co-administered with the nucleic acid or protein in a subject in vivo or ex vivo. Uses HES-oligonucleotide complex to produce a combination- of the compositions in treating exemplary diseases or 50 al effect. In further embodiments, the additional agent is disorders selected from: co-administered with the HES-oligonucleotide complex to produce a synergistic effect. In certain embodiments, an infectious disease, cancer, a proliferative disease the additional agent is administered to treat an undesired or disorder, a .neurological disease or disorder, and side effect of an HES-oligonucleotide complex. In some inflammatory disease or disorder, a disease or dis- 55 embodiments, the HES-oligonucleotide complex is ad- order of the immune system, a disease or disorder ministered at the same time as the additional agent. In of the cardiovascular system, a metabolic disease some embodiments, the HES-oligonucleotide and addi- or disorder, a disease or disorder of the skeletal sys- tional agent are prepared together in a single pharma-

50 97 EP 2 790 736 B1 98 ceutical formulation. In other embodiments, the HES-ol- an intramolecular HES in the oligonucleotide was con- igonucleotide and additional agent are prepared sepa- firmed by absorbance spectrometry and fluorometry. All rately. In further embodiments, the additional agent is measurements were carried out in phosphate buffered administered at a different time from the HES-oligonu- saline (PBS) in which the labeled oligonucleotide was cleotide complex. 5 readily solubilized. [0341] As used in this specification and the appended [0345] A volume of two hundred microliters of the la- claims, the singular forms "a," "an," and "the" include plu- beled oligonucleotide at a concentration of 5 micromolar ral references unless the context clearly dictates other- in PBS was injected into the tail vein of a six week old wise. Thus for example, references to "the method" in- C57BL/6mouse (464 micrograms/kilogram). After 18 cludes one or more methods, and/or steps of the type 10 hours, the mouse was sacrificed by cervical dislocation; described herein and/or which will become apparent to blood was immediately withdrawn from the heart and the those persons skilled in the art upon reading this disclo- spleen was removed. The blood was diluted with PBS, sure and so forth. In addition, the term ’cell’ can be con- placed over Hypaque-Ficoll, and centrifuged at 1300 rpm strued as a cell population, which can be either hetero- for 30 minutes. Cells at the interface between the Hy- geneous or homogeneous in nature, and can also refer 15 paque-Ficoll and PBS were collected, washed with PBS, to an aggregate of cells. Moreover, each of the limitations placed on a #0 borosilicate glass surface in a Mattek of the invention can encompass various embodiments of glass bottom microwell dish (P35G-0-10-C), allowed to the invention. It is, therefore, envisioned that each of the settle (ca. 10 minutes), and then imaged with a Leica limitations of the invention involving any one element or DMIRE2 confocal microscope. In parallel a single cell combinations of elements can be included in each em- 20 suspension from the spleen was made by applying the bodiment of the invention. end of a syringe to the resected organ and then triturating [0342] It is understood that the foregoing detailed de- the suspension. The splenocytes in PBS were then ex- scription and the following examples are illustrative only posed to an equal volume of ACK lysis buffer for 3 min- and are not to be taken as limitations upon the scope of utes, diluted further with PBS, and centrifuged. Cells in the invention. Various changes and modifications to the 25 the pellet were then resuspended in PBS, placed in a disclosed embodiments, which will be apparent to those Mattek glass bottom microwell dish (P35G-0-10-C), al- of skill in the art, may be made without departing from lowed to settle (ca. 10 minutes), and finally imaged with the spirit and scope of the present invention. a confocal microscope. [0346] Imaging of the blood and splenocyte samples EXAMPLES 30 was carried out by acquiring a series of stacks of 1 micron sections in both the fluorescence and brightfield (differ- [0343] The following examples which are offered to il- ential interference contrast (DIC)) channels. Images lustrate, but not to limit, the claimed invention, clearly were reconstructed by overlaying the sections of each show: (1) the presence of an HES allows delivery of oli- channel to produce a condensed stack then overlaying gonucleotides inside live cells without toxicity in a living 35 the condensed images from fluorescence and DIC chan- organism (2) the formation of an HES in a double-strand- nels. ed RNA (3) the absence of inhibition by an HES of [0347] Images showed the fluorescence channels processing of a double-stranded RNA (dsRNA) by the overlayed on the DIC images indicated all splenocytes endonuclease Dicer and (4) the knockdown of a gene by and blood cells took up the HES-containing oligonucle- a dsRNA containing an H-type excitonic structure. 40 otide. The presence of oligonucleotide inside live cells was confirmed by examination of each 1 micron section. Example 1 As was also evident, particularly from the DIC images, cells from both blood and spleen were healthy, a point In vivo delivery of an oligonucleotide containing an H- further substantiated by the lack of uptake of trypan blue type excitonic structure 45 or propidium iodide by cells in these same samples.

[0344] In order to show that oligonucleotides can be Example 2 delivered inside live cells without toxicity in a live organ- ism, a strand of DNA containing a sequence of 24 nucleic Quantitation of in vivo delivery of an oligonucleotide con- acids complementary to β-actin (CCC GGC GAT ATC 50 taining an H-type excitonic structure ATC ATC CAT AAC (SEQ ID NO:1) (Sokol et al. Proc. Natl. Acad. Sci. USA 95:11538-43 (1998)) was synthe- [0348] In order to quantitate the in vivo delivery of oli- sized and covalently labeled on opposite ends of the gonucleotides inside live cells without toxicity in a live strand with the fluorophore (N-Ethyl-N’-[5-(N"-succinim- organism, a Dicer substrate was prepared as described idyloxycarbonyl)pentyl]-3,3,3’,3’,-tetramethyl-2,2’-in- 55 in Example 3. The sequence for the Dicer substrate, i.e., dodicarbocyanine chloride). The labeled oligonucleotide the sense strand and antisense strand for eGFP (Kim et was purified by reverse phase high pressure liquid chro- al. Nature Biotech. 22:321-5 (2004)), was chosen so that matography (hplc) and then lyophilized. The presence of no complementary pairing in the subject mice (standard,

