(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`24 May 2007 (24.05.2007)
`
` (10) Intemational Publication Number
`
`WO 2007/058894 A2
`
`(51) International Patent Classification:
`C12N15/11 (2006.01)
`A61P 1/16 (2006.01)
`A61K 31/712 (2006.01)
`A6IP 29/00 (2006.01)
`A61K 31/7125 (2006.01)
`A6IP 31/00 (2006.01)
`C07H 21/00 (2006.01)
`
`(21) International Application Number:
`PCT/USZOO6/O43651
`
`(22) International Filing Date:
`10 November 2006 (10.1 1.2006)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/735,429
`60/862,350
`
`10 November 2005 (10.1 1.2005)
`20 October 2006 (20. 10.2006)
`
`US
`US
`
`(71) Applicants (for all designated States except US): THE
`UNIVERSITY OF NORTH CAROLINA AT CHAPEL
`HILL [US/US]; Chapel Hill, NC 27599 (US). ERCOLE
`BIOTECH,
`INC.
`[US/US]; 79 TW Alexander Dr.,
`Research Triangle Park, NC 27709 (US). SANTARIS
`PHARMA A/S [DK/DK]; Boge AlIe 3, DK—2970 Hor—
`sholm (DK).
`
`(72)
`(75)
`
`Inventors; and
`(for US only): SAZANI, Peter,
`Inventors/Applicants
`L.
`[US/US]; 5 Tupelo Lane, Chapel Hill, NC 27514
`(US). KOLE, Ryszard [US/US]; 203 Longwood Dr.,
`
`Chapel Hill, NC 27514 (US). ORUM, Henrik [DK/DK];
`Anemonevej 4, DK—3500 Vaerlose (DK).
`
`(74) Agents: BAER, Madeline et al.; Brown Raysman Mill—
`stein Felder & Steiner, 900 Third Ave., New York, NY
`10022 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind 9‘ national protection available): AE, AG, AL, AM,
`AT,AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS,
`JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LV,LY,MA, MD, MG, MK, MN, MW, MX, MY,
`MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW
`
`(84) Designated States (unless otherwise indicated, for every
`kind 9‘ regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT,LU, LV,MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`international search report and to be republished
`without
`upon receipt (f that report
`
`refer to the "Guid—
`For two—letter codes and other abbreviations,
`ance Notes on Codes andAbbreviations” appearing at the begin—
`ning g" each regular issue if the PCT Gazette.
`
`(54) Title: SPLICE SWITCHING OLIGOMERS FOR TNF SUPERFAMILY RECEPTORS AND THEIR USE IN TREATMENT
`OF DISEASE
`
`(57) Abstract: Methods and compositions are disclosed for controlling expression of TNF receptors (TNFRl and TNFR2) and of
`other receptors in the TNFR superfamily using compounds that modulate splicing of pre—mRNA encoding these receptors. More
`specifically these compounds cause the removal of the transmembrane domains of these receptors and produce soluble forms of the
`receptor which act as an antagonist to reduce TNF— on activity or activity of the relevant ligand. Reducing TNF— oc activity provides
`a method of treating or ameliorating inflammatory diseases or conditions associated with TNF—Oc activity. Similarly, diseases asso—
`ciated with other ligands can be treated in like manner.
`In particular, the compounds of the invention are splice—splice switching
`oligomers (SSOs) which are small molecules that are stable in vivo, hybridize to the RNA in a sequence specific manner and, in
`conjunction with their target, are not degraded by RNAse H.
`
`
`
`W02007/058894A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`WO 2007/058894
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`PCT/US2006/043651
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`SPLfCE SWITCHING OLIGOMERS FOR TNF SUPERFAMILY RECEPTORS AND
`
`THEIR USE IN TREATMENT OF DISEASE
`
`[0001]
`
`This application claims priority to US. Provisional application Ser. No.
`
`60/862,350, filed October 20, 2006 and US. Provisional application Ser. No. 60/735,429,
`
`filed November 10, 2005 which are incorporated by reference herein in their entirety.
`
`FIELD OF THE INVENTION
`
`[0002]
`
`The present invention relates to compositions and methods for controlling splicing
`
`of pre—mRNA molecules and regulating protein expression with splice switching
`
`oligonucleotides or splice switching oligomers (SSOs). 880s are not limited to nucleotides
`
`but include any polymer or molecule that is able to hybridize to a target RNA with sequence
`
`specificity and does not activate RNase H or otherwise lead to degradation of the target RNA.
