HK1150850B - Splice switching oligomers for tnf superfamily receptors and their use in treatment of disease - Google Patents

Description

Splice switching oligomers of TNF superfamily receptors and their use for treating diseases

Priority to US 60/862,350, PCT/US2006/043651 and US 11/595,485, which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to compositions and methods for making splice variants of the TNF α receptor (TNFR) in vivo or in vitro, and the resulting TNFR protein variants. Such variants can be made using splice switching (splice switching) oligonucleotides or Splice Switching Oligomers (SSOs) to control splicing of pre-messenger RNA molecules and to regulate protein expression. Preferred SSOs according to the invention target exon 7 or 8 of TNFR1(TNFRSF1A) or TNFR2(TNFRSF1A) messenger RNA (pre-mRNA), typically results in the production of TNFR variants comprising a deletion of part or all of exon 7 or 8, respectively. SSOs targeting exon 7 were found to produce soluble forms of TNFR for therapeutic use in the treatment of inflammatory diseases. SSOs are characterized in that they are essentially incapable, or incapable, of supplementing RNaseH.

Background

WO2007/05889, incorporated herein by reference, describes background related to pre-messenger RNA splicing, the role of TNF- α in inflammation and inflammatory disorders, and the mediation of TNF- α activity mediated via TNF1 and TNF 2.

TNF- α is a pro-inflammatory cytokine in the form of a membrane-bound homotrimer, which is released into the circulation by the protease TNF- α converting enzyme (TACE). TNF- α, which is introduced into the circulation, is a mediator of the inflammatory response caused by injury and infection. The progression of inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis and psoriatic arthritis all involve the action of TNF- α (Palladino, m.a., et al, 2003, nat. rev. drug discov.2: 736-46). Short time (acute) exposure to high levels of TNF- α will lead to the development of sepsis, a phenomenon that has already occurred during heavy infections; symptoms of sepsis include shock, hypoxia, multiple organ failure and death. Chronic (chronic) low doses of TNF- α can lead to a malignancy-associated disease characterized by weight loss, dehydration and fat loss, cachexia.

The activity of TNF- α is mediated primarily by two receptors encoded by two distinct genes, TNFR1 and TNFR 2. TNFR1 is a membrane-bound protein with a molecular weight of approximately 55 kilodaltons (kDa), while TNFR2 is a membrane-bound protein with a molecular weight of 75 kDa. The soluble extracellular domains of both receptors are to some extent shed from the cell membrane by metalloproteases. In addition, alternative splicing of the pre-messenger RNA of TNFR2 can result in an active full-length membrane-bound receptor (mTNFR2) or a secreted pseudoreceptor (sTNFR2) lacking the coding sequence for exons 7 and 8, including the transmembrane region. sTNFR2 binds TNF- α but does not elicit a physiological response and therefore reduces TNF- α activity. Although endogenous secreted TNFR1 splice variants have not been identified, the similarity of the gene structures of these two receptors clearly indicates the possibility of producing this TNFR1 isoform (isotype).

The effects of over-activation of TNF superfamily members have made it useful to control alternative splicing of TNFR receptors, so that the amount of secreted forms is increased and the amount of intact membrane forms is decreased. The present invention provides splice switching oligonucleotides or Splice Switching Oligomers (SSOs) that achieve this goal. SSOs resemble antisense oligonucleotides (ASONs). However, in contrast to ASONs, SSOs hybridize to target RNA but do not result in degradation of the target RNA by RNase H.

SSOs have been used to modify aberrant splicing found in certain thalassemias (KoIe, U.S. Pat. No.5,976,879; Lacerra, G., et al., 2000, Proc. Natl. Acad. Sci.97: 9591). Studies on the IL-5 receptor alpha-chain (IL-5R alpha) have shown that SSOs targeting transmembrane exons increase synthesis of secreted forms while inhibiting synthesis of intact membrane forms (Bennett, U.S. Pat. No.6,210,892; Karras, J.G., et al., 2000, MoL Pharm, 58: 380). WO00/58512 also discloses examples of splicing IL-5R to a soluble form (examples 25 and 30).

SSOs have been used to generate the major CD40 splice variant detected in Tone, where the deletion of exon 6 upstream of the transmembrane region results in an altered reading frame for the protein. When SSO produces the expected mRNA splice variant, the translation product of the variant mRNA also becomes unstable, as no secreted receptor is detected (Siwkowski, A.M., et al, 2004, Nucleic Acids Res.32; 2695). Tone et al, PNAS, 2001, 98 (4): 1751-1756 mouse splice variants lacking exon 6 are not predicted to be stable secreted forms of CD40 (see page 1756, right panel).

WO02/088393 discloses gapmer oligonucleotides targeting mouse TNFR2 with 2' MOE wings and a deoxy notch, which are used to complement (recurait) RNAseH to degrade TNFR2mRNA (mRNA down-regulation). The SSO oligonucleotides of the invention are not designed to complement RNaseH, but to disrupt processing of TNFR pre-messenger RNA, thus resulting in the production of stable secreted forms of ligand-binding TNFR splice variants.

US2005/202531 teaches that antisense oligonucleotides can be used to alter the alternative splicing pattern of CD40, but it does not teach or provide any hint as to the CD40 splicing element or region that should be targeted by SSOs, nor which sequence should be used.

Summary of The Invention

The present invention uses splice switching oligonucleotides or Splice Switching Oligomers (SSOs) to control alternative splicing from TNFR superfamily receptors, such that the number of secreted stable soluble ligand-bound forms is increased and the number of intact membrane forms is decreased.

The present invention provides an oligomer of between 8 and 16 nucleotide bases in length comprising a contiguous (contiguous) nucleobase sequence consisting of nucleotides of between 8 and 16 bases in length, wherein said contiguous nucleobase sequence is complementary to a corresponding region of the contiguous nucleotides present in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4 and said contiguous nucleobase sequence does not comprise 5 or more than 5 contiguous DNA (2' -deoxyribonucleoside) monomer units comprising at least one nucleotide analogue selected from the group consisting of β -D-oxolna, thio-LNA, amino-LNA and ena-LNA.

Optionally, in the above embodiments, the contiguous nucleobase sequence comprises or consists of at least one still further additional (further) nucleotide analogue (X).

In one embodiment, the further nucleotide analogue units are independently selected from the group consisting of 2 ' -OMe-RNA units, 2 ' -fluoro-DNA units, 2 ' -MOE RNA units and LNA.

In one embodiment, the oligomer or contiguous nucleobase sequence consists of a nucleobase sequence between 8 and 15 bases in length, e.g., consisting of 9, 10, 11, 12, 13 or 14 nucleobases.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence or nucleobase motif selected from the group consisting of SEQ ID NO 131-SEQ ID NO 145, SEQ ID NO 147-SEQ ID NO 161 and SEQ ID NO 163-177.

In one embodiment, the oligomer is selected from the group consisting of SEQ ID NO 245-SEQ ID NO 246, SEQ ID NO 251-263, SEQ ID NO 264-SEQ ID NO 279 and SEQ ID NO280-SEQ ID NO 295.

In one embodiment, said contiguous nucleobase sequence is identical to or present in a nucleobase sequence or a nucleobase motif selected from the group consisting of SEQ ID NO 130, SEQ ID NO 146 and SEQ ID NO 162.

In one embodiment, the oligomer is selected from the group consisting of SEQ ID NO 244, SEQ ID NO 264 and SEQ ID NO 280.

The present invention relates to splice switching oligonucleotides or Splice Switching Oligomers (SSOs). Preferred SSOs according to the invention target exon 7 of TNFR1(TNFRSF1A) or TNFR2(TNFRSF1A) pre-messenger RNA, typically resulting in TNFR variants comprising a deletion of part or all of exon 7, respectively. SSOs targeting exon 7 were found to produce soluble forms of TNFR for therapeutic use in the treatment of inflammatory diseases. SSOs are characterized in that they are essentially incapable of supplementing (recovering) RNaseH.

The present invention provides an oligomer of between 8 and 50 nucleotide bases in length, for example an oligomer of between 8 and 16 nucleotide bases in length, comprising (or consisting of) a contiguous nucleobase sequence of between 8 and 50 nucleotides in length, wherein said contiguous nucleobase sequence is complementary, preferably fully complementary, to a corresponding region of a contiguous nucleotide present in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4, and said contiguous nucleobase sequence does not comprise 5 or more than 5 contiguous DNA (2' -deoxyribonucleoside) monomer units.

SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4 are identical to SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4 in PCT/US 2006/043651.

SEQ ID NO 247 is reverse complementary to SEQ ID NO 1. SEQ ID NO 248 is reverse complementary to SEQ ID NO 2. SEQ ID NO249 is reverse complementary to SEQ ID NO 3. SEQ ID NO 250 is reverse complementary to SEQ ID NO 4.

Thus, preferred according to the invention are oligomers comprising or consisting of a contiguous nucleobase sequence which is homologous (preferably 100% homologous) to a corresponding region (i.e. a part) of SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO249 or SEQ ID NO 250.

The present invention provides an oligomer of between 8 and 50 nucleotide bases in length comprising (or consisting of) a contiguous nucleobase sequence of between 8 and 50 nucleotides in length, wherein said contiguous nucleobase sequence is present in the (corresponding) region of contiguous nucleotides present in SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO249 or SEQ ID NO 250, and said contiguous nucleobase sequence does not comprise 5 or more than 5 contiguous monomeric units of DNA (2' -deoxyribonucleoside).

The present invention provides an oligomer of between 8 and 50 nucleotide bases (nucleobase) in length comprising (or consisting of) a contiguous nucleobase sequence of between 8 and 50 nucleotides in length, wherein said contiguous nucleobase (nucleobase) sequence is complementary, preferably fully complementary, to a corresponding region of contiguous nucleotides present in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4, wherein said oligomer is substantially incapable, or incapable, of complementing RNAseH when formed in a duplex (duplex) with a complex of complementary mRNA molecules.

The present invention provides an oligomer of between 8 and 50 nucleobases in length comprising (or consisting of) a contiguous nucleobase sequence consisting of nucleotides of between 8 and 50 bases in length, wherein said contiguous nucleobase sequence is present in the (corresponding) region of contiguous nucleotides present in SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO249 or SEQ ID NO 250, wherein said oligomer is substantially incapable, or incapable, of complementing RNAseH when formed in a duplex with a complex of complementary mRNA molecules.

Still further, the invention provides a conjugate comprising an oligomer of the invention and at least one non-nucleotide moiety covalently attached to the oligomer.

Further, the invention provides a pharmaceutical composition comprising the oligomer or conjugate of the invention, and a pharmaceutically acceptable carrier.

Still further, the present invention provides a method of altering the splicing of a TNF α receptor pre-messenger RNA mRNA selected from TNFRSF1A TNFRSF1A or TNFRSF1A in a mammalian cell expressing a TNFRSF1A TNF α receptor or a TNFRSF1BTNF α receptor, comprising administering to the cell an oligomer, conjugate or pharmaceutical composition of the present invention.

The invention also relates to a method of preparing TNFRSF1A TNF α receptor or a soluble form of TNFRSF1B TNF α receptor in a mammalian cell expressing said TNF α receptor, said method comprising administering to the cell an oligomer, conjugate or pharmaceutical composition of the invention.

The above method may further comprise the step of purifying TNFRSF1A TNF α receptor or a soluble form of TNFRSF1BTNF α receptor.

The invention also provides a method of increasing the expression of TNFRSF1A TNF α receptor or a soluble form of TNFRSF1B TNF α receptor in a mammalian cell expressing said TNF α receptor, said method comprising administering to the cell an oligomer, conjugate or pharmaceutical composition of the invention.

The above method may be carried out in vivo or in vitro.

The present invention provides the use of an oligomer according to the invention for the preparation of a pharmaceutical formulation for the treatment of an inflammatory disease or condition (condition).

The present invention provides conjugates for the treatment of inflammatory diseases or conditions.

The present invention provides a method of treatment or prophylaxis of an inflammatory disease or condition, said method comprising the step of administering to a patient suffering from or likely to suffer from said inflammatory disease a pharmaceutical composition of the present invention.

The present invention provides an isolated or purified soluble form of a TNF α receptor comprising a deletion in the transmembrane binding domain encoded by exon 7, wherein the TNF α receptor is selected from the group consisting of TNF α receptor TNFRSF1A or TNFRSF 1B.

The present invention provides an isolated or purified soluble form of a TNF α receptor lacking a transmembrane binding domain encoded by exon 7, wherein the TNF α receptor is selected from the group consisting of TNF α receptor TNFRSF1A or TNFRSF 1B.

The invention still further provides nucleic acids encoding soluble forms of TNF α receptors.

The invention further provides vectors, e.g., expression vectors, comprising the nucleic acids of the invention.

The invention further provides a host cell comprising a nucleic acid or vector of the invention.

The present invention still further provides a method of producing a soluble form of a TNF α receptor, said method comprising the steps of culturing a host cell of the invention under conditions permitting expression of a nucleic acid of the invention, and subsequently isolating said soluble form of a TNF α receptor from said host cell.

The present invention still further provides a pharmaceutical composition comprising an isolated or purified soluble form of a TNF α receptor of the present invention or a TNF α receptor prepared according to the methods of the present invention, and a pharmaceutically acceptable carrier.

The present invention further provides the use of an isolated or purified soluble form of a TNF α receptor of the present invention or a TNF α receptor prepared according to a method of the present invention for the preparation of a pharmaceutical formulation for the treatment of an inflammatory disease or condition.

The invention still further provides the use of an isolated or purified soluble form of a TNF α receptor of the invention or a TNF α receptor prepared according to a method of the invention for the treatment of an inflammatory disease or condition.

The relevant cases PCT/US2006/043651, PCT/US2007/10557, US 11/595,485 and US 11/799,117 are all hereby incorporated by reference in their entirety.

Brief Description of Drawings

The following figures are the same as those described in PCT/US 2007/10557. Fig. 20 is new to the present application.

Figure 1 schematically depicts the structure of human TNFR 2. The corresponding exons and introns are represented by boxes (box) and lines, respectively. The signal sequence and transmembrane regions are shaded. The residues that form the boundaries of the signal sequence and transmembrane region, and the last residues, are indicated below the figure. Exon boundaries are marked above the figure; if the 3 'end of an exon and the 5' end of the following exon have the same residue number, then the splice junction will be located within the codon encoding that residue.

Figure 2A graphically illustrates the amount of soluble TNFR2 from SSO-treated primary (primary) human hepatocytes. SSO indicated by 5A concentration of 0nM was transfected into primary human hepatocytes. After 48 hours, use is made of&Obtained by D system (Minneapolis, MN)Human sTNFRII ELISA kit (quantikine (r) Human stnrii ELISA kit) soluble TNFR2 in extracellular medium was analyzed by enzyme-linked immunosorbent assay (ELISA). Error bars represent standard deviations of 3 independent experiments. FIG. 2B splice switching of TNFR2 in total RNA was analyzed by RT-PCR using primers specific for human TNFR 2. SSOs targeting exon seven resulted in a transition from full-length TNFR2mRNA (FL) to TNFR2 Δ 7mRNA (Δ 7). SSO 3083 is a control SSO without TNFR2 splicing switching ability.

FIG. 3 shows the splice product of L929 cells treated with SSO 10-mers targeting exon 7 of mouse TNFR 2. L929 cells were transfected with the indicated SSO concentrations (50 or 100nM) and TNFR2 splicing transitions were assessed after 24 hours by RT-PCR. PCR primers were used to amplify exon 5 to exon 9, so that a 486bp band could represent "full length" (FL) TNFR 2. The 408bp band represents the transcript lacking exon 7 (. DELTA.7).

FIGS. 4A and 4B show splice products from mice treated with SSO 10-mers targeting exon 7 of mouse TNFR 2. The indicated SSOs were resuspended in saline and then injected intraperitoneally (i.p.) at a dose of 25 mg/kg/day into mice for a total of 5 days. Mice were pre-bled prior to SSO injection, and 10 days after the last SSO injection, and sacrificed. At sacrifice, TNFR2 splice switching in total RNA from liver was analyzed by RT-PCR. FL-full length TNFR 2; Δ 7-TNFR2 Δ 7 (FIG. 4A). Using from R&Obtained by D system (Minneapolis, MN)Mouse stnfrii ELISA kit (quantikine (r) Mouse stnfilieisa kit) the concentration of TNFR2 Δ 7 in sera obtained before (before) and after (after) SSO injection was determined by ELISA (fig. 4B). Error bars represent the standard deviation of 3 independent readings from the same sample.

FIG. 5 depicts the splicing convertibility of SSOs of varying lengths. As in fig. 2, primary human hepatocytes were transfected with the indicated SSO and analyzed for expression of TNFR2 by RT-PCR (upper grid) and ELISA (lower grid). Error bars represent standard deviations from 2 independent experiments.

FIGS. 6A and 6B illustrate the induction of TNFR2 Δ 7mRNA in the liver of SSO-treated mice. FIG. 6A: total RNA from SSO 3274-treated mouse liver was analyzed by RT-PCR and the products visualized on a 1.5% agarose gel. The sequence of the exon 6-exon 8 junction is shown in FIG. 6B.

Fig. 7A and 7B illustrate induction of TNFR2 Δ 7mRNA in SSO-treated primary human hepatocytes. FIG. 7A: total RNA from SSO 3379 treated cells was analyzed by RT-PCR and the products visualized on a 1.5% agarose gel. The sequence of the exon 6-exon 8 junction is shown in FIG. 7B.

FIGS. 8A and 8B graphically illustrate the dose dependence of TNFR2 pre-messenger RNA splice switching produced by SSOs 3378, 3379 and 3384. Primary human hepatocytes were transfected with SSO at 1-150nM as indicated. After 48 hours, cells were harvested to obtain total RNA, and the extracellular medium was collected. FIG. 8A: splicing transitions of TNFR2 in total RNA were analyzed by RT-PCR using primers specific for human TNFR 2. For each SSO, the number of splice transitions was plotted as a function of SSO concentration. FIG. 8B: the concentration of soluble TNFR2 in the extracellular medium was determined by ELISA and plotted as a function of SSO. Error bars represent standard deviations of at least 2 independent experiments.

FIG. 9 illustrates the detection of secreted TNFR2 splice variant from L929 cells. Cells were transfected with the indicated SSOs. After 72 hours, the extracellular medium was removed and analyzed by ELISA. Data are expressed as pg soluble TNFR 2/mL.

Figure 10 shows the splicing products in mice injected intraperitoneally (i.p.) with SSO 3274 (top) and 3305 (bottom). SSO was injected intraperitoneally at a dose of 25 mg/kg/day for either 32744 (4/1 and 4/10) or 10 days (10/1). Mice were sacrificed 1 day (4/1 and 10/1) or 10 days (4/10) after the last injection and total RNA from liver was analyzed by RT-PCR for TNFR2 splice switching. SSO 3305 was injected daily at the indicated dose for a total of 4 days. The following day, mice were sacrificed and livers were analyzed using the same method as 3274 treated animals.

Figure 11A illustrates the amount of soluble TNFR2 in mouse serum 10 days after SSO treatment. Mice were injected intraperitoneally with the indicated SSO or saline (n ═ 5 per group) at a dose of 25 mg/kg/day for 10 days. Sera were collected 4 days before the start of injection and the indicated days after the last injection. On day 10, sera were analyzed by ELISA as described in figure 22, mice were sacrificed and liver splicing transitions of TNFR2 were analyzed by RT-PCR as described in figure 30 (figure 11B).

Figure 12A illustrates the amount of soluble TNFR2 in mouse serum 27 days after SSO treatment. Mice were treated as described in fig. 11, except that serum samples were collected on day 27 after the last injection. SSOs 3083 and 3272 are controls without TNFR2 splicing switching ability. On day 27, mice were sacrificed and liver splicing transitions of TNFR2 were analyzed by RT-PCR as described in figure 11 (figure 11B).

Figures 13A and 13B depict anti-TNF- α activity of sera from SSO-treated mice in a cell-based assay, where serum samples were collected at day 5 (figure 16A) and day 27 (figure 16B) after SSO treatment. L929 cells were treated with 0.1ng/mL TNF-. alpha.or TNF-. alpha.and 10% serum obtained from mice treated with the indicated SSO. Cell viability was measured after 24 hours and normalized to untreated cells.

FIG. 14 compares anti-TNF-alpha activity from sera from mice treated with labeled SSO oligonucleotides to that from mice treated with labeled SSO oligonucleotides using the cell survival assay described in FIG. 13Andby recombination ofActivity of the soluble TNFR2(rsTNFR2) extracellular domain.

FIGS. 15A and 15B compare the stability of muTNFR2 Δ 7 protein (FIG. 15A) and mRNA (FIG. 15B). Mice were injected daily with SSO 3272, SSO 3274 or SSO 3305 (n-5) at a dose of 25 mg/kg/day. After the last injection, mice were bled on the indicated day and the serum TNFR2 concentration was measured. Mice were sacrificed the day indicated after the last injection of SSO and the total RNA obtained was analyzed by RT-PCR as described in figure 10.

Figure 16 depicts the TNFR2 Δ 7 protein (dashed line) and mRNA (solid line) levels as a percentage of protein or mRNA content, respectively, as a function of time 10 days after the last injection.

FIG. 17 illustrates the dose dependence of anti-TNF-alpha activity of TNFR2 Δ 7 expressed by HeLa cells following transfection with a TNFR2 Δ 7 mammalian expression plasmid. HeLa cells were transfected with the indicated mouse or human TNFR2 Δ 7 and the extracellular medium was collected after 48 hours. The concentration of TNFR2 Δ 7 in the medium (medium) was determined by ELISA and serial dilutions were made. The anti-TNF-alpha activity of these dilutions was determined by the L929 cytotoxicity assay in FIG. 14.

FIG. 18 shows the expressed mouse (A) and human (B) TNFR2D7 proteins separated by polyacrylamide gel electrophoresis (PAGE). Hela cells were transfected with the indicated plasmids. After 48 hours, the extracellular medium was collected and concentrated, and the cells were collected in RIPA lysis buffer. Proteins in the samples were separated by PAGE and western blotted using TNFR2 primary antibody (Abcam) which recognizes the C-terminus of human and mouse TNFR2D7 proteins. Medium, extracellular medium samples of Hela cells transfected with marker plasmids; lysate, cell lysate of Hela cells transfected with the marker plasmid. CM, control medium from untransfected Hela cells; CL, control cell lysate from untransfected Hela cells. Molecular weight marker (kDal).

Figure 19 shows purified His-tagged human and mouse TNFR2D 7. An unconcentrated extracellular medium containing the indicated TNFR2D7 protein was prepared according to fig. 18. Approximately 32mL of the medium was applied to a 1mL HisPur cobalt spin column (Pierce) and bound protein was eluted with 1mL of buffer containing 150mM imidazole. Each sample was analyzed by PAGE and western blot analysis was performed as per FIG. 18. Multiple bands in lanes 1144-4 and 1319-1 indicate the presence of a different glycosylated form of TNFR2D 7.

FIG. 20 alignment of oligomer motifs of the invention with their target sequences SEQ ID NO1 (FIG. 20A), SEQ ID NO2 (FIG. 20B), SEQ ID NO3 (FIG. 20C) and SEQ ID NO4 (FIG. 20D).

Detailed Description

The present invention provides compositions and methods for controlling the expression of TNF receptors (TNFR1 and TNFR2) by controlling the splicing of pre-messenger RNAs encoding said receptors and other cytokine receptors from the TNFR superfamily. More specifically, the invention results in increased expression of the secreted form and decreased expression of the intact membrane form. In addition, the invention is useful for treating diseases associated with excessive cytokine activity.

An exon that is present in the intact membrane form of an mRNA, but is removed from the primary transcript (pre-messenger RNA) to produce the secreted form of the mRNA is called the "transmembrane exon". The present invention relates to nucleic acids and nucleic acid analogs that are complementary to either of a transmembrane exon and/or an adjacent intron of a pre-receptor messenger RNA. Complementarity may be based on sequences present in the pre-messenger RNA sequence covering the splice site, including, but not limited to, sequences covering (or spanning a span) exon-intron junctions, or based solely on sequences of introns or sequences of exons.

There are several alternative chemical processes () known to the person skilled in the art. An important feature is the ability to hybridize to the target RNA without causing degradation of the target to 2' -deoxyoligonucleotides by RNase H ("antisense oligonucleotides" hereinafter referred to as "ASON"). For clarity, such compounds will be referred to as splice-switch oligomers (SSOs). One skilled in the art will appreciate that SSOs include, but are not limited to, 2' O-modified oligonucleotides, ribonucleoside phosphorothioates, and peptide nucleic acids and other polymers lacking ribofuranosyl-based linkages.

One embodiment of the present invention relates to methods of administering SSOs to a patient or living subject for the treatment of an inflammatory disease or condition. The administered SSOs alter the splicing of pre-messenger RNA, produce splice variants that encode stable secreted ligand-binding forms of TNFR superfamily receptors, and thus reduce the activity of ligand-binding receptors. In another embodiment, the invention relates to methods of producing a stably secreted ligand-binding form of a TNFR superfamily receptor in a cell by administering SSOs to the cell.

One embodiment of the invention relates to a full-length or mature protein that can bind TNF encoded by a cDNA derived from a mammalian TNFR gene in which exon 6 is directly followed by exon 8, thus lacking exon 7 ("TNFR. delta.7"). In another embodiment, the invention relates to a pharmaceutical composition comprising TNFR δ 7. In a still further embodiment, the invention relates to a method of treating an inflammatory disease or condition by administering a pharmaceutical composition comprising TNFR δ 7.

In another embodiment, the invention relates to a nucleic acid encoding TNFR δ 7. In a still further embodiment, the present invention relates to a pharmaceutical composition comprising a nucleic acid encoding TNFR δ 7.

In another embodiment, the invention relates to an expression vector comprising a nucleic acid encoding TNFR δ 7.

In a still further embodiment, the invention relates to a method of increasing the level of soluble TNFR in the serum of a mammal by transforming a mammalian cell with an expression vector comprising a nucleic acid encoding TNFR δ 7. In another embodiment, the invention relates to a cell transformed with an expression vector comprising a nucleic acid encoding TNFR δ 7.

In a still further embodiment, the invention relates to a method of producing TNFR δ 7 by culturing a cell transformed with an expression vector comprising a nucleic acid encoding TNFR 57 under conditions suitable for expression of TNFR δ 7. In another embodiment, the invention relates to a method of treating an inflammatory disease or condition (condition) by administering an expression vector comprising a nucleic acid encoding TNFR δ 7.