51 99 EP 2 790 736 B1 100 nontransfected BALB/C strain) could take place. The by reverse phase hplc is also shown in the corresponding double-labeled lyophilized dsRNA was solubilized in panels on the right side of Figure 2. phosphate buffered saline (PBS). The presence of an [0353] With a data acquisition rate of 1 datum/sec the intramolecular HES in the oligonucleotide was confirmed center section shows, first, the fluorescence intensity of by absorbance spectrometry and fluorometry. 5 the sense solution as a function of time (from 0 to ca. 80 [0349] A volume of two hundred microliters of the la- sec.) to be ca. 7000 Counts. When the shutter is closed beled oligonucleotide at a concentration of 5 micromolar at 80 sec. in order to add the antisense solution, the in- or 10 micromolar in PBS was injected into the tail vein or tensity drops to the zero. Upon re-opening the shutter, the peritoneum of each 10-12 week old BALB/C mouse the intensity is recorded at ca. 1100 Counts and remains (0.75 or 1.5 milligrams/kilogram). After 3 hours, blood 10 steady at this level due to the tight complex formed be- was drawn in the presence of heparin from the heart of tween the sense and antisense strands. each mouse. The blood was diluted with PBS, placed [0354] The lowest panels on the right and left sides over Hypaque-Ficoll, and centrifuged at 1300 rpm for 30 show the emission spectrum and hplc chromatogram of minutes. Cells at the interface between the Hypaque- the sense-antisense complex, respectively. Ficoll and PBS were collected; the fluorescence of indi- 15 vidual cells was measured with a Cytek-modified Becton- Example 4 Dickinson Caliber flow cytometer, [0350] FIG. 1 shows histograms of blood cells isolated Recognition of the a double-stranded sense-antisense from mice three hours after an injection of 200 microliters RNA complex by Dicer of buffer (PBS) or a Dicer substrate. In Panel a, fluores- 20 cence from cells which were isolated after a singleip [0355] Dicer is an endonuclease that cleaves double- injection of PBS or the Dicer substrate (1.5 mg/kg) is stranded RNA (dsRNA) and preMiRNA (MiRNA) into shown in histogram format. The increase in fluorescence short double-stranded RNA fragments called small inter- intensity of ca. 2 logs in the cells exposed to the Dicer fering siRNA. Since one of the embodiments of this in- substrate relative to those from the animal that had re- 25 vention is the delivery of oligonucleotides for silencing ceived an injection of PBS indicates significant uptake of RNA, it is essential that an HES-containing dsRNA be the Dicer substrate. Moreover, the light scattering prop- recognizable and cleavable by Dicer. Therefore, the dsR- ertiesof both groupsindicated highly viable cells.In Panel NA described in Example 3 which contains an HES on b, histograms show the fluorescence of cells isolated af- theend ofthe duplexwas exposed to a recombinant Dicer ter an iv injection of PBS, the Dicer substrate at a con- 30 (Recombinant Turbo Dicer Cat (# T520002) from Gen- centration of 1.5 mg/kg, or the Dicer substrate at a con- lantis). Using the digestion conditions in the instructions centration of 0.75 mg/kg. As with the ip route, cells from from the reagent supplier the fluorescence of the dsRNA- iv-injected animals that had received the Dicer substrate containing solution was measured after addition of this at either dose also showed ca. a two log increase in flu- endonuclease. orescence intensity per cell relative to those from the35 [0356] Two Dicer substrates derivatized with an HES PBS animal with the higher concentration resulting in a were synthesized: one was comprised of two strands of slightly higher average intensity per cell. And, again, no unmodified ribonucleotides (25 and 27 bases) and a sec- signs of toxicity were observed. ond with the same two strands but with the 25 nucleotide chain extended with two O-methylated nucleotides on Example 3 40 the end. Terminal O-methylation has been shown to pro- tect oligonucleotides from exonucleases present in plas- Formation of an intramolecular HES in real-time ma. As shown in Figure 3, the fluorescence of the solu- tions of both dsRNAs increased as a function of time after [0351] The formation of an HES is associated with addition of Dicer, thus confirming the absence of inhibi- quenching of fluorescence; specifically, the fluorescence 45 tion of the HES for processing by this endonuclease. Ad- of the dimer is reduced relative to that of the individual ditionally, the dsRNA with the O-methylation showed a components. Therefore, in order to illustrate the process slightly slower rate of digestion, consistent with the pro- of HES formation, two complementary strands of RNA, tective effect of this modification. i.e., the sense strand and antisense strand (Kim et al. Nature Biotech. 22:321-5 (2004)), were each labeled with 50 Example 5 N-Ethyl-N’-[5-(N"-succinimidyloxycarbonyl)pentyl]- 3,3,3’,3’,-tetramethyl-2,2’-indodicarbocyanine chloride Knockdown of a gene by a dsRNA containing an H-type and then added together; the fluorescence intensity of excitonic structure the latter solution was then compared with those of the components, i.e., the single strands alone. 55 [0357] In order to show the functionality of an oligonu- [0352] The fluorescence spectra of the two singly-la- cleotide linked to an H-type excitonic structure, the fluo- beled strands are shown in the top two panels on the left rescence per cell from blood cells of mice transgenic for side of Figure 2. The purity of each strand as measured expression of eGFP was measured after exposure to a

52 101 EP 2 790 736 B1 102 double-stranded RNA (dsRNA) derivatized with an H- the nucleic acid sequence and including 1, 2 or 3 type excitonic structure, as described in Figure 2, and base substitutions, wherein the composition is for containing the sense and antisense strands coding for use in the treatment of disease or disorder selected eGFP (Kim et al. Nature Biotech. 22:321-5 (2004)). from: an infectious disease, cancer, a proliferative Measurements were made by flow cytometry from the 5 disease or disorder, a neurological disease or disor- blood of mice after separation of mononuclear cells. der, and inflammatory disease or disorder, a disease [0358] Figure 4 shows the superimposed histograms or disorder of the immune system, a disease or dis- of both the control and Dicer-treated populations. The order of the cardiovascular system, a metabolic dis- control cells show two populations: ca. 67% of cells with ease or disorder, a disease or disorder of the skeletal >102 fluorescence units per cell than a second nonfluo- 10 system, and a disease or disorder of the skin or eyes. rescent population. Treatment with the Dicer substrate results in a single population with an average fluores- 2. The composition for use according to claim 1, where- cence just slightly above that of the nonfluorescent con- in the therapeutic oligonucleotide is from 8 nucle- trol cells. otides to 750 nucleotides. 15 SEQUENCE LISTING 3. The composition for use according to claim 1 or claim 2, wherein the HES-oligonucleotide comprises 3 or [0359] more fluorophores capable of forming one or more HES. <110> ONCOIMMUNIN INC. Packard, Beverly Ko- 20 moriya, Akira 4. The composition for use according to claim 1, 2 or 3, wherein the therapeutic oligonucleotide is single <120> IN VIVO DELIVERY OF OLIGONUCLE- stranded or is double stranded. OTIDES 25 5. The composition for use according to any preceding <130> 3288.001PC01/TJS/KKH claim, wherein the therapeutic oligonucleotide is a member selected from: siRNA, shRNA, miRNA, a <140> To be assigned Dicer substrate, a decoy and an antisense. <141> Herewith 30 6. The composition for use according to claim 5, where- <160> 1 in the therapeutic oligonucleotide is an antisense ol- igonucleotide that has at least 85% sequence com- <170> PatentIn version 3.5 plementarity or is complementary and includes 1, 2 or 3 base substitutions, and specifically hybridizes <210> 1 35 to an RNA. <211> 24 <212> DNA 7. The composition for use according to claim 6, where- <213> Artificial Sequence in the therapeutic antisense oligonucleotide