`
`Specifically described embodiments concern receptors for the tumor necrosis factor (TNF)
`
`superfamily.
`
`BACKGROUND OF THE INVENTION
`
`[0003]
`
`The production of mRNA by eukaryotic cells is a two—stage process. First, a long
`
`contiguous transcript, prc—mcsscngcr RNA (prc—mRNA), is formcd. Thc prc—mRNA contains
`
`sequences that code for protein (exons) interspersed with sequences that do not code for
`
`protein (introns). Second, the introns of the transcript are removed and the exons are joined
`
`by aprocess called splicing. This process is a key step in generation of mature, functional
`
`mRNA. The 5' end of each intron contains a splice—donor site or 5‘ splice site, and the 3' end
`
`of each intron contains a splice acceptor or 3' splice site. Processing of pre—mRNA involves a
`
`complex containing protein and RNA molecules, referred to collectively as the spliceosome,
`
`which carries out splicing and transport of mRNA from the nucleus.
`
`[0004]
`
`When alternative splice sites are present, the splicing step permits the synthesis of
`
`two or more (related) proteins from a single gene (See, e. g., Gist, A., 2005, Scientific
`
`American, April, p.60). Among the genes that employ alternative splicing as aphysiological
`
`mechanism are the cell— surface receptors for protein cytokines that influence the
`
`inflammatory and immunc system. These protcins are cxprcsscd in an integral mcmbranc
`
`form and transduce signals in response to cytokine ligand binding. Such cytokine receptors
`
`also exist as a secreted form that can bind cytokine and prevent signal transduction. These
`
`two receptor forms are produced by alternative splicing and differ by the deletion of the one
`
`or more exons needed to encode the membrane—spanning domain of the molecule. For some
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`produced by proteolytic cleavage of the extracellular domain from the integral membrane
`
`bound receptors.
`
`[0005]
`
`One such family of receptors is the TNF receptor (TNFR) superfamily. The
`
`TNFR superfamily currently consists of 29 receptors that mediate cellular signaling as a
`
`consequence of binding to one or more of the 19 ligands currently identified in the TNF
`
`superfamily. The TNFR superfamily is a group of type I transmembrane proteins, with a
`
`carboxy—terminal intracellular domain and an amino-terminal extracellular domain
`
`characterized by a common cysteine rich domain (CRD). The TNFR superfamily can be
`
`divided into two subgroups: receptors containing the intracellular death domain (DD) and
`
`those lacking it. The DD is an 80 amino acid motif that is responsible for the induction of
`
`apoptosis following receptor activation. Additionally, TNF—ocreceptor type I ( TNFSFRlA ,
`
`hereafter ”TNFRl", exemplified by GenBank accession number X553 13 for human mRNA)
`
`and TNF—ocreceptor type II (TNFSFlB, hereafter "TNFR2", exemplified by GenBank
`
`accession number NM_001066 for human mRNA) have a unique domain in common, called
`
`the pre—ligand—binding assembly domain (PLAD) that is required for assembly of multiple
`
`receptor subunits and subsequent binding to TNF—oc. Most members of the TNFR
`
`superfamily activate signal transduction by associating with TNFR-associated factors
`
`(TRAFs). The association is mediated by specific motifs in the intracellular domain of TNFR
`
`superfamily members. (Palladino, M.A., et al., 2003, Nat. Rev. Drug Discov. 22736—46).
`
`Other members of the TNFR superfamily include RANK (TNFRSFl IA), CD40 (TNFRSFS),
`
`CD30 (TNFRSFS), and LT—BR (TNFRSF3).
`
`[0006]
`
`TNF-Otis a pro—inflammatory cytokine that exists as a membrane—bound
`
`homotrimer and is released into the circulation by the protease TNF—occonverting enzyme
`
`(TACE). TNF—Otis introduced into the circulation as a mediator of the inflammatory response
`
`to injury and infection. TNF—06 activity is implicated in the progression of inflammatory
`
`diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis and
`
`psoriatic arthritis (Palladino, M.A., et al., 2003, Nat. Rev. Drug Discov. 22736—46). The acute
`
`exposure to high levels of TNF-Qt, as experienced during a massive infection, results in
`
`sepsis; its symptoms include shock, hypoxia, multiple organ failure, and death. Chronic low
`
`doses of TNF-Cc can cause cachexia, a disease characterized by weight loss, dehydration and
`
`fat loss, and is associated with malignancies.