In another embodiment, Splice Switching Oligomers (SSOs) are disclosed that alter the splicing of mammalian TNFR2 pre-messenger RNA to produce a mammalian TNFR2 protein, said TNFR2 protein binds TNF and is directly linked to exon 8 following exon 6, thereby lacking exon 7 ("TNFR 2. delta.7"). One embodiment of the present invention relates to methods of administering SSOs to a patient or living subject for the treatment of an inflammatory disease or condition. The administered SSOs are capable of altering splicing of mammalian TNFR2 pre-messenger RNA to produce TNFR2 δ 7. In another embodiment, the invention relates to methods of producing TNFR2 δ 7 in a cell by administering SSOs to the cell.

The above objects and other aspects of the present invention are discussed in detail in the drawings herein and the following description.

Oligomer

In one embodiment, the oligomer consists of contiguous nucleobase sequences.

However, it is also conceivable that the oligomer may comprise other nucleobase sequences flanking either the 5 'or 3' end of the contiguous nucleobase sequence, or more distal nucleobase sequences flanking both the 5 'and 3' ends. Suitable lengths of these 5 'and/or 3 "flanking' regions are 1, 2,3, 4, 5 or 6 nucleobases. For in vivo use, it is contemplated that the DNA or RNA nucleobases at the ends of the oligomers of the invention may be cleaved from the oligomer by endogenous exonucleases, and thus, the inclusion of flanking DNA or RNA units does not affect the in vivo performance of the oligomer.

In one embodiment, adjacent to the nucleobase sequence 3' terminal side connected to 1, 2 or 3 DNA or RNA units. 3' DNA units are often used during solid phase synthesis of oligomers.

In one embodiment, adjacent to the nucleobase sequence of the 5' terminal side connected to 1, 2 or 3 DNA or RNA units.

In one embodiment, the invention provides an oligomer of between 8 and 50 nucleobases in length comprising a contiguous nucleobase sequence consisting of nucleotides of between 8 and 50 bases in length, wherein said contiguous nucleobase sequence is complementary to a corresponding region of a contiguous nucleotide present in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4 (i.e. said contiguous nucleobase sequence is complementary to a corresponding region (' corresponding ' or part) of a contiguous nucleotide present in SEQ ID NO 247, SEQ ID NO 248, SEQ ID NO249 or SEQ ID NO 250), and said contiguous nucleobase sequence does not comprise 5 or more than 5 contiguous monomeric units of DNA (2 ' -deoxyribonucleoside).

In one embodiment, the oligomer is substantially incapable of replenishing RNAseH when formed in a duplex with a complex of complementary mRNA molecules.

In one embodiment, the contiguous nucleobase sequence comprises or consists of the nucleotide analogue (X).

In one embodiment, the nucleotide analogues (X) are independently selected from the group consisting of 2 '-O-alkyl-RNA units, 2' -OMe-RNA units, 2 '-amino-DNA units, 2' -fluoro-DNA units, LNA units, PNA units, HNA units, INA units.

In one embodiment, the contiguous nucleobase sequence comprises a nucleotide analogue (X) and a nucleotide (X).

In one embodiment, the contiguous nucleobase sequence does not comprise a region of more than 7 contiguous nucleobase analog units (X), such as no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 contiguous nucleobase analog units (X).

In one embodiment, the nucleobase closest to the 5 'end (5' most) of the contiguous nucleobase sequence is a nucleotide analogue (X).

In one embodiment, the nucleobase which adjoins the nucleobase sequence closest to the 5 'end is a nucleotidic unit (x), such as a DNA (2' - -deoxyribonucleoside) monomeric unit.

In one embodiment, the nucleobase which adjoins the nucleobase sequence closest to the 3' end is a nucleotide analogue (X).

In one embodiment, the nucleobase which adjoins the nucleobase sequence closest to the 3 'end is a nucleotidic unit (x), such as a DNA (2' - -deoxyribonucleoside) monomeric unit. In one embodiment, the contiguous nucleobase sequence comprises or consists of an alternating sequence of nucleotides and nucleobases. In one embodiment, the alternating sequence of nucleotides and nucleobases is selected from the group consisting of Xx, xX, Xxx, xXx, xxX, XXx, XxX, xXX, XXXX, XXxX, XxXXX, xXXX, xxxX, xxXx, xXXx, Xxxx, XXXXX, XXXXXXX, XXXXXX, xXXXXX, xxXXX, xxXxx, xXXX, Xxxxx, Xxxxxxx, wherein the alternating sequence may be arbitrarily repeated.

In one embodiment, adjacent to the nucleobase sequence within the full length of the repeat sequence, which can be any truncated 5 'and/or 3' repeat.

In one embodiment, the single stranded oligonucleotide comprises said at least one LNA analogue unit and at least one further nucleotide analogue unit different from LNA.

In one embodiment, the single stranded oligonucleotide is comprised of at least one X¹X²X¹Or X²X¹X²Sequence composition of wherein X¹Is LNA, X²Are nucleotide analogues other than LNA, such as 2 '-OMe RNA units and 2' -fluoro DNA units.

In one embodiment, the nucleobase sequence of the single stranded oligonucleotide consists of alternating (alternative) X¹And X²And (4) unit composition.

In one embodiment, the nucleotide analogue units, such as X, are independently selected from the group consisting of 2 '-ome rna units, 2' -fluoro-DNA units and LNA units.

In one embodiment, the nucleotide analogue unit (X) is a LNA unit.

In one embodiment, the LNA unit is selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA and ena-LNA.

In one embodiment, the contiguous sequence of nucleobases does not comprise a contiguous subsequence of 5 or more than 5 contiguous nucleobases independently selected from DNA and LNA units, wherein the LNA present in the contiguous subsequence is in the alpha-L-configuration.

In one embodiment, the contiguous sequence of nucleobases does not comprise a contiguous subsequence of 5 or more than 5 contiguous nucleobases independently selected from units of DNA and LNA, wherein LNA present in the contiguous subsequence is alpha-L-oxy-LNA.

In one embodiment, all LNA units are in the β -D configuration.

In one embodiment, the contiguous nucleobase sequence consists solely of LNA and DNA units.

In one embodiment, the contiguous nucleobase sequence consists solely of LNA and DNA units. Preference is given to LNA units of the beta-D configuration, for example beta-D-oxy or beta-D-thio or beta-D-amino.

In one embodiment the LNA is selected from the group consisting of β -D-oxy-LNA, β -D-thio-LNA, β -D-amino-LNA, ena-LNA, optionally including the group consisting of α -L-oxy-LNA, α -L-thio-LNA or α -L-amino-LNA.

In one embodiment, the contiguous nucleobase sequence is between 8 and 16, for example 9, 10, 11, 12, 13, 14, 15 or 16 nucleobases in length, or between 10-14, 11-14 or 12-14.

In one embodiment, the contiguous nucleobase sequence is between 8 and 15, e.g. 8, 9, 10, 11, 12, 13, 14 or 15 nucleobases in length.

In one embodiment, the contiguous nucleobase sequence comprises nucleobases complementary to a corresponding region present in SEQ ID NO1 or SEQ ID NO3, i.e. the nucleobases of the (corresponding) region of the contiguous nucleotide present in SEQ ID NO 247 or SEQ ID NO 249.

In one embodiment, the contiguous nucleobase sequence is complementary to a corresponding region of contiguous nucleotides present in a sequence selected from the group consisting of 51-164 of SEQ ID NO1, 51-79 of SEQ ID NO2, 51-127 of SEQ ID NO3 and 51-85 of SEQ ID NO 4. In one embodiment, the contiguous nucleobase sequence is complementary to a corresponding region of contiguous nucleotides present in a sequence selected from the group consisting of 1-50 of SEQ ID NO1, 165-215 of SEQ ID NO1, 1-50 of SEQ ID NO2, 80-130 of SEQ ID NO2, 1-50 of SEQ ID NO3, 128-178 of SEQ ID NO3, 1-50 of SEQ ID NO4 and 86-136 of SEQ ID NO 4.

In one embodiment, the contiguous nucleobase sequence comprises a nucleobase sequence complementary to a 5 'exon/intron 3' or 3 'intron/exon 5' border.

In one embodiment, the 5 'exon/intron 3' or 3 'intron/exon 5' boundary is selected from the group consisting of nucleobases 50-51 of SEQ ID NO1, 164-165 of SEQ ID NO1, 50-51 of SEQ ID NO2, 79-80 of SEQ ID NO2, 51-52 of SEQ ID NO3, 129-139 of SEQ ID NO3, 50-51 of SEQ ID NO4, 81-82 of SEQ ID NO 4.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence present in a sequence selected from the group consisting of SEQ ID NO 74 to SEQ ID NO 105.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence selected from the group consisting of SEQ ID NO 74, SEQ ID NO75, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 82 and SEQ ID NO 84.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence selected from the group consisting of SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88 and SEQ ID NO 89.

In one embodiment, the oligomer is selected from the group consisting of SEQ ID NO 74, SEQ ID NO75, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 82 and SEQ ID NO 84.

In one embodiment, the oligomer is selected from the group consisting of SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88 and SEQ ID NO 89.

In one embodiment, the contiguous nucleobase sequence comprises a nucleobase sequence complementary to the nucleotide region 47-49, 54-56 and 122-124 selected from SEQ ID No 3.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence or nucleobase motif selected from the group consisting of SEQ ID NO 130-SEQ ID NO 145, SEQ ID NO 146-SEQ ID NO 161 and SEQ ID NO 162-177.

In one embodiment, the contiguous nucleobase sequence is identical to or present in a nucleobase sequence or nucleobase motif selected from the group consisting of SEQ ID NO 131-SEQ ID NO 145, SEQ ID NO 147-SEQ ID NO 161 and SEQ ID NO 163-177.

In one embodiment, the oligomer is selected from the group consisting of SEQ ID NO 243, SEQ ID NO 244, SEQ ID NO 245 or SEQ ID NO 246.

In one embodiment, the oligomer comprises at least one non-nucleotide moiety covalently attached to the oligomer.

Splice Switching Oligomers (SSOs):

in another aspect, the invention uses splice switching oligonucleotides or Splice Switching Oligomers (SSOs) to control alternative splicing of TNFR2 such that the number of soluble ligand-bound forms lacking exon 7 is increased and the number of intact membrane forms is decreased. The methods and compositions of the invention are useful for treating diseases associated with excess tnf activity.

Accordingly, one embodiment of the present invention relates to methods of administering SSOs to a patient for the treatment of an inflammatory disease or condition. The administered SSOs are capable of altering splicing of pre-messenger RNA to produce a mammalian TNFR2 protein lacking exon 7.

In another embodiment, the invention relates to methods of producing a mammalian TNFR2 protein lacking exon 7 in a cell by administering SSOs to the cell.

The length of the SSO (i.e., the number of monomers in the oligomer) is similar to that of an antisense oligonucleotide (ASON), typically between about 8-30. In a preferred embodiment, the SSO is between about 10-16 nucleotides. The present invention can be practiced using several chemistries that SSOs hybridize to RNA but do not activate RNAseH-induced RNA destruction as do conventional antisense 2' -deoxyoligonucleotides. Nucleic acid oligomers which can be modified with 2 'O, e.g.2' O by-O-CH₃、-O-CH₂-CH₂-O-CH₃、-O-CH₂-CH₂-CH₂-NH₂、-O-CH₂-CH₂-CH₂-OH or-F instead of (relocated with) performing the invention, preferably a 2 'O-methyl or 2' O-methoxyethyl modified nucleic acid oligomer. The nucleobase need not be linked to a sugar; so-called peptide nucleic acid oligomers or morpholine-based oligomers may be used. In Sazani, p.et al, 2001, nucleic acids res.29: a comparison of these different chemical ligation processes is found in 3695. The term splice switching oligonucleotide is intended to include such forms. The relationship between antisense oligonucleotide gapmers and SSOs can be understood by those skilled in the art. Gapmers are AS's that contain an RNAse H activation region, typically a 2' -deoxyribonucleoside phosphorothioate (deoxyriboside)ON, wherein the activated region is flanked by nuclease resistant oligomers that are not activated. In general, any chemistry suitable for the flanking sequences in gapperason can be used in SSO.

The SSOs of the present invention can be prepared by well-known solid phase synthesis techniques. Any other means suitable for such synthesis may additionally or alternatively be used. It is known that oligonucleotides can be prepared using similar methods, such as phosphorothioates and alkyl derivatives.

The bases of the SSO can be conventional cytosine, guanine, adenine and uracil or thymidine. Alternatively, modified bases may be used. Of particular interest are modified bases with increased binding affinity. Non-limiting examples of preferred modified bases are nucleotides known as g-clamp or 9- (aminoethoxy) phenoxazine (phenoxazine), cytosine analogs that form 4 hydrogen bonds with guanosine. (Flanagan, W.M., et al, 1999, proc.Natl.Acad.Sci.96: 3513; Holmes, S.C, 2003, Nucleic Acids Res.31: 2759). Specific examples of other bases include, but are not limited to, 5-methylcytosine (C: (C))^MeC) Isocytosine, pseudocytosine, 5-bromouracil, 5-propynyluracil (propyluracil), 5-propynyl (propylny) -6, 5-methylthiazoluracils, 6-aminopurine, 2-aminopurine, inosine, 2, 6-diaminopurine, 7-propynyl-7-deazaadenine (deazaadenine), 7-propynyl (propylne) -7-deazaguanine (deazaguanine), and 2-chloro 6-aminopurine.

When LNA nucleotides are used in SSO, it is preferred that non-LNA nucleotides are also present. LNA nucleotides have such a high affinity in hybridizations with very pronounced non-specific binding that the effective concentration of free SSO can be reduced. When LNA nucleotides are used, they can also be conveniently replaced (alternate) with 2' -deoxynucleotides. Alternative (hybridizing) nucleotides, alternative dinucleotides or mixtures thereof may be used, for example LDLDLD, LDDLDD or LLDLLD. For example, in one embodiment, the nucleotide comprises a nucleotide sequence selected from the group consisting of LDLDDLLDDLDLDLL, LDLDLLLDDLLLDLL, LMLMMLLMMLMLMLL, LMLMLLLMMLLLMLL, LFLFFLLFFLFLFLL, LFLFLLLFFLLLFLL, LDDLDDLDDL, DLDDLDDLDD, DDLDDLDDLD, LMMLMMLMML, MLMMLMMLMM, MMLMMLMMLM, LFFLFFLFFL, FLFFLFFLFF, FFLFFLFFLF, DLDLDLDLDL, LDLDLDLDL, MLMLMLMLML, LMLMLMLML, FLFLFLFLFL, LFLFLFLFL, wherein L is an LNA unit, D is a DNA unit, M is 2 'Moe, and F is 2' fluoro.

When 2 '-deoxynucleotides or 2' -deoxynucleoside phosphorothioates are mixed with LNA nucleotides, it is important to avoid RNAse H activation. LNA nucleotides for SSO are expected to be between about one-third and two-thirds suitable. When affinity enhancing modifications are used, including but not limited to LNA or g-clamp nucleotides, one skilled in the art will recognize that it is necessary to increase the proportion of such affinity enhancing modifications.

A number of alternative chemistries that do not activate RNAse H can be used. For example: suitable SSOs can be oligonucleotides in which at least one internucleotide bridging phosphate residue is a modified phosphate, such as methyl phosphonate, methyl thiophosphate, phosphomorpholinonate, phosphoperazidate and phosphoramidate.

For example: phosphate residues bridging between nucleotides may be modified one every other (every other) as described. In another non-limiting example, such an SSO is a lower alkyl moiety (e.g., C) wherein at least one nucleotide comprises the 2' position₁-C₄Linear or branched, saturated or unsaturated alkyl groups, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl and isopropyl). For example: every other nucleotide can be modified as described (see column 4 of U.S. Pat. No. 4, 5,976,879). For in vivo use, phosphorothioate linkages are preferred.

The SSO is about 8-30 bases in length. It will be appreciated by those skilled in the art that when chemical modifications are used that increase affinity, the SSO can be shorter, but specificity is still maintained. It will be further appreciated by those skilled in the art that the upper limit of the length of the SSO is limited by the limitations of maintaining specific recognition of the target sequence, avoiding self-hybridization of secondary structures to form the SSO, and access to the cell. These limitations mean that SSOs of increased length (beyond a certain length that (above and beyond) depends on SSO affinity) are often found to be less specific, inactive or poorly active SSOs.

SSOs of the invention include, but are not limited to, SSO modifications that involve chemically linking to the SSO one or more moieties or conjugates that enhance SSO activity, cellular distribution, or cellular uptake. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acids, thioethers such as hexyl-thio-trityl mercaptan (tritylthiol), thiocholesterol, fatty chains such as dodecyl or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium-di-oxy-hexadecyl-rac-glycero-3-h-phosphate compounds (phosphonates), polyamine or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine or hexanamino-carbonyl-hydroxycholesterol moieties.

Not all positions in a given SSO need to be uniformly changed, and indeed more than one of the above-described modifications can be introduced in a single compound, or even a single nucleoside, within an SSOs.

The SSOs can be mixed with, encapsulated, coupled to, or associated with other molecules, molecular structures, or mixtures of compounds, e.g., to form liposomes, receptor-targeted molecules that facilitate absorption, distribution, and/or absorption, oral dosage forms, rectal dosage forms, topical dosage forms, or other dosage forms.

It will be understood by those skilled in the art that cell differentiation includes, but is not limited to, the differentiation of spliceosomes. Thus, the activity of any particular SSO depends on the cell type into which it is introduced. For example, SSOs that are effective in one cell type may not be effective in another cell type.

The methods, oligonucleotides and formulations (formulations) of the invention are also useful in vitro or in vivo tools for investigating gene splicing in humans or animals. Such methods may be practiced by modification of the procedures described herein or those procedures known to those skilled in the art.

The SSOs disclosed herein can be used to treat any condition in which a practitioner intends to limit the signal pathway for tnf action or tnf activation. In particular, the invention is useful for the treatment of inflammatory diseases. In one embodiment, the condition is an inflammatory systemic disease, such as rheumatoid arthritis or psoriatic arthritis. In one embodiment, the disease is inflammatory liver disease. Examples of inflammatory liver diseases include, but are not limited to, hepatitis associated with hepatitis a, b or c virus, alcoholic liver disease and non-alcoholic steatosis. In another embodiment, the inflammatory disease is a skin disease, such as psoriasis.

Complementing RNAseH

The oligomers of the invention do not mediate cleavage of complementary single stranded RNA molecules based on RNAseH. Oligonucleotides that are effective for replenishing RNAseH require at least 5 contiguous stretches of DNA nucleobases (stretch).

EP 1222309 provides an in vitro method for determining RNaseH activity, which can be used to determine the ability to supplement RNaseH. When provided with a complementary RNA target, a compound is considered to be capable of supplementing RNase H if it has an initial rate in pmol/L/min of at least 1%, such as at least 5%, such as at least 10% or less than 20% of that of an equivalent DNA oligonucleotide, which does not have a 2' site substitution, all nucleotides in the oligonucleotide being phosphodiester linked, as determined using the method provided in examples 91-95 of EP 1222309.

When provided with a complementary RNA target and RNaseH, as determined using the methods provided in examples 91-95 of EP 1222309, a compound is considered to be substantially not complementing RNase H if its initial rate of RNaseH in pmol/L/min is less than 20%, such as less than 10%, such as less than 5% or preferably less than 0.1% (even less than 0.1%) of the initial rate determined using an equivalent DNA oligonucleotide which does not have a 2' site substitution and which has a phosphodiester linker between all nucleotides in the oligonucleotide.

Nucleotide analogs

When referring to a preferred nucleotide sequence motif or nucleotide sequence consisting of only nucleotides, it will be appreciated that an oligomer of the invention defined by that sequence may comprise a corresponding nucleotide analogue, such as LNA units or other nucleotide analogues, in place of one or more of the nucleotides present in the sequence, which nucleotide analogues increase duplex stability/Tm (i.e. affinity enhancing nucleotide analogues) of the oligomer/target duplex.

In addition, the nucleotide analogs can enhance oligomer stability in vivo.

In one embodiment, the nucleotide analogues (X) are independently selected from the group consisting of 2 ' -oxy-alkyl-RNA units, 2 ' -OMe-RNA units, 2 ' -MOE RNA units, 2 ' -amino-DNA units, 2 ' -fluoro-DNA units, LNA units, PNA units, HNA units and INA units.

In one embodiment, the contiguous nucleobase sequence does not comprise a 2 'OMe ribonucleotide analog or a 2' -MOE ribonucleotide analog.

In one embodiment, the nucleotide analog is 2 'MOE, i.e., 2' oxy-2 methoxyethyl RNA. Thus, in one embodiment, reference is made herein to X in the nucleobase motif²Or M may be MOE.

Incorporation of affinity-enhancing nucleotide analogues, such as LNA or T-substituted sugars, into the oligomer may allow for a reduction in the size of the specifically bound oligomer, and may also reduce the upper limit on the size of the oligomer before non-specific or aberrant binding occurs.

It is also suitable that the nucleobase sequence or contiguous nucleobase sequence of the oligomer is not fully complementary to the corresponding region of the TNFR target sequence, and in one embodiment, when the oligomer comprises an affinity enhancing nucleotide analog, such nucleotide analog forms a complement with the corresponding nucleotide in the TNFR target.

Thus the oligomer may comprise or consist of a simple sequence of natural nucleotides, preferably 2' -deoxynucleotides (generally referred to herein as "DNA"), but possibly also ribonucleotides (generally referred to herein as "RNA"), or it may comprise (and possibly consist entirely of) one or more nucleotide "analogues".

Nucleotide analogs are variants of natural DNA or RNA nucleotides resulting from modifications of sugar, and/or base and/or phosphate moieties. The term "nucleobase" includes natural nucleotides (of the DNA or RNA type), as well as such "analogs" thereof. In principle an analogue may be simply "silent", or "identical" to a natural nucleotide in the context of an oligonucleotide, i.e.have no functional effect on the way in which the oligonucleotide inhibits the expression of β -catenin. Such "equivalent" analogs are useful in any event if, for example, they are easier or cheaper to manufacture, or more stable during storage or manufacture, or carry a label or tag. However, those analogs are preferred which have a functional effect on the mode of action of the oligomer in inhibiting expression; for example, analogs that affect by increasing binding affinity to a target, increasing resistance to intracellular nucleases, and/or increasing ease of transport into a cell.

Examples of such nucleotide modifications include altering the sugar moiety to provide a 2' -substituent or to create bridging (locked nucleic acid) structures that enhance binding affinity and possibly provide some enhanced nuclease resistance; the internucleotide linkage is changed from the normal phosphodiester bond to a linkage more resistant to nuclease attack, such as phosphorothioate or boranophosphate (boranophosphate).

Preferred nucleotide analogues are LNAs, such as beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, with beta-D-oxy-LNA being most preferred.

In some embodiments, the oligomer comprises 3-8 nucleotide analogs, such as 6 or 7 nucleotide analogs. In a most preferred embodiment so far, at least one of said nucleotide analogues is a Locked Nucleic Acid (LNA); for example, at least 3 or at least 4, or at least 5, or at least 6, or at least 7 or 8 nucleotide analogues are LNA. In some embodiments, all of the nucleotide analogs are LNAs.

In some embodiments, the nucleotide analogues present inside the oligomer of the invention are independently selected from, for example, 2 ' -oxy-alkyl-RNA units, 2 ' -amino-DNA units, 2 ' -fluoro-DNA units, LNA units, Arabinose Nucleic Acid (ANA) units, 2 ' -fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units and 2 ' -MOE RNA units.

2 ' -O-methoxyethyl-RNA (2 ' MOE), 2 ' -fluoro-DNA monomer and LNA are preferred nucleotide analogues, and thus the oligonucleotide of the invention may comprise nucleotide analogues independently selected from these three analogues, or may comprise only one analogue selected from these three.

The oligomer according to the invention preferably comprises at least one Locked Nucleic Acid (LNA) unit, such as 1, 2,3, 4, 5,6, 7 or 8 LNA units, preferably 4-8 LNA units, most preferably 4, 5 or 6LNA units. The oligomer comprises β -D-oxy-LNA and one or more of the following LNA units: thio-LNAs, amino-LNAs, oxy-LNAs, ena-LNAs and/or alpha-LNAs of the D-beta, L-alpha configuration or combinations thereof are suitable.

In one embodiment of the invention, the oligomer may comprise LNA and DNA units. The total number of LNA and DNA unit combinations is preferably 8-24, such as 8-15, or 10-25, or 10-20 or 12-16.

In one embodiment of the invention, the nucleobase sequence of the oligomer, e.g. the contiguous nucleobase sequence, consists of at least one LNA, the remaining nucleobase units being DNA units.

In some embodiments of the oligomer according to the invention, e.g. an antisense oligonucleotide comprising LNA, all LNA C units are 5-methylcytosine. In some embodiments, all of the nucleotide analogs are LNAs.

In a most preferred embodiment, the oligomer comprises only LNA nucleotide analogues and nucleotides (RNA or DNA, most preferably DNA nucleotides, with optionally modified inter-nucleobase linkages, such as phosphorothioates).

In some embodiments, at least one of the nucleotide analogs is a 2 '-MOE-RNA, e.g., 2,3, 4, 5,6, 7, or 8 2' -MOE-RNA nucleobase units.

In some embodiments, at least one of the nucleotide analogs is 2 '-fluoro-DNA, e.g., 2,3, 4, 5,6, 7, or 8 2' fluoro-DNA nucleobase units.

Freeer & Altmann; nucleic acids res, 1997, 25, 4429-; current in Drug Development, 2000, 3(2), 293-213, and outline 1 describe specific examples of nucleoside analogs.

Sketch 1

The term "LNA" refers to a bicyclic nucleotide analog referred to as a "locked nucleic acid". It may refer to LNA monomers and when used in the context of "LNA oligonucleotides" may refer to oligonucleotides comprising one or more such bicyclic nucleotide analogues.

A particularly preferred chemistry is provided by Locked Nucleic Acids (LNA) (Koshkin, A.A., et al., 1998, Tetrahedron 54: 3607; Obika, S.et al., 1998, Tetrahedron Lett.39: 5401). The terms "LNA unit", "LNA monomer", "LNA residue", "locked nucleic acid unit", "locked nucleic acid monomer" or "locked nucleic acid residue" as used herein refer to bicyclic nucleoside analogs. LNA units and methods for their synthesis are described in WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO 03/095467. LNA units can also be defined according to a chemical formula. Thus, the "LNA unit" as used herein has the chemical structure shown in formula 1 below.

