(12) Patent Application Publication (10) Pub. No.: US 2012/0094934 A1 Collard Et Al
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US 20120094.934A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0094934 A1 Collard et al. (43) Pub. Date: Apr. 19, 2012 (54) TREATMENT OF TUMOR NECROSIS A 6LX 3L/75 (2006.01) FACTOR RECEPTOR 2 (TNFR2) RELATED A63L/72 (2006.01) DISEASES BY INHIBITION OF NATURAL C7H 2L/00 (2006.01) ANTISENSE TRANSCRIPT TO TNFR2 C07K 2/00 (2006.01) C7H 2L/04 (2006.01) (75) Inventors: Joseph Collard, Delray Beach, FL GOIN 25/04 (2006.01) (US): Olga Khorkova Sheman, A6IP35/00 (2006.01) Tequesta, FL (US) A6IP 25/00 (2006.01) A6IP37/00 (2006.01) (73) Assignee: OPKO CuRNA, LLC, Miami, FL A6IP 29/00 (2006.01) (US) A6IPI/00 (2006.01) A6IP3/00 (2006.01) (21) Appl. No.: 13/379,728 A6IPL/I6 (2006.01) A6IPI3/2 (2006.01) (22) PCT Filed: Jun. 24, 2010 A6IP 9/00 (2006.01) A6IP 9/10 (2006.01) (86). PCT No.: PCT/US 10/39824 A6IP 9/02 (2006.01) CI2N 5/071 (2010.01) S371 (c)(1), (2), (4) Date: Dec. 21, 2011 (52) U.S. Cl. ...... 514/20.9; 435/375; 514/44. A:536/24.5; 530/322:436/501 Related U.S. Application Data (57) ABSTRACT (60) Provisional application No. 61/219,911, filed on Jun. The present invention relates to antisense oligonucleotides 24, 2009. that modulate the expression of and/or function of Tumor Necrosis Factor Receptor 2 (TNFR2), in particular, by target Publication Classification ing natural antisense polynucleotides of Tumor Necrosis Fac (51) Int. Cl. tor Receptor 2 (TNFR2). The invention also relates to the A6 IK 38/14 (2006.01) identification of these antisense oligonucleotides and their A6 IK3I/7088 (2006.01) use in treating diseases and disorders associated with the A 6LX 3L/725 (2006.01) expression of TNFR2. Patent Application Publication Apr. 19, 2012 US 2012/0094934 A1 US 2012/0094934 A1 Apr. 19, 2012 TREATMENT OF TUMIOR NECROSS derivatives, fragments and complementary sequences FACTOR RECEPTOR 2 (TNFR2) RELATED thereto. Examples of antisense oligonucleotides are set forth DISEASES BY INHIBITION OF NATURAL as SEQID NOS: 4 to 10. ANTISENSE TRANSCRIPT TO TNFR2 0008 Another embodiment provides a method of modu lating function and/or expression of an TNFR2 polynucle otide in patient cells or tissues in vivo or in vitro comprising 0001. The present application claims the priority of U.S. contacting said cells or tissues with an antisense oligonucle provisional patent application No. 61/219,911 filed Jun. 24. otide 5 to 30 nucleotides in length wherein said oligonucle 2009 which is incorporated herein by reference in its entirety. otide has at least 50% sequence identity to a reverse comple ment of the an antisense of the TNFR2 polynucleotide; FIELD OF THE INVENTION thereby modulating function and/or expression of the TNFR2 0002 Embodiments of the invention comprise oligonucle polynucleotide in patient cells or tissues in vivo or in vitro. otides modulating, expression and/or function of TNFR2 and 0009. Another embodiment provides a method of modu associated molecules. lating function and/or expression of an TNFR2 polynucle otide in patient cells or tissues in vivo or in vitro comprising BACKGROUND contacting said cells or tissues with an antisense oligonucle 0003 DNA-RNA and RNA-RNA hybridization are otide 5 to 30 nucleotides in length wherein said oligonucle important to many aspects of nucleic acid function including otide has at least 50% sequence identity to an antisense oli DNA replication, transcription, and translation. Hybridiza gonucleotide to an TNFR2 antisense polynucleotide; thereby tion is also central to a variety of technologies that either modulating function and/or expression of the TNFR2 poly detect a particular nucleic acid or alter its expression. Anti nucleotide in patient cells or tissues in vivo or in vitro. sense nucleotides, for example, disrupt gene expression by 0010. In a preferred embodiment, a composition com hybridizing to target RNA, thereby interfering with RNA prises one or more antisense oligonucleotides which bind to splicing, transcription, translation, and replication. Antisense sense and/or antisense TNFR2 polynucleotides. DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is 0011. In another preferred embodiment, the oligonucle present in most cell types. Antisense molecules can be deliv otides comprise one or more modified or Substituted nucle ered into cells, as is the case for oligodeoxynucleotides otides. (ODNs), or they can be expressed from endogenous genes as 0012. In another preferred embodiment, the oligonucle RNA molecules. The FDA recently approved an antisense otides comprise one or more modified bonds. drug, VITRAVENETM (for treatment of cytomegalovirus 0013. In yet another embodiment, the modified nucle retinitis), reflecting that antisense has therapeutic utility. otides comprise modified bases comprising phosphorothio ate, methylphosphonate, peptide nucleic acids, 2'-O-methyl, SUMMARY fluoro- or carbon, methylene or other locked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are 0004. This Summary is provided to present a summary of locked nucleic acid molecules, including C-L-LNA. the invention to briefly indicate the nature and substance of 0014. In another preferred embodiment, the oligonucle the invention. It is submitted with the understanding that it otides are administered to a patient Subcutaneously, intramus will not be used to interpret or limit the scope or meaning of cularly, intravenously or intraperitoneally. the claims. 