`
`[0007]
`
`TNF—06 activity is mediated primarily through two receptors coded by two
`
`different genes, TNFRl and TNFR2. TNFRl is a membrane—bound protein with a molecular
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`weight 'of “approximately 55 kilodaltons (kDal), while TNFR2 is a membrane—bound protein
`
`with a molecular weight of 75 kDal. The soluble extracellular domains of both receptors are
`
`shed to some extent from the cell membrane by the action of metalloproteases. Moreover,
`
`the pre—mRNA of TNFR2 undergoes alternative splicing, creating either a full length, active
`
`membrane—bound receptor (mTNFR2), or a secreted decoy receptor (sTNFR2) that lacks
`
`exons 7 and 8 which encompasses the coding sequences for the transmembrane (Lainez et al,
`
`2004, Int. Immunol, 162169). The sTNFR2 binds TNF-ocbut does not elicit aphysiological
`
`response, thus reducing TNF—oc activity. Although an endogenous, secreted splice variant of
`
`TNFRl has not yet been identified, the similar gene structures of the two receptors strongly
`
`suggest the potential to produce this TNFRl
`
`isoform.
`
`[0008]
`
`Knockout mice lacking both TNFRl and TNFR2 treated with drugs that target the
`
`TNF signaling pathways indicate such drugs may be beneficial in treating stroke or traumatic
`
`brain injury (Bruce, et al., 1996, Nat. Med. 2:788). TNFR2 knockout mice were also used to
`
`establish a role for TNFR2 in experimentally—induced cerebral malaria (Lucas, R., et al.,
`
`1997, Eur. J. Immunol. 2721719) and autoimmune encephalomyelitis (Suvannavejh, G.C., et
`
`al., 2000, Cell. Immunol,, 205:24), models for human cerebral malaria and multiple sclerosis,
`
`respectively.
`
`[0009]
`
`TNFR2 is present at high density on T cells and appears to play a role in the
`
`immune responses that lead to alveolitis in the pulmonary microenvironment of interstitial
`
`lung disease (Agostini, C , et al., 1996, Am. J. Respir. Crit. Care Med, 15321359). TNFR2 is
`
`also implicated in human metabolic disorders of lipid metabolism and has been associated
`
`with obesity and insulin resistance (Fernandez—Real, et al., 2000, Diabetes Care, 232831),
`
`familial combined hyperlipidemia (Geurts, et al., 2000, Hum. Mol. Genet. 922067; van
`
`Greevenbroek, et al., 2000, Atherosclerosis, 153:1), hypertension and hypercholesterolemia
`
`(Glenn, et al., 2000, Hum. Mol. Genet, 921943). TNFR2 has recently been associated with
`
`human narcolepsy (Komata, T., et al., 1999, Tissue Antigens, 532527). In addition, TNFR2
`
`polymorphism appears to lead to susceptibility to systemic lupus erythematosus (Hohjoh, H.,
`
`et al., 2000, Tissue Antigens, 562446).
`
`[0010]
`
`Splice variants of CD40 (Tone, M., et al., 2001, Proc. Natl. Acad. Sci. 9821751)
`
`("Tone"), and CD95 (FAS) (Shen, L., et al., 2002, Am. J. Path. 161 22123), have been found
`
`in malignancies. Several of these splice variants result in loss of the transmembrane region
`
`due to deletion or due to mutations affecting the reading frame of exon 7. Whether these
`
`represent aberrant variants resulting from malignant transformation or physiological
`
`alternatives is not yet known.
`
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`[00] Tf '
`
`Because ot tfte role played by excessive activity by TNF superfamily members, it
`
`would be useful to control the alternative splicing of TNFR receptors so that the amount of
`
`the secreted form is increased and the amount of the integral membrane form is decreased.