Formula 1

Wherein

X is selected from the group consisting of O, S and NRH, R is H or C₁-C₄An alkyl group;

y is (-CH)₂)_rR is an integer from 1 to 4;

b is a base of natural or unnatural origin as described above.

In a preferred embodiment, r is 1 or 2, and in a more preferred embodiment, r is 1.

The LNA used in the oligonucleotide compound of the present invention preferably has a structure represented by the following general formula

Wherein X and Y are independently selected from the group consisting of-O-, -S-, -N (H) -, N (R) -, -CH₂-or-CH- (if part of a double bond), -CH₂-O-、-CH₂-S-、-CH₂-N(H)-、-CH₂-N(R)-、-CH₂-CH₂-or-CH₂-CH- (if part of a double bond), -CH ═ CH-, wherein R is selected from hydrogen and C_1-4-an alkyl group; z and Z^*Independently selected from an internucleotide linkage, a terminal group or a protecting group; b is natural or non-naturalA nucleotide base portion; asymmetric groups can be found in either of two directions.

The LNA in the oligomer for use in the present invention preferably comprises at least one LNA unit corresponding to any one of the following formulae

Wherein Y is-O-, -S-, -N (H) -, N (R)^H) -; z and Z^*Independently selected from an internucleotide linkage, a terminal group or a protecting group; b constitutes a natural or non-natural nucleotide base moiety, R^HSelected from hydrogen and C_1-4-an alkyl group.

The LNA used in the oligomer of the invention preferably comprises an amino acid selected from the group consisting of-O-P (O)₂-O-，-O-P(O，S)-O-，-O-P(S)₂-O-，-S-P(O)₂-O-，-S-P(O，S)-O-，-S-P(S)₂-O-，-O-P(O)₂-S-，-O-P(O，S)-S-，-S-P(O)₂-S-，-O-PO(R^H)-O-，O-PO(OCH₃)-O-，-O-PO(NR^H)-O-，-O-PO(OCH₂CH₂S-R)-O-，-O-PO(BH₃)-O-，-O-PO(NHR^H)-O-，-O-P(O)₂-NR^H-，-NR^H-P(O)₂-O-，-NR^H-CO-O-, wherein R^HSelected from hydrogen and C_1-4An alkyl group.

Specific preferred LNA units are shown in fig. 2:

beta-D-amino acid-LNA

Sketch (scheme)2

The term "thio-LNA" encompasses compounds wherein at least one of X or Y in the above formula is selected from S or-CH₂of-S-)The nucleotide is locked. The thioalcos may be in the β -D and α -L configurations.

The term "amino-LNA" encompasses those in which at least one of X or Y in the above formula is selected from the group consisting of-N (H) -, N (R) -, CH₂Locked nucleotides of-N (H) -and-CH 2-N (R) -wherein R is selected from the group consisting of hydrogen and C_1-4-an alkyl group. The amino-LNA may be in the beta-D and alpha-L configurations.

The term "oxy-LNA" encompasses compounds in which at least one of X or Y in the above formula represents-O-or-CH₂-locked nucleotides of O-. The oxygen LNA may be in the beta-D and alpha-L configurations.

The term "ena-LNA" encompasses the compounds wherein Y in the above formula is-CH₂-O- (wherein-CH)₂The oxygen atom of O-is attached to the 2' -position opposite to the base B).

In a preferred embodiment the LNA is selected from the group consisting of beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio, in particular beta-D-oxy-LNA.

The oligomer according to the invention preferably comprises at least one nucleotide analogue, e.g. a Locked Nucleic Acid (LNA) unit, such as 1, 2,3, 4, 5,6, 7, 8, 9 or 10 nucleotide analogue, e.g. a Locked Nucleic Acid (LNA) unit, preferably 3-9 nucleotide analogues, e.g. an LNA unit, such as 4-8 nucleotide analogues, e.g. an LNA unit, such as 6-9 nucleotide analogues, e.g. an LNA unit, preferably 6, 7 or 8 nucleotide analogues, e.g. an LNA unit.

The oligomer according to the invention, e.g. an antisense oligonucleotide, may comprise 1, 2,3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide analogues, e.g. LNA units, in particular 3, 4, 5,6, 7, 8, 9 or 10 nucleotide analogues, e.g. LNA units.

Preferred LNA units comprise at least one β -D-oxy-LNA unit, for example 2,3, 4, 5,6, 7, 8, 9 or 10 β -D-oxy-LNA units.

Oligomers of the invention, such as antisense oligonucleotides, may comprise more than one LNA unit. The compounds comprise β -D-oxy-LNA and one or more of the following LNA units: thio-LNAs, amino-LNAs, oxy-LNAs, ena-LNAs and/or alpha-LNAs of the D-beta, L-alpha configuration or combinations thereof are suitable.

Preferred oligomers, such as antisense oligonucleotides, may comprise or consist of nucleotide analogues, such as LNA and DNA units.

LNA and DNA are preferred, but MOE, 2 '-O-Me, and other 2' substituted analogs and RNA may also be used.

Preferred DNA analogs include those in which 2' -H is replaced by a group other than OH (RNA), for example by-O-CH 3, -O-CH2-CH2-O-CH3, -O-CH2-CH2-CH2-NH2, -O-CH2-CH2-CH2-OH or-F.

Preferred RNA analogs include those in which the 2' -OH group has been altered, for example by a group other than-H (DNA), -O-CH3, -O-CH2-CH2-O-CH3, -O-CH2-CH2-CH2-NH2, -O-CH2-CH2-CH2-OH or-F.

In one embodiment, the nucleotide analog is "ENA".

In one embodiment, the oligomer of the invention does not comprise any RNA units.

High affinity nucleotide analogs are nucleotide analogs that yield oligonucleotides that form duplexes with complementary RNA nucleotides that have higher thermal stability than the binding affinity of an equivalent DNA nucleotide. Typically by measuring T_mTo determine this.

Nucleotide analogs that increase the Tm of the oligomer/target nucleic acid target are preferred (affinity-enhancing nucleotide analogs) over equivalent nucleotides. The oligomer can suitably hybridize to a target nucleic acid, e.g., TNFR mRNA, forming a duplex having a Tm of at least 30 ℃, e.g., 37 ℃, e.g., at least 40 ℃, at least 50 ℃, at least 55 ℃, or at least 60 ℃. For example, in one embodiment, the Tm is between 30-80 ℃, e.g., between 40-70 ℃.

In one embodiment, at least 30%, such as at least 33%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 66%, such as at least 70%, such as at least 80%, such as at least 90% of the nucleobases of the oligomer of the invention are nucleotide analogue nucleobases, such as LNA. In one embodiment, all nucleobases of the oligomer of the invention are nucleotide analogue nucleobases, such as LNA.

It can be appreciated that for shorter oligonucleotides, it may be desirable to increase the proportion of (high affinity) nucleotide analogues, such as LNA.

The term "oligonucleotide (or simply" oligo ") used interchangeably with the term" oligomer "in the context of the present invention refers to a molecule formed by a covalent bond of two or more nucleobases. When used in the context of an oligonucleotide (also referred to as a single stranded oligonucleotide) of the invention, the term "oligonucleotide" may in one embodiment have, for example, 8 to 26 nucleobases, such as 12 to 26 nucleobases. In the preferred embodiments detailed herein, the oligonucleotides of the invention have a length of 10-16 nucleobases or 8-15 nucleobases.

Variation of oligomer Length

The length of the oligonucleotides of the invention may vary. Even shorter oligonucleotides, such as 10-17 or 10-15 bases, are considered more advantageous.

In such embodiments, the oligonucleotides of the invention have a length of 10, 11, 12, 13, 14, 15 or 16 nucleobases.

In one embodiment, the oligonucleotide according to the invention is 8-24 nucleobases in length, such as between 10-24, 12-24 nucleotides, such as between 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length, preferably 10-22, such as between 12-22 nucleotides in length, such as between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides in length, more preferably 10-20, such as between 12-20 nucleotides in length, even more preferably 10-19, such as between 12-19 nucleotides in length, such as between 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length, such as between 10-18, such as between 12-18 nucleotides in length, for example 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length, more preferably 10-17, for example 12-17 nucleotides in length, for example 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides in length, most preferably 10-16, for example 12-16 nucleotides in length, for example 10, 11, 12, 13, 14, 15 or 16 nucleotides in length.

Internucleotide linking group

The term "internucleotide linkage" is used to refer to a group capable of covalently linking two nucleobases together, e.g., between DNA units, between a DNA unit and a nucleotide analog, between two non-LNA units, between a non-LNA unit and an LNA unit, between two LNA units, and the like. Preferred examples include phosphate, phosphodiester groups and phosphorothioate groups.

The internucleotide linking group may be selected from the group consisting of: -O-P (O)2-O-, -O-P (O, S) -O-, -O-P (S)2-O, -S-P (O)2-O, -S-P (CS) -O-, -S-P (S)2-O, -O-P (O)2-S-, -O-P (CS) -S-, -S-P (O)2-S-, -O-PO (RH) -O-, O-PO (OCH3) -O-, -O-PO (NRH) -O-, -O-PO (OCH2CH2S-R) -O-, -O-PO (BH3) -O-, -O-PO (NHRH) -O-, -O-P (O)2-NRH-, -NRH-P (O)2-O-, -NRH-CO-O-, -NRH-CO-NRH-, and/or internucleotide linkages may be selected from the group consisting of: -O-CO-O-, -O-CO-NRH-, -NRH-CO-CH2-, -O-CH2-CO-NRH-, -O-CH2-CH2-NRH-, -CO-NRH-CH2-, -CH2-NRH-CO-, -O-CH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NRH-, -O-CH2-CH2-NRH-CO-, -CH2-NCH3-O-CH2-, wherein RH is selected from hydrogen and C1-4 alkyl. Suitably, in some embodiments, the internucleotide linkages comprising sulfur provided above are preferred.

Modification of internucleotide linkages

The internucleotide linkages in the oligonucleotide are phosphate groups, but these phosphate groups may be replaced with internucleotide linkages other than phosphate. In an interesting further embodiment of the invention, the structure of the internucleotide linkage of the oligonucleotide of the invention is altered, i.e. the modified oligonucleotide comprises an internucleotide linkage different from phosphate. Thus, in a preferred embodiment, the oligonucleotide of the invention comprises at least one internucleotide linkage group different from phosphate.

Specific examples of internucleotide linkages other than phosphate (-O-P (O)2-O-) include-O-P (O, S) -O-, -O-P (S)2-O-, -S-P (O, S) -O-, -S-P (S)2-O-, -O-P (O)2-S-, -O-P (O, S) -S-, -S-P (O)2-S-, -O-PO (RH) -O-, O-PO (OCH3) -O-, -O-PO (NRH) -O-, -O-PO (OCH2CH2S-R) -O-, -O-PO (BH3) -O-, -O-PO (NHRH) -O-, -O-P (O)2-NRH-, -NRH-P (O)2-O-, -NRH-CO-O-, -NRH-CO-NRH-, -O-CO-O-, -O-CO-NRH-, -NRH-CO-CH2-, -O-CH2-CO-NRH-, -O-CH2-CH2-NRH-, -CO-NRH-CH2-, -CH2-NRH-CO-, -O-CH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NRH-, -O-CH2-CH2-NRH-CO-, -CH2-NCH3-O-CH2, wherein RH is hydrogen or C1-4-alkyl.

When the internucleotide linkage group is modified, the preferred internucleotide linkage group is a phosphorothioate group (-O-P (O, S) -O-). In a preferred embodiment, all internucleotide linkages of the oligonucleotide according to the invention are phosphorothioate.

Complete phosphorothioate formation of the oligonucleotide is preferred in most therapeutic applications, except for therapeutic oligonucleotides used in the CNS, e.g. the brain or spine (spine) where phosphorothioate formation may be toxic, because of the lack of nucleases, phosphodiester bonds may even be used between adjacent DNA units.

In one embodiment, the oligomer comprises alternating LNA and DNA units (Xx) or (Xx).

In one embodiment, the oligomer comprises an alternating LNA motif (Xxx), xXx, or xxX followed by 2 DNA units.

In one embodiment, at least one DNA or non-LNA nucleotide analogue unit is substituted with an LNA nucleobase at a position selected from any one of the embodiments described above determined to be an LNA nucleobase unit.

In one embodiment, "X" represents an LNA unit.

In one embodiment, the oligomer comprises at least 3 nucleotide analogue units, such as at least 4 nucleotide analogue units, such as at least 5 nucleotide analogue units, such as at least 6 nucleotide analogue units, such as at least 7 nucleotide analogue units, such as at least 8 nucleotide analogue units, such as at least 9 nucleotide analogue units, such as at least 10, such as at least 11, such as at least 12 nucleotide analogue units.

In one embodiment, the oligomer comprises at least 3 LNA units, such as at least 4 LNA units, such as at least 5 LNA units, such as at least 6LNA units, such as at least 7 LNA units, such as at least 8 LNA units, such as at least 9 LNA units, such as at least 10 LNA units, such as at least 11 LNA units, such as at least 12 LNA units.

In one embodiment, at least one of the nucleotide analogues, e.g. LNA units, is cytosine or guanine, e.g. a nucleotide analogue, e.g. 1-10 of the LNA units are cytosine or guanine, e.g. a nucleotide analogue, e.g. 2,3, 4, 5,6, 7, 8 or 9 of the LNA units are cytosine or guanine.

In one embodiment, the at least two nucleotide analogues, e.g. LNA units, are cytosine or guanine. In one embodiment, the at least three nucleotide analogues, e.g. LNA units, are cytosine or guanine. In one embodiment, the at least four nucleotide analogues, e.g. LNA units, are cytosine or guanine. In one embodiment, at least five nucleotide analogues, e.g. LNA units, are cytosine or guanine. In one embodiment, the at least six nucleotide analogues, e.g. LNA units, are cytosine or guanine. In one embodiment, the at least seven nucleotide analogue, e.g. the LNA unit, is cytosine or guanine. In one embodiment, at least eight nucleotide analogues, e.g., LNA units, are cytosine or guanine.

In a preferred embodiment, the nucleotide analog forms a duplex with a complementary RNA nucleotide having a higher thermostability compared to the binding affinity of an equivalent DNA nucleotide to the complementary RNA nucleotide.

In one embodiment, the nucleotide analogs confer enhanced serum stability to the single-stranded oligonucleotide.

Further design of the oligomers of the invention

In one embodiment, the first nucleobase of the oligomer according to the invention, counted from the 3' end, is a nucleotide analogue, e.g. a LNA unit.

In one embodiment, the second nucleobase of the oligomer according to the invention, counted from the 3' end, is a nucleotide analogue, such as a LNA unit.

In one embodiment, "x" represents a unit of DNA.

In one embodiment, the oligomer comprises a nucleotide analogue unit, such as an LNA unit, at the 5' end. In one embodiment, the nucleotide analogue units, e.g. X, are independently selected from the group consisting of 2 ' -oxy-alkyl-RNA units, 2 ' -OMe-RNA units, 2 ' -amino-DNA units, 2 ' -fluoro-DNA units, 2 ' -MOE RNA units, LNA units, PNA units, HNA units and INA units.

In one embodiment, all nucleobases of the oligomer of the invention are nucleotide analogue units.

In one embodiment, the nucleotide analogue units, such as X, are independently selected from the group consisting of 2 '-OMe-RNA units, 2' -fluoro-DNA units and LNA.

In one embodiment, the oligomer comprises said at least one LNA analog unit and at least one further nucleotide analog unit different from LNA.

In one embodiment, the non-LNA nucleotide analogue units are independently selected from 2 '-OMe RNA units and 2' -fluoro-DNA units.

In one embodiment the oligomer consists of X¹X²X¹Or X²X¹X²Wherein X is¹Is LNA, X²Is a 2 '-OMe RNA unit or a 2' -fluoro-DNA unit.

In one embodiment, the nucleobase sequence of the oligomer consists of alternating xs¹And X²And (4) unit composition.

In one embodiment, the oligomer according to the invention does not comprise a region of more than 5 contiguous DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 6 contiguous DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 7 contiguous DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 8 contiguous DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 3 contiguous DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 2 contiguous DNA nucleotide units.

In one embodiment, the oligomer comprises at least one region consisting of at least two contiguous nucleotide analogue units, for example at least two contiguous LNA units.

In one embodiment, the oligomer comprises at least one region consisting of at least three contiguous nucleotide analogue units, for example at least three contiguous LNA units.

In one embodiment, the oligomer according to the invention does not comprise a region of more than 7 contiguous nucleotide analogue units, such as LNA units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 6 contiguous nucleotide analogue units, such as LNA units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 5 contiguous nucleotide analogue units, such as LNA units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 4 contiguous nucleotide analogue units, such as LNA units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 3 contiguous nucleotide analogue units, such as LNA units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 2 contiguous nucleotide analogue units, such as LNA units.

In one embodiment, the oligonucleotide of the invention comprises at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or e.g. 100% of the nucleobase units of the oligomer (preferably with high affinity) are nucleotide analogues, such as Locked Nucleic Acid (LNA) nucleobase units,

tables 3 and 4 below provide non-limiting examples of short microRNA sequences that can be conveniently targeted by the oligonucleotides of the invention.

In one embodiment, the oligonucleotide according to the invention has a 5 '-3' oriented nucleobase sequence selected from the group consisting of the following motifs:

LxLxxLLxxLL

LxLxLLLxxLL

LxxLxxLxxL

xLxxLxxLxx 'three each' (third)

xxLxxLxxLxxLx 'three each'

xLxLxLxL 'two each' (second)

LXRLXL two's per two'

Ld Ld d LLd d LL

LdLdLLLddLLL

LMLMMLLMMLL

LMLMLLLMMLL

LFLFFLLFFLL

LFLFLLLFFLLL

LLLLLL

LLLLLLL

LLLLLLLL

LLLLLLLLL

LLLLLLLLLL

LLLLLLLLLLL

LLLLLLLLLLLL

LMMLMMLMML

MLMMLMMLMM 'every three'

MMLMMLMMLM 'every three'

LFFLFFLFFL 'every three'

FLFFLFFLFF 'every three'

FFLFFLFFLF 'every three'

dLdLdLdLdL 'two per'

LdLdLdLdL 'two per'

MLMLMLMLML 'two per'

LMLMLMLML 'two per'

FLFLFLFLFL 'two per'

LFLFLFLFL 'two per'

Ld Ld d LLd d Ld Ld LL

LdLdLLLddLLLdLL

LMLMMLLMMLMLMLL

LMLMLLLMMLLLMLL

LFLFFLLFFLFLFLL

LFLFLLLFFLLLFLL

LddLddLddL(d)(d)(L)(d)(d)(L)(d)

dLddLddLdd(L)(d)(d)(L)(d)(d)(L)

ddLddLddLd(d)(L)(d)(d)(L)(d)(d)

LMMLMMLMML(M)(M)(L)(M)(M)(L)(M)

MLMMLMMLMM(L)(M)(M)(L)(M)(M)(L)

MMLMMLMMLM(M)(L)(M)(M)(L)(M)(M)

LFFLFFLFFL(F)(F)(L)(F)(F)(L)(F)

FLFFLFFLFF(L)(F)(F)(L)(F)(F)(L)

FFLFFLFFLF(F)(L)(F)(F)(L)(F)(F)

dLdLdLdLdL(d)(L)(d)(L)(d)(L)(d)

LdLdLdLdL(d)(L)(d)(L)(d)(L)(d)(L)

MLMLMLMLML(M)(L)(M)(L)(M)(L)(M)

LMLMLMLML(M)(L)(M)(L)(M)(L)(M)(L)

FLFLFLFLFL(F)(L)(F)(L)(F)(L)(F)

LFLFLFLFL(F)(L)(F)(L)(F)(L)(F)(L)

Where L ═ LNA units, D ═ DNA units, M ═ 2 'MOE RNA, F ═ 2' fluoro, 'x' is a form defined herein. It will be appreciated that the above pattern may be repeated for longer oligomers, and that for shorter oligomers, the corresponding portions of the above motifs may be used from the 5 'end or the 3' end, with the residues in parentheses being optional.

In one embodiment, the invention further provides an oligomer wherein the oligomer (or contiguous nucleobase sequence) comprises at least one phosphorothioate linkage, and/or at least one 3 'terminal LNA unit, and/or at least one 5' terminal LNA unit.

Protein

The present invention still further provides an isolated or purified soluble form of a TNF α receptor comprising a deletion in the transmembrane binding domain encoded by exon 7, wherein said TNF α receptor is selected from the group consisting of TNF α receptor TNFRSF1A or TNFRSF1B, variants, fragments or homologs thereof.

In one embodiment, an isolated or purified soluble form of a TNF α receptor according to the present invention lacks the transmembrane binding domain encoded by exon 7.

In one embodiment, the isolated or purified soluble form of the TNF α receptor is human TNFR1TNF α receptor (residues 1-455 of SEQ ID NO 123, residues 30-455, variants, fragments or homologs thereof), wherein the deletion is between residues 209 and 246 (or the region corresponding to residues 209 and 246 of SEQ ID NO 123).

In one embodiment, the isolated or purified soluble form of TNF α has a sequence consisting of residues 1-208, residues 30-208 of SEQ ID NO119, variants, fragments or homologs thereof.

In one embodiment, the isolated or purified soluble form of the TNF α receptor is human TNFR2TNF α receptor (residues 1-435 of SEQ ID NO 127, residues 23-435, variants, fragments, or homologs thereof, wherein the deletion is between residues 263 and 289 (or a region corresponding to residues 209 and 246 of SEQ ID NO 123).

In one embodiment, the isolated or purified soluble form of the TNF α receptor has a sequence consisting of residues 1-262 or 23-262 of SEQ id no 127, or a variant, fragment or homolog thereof.

In a preferred embodiment, the soluble form of the TNF α receptor is isolated and purified.

One embodiment of the present invention is a full-length or mature protein encoded by a cDNA derived from a mammalian TNFR gene, wherein exon 6 of the cDNA is directly followed by exon 8, and therefore lacks exon 7. In addition, the protein may bind TNF, preferably TNF-alpha, and may act as an antagonist of TNF, preferably TNF-alpha. TNFRs of the invention are preferably capable of inhibiting TNF mediated cytotoxicity to a greater extent than soluble extracellular domain alone, more preferably to a level comparable to that of TNFR: fc to comparable or exceeding its extent. Mammalian TNFRs disclosed herein include, but are not limited to, human, primate, murine, canine, feline, bovine, ovine, equine, and porcine TNFRs. In addition, the mammalian TNFRs disclosed herein include, but are not limited to, protein sequences produced by one or more single nucleotide polymorphisms, such as those disclosed in EP patent application 1,172,444, so long as the protein retains comparable biological activity to the reference sequence used for comparison.

In one embodiment, the mammalian TNFR is mammalian TNFR1, preferably human TNFR 1. For human TNFR1, as shown in SEQ ID NO: the huTNFR1 Δ 7 comprising a signal sequence and the mature huTNFR1 Δ 7 lacking a signal sequence shown at 122 (amino acids 30-417 of SEQ ID NO: 122) give two non-limiting examples of embodiments. The sequence of these huTNFR1 Δ 7 proteins is that of amino acids 1-208 of wild-type human TNFR1(SEQ ID No: 118) comprising the signal sequence or of mature huTNFR1 Δ 7 lacking the signal sequence, i.e., amino acids 30-208 of wild-type human TNFR1, in both cases directly linked to amino acids 247-455 of wild-type human TNFR 1.

In another preferred embodiment, the mammalian TNFR is mammalian TNFR2, most preferably human TNFR 2. For human TNFR2, as shown in SEQ ID NO: the huTNFR2 Δ 7 comprising a signal sequence or mature huTNFR2 Δ 7 lacking a signal sequence shown at 126 (amino acids 23-435 of SEQ ID NO: 126) give two non-limiting examples of embodiments. The sequences of these huTNFR2 Δ 7 proteins are the sequences of amino acids 1-262 of wild-type human TNFR2(SEQ ID No: 120) comprising the signal sequence or of mature huTNFR2 Δ 7 lacking the signal sequence, i.e., amino acids 23-262 of wild-type human TNFR2, both of which are followed by the amino acid glutamic acid directly due to the formation of a unique codon at the junction of exons 6-8, and the amino acid 290-461 of wild-type human TNFR 2.

The proteins of the present invention also include those that are chemically modified. Chemical modification of a protein refers to a protein in which at least one amino acid residue is altered by natural processes, such as processing or other post-translational modifications, or by chemical modification methods known in the art. Such modifications include, but are not limited to, acetylation, acylation, amidation, ADP ribosylation, glycosylation, methylation, pegylation, prenylation, phosphorylation, or coupling to cholesterol.

In one embodiment, the proteins of the invention may also include variants, fragments and homologs of the proteins of the invention. However, this protein contains a deletion in the amino acid sequence encoded by exon 7 or exon 8 as explained herein.

Nucleic acids

The invention further provides nucleic acids encoding soluble forms of the TNF α receptors of the invention.

In one embodiment, the nucleic acid is selected from the group consisting of: nucleotides 1-1251 of SEQ ID NO 121, 88-1251 of SEQ ID NO 121, 1-1305 of SEQ ID NO 125 and 67-1305 of SEQ ID NO 125, or variants, homologs or fragments thereof, including nucleic acids which encode the same original amino acid sequence as the nucleic acid due to the degeneracy of the genetic code.

One embodiment of the invention is a nucleic acid encoding a full-length or mature protein encoded by a cDNA derived from a mammalian TNFR gene, wherein exon 6 of the cDNA is directly followed by exon 8, and therefore lacks exon 7.

Such sequences are preferably provided in a form in which the open reading frame is not interrupted by internal untranslated sequences or introns typically present in eukaryotic genes. Genomic DNA containing related sequences may also be used. In one embodiment, the nucleic acid is mRNA or cDNA. In another embodiment, it is genomic DNA.