<220> 40 (i) is a substrate for RNAse H when hybridized <223> synthesized complement of Beta-actin to the RNA, optionally a gapmer; (ii) is not a substrate for RNAse H when hybrid- <400> 1 ized to the RNA, optionally wherein each nucl- cccggcgata tcatcatcca taac 24 eoside of therapeutic antisense oligonucleotide 45 comprises a modified sugar moiety comprising a modification at the 2’-position, a PNA motif, or Claims a morpholino motif; or (iii) is DNA or a DNA mimic; or 1. A composition for delivering a therapeutic oligonu- (iv) has a least 85% sequence complementarity cleotide to a subject, said composition comprising a 50 or is complementary and includes 1, 2 or 3 base therapeutically effective amount of an H-type exci- substitutions, and is specifically hybridizable to tonic structure (HES)- oligonucleotide containing a a target region of the RNA selected from the therapeutic oligonucleotide that specifically hybrid- group consisting of: izes in vivo, to a nucleic acid sequence and modu- lates the level of a protein encoded or regulated by 55 (a) a sequence within 30 nucleotides of the the nucleic acid, the therapeutic oligonucleotide hav- AUG start codon of an mRNA; ing at least 85% sequence complementarity to the (b) nucleotides 1-10 of a miRNA; nucleic acid sequence or being complementary to (c) a sequence in the 5’ untranslated region

53 103 EP 2 790 736 B1 104

of an mRNA; thomyoviruses (e.g., influenza viruses A, B (d) a sequence in the 3’ untranslated region and C and PIV), papovaviruses (e.g., pap- of an mRNA; illomavirues), picornaviruses (e.g., rhinovi- (e) an intron/exon junction of an mRNA; ruses, enteroviruses and hepatitis A virus- (f) a sequence in a precursor-miRNA (pre- 5 es), poxviruses, reoviruses (e.g., rota- miRNA)or primary-miRNA (pri-miRNA) that virues),togaviruses (e.g., rubellavirus), and when bound by the oligonucleotide blocks rhabdoviruses (e.g., rabies virus); or miRNA processing; and (g) an intron/exon junction and a region 1 (ii) a bacterial infection associated with a mem- to 50 nucleobases 5’ of an intron/exon junc- 10 ber of a bacterial genus selected from: tion of an RNA. Salmonella, Shigella, Chlamydia, Helico- 8. The composition for use according to claim 1, where- bacter, Yersinia, Bordatella, Pseu- in the therapeutic oligonucleotide can induce RNA domonas, Neisseria, Vibrio, Haemophilus, interference (RNAi), and is optionally siRNA, shRNA 15 Mycoplasma, Streptomyces, Treponema, or a Dicer substrate which is optionally 18-35 nucle- Coxiella, Ehrlichia, Brucella, Streptobacil- otides in length. lus, Fusospirocheta, Spirillum, Ureaplas- ma, Spirochaeta, Mycoplasma, Actinomyc- 9. The composition for use according to any preceding etes, Borrelia, Bacteroides, Trichomoras, claim, that contains one or more modified nucleoside 20 Branhamella, Pasteurella, Clostridium, Co- motifs selected from: 2’OME, locked nucleic acid rynebacterium, Listeria, Bacillus, Erysip- (LNA), alpha LNA, 2’-Fluoro (2’F), 2’- elothrix, Rhodococcus, Escherichia, Kleb-

O(CH2)2OCH3(2’-MOE), 2’-OCH3(2’-O-methyl), siella, Pseudomanas, Enterobacter, Serra- PNA and morpholino ,wherein the LNAor alpha LNA tia, Staphylococcus, Streptococcus, Le- 25 optionally has a methylene (--CH2-) n group bridging gionella, Mycobacterium, Proteus, Campy- the 2’ oxygen atom and the 4’ carbon atom wherein lobacter, Enterococcus, Acinetobacier, n is 1 or 2, optionally with a methyl group at the 5’ Morganella, Moraxella, Citrobacter, Rick- position. ettsia and Rochlimeae.

10. The composition for use according to any preceding 30 12. The composition for use according to any one of claim, that contains one or more modified internucl- claims 1-10, wherein the cancer is selected from: eoside linkages selected from: phosphorothioate, phosphorodithioate, phosphoramide, 3’-methylene squamous cell cancer, small-cell lung cancer, phosphonate, O-methylphosphoroamidiate, PNA non-small cell lung cancer, adenocarcinoma of and morpholino and/or one or more modified nucle- 35 the lung, squamous carcinoma of the lung, can- obases selected from C-5 propyne and 5-methyl C. cer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, 11. The composition for use according to any preceding glioblastoma, cervical cancer, ovarian cancer, claim, wherein the infectious disease is liver cancer, bladder cancer, hepatoma, breast 40 cancer, colon cancer, colorectal cancer, en- (i) a viral infection caused by a virus selected dometrial or uterine carcinoma, salivary gland from: carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic retroviruses (e.g., human T-cell lympho- carcinoma, various types of head and neck can- trophic virus (HTLV) types I and II and hu- 45 cers, and a hematologic cancer selected from: man immunodeficiency virus (HIV)), herpes lymphoma, such as, Burkitt’s lymphoma, diffuse viruses (e.g., herpes simplex virus (HSV) large cell lymphoma, follicular lymphoma, Hodg- types I and II, Epstein-Barr virus, HHV6- kin’s lymphoma, mantle cell lymphoma, margin- HHV8, and cytomegalovirus), arenavirus al zone lymphoma, mucosa-associated-lym- (e.g., lassa fever virus), paramyxoviruses 50 phoid tissue B cell lymphoma, non-Hodgkin’s (e.g., morbiUivirus virus, human respiratory lymphoma, small lymphocytic lymphoma, and a syncytial virus, mumps, hMPV, and pneu- T cell lymphoma, leukemia, such as, chronic movirus), adenoviruses, bunyaviruses lymphocytic leukemia, B cell leukemia (CD5+ B (e.g., hantavirus), cornaviruses, filoviruses lymphocytes), chronic myeloid leukemia, lym- (e.g., Ebola virus), flaviviruses (e.g., hepa- 55 phoid leukemia, acute lymphoblastic leukemia, titis C virus (HCV), yellow fever virus, and myelodysplasia, myeloid leukemia, acute mye- Japanese encephalitis virus), hepadnavi- loid leukemia, and secondary leukemia, myelo- ruses (e.g., hepatitis B viruses (HBV)), or- ma, lymphoid malignancy, cancer of the spleen,