`
`The present invention provides splice switching oligonucleotides or splice switching
`
`oligomers (SSOs) to achieve this goal. 880s are similar to antisense oligonucleotides
`
`(ASONs). However, in contrast to ASON, 880s are able to hybridize to a target RNA
`
`without causing degradation of the target by RNase H
`
`[0012]
`
`SSOs have been used to modify the aberrant splicing found in certain thalassemias
`
`(US. Pat. No. 5,976,879 to Kole; Lacerra, G., et al., 2000, Proc. Natl. Acad. Sci. 97:9591).
`
`Studies with the IL—5 receptor oc—chain (IL-5Roc) demonstrated that SSOs directed against the
`
`membrane—spanning exon increased synthesis of the secreted form and inhibited synthesis of
`
`the integral membrane form (US. Pat. No. 6,210,892 to Bennett; Karras, J .G., et al., 2000,
`
`MoL Pharm, 582380).
`
`[0013]
`
`The lL—5 receptor is a member of a receptor type that occurs as a hctcrodimcr.
`
`The interleukin 5 receptor (IL-5R) is a member of the IL—3R family of receptors, which also
`
`includes interleukin 3 receptor (IL—3R) and GM—CSF.
`
`lL—3R family members are
`
`multisubunit receptors consisting of a shared common [3 chain, and a unique occhain that
`
`conveys cytokine ligand specificity.
`
`IL—3R family members are expressed in the
`
`hematopoietic system.
`In particular, IL—5 is expressed exclusively in eosinophils, basophils
`and B cells (Adachiand, T. & Alam, R., 1998, Am. J. Physiol. 2752C623—33). These
`
`'
`
`receptors and the TNFR superfamily of the present invention have no sequence homology
`
`and operate in distinct signaling pathways.
`
`[0014]
`
`SSOs have been used to produce the major CD40 splice variant detected in Tone,
`
`in which deletion of exon 6, which is upstream of the transmembrane region, resulted in an
`
`altered reading frame of the protein. While the 880 resulted in the expected mRN A splice
`
`variant, the translation product of the variant mRNA appeared to be unstable because the
`
`secreted receptor could not be detected (Siwkowski, A.M., et al., 2004, Nucleic Acids Res.
`
`32; 2695).
`
`SUMMARY OF THE INVENTION
`
`[0015]
`
`The present invention provides compositions and methods for controlling
`
`expression of TNF receptors (TNFRl and TNFR2) and of other cytokine receptors from the
`
`TNFR superfamily by controlling the splicing of pre—mRNA that codes for the said receptors.
`
`More specifically, the invention causes the increased expression of the secreted form and the
`
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`06creaseoc expression ot the integral—membrane form. Furthermore, the invention can be used
`
`in the treatment of diseases associated with excessive cytokine activity.
`
`[0016]
`
`The exon or exons that are present in the integral membrane form mRNA but are
`
`removed from the primary transcript (the "pre—mRNA") to make a secreted form mRNA are
`
`termed the "transmembrane exons. The invention involves nucleic acids and nucleic acid
`
`analogs that are complementary to either of the transmembrane exons and/or adjacent introns
`
`of a receptor pre—mRNA. Complementarity can be based on sequences in the sequence of
`
`pre—mRNA that spans the splice site, which would include, but is not limited to,
`
`complcmtarity based on sequences that span the cxon—intron junction, or complementarity
`
`can be based solely on the sequence of the intron, or complementarity can be based solely on
`
`the sequence of the exon.
`
`[0017]
`
`There are several alternative chemistries available and known to those skilled in
`
`the art. One important feature is the ability to hybridize to a target RNA without causing
`
`degradation of the target by RNase H as do 2'—deoxy oligonucleotides ("antisense
`
`oligonucleotides" hereafter "ASON"). For clarity, such compounds will be termed splice-
`
`switching oligomers (SSOs). Those skilled in the art appreciate that SSO include, but are not
`
`limited to, 2' O—modified oligonucleotides and ribonucleosidephosphorothioates as well as
`
`peptide nucleic acids and other polymers lacking ribofuranosyl—based linkages.
`
`[0018]
`
`One embodiment of the invention is a method of treating an inflammatory disease
`
`or condition by administering SSOs to apaticnt or a live subject. The SSOs that are
`
`administered alter the splicing of a pre—mRNA to produce a splice variant that encodes a
`
`stable, secreted, ligand—binding form of a receptor of the TNFR superfamily, thereby
`
`decreasing the activity of the ligand for that receptor.