In one embodiment, the mammalian TNFR is mammalian TNFR 1. Mammalian TNFR1 suitable for this embodiment is preferably human TNFR 1. For human TNFR1, as shown in SEQ ID NO: 122 encoding huTNFR1 Δ 7 and mature huTNFR1 Δ 7 lacking a signal sequence (amino acids 30-417 of SEQ ID NO: 122) give two non-limiting examples of embodiments. The sequence of these huTNFR1 Δ 7 nucleic acids is preferably SEQ ID NO: 121 and nucleotides 1-1251 of SEQ ID NO: 121, nucleotides 88-1251. The sequence of these huTNFR1 Δ 7 nucleic acids is nucleotides 1-625 of wild-type human TNFR1(SEQ ID NO: 117) or nucleotides 88-625 of wild-type human TNFR1, i.e., mature huTNFR2 Δ 7 lacking the signal sequence, which in both cases is directly followed by nucleotide 740-1368 of wild-type human TNFR 1.

In another preferred embodiment, the mammalian TNFR is mammalian TNFR2, most preferably human TNFR 2. For human TNFR2, as shown in SEQ ID NO: 126 encoding huTNFR2 Δ 7 or mature huTNFR2 Δ 7 lacking a signal sequence (amino acids 23-435 of SEQ ID NO: 126) to give two non-limiting examples of embodiments. The sequence of these huTNFR2 Δ 7 nucleic acids is preferably SEQ ID NO: 115 and SEQ ID NO: 115, nucleotides 67-1305. The sequences of these huTNFR2 Δ 7 nucleic acids are nucleotides 1-787 of wild-type human TNFR2(SEQ ID No: 119) or nucleotides 67-787 of wild-type human TNFR2, i.e., mature huTNFR2 Δ 7 lacking the signal sequence, which in both cases are directly followed by amino acids 866-1386 of wild-type human TNFR 2.

The bases of the nucleic acids of the invention may be conventional cytosine, guanine, adenine and uracil or thymine deoxynucleoside bases. Alternatively, modified bases may be used. Other suitable bases include, but are not limited to, 5-methylcytosine (C^MeC) Isocytosine, pseudocytosine, 5-bromouracil, 5-propynyluracil, 5-propyne-6, 5-methylthiazolyluracil, 6-aminopurine, 2-aminopurine, inosine, 2, 6-diaminopurine, 7-propyne-7-deazaadenine (deazaadenine), 7-propyne-7-deazaguanine (deazaguanine), 2-chloro (aminoethoxy) -6-aminopurine and 9- (aminoethoxy) phenoxazine.

Suitable nucleic acids of the invention include a number of selective chemical changes. For example: suitable nucleic acids of the invention include, but are not limited to, nucleic acids in which at least one internucleotide bridging phosphate residue is a modified phosphate, such as phosphorothioate, methylphosphonate, methylphosphonothioate, phosphoromorpholate, phosphoropiperazidate and phosphoramidate (phosphoroamidate). In another non-limiting example, suitable nucleic acids of the invention include those wherein at least one nucleotide comprises a lower alkyl moiety at the 2' position (e.g., C)₁-C₄Linear or branched, saturated or unsaturated alkyl groups, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl and isopropyl).

Nucleic acids of the invention also include, but are not limited to, those in which at least one nucleotide is a nucleic acid analog. Examples of such analogs include, but are not limited to Hexitol (HNA) nucleotides, 2 'O-4' C-linked bicyclic furonitrosourea (LNA) nucleotides, Peptide Nucleic Acid (PNA) analogs, phosphoramidate analogs of N3 '→ P5', phosphorodiamidite morpholino nucleotide analogs, and combinations thereof.

Nucleic acids of the invention include, but are not limited to, nucleic acid modifications involving chemical attachment of one or more moieties or conjugates to the nucleic acid. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acids, thioethers such as hexyl-thio-trityl mercaptan (tritylthiol), thiocholesterol, fatty chains such as dodecyl or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium, 1, 2-di-oxy-hexadecyl-rac-glycerol-thio-H-phosphate compounds, polyamine or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine or hexaneamine-carbonyl-hydroxycholesterol moieties.

Expression vectors and host cells

The invention also provides vectors comprising the nucleic acids of the invention.

In one embodiment, the vector comprises an expression cassette capable of driving expression of said nucleic acid in a host cell.

The invention also provides host cells comprising a nucleic acid or vector of the invention.

The invention also provides a method of producing a soluble form of TNF α receptor, said method comprising the steps of culturing a host cell of the invention under conditions permitting expression of said nucleic acid, and subsequently isolating said soluble form of TNF α receptor from said host cell.

The expression vector provided by the present invention is capable of amplifying or expressing a DNA encoding the mammalian TNFR of the present invention. The present invention also provides a host cell transformed with the above expression vector. Expression vectors are replicable DNA constructs having a synthetic or cDNA-derived DNA segment encoding a mammalian TNFR or bioequivalent analog operably linked to appropriate transcriptional or translational regulatory elements derived from a mammalian, bacterial, viral or insect gene. A transcriptional unit typically comprises a combination of (a) genetic elements that have a regulatory role in gene expression, such as transcriptional promoters or enhancers, (b) structural or coding sequences that are transcribed into mRNA and translated into protein, and (c) appropriate transcriptional and translational initiation and termination sequences. Such regulatory elements include the control sequences for transcription, sequences encoding the appropriate mRNA ribosome binding sites. An origin of replication that generally confers replication ability in the host cell and a selection gene that helps in the recognition of transformants may be additionally integrated.

When the DNA regions are functionally related to each other, they may be operably linked. For example: if the expression sequence is involved in a precursor of polypeptide secretion, then DNA encoding a signal peptide (secretion guide) is operably linked to the DNA encoding the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally operably linked means contiguous and, for the secretion guide, contiguous and in reading frame. The structural elements designed for use in the yeast expression system preferably include a leader sequence that allows the host cell to secrete the translated protein outside the cell. In other words, the expression of the recombinant protein has no leader or transporter sequence, and it may contain an N-terminal methionine residue. This residue can then be optionally cleaved from the expressed protein to provide the final product.

Mammalian TNFR DNA may be expressed or amplified in a recombinant expression system comprising essentially a homogeneous single culture of a suitable host microorganism, e.g., a bacterium, e.g., E.coli, or a yeast, e.g., Saccharomyces cerevisiae, which has stably integrated (by transformation or transfection) the recombinant transcription unit into chromosomal DNA, or which carries the recombinant transcription unit as an inherent plasmid component. The recombinant expression systems defined herein will express heterologous proteins constitutively or under the induction of regulatory elements associated with the expressed DNA sequences or artificial genes.

The transformed host cell is a cell transformed or transfected with a mammalian TNFR vector constructed using recombinant DNA techniques. The transformed host cell typically expresses TNFR, but the transformed host cell need not express TNFR for cloning or amplifying TNFR DNA. Suitable host cells for expression of mammalian TNFR include prokaryote, yeast, fungus or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E.coli or Bacillus. Higher eukaryotic cells include, but are not limited to, established insect and mammalian cell lines. Using RNAs derived from the DNA constructs of the present invention, mammalian TNFR can also be produced using a cell-free translation system. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are well known in the art.

Prokaryotic expression hosts can be used to express TNFRs that do not require extensive proteolysis and disulfide processing. Prokaryotic expression vectors typically contain one or more phenotypic selectable markers, such as genes encoding proteins that confer antibiotic resistance or supply autotrophic requirements, and origins of replication recognized by the host to protect amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, B.subtilis, Salmonella typhimurium, and various species of bacteria within the genera Pseudomonas, Streptomyces, and Staphylolococcus, although other bacteria may be used as hosts of choice.

Useful expression vectors for bacterial use can contain a selectable marker and a bacterial origin of replication derived from a commercially available plasmid (ATCC37017) containing the genetic components of the well-known cloning vector pBR 322. These pBR322 "backbone" portions are combined with appropriate promoters and structural sequences to be expressed. pBR322 contains genes resistant to ampicillin and tetracycline, and therefore provides a simple means for identifying transformed cells. Such commercial vectors include, for exampleET vector series (EMD Biosciences, inc., Madison, Wis.).

Promoters commonly used in recombinant microbial expression vectors include the lactose promoter system, λ P_LPromoter, T7 promoter and T7lac promoter. Particularly useful are bacterial expression systems which are capable of,ET System (EMD Biosc)ies, inc., Madison, Wis.) used a T7 or T7lac promoter and a strain of e.coli, such as BL21(DE3), which contains a chromosomal copy of the T7RNA polymerase gene.

TNFR proteins may also be expressed in yeast and fungal hosts, preferably in Saccharomyces species, such as Saccharomyces cerevisiae of other genera, for example Pichia or Kluyveromyces may also be used. Yeast vectors typically comprise an origin of replication from a 2 μ yeast plasmid or Autonomously Replicating Sequence (ARS), a promoter, DNA encoding TNFR, polyadenylation and transcription termination sequences, and a selection gene. The yeast vector preferably includes a replication origin allowing transformation of yeast and E.coli and a selectable marker, such as ampicillin resistance gene of E.coli, TRP1 or URA3 gene of lager brewing yeast providing a selectable marker for a yeast mutant lacking the ability to grow in tryptophan or uracil, respectively, and a promoter for inducing transcription of a downstream structural gene derived from a highly expressed yeast gene. The presence of TRP1 or URA3 lesions in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan or uracil, respectively.

Suitable promoter sequences in yeast vectors include the promoters of metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are well known in the art.

DNA sequences from pUC18 for selection and replication in E.coli (Amp) may be used^rGenes and origins of replication), and glucose-repressible ADH2 promoter and [ alpha ]]-yeast DNA sequences of factor secretion guides to assemble preferred yeast vectors. By inserting yeast [ alpha ] directing secretion of heterologous proteins between a promoter and a structural gene to be expressed]-a factor leader (leader). Can modify the guideSequences such that they contain one or more useful restriction enzyme sites near the 3' end to facilitate fusion of the leader sequence to the foreign gene. Suitable yeast transformation procedures are known to those skilled in the art.

Host strains transformed with vectors containing the ADH2 promoter can be grown for expression in rich medium consisting of 1% yeast extract, 2% peptone, and 1% or 4% glucose supplemented with 80mg/ml adenine and 80mg/ml uracil. As soon as the glucose is exhausted from the medium, the ADH2 promoter is derepressed. Before further purification, the crude yeast supernatant was harvested by filtration and kept at 4 ℃.

TNFR proteins may also be conveniently expressed using a variety of mammalian or insect cell culture systems. Expression of recombinant proteins in mammalian cells is particularly preferred, as such proteins are generally capable of proper folding, are suitably modified, and are fully functional. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells, as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, such as the L929, C127, 3T3, Chinese Hamster Ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may contain non-transcriptional elements such as an origin of replication, a suitable promoter such as the CMVie promoter, the chicken β -actin promoter or the synthetic hEF1-HTLV promoter, an enhancer associated with the gene to be expressed, and other 5 'or 3' flanking non-transcribed sequences, 5 'or 3' flanking non-translated sequences such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for the production of heterologous proteins in insect cells are known to those skilled in the art.

Transcriptional and translational control sequences for expression vectors used in transforming vertebrate cells can be provided by a viral source. For example, commonly used promoters and enhancers are derived from polyoma, adenovirus 2, monkey kidney virus 40(SV40), human cytomegalovirus, e.g., CMVie promoter, HTLV, e.g., the synthetic hEF1-HTLV promoter. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early and late promoters, enhancers, splicing and polyadenylation sites may be used to provide additional genetic elements required for expression of heterologous DNA sequences.

Still further, mammalian genomic TNFR promoters can be used, such as control and/or signal sequences, if such control sequences are compatible with the host cell of choice.

In a preferred embodiment of the present invention, the recombinant expression vector comprising TNFR cDNAs is stably integrated into the DNA of the host cell.

Protein expression and purification:

when mammalian or insect cells are used, the appropriately expressed TNFR protein will be secreted into the extracellular medium. The protein is recovered from the culture medium using standard biochemical techniques, concentrated and purified. Following expression by lentivirus (lentivirus) transduction, AAV transduction, plasmid transfection or any similar procedure in mammalian cells, or by baculovirus transduction in insect cells, concentration filters of appropriate filtration molecular weight are employed, e.g.The filtration unit concentrates the extracellular medium of these cells and in order to avoid loss of TNFR protein, the filter should allow protein flow through at or below 50 kDal.

When expressing the TNFR protein in bacterial culture, purification can be performed by standard biochemical techniques. The bacteria were lysed and the cell extract containing TNFR was desalted and concentrated.

In both cases, the TNFR protein is preferably purified by affinity chromatography. Preferably, column chromatography with an affinity matrix comprising TNF-alpha is used. In other words, an affinity purification tag may be added to the N-or C-terminus of the TNFR protein. For example: a polyhistidine tag (His-tag) can be used to achieve this, where the polyhistidine tag is an amino acid motif with at least six histidines (Hengen, P., 1995, Trends biochem. Sci.20: 285-86). The addition of the His-tag can be accomplished by in-frame addition of a nucleotide sequence encoding the His-tag directly to the 5 'or 3' end of the TNFR open reading frame in the expression vector. SEQ ID NO: 126 such a nucleotide sequence suitable for the addition of a carboxy-terminal His tag is given. When the His-tag is incorporated into the protein, the tagged TNFR is purified using a nickel or cobalt affinity chromatography column, and then the His-tag may be optionally cleaved off. Other suitable affinity purification tags and methods of purification of proteins having such tags are well known in the art.

Alternatively, a non-affinity based purification scheme can be used, including fractionation of TNFR extracts on a series of columns based on size (molecular sieve chromatography), charge (anion and cation exchange chromatography) and hydrophobicity (reverse phase chromatography). High performance liquid chromatography can be used to simplify these steps.

Other methods of expressing and purifying TNFR proteins are well known in the art (see, e.g., U.S. Pat. No.5,605,690 to Jacobs).

Definition of

The term "internucleotide linkage" is used to refer to a group capable of covalently linking two nucleobases together, e.g., between DNA units, between a DNA unit and a nucleotide analog, between two non-LNA units, between a non-LNA unit and an LNA unit, between two LNA units, and the like. Preferred examples include phosphate, phosphodiester groups and phosphorothioate groups.

As used herein, the term "nitrogenous base" (nitrogenes base) includes purines and pyrimidines, such as DNA nucleobases A, C, T and G, RNA nucleobases A, C, U and G, and non-DNA/RNA nucleobases, such as 5-methylcytosine ((R))^MeC) Isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-fluorouracil, 5-methylthiazolyluracil, 6-aminouracilAlkylpurine, 2-aminopurine, inosine, 2, 6-diaminopurine, 7-propyne-7-deazaAdenine (d)eazaadenine), 7-propyn-7-deazaguanine (deazaguanine) and 2-chloro 6-aminopurine, in particular^MeC. It will be appreciated that the actual choice of non-DNA/RNA nucleobases will depend on the corresponding (or matching) nucleotides present in the small RNA strand to which the oligonucleotide is targeted. For example, if the corresponding nucleotide is G, it is often necessary to select a non-DNA/RNA nucleobase that is capable of establishing a hydrogen bond with G. In this particular case, where the corresponding nucleotide is G, a typical example of a preferred non-DNA/RNA nucleobase is^MeC。

The terms "tumor necrosis factor receptor", "TNF receptor" and "TNFR" as used herein refer to proteins having the amino acid sequence of a native mammalian TNF receptor sequence or proteins substantially similar to a native mammalian TNF receptor sequence, which are capable of binding TNF molecules. In this context, a "native" receptor or gene for such a receptor refers to a naturally occurring receptor or gene, as well as allelic variations of such a receptor and gene that occur in nature.

The term "mature" used in conjunction with TNFR refers to a protein expressed in a form lacking a leader or signal sequence that may be present in the full-length transcript of the native gene.

The nomenclature of TNFR proteins used herein follows the convention of placing the designation of proteins (e.g., TNFR2) before the designation of species, e.g., hu (for humans) or mu (for murine animals), followed by Δ (meaning deletion) and the number of exons deleted. For example: huTNFR2 Δ 7 refers to human TNFR2 that lacks exon 7. In the absence of any species designation, TNFR is generically mammalian TNFR.

The term "secreted" means that the protein is soluble, that is, it does not bind to the cell membrane. In this context, a form is soluble if, using routine tests known to those skilled in the art, the majority of the form is detectable in components not associated with the membrane, such as cell supernatants or sera.

The term "stable" refers to a secreted form of TNFR detectable in harvested cells, cell supernatants or serum using conventional assays known to those skilled in the art, such as western blots, ELISA assays.

The terms "tumor necrosis factor" and "TNF" as used herein refer to a naturally occurring protein ligand that binds to a TNF receptor. TNF includes, but is not limited to TNF-alpha and TNF-beta.

The term "inflammatory disease or condition" as used herein refers to a disease, disorder or other medical condition that is caused, at least in part, by the binding of TNF to its receptor, or is exacerbated by such binding. Such diseases or conditions include, but are not limited to, those associated with elevated TNF levels, elevated TNF receptor levels, enhanced sensitization, or dysregulation of the corresponding signaling pathway. The term also includes diseases and conditions in which known TNF antagonists have been shown to be useful. Inflammatory diseases or conditions include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis (ankylosing spondylitis), inflammatory bowel disease (including Crohn's disease and ulcerative colitis), hepatitis, sepsis, alcoholic liver disease, and non-alcoholic steatosis.

The term "hepatitis" as used herein refers to a gastrointestinal disease, condition or disorder characterized by inflammation of at least a portion 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.

The term "TNF antagonist" as used herein refers to a protein capable of measurably inhibiting TNF-mediated cytotoxicity as measured by standard assays well known in the art (see, e.g., the L929 cytotoxicity assay described in the examples below).

The term "binds TNF" means that a protein can bind detectable levels of TNF, preferably TNF- α, as determined by standard binding assays well known in the art (see, e.g., columns 16-17 of Smith, U.S. Pat. No.5,945,397). Each nanomolar receptor of the present invention is preferably capable of binding greater than 0.1 nanomolar TNF-alpha, and more preferably capable of binding greater than 0.5 nanomolar TNF-alpha, when tested using standard binding assays.

The term "regulatory element" as used herein refers to a nucleotide sequence involved in molecular interactions that contribute to the regulation of nucleic acid function, including but not limited to regulation of nucleic acid replication, transcription, splicing, translation or degradation. In nature, modulation may be enhancement or inhibition. Regulatory elements known in the art include, for example, translational regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that, under certain conditions, helps initiate transcription of a coding region, which is usually located downstream (3' to) the promoter. Expression vectors typically comprise such regulatory elements operably linked to a nucleic acid of the invention.

The terms "oligomer", "splice switching oligomer" and "oligonucleotide" are used interchangeably herein.

The term "operably linked" as used herein refers to the juxtaposition of genetic elements wherein the relationship of the elements allows them to function in the intended manner. For example, a promoter is operably linked to a coding region (e.g., in an expression vector) if the promoter helps initiate transcription of the coding sequence. Residues may be inserted between the promoter and coding region as long as this functional relationship is maintained.

The term "transformation" or "transfection" as used herein refers to the insertion of foreign nucleic acid into a cell, regardless of the method used for insertion, such as lipofection, transduction, infection, or electroporation. The exogenous nucleic acid may be maintained in a non-integrating vector, such as a plasmid, or may be integrated into the genome of the cell.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to an already linked molecule. One vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA sequences may be ligated. Another vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell when introduced into the host cell, and are therefore capable of replication along with the host genome. In addition, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids or viral vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses).

The term "isolated protein" as used herein refers to a protein or polypeptide that does not occur in nature, or that is isolated from one or more components with which it is naturally associated.

The term "isolated nucleic acid" as used herein refers to a non-naturally occurring nucleic acid, and/or a nucleic acid in the form of an isolated fragment or as a component of a larger construct, wherein the fragment or component is derived from a substantially pure nucleic acid that has been isolated at least once, i.e., free of contaminating endogenous material, and in an amount or concentration that permits identification and manipulation by standard biochemical methods, such as those using cloning vectors.

The term "purified protein" as used herein refers to a protein that is substantially free of other proteins. However, such purified proteins may comprise other proteins added as stabilizers, carriers, excipients, or co-therapeutics. The term "purified" as used herein preferably means at least 50%, such as at least 80%, more preferably 95-99%, most preferably 99.8% by weight of the protein present based on the dry weight of the protein present, wherein the protein present excludes proteins added as stabilizers, carriers, excipients or co-therapeutic agents.

The term "altering the splicing of pre-messenger RNA" as used herein refers to altering the splicing of a cellular pre-messenger RNA target resulting in an altered ratio of spliced products. Such splicing changes can be detected by a variety of techniques well known to those skilled in the art. For example, RT-PCR of total cellular RNA can be used to detect the ratio of spliced products in the presence and absence of SSO.

The term "complementary" as used herein is intended to mean that the complementarity is such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA comprising the target sequence, or that the pairing is so precise. It is understood in the art that the oligonucleotide sequence need not be 100% complementary to the target sequence. For example, for SSO, where splicing is permitted, if sufficient complementarity exists, binding to the target will occur and non-specific binding will be avoided. However, oligonucleotides or contiguous nucleobase sequences which are fully complementary (i.e.complete) to the target sequence (e.g.the region of SEQ ID NO1-4 mentioned herein) are preferred.

The terms "corresponding" and "corresponding" as used in the context of an oligonucleotide refer to a comparison between a nucleobase of a compound of the invention and its reverse complement, in one embodiment a nucleobase sequence and an equivalent (identical) nucleobase sequence, which may comprise, for example, other nucleobases, but retains the same base sequence, or a sequence complementary thereto. Nucleotide analogs can be compared directly to their equivalents or to the corresponding natural nucleotides. A sequence that forms a reverse complement to a sequence is referred to as a complementary sequence of the sequence.

When referring to the length of a nucleotide molecule as referred to herein, the length corresponds to the number of monomeric units, i.e., nucleobases, irrespective of whether those monomeric units are nucleotides or nucleotide analogs. In the case of nucleobases, the terms monomer and unit are used interchangeably herein.

It is to be understood that when the term "about" is used in the context of a ratio or a numerical range, the read disclosure should include the ratio or range mentioned.

The term "variant" as used herein in the context of a protein or polypeptide (sequence) refers to a polypeptide prepared from said polypeptide by insertion, deletion or substitution of one or more amino acids, i.e. at least one amino acid, but preferably less than 50 amino acids, such as less than 40, less than 30, less than 20 or less than 10 amino acids, such as 1 amino acid, 1-2 amino acids, 1-3 amino acids, 1-4 amino acids, 1-5 amino acids, in the original (parent) polypeptide, or a polypeptide prepared using sequence information from said polypeptide.

The term "homologue" as used herein in the context of a protein or polypeptide (sequence) refers to a polypeptide which is at least 70% homologous, such as at least 80% homologous, e.g. at least 85% homologous or at least 90% homologous, such as at least 95%, 96%, 97%, 98% or 99% homologous to the polypeptide sequence in question. Homology between two polypeptides can be determined using the Blosum62 algorithm with the ClustalW alignment algorithm, where the degree of Gap (Gap extend) is 0.5 and the Gap open (Gap open) is 10 (see http:// www.ebi.ac.uk/embos/align/index. The alignment may be a local alignment (water) in one embodiment, or a global alignment (needle) in another embodiment, separate. Since the exon-deleted homologs of the TNFR proteins mentioned herein include deletions in the respective exons, the global alignment is more preferred.

The term "fragment" as used herein in the context of a protein or polypeptide (sequence) refers to a polypeptide that consists only of a portion of the polypeptide sequence. Thus a fragment may comprise at least 5% of the polypeptide sequence, for example at least 10%, including at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the polypeptide sequence.

The definitions of variants, fragments and homologues described above also apply to nucleic acid sequences, but the homology algorithm uses DNAfull. Obviously, when referring to nucleic acid variants, fragments or homologues, the terms protein, polypeptide and amino acid should be replaced by nucleic acids, polynucleotides or nucleobases/nucleotides accordingly.

The term "membrane-bound form" or "whole membrane form" as used herein refers to a protein having an amino acid sequence spanning the cell membrane, the amino acid sequence being on both sides of the membrane.

The term "stably secreted ligand-binding form" or sometimes referred to as "stably soluble ligand-binding form" (the terms "secreted" and "soluble" are synonymous and used interchangeably herein) as used herein refers to a protein that associates with a receptor in its native membrane-bound form by being secreted, remaining stable and still being able to bind the corresponding ligand. It should be noted that these forms are not limited by whether the secreted form is physiologically active, as long as the splice variant product is secreted, stable, and still binds the ligand when produced.

The term "secreted" refers to a soluble form, that is, it is no longer bound to the cell membrane. In this context, if a form is found to be soluble using routine tests known to those skilled in the art, the majority of the form can be detected in parts not associated with the membrane, such as cell supernatant or serum.

The term "stable" refers to a secreted form that is detectable by one of skill in the art using routine assays. For example, western blot, ELISA assays can be used to detect such forms in harvested cells, cell supernatants, or patient sera.

The term "binding partner" means that the form retains at least some significant level of specific ligand binding activity, although not necessarily all of the specific ligand binding activity of the corresponding monolithic membrane form.

The term "decreasing the activity of a ligand" as used herein refers to any effect that results in a decrease in intracellular signaling resulting from ligand binding or interaction with a receptor. For example, binding of a ligand to a soluble form of its receptor or reducing the number of membrane forms of the receptor that effectively bind the ligand may reduce activity.

Pharmaceutical compositions and formulations

Other embodiments of the invention relate to pharmaceutical compositions comprising oligomers, proteins and nucleic acids of the invention.

The oligomers, nucleic acids and proteins of the invention may be incorporated into other molecules, molecular structures or mixtures of compounds by blending, encapsulation, conjugation or other means, examples being liposomes, receptor targeting molecules, oral dosage forms, rectal dosage forms, topical dosage forms or other dosage forms that facilitate uptake, distribution and/or absorption.

The dosage form of the invention comprises the oligomer, nucleic acid or protein of the invention in a physiologically or pharmaceutically acceptable carrier, e.g. an aqueous carrier. Thus, dosage forms for use in the present invention include, but are not limited to, those suitable for parenteral administration, including intra-articular, intraperitoneal, intravenous, intraarterial, subcutaneous, intramuscular injection or infusion, as well as those suitable for topical, ophthalmic, vaginal, oral, rectal or pulmonary administration, including inhalation or insufflation of powders or aerosols, including those delivered by nebulizer, intratracheal, and intranasal. The formulations may conveniently be presented in unit dosage form or may be prepared by any of the methods well known in the art. The most appropriate route of administration in any given case will depend on the nature and severity of the condition being treated in the subject, and the particular active compound used.

The pharmaceutical compositions of the present invention include, but are not limited to, physiologically and pharmaceutically acceptable salts thereof, i.e., salts which retain the desired biological activity of the parent compound and which do not produce undesired toxic effects. Examples of such salts are (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, and the like; (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid (naphthalene disulfonic acid), polygalacturonic acid, and the like.