54 105 EP 2 790 736 B1 106

and cancer of the lymph nodes. eration, hearing loss due to degeneration of in- ner ear sensory cells or neurons, epilepsy, Bell’s 13. The composition for use according to any one of palsy, frontotemporal dementia with parkinson- claims 1-10, wherein the disease or disorder of the ism linked to chromosome 17 (FTDP-17), mul- immune system is selected from: 5 tiple sclerosis, diffuse cerebral cortical atrophy, Lewy- body dementia, Pick disease, trinucle- autoimmune hemolytic anemia, autoimmune otide repeat disease, prion disorder, and Shy- neonatal thrombocytopenia, , autoimmune neu- Drager syndrome. tropenia, autoimmunocytopenia, hemolytic ane- mia,antiphospholipid syndrome, dermatitis, glu- 10 15. The composition for use according to any one of ten-sensitive enteropathy, allergic encephalo- claims 1-10, wherein the cardiovascular disease or myelitis, myocarditis, relapsing polychondritis, disorder is selected from: rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neu- clottingdisorders, coronary artery disease, amy- ritis, Uveitis Ophthalmia, Polyendocrinopathies, 15 loidosis,hemophilia, anemia,hemoglobulinopa- Purpura (e.g., Henloch Scoenlein purpura, au- thies, high cholesterol, high tryglycerides, toimmune thrombocytopenicpurpura, idiopathic stroke, heart disease, atherosclerosis, resteno- thrombocytopenia purpura), Reiter’s Disease, sis, thrombosis, leucopenia, neutropenia, Stiff-Man Syndrome, Autoimmune Pulmonary thrombocytopenia, granulocytopenia, pancy- Inflammation, myocarditis, IgA glomerulone-20 toia and idiopathic thrombocytopenic purpura phritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, diabetes (e.g. insulin dependent diabetes me llitus, diabe- Patentansprüche tes mellitus), autoimmune inflammatory eye, au- toimmune thyroiditis, hypothyroidism (i.e.,25 1. Zusammensetzung zur Verabreichung eines thera- Hashimoto’s thyroiditis), systemic lupus ery- peutischen Oligonukleotids an ein Subjekt, wobei die thematous, discoid lupus, Goodpasture’s syn- Zusammensetzung eine therapeutisch wirksame drome, Pemphigus, Receptor autoimmunities Menge eines Oligonukleotids mit exzitonischer (e.g., Graves’ Disease, Myasthenia Gravis, in- Struktur vom H-Typ (HES) umfasst, das ein thera- sulin resistance), autoimmune hemolytic ane- 30 peutisches Oligonukleotid enthält, das spezifisch in mia, rheumatoid arthritis, scleroderma with anti- vivo an eine Nukleinsäuresequenz hybridisiert und collagen antibodies, mixed connective tissue den Spiegel eines Proteins moduliert, für das die Nu- disease, polymyositis/dermatomyositis, perni- kleinsäure kodiert oder das durch die Nukleinsäure cious anemia, idiopathic Addison’s disease, in- reguliert wird, wobei das therapeutische Oligonuk- fertility, glomerulonephritis such as primary35 leotid mindestens 85 % Sequenzkomplementarität glomerulonephritis and IgA nephropathy, bul- zu der Nukleinsäuresequenz aufweist oder komple- lous pemphigoid, Sjogren’s syndrome, adrener- mentär zu der Nukleinsäuresequenz ist und 1, 2 oder gic drug resistance (including adrenergic drug 3 Basensubstitutionen einschließt, wobei die Zu- resistance with asthma or cystic fibrosis), chron- sammensetzung zur Verwendung in der Behand- ic active hepatitis, primary biliary cirrhosis, other 40 lung einer Erkrankung oder Störung ausgewählt aus endocrine gland failure, vitiligo, vasculitis, post- einer Infektionskrankheit, Krebs, einer proliferativen MI, cardiotomy syndrome, urticaria, atopic der- Erkrankung oder Störung, einer neurologischen Er- matitis, asthma, inflammatory myopathies, krankung oder Störung und entzündlichen Erkran- Crohn’s disease, inflammatory bowel disease, kung oder Störung, einer Erkrankung oder Störung psoriasis and ulcerative colitis. 45 des Immunsystems, einer Erkrankung oder Störung des Herz-Kreislauf-Systems, einer Stoffwechseler- 14. The composition for use according to any one of krankung oder - Störung, einer Erkrankung oder Stö- claims 1-10, wherein the neurological disease or dis- rung des Skelettsystems und einer Erkrankung oder order is selected from: Störung der Haut oder der Augen ist. 50 familial and sporadic amyotrophic lateral scle- 2. Zusammensetzung zur Verwendung nach Anspruch rosis (FALS and ALS, respectively), familial and 1, wobei das therapeutische Oligonukleotid aus 8 sporadic Parkinson’s disease, Huntington’s dis- Nukleotiden bis 750 Nukleotiden ist. ease (Huntington’s chorea), familial and sporad- ic Alzheimer’s disease, Spinal Muscular Atrophy 55 3. Zusammensetzung zur Verwendung nach Anspruch (SMA), optical neuropathies such as glaucoma 1 oder Anspruch 2, wobei das HES-Oligonukleotid or associated disease involving retinal degen- 3 oder mehr Fluorophore umfasst, die in der Lage eration, diabetic neuropathy, or macular degen- sind, eine oder mehrere HES zu bilden.

55 107 EP 2 790 736 B1 108

4. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das therapeutische Oligonukleotid RNA-In- 1, 2 oder 3, wobei das therapeutische Oligonukleotid terferenz (RNAi) induzieren kann und gegebenen- einsträngig oder doppelsträngig ist. falls siRNA, shRNA oder ein Dicer-Substrat ist, das gegebenenfalls eine Länge von 18 bis 35 Nukleoti- 5. Zusammensetzung zurVerwendung nacheinem der 5 den hat. vorhergehenden Ansprüche, wobei das therapeuti- scheOligonukleotid ein Mitgliedausgewählt aus siR- 9. Zusammensetzungzur Verwendung nach einemder NA, shRNA, miRNA, einem Dicer-Substrat, einem vorhergehenden Ansprüche, die ein oder mehrere Decoy und einem Antisense ist. modifizierte Nukleosidmotive enthält, die ausge- 10 wählt sind aus: 2’OME, verschlossener Nukleinsäu- 6. Zusammensetzung zur Verwendung nach Anspruch re (Locked Nucleic Acid; LNA), alpha-LNA, 2’-Fluor