`
`In another embodiment, the invention
`
`is a method of producing a stable, secreted, ligand—binding form of a receptor of the TNFR
`
`superfamily in a cell by administering SSOs to the cell.
`
`[0019]
`
`The foregoing and other objects and aspects of the present invention are discussed
`
`in detail in the drawings herein and the specification set forth below.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0020]
`
`FIG. 1 depicts the structure of aportion of the tumor necrosis factor receptor pre—
`
`mRNA and spliced products for TNFRl and TNFR2. These transcripts normally contain
`
`exon 7 and exon 8, which code for the transmembrane domain of the receptors. SSOs (bars)
`
`directed towards either or both of these exons elicit alternative splicing events, resulting in
`
`transcripts that lack the full transmembrane domain.
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`LU021]"
`
`‘FlU. I SHthBS splicing products of SSOs for murine TNFRl
`
`in cell culture.
`
`NIH—3T3 cells were mock transfected [Lipofectamine® 2000 (LFA2000 Only)] or transfected
`
`with the indicated concentration of either an exon 7 skipping TNFRl SSO, A7—5 or A7—10,
`
`alone or a combination of exon 7 skipping SSO and an exon 8 skipping SSO, A8—3. Total
`
`RNA was isolated and RT-PCR performed 24 hours later. The PCR primers were used to
`
`amplify from Exon 5 to Exon 9, so that "Full Length" TNFRl
`
`is represented by a 475 bp
`
`band. Transcripts lacking exon 7 (A Exon 7) and lacking both exon 7 and exon 8 (A Exon
`
`7/8) are represented by 361 bp and 332 bp bands, respectively.
`
`[0022]
`
`FIG. 3 shows the splicing products of SSOs for murinc TNFR2 in cell culture.
`
`NIH—3T3 cells were mock transfected (LFA2000 Only) or transfected with the indicated
`
`concentration of either an exon 7 skipping TNFR2 SSO, B7—6 or B7—] , alone or a
`
`combination of exon 7 skipping oligonucleotide and an exon 8 skipping oligonucleotide, B8-
`
`4. Total RNA was isolated and RT—PCR performed 24 hours later. The PCR primers were
`
`used to amplify from Exon 5 to Exon 9, so that "Full Length" TNFRZ is represented by a 486
`
`bp band. Transcripts lacking exon 7 (A Exon 7) and lacking both exon 7 and exon 8 (A Exon
`
`7/8) are represented by 408 bp and 373 bp bands, respectively.
`
`[0023]
`
`FIGs. 4A and 4B present the sequences of exons 7 (4A) and 8 (4B) of murine
`
`TNFRl and of the flanking introns. Also shown are the sequences of 20—Me—
`
`oligoribonucleoside—phosphorothioate SSOs that were assayed for splice switching activity.
`
`[0024]
`
`FIGs. 5A and 5B present the sequences of exons 7 (5A) and 8 (5B) of murine
`
`TNFR2 and of the flanking introns. Also shown are the sequences of 20—Me—
`
`oligoribonucleoside—phosphorothioate SSOs that were assayed for splice switching activity.
`
`[0025]
`
`FIG. 6 provides an alignment of the human and murine TNF receptor genes in the
`
`regions that encode the transmembrane exons. The murine sequences, SEQ ID Nos: 107,
`
`108, 109, and 110, are homologous to the human sequences, SEQ ID Nos: 1, 2, 3, and 4,
`
`respectively.
`
`[0026]
`
`FIG. 7 shows the splicing products of SSOs for primary mouse hepatocyte
`
`cultures, in assays conducted as described in Figures 2 and 3.
`
`[0027]
`
`FIGs. 8A—8D provide mouse and human TNFRZ (TNFRSFlB)
`
`(8A and 8B) and
`
`TNFR1 (TNFRSFlA) (8C and 8D) LNA SSO sequences from Tables 2 and 3. Figures 8A
`
`and 8C schematically illustrate the position of each SSO relative to the targeted exon.
`
`Figures 8B and 8D show the pre—mRNA sequence (5' to 3') and the 880s (3' to 5') hybridized
`
`to it.
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`[UOZ8]
`
`FiU. y shows tne splicing products for L929 murine cells treated with LNA SSOs.