0015. In another preferred embodiment, the oligonucle 0005. In one embodiment, the invention provides methods otides are administered in a pharmaceutical composition. A for inhibiting the action of a natural antisense transcript by treatment regimen comprises administering the antisense using antisense oligonucleotide(s) targeted to any region of compounds at least once to patient, however, this treatment the natural antisense transcript resulting in up-regulation of can be modified to include multiple doses over a period of the corresponding sense gene. It is also contemplated herein time. The treatment can be combined with one or more other that inhibition of the natural antisense transcript can be types of therapies. achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention. 0016. In another preferred embodiment, the oligonucle 0006. One embodiment provides a method of modulating otides are encapsulated in a liposome or attached to a carrier function and/or expression of an TNFR2 polynucleotide in molecule (e.g. cholesterol, TAT peptide). patient cells or tissues in vivo or in vitro comprising contact 0017. Other aspects are described infra. ing said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at BRIEF DESCRIPTION OF THE DRAWINGS least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides 0018 FIG. 1 is a graph of real time PCR results showing within nucleotides 1 to 413 of SEQID NO: 2 and 1 to 413 of the fold change+standard deviation in TNFR2 mRNA after SEQ ID NO: 3 thereby modulating function and/or expres treatment of HepG2 cells with phosphorothioate oligonucle sion of the TNFR2 polynucleotide in patient cells or tissues in otides introduced using Lipofectamine 20100, as compared vivo or in vitro. to control. Real time PCR results show that the levels of 0007. In another preferred embodiment, an oligonucle TNRSF1B mRNA in HepG2 cells are significantly increased otide targets a natural antisense sequence of TNFR2 poly 48 h after treatment with 1 of the oliogs designed to nucleotides, for example, nucleotides set forth in SEQ ID TNRSF1B antisense Hs.679340 (CUR-0792) and 1 oligo NOS: 2 and 3, and any variants, alleles, homologs, mutants, designed to Hs.639108 (CUR-0787). Bars denoted as CUR US 2012/0094934 A1 Apr. 19, 2012 0790, CUR-0792, CUR-0788, CUR-0786, CUR-0789, CUR and more preferably still up to 1% of a given value. Alterna 0787 and CUR-0791 correspond to samples treated with SEQ tively, particularly with respect to biological systems or pro ID NOS: 4 to 10 respectively. cesses, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, SEQUENCE LISTING DESCRIPTION of a value. Where particular values are described in the appli 0019 SEQID NO: 1: Homo sapiens tumor necrosis factor cation and claims, unless otherwise stated the term “about receptor superfamily, member 1B (TNFRSF1B), mRNA. meaning within an acceptable error range for the particular (NCBI Accession No.: NM 001066); SEQID NO: 2: Natu value should be assumed. ral TNFR2 antisense sequence (Hs.639108); SEQID NO:3: 0024. As used herein, the term “mRNA' means the pres Natural TNFR2 antisense sequence (Hs.679340); SEQ ID ently known mRNA transcript(s) of a targeted gene, and any NOs: 4 to 10: Antisense oligonucleotides. * indicates phos further transcripts which may be elucidated. phothioate bond. 0025 By “antisense oligonucleotides’ or “antisense com DETAILED DESCRIPTION pound is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it 0020 Several aspects of the invention are described below is an RNA oligonucleotide it binds to another RNA target by with reference to example applications for illustration. It means of RNA-RNA interactions and alters the activity of the should be understood that numerous specific details, relation target RNA. An antisense oligonucleotide can upregulate or ships, and methods are set forth to provide a full understand downregulate expression and/or function of a particular poly ing of the invention. One having ordinary skill in the relevant nucleotide. The definition is meant to include any foreign art, however, will readily recognize that the invention can be RNA or DNA molecule which is useful from a therapeutic, practiced without one or more of the specific details or with diagnostic, or other viewpoint.