5, wobei das therapeutische Oligonukleotid ein An- (2’F), 2’-O(CH2)2OCH3 (2’-MOE), 2’-OCH3 (2’-O- tisense-Oligonukleotid ist, das mindestens 85 % Se- Methyl), PNA und Morpholino, wobei die LNA oder quenzkomplementarität aufweist oder komplemen- alpha-LNA gegebenenfalls eine Methylengruppe (-- 15 tär ist und 1, 2 oder 3 Basensubstitutionen ein- CH2-)n aufweist, die das 2’-Sauerstoffatom und das schließt und spezifisch an eine RNA hybridisiert. 4’-Kohlenstoffatom verbrückt, wobei n 1 oder 2 ist, gegebenenfalls mit einer Methylgruppe an der 5’- 7. Zusammensetzung zur Verwendung nach Anspruch Position. 6, wobei das therapeutische Antisense-Oligonukle- otid 20 10. Zusammensetzungzur Verwendung nach einemder vorhergehenden Ansprüche, die ein oder mehrere (i) ein Substrat für RNAse H ist, wenn es an die modifizierte Internukleosidbindungen ausgewählt RNA hybridisiert ist, gegebenenfalls ein Gap- aus: Phosphorthioat, Phosphordithioat, Phosphora- mer; mid, 3’-Methylenphosphonat, O-Methylphosphora- (ii) kein Substrat für RNAse H ist, wenn es an 25 midiat, PNA und Morpholino und/oder eine oder die RNA hybridisiert ist, wobei gegebenenfalls mehrere modifizierte Nukleobasen ausgewählt aus jedes Nukleosid des therapeutischen Antisen- C-5-Propin und 5-Methyl-C enthält. se-Oligonukleotids einen modifizierten Zucker- anteil umfasst, der eine Modifikation an der 2’- 11. Zusammensetzungzur Verwendung nach einemder Position, ein PNA-Motiv oder ein Morpholino- 30 vorhergehenden Ansprüche, wobei die Infektions- Motiv umfasst; oder krankheit (iii) DNA oder ein DNA-Imitator ist; oder (iv) mindestens 85 % Sequenzkomplementari- (i) eine Virusinfektion, die durch ein Virus her- tät aufweist oder komplementär ist und 1, 2 oder vorgerufen wird, das ausgewählt ist aus: 3 Basensubstitutionen einschließt, und spezi- 35 fisch an eine Zielregion der RNA hybridisierbar Retroviren (z. B. humanem T-Zell-lympho- ist, die ausgewählt ist aus der Gruppe beste- tropem Virus (HTLV) Typen I und II und hu- hend aus: manem Immunschwächevirus (HIV)), Her- pesviren (z. B. Herpes simplex-Virus (HSV) (a) einer Sequenz innerhalb von 30 Nukle- 40 Typen I und II, Epstein-Barr-Virus, HHV6- otiden des AUG-Startcodons einer mRNA; HHV8 und Zytomegalovirus), Arenavirus (z. (b) Nukleotiden 1-10 einer miRNA; B. Lassafiebervirus), Paramyxoviren (z. B. (c) einer Sequenz in der 5’-untranslatierten Morbillivirus, humanem respiratorischem Region einer mRNA; Synzytialvirus, Mumps, hMPV und Pneu- (d) einer Sequenz in der 3’-untranslatierten 45 movirus), Adenoviren, Bunyaviren (z. B. Region einer mRNA; Hantavirus), Cornaviren, Filoviren (z. B. (e) einer Intron/Exon-Verknüpfung einer Ebolavirus), Flaviviren (z. B. Hepatitis C-Vi- mRNA; rus (HCV), Gelbfiebervirus und japani- (f) einer Sequenz in einer Vorläufer-miRNA schem Enzephalitisvirus), Hepadnaviren (PrämiRNA) oder primären miRNA (pri-50 (z. B. Hepatitis B-Viren (HBV)), Orthomyo- miRNA), die, wenn sie durch das Oligonu- viren (z. B. Influenzaviren A, B und C und kleotid gebunden wird, die Verarbeitung der PIV), Papovaviren (z. B. Papillomaviren), miRNA blockiert; und Picornaviren (z. B. Rhinoviren, Enteroviren (g) einer Intron/Exon-Verknüpfung und ei- und Hepatitis A-Viren), Pockenviren, Reo- ner Region von 1 bis 50 Nukleobasen 5’ zu 55 viren (z. B. Rotaviren), Togaviren (z. B. Ru- einer Intron/ Exon-Verknüpfung einer RNA. bellavirus) und Rhabdoviren (z. B. Tollwut- virus); oder 8. Zusammensetzung zur Verwendung nach Anspruch