`
`Cells were transfected with the indicated LNA SSO at a final concentration of 50 nM. After
`
`24 hours, the cells were lysed and analyzed for splice switching by RT—PCR. Top panel,
`
`SSOs targeted to exon 7; bottom panel, SSOs targted to exon 8. FLSfull length TNFR2
`
`amplicon; A7, A8, A7/8, amplicons of the respective TNFR2 splice variants.
`
`[0029]
`
`FIG. 10 shows the splicing products for L929 murine cells using LNA SSO
`
`combinations targeted to TNFR2. L929 cells were treated with the indicated single or
`
`multiple LNA SSOs at 50 nM each and analyzed 24 hours later as described in Figure 9.
`
`[0030]
`
`FIG. 11 the splicing products for L929 murine cells using LNA SSO combinations
`
`targeted to TNFRl. L929 cells were treated with the indicated single or multiple LNA SSOs
`
`at 50 nM each and analyzed 24 hours later as described in Figure 9.
`
`[0031]
`
`FIG. 12 shows the splicing products for primary mousc hcpatocytcs treated with
`
`LNA SSOs. Primary mouse hepatocytes were transfected with 33 nM each final
`
`concentration of the indicated single or multiple LNA 880s and analyzed as described in
`
`Figure 9.
`
`[0032]
`
`FIG. 13 graphically illustrates detection of secreted TNFR2 splice variants from
`
`L929 cells (left) and primary mouse hepatocytes (right). Cells were transfected with the
`
`indicated LNA 8803. After 72 hours, the extracellular media was removed and analyzed by
`
`enzyme linked immunosorbant assay (ELISA) using antibodies from the Quantikine® Mouse
`
`sTNF RII ELISA kit from R&D Systems (Minneapolis, MN). The data are expressed as pg
`
`soluble TNFR2 per mL.
`
`[0033]
`
`FIG. 14 shows the splicing products for primary human hepatocytes treated with
`
`LNA SSOs targeted to TNFR2. Primary human hepatocytes were transfected with the
`
`indicated LNA SSO and analyzcd for splicc switching by RT—PCR aftcr 24 hours as
`
`described in Figure 9. The PCR primers were used to amplify from Exon 5 to Exon 9, so that
`
`"Full Length"(FL) TNFR2 is represented by a 463 bp band. Transcripts lacking exon 7 (A
`
`Exon 7), lacking exon 8 (A Exon 8), and lacking both exon 7 and exon 8 (A exon 7/8) are
`
`represented by 385 bp, 428 bp, and 350 bp bands, respectively.
`
`[0034]
`
`FIG. 15 shows the splicing products for intraperitoneal (i.p.) injection of LNA
`
`3274 (top) and 3305 (bottom) in mice. LNA 3274 was injected i.p. at 25mg/kg/day for either
`
`4 days (4/1 and 4/10) or 10 days (10/1). Mice were sacrificed either 1day (4/1 and 10/1) or
`
`10 (4/ 10) days after the last injection and total RNA from liver was analyzed for splice
`
`switching of TNFR2 by RT-PCR. LNA 3305 was injected at the indicated dose per day for 4
`
`days. Mice were sacrificed the next day and the livers analyzed as with 3274 treated animals.
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`"[0035J
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`FIG. '16 (top panel) graphically illustrates the amount of soluble TNFR2 in mouse
`
`serum 10 days after SSO treatment. Mice were injected i.p. with the indicated SSO or saline
`
`(n=5 per group) at 25 mg/kg/day for 10 days. Serum collected 4 days before injections began
`
`and the indicated number of days after the last injection. Sera was analyzed by ELISA as
`
`described in Figure 13. At day 10, mice were sacrificed and livers were analyzed for TNFR2
`
`splice switching by RT—PCR (bottom panel) as described in Figure 9.
`
`[0036]
`
`FIG. 17 graphically illustrates the amount of soluble TNFRl
`
`in the serum after
`
`TNFR2 SSO treatment. Mouse serum from Figure 16 was analyzed for soluble TNFRl by
`
`ELISA using antibodies from the Quantikine® Mouse sTNF RI ELISA kit from R&D
`
`Systems (Minneapolis, MN).