The present invention provides the use of oligomers, proteins and nucleic acids as described above for the preparation of a pharmaceutical formulation for the treatment of a patient suffering from an inflammatory condition involving TNF hyperactivity as discussed below. In the manufacture of the medicament according to the invention, the oligomers, nucleic acids and proteins of the invention are typically mixed with other acceptable carriers. Of course, the carrier must be acceptable, i.e., compatible with any other ingredients in the dosage form, and must not be deleterious to the patient. The carrier may be a solid or a liquid. The oligomers, nucleic acids and proteins of the invention are incorporated into the formulations of the invention prepared by any of the well-known pharmaceutical techniques consisting essentially of mixing the components, wherein the formulation optionally includes one or more additional therapeutic ingredients.

The formulations of the present invention include sterile aqueous and non-aqueous injection solutions of the active compound, which are preferably isotonic with the blood of the intended recipient and substantially pyrogen-free. These formulations may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include, but are not limited to, suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example, saline or water for injection, immediately prior to use.

In such formulations, the oligomers, nucleic acids and proteins of the invention may be contained within particles or vesicles, such as liposomes or microcrystals suitable for parenteral administration. The particles may be of any suitable structure, such as a monolayer or multilayer of particles, so long as the oligomers, nucleic acids and proteins of the invention are contained therein. Such particles and vesicles are particularly preferred to be positively charged lipids, such as N- [1- (2, 3-diolyoxy) propyl ] -N, N, N-trimethyl-aminomethylsulfate or "DOTAP". The preparation of such lipid particles is well known (see column 6 of reference U.S. Pat. No.5,976,879).

Accordingly, one embodiment of the invention relates to methods of treating an inflammatory disease or condition by decreasing the TNF activity of a stably secreted ligand-binding form of a TNF receptor by administering the receptor. In another embodiment, the invention relates to methods of treating an inflammatory disease or condition by decreasing the TNF activity of a stably secreted ligand-binding form of a TNF receptor by administering an oligonucleotide encoding the receptor. In another embodiment, the invention relates to a method of producing a stable secreted ligand-binding form of a TNF receptor.

The following aspects of the invention discussed below are applicable to the embodiments described above.

The methods, nucleic acids, proteins and dosage forms of the invention are also useful in vitro or in vivo tools.

Embodiments of the invention may be used in any state where a treatment practitioner intends to limit the signaling pathway of TNF action or TNF activation. In particular, the invention is useful for the treatment of inflammatory diseases. In one embodiment, the condition is an inflammatory systemic disease, such as rheumatoid arthritis or psoriatic arthritis. In another embodiment, the disease is an inflammatory liver disease. Examples of inflammatory liver diseases include, but are not limited to, hepatitis associated with hepatitis a, b or c virus, alcoholic liver disease and non-alcoholic steatosis. In another embodiment, the inflammatory disease is a skin disease, such as psoriasis.

Uses of the invention include, but are not limited to, the treatment of diseases for which known TNF antagonists have been shown to be useful. Three specific TNF antagonists are currently approved by the FDA. These drugs are etanerceptinfliximabAnd adalimumabOne or more of these drugs have been approved for the treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease (Crohn's disease or ulcerative colitis).

Treatment of inflammatory diseases with proteins:

one embodiment of the invention relates to methods of treating inflammatory diseases or conditions by administering to a patient SSOs that alter the splicing of pre-messenger RNA to produce a splice variant encoding a stably secreted ligand-binding form of a TNFR superfamily receptor, thereby reducing the activity of the ligand at that receptor. In another embodiment, the invention relates to methods of producing a stably secreted ligand-binding form of a TNFR superfamily receptor in a cell by administering SSOs to the cell.

For therapeutic use, the purified TNFR proteins of the invention are administered to a patient, preferably a human, to treat TNF-dependent inflammatory diseases, such as arthritis. In the treatment of humans, huTNFRs are preferably used. TNFR proteins of the present invention may be administered by bolus injection, by contiguous infusion, by sustained release from an implant, or by other suitable techniques. Typically, the TNFR therapeutic protein is administered in the form of a composition comprising the purified protein and a physiologically acceptable carrier, excipient or diluent. The dosage and concentration of such carriers used are non-toxic to recipients. Typically, such compositions are prepared by combining the TNFR with buffers, antioxidants, such as ascorbic acid, polypeptides, proteins, amino acids, sugars including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Suitable diluents as examples are neutral buffered saline or saline mixed with the same serum albumin. The product is preferably formulated as a lyophilizate using a suitable excipient solution, such as sucrose as a diluent. Preservatives, such as benzyl alcohol, may also be added. Of course, the number and frequency of administration will depend on the nature of such factors, the severity of the indication to be treated, the desired response, the condition of the patient, and the like.

A preferred range of therapeutically effective amounts of the TNFR proteins of the present invention for systemic administration is from 0.1 mg/kg/week to about 100 mg/kg/week. In a preferred embodiment, the amount of TNFR administered is from about 0.5 mg/kg/week to about 50 mg/kg/week. For topical application, the preferred dosage is from about 0.01mg/kg to about 1.0mg/kg per injection.

Use of expression vectors to elevate levels of TNF antagonists in mammals

The invention provides methods for increasing the level of a TNF antagonist in a mammal. The method comprises the step of transforming a mammalian cell with an expression vector as described herein, wherein the expression vector drives the expression of a TNFR as described herein. The method is particularly useful for large mammals, such as domestic pets, mammals and primates used for food production. Examples of large mammals are dogs, cats, horses, cows, sheep, deer and pigs. Examples of primates are monkeys, apes, and humans.

Mammalian cells can be transformed in vivo or in vitro. When transformed in vivo, the expression vector is administered directly, e.g., by injection, to a mammal. Means for transforming cells in vivo are well known in the art. When transforming in vitro, cells are first obtained from a mammal, transformed in vitro, and then the transformed cells are transplanted into the mammal.

Uses of the invention include, but are not limited to, the treatment of diseases for which known TNF antagonists have been shown to be useful. Three specific TNF antagonists are currently approved by the FDA. These drugs are etanerceptinfliximabAnd adalimumabThese drugsOne or more of which have been approved for the treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease (Crohn's disease or ulcerative colitis).

The process of administering SSO to a subject can be accomplished using the steps developed for ASONs. ASON in saline has been successfully administered to experimental animals and human subjects at doses up to 6mg/kg three times per week by intravenous administration (Yacysyhn, B.R., et al, 2002, Gut 51: 30 (anti-ICAM-1 ASON for Crohn's disease); Stevenson, J.et al, 1999, J.ClinicalOncology 17: 2227 (anti-RAF-1 ASON targeting PBMC)). Pharmacokinetics of 2' o-MOE phosphorothioate ASON targeting TNF- α have been reported (Geary, R.S., et al, 2003, drug metabolism and Disposition 31: 1419). The systematic effectiveness of mixed LNA/DNA molecules has also been reported (fluid, K., et al., 2003, Nucleic Acids Res.31: 953).

The systemic activity of SSO in a mouse model system was investigated using 2' O-MOE phosphorothioate and PNA chemistry. Significant activity was observed in all tissues studied except brain, stomach and dermis (Sazani, p., et al., 2002, Nature Biotechnology 20, 1228).

In general, any method of administration useful in conventional antisense therapy can be used to administer the SSOs of the invention. To test for SSO in cultured cells, any technique that has been developed to test for ASON or SSO can be used.

The dosage forms of the invention comprise the SSOs in a physiologically or pharmaceutically acceptable carrier, e.g., an aqueous carrier. Thus, dosage forms for use in the present invention include, but are not limited to, those suitable for parenteral administration, including intraperitoneal, intraarticular, intravenous, intraarterial, subcutaneous, intramuscular injection or infusion, as well as those suitable for topical, ophthalmic, vaginal, oral, rectal or pulmonary (including inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal and intranasal delivery) administration. The formulations may conveniently be presented in unit dosage form or may be prepared by any of the methods well known in the art. The most appropriate route of administration in any given case will depend on the nature and severity of the condition being treated in the subject, and the particular active compound used.

The pharmaceutical compositions of the present invention include, but are not limited to, physiologically and pharmaceutically acceptable salts thereof, i.e., salts which retain the desired biological activity of the parent compound and which do not produce undesired toxic effects. Examples of such salts are (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, and the like; (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like.

The present invention provides the use of SSOs having the above characteristics for the preparation of a medicament for increasing the ratio of mammalian TNFR2 protein lacking exon 7/its corresponding membrane bound form in a patient afflicted with an inflammatory disorder in which TNF-alpha as discussed above is implicated. In the manufacture of medicaments according to the invention, the SSOs are typically mixed with an acceptable carrier. Of course, the carrier must be acceptable, i.e., compatible with any other ingredients in the dosage form, and must not be deleterious to the patient. The carrier may be a solid or a liquid. The SSOs are incorporated into the formulations of the present invention prepared by any of the well-known pharmaceutical techniques consisting essentially of mixing the components, wherein the formulations optionally include one or more additional therapeutic ingredients.

The formulations of the present invention include sterile aqueous and non-aqueous injection solutions of the active compound, which are preferably isotonic with the blood of the intended recipient and substantially pyrogen-free. These formulations may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include, but are not limited to, suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example, saline or water for injection, immediately prior to use.

In such formulations, the SSOs can be contained within particles or vesicles, such as liposomes or microcrystals suitable for parenteral administration. The particles may be of any suitable structure, such as monolayer or multilayer particles, so long as the SSOs are contained therein. Such particles and vesicles are particularly preferred to be positively charged lipids, such as N- [1- (2, 3-diolyoxy) propyl ] -N, N, N-trimethyl-aminomethylsulfate or "DOTAP". The preparation of such lipid particles is well known (see column 6 of reference U.S. Pat. No.5,976,879).

The SSO can target any element or combination of elements that modulate splicing, including a3 'splice site, a 5' splice site, a branch point, a polypyrimidine tract, an exon splicing enhancer, an exon splicing silencer, an intron splicing enhancer, an intron splicing silencer.

It will be appreciated by those skilled in the art that the invention targeting human TNF TNFR2 can be practiced using SSOs having a sequence complementary to at least 8 nucleotides, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, preferably 10-16 nucleotides of the portion of the TNFR1 or TNFR2 gene that contains exon 7 and its adjacent intron.

SEQ ID NO: 3 comprises the sequence of exon 7 of TNFR2 and 50 contiguous nucleotides flanking the intron. For example, an SSO targeting human TNFR2 can have a nucleobase sequence selected from the sequences listed in table 4. When affinity enhancing modifications are used, including but not limited to LNA or g-clamp nucleotides, one skilled in the art will recognize that the length of SSO can be reduced accordingly.

Those skilled in the art will also recognize that selection of the SSO sequence must be considered to avoid self-complementary SSO, which may lead to the formation of a partial "hairpin" double-stranded structure. In addition, high GC content should be avoided to minimize the possibility of non-specific base pairing. Furthermore, the use of SSOs that pair with the off-target gene as shown by BLAST should also be avoided.

In some cases, it is preferred to select SSO sequences that target humans and at least one other species. These SSOs can be used to test and optimize the present invention in the other species described before use in humans, thus helping to adjust approval and drug development objectives. For example, SEQ id nos: 14, 30, 46, 70 and 71 are also 100% complementary to the corresponding macaque Mullata sequence. Thus, experimental treatments with these sequences may be performed in monkeys prior to use in humans.

The following aspects of the invention discussed below are applicable to the embodiments described above.

The length of the SSO is similar to that of an antisense oligonucleotide (ASON), typically between about 10-24 nucleotides. SSOs can be used with RNA hybridization, unlike conventional antisense 2^rSeveral chemistries like deoxyoligonucleotides that activate RNAse H induced RNA destruction. The invention may be practiced using 2 ' O modified nucleic acid oligomers, such as 2 ' O-methyl or 2 ' O-methoxyethyl phosphorothioates. The nucleobase need not be linked to a sugar; so-called peptide nucleic acid oligomers or morpholine-based oligomers may be used. In Sazani, p.et al, 2001, nucleic acids res.29: a comparison of these different chemical ligation processes is found in 3695. The term splice switching oligonucleotide is intended to include such forms. The relationship between antisense oligonucleotide gapmers and SSOs can be understood by those skilled in the art. Gapmers are ASON's that contain an RNAse H activation region (typically a 2' -deoxyribonucleoside phosphorothioate), which is flanked by a nuclease resistant oligomer that is not activated. In general, suitable gapmers can be used in SSOAny chemistry of flanking sequences in ASONs.

The SSOs of the present invention can be prepared by well-known solid phase synthesis techniques. Any other means suitable for such synthesis may additionally or alternatively be used. It is known that oligonucleotides can be prepared using similar methods, such as phosphorothioates and alkyl derivatives.

Locked Nucleic Acids (LNA) provide a particularly preferred chemistry (Koshkin, A.A., et al., 1998, Tetrahedron 54: 3607; Obika, S.et al., 1998, Tetrahedron Lett.39: 5401). LNA is a conventional phosphodiester-linked ribonucleotide, except that the ribofuranonitrosourea moiety is a bridge between 2 'O and 4' C forming a bicyclic ring. This bridging forces the nitrosourea loop conformation to change to the V-bridge (endo) conformation that is used when the oligonucleotide hybridizes to complementary RNA. Recent advances in synthetic LNAs are described in WO 03/095467. Most typical bridges are methylene or vinyl groups. Morita, et al, 2003, bioorg.and med.chem.11: 2211 describes the synthesis of 2 'O, 4' C-ethylene-bridged nucleic acids (ENA) and other LNAs. However, alternative chemistries may be used to replace 2 'O with 2' N. LNA and conventional nucleotides can be mixed to form chimeric SSO. For example, chimeric SSOs formed from alternating LNAs and 2 ' deoxynucleotides or alternating LNAs and 2 ' O-Me or 2 ' O-MOE can be used. Any alternative to these chemistries, not just 2' -deoxynucleotides, can be phosphothioate-based linkages replacing phosphodiesters. For in vivo use, phosphorothioate linkages are preferred.

When LNA nucleotides are used in SSO, it is preferred that non-LNA nucleotides are also present. LNA nucleotides have such a high affinity in hybridizations with very pronounced non-specific binding that the effective concentration of free SSO can be reduced. When LNA nucleotides are used, they can also be conveniently replaced with 2' deoxynucleotides. Alternating nucleotides, alternating dinucleotides or mixed forms, such as LDLDLD or LDDLDD, may be used. When 2 '-deoxynucleotides or 2' -deoxynucleoside phosphorothioates are mixed with LNA nucleotides, it is important to avoid RNase H activation. LNA nucleotides for SSO are expected to be between about one-third and two-thirds suitable. For example, if the SSO is a 12 mer, then there will be at least four LNA nucleotides and four conventional nucleotides.

The bases of the SSO can be conventional cytosine, guanine, adenine and uracil or thymidine. Alternatively, modified bases may also be used. Of particular interest are modified bases with increased binding affinity. A non-limiting example of a preferred modified base is a nucleotide known as G-clamp or 9- (aminoethoxy) phenoxazine, a cytosine analogue that forms 4 hydrogen bonds with guanosine (Flanagan, W.M., et al., 1999, Proc. Natl. Acad. Sci.96: 3513; Holmes, S.C., 2003, Nucleic Acids Res.31: 2759).

A variety of alternative chemistries that do not activate RNase H can be utilized. For example: suitable SSOs may be oligonucleotides in which at least one or all of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl thiophosphonates, phosphoromorpholates, phosphoropiperazidates and phosphoramidates. For example: phosphate residues bridging between nucleotides may be modified every other according to what is described. In another non-limiting example, such an SSO is an oligonucleotide in which at least one or all of the nucleotides comprise a lower alkyl moiety at the 2' position (e.g., C1-C4, linear or branched, saturated or unsaturated lower alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl nucleisopropyl). For example, every other nucleotide may be modified in accordance with what is described. [ see column 4 of U.S. Pat. No.5,976,879 ].

The length of the SSO (i.e., the number of monomers in the oligomer) is from about 10 to about 30 bases. In one embodiment, a 20 base 2' O-Me-ribonucleoside phosphorothioate is effective. It will be appreciated by those skilled in the art that when chemical modifications are used that increase affinity, the SSO can be shorter, but specificity is still maintained. It will be further appreciated by those skilled in the art that the upper limit of the length of the SSO is limited by the limitations of maintaining specific recognition of the target sequence, avoiding self-hybridization of secondary structures to form the SSO, and access to the cell. These limitations mean that SSOs of increased length (beyond a certain length that depends on SSO affinity) are often found to be less specific, inactive or poorly active SSOs.

SSOs of the invention include, but are not limited to, SSO modifications that involve chemically linking to the SSO one or more moieties or conjugates that enhance SSO activity, cellular distribution, or cellular uptake. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acids, thioethers such as hexyl-thio-trityl mercaptan (tritylthiol), thiocholesterol, fatty chains such as dodecyl or undecyl residues, phospholipids such as dicetyl-rac-glycerol or triethylammonium-1, 2-di-oxo-hexadecyl-rac-glycero-3-hydro-phosphate compounds, polyamine or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine or hexaneamine-carbonyl-hydroxycholesterol moieties.

Not all positions in a given SSO need to be uniformly changed, and indeed more than one of the above-described modifications can be introduced in a single compound, or even a single nucleoside, within an SSOs. The SSOs can be mixed with, encapsulated, coupled to, or associated with other molecules, molecular structures, or mixtures of compounds, e.g., to form liposomes, receptor-targeted molecules that facilitate absorption, distribution, and/or absorption, oral dosage forms, rectal dosage forms, topical dosage forms, or other dosage forms.

It will be understood by those skilled in the art that cell differentiation includes, but is not limited to, the differentiation of spliceosomes. Thus, the activity of any particular SSO of the invention depends on the cell type into which it is introduced. For example, SSOs that are effective in one cell type may not be effective in another cell type.

The methods, oligonucleotides and dosage forms of the invention are also useful in vitro or in vivo tools for investigating human or animal gene splicing. Such methods may be practiced by modification of the procedures described herein or those procedures known to those skilled in the art.

The present invention may be used in any state where a treatment practitioner intends to limit the action of a TNF superfamily ligand or the signaling pathway activated by such a ligand. In particular, the invention is useful for the treatment of inflammatory diseases. In one embodiment, the condition is an inflammatory systemic disease, such as rheumatoid arthritis or psoriatic arthritis. In another embodiment, the disease is an inflammatory liver disease. Examples of inflammatory liver diseases include, but are not limited to, hepatitis associated with hepatitis a, b or c virus, alcoholic liver disease and non-alcoholic steatosis. In another embodiment, the inflammatory disease is a skin disease, such as psoriasis.

Uses of the invention include, but are not limited to, the treatment of diseases for which known TNF antagonists have been shown to be useful. Three specific TNF antagonists are currently approved by the FDA. These drugs are etanerceptinfliximabAnd adalimumabOne or more of these drugs have been approved for the treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease (Crohn's disease or ulcerative colitis).

In a preferred embodiment, the receptor is the TNFR1 or TNFR2 receptor. In other embodiments, the receptor is a member of the TNFR superfamily that is substantially homologous to TNFR1 and TNFR2, such as TNFRSF3, TNFRSF5 or TNFRSFI IA, such that deletion of one or both of the two exons homologous to exons 7 and 8 results in a secreted form of the receptor. It will be appreciated by those skilled in the art that the operability of the present invention is not dependent on whether such secreted forms are physiologically active, as long as the product of such splice variants is secreted, stable, and capable of binding a ligand.

The process of administering SSO to a subject can be accomplished using the steps developed for ASONs. ASON in saline has been successfully administered to experimental animals and human subjects at doses up to 6mg/kg three times per week by intravenous administration (Yacysyhn, B.R., et al, 2002, Gut 51: 30 (anti-ICAM-1 ASON for Crohn's disease); Stevenson, J.et al, 1999, J.ClinicalOncology 17: 2227 (anti-RAF-1 ASON targeting PBMC)). Pharmacokinetics of 2' o-MOE phosphorothioate ASON targeting TNF- α have been reported (Geary, R.S., et al, 2003, drug metabolism and Disposition 31: 1419). The systematic effectiveness of mixed LNA/DNA molecules has also been reported (fluid, K., et al., 2003, Nucleic Acids Res.31: 953).

The systemic activity of SSO in a mouse model system was investigated using 2' O-MOE phosphorothioate and PNA chemistry. Significant activity was observed in all tissues studied except brain, stomach and dermis (Sazani, p., et al., 2002, Nature Biotechnology 20, 1228).

In general, any method of administration useful in conventional antisense therapy can be used to administer the SSOs of the invention. To test for SSO in cultured cells, any technique that has been developed to test for ASON or SSO can be used.

The dosage forms of the invention comprise the SSOs in a physiologically or pharmaceutically acceptable carrier, e.g., an aqueous carrier. Thus, dosage forms for use in the present invention include, but are not limited to, those suitable for parenteral administration, including intraperitoneal, intravenous, intraarterial, subcutaneous, intramuscular injection or infusion, as well as those suitable for topical administration (including ophthalmic and for mucosal, including vaginal delivery), oral, rectal or pulmonary (including inhalation or insufflation including powders or aerosols, including by nebulizer, intratracheal, intranasal delivery). The formulations may conveniently be presented in unit dosage form or may be prepared by any of the methods well known in the art. The most appropriate route of administration in any given case will depend on the nature and severity of the condition being treated in the subject, and the particular active compound used.

The pharmaceutical compositions of the present invention include, but are not limited to, physiologically and pharmaceutically acceptable salts thereof, i.e., salts which retain the desired biological activity of the parent compound and which do not produce undesired toxic effects. Examples of such salts are (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, and the like; (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed with basic anions such as chlorine, bromine, and iodine.

The present invention provides the use of SSOs having the above characteristics for the preparation of a medicament for increasing the ratio of soluble form of a member of the TNFR superfamily/its corresponding membrane bound form in a patient afflicted with an inflammatory disorder involving hyperactivity of a cytokine, e.g., TNF-alpha as discussed above. In the manufacture of medicaments according to the invention, the SSOs are typically mixed with an acceptable carrier. Of course, the carrier must be acceptable, i.e., compatible with any other ingredients in the dosage form, and must not be deleterious to the patient. The carrier may be a solid or a liquid. The SSOs are incorporated into the formulations of the present invention prepared by any of the well-known pharmaceutical techniques consisting essentially of mixing the components, wherein the formulations optionally include one or more additional therapeutic ingredients.

The formulations of the present invention include sterile aqueous and non-aqueous injection solutions of the active compound, which are preferably isotonic with the blood of the intended recipient and substantially pyrogen-free. These formulations may contain antioxidants, buffers, bacteriostats, and solutes (solutes) that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include, but are not limited to, suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water for injections, immediately prior to use.

In such formulations, the SSOs may be contained within lipid particles or vesicles, such as liposomes or microcrystals suitable for parenteral administration. The particles may be of any suitable structure, such as monolayer or multilayer particles, so long as the SSOs are contained therein. Such particles and vesicles are particularly preferred to be positively charged lipids, such as N- [1- (2, 3-diolyoxal) propyl ] -N, N, N-trimethyl-aminomethylsulfate or "DOTAP". The preparation of such lipid particles is well known. [ see column 6 of reference U.S. Pat. No.5,976,879 ]

The SSO can target any element or combination of elements that modulate splicing, including a3 'splice site, a 5' splice site, a branch point, a polypyrimidine tract, an exon splicing enhancer, an exon splicing silencer, an intron splicing enhancer, an intron splicing silencer. The sequence of SSO can be determined at the guide of the following table showing SSOs activity, wherein the sequence and position of SSOs are found to be depicted in fig. 20. Those skilled in the art will note that: 1) SSOs complementary to exons need not be complementary to a splice acceptor site or a splice donor site, noting SSOs A7-10, B7-7, and B7-9 of Table 1; 2) SSOs complementary to intron sequences and SSOs of as little as one nucleotide to an exon are operable, Note A8-5 and B7-6 (Table 1);

3) SSOs that complement introns immediately adjacent to exons are also effective, noting 3312 of table 2; and 4) the potency of the oligonucleotides alone may generally be predictive of the potency of SSO in combination with other SSOs.

It will be appreciated by those skilled in the art that the present invention targeting the human TNF-alpha receptor can be practiced using SSOs having a sequence complementary to at least 10 nucleotides, preferably 15-20 nucleotides, of the TNFR1 or TNFR2 gene portions comprising exons 7 or 8 and their adjacent introns. More preferably, at least one nucleotide of the exon itself is contained within the complementary sequence. SEQ ID Nos: 1-4 comprise the sequences of exons 7 and 8 of TNFR1(SEQ ID Nos: 1 and 2) and TNFR2(SEQ ID Nos: 3 and 4), and 50 contiguous nucleotides of the flanking introns. When affinity enhancing modifications are used, including, but not limited to, LNA or G-clamp nucleotides, the skilled artisan will recognize that the length of SSO may be reduced accordingly. A length of 16 nucleotides is effective when alternating regular and LNA nucleotides are used.

Those skilled in the art will also recognize that selection of the SSO sequence must be considered to avoid self-complementary SSO, which may lead to the formation of a partial "hairpin" double-stranded structure. In addition, high GC content should be avoided to minimize the possibility of non-specific base pairing. Furthermore, the use of SSOs that pair with the off-target gene as shown by BLAST should also be avoided.

In some cases, it is preferred to select SSO sequences that target humans and at least one other species. These SSOs can be used to test and optimize the present invention in the other species described before use in humans, thus helping to adjust approval and drug development objectives. For example, SEQ id nos: 74, 75, 77, 78, 80 and 89 are also 100% complementary to the corresponding Macaca mullta macaque sequence. Thus, experimental treatments with these sequences may be performed in monkeys prior to use in humans.

Those skilled in the art will appreciate that various omissions, additions and modifications may be made to the invention as described above without departing from the scope thereof, and all such modifications and changes are intended to be within the scope of the invention as defined in the appended claims. All cited references, patents, patent applications, or other documents are incorporated herein by reference.

In the sequence listing below, WO2007/058894 discloses SEQ ID NOs 1-116. SEQ ID NOs 117-242 is the SEQ ID NOs 1-126 disclosed in PCT/US 2007/10557. SEQ IDsNOs 243-246 are novel sequences in the present application and are preferred oligomers of the present invention.