56 109 EP 2 790 736 B1 110

(ii) eine bakterielle Infektion ist, die im Zusam- rung des Immunsystems ausgewählt ist aus: menhang mit einer Bakteriengattung steht, die ausgewählt ist aus: autoimmuner hämolytischer Anämie, autoim- muner Thrombozytopenie des Neugeborenen, Salmonella, Shigella, Chlamydia, Helico- 5 autoimmuner Neutropenie, Autoimmunzytope- bacter, Yersinia, Bordatella, Pseudomo- nie, hämolytischer Anämie, Antiphospholipid- nas, Neisseria, Vibrio, Haemophilus, Myco- syndrom, Dermatitis, gluten-sensitiver Entero- plasma, Streptomyces, Treponema, Coxi- pathie, allergischer Enzephalomyelitis, Myokar- ella, Ehrlichia, Brucella, Streptobacillus, Fu- ditis, rezidivierender Polychondritis, rheumati- sospirocheta, Spirillum, Ureaplasma, Spi- 10 scher Herzerkrankung, Glomerulonephritis (z. rochaeta, Mycoplasma, Actinomycetes, B. IgA-Nephropathie), multipler Sklerose, Neu- Borrelia, Bacteroides, Trichomoras, Bran- ritis, Uveitis ophthalmia, Polyendokrinopathien, hamella, Pasteurella, Clostridium, Coryne- Purpura (z. B. Purpura Henloch-Schönlein, au- bacterium, Listeria, Bacillus, Erysipelothrix, toimmuner thrombozytopenischer Purpura, idi- Rhodococcus, Escherichia, Klebsiella,15 opathischer thrombozytopenischer Purpura), Pseudomonas, Enterobacter, Serratia, Sta- Reiter’scher Krankheit, Stiff-Man-Syndrom, au- phylococcus, Streptococcus, Legionella, toimmuner Lungenentzündung, Myokarditis, Mycobacterium, Proteus, Campylobacter, IgA-Glomerulonephritis, membranoproliferati- Enterococcus, Acinetobacter, Morganella, ver Glomerulonephritis Typ II, rheumatischer Moraxella, Citrobacter, Rickettsia und 20 Herzerkrankung, Guillain-Barre-Syndrom, Dia- Rochlimeae. betes (z. B. insulinabhängigem Diabetes melli- tus, Diabetes mellitus), autoimmunem entzün- 12. Zusammensetzung zurVerwendung nacheinem der detem Auge, Autoimmun-Thyroiditis, Hypothy- Ansprüche 1 bis 10, wobei der Krebs ausgewählt ist roidismus (d. h. Hashimoto-Thyroiditis), syste- aus: 25 mischem Lupus erythematous, diskoidem Lu- pus, Goodpasture-Syndrom, Pemphigus, Re- Plattenepithelkrebs, kleinzelligem Lungen- zeptor-Autoimmunerkrankungen (e.g. Morbus krebs, nicht-kleinzelligem Lungenkrebs, Adeno- Basedow, Myasthenia gravis, Insulinresistenz), karzinom der Lunge, Epithelkarzinom der Lun- autoimmunerhämolytischer Anämie,rheumato- ge, Krebs des Peritoneums, hepatozellulärem 30 ider Arthritis, Sklerodermie mit Antikörpern ge- Krebs, Gastrointestinalkrebs, Pankreaskrebs, gen Kollagen, gemischter Bindegewebserkran- Glioblastom, Zervixkrebs, Eierstockkrebs, Le- kung, Polymyositis/Dermatomyositis, perniziö- berkrebs, Blasenkrebs, Hepatom, Brustkrebs, ser Anämie, idiopathischem Morbus Addison, Darmkrebs, Kolorektalkrebs, Endometrium- Infertilität, Glomerulonephritis, wie primärer oder Uteruskarzinom, Speicheldrüsenkarzi-35 Glomerulonephritis und IgA-Nephropathie, bul- nom, Nierenkrebs, Leberkrebs, Prostatakrebs, lösem Pemphigoid, Sjögren-Syndrom, Resis- Vulvakrebs, Schilddrüsenkrebs, Leberkarzi- tenz gegen adrenerge Arzneimittel nom, verschiedenen Typen von Krebserkran- (einschließlich Resistenz gegen adrenerge Arz- kungen von Kopf und Hals, und hämatologi- neimittel mit Asthma oder Mukoviszidose), chro- schem Krebs ausgewählt aus: Lymphom, wie 40 nischer aktiver Hepatitis, primärer biliärer Zir- Burkitt-Lymphom, diffusem großzelligem Lym- rhose, anderem Versagen von endokrinen Drü- phom, follikulärem Lymphom, Hodgkin-Lym- sen, Vitiligo, Vaskulitis, Post-MI, Kardiotomie- phom, Mantelzelllymphom, Marginalzonenlym- syndrom, Urticaria, atopischer Dermatitis, Asth- phom, Mukosa-assoziiertem Lymphoidgewebe ma, entzündlichen Myopathien, Morbus Crohn, B-Zell-Lymphom, Non-Hodgkin-Lymphom, klei- 45 entzündlicher Darmerkrankung, Psoriasis und nem lymphozytischem Lymphom und einem T- Colitis ulcerosa. Zell-Lymphom, Leukämie, wie chronischer lym- phozytischer Leukämie, B-Zellen-Leukämie 14. Zusammensetzungzur Verwendung nach einemder (CD5+ B-Lymphozyten), chronischer myeloi- Ansprüche 1 bis 10, wobei die neurologische Erkran- scher Leukämie, lymphoider Leukämie, akuter 50 kung oder Störung ausgewählt ist aus: lymphoblastischer Leukämie, Myelodysplasie, myeloischerLeukämie, akuter myeloischer Leu- familiär bedingter und sporadischer amyotro- kämie und sekundärer Leukämie, Myelom, lym- pher Lateralsklerose (FALS beziehungsweise phoiden bösartigen Erkrankungen, Krebs der ALS), familiär bedingtem und sporadischem Milz und Krebs der Lymphknoten. 55 Morbus Parkinson, Huntington-Erkrankung (Chorea Huntington), familiär bedingtem und 13. Zusammensetzung zurVerwendung nacheinem der sporadischem Morbus Alzheimer, spinaler Mus- Ansprüche 1 bis 10, wobei die Erkrankung oder Stö- kelatrophie (SMA), optischen Neuropathien, wie

57 111 EP 2 790 736 B1 112

Glaukom oder damit zusammenhängender Er- 3. Composition destinée à être utilisée selon la reven- krankung, die Netzhautdegeneration, diabeti- dication 1 ou la revendication 2, dans laquelle le scheNeuropathie oderMakuladegeneration be- HES-oligonucléotide comprend 3 ou plus de 3 fluo- inhalten, Hörverlust infolge von Degeneration rophores capables de former une ou plusieurs HES. der Sinneszellen des Innenohrs oder Neuronen, 5 Epilepsie, Bell’scher Parese, frontotemporaler 4. Composition destinée à être utilisée selon la reven- Demenz mit Parkinsonismus, der mit Chromo- dication 1, 2 ou 3, dans laquelle l’oligonucléotide thé- som 17 in Zusammenhang steht (FTDP-17), rapeutique est simple brin ou est double brin. multipler Sklerose, diffuser Hirnrindenatrophie, Lewy-Körper-Demenz, Morbus Pick, Trinukleo- 10 5. Composition destinée à être utilisée selon une quel- tid-Repeat-Erkrankung, Prionenerkrankung conque revendication précédente, dans laquelle und Shy-Drager-Syndrom. l’oligonucléotide thérapeutique est un élément choisi entre : un ARNsi, un ARNsh, un miARN, un substrat 15. Zusammensetzung zurVerwendung nacheinem der de Dicer, un leurre et un antisens. Ansprüche 1 bis 10, wobei die Herz-Kreislauf-Er- 15 krankung oder Störung ausgewählt ist aus: 6. Composition destinée à être utilisée selon la reven- dication 5, dans laquelle l’oligonucléotide thérapeu- Gerinnungsstörungen, koronarer Arteriener- tique est un oligonucléotide antisens qui a une com- krankung, Amyloidose, Hämophilie, Anämie, plémentarité de séquence d’au moins 85% ou qui Hämoglobulinopathien, hohem Cholesterins- 20 est complémentaire et qui comprend 1, 2 ou 3 subs- piegel, hohem Triglyceridspiegel, Schlaganfall, titutions de bases et qui s’hybride spécifiquement à Herzerkrankung, Atherosklerose, Restenose, un ARN. Thrombose, Leukopenie, Neutropenie, Throm- bozytopenie, Granulozytopenie, Panzytopenie 7. Composition destinée à être utilisée selon la reven- und idiopathischer thrombozytopenischer Pur- 25 dication 6, dans laquelle l’oligonucléotide antisens pura. thérapeutique