`
`[0037]
`
`FIG. 18 (top panel) graphically illustrates the amount of soluble TNFR2 in mouse
`
`serum 27 days after SSO treatment. Mice were treated as in Figure 16, except that serum
`
`samples were collected until day 27 after the last injection. LNA 3083 and 3272 are control
`
`SSOs with no TNFR2 splice switching ability. At day 27, mice were sacrificed and livers
`
`were analyzed for TNFR2 splice switching by RT—PCR (bottom panel) as described in Figure
`
`9.
`
`[0038]
`
`FIG. 19 graphically depicts the anti—TNF—oc activity in serum from LNA
`
`oligonucleotide—treated mice. L929 cells were treated with either 0.1 ng/mL TNF—0L (TNF),
`
`or TNF—ocplus 10% serum from mice treated with the indicated oligonucleotide (see also
`
`Figure 18). Cell viability was measured 24 hours later and normalized to untreated cells
`
`(Untreated).
`
`[0039]
`
`FIG. 20 graphically compares the anti—TNF—Ocactivity of serum from LNA
`
`oligonucleotide—treated mice to recombinant soluble TNFR2 (rsTNFR2) and to that of
`
`Enbrel® using the cell survival assay described in Figure 19.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0040]
`
`As used herein, the terms "tumor necrosis factor receptor superfamily" or "TNFR
`
`superfamily" or "TNFRSF" refer to a group of type I transmembrane proteins, with a
`
`carboxy—terminal intracellular domain and an amino—terminal extracellular domain
`
`characterized by a common cysteine rich domain (CRD). The TNFR superfamily consists of
`
`receptors, mediate cellular signaling as a consequence of binding to one or more ligands in
`
`the TNF superfamily. The TNFR superfamily can be divided into two subgroups: receptors
`
`containing the intracellular death domain (DD) and those lacking it. The DD is an 80 amino
`
`acid motif that is responsible for the induction of apoptosis following receptor activation.
`
`Members of the TNFR superfamily include, but are not limited to, TNFRl (TNFRSFlA) 3
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`TNFR2 (‘t 'I'K’l‘FRSFTBX’KANK (TNFRSFIlA), CD40 (TNFRSFS), CD30 (TNFRSFS), and
`
`LT— BR (TNFRSFS).
`
`[0041]
`
`As used herein, the terms "tumor necrosis factor superfamily" or "TNF
`
`superfamily" refer to the group of ligands that bind to one or more receptors in the TNFR
`
`superfamily, The binding of a TNF family ligand to its corresponding receptor or receptors
`
`mediate cellular signaling. Members of the TNF superfamily include, but are not limited to,
`
`TN F—Oc, RAN KL, CD4OL, LT—0(, or LT— B.
`
`[0042]
`
`As used herein, the term "an inflammatory disease or condition" refers to a
`
`disease, disorder, or other medical condition that at least in part results from or is aggravated
`
`by the binding ‘of a ligand from the TNF superfamily to its corresponding receptor or
`
`receptors. Such diseases or conditions include, but are not limited to, those associated with
`
`increased levels of the TNF superfamily ligand, increased levels of TNFR superfamily
`
`receptor levels, or increased sensitization of the corresponding signaling pathway. Examples
`
`of inflammatory diseases or conditions include, but are not limited to, rheumatoid arthritis,
`
`juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis,
`
`inflammatory bowel disease (including Crohn's disease or ulcerative colitis), hepatitis, sepsis,
`
`alcoholic liver disease, and non—alcoholic steatosis.
`
`[0043]
`
`As used herein, the term "hepatitis" refers to a gastroenterological disease,
`
`condition, or disorder that is characterized, at least in part, by inflammation of the liver.
`
`Examples of hepatitis include, but are not limited to, hepatitis associated with hepatitis A
`
`virus, hepatitis B virus, hepatitis C virus, or liver inflammation associated with
`
`ischemia/reperfusion.
`
`[0044]
`
`As used herein, the terms "membrane bound form" or "integral membrane form"
`
`refer to proteins having amino acid sequences that span a cell membrane, with amino acid
`
`sequences on each side of the membrane.
`
`[0045]
`
`As used herein, the term "stable, secreted, ligand—binding form" or as it is
`
`sometimes known "stable, soluble, ligand—binding form." (where the terms "secreted" and
`
`"soluble" are synonymous and interchangeable herein) refer to proteins that are related to the
`
`native membrane bound form receptors, in such a way that they are secreted and stable and
`
`still capable of binding to the corresponding ligand.
