Patent Publication Number: US-7897582-B2

Title: Oligonucleotide compositions and methods for the modulation of the expression of B7 protein

Description:
RELATED APPLICATIONS 
     This application is a Continuation of U.S. application Ser. No. 10/641,962, filed Aug. 15, 2003, now allowed, which is a application claiming the benefit under 35 USC 119(e) of U.S. Provisional Patent Application Ser. No. 60/651,504, filed May 23, 2003, now expired; each of which is herein incorporated by reference in its entirety. 
    
    
     SEQUENCE LISTING 
     The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0020US.C1, created on May 14, 2007 which is 180 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. 
     FIELD OF THE INVENTION 
     This invention relates to diagnostics, research reagents and therapeutics for disease states which respond to modulation of T cell activation. In particular, this invention relates to antisense oligonucleotide interactions with certain messenger ribonucleic acids (mRNAs) or DNAs involved in the synthesis of proteins that modulate T cell activation. Antisense oligonucleotides designed to hybridize to nucleic acids encoding B7 proteins are provided. These oligonucleotides have been found to lead to the modulation of the activity of the RNA or DNA, and thus to the modulation of T cell activation. Palliative, therapeutic and prophylactic effects result. 
     BACKGROUND OF THE INVENTION 
     Inflammation is a localized protective response mounted by tissues in response to injury, infection, or tissue destruction resulting in the destruction of the infectious or injurious agent and isolation of the injured tissue. A typical inflammatory response proceeds as follows: recognition of an antigen as foreign or recognition of tissue damage, synthesis and release of soluble inflammatory mediators, recruitment of inflammatory cells to the site of infection or tissue damage, destruction and removal of the invading organism or damaged tissue, and deactivation of the system once the invading organism or damage has been resolved. In many human diseases with an inflammatory component, the normal, homeostatic mechanisms which attenuate the inflammatory responses are defective, resulting in damage and destruction of normal tissue. 
     Cell-cell interactions are involved in the activation of the immune response at each of the stages described above. One of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. In general, the first inflammatory cells to appear at the site of inflammation are neutrophils, followed by monocytes and lymphocytes. Cell-cell interactions are also critical for activation of both B-lymphocytes (B cells) and T-lymphocytes (T cells) with resulting enhanced humoral and cellular immune responses, respectively. 
     The hallmark of the immune system is its ability to distinguish between self (host) and nonself (foreign invaders). This remarkable specificity exhibited by the immune system is mediated primarily by T cells. T cells participate in the host&#39;s defense against infection but also mediate organ damage of transplanted tissues and contribute to cell attack in graft-versus-host disease (GVHD) and some autoimmune diseases. In order to induce an antigen-specific immune response, a T cell must receive signals delivered by an antigen-presenting cell (APC). T cell-APC interactions can be divided into three stages: cellular adhesion, T cell receptor (TCR) recognition, and costimulation. At least two discrete signals are required from an APC for induction of T cell activation. The first signal is antigen-specific and is provided when the TCR interacts with an antigen in the context of a major histocompatibility complex (MHC) protein, or an MHC-related CD1 protein, expressed on the surface of an APC (“CD,” standing for “cluster of differentiation,” is a term used to denote different T cell surface molecules). The second (costimulatory) signal involves the interaction of the T cell surface antigen, CD28, with its ligand on the APC, which is a member of the B7 family of proteins. 
     CD28, a disulfide-linked homodimer of a 44 kilodalton polypeptide and a member of the immunoglobulin superfamily, is one of the major costimulatory signal receptors on the surface of a resting T cell for T cell activation and cytokine production (Allison,  Curr. Opin. Immunol.,  1994, 6, 414; Linsley and Ledbetter,  Annu. Rev. Immunol.,  1993, 11, 191; June et al.,  Immunol. Today,  1994, 15, 321). Signal transduction through CD28 acts synergistically with TCR signal transduction to augment both interleukin-2 (IL-2) production and proliferation of naive T cells. B7-1 (also known as CD80) was the first ligand identified for CD28 (Liu and Linsley,  Curr. Opin. Immunol.,  1992, 4, 265). B7-1 is normally expressed at low levels on APCs, however, it is upregulated following activation by cytokines or ligation of cell surface molecules such as CD40 (Lenschow et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1993, 90, 11054; Nabavi et al.,  Nature,  1992, 360, 266). Initial studies suggested that B7-1 was the CD28 ligand that mediated costimulation (Reiser et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1992, 89, 271; Wu et al.,  J. Exp. Med.,  1993, 178, 1789; Harlan et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1994, 91, 3137). However, the subsequent demonstration that anti-B7-1 monoclonal antibodies (mAbs) had minimal effects on primary mixed lymphocyte reactions and that B7-1-deficient mice responded normally to antigens (Lenschow et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1993, 90, 11054; Freeman et al.,  Science,  1993, 262, 909) resulted in the discovery of a second ligand for the CD28 receptor, B7-2 (also known as CD86). In contrast with anti-B7-1 mAbs, anti-B7-2 mAbs are potent inhibitors of T cell proliferation and cytokine production (Wu et al.,  J. Exp. Med.,  1993, 178, 1789; Chen et al.,  J. Immunol.,  1994, 152, 2105; Lenschow et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1993, 90, 11054). B7:CD28 signaling may be a necessary component of other T cell costimulatory pathways, such as CD40:CD40L (CD40 ligand) signaling (Yang et al.,  Science,  1996, 273, 1862). 