Table 4: splice switching oligomers targeting human TNFR 2: upper case LNA, lower case DNA) -note that SEQ ID No 243 targets mouse TNFR 2.

3378

SEQID	Name (R)	Sequence (5 '-3')	Description of the invention	Nucleobase motifs
					130	SK100	CcA cAa TcA gTc CtA g	3378 full length	CCA CAA TCA GTC CTA G
131	SK101	A cAa TcA gTc CtA g	-2nt 5′(14mer)	A CAA TCA GTC CTA G
					132	SK102	Aa TcA gTc CtA g	-4nt 5′(12mer)	AA TCA GTC CTA G
133	SK103	TcA gTc CtA g	-6nt 5′(10mer)	TCA GTC CTA G
					134	SK104	CcA cAa TcA gTc Ct	-2nt 3′(14mer)	CCA CAA TCA GTC CT
135	SK105	CcA cAa TcA gTc	-4nt 3′(12mer)	CCA CAA TCA GTC
					136	SK106	CcA cAa TcA g	-6nt 3′(10mer)	CCA CAA TCA G
137	SK107	Ca CaA tCa GtC cTa	-1nt 5′；-1nt 3′(14mer)	CA CAA TCA GTC CTA
					138	SK108	Ca CaA tCa GtC c	-1nt 5′；-3nt 3′(12mer)	CA CAA TCA GTC C
139	SK109	A cAa TcA gTc Ct	-2nt 5′；-2nt 3′(12mer)	A CAA TCA GTC CT
					140	SK110	CaA tCa GtC cTa	-3nt 5′；-1nt 3′(12mer)	CAA TCA GTC CTA
141	SK111	Ca CaA tCa Gt	-1nt 5′；-5nt 3′(10mer)	CA CAA TCA GT
					142	SK112	A cAa TcA gTc	-2nt 5′；-4nt 3′(10mer)	A CAA TCA GTC
143	SK113	CaA tCa GtC c	-3nt 5′；-3nt 3′(10mer)	CAA TCA GTC C
					144	SK114	Aa TcA gTc Ct	-4nt 5′；-2nt 3′(10mer)	AA TCA GTC CT
145	SK115	AtCa GtC cTa	-5nt 5′；-1nt 3′(10mer)	A TCA GTC CTA

3379

SEQID	Name (R)	Sequence (5 '-3')	Description of the invention	Nucleobase motifs
					146	SK116	CaG tCc TaG aAa GaA a	3379 full length	CCA CAA TCA GTC CTA G
147	SK117	G tCc TaG aAa GaA a	-2nt 5′(14mer)	G TCC TAG AAA GAA A
					148	SK118	Cc TaG aAa GaA a	-4nt 5′(12mer)	CC TAG AAA GAA A
149	SK119	TaG aAa GaA a	-6nt 5′(10mer)	TAG AAA GAA A

150	SK120	CaG tCc TaG aAa Ga	-2nt 3′(14mer)	CAG TCC TAG AAA GA
					151	SK121	CaG tCc TaG aAa	-4nt 3′(12mer)	CAG TCC TAG AAA
152	SK122	CaG tCc TaG a	-6nt 3′(10mer)	CAG TCC TAG A
					153	SK123	Ag TcC tAg AaA gAa	-1nt 5′；-1nt3′(14mer)	AG TCC TAG AAA GAA
154	SK124	Ag TcC tAg AaA g	-1nt 5′；-3nt 3′(12mer)	AG TCC TAG AAA G
					155	SK125	G tCc TaG aAa Ga	-2nt 5′；-2nt 3′(12mer)	G TCC TAG AAA GA
156	SK126	TcC tAg AaA gAa	-3nt 5′；-1nt 3′(12mer)	TCC TAG AAA GAA
					157	SK127	Ag TcC tAg Aa	-1nt5′；-5nt 3′(10mer)	AG TCC TAG AA
158	SK128	G tCc TaG aAa	-2nt 5′；-4nt 3′(10mer)	G TCC TAG AAA
					159	SK129	TcC tAg AaA g	-3nt 5′；-3nt3′(10mer)	TCC TAG AAA G
160	SK130	Cc TaG aAa Ga	-4nt 5′；-2nt 3′(10mer)	CC TAG AAA GA
					161	SK131	C tAg AaA gAa	-5nt 5′；-1nt 3′(10mer)	C TAG AAA GAA

3384

SEQID	Name (R)	Sequence (5 '-3')	Description of the invention	Nucleobase motifs
					162	SK132	AcT tTt CaC cTg GgT c	3384 full Length	CCA CAA TCA GTC CTA G
163	SK133	T tTt CaC cTg GgT c	-2nt 5′(14mer)	T TTT CAC CTG GGT C
					164	SK134	Tt CaC cTg GgT c	-4nt 5′(12mer)	TT CAC CTG GGT C
165	SK135	CaC cTg GgT c	-6nt 5′(10mer)	CAC CTG GGT C
					166	SK136	AcT tTt CaC cTg Gg	-2nt 3′(14mer)	ACT TTT CAC CTG GG
167	SK137	AcT tTt CaC cTg	-4nt 3′(12mer)	ACT TTT CAC CTG
					168	SK138	AcT tTt CaC c	-6nt 3′(10mer)	ACT TTT CAC C
169	SK139	Ct TtT cAc CtG gGt	1nt5′；-1nt 3′(14mer)	CT TTT CAC CTG GGT
					170	SK140	Ct TtT cAc CtGg	-1nt 5′；3nt 3′(12mer)	CT TTT CAC CTG G
171	SK141	T tTt CaC cTg Gg	-2nt 5′；-2nt 3′(12mer)	T TTT CAC CTG GG
					172	SK142	TtT cAc CtG gGt	-3nt 5′；-1nt 3′(12mer)	TTT CAC CTG GGT
173	SK143	Ct TtT cAc Ct	-1nt 5′；-5nt 3′(10mer)	CT TTT CAC CT
					174	SK144	T tTt CaC cTg	-2nt 5′；-4nt 3′(10mer)	T TTT CAC CTG
175	SK145	TtT cAc CtGg	-3nt 5′；-3nt 3′(10mer)	TTT CAC CTG G
					176	SK146	Tt CaC cTgGg	-4nt5′；-2nt 3′(10mer)	TT CAC CTG GG
177	SK147	T cAc CtG gGt	-5nt 5′；-1nt 3′(10mer)	T CAC CTG GGT

Upper case LNAs, preferably oxygen LNAs (superscript o), preferably phosphorothioate linkages subscript s, lower case DNA).^mC ═ preferably 5-methylcytosine.

Tables 1-3 are according to tables 1-3 of WO2007/058894 expressly incorporated herein.

Further embodiments of the invention:

the present invention provides methods of treating an inflammatory disease or condition comprising administering to a subject one or more Splice Switching Oligomers (SSOs) in an amount that reduces the activity of a ligand of the Tumor Necrosis Factor Receptor (TNFR) superfamily for a period of time, wherein said one or more SSOs are capable of altering the splicing of pre-messenger RNAs encoding said receptor, thereby increasing the production of a stably secreted ligand-bound form of said receptor.

In one embodiment, the mammalian receptor is selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF5, TNFRSF8 and TNFRSF 1A.

In one embodiment, the receptor is human TNFRSF1A or human TNFRSF 1B. In one embodiment, the receptor is human TNFRSF 1B.

In one embodiment, the ligand is TNF- α, RANKL, CD40L LT- α, or LT- β.

In one embodiment, the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), hepatitis, sepsis, alcoholic liver disease, and non-alcoholic steatosis.

In one embodiment of the method of treating an inflammatory disease or condition, two or more SSOs are administered.

In one embodiment the receptor is TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF5 or TNFRSF1A and said altering splicing of said pre-messenger RNA comprises excising exon 7 or exon 8, or both, from said pre-messenger RNA.

In one embodiment, said altering splicing of said pre-messenger RNA comprises excision of exon 7.

In one embodiment, the receptor is human TNFRSF1A or human TNFRSF1B, and the SSO comprises a peptide sequence that is identical to a sequence from SEQ ID Nos: 1, 2,3 or 4, from at least 10 to at least 20 nucleotides complementary to the contiguous sequence.

In one embodiment, the SSO sequence comprises a sequence selected from the group consisting of SEQ ID Nos: 74, 75, 77, 78, 80, 82, 84 and 86-89.

In one embodiment, the SSOs comprise one or more nucleotides or nucleosides independently selected from the group consisting of: 2 ' -deoxyribonucleotides, 2 ' O-Me ribonucleotides, 2 ' O-MOE ribonucleotides, Hexitol (HNA) nucleotides or nucleosides, 2 ' O-4 ' C-linked bicyclic furonitrosourea (LNA) nucleotides or nucleosides, any phosphorothioate analogue described above, any Peptide Nucleic Acid (PNA) analogue described above; any methylphosphonate analog described above, any peptide nucleic acid analog described above; any of the above N3 '→ P5' (N3 'fwdarw P5') phosphoramidate analogs, any of the above phosphorodiamidite morpholino nucleotide analogs, and combinations thereof.

In one embodiment, the SSOs comprise one or more nucleotides or nucleosides independently selected from the group consisting of 2 ' O-Me ribonucleotides and 2 ' O-4 ' C-linked bicyclic furonitrosourea (LNA) nucleotides or nucleosides. In one embodiment, the administration is parenteral, topical, oral, rectal, or pulmonary.

In one embodiment, the present invention provides a method of increasing production of a stably secreted ligand-binding form of a receptor from the TNFR superfamily in a cell, comprising administering to the cell one or more Splice Switching Oligomers (SSOs), wherein the one or more SSOs are capable of altering splicing of a pre-messenger RNA encoding the receptor, thereby increasing production of the stably secreted ligand-binding form of the receptor.

In one embodiment, the method is performed in vivo.

In one embodiment, the receptor is a mammalian receptor selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF5, TNFRSF8 and TNFRSF 1A.

In one embodiment, the receptor is human TNFRSF1A or human TNFRSF 1B.

In one embodiment, the receptor is human TNFRSF 1B.

In one embodiment, the SSO comprises a peptide derived from SEQ ID Nos: 1, 2,3 or 4, from at least 10 to at least 20 nucleotides complementary to the contiguous sequence.

In one embodiment, the present invention provides SSOs of at least 10 to at least 20 nucleotides, which are capable of altering the splicing of pre-messenger RNAs encoding receptors from the TNFR superfamily, and thus capable of increasing the production of a stably secreted ligand-binding form of said receptors.

In one embodiment, the receptor is a mammalian receptor selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF5, TNFRSF8 and TNFRSF 1A.

In one embodiment, the receptor is human TNFSF1A or human TNFRSF 1B. In one embodiment, the receptor is human TNFRSF 1B.

In one embodiment, the SSO comprises a sequence identical to a sequence from SEQ ID Nos: 1, 2,3 or 4, from at least 10 to at least 20 nucleotides complementary to the contiguous sequence.

In one embodiment, the SSOs comprise one or more nucleotides or nucleosides independently selected from the group consisting of: 2 ' -deoxyribonucleotides, 2 ' O-Me ribonucleotides, 2 ' O-MOE ribonucleotides, Hexitol (HNA) nucleotides or nucleosides, 2 ' O-4 ' C-linked bicyclic furonitrosourea (LNA) nucleotides or nucleosides, any phosphorothioate analogue described above, any Peptide Nucleic Acid (PNA) analogue described above; any methylphosphonate analog described above, any peptide nucleic acid analog described above; any of the N3 '→ P5' phosphoramidate analogs described above, any phosphorodiamidite morpholino nucleotide analogs described above, and combinations thereof.

In one embodiment, the 2 'O-4' C-linked bicyclic furonitrosourea (LNA) nucleotide or nucleoside is a 2 'O-4' C- (methylene) -furonitrosourea nucleotide or nucleoside, or a 2 'O-4' C- (ethylene) furourea nucleotide or nucleoside, respectively. In one embodiment, the SSOs comprise one or more nucleotides or nucleosides independently selected from the group consisting of 2 ' O-Me ribonucleotides and 2 ' O-4 ' C-linked bicyclic furonitrosourea (LNA) nucleotides or nucleosides.

In one embodiment, the SSO sequence comprises a sequence selected from the group consisting of SEQ ID Nos: 8, 9, 14, 17-21, 24-29, 32, 33, 38-42, 44-46, 50-52, 55-57, 60, 68-71, 74, 75, 77, 78, 80, 82, 84, and 86-89.

In one embodiment, the present invention provides a pharmaceutical composition comprising SSO and a pharmaceutically acceptable carrier.

In one embodiment, the SSO comprises a peptide derived from SEQ ID Nos: 1, 2,3 or 4, from at least 10 to at least 20 nucleotides complementary to the contiguous sequence.

In one embodiment, the present invention provides an isolated protein capable of binding Tumor Necrosis Factor (TNF), said protein having a sequence comprising amino acids encoded by cDNA derived from a mammalian Tumor Necrosis Factor Receptor (TNFR) gene, wherein the cDNA comprises, in a contiguous arrangement from 5 'to 3', a codon encoding a first amino acid following the cleavage site of the signal sequence of said gene, which crosses (through) exon 6 of said gene, exon 8 of said gene and exon 10 of said gene; or a codon encoding the first amino acid of the open reading frame of the gene, which crosses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene.

In one embodiment, the TNF is TNF-alpha.

In one embodiment, the protein comprises at least one chemical processing or post-translational modification, wherein the modification is selected from the group consisting of acetylation, acylation, amidation, ADP-ribosylation, glycosylation, methylation, pegylation, prenylation, phosphorylation, or cholesterol binding.

In one embodiment, the receptor is TNFR1, such as human TNFR1, and in one embodiment, the receptor is TNFR2, such as human TNFR 2. In one embodiment, the protein comprises a sequence selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 6, amino acids 30-417 of SEQ ID NO: 8. SEQ ID NO: 8, amino acids 30-416 of SEQ ID NO: 10. SEQ ID NO: 10, amino acids 23-435 of SEQ ID NO: 12 and SEQ ID NO: 12 amino acids 23-448.

In one embodiment, the invention provides a pharmaceutical composition comprising a protein of the invention in admixture with a pharmaceutically acceptable carrier. In one embodiment, the invention provides a composition comprising a purified protein of the invention.

In one embodiment, the invention provides a method of treating an inflammatory disease or condition comprising administering to a subject a pharmaceutical composition of the invention for a time and in an amount effective to reduce the activity of TNF.

In one embodiment, the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), hepatitis associated with hepatitis a virus, hepatitis associated with hepatitis b virus, hepatitis associated with hepatitis c virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease and non-alcoholic steatosis. In one embodiment, the present invention provides an isolated nucleic acid derived from a mammalian Tumor Necrosis Factor Receptor (TNFR) gene and encoding a protein capable of binding Tumor Necrosis Factor (TNF), wherein the cDNA of said protein comprises, in a contiguous arrangement from 5 'to 3', a codon encoding a first amino acid following the cleavage site of the signal sequence of said gene, which crosses exon 6 of said gene, exon 8 of said gene and exon 10 of said gene; or a codon encoding the first amino acid of the open reading frame of the gene, which crosses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene. In this embodiment, the sequence of the protein comprises a sequence selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO: 6, amino acids 30-417 of SEQ ID NO: 8. SEQ ID NO: 8, amino acids 30-416 of SEQ ID NO: 10. SEQ ID NO: 10, amino acids 23-435 of SEQ ID NO: 12 and SEQ ID NO: 12 amino acids 23-448. In one embodiment, the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 5, nucleotides 1-1251 of SEQ ID NO: 5, nucleotides 88-1251 of SEQ ID NO: 7, nucleotides 1-1248 of SEQ ID NO: 7, nucleotides 88-1248 of SEQ ID NO: 9, nucleotides 1-1305 of SEQ ID NO: 9, nucleotides 67-1305, SEQ ID NO: 11, nucleotides 1-1344 of SEQ ID NO: 11 from nucleotide 67 to nucleotide 1344. In one embodiment, the invention provides an expression vector comprising a nucleic acid of the invention operably linked to a regulatory sequence.

In one embodiment, the invention provides a method of enhancing the level of a TNF antagonist in a mammal comprising transforming cells of said mammal with an expression vector of the invention such that they express said TNF antagonist, wherein said vector drives the expression of said TNFR. In one embodiment, the mammal is a human, e.g., the human is an individual having an inflammatory disease or condition.

In one embodiment, the expression vector is a plasmid or virus.

In one embodiment, the cell is transformed in vivo. In one embodiment, the cell is transformed in vitro.

In one embodiment, the expression vector comprises a tissue-specific promoter, which may be derived from, for example, hepatocytes or macrophages.

In one embodiment, the cell is selected from the group consisting of a liver cell, a hematopoietic cell, a spleen cell, and a muscle cell.

The present invention provides cells, such as mammalian cells, insect cells, or microbial cells, transformed with the expression vectors of the invention.

The present invention provides a method of producing a protein capable of binding Tumor Necrosis Factor (TNF), comprising culturing a cell of the invention under conditions suitable for expression of the protein, and harvesting the protein.

The invention provides a pharmaceutical composition comprising a nucleic acid or vector of the invention in admixture with a pharmaceutically acceptable carrier. The invention provides methods of treating an inflammatory disease or condition comprising administering to a subject an expression vector of the invention for a time and in an amount sufficient to reduce the activity of TNF, e.g., the activity of TNF-alpha.

In one embodiment, the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), hepatitis associated with hepatitis a virus, hepatitis associated with hepatitis b virus, hepatitis associated with hepatitis c virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease and non-alcoholic steatosis.

In one embodiment, the invention provides a method of treating an inflammatory disease or condition comprising administering to a subject one or more Splice Switching Oligomers (SSOs) of the invention for a period of time, in an amount effective to reduce TNF activity, wherein said one or more SSOs alter splicing of pre-messenger RNA encoding mammalian tumor necrosis factor receptor 2(TNFR2) (or TNFR1), thus being capable of increasing the production of a protein capable of binding to Tumor Necrosis Factor (TNF), the protein having a sequence comprising amino acids encoded by cDNA derived from the receptor gene, wherein the cDNA comprises from 5 'to 3' of a contiguous arrangement of codons encoding the first amino acid after the cleavage site of the signal sequence of said gene, it crosses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene; or a codon encoding the first amino acid of the open reading frame of the gene, which crosses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene.

In one embodiment, the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), hepatitis associated with hepatitis a virus, hepatitis associated with hepatitis b virus, hepatitis associated with hepatitis c virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease and non-alcoholic steatosis. In one embodiment, the administration is parenteral, topical, oral, rectal, or pulmonary.

In one embodiment, the present invention provides a Splice Switching Oligomer (SSO) comprising at least 8 nucleotides, wherein said SSOs are capable of altering the splicing of pre-messenger RNA encoding mammalian tumor necrosis factor receptor 2(TNFR2) (or TNFR1) to increase the production of a protein capable of binding Tumor Necrosis Factor (TNF), said protein having a sequence comprising amino acids encoded by a cDNA derived from said acceptor gene, wherein the cDNA comprises from 5 'to 3' in contiguous arrangement, following the cleavage site of the signal sequence of said gene, a codon encoding a first amino acid that traverses exon 6 of said gene, exon 8 of said gene and exon 10 of said gene; or a codon encoding the first amino acid of the open reading frame of the gene, which crosses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene.

In one embodiment, the present invention provides a polypeptide comprising a sequence identical to a sequence from SEQ ID NO: 13 is a SSO of at least 8 nucleotides complementary to the contiguous sequence of seq id no.

In one embodiment, the SSO sequence comprises a sequence selected from the group consisting of SEQ ID Nos: 14, 30, 46, 70, 71, 72 and 73, and subsequences of at least 8 nucleotides thereof.

In one embodiment, the SSO sequence comprises a sequence selected from the group consisting of SEQ ID Nos: 14-61.

The present invention provides a method of increasing production of a protein capable of binding Tumor Necrosis Factor (TNF) in a cell, comprising administering to the cell one or more Splice Switching Oligomers (SSOs), wherein the protein has a sequence comprising amino acids encoded by a cDNA derived from a mammalian tumor necrosis factor receptor 2(TNFR2) (or TNFR1) gene, wherein the cDNA comprises, in a 5 'to 3' contiguous arrangement, a codon encoding a first amino acid following the cleavage site of the signal sequence of the gene, that traverses exon 6 of the gene, exon 8 of the gene and exon 10 of the gene; or a codon encoding the first amino acid of the open reading frame of said gene that crosses exon 6 of said gene, exon 8 of said gene and exon 10 of said gene, wherein said one or more SSOs are capable of altering splicing of a pre-messenger RNA encoding said receptor such that production of said protein is increased. In one embodiment, the method is performed in vivo.

The present invention provides a pharmaceutical composition comprising the SSO of the invention and a pharmaceutically acceptable carrier.

Examples

The following examples are equivalent to those described in PCT/US 2007/10557.

Example 1

An oligonucleotide. Table 6 lists chimeric Locked Nucleic Acid (LNA) SSOs having alternating 2 ' deoxy-and 2 ' O-4 ' - (methylene) -bicyclic-ribonucleoside phosphorothioates and sequences as described above. These sequences were synthesized by Santaris Pharma, Denmark. For each SSO, the nucleoside at the 5 ' -terminus is a 2 ' oxy-4 ' -methylene-ribonucleoside and the nucleoside at the 3 ' -terminus is a 2 ' deoxyribonucleoside. Table 7 shows the sequences of chimeric LNA SSOs with alternating 2 '-oxy-methyl-ribonucleoside-phosphorothioate (2' -OMe) and 2 'O-4' - (methylene) -bicyclic-ribonucleoside-phosphorothioate. These sequences were synthesized by Santaris Pharma, Denmark. LNA is shown in upper case and 2' -OME in lower case.

Cell culture and transfection. L929 cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (37 ℃, 5% CO 2). For transfection, L929 cells were seeded into 24-well plates, 10 per well⁵Individual cells, 24 hours later were transfected. The oligonucleotide at the indicated concentration was mixed with 2. mu.l Lipofectamine according to the manufacturer's instructions^TM2000 transfection reagent (Invitrogen) formed a complex. The nucleotide/lipid complexes are then applied to the cells and incubated for 24 hours. Aspirating the medium and applying TRI-reagent^TM(MRC, Cincinnati, OH) cells were harvested.

RT-PCR. Total RNA was isolated using TRI reagent (MRC, Cincinnati, OH),and passed using rTth polymerase (Applied Biosystems) according to the supplier's instructionsmRNA for TNFR1 or TNFR2 was amplified by RT-PCR. Approximately 200ng of RNA was used per reaction. The primers used in the examples described herein are included in table 2. Performing PCR circulation: 60 seconds at 95 ℃; 56 ℃ for 30 seconds; at 72 c, 60 seconds, for a total of 22-30 cycles.

In some cases, Cy 5-labeled dCTP (GE healthcare) was included in the PCR step for visualization (0.1. mu.l per 50. mu.l PCR reaction). The PCR products were separated on a 10% native polyacrylamide gel and Typhoon was used^TMA 9400 scanner (GE Healthcare) visualized a Cy 5-labeled strip. Using ImageQuant^TMThe (GE Healthcare) software quantifies the scans. Alternatively, in the absence of Cy 5-labeled dCTP, the PCR products were separated on a 1.5% agarose gel containing traces of ethidium bromide for visualization.

PCR

According to the instructions of the manufacturerPCR was performed with Taq DNA polymerase (Invitrogen). A50. mu.l reaction was performed using about 30pmol of forward and reverse primers. Primers used in the examples described herein are included in table 5. Unless otherwise stated, the thermocycling reaction was carried out according to the following conditions: 94 ℃ for 3 minutes; then 30-40 cycles of 94 ℃, 30 seconds, 55 ℃, 30 seconds, and 72 ℃, 105 seconds; followed by 72 ℃ for 3 minutes. The products were analyzed on a 1.5% agarose gel and visualized with ethidium bromide.