`
`It should be noted that these forms are
`
`not defined by whether or not such secreted forms are physiological, only that the products of
`
`such splice variants would be secreted, stable, and still capable of ligand—binding when
`
`produced.
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`[664G
`
`TKe term""se"cre'|'ed" means that the form is soluble, i.e., that it is no longer bound
`
`to the cell membrane.
`
`In this context, a form will be soluble if using conventional assays
`
`known to one of skill in the art most of this form can be detected in fractions that are not
`
`associated with the membrane, e.g., in cellular supernatants or serum.
`
`[0047]
`
`The term ”stable" means that the secreted form is detectable using conventional
`
`assays by one of skill in the art. For example, western blots, ELISA assays can be used to
`
`detect the form from harvested cells, cellular supernatants, or serum from patients.
`
`[0048]
`
`The term ”ligand—binding" means that the form retains at least some significant
`
`level, although not necessarily all, of the specific ligand—binding activity of the corresponding
`
`integral membrane form.
`[0049]
`As used herein, the term "to reduce the activity of a ligand" refers to any action
`
`that leads to a decrease in transmission of an intracellular signal resulting from the ligand
`
`binding to or interaction with the receptor. For example, activity can be reduced by binding
`
`of the ligand to a soluble form of its receptor or by decreasing the quantity of the membrane
`
`form of its receptor available to bind the ligand.
`
`[0050]
`
`As used herein, the term ”altering the splicing of a pre—mRNA" refers to altering
`
`the splicing of a cellular pre—mRNA target resulting in an altered ratio of splice products.
`Such an alteration of splicing can be detected by a variety of techniques well known to one of
`
`skill in the art. For example, RT—PCR on total cellular RNA can be used to detect the ratio of
`
`splice products in the presence and the absence of an SSO.
`
`[0051]
`
`As used herein, the term ”complementary" is used to indicate a sufficient degree
`
`of complementarity or precise pairing such that stable and specific binding occurs between an
`
`SSO and a DNA or RNA containing the target sequence.
`
`It is understood in the art that the
`
`sequence of an SSO need not be 100% complementary to that of its target. There is a
`
`sufficient degree of complementarity when, under conditions which permit splicing, binding
`
`to the target will occur and non—specific binding will be avoided.
`
`[0052]
`
`The present invention employs splice switching oligonucleotides or splice
`
`switching oligomers (SSOs) to control the alternative splicing of receptors from the TNFR
`
`superfamily so that the amount of a soluble, stable, secreted, ligand—binding form is increased
`
`and the amount of the integral membrane form is decreased. The methods and compositions
`
`of the present invention can be used in the treatment of diseases associated with excessive
`
`TNF superfamily activity.
`
`[0053]
`
`Accordingly one embodiment of the invention is a method of treating an
`
`inflammatory disease or condition by administering SSOs to a patient, The SSOs that are
`
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`aiimihTstereO "alter triemsplicihg of a pre-mRNA to produce a splice variant that encodes a
`
`stable, secreted, ligand-binding form of areceptor of the TNFR superfamily,
`
`thereby
`
`decreasing the activity of the ligand for that receptor.
`
`In another embodiment,
`
`the invention
`
`is a method of producing a stable, secreted,
`
`ligand-binding form of a receptor of the TNFR
`
`superfamily in a cell by administering $805 to the cell.
`
`[0054]
`
`The following aspects of the present invention discussed below apply to the
`
`foregoing embodiments.
`
`[0055]
`
`The length of the SSO is similar to an antisense oligonucleotide (ASON),
`
`typically between about 10 and 24 nucleotides. The invention can be practiced with SSOs of
`
`several chemistries that hybridize to RNA, but that do not activate the destruction of the RNA
`
`by RNase H, as do conventional antisense 2r-deoxy oligonucleotides. The invention can be
`
`practiced using 2'0 modified nucleic acid oligomers, such as 2'O—methyl or 2'0-
`
`methyloxyethyl phosphorothioate. The nucleobases do not need to be linked to sugars;
`
`so—
`
`called peptide nucleic acid oligomers or morpholine-based oligomers can be used. A
`
`comparison of these different link