     In addition to binding CD28, B7-1 and B7-2 bind the cytolytic T-lymphocyte associated protein CTLA4. CTLA4 is a protein that is structurally related to CD28 but is expressed on T cells only after activation (Linsley et al.,  J. Exp. Med.,  1991, 174, 561). A soluble recombinant form of CTLA4, CTLA4-Ig, has been determined to be a more efficient inhibitor of the B7:CD28 interaction than monoclonal antibodies directed against CD28 or a B7 protein. In vivo treatment with CTLA4-Ig results in the inhibition of antibody formation to sheep red blood cells or soluble antigen (Linsley et al.,  Science,  1992, 257, 792), prolongation of cardiac allograft and pancreatic islet xenograft survival (Lin et al.,  J. Exp. Med.,  1993, 178, 1801; Lenschow et al., 1992 , Science,  257, 789; Lenschow et al.,  Curr. Opin. Immunol.,  1991, 9, 243), and significant suppression of immune responses in GVHD (Hakim et al.,  J. Immun.,  1995, 155, 1760). It has been proposed that CD28 and CTLA4, although both acting through common B7 receptors, serve opposing costimulatory and inhibitory functions, respectively (Allison et al.,  Science,  1995, 270, 932). CTLA4-Ig, which binds both B7-1 and B7-2 molecules on antigen-presenting cells, has been shown to block T-cell costimulation in patients with stable psoriasis vulgaris, and to cause a 50% or greater sustained improvement in clinical disease activity in 46% of the patients to which it was administered. This result was dose-dependent. Abrams et al.,  J. Clin. Invest.,  1999, 9, 1243-1225. 
     European Patent Application No. EP 0 600 591 discloses a method of inhibiting tumor cell growth in which tumor cells from a patient are recombinantly engineered ex vivo to express a B7-1 protein and then reintroduced into a patient. As a result, an immunologic response is stimulated against both B7-transfected and nontransfected tumor cells. 
     International Publication No. WO95/03408 discloses nucleic acids encoding novel CTLA4/CD28 ligands which costimulate T cell activation, including B7-2 proteins. Also disclosed are antibodies to B7-2 proteins and methods of producing B7-2 proteins. 
     International Publication No. WO95/05464 discloses a polypeptide, other than B7-1, that binds to CTLA4, CD28 or CTLA4-Ig. Also disclosed are methods for obtaining a nucleic acid encoding such a polypeptide. 
     International Publication No. WO 95/06738 discloses nucleic acids encoding B7-2 (also known as B70) proteins. Also disclosed are antibodies to B7-2 proteins and methods of producing B7-2 proteins. 
     European Patent Application No. EP 0 643 077 discloses a monoclonal antibody which specifically binds a B7-2 (also known as B70) protein. Also disclosed are methods of producing monoclonal antibodies which specifically bind a B7-2 protein. 
     U.S. Pat. No. 5,434,131 discloses the CTLA4 protein as a ligand for B7 proteins. Also disclosed are methods of producing CTLA4 fusion proteins (e.g., CTLA4-Ig) and methods of regulating immune responses using antibodies to B7 proteins or CTLA4 proteins. 
     International Publication No. WO95/22619 discloses antibodies specific to B7-1 proteins which do not bind to B7-2 proteins. Also disclosed are methods of regulating immune responses using antibodies to B7-1 proteins. 
     International Publication No. WO95/34320 discloses methods for inhibiting T cell responses using a first agent which inhibits a costimulatory agent, such as a CTLA4-Ig fusion protein, and a second agent which inhibits cellular adhesion, such as an anti-LFA-1 antibody. Such methods are indicated to be particularly useful for inhibiting the rejection of transplanted tissues or organs. 