(i) est un substrat pour l’ARNase H lorsqu’il est Revendications hybridé à l’ARN, éventuellement un gapmer ; 30 (ii) n’est pas un substrat pour l’ARNase H lors- 1. Composition pour l’administration d’un oligonucléo- qu’il est hybridé à l’ARN, éventuellement cha- tide thérapeutique à un sujet, ladite composition que nucléoside d’oligonucléotide antisens thé- comprenant une quantité thérapeutiquement effica- rapeutique comprenant une fraction de sucre ce d’un HES (structure excitonique de type H)-oligo- modifiée comprenant une modification en posi- nucléotide contenant un oligonucléotide thérapeuti- 35 tion 2’, un motif ANP ou un motif morpholino ; ou que qui s’hybride spécifiquement in vivo à une sé- (iii) est un ADN ou un mimétique d’ADN ; ou quence d’acide nucléique et qui module le taux d’une (iv) a une complémentarité de séquence d’au protéine codée ou régulée par l’acide nucléique, l’oli- moins 85 % ou est complémentaire et comprend gonucléotide thérapeutique ayant une complémen- 1, 2 ou 3 substitutions de bases et peut s’hybri- tarité de séquence d’au moins 85% avec la séquen- 40 der spécifiquement à une région cible de l’ARN ce d’acide nucléique ou étant complémentaire à la choisie dans le groupe constitué par : séquence d’acide nucléique et comprenant 1, 2 ou 3 substitutions de bases, la composition étant des- (a)une séquence à moinsde 30 nucléotides tinée à être utilisée dans le traitement d’une maladie du codon de départ AUG d’un ARNm ; ou d’un trouble choisis entre : une maladie infectieu- 45 (b) les nucléotides 1-10 d’un miARN ; se, un cancer, une maladie ou un trouble prolifératifs, (c) une séquence dans la région non tradui- une maladie ou un trouble neurologiques et une ma- te 5’ d’un ARNm ; ladie ou un trouble inflammatoires, une maladie ou (d) une séquence dans la région non tradui- un trouble du système immunitaire, une maladie ou te 3’ d’un ARNm ; untrouble dusystème cardiovasculaire, une maladie 50 (e) une jonction intron/exon d’un ARNm ; ou un trouble métaboliques, une maladie ou un trou- (f) une séquence dans un précurseur de ble du système squelettique et une maladie ou un miARN (pré-miARN) ou un miARN primaire trouble de la peau ou des yeux. (pri-miARN) qui lorsqu’elle est liée par l’oli- gonucléotide bloque le traitement du 2. Composition destinée à être utilisée selon la reven- 55 miARN ; et dication 1, dans laquelle l’oligonucléotide thérapeu- (g) une jonction intron/exon et une région tique a de 8 nucléotides à 750 nucléotides. de 1 à 50 nucléobases 5’ d’une jonction in- tron/exon d’un ARN.

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8. Composition destinée à être utilisée selon la reven- réovirus (par exemple les rotavirus), les to- dication 1, dans laquelle l’oligonucléotide thérapeu- gavirus (par exemple le virus de la rubéole) tique peut induire une interférence par ARN (ARNi) et les rhabdovirus (par exemple le virus et est éventuellement un ARNsi, un ARNsh ou un rabique) ; ou substrat de Dicer qui a éventuellement une longueur 5 de 18-35 nucléotides. (ii) une infection bactérienne associée à un élé- ment d’un genre bactérien choisi entre : 9. Composition destinée à être utilisée selon une quel- conque revendication précédente, qui contient un ou Salmonella, Shigella, Chlamydia, Helico- plusieurs motifs nucléosidiques modifiés choisis10 bacter, Yersinia, Bordatella, Pseudomo- entre : 2’OME, acide nucléique bloqué (LNA), LNA nas, Neisseria, Vibrio, Haemophilus, Myco-

alpha, 2’-fluoro (2’F), 2’-O(CH 2)2OCH3 (2’-MOE), 2’- plasma, Streptomyces, Treponema, OCH3 (2’-O-méthyl), ANP et morpholino, le LNA ou Coxiella, Ehrlichia, Brucella, Streptobacil- LNA alpha ayant éventuellement un groupe méthy- lus, Fusospirocheta, Spirillum, Ureaplas- 15 lène (--CH2-)n pontant l’atome d’oxygène en 2’ et ma, Spirochaeta, Mycoplasma, Actinomy- l’atome de carbone en 4’, n valant 1 ou 2, éventuel- cetes, Borrelia, Bacteroides, Trichomoras, lement avec un groupe méthyle en position 5’. Branhamella, Pasteurella, Clostridium, Co- rynebacterium, Listeria, Bacillus, Erysipe- 10. Composition destinée à être utilisée selon une quel- lothrix, Rhodococcus, Escherichia, Kleb- conque revendication précédente, qui contient une 20 siella, Pseudomonas, Enterobacter, Serra- ou plusieurs liaisons internucléosidiques modifiées tia, Staphylococcus, Streptococcus, Legio- choisies entre : phosphorothioate, phosphorodi- nella, Mycobacterium, Proteus, Campylo- thioate, phosphoramide, 3’-méthylènephosphonate, bacter, Enterococcus, Acinetobacter, Mor- O-méthylphosphoramidate, ANP et morpholino ganella, Moraxella, Citrobacter, Rickettsia et/ou une ou plusieurs nucléobases modifiées choi- 25 et Rochlimeae. sies entre C-5 propyne et 5-méthyl C. 12. Composition destinée à être utilisée selon l’une quel- 11. Composition destinée à être utilisée selon une quel- conque des revendications 1-10, le cancer étant conque revendication précédente, la maladie infec- choisi entre : tieuse étant 30 le cancer à cellules squameuses, le cancer du (i) une infection virale provoquée par un virus poumon à petites cellules, le cancer du poumon choisi parmi : non à petites cellules, un adénocarcinome du poumon, un carcinome squameux du poumon, les rétrovirus (par exemple le virus T-lym- 35 un cancer du péritoine, un cancer hépatocellu- photrophique humain (HTLV) des types I et laire, un cancer gastrointestinal, un cancer du II et le virus de l’immunodéficience humaine pancréas, un glioblastome, un cancer du col de (VIH)), les virus de l’herpès (par exemple le l’utérus, un cancer de l’ovaire, un cancer du foie, virus de l’herpès simplex (HSV) des types un cancer de la vessie, un hépatome, un cancer I et II, le virus d’Epstein-Barr, les HHV6- 40 du sein, un cancer du côlon, un cancer colorec- HHV8 et un cytomégalovirus), un arénavi- tal, un carcinome de l’endomètre ou de l’utérus, rus (par exemple le virus de la fièvre de Las- un carcinome des glandes salivaires, un cancer sa), les paramyxovirus (par exemple un vi- du rein, un cancer du foie, un cancer de la pros- rus morbillivirus, le virus respiratoire syncy- tate, un cancer de la vulve, un cancer de la thy- tial humain, le virus des oreillons, le hMPV 45 roïde, un carcinome hépatique, divers types de et un pneumovirus), les adénovirus, les bu- cancers de la tête et du cou et un cancer héma- nyavirus (par exemple un hantavirus), les tologique choisi entre : un lymphome, tel que le coronavirus, les filovirus (par exemple le vi- lymphome de Burkitt, le lymphome diffus à gran- rus Ebola), les flavivirus (par exemple le vi- des cellules, le lymphome folliculaire, un lym- rusde l’hépatite C (VHC), le virus dela fièvre 50 phome hodgkinien, le lymphome à cellules du jaune et le virus de l’encéphalite japonaise), manteau, le lymphome de la zone marginale, le les hépadnavirus (par exemple les virus de lymphome à cellules B du tissu lymphoïde as- l’hépatite B (VHB)), les orthomyovirus (par socié aux muqueuses, un lymphome non hod- exemple les virus de la grippe A, B et C et gkinien, le lymphome lymphocytaire à petites les PIV), les papovavirus (par exemple les 55 cellules et un lymphome à cellules T, une leu- papillomavirus), les picornavirus (par cémie, telle que la leucémie lymphocytaire chro- exemple les rhinovirus, les entérovirus et nique, la leucémie à cellules B (lymphocytes B les virus de l’hépatite A), les poxvirus, les CD5+), la leucémie myéloïde chronique, la leu-