Table 5: RT-PCR and PCR primers

SEQID.	Name (R)	Sequence 5 'to 3'
					Human beingTNFR2
190	TR001	ACT GGG CTT CAT CCC AGC ATC
			191	TR002	CAC CAT GGC GCC CGT CGC CGT CTG G
192	TR003	CGA CTT CGC TCT TCC AGT TGA GAA GCC CTT GTG CCT GCA G
			193	TR004	TTA ACT GGG CTT CAT CCC AGC ATC
194	TR005	CTG CAG GCA CAA GGG CTT CTC AAC TGG AAG AGC GAA GTC G
			195	TR026	TTA ACT GGG CTT CAT CCC AGC
196	TR027	CGA TAG AAT TCA TGG CGC CCG TCG CCG TCT GG
			197	TR028	CCT AAC TCG AGT TAA CTG GGC TTC ATC CCA GC
198	TR029	GAC TGA GCG GCC GCC ACC ATG GCG CCC GTC GCC GTC TGG
			199	TR030	CTA AGC GCG GCC GCT TAA CTG GGC TTC ATC CCA GCA TC
200	TR047	CGT TCT CCA ACA CGA CTT CA
			201	TR048	CTT ATC GGC AGG CAA GTG AGG
202	TR049	ACT GAA ACA TCA GAC GTG GTG TGC
			203	TR050	CCT TAT CGG CAG GCA AGT GAG
		Human beingTNFR1
			204	TR006	CCT CAT CTG AGA AGA CTG GGC G
205	TR007	GCC ACC ATG GGC CTC TCC ACC GTG C
			206	TR008	GGG CAC TGA GGA CTC AGT TTG TGG GAA ATC GAC ACC TG
207	TR009	CAG GTG TCG ATT TCC CAC AAA CTG AGT CCT CAG TGC CC
			208	TR010	CAC CAT GGG CCT CTC CAC CGT GC
209	TR011	TCT GAG AAG ACT GGG CG
			210	TR031	CGA TAG GAT CCA TGG GCC TCT CCA CCG TGC
211	TR032	CCT AAC TCG AGT CAT CTG AGA AGA CTG GGC G
			212	TR033	GAC TGA GCG GCC GCC ACC ATG GGC CTC TCC ACC GTG C
213	TR034	CTA AGC GCG GCC GCT CAT CTG AGA AGA CTG GGC G
					MouseTNFR2
214	TR012	GGT CAG GCC ACT TTG ACT GC
			215	TR013	CAC CGC TGC CCC TAT GGC G
216	TR014	CAC CGC TGC CAC TAT GGC G

217	TR015	GGT CAG GCC ACT TTG ACT GCA ATC
			218	TR016	GCC ACC ATG GCG CCC GCC GCC CTC TGG
219	TR017	GGC ATC TCT CTT CCA ATT GAG AAG CCC TCC TGC CTA CAA AG
			220	TR018	CTT TGT AGG CAG GAG GGC TTC TCA ATT GGA AGA GAG ATG CC
221	TR019	GGC CAC TTT GAC TGC AAT CTG
			222	TR035	CAC CAT GGC GCC CGC CGC CCT CTG G
223	TR036	TCA GGC CAC TTT GAC TGC AAT C
			224	TR037	CGA TAG AAT TCA TGG CGC CCG CCG CCC TCT GG
225	TR038	CCT AAC TCG AGT CAG GCC ACT TTG ACT GCA ATC
			226	TR039	GAC TGA GCG GCC GCC ACC ATG GCG CCC GCC GCC CTC TGG
227	TR040	CTA AGC GCG GCC GCT CAG GCC ACT TTG ACT GCA ATC
			228	TR045	GAG CCC CAA ATG GAA ATG TGC
229	TR046	GCT CAA GGC CTA CTG CAT CC
					MouseTNFR1
230	TR020	GGT TAT CGC GGG AGG CGG GTC G
			231	TR021	GCC ACC ATG GGT CTC CCC ACC GTG CC
232	TR022	CAC AAA CCC CCA GGA CTC AGT TTG TAG GGA TCC CGT GCC T
			233	TR023	AGG CAC GGG ATC CCT ACA AAC TGA GTC CTG GGG GTT TGT G
234	TR024	CAC CAT GGG TCT CCC CAC CGT GCC
			235	TR025	TCG CGG GAG GCG GGT CGT GG
236	TR041	CGA TAG TCG ACA TGG GTC TCC CCA CCG TGC C
			237	TR042	CCT AAG AAT TCT TAT CGC GGG AGG CGG GTC G
238	TR043	GAC TGA GCG GCC GCC ACC ATG GGT CTC CCC ACC GTG CC
			239	TR044	CTA AGC GCG GCC GCT TAT CGC GGG AGG CGG GTC G

And (3) culturing human liver cells. Human hepatocytes in suspension were obtained from ADMET technology or UNC cell Metabolism and Transport center (UNC Cellular Metabolism and Transport Core) of UNC-Chapel Hill. Cells were washed and suspended in RPMI 1640 supplemented with 10% FBS, 1mg/mL human insulin and 13nM DexamethaSONe. Hepatocytes were plated in 6-well plates with 3ml of medium per plate, 10⁵And (4) cells. After 1-1.5 hours, non-adherent cells were removed and the medium was replaced with RPMI 1640 without FBS supplemented with 1mg/mL human insulin and 130nM DexamethaSONe.

For delivery of SSOs into hepatocytes in 6-well plates, 10ml of 5mM stock SSO was diluted to 100ml of OPTI-MEM^TM，4ml Lipofectamine^TM2000 was diluted to 100ml OPTI-MEM^TM. 200ml of complex solution was then applied to cells in 6-well plates containing 2800ml of medium, for a total of 3000ml of liquid. The final SSO concentration was 17 nM. After 24 hours, in TRI-Reagent^TMHarvesting the cells. Total RNA was isolated according to the manufacturer's instructions. Approximately 200ng of total RNA was subjected to reverse transcription-PCR (RT-PCR).

And (4) ELISA. To determine the level of soluble TNFR2 in cell culture medium or serum, slave R was used&Obtained by D system (Minneapolis, MN)Mouse sTNF RII ELISA kit or slave R&Obtained by D system (Minneapolis, MN)Human stnfriielisa kit. Detect and makeThe antibodies used may also detect the protease cleaved form of the receptor. The ELISA plates were read using a fully automatic quantitative plotter set at 450nm with a wavelength correction set at 570 nm.

For in vivo mouse experiments, blood from animals was coagulated at 37 ℃ for 1 hour and centrifuged at 4 ℃ at 14,000rpm (Joua BRA4i centrifuge) for 10 minutes. Sera were collected and tested using 50ml mouse sera diluted 1: 10 according to the manufacturer's instructions.

L929 cytotoxicity assay. Treated with 0.1ng/mL of TNF-. alpha.and 1mg/mL of actinomycin D in the presence of 10% serum from mice plated on 96-well plates, 10 per well⁴Individual L929 cells, cells at 37 degrees C growth-24 hours, wherein the mouse with a total of 100mL complete MEM medium (containing 10% qualified FBS) in the marker oligonucleotide treatment. The control group was plated in 10% serum from untreated mice. After 24 hours, by adding 20mL CellTiterAQ_ueousOne Solution Reagent (Promega) and the absorbance at 490nm measured with a fully automatic quantitative plotter microplate reader. Cell viability was normalized to untreated cells.

Protein spotting (Western blots). Twenty ml of medium or 20mg of lysate were loaded to 4-12%Polyacrylamide gel (Invitrogen) in each well. The gel was run at 200V for 40 minutes. 30V transfer for 1 hour, proteins were transferred to Invitroron^TMPVDF membrane (Invitrogen) and thenBlocking Buffer (Blocking Buffer) (Pierce) the membrane was blocked for 1 hour. The membranes were incubated with rabbit polyclonal antibody (Abeam) recognizing the C-terminus of human and mouse TNFR2 at room temperatureAfter washing three times with PBS-T buffer (1xPBS, 0.1% Tween-20) for 3 hours, the membrane was incubated with a secondary goat anti-rabbit antibody (Abeam) for 1 hour at room temperature, followed by washing three more times with PBS-T buffer. Finally, ECL Plus is used according to the introduction of the manufacturer^TM(GEHealthcare) protein was detected and photographed.

Example 2 splicing conversion Activity of SSO on TNFR mRNA

Table 6 shows the splice switching activity of SSOs having the sequence as described in U.S. application No.11/595,485 and targeting mouse and human TNFRs. At least 8 of the SSOs targeting exon 7 of mouse TNFR2 produced some mutfr 2 Δ 7 mRNA. In particular, SSO 3312 and 3305 induced at least 50% of exon 7 deletions; SSO 3305 treatment resulted in almost complete deletions. Transfected into primary human hepatocytes, and at least 7 of the SSOs targeting exon 7 of human TNFR2 produced some huTNFR2 Δ 7 mRNA. In particular, SSOs 3378, 3379, 3384 and 3459 induced at least 75% of exon 7 deletion (fig. 2B), and huTNFR2 Δ 7 was significantly induced into the extracellular medium (fig. 2A).

Table 6: splicing conversion Activity of SSO

Table 7 contains sequences of 10-nucleotide chimeric SSOs with alternating 2 '-oxo-methyl-ribonucleoside-phosphorothioate (2' -OMe) and 2 'oxo-4' - (methylene) -bicyclic-ribonucleoside-phosphorothioate. These SSOs target exon 7 of mouse TNFR 2.

Table 7: mouse-targeted SSO of LNA/2' -OMe-ribonucleoside-phosphorothioate chimera

SEQ ID.	Name (R)	Sequence 5 'to 3'*
			178	3274	AgAgCaGaAcCtTaCt
179	3837	gAaCcTuAcT
			180	3838	aGaGcAgAaC
181	3839	gAgCaGaAcC
			182	3840	aGcAgAaCcT
183	3841	gCaGaAcCuT
			184	3842	cAgAaCcTuA
185	3843	aGaAcCuTaC

^*The capital letter is 2 'O-4' - (methylene) -bicyclic-ribonucleoside; the lower case letters being 2' -OMe

To analyze the in vitro splicing switching activity of the SSOs listed in table 7, L929 cells can be cultured and seeded as described in example 1. To deliver each of the SSOs in Table 7 to L929 cells, the SSOs were diluted to 50ml OPTI-MEM^TMThen 50ml Lipofectamine was added^TM2000 mixture (1 part Lipofectamine)^TM2000 and 25 parts of OPTI-MEM^TM) And incubated for 20 minutes. Next, 400ml of serum-free medium was added to the SSOs and administered to the cells in the 24-well plate. The final concentration of SSO was 50 or 100 nM. After 24 hours, the cells were harvested to 800ml TRI-Reagent^TMIn (1). Total RNA was isolated according to the manufacturer's instructions and analyzed by RT-PCR using the forward primer TR045(SEQ ID NO: 228) and the reverse primer TR046(SEQ ID NO: 229) (FIG. 3).

To analyze the in vivo splicing switching activity of the SSOs listed in Table 7, mice were intraperitoneally injected with the SSOs listed in Table 4 at a dose of 25 mg/kg/day for a total of 5 days. Mice were bled prior to injection and again 1, 5, 10 days after the last injection. The concentration of soluble TNFR2 Δ 7 in serum was measured by ELISA before the first injection and 10 days after the last injection (fig. 4B). Mice were sacrificed on day 10 and total RNA obtained from 5-10mg liver was analyzed by RT-PCR using the forward primer TR045(SEQ ID NO: 228) and the reverse primer TR046(SEQ ID NO: 229).

All in vitro tested SSO 3274 SSOs subsequences of 10 nucleotides produced at least some mutfr 2 Δ 7mRNA (fig. 3). In particular, SSOs 3839, 3840 and 3841 showed greater splicing switching activity than the longer 16 nucleotide SSO 3274 from which they were derived. Three 10-nucleotide SSOs 3839, 3840, 3841, which showed the greatest activity in vitro, also produced large amounts of mutFRNR 2 Δ 7mRNA (FIG. 4A) and soluble mutFRNR 2 Δ 7 protein (FIG. 4B) in mice.

To evaluate the effect of SSO length on the splicing turnover activity of human TNFR2, cells were treated with SSOs of different lengths. Primary human hepatocytes were transfected with labeled SSOs selected from table 4. These SSOs were synthesized by Santaris Pharma, Denmark, using alternating 2 ' deoxy and 2 ' O-4 ' - (methylene) -bicyclic-ribonucleoside phosphorothioates. The 5 ' -terminal nucleoside of each SSO is a 2 ' O-4 ' -methylene-ribonucleoside and the 3 ' -terminal nucleoside is a 2 ' deoxyribonucleoside. These SSOs are 10-, 12-, 14-, or 16-nucleotides in length. The concentration of soluble TNFR2 Δ 7 was determined by ELISA (fig. 5, upper grid). Total RNA was analyzed for splicing conversion activity by RT-PCR (FIG. 5, bottom grid).

Example 3 analysis of SSO-induced splicing junctions of TNFR2 splice variants

To confirm that the splicing transition of SSO produced the expected TNFR2 Δ 7mRNA in mouse and human cells, SSO-induced TNFR2 Δ 7mRNA was analyzed by RT-PCR and sequenced.

A mouse. Mice were injected intraperitoneally with SSO 3274 at a dose of 25 mg/kg/day for 10 days. The mice were then sacrificed and total RNA from the liver was analyzed by RT-PCR using the forward primer TR045(SEQ ID NO: 228) and the reverse primer TR046(SEQ ID NO: 229). The products were analyzed on a 1.5% agarose gel and the product encoding TNFR2 Δ 7 was isolated using standard molecular biology techniques. The isolated TNFR2 Δ 7 product was amplified by PCR using the same primers and sequenced (fig. 6B). The sequence data contained sequence CTCTCTTCCAATTGAGAAGCCCTCCTGC (nt 777-804 of SEQ ID NO: 127), which also confirmed that SSO-induced TNFR 2. delta.7 mRNA lacks exon 7, with exon 6 ligated directly to exon 8.

Human hepatocytes. Primary human hepatocytes were transfected with SSO 3379 as described in example 1. Total RNA was isolated 48 hours after transfection. Random hexamer primers were used with Superscript according to the manufacturer's instructions^TMII reversionThe RNA was converted to cDNA by a transcriptase (Invitrogen). PCR was performed on the cDNA using the forward primer TR049(SEQ ID NO: 202) and the reverse primer TR050(SEQ ID NO: 203). The products were analyzed on a 1.5% agarose gel (FIG. 7A). Bands corresponding to TNFR2 Δ 7 were isolated and sequenced using standard molecular biology techniques (fig. 7B). The sequence data contained sequence CGCTCTTCCAGTTGAGAAGCCCTTGTGC (nucleotide 774 and 801 of SEQ ID NO: 125), which also confirmed that SSO-induced TNFR 2. delta.7 mRNA lacked exon 7, with exon 6 ligated directly to exon 8.

Example 4-SSO Induction of dose-dependent production of TNFR2 Δ 7 protein in Primary human hepatocytes

The dose response of the splicing switching activity of SSOs was tested in primary human hepatocytes. Human hepatocytes in suspension from ADMET technology were obtained. Cells were washed three times and suspended in inoculation medium (supplemented with L-glutamic acid, 10% FBS, penicillin, streptomycin and 12 nmdexemethanose). Viability of hepatocytes was assessed and plated on collagen-coated 24-well plates, 1.0x10 per well⁵And (4) cells. Typical cell viability is 85-93%. After about 24 hours, the medium was replaced with maintenance medium (inoculation medium without FBS).

To deliver each of the SSOs to hepatocytes, the SSOs were diluted to 50ml of OPTI-MEM^TMThen 50ml Lipofectamine was added^TM2000 mixture (1 part Lipofectamine)^TM2000 and 25 parts of OPTI-MEM^TM) And incubated for 20 minutes. The SSOs were then administered to cells in 24-well plates. The final SSO concentration was 1-150 nM. After 48 hours, the cells were harvested to 800ml TRI-Reagent^TMIn (1).

Total RNA from cells was analyzed by RT-PCR using the forward primer TR047(SEQ ID NO: 200) and the reverse primer TR048(SEQ ID NO: 201) (FIG. 8A). The concentration of soluble TNFR2 Δ 7 in serum was determined by ELISA (fig. 8B). Both huTNFR2 Δ 7mRNA (fig. 8A) and secreted huTNFR2 Δ 7 protein (fig. 8B) showed dose-dependent increases.

Example 5 secretion of TNFR2 splice variant from murine cells

SSOs were tested for their ability to induce production and secretion of soluble TNFR2 protein into extracellular medium. L929 cells were treated with SSOs as described in example 1 and extracellular media samples were collected 48 hours after transfection. The concentration of soluble TNFR2 in the samples was measured by ELISA (figure 9). SSOs that induce the best RNA splice transfer also secrete the most protein into the extracellular medium. In particular, SSOs 3305, 3312, and 3274 can increase soluble TNFR2 by at least 3.5-fold relative to background. Thus, induction of splice variant mRNA is correlated with production and secretion of soluble TNFR 2.

Example 6 in vivo injection of SSOs in mice MuTNFR2 Delta 7mRNA

SSO 3305 in saline was injected intraperitoneally (i.p.) into mice at a dose of 3mg/kg to 25mg/kg daily for 4 days. Mice were sacrificed on day 5 and total RNA from liver was analyzed by RT-PCR. The data show splicing switching potency similar to that found in cell culture. At a maximum dose of 25mg/kg, SSO 3305 treatment induced almost complete conversion to Δ 7mRNA (fig. 10, bottom grid).

Similar experiments performed on SSO 3274 induced approximately 20% conversion to Δ 7 mRNA. To optimize the process of SSO 3274 induction of Δ 7mRNA, the method of administration and the time from the last injection to the dead animals were varied. SSO 3274 was injected (i.p) into mice daily for 4 days. SSO treatment induced approximately 30% conversion to Δ 7mRNA in mice analyzed on day 15, while a 20% conversion was observed in mice analyzed on day 5 (fig. 10, upper grid). In addition, mice sacrificed on day 11 by day 710 of injection showed induction of 50% mRNA (fig. 10, top). These in vivo data indicate that TNFR2SSOs can produce mutfr 2 Δ 7mRNA at least 10 days after administration.

Example 7 circulating TNFR2 Δ 7

Mice were injected intraperitoneally with (i.p) SSO 3274, 3305 or control 3083 at a dose of 25 mg/kg/day for 10 days. Mice were bled prior to injection and again 1, 5, 10 days after the last injection. The concentration of soluble TNFR2 Δ 7 in serum was determined. SSO treatment can induce soluble TNFR2 Δ 7 protein levels above background for at least 10 days (fig. 11).

To test the effect of the longer time points, the experiment can be repeated except that serum samples are collected until day 27 after the last injection. The results showed that the level of soluble TNFR2 Δ 7 decreased only slightly on day 27 after the last injection (fig. 12).

Example 8 anti-TNF-alpha Activity in mouse serum

Sera from SSO 3274-treated mice were tested for anti-TNF- α activity in an L929 cytotoxicity assay. In this assay, the ability of serum to protect cultured L929 cells from the cytotoxic effects of a fixed concentration of TNF-. alpha.was determined as described in example 1. Sera from mice treated with SSO 3274, but not with control SSOs (3083 or 3272), enhanced the viability of L929 cells exposed to TNF-. alpha.at 0.1 ng/mL. Thus, SSO 3274 serum contains sufficient TNF- α antagonist to bind to and activate TNF- α, and thus can protect cells from the cytotoxic effects of TNF- α. This anti-TNF- α activity was present in animal sera 5 days and 27 days after the last injection of SSO 3274.

Example 9 comparison of TNFR2 Δ 7 produced by SSO with other anti-TNF- α antagonists

L929 cells were seeded according to the method described above. Preparation of a serum-free MEM containing 90. mu.l, 0.1ng/ml TNF-. alpha.and 1ug/ml actinomycin D, and (i) a serum-free MEM from(ii) serum from an SSO 3274 or SSO 3305 treated mouse (1.25-10%, diluted in the serum of an untreated mouse; the concentration of TNFR 2. delta.7 is determined by ELISA) or (iii)(0.45-150pg/ml) to a final volume of 100. mu.l and a final mouse serum concentration of 10%. The samples were incubated at room temperature for 30 minutes. Subsequently, the samples were applied to the plated cells and incubated at 37 ℃ for 24 hours in humid air containing 5% CO 2. By adding 20. mu.l CellTiterAQ_ueousCell viability was measured by One Solution Reagent (Promega) and absorbance at 490nm measured using a fully automatic quantitative plotter microplate reader. Cell viability was normalized to untreated cells and plotted as a function of TNF antagonist concentration (figure 14).

Example 10 stability of TNFR2 Δ 7mRNA and protein

Mice were injected intraperitoneally daily with SSO 3274 or 3272 (control) (n-5) at a dose of 25 mg/kg/day for a total of 5 days. Mice were bled prior to the start of the injection and 5, 15, 22, 27 and 35 days after the last injection. The concentration of soluble TNFR2 Δ 7 in serum was measured (fig. 15A). At sacrifice, the splicing transition of TNFR2 in liver was also determined by RT-PCR of total RNA from liver. Combining the data from example 7, it was found that the time course of TNFR2mRNA levels after SSO treatment was constructed together with the time course of TNFR2 Δ 7 protein in serum and the two were compared (FIG. 16). The data show that TNFR2 Δ 7mRNA decays at a rate approximately 4-fold faster in vivo than TNFR2 Δ 7 protein in serum. On day 35, only trace amounts of TNFR2 Δ 7mRNA were detected, but TNFR2 Δ 7 protein decreased only 20% from the highest concentration.

Example 11 Generation of human TNFR2 Δ 7

A cDNA plasmid containing the full-length human TNFR2cDNA was obtained by purchasing from OriGene (catalog No: TC119459, NM-001066.2). The cDNA was obtained by performing PCR on the plasmid using a reverse primer TR001(SEQ ID NO: 116) and a forward primer TR002(SEQ ID NO: 117). The PCR product, which contained the 1383bp TNFR2 open reading frame without a stop codon, was isolated and purified using standard molecular biology techniques. Alternatively, the full-length human TNFR2cDNA was obtained by performing RT-PCR on total RNA from human monocytes using TR001 reverse primer and TR002 forward primer. The PCR product was isolated and purified using standard molecular biology techniques.

To generate human TNFR2 Δ 7cDNA, two separate PCR reactions were performed on full-length human TNFR2cDNA to generate overlapping fragments of TNFR2 Δ 7 cDNA. In one reaction, PCR was performed on the full-length TNFR2cDNA using forward primer TR003(SEQ ID NO: 190) and reverse primer TR004(SEQ ID NO: 191). In one reaction, PCR was performed on the full-length TNFR2cDNA using reverse primer TR005(SEQ ID NO: 192) and forward primer TR 002. Finally, 2 overlapping fragments were pooled and PCR was performed using forward primer TR002 and reverse primer TR 004. PCR products predicted to contain a 1308bp TNFR2 Δ 7 open reading frame with a stop codon (SEQ ID NO: 125) were isolated and purified using standard molecular biology techniques.

Similarly, in these PCR reactions, the 1305bp open reading frame of human TNFR2 Δ 7 with a stop codon was generated using reverse primer TR001 instead of reverse primer TR 004. This also allows the addition of an in-frame C-terminal affinity purification tag, such as a His tag, when the final PCR product is inserted into an appropriate vector.

Example 12 Generation of human TNFR1 Δ 7

A cDNA plasmid containing the full-length human TNFR2cDNA was obtained by purchasing from OriGene (catalog No: TC127913, NM-001065.2). The cDNA was obtained by performing PCR on the plasmid using the reverse primer TR006(SEQ ID NO: 204) and the forward primer TR007(SEQ ID NO: 205). The PCR product of the full-length human TNFR1cDNA was isolated and purified using standard molecular biology techniques.

Alternatively, full-length human TNFR1cDNA was obtained by performing RT-PCR on total RNA from human monocytes using reverse primer TR006 and forward primer TR 007. The PCR product of the full-length human TNFR1cDNA was isolated and purified using standard molecular biology techniques.

To generate human TNFR1 Δ 7cDNA, two separate PCR reactions were performed on full-length human TNFR1cDNA to generate overlapping fragments of TNFR1 Δ 7 cDNA. In one reaction, PCR was performed on the full length TNFR1cDNA using the forward primer TR008(SEQ ID NO: 206) and the reverse primer TR 006. In one reaction, PCR was performed on the full-length TNFR1cDNA using reverse primer TR009(SEQ ID NO: 207) and forward primer TRO1O (SEQ ID NO: 208). Finally, 2 overlapping fragments were pooled and PCR was performed using the forward primer TRO1O and the reverse primer TR 006. A PCR product comprising the 1254bp human TNFR1 Δ 7 open reading frame with a stop codon (SEQ ID NO: 121) was isolated and purified using standard molecular biology techniques.

Alternatively, in these PCR reactions, the open reading frame of 1251bp human TNFR1 Δ 7 without a stop codon can be generated using reverse primer TR011 instead of reverse primer TR 006. This also allows the addition of an in-frame C-terminal affinity purification tag, such as a His tag, when the final PCR product is inserted into an appropriate vector.

Example 13 Generation of murine TNFR2 Δ 7cDNA

To generate full-length murine TNFR2cDNA, a commercially available FirstChoice was paired with a reverse primer TR012(SEQ ID NO: 214) and a forward primer TR013(SEQ ID NO: 215)^TMThe Mouse Liver cDNA (PCR-Ready Mouse Liver cDNA) prepared for PCR (Ambion, catalog No: AM3300) was subjected to PCR. PCR products of full-length murine TNFR2cDNA were isolated and purified using standard molecular biology techniques. Then, an appropriate Kozak sequence was introduced thereto by PCR of the resulting product using TR014 forward primer (SEQ ID NO: 216) and reverse primer TR 012.

Alternatively, the full-length mouse TNFR2cDNA was obtained by performing RT-PCR on total RNA from human monocytes or mouse hepatocytes using reverse primer TR015(SEQ ID NO: 217) and forward primer TR016(SEQ ID NO: 218). PCR products of full-length murine TNFR2cDNA were isolated and purified using standard molecular biology techniques.

To generate the murine TNFR2 Δ 7cDNA, two separate PCR reactions were performed on the full length murine TNFR2cDNA to generate overlapping fragments of the TNFR2 Δ 7 cDNA. In one reaction, PCR was performed on the full length TNFR2cDNA using forward primer TR017(SEQ ID NO: 219) and reverse primer TR 015. In one reaction, PCR was performed on the full length TNFR2cDNA using reverse primer TR018(SEQ ID NO: 220) and forward primer TR 016. Finally, 2 overlapping fragments were pooled and PCR was performed using the forward primer TR016 and the reverse primer TR 015. A PCR product predicted to contain the 1348bp murine TNFR2 Δ 7 open reading frame with a stop codon (SEQ ID NO: 127) was isolated and purified using standard molecular biology techniques.

Alternatively, in these PCR reactions, the 1345bp open reading frame for murine TNFR 2. delta.7 without a stop codon can be generated using reverse primer TR019(SEQ ID NO: 221) in place of reverse primer TR 015. This also allows the addition of an in-frame C-terminal affinity purification tag, such as a His tag, when the final PCR product is inserted into an appropriate vector.

Example 14 Generation of murine TNFR1 Δ 7cDNA

To generate full-length murine TNFR1cDNA, a commercially available first Choice pair was prepared using a reverse primer TR020(SEQ ID NO: 230) and a forward primer TR021(SEQ ID NO: 231)^TMThe Mouse Liver cDNA (PCR-Ready Mouse Liver cDNA) prepared for PCR (Ambion, catalog No: AM3300) was subjected to PCR. PCR products of the full-length murine TNFR1cDNA were isolated and purified using standard molecular biology techniques.

Alternatively, the full-length murine TNFR1cDNA was obtained by performing RT-PCR on total RNA from mouse monocytes using reverse primer TR020 and forward primer TR 021. PCR products of the full-length murine TNFR1cDNA were isolated and purified using standard molecular biology techniques. To generate the murine TNFR1 Δ 7cDNA, two separate PCR reactions were performed on the full-length human TNFR1cDNA to generate overlapping fragments of the TNFR1 Δ 7 cDNA. In one reaction, PCR was performed on the full-length TNFR1cDNA using a forward primer TR022(SEQ ID NO: 232) and a reverse primer TR 020. In another reaction, PCR was performed on the full length TNFR1cDNA using reverse primer TR023(SEQ ID NO: 233) and forward primer TR024(SEQ ID NO: 234). Finally, 2 overlapping fragments were pooled and PCR was performed using the forward primer TR024 and the reverse primer TR 020. A1259 bp PCR product containing the 1251bp murine TNFR1 Δ 7 open reading frame with a stop codon (SEQ ID NO: 123) was isolated and purified using standard molecular biology techniques.