59 115 EP 2 790 736 B1 116

cémie lymphoïde, la leucémie lymphoblastique 14. Composition destinée à être utilisée selon l’une quel- aiguë, une myélodysplasie, la leucémie myéloï- conque des revendications 1-10, la maladie ou le de, la leucémie myéloïde aiguë et une leucémie trouble neurologiques étant choisis entre : secondaire, un myélome, une malignité lym- phoïde, un cancer de la rate et un cancer des 5 la sclérose latérale amyotrophique familiale et ganglions lymphatiques. sporadique (SLAF et SLAS, respectivement), la maladie de Parkinson familiale et sporadique, 13. Composition destinée à être utilisée selon l’une quel- la maladie de Huntington (la chorée de Hunting- conque des revendications 1-10, la maladie ou le ton), la maladie d’Alzheimer familiale et spora- trouble du système immunitaire étant choisis entre : 10 dique, l’amyotrophie spinale (AS), les neuropa- thies optiques telles que le glaucome ou une l’anémie hémolytique auto-immune, la thrombo- maladie associée impliquant une dégénéres- cytopénie néonatale auto-immune, la neutropé- cence rétinienne, une neuropathie diabétique nie auto-immune, la cytopénie auto-immune, ou une dégénérescence maculaire, une perte l’anémie hémolytique, le syndrome des anti-15 auditive due à la dégénérescence de cellules phospholipides, la dermatite, l’entéropathie sensorielles ou de neurones de l’oreille interne, sensible au gluten, l’encéphalomyélite allergi- l’épilepsie, la paralysie de Bell, la démence fron- que, la myocardite, la polychondrite récidivante, to-temporale avec parkinsonisme liée au chro- une cardiopathie rhumatismale, une glomérulo- mosome 17 (DFTP-17), la sclérose en plaques, néphrite (par exemple la néphropathie à IgA), la 20 l’atrophie corticale cérébrale diffuse, la démen- sclérose en plaques, la neurite, l’uvéite, les po- ce à corps de Lewy, la maladie de Pick, la ma- lyendocrinopathies, le purpura (par exemple le ladie à triplets répétés, un trouble à prion et le purpura de Henoch-Schonlein, le purpura syndrome de Shy-Drager. thrombocytopénique auto-immun, le purpura thrombocytopénique idiopathique), la maladie 25 15. Composition destinée à être utilisée selon l’une quel- de Reiter, le syndrome de Stiff-Man, une inflam- conque des revendications 1-10, la maladie ou le mation pulmonaire auto-immune, la myocardite, trouble cardiovasculaires étant choisis entre : la glomérulonéphrite à IgA, la maladie de dépôt dense, une cardiopathie rhumatismale, le syn- les troubles de la coagulation, une coronaropa- drome de Guillain-Barré, le diabète (par exem- 30 thie, l’amyloïdose, l’hémophilie, une anémie, les ple le diabète sucré insulinodépendant, le dia- hémoglobinopathies, un taux élevé de choles- bète sucré), une maladie inflammatoire auto-im- térol, un taux élevé de triglycérides, un accident mune de l’oeil, une thyroïdite auto-immune, l’hy- vasculaire cérébral, une cardiopathie, l’athéro- pothyroïdie (par exemple la thyroïdite de Hashi- sclérose, la resténose, une thrombose, la leu- moto), le lupus érythémateux disséminé, le lu- 35 copénie, la neutropénie, la thrombocytopénie, pus discoïde, le syndrome de Goodpasture, le la granulocytopénie, la pancytopénie et le pur- pemphigus, les auto-immunités contre les ré- pura thrombocytopénique idiopathique. cepteurs (par exemple la maladie de Grave, la myasthénie grave, l’insulinorésistance), l’ané- mie hémolytique auto-immune, la polyarthrite 40 rhumatoïde, la sclérodermie à anticorps anti- collagène, la maladie du tissu conjonctif mixte, la polymyosite/dermatomyosite, l’anémie perni- cieuse, la maladie d’Addison idiopathique, la stérilité, une glomérulonéphrite telle que la glo- 45 mérulonéphrite primitive et la néphropathie à IgA, la pemphigoïde bulleuse, le syndrome de Sjögren, la résistance aux médicaments adré- nergiques (notamment la résistance aux médi- caments adrénergiques avec l’asthme ou la mu- 50 coviscidose), l’hépatite chronique active, la cir- rhose biliaire primitive, une insuffisance d’une autre glande endocrine, le vitiligo, la vasculite, un post-IM, le syndrome post-cardiotomie, l’ur- ticaire, la dermatite atopique, l’asthme, les myo- 55 pathies inflammatoires, la maladie de Crohn, une maladie intestinale inflammatoire, le psoria- sis et la rectocolite hémorragique.

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