Alternatively, in these PCR reactions, the open reading frame for murine TNFR1 Δ 7 of 1248bp without a stop codon can be generated using reverse primer TR025(SEQ ID NO: 235) instead of reverse primer TR 020. This also allows the addition of an in-frame C-terminal affinity purification tag, such as a His tag, when the final PCR product is inserted into an appropriate vector.

Example 15 construction of vectors for expression of human TNFR2 Δ 7 in mammalian cells

To express the human TNFR2 Δ 7 protein in mammalian cells, the human TNFR2 Δ 7cDNA PCR product from example 12 was integrated into an appropriate mammalian expression vector. PCR products of TNFR2 Δ 7cDNA with and without a stop codon from example 12 were mixed with pcDNA according to the manufacturer's instructions^TM3.1D V5-HisThe expression vector (Invitrogen) was blunt-ended and isolated. Transformation with a plasmid containing an insert encoding human TNFR2 Δ 7 was performed according to the supplier's instructionsTop10 competent cells (Invitrogen). Fifty mL of the transformation mixture was plated on LB medium with 100mg/mL ampicillin and incubated overnight at 37 ℃. The single colony was inoculated to LB medium with 100mg/mL ampicillin and incubated overnight at 37 ℃. The culture was then inoculated into 200mL LB medium with 100mg/mL ampicillin and grown overnight at 37 ℃. Using GenElute according to the manufacturer's instructions^TMPlasmid Maxiprep kit (plasmid)id Maxiprep kit) (Sigma) plasmid was isolated. The purification efficiency was 0.5-1.5mg plasmid per preparation.

Three human TNFR2 Δ 7 clones (1319-1, 1138-5 and 1230-1) were generated and sequenced. Clone 1319-1 contained the human TNFR2 Δ 7 open reading frame without a stop codon directly followed by an in-frame His tag from the plasmid; while clones 1138-5 and 1230-1 contained the TNFR2 Δ 7 open reading frame immediately followed by a stop codon. SEQ ID NO: 242 the His-tagged sequence from the plasmid is given. The sequences of the TNFR2 Δ 7 open reading frames of clones 1230-1 and 1319-1 were compared to SEQ ID NOs with and without stop codons, respectively: 125 are identical. However, relative to SEQ id no: 125, TNFR2 Δ 7 open reading frame sequence of clone 1138-5 (SEQ ID NO: 231) differs by one nucleotide at position 1055 of exon 10, the former being A and the latter being G. This single nucleotide change results in amino acid 352 being changed from glutamine to arginine.

Example 16 expression of human TNFR2 Δ 7 in E.coli

For expression of human TNFR2 Δ 7 protein in bacteria, the human TNFR2 Δ 7cDNA from example 12 was integrated into an appropriate expression vector, such as pETDirection alExpression vector (Invitrogen). The primer sequence was determined using a forward primer (TR002) (SEQ ID NO: 191) and a reverse primer (TR026) SEQ ID NO: 195 PCR was performed on the PCR fragment from example 12 to introduce homologous recombination sites for the appropriate vector. Using pET101/D-The resulting PCR fragment was incubated with vector (Invitrogen) to generate the human TNFR2 Δ 7 bacterial expression vector. The resulting vector was transformed into E.coli strain BL21(DE 3). Human TNFR2 Δ 7 was then expressed from bacterial cells according to the manufacturer's instructions.

Example 17 expression of human TNFR2 Δ 7 in insect cells

To express the human TNFR2 Δ 7 protein in insect cells, the human TNFR2 Δ 7cDNA from example 12 was integrated into a baculovirus vector. Human TNFR 2. delta.7 cDNA from example 12 was PCR with forward primer (TR027) (SEQ ID NO: 196 and reverse primer (TR028) (SEQ ID NO: 197.) the resulting PCR product was digested with restriction enzymes EcoRI and Xhol^TMVectors (Invitrogen), such as any of pENTR (TM)1A, pENTR (TM)2B, pENTR (TM)3C, pENTR (TM)4 or pENTR (TM)11 vectors, are ligated to produce the import vector. The product is then isolated, amplified and purified using standard molecular biology techniques.

Using LRClonase according to the manufacturer's instructions^TM(Invitrogen) with BaculoDirect via the input vector^TMHomologous recombination of linear dna (invitrogen) produced a baculovirus vector containing human TNFR2 Δ 7 cDNA. The reaction mixture was then used to infect Sf9 cells to produce recombinant baculovirus. After harvesting the recombinant baculovirus, it was confirmed that human TNFR2 Δ 7 was expressed. Amplification of recombinant baculovirus produces high titer viral stocks. Sf9 cells were infected with high titer viral stocks, expressing human TNFR2 Δ 7 protein.

Example 18 production of adeno-associated viral vectors expressing human TNFR2 Δ 7

For delivery of the human TNFR2 Δ 7 gene to mammalian cells for expression in the mammalian cells in vitro or in vivo, the expression of the TNFR2 Δ 7 gene in mammalian cells was determined according to Grieger, j., et al, 2006, Nature Protocols 1: 1412 describes the generation of recombinant adeno-associated virus (rAAV) vectors using a three-plasmid transfection system. The purified human TNFR2D7PCR product of example 12 was PCR-performed using a forward primer (TR029) (SEQ ID NO: 198) and a reverse primer (TR030) (SEQ ID NO: 199) to introduce a unique flanking NotI restriction site. The resulting PCR product was digested with Notl restriction enzyme and isolated by standard molecular biology techniques. The Notl digested fragment was then ligated to Notl digested pTR-UF2 (vector center laboratory, University of North Carolina (UNC), USA) to generate a plasmid containing the human TNFR2D7 open reading frame operably linked to the CMVie promoter, flanked by inverted terminal repeats. The resulting plasmids were then transfected into HEK-293 cells as described by Grieger, j. et al using plasmids pXX680 and phepper (UNC vector center laboratory) to generate rAAV particles comprising the human TNFR2 Δ 7 gene, with expression of the human TNFR2 Δ 7 gene driven by a strong constitutive CMVie promoter. Virions were harvested and purified as described in Grieger, j.

Example 19 expression of human TNFR1 Δ 7 in E.coli

To express the human TNFR1 Δ 7 protein in bacteria, the cDNA is integrated into an appropriate expression vector, such as pETDirectionalalExpression vector (Invitrogen). PCR of the cDNA using the forward primer (TR010) (SEQ ID NO: 208 and reverse primer (TR006) (SEQ ID NO: 204) to introduce homologous recombination sites into the appropriate vector according to the manufacturer's instructions, pET101/D-The resulting PCR fragment was incubated with vector (Invitrogen) to generate the human TNFR1 Δ 7 bacterial expression vector. The resulting vector was transformed into E.coli strain BL21(DE 3). Human TNFR1 Δ 7 was then expressed from bacterial cells according to the manufacturer's instructions.

Example 20 expression of human TNFR1 Δ 7 in mammalian cells

To express the human TNFR1 Δ 7 protein in mammalian cells, the human TNFR1 Δ 7cDNAPCR product was integrated into an appropriate mammalian expression vector, and the human TNFR1 Δ 7cDNA PCR product was combined with pcDNA according to the manufacturer's instructions^TM3.1D V5-HisThe expression vector (Invitrogen) was blunt-ended. Then, the product is separated and expanded by standard molecular biology techniquesAnd (c) amplifying and purifying the product to produce a mammalian expression vector. The vector was then transfected into mammalian cells, where expression of human TNFR1 Δ 7 protein was driven by a stronger constitutive CMVie promoter.

Example 21 expression of human TNFR1 Δ 7 in insect cells

To express the human TNFR1 Δ 7 protein in insect cells, the cDNA from example 12 was integrated into a baculovirus vector. The cDNA from example 12 was subjected to PCR using a forward primer (TR031) (SEQ ID NO: 210) and a reverse primer (TR032) (SEQ ID NO: 211). The resulting PCR product was digested with restriction enzymes EcoRI and Xhol. The digested PCR product was combined with EcoRI and Xhol digested pENTR^TMVectors (Invitrogen), e.g. pENTR^TM1A，pENTR^TM2B，pENTR^TM3C，pENTR^TM4 or pENTR^TM11 to create an input carrier. The product is then isolated, amplified and purified using standard molecular biology techniques.

LR clone was used according to the manufacturer's instructions^TM(Invitrogen) with BaculoDirect via the input vector^TMHomologous recombination of linear dna (invitrogen) produced a baculovirus vector containing human TNFR1 Δ 7 cDNA. The reaction mixture was then used to infect Sf9 cells to produce recombinant baculovirus. After harvesting the recombinant baculovirus, it was confirmed that human TNFR1 Δ 7 was expressed. Amplification of recombinant baculovirus produces high titer viral stocks. Sf9 cells were infected with high titer viral stocks, expressing human TNFR1 Δ 7 protein.

Example 22-Generation of adeno-associated viral vectors expressing human TNFR1 Δ 7

For delivery of the human TNFR1 Δ 7 gene to mammalian cells for expression in the mammalian cells in vitro or in vivo, the expression of the TNFR1 Δ 7 gene in mammalian cells was determined according to Grieger, j., et al, 2006, Nature Protocols 1: 1412 describes the generation of recombinant adeno-associated virus (rAAV) vectors using a three-plasmid transfection system. The purified human TNFR1D7PCR product was PCR-performed using a forward primer (TR033) (SEQ ID NO: 212) and a reverse primer (TR034) (SEQ ID NO: 213) to introduce a flanking Notl restriction site. The resulting PCR product was digested with Notl restriction enzyme and isolated by standard molecular biology techniques. The Notl digested fragment was then ligated to Notl digested pTR-UF2 (vector center laboratory, University of North Carolina (UNC), USA) to generate a plasmid containing the human TNFR1D7 open reading frame operably linked to the CMVie promoter, flanked by inverted terminal repeats. The resulting plasmids were then transfected into HEK-293 cells as described by Grieger, j. et al using plasmids pXX680 and phepper (UNC vector center laboratory) to generate rAAV particles comprising the human TNFR1 Δ 7 gene, with expression of the human TNFR1 Δ 7 gene driven by a strong constitutive CMVie promoter. Virions were harvested and purified as described in Grieger, j.

Example 23 construction of vectors for expression of mouse TNFR2 Δ 7 in mammalian cells

To express the murine TNFR2 Δ 7 protein in mammalian cells, the murine TNFR2 Δ 7cDNA PCR product from example 14 was integrated into an appropriate mammalian expression vector. TNFR2 Δ 7cDNAPCR products from example 14 with and without a stop codon were mixed with pcDNA according to the manufacturer's instructions^TM3.1D V5-HisThe expression vector (Invitrogen) was blunt-ended and isolated. Transformation with a plasmid containing an insert encoding murine Δ 7TNFR2 was performed according to the supplier's instructionsTop10 competent cells (Invitrogen). Fifty mL of the transformation mixture was plated on LB medium with 100mg/mL ampicillin and incubated overnight at 37 ℃. The single colony was inoculated to LB medium with 100mg/mL ampicillin and incubated overnight at 37 ℃. The culture was then inoculated into 200mL LB medium with 100mg/mL ampicillin and grown overnight at 37 ℃. In accordance withGenElute was used according to the manufacturer's instructions^TMPlasmid Maxiprep kit (Plasmid Maxiprep kit) (Sigma) isolated plasmids. The purification efficiency was 0.5-1.5mg plasmid per preparation.

Two murine TNFR2 Δ 7 clones (1144-4 and 1145-3) were generated and sequenced. Clone 1144-4 comprises the murine TNFR2 Δ 7 open reading frame without a stop codon directly followed by an in-frame His tag from the plasmid; clone 1145-3 contained the TNFR2 Δ 7 open reading frame immediately followed by a stop codon. SEQ ID NO: 242 the His-tagged sequence from the plasmid is given. Relative to SEQ ID NO: 127, TNFR2 Δ 7 open reading frames of two clones 1144-4 and 1145-3 differ in sequence by one nucleotide at position 11. These single nucleotide changes result in a single nucleotide sequence relative to SEQ ID NO: 128 appear to be four amino acids different.

Example 24 expression of murine TNFR2 Δ 7 in E.coli

To express the mouse TNFR2 Δ 7 protein in bacteria, the mouse TNFR2 Δ 7cDNA from example 14 was integrated into an appropriate expression vector, such as pET directExpression vector (Invitrogen). The PCR fragment from example 14 was subjected to PCR using a forward primer (TR035) (SEQ ID NO: 222) and a reverse primer (TR036) (SEQ ID NO: 223) to introduce a homologous recombination site for the appropriate vector. Using pET101/D-The resulting PCR fragment was incubated with vector (Invitrogen) to generate the murine TNFR2 Δ 7 bacterial expression vector. The resulting vector was transformed into E.coli strain BL21(DE 3). Murine TNFR2 Δ 7 was then expressed from bacterial cells according to the manufacturer's instructions.

Example 25 expression of mouse TNFR2 Δ 7 in insect cells

For expression of the murine TNFR2 Δ 7 protein in insect cells, cells from Glycine max (L.) Merr were culturedThe cDNA of example 14 was integrated into a baculovirus vector. PCR was performed on the cDNA from example 14 using a forward primer (TR037) (SEQ ID NO: 224) and a reverse primer (TR038) (SEQ ID NO: 225). The resulting PCR product was digested with restriction enzymes EcoRI and Xhol. The digested PCR product was combined with EcoRI and Xhol digested pENTR^TMVectors (Invitrogen), e.g. pENTR^TM1A，pENTR^TM2B，pENTR^TM3C，pENTR^TM4 or pENTR^TM11 to create an input carrier. The product is then isolated, amplified and purified using standard molecular biology techniques. LR clone was used according to the manufacturer's instructions^TM(Invitrogen) with BaculoDirect via the input vector^TMHomologous recombination of linear dna (invitrogen) produced a baculovirus vector containing murine TNFR2 Δ 7 cDNA. The reaction mixture was then used to infect Sf9 cells to produce recombinant baculovirus. After harvesting the recombinant baculovirus, it was confirmed that murine TNFR2 Δ 7 was expressed. Amplification of recombinant baculovirus produces high titer viral stocks. Sf9 cells were infected with high titer viral stocks to express the murine TNFR2 Δ 7 protein.

Example 26 Generation of adeno-associated viral vectors expressing murine TNFR2 Δ 7

For in vitro or in vivo delivery of the murine TNFR2 Δ 7 gene to mammalian cells for expression in said mammalian cells, the expression of the TNFR2 Δ 7 gene was determined according to Grieger, j., et al, 2006, Nature Protocols 1: 1412 describes the generation of recombinant adeno-associated virus (rAAV) vectors using a three-plasmid transfection system. The purified murine TNFR2D7PCR product of example 14 was PCR-performed using a forward primer (TR039) (SEQ ID NO: 226) and a reverse primer (TR040) (SEQ ID NO: 227) to introduce a unique flanking Notl restriction site. The resulting PCR product was digested with Notl restriction enzyme and isolated by standard molecular biology techniques. The Notl digested fragment was then ligated to Notl digested pTR-UF2 (vector center laboratory, University of North Carolina (UNC), USA) to generate a plasmid containing the murine TNFR2D7 open reading frame operably linked to the CMVie promoter, flanked by inverted terminal repeats. The resulting plasmids were then transfected into HEK-293 cells as described by Grieger, j. et al using plasmids pXX680 and phepper (UNC vector center laboratory) to generate rAAV particles containing the murine TNFR2 Δ 7 gene, with expression of the murine TNFR2 Δ 7 gene driven by a strong constitutive CMVie promoter. Virions were harvested and purified as described in Grieger, j.

Example 27 expression of murine TNFR1 Δ 7 in E.coli

To express the mouse TNFR 1. delta.7 protein in bacteria, the cDNA from example 15 was integrated into an appropriate expression vector, such as pET directiveExpression vector (Invitrogen). The complementary DNA from example 15 was subjected to PCR using a forward primer (TR024) (SEQ ID NO: 234) and a reverse primer (TR020) (SEQ ID NO: 235) to introduce a site of homologous recombination into the appropriate vector. Using pET101/D-The resulting PCR fragment was incubated with vector (Invitrogen) to generate the murine TNFR1 Δ 7 bacterial expression vector. The resulting vector was transformed into E.coli strain BL21(DE 3). Murine TNFR1 Δ 7 was then expressed from bacterial cells according to the manufacturer's instructions.

Example 28 expression of mouse TNFR1 Δ 7 in mammalian cells

To express the murine TNFR1 Δ 7 protein in mammalian cells, the murine TNFR1 Δ 7cDNA PCR product from example 15 was integrated into an appropriate mammalian expression vector. PCR product of murine TNFR1 Δ 7cDNA from example 15 with pcDNA according to the manufacturer's instructions^TM3.1DV5-HisThe expression vector (Invitrogen) was blunt-ended. Then, using the standard scoreThe product is isolated, amplified and purified by sub-biological techniques to produce a mammalian expression vector. The vector was then transfected into mammalian cells, where expression of the murine TNFR1 Δ 7 protein was driven by a stronger constitutive CMVie promoter.

Example 29 expression of mouse TNFR1 Δ 7 in insect cells

To express the murine TNFR1 Δ 7 protein in insect cells, the cDNA from example 15 was integrated into a baculovirus vector. PCR was performed on the cDNA from example 15 using the forward primer (TR041) (SEQ ID NO: 236) and the reverse primer (TR042) (SEQ ID NO: 237). The resulting PCR product was digested with restriction enzymes EcoRI and Xhol. The digested PCR product was combined with EcoRI and Xhol digested pENTR^TMVectors (Invitrogen), e.g. pENTR^TM1A，pENTR^TM2B，pENTR^TM3C，pENTR^TM4 or pENTR^TM11 to create an input carrier. The product is then isolated, amplified and purified using standard molecular biology techniques. Using LRClonase according to the manufacturer's instructions^TM(Invitrogen) with BaculoDirect via the input vector^TMHomologous recombination of linear dna (invitrogen) produced a baculovirus vector containing murine TNFR1 Δ 7 cDNA. The reaction mixture was then used to infect Sf9 cells to produce recombinant baculovirus. After harvesting the recombinant baculovirus, it was confirmed that murine TNFR1 Δ 7 was expressed. Amplification of recombinant baculovirus produces high titer viral stocks. Sf9 cells were infected with high titer viral stocks to express the murine TNFR1 Δ 7 protein.

Example 30 Generation of adeno-associated viral vectors expressing murine TNFR1 Δ 7

For in vitro or in vivo delivery of the murine TNFR1 Δ 7 gene to mammalian cells for expression in said mammalian cells, the expression of the TNFR1 Δ 7 gene was determined according to Grieger, j., et al, 2006, Nature Protocols 1: 1412 describes the generation of recombinant adeno-associated virus (rAAV) vectors using a three-plasmid transfection system. The purified murine TNFR1D7PCR product of example 14 was PCR-performed using a forward primer (TR043) (SEQ ID NO: 238) and a reverse primer (TR044) (SEQ ID NO: 239) to introduce a unique flanking Notl restriction site. The resulting PCR product was digested with Notl restriction enzyme and isolated by standard molecular biology techniques. The Notl digested fragment was then ligated to Notl digested pTR-UF2 (vector center laboratory, University of North Carolina (UNC), USA) to generate a plasmid containing the murine TNFR1D7 open reading frame operably linked to the CMVie promoter, flanked by inverted terminal repeats. The resulting plasmids were then transfected into HEK-293 cells as described by Grieger, j. et al using plasmids pXX680 and phepper (UNC vector center laboratory) to generate rAAV particles containing the murine TNFR2 Δ 7 gene, with expression of the murine TNFR1 Δ 7 gene driven by a strong constitutive CMVie promoter. Virions were harvested and purified as described in Grieger, j.

EXAMPLE 31 Generation of a Lentiviral vector expressing TNFR. DELTA.7

To deliver the TNFR Δ 7 gene to mammalian cells for expression in the mammalian cells in vitro or in vivo, a replication incompetent lentiviral vector is generated. The PCR products from examples 27, 30, 35 and 38 were mixed with pLenti6/V5-D-Vectors (Invitrogen) were blunt-ended. The resulting plasmid was transformed into E.coli using standard molecular biology techniques, and amplified and purified. This plasmid was transfected into 293FT cells (Invitrogen) according to the manufacturer's instructions to produce lentiviral particles comprising the TNFR Δ 7 gene, where expression of the TNFR Δ 7 gene is driven by a stronger constitutive CMVie promoter. According to tisscornia, g., et al, 2006, Nature Protocols 1: 241 to provide a lentiviral source material suitable for transducing mammalian cells.

Example 32 expression of TNFR2 Δ 7 in mammalian cells

The plasmids generated in examples 26 and 34 were used to express active proteins in mammalian HeLa cells, and the resulting proteins were tested for anti-TNF-alpha activity. HeLa cells were seeded in 24-well plates containing SMEM medium containing L-glutamine, gentamicin, kanamycin, 5% FBS, and 5% HS at 1.0X10 per well⁵And (4) cells. Cells were grown overnight at 37 ℃ in humidified air containing 5% CO 2. Approximately 250ng of plasmid DNA was added to 50ml of OPTI-MEM^TMThen 50ml Lipofectamine was added^TM2000 mixture (1 part Lipofectamine)^TM2000 and 25 parts of OPTI-MEM^TM) And incubated for 20 minutes. Next, 400ml of serum-free medium was added and administered to the cells in 24-well plates. After 48 hours incubation at 37 ℃ in humid air containing 5% CO2, the medium was collected and the cells were harvested in 800mL TRI-Reagent^TMIn (1). Total RNA was isolated from cells according to the manufacturer's instructions and analyzed by RT-PCR using the forward primer TR047(SEQ ID NO: 200) and reverse primer TR048(SEQ ID NO: 201) for human TNFR2 Δ 7 or the forward primer TR045(SEQ ID NO: 228) and reverse primer TR046(SEQ ID NO: 229) for mouse TNFR2 Δ 7. The concentration of soluble TNFR2 in the medium was determined by ELISA.

The above media were tested for anti-TNF-alpha activity in the L929 cytotoxicity assay. L929 cells were seeded in 96-well plates containing 10% conventional FBS, penicillin and streptomycin in MEM medium at 2X10 per well⁴Cells were grown overnight at 37 ℃ in humidified air containing 5% CO 2. Media samples were diluted 1, 2,4, 8 and 16 fold with media from untransfected Hela cells. 90. mu.l of each of these samples was added to 10. mu.l of serum-free medium containing 1.0ng/ml TNF-alpha and 1ug/ml actinomycin D. The cell culture medium was removed and replaced with 100. mu.l of the sample. Cells were then grown overnight at 37 ℃ in humidified air containing 5% CO 2. Then 20mLCellTiterAQ_ueousOne Solution Reagent (Promega) was added to each wellIn (1). After 4 hours, the viability of the cells was measured by measuring the absorbance at 490nm with a fully automatic quantitative plotter microplate reader. Cell viability was normalized to untreated cells and plotted as a function of concentration of TNF antagonist (figure 17).

Using GraphPadThe software analyzes the data from this example and example 9 to determine the EC of the 3 antagonist₅₀The value is obtained. To pairFor each of the antagonists of these examples, sigmoidal dose-response curves were fitted by non-linear regression with maximum and minimum responses fixed at 100% and 0%, respectively. EC shown in Table 8₅₀The values correspond to 95% confidence levels, each curve having r from 0.7 to 0.9²The value is obtained.

Table 8: activity of TNF-alpha antagonists

TNF-alpha antagonists	EC50(ng/mL)
		Etanercept	1.1±0.5
Recombinant soluble TNFR2(rsTNFR2)	698±180
		SSO 3305-treated mouse serum (mouse TNFR 2. DELTA.7)	0.6±0.2
SSO 3274-treated mouse serum (mouse TNFR 2. DELTA.7)	0.8±0.3
		Extracellular Medium from 1144-4 transfected Hela cells (mouse TNFR 2. DELTA.7)	2.4±1.4
Extracellular Medium from 1145-3 transfected Hela cells (mouse TNFR 2. DELTA.7)	2.4±0.8
		Extracellular Medium from 1230-1 transfected Hela cells (human TNFR 2. DELTA.7)	1.4±1.1
Extracellular Medium from 1319-1 transfected Hela cells (human TNFR 2. DELTA.7)	1.7±1.0
		Extracellular Medium from 1138-5 transfected Hela cells (human TNFR 2. DELTA.7)	1.8±1.1

Example 33 expression and purification of TNFR2 Δ 7 in mammalian cells

The plasmids generated in example 15 and examples were used to express and purify TNFR2 Δ 7 from mammalian HeLa cells. HeLa cells were seeded in 6-well plates at 5X10 per well⁵Cells were grown overnight at 37 ℃ in humidified air containing 5% CO 2. Then, 1144-4 (mouse TNFR 2. delta.7 with His-tag), 1145-1 (mouse TNFR 2. delta.7 without His-tag), 1230-1 (human TNFR 2. delta.7 without His-tag) or 1319-1 (human TNFR 2. delta.7 with His-tag) plasmids were used, 1.5mg of plasmid DNA was transfected into each well. Media was collected 48 hours post transfection and concentrated approximately 40-fold using Amicon MWCO 30,000 filters. Cells were lysed in 120mL RIPA lysis buffer (Invitrogen) with protease inhibitor (Sigma-aldrich) and kept on ice for 5 minutes. Protein concentration was determined by Bradford assay. Proteins were isolated from a portion of the cell lysate and the extracellular medium was analyzed by western blot for TNFR2 as described in example 1 (figure 18).

His-tagged human and mouse TNFR2D7 (clones 1319-1 and 1144-4, respectively) were purified from the above medium by affinity chromatography. HisPur^TMCobalt spin columns (Pierce) were used to purify His-tagged mouse and human TNFR2 Δ 7 from the above medium. The manufacturer recommends 1ml HisPur equilibrated with 50mM sodium phosphate, 300mM sodium chloride, 10mM imidazole buffer (pH 7.4)^TMThe column can purify approximately 32ml of medium. Then, the column was washed with two column volumes of the same buffer, and the protein was eluted with 1ml of 50mM sodium phosphate, 300mM sodium chloride, 150mM imidazole buffer (pH 7.4). Each eluate was analyzed by western blot analysis as described above for 5 ml. TNFR2 Δ 7 appeared in the eluate, with multiple bands representing variable glycosylation forms of TNFR2D 7. TNFR2D7 protein expressed from plasmid 1230-1 or 1145-1 not containing a His tag, which was subjected to the above purification step, was used as a negative control. These proteins did not bind to the affinity column and were not present in the eluate (fig. 19).

Claims (1)

1. As shown in SEQ ID NO: 245.