     International Publication No. WO95/32734 discloses Fc RII bridging agents which either prevent the upregulation of B7 molecules or impair the expression of ICAM-3 on antigen presenting cells. Such FcRII bridging agents include proteins such as aggregated human IgG molecules or aggregated Fc fragments of human IgG molecules. 
     International Publication No. WO96/11279 discloses recombinant viruses comprising genetic sequences encoding (1) one or more immunostimulatory agents, including B7-1 and B7-2, and (2) antigens from a disease causing agent. Also disclosed are methods of treating diseases using such recombinant viruses. 
     To date, there are no known therapeutic agents which effectively regulate and prevent the expression of B7 proteins such as B7-1 and B7-2. Thus, there is a long-felt need for compounds and methods which effectively modulate critical costimulatory molecules such as the B7 proteins. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, oligonucleotides are provided which specifically hybridize with nucleic acids encoding B7-1 or B7-2. Certain oligonucleotides of the invention are designed to bind either directly to mRNA transcribed from, or to a selected DNA portion of, the B7-1 or B7-2 gene, thereby modulating the amount of protein translated from a B7-1 or B7-2 mRNA or the amount of mRNA transcribed from a B7-1 or B 7-2 gene, respectively. 
     Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. Such oligonucleotides are commonly described as “antisense.” Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents. 
     It has been discovered that the B7-1 and B7-2 genes, encoding B7-1 and B7-2 proteins, respectively, are particularly amenable to this approach. As a consequence of the association between B7 expression and T cell activation and proliferation, inhibition of the expression of B7-1 or B7-2 leads to inhibition of the synthesis of B7-1 or B7-2, respectively, and thereby inhibition of T cell activation and proliferation. Additionally, the oligonucleotides of the invention may be used to inhibit the expression of one of several alternatively spliced mRNAs of a B7 transcript, resulting in the enhanced expression of other alternatively spliced B7 mRNAs. Such modulation is desirable for treating various inflammatory or autoimmune disorders or diseases, or disorders or diseases with an inflammatory component such as asthma, juvenile diabetes mellitus, myasthenia gravis, Graves&#39; disease, rheumatoid arthritis, allograft rejection, inflammatory bowel disease, multiple sclerosis, psoriasis, lupus erythematosus, systemic lupus erythematosus, diabetes, multiple sclerosis, contact dermatitis, rhinitis, various allergies, and cancers and their metastases. Such modulation is further desirable for preventing or modulating the development of such diseases or disorders in an animal suspected of being, or known to be, prone to such diseases or disorders. 
     In one embodiment, the invention provides methods of inhibiting the expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering to said individual a compound of the invention targeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein said compound specifically hybridizes with and inhibits the expression of a nucleic acid molecule encoding B7-1 or B7-2. 
     The invention further provides methods of inhibiting expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering to an individual a compound of the invention which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding B7-1 or B7-2. Regions in the nucleic acid which when hybridized to a compound of the invention effect significantly lower B7-1 or B7-2 expression compared to a control, are referred to as active sites. 
     The invention also provides methods of inhibiting expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering a compound of the invention targeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein the compound specifically hybridizes with the nucleic acid and inhibits expression of B7-1 or B7-2. 
     In another aspect the invention provides methods of inhibiting expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering a compound of the invention targeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein the compound specifically hybridizes with the nucleic acid and inhibits expression of B7-1 or B7-2, said compound comprising at least 8 contiguous nucleobases of any one of the compounds of the invention. 
     The invention also provides methods of inhibiting the expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering a compound of the invention targeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein the compound specifically hybridizes with an active site in the nucleic acid and inhibits expression of B7-1 or B7-2, and the compound comprises at least 8 contiguous nucleobases of any one of the compounds of the invention. 
     In another aspect the invention provides methods of inhibiting expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual, comprising the step of administering an oligonucleotide mimetic compound targeted to a nucleic acid molecule encoding B7-1 or B7-2, wherein the compound specifically hybridizes with the nucleic acid and inhibits expression of B7-1 or B7-2, and the compound comprises at least 8 contiguous nucleobases of a compound of the invention. 
     In another aspect, the invention provides methods of inhibiting the expression of a nucleic acid molecule encoding B7-1 or B7-2 in an individual comprising the step of administering a compound of the invention targeted target to a nucleic acid encoding B7-1 or B7-2, wherein the compound inhibits B7-1 or B7-2 mRNA expression by at least 5% in 80% confluent HepG2 cells in culture at an optimum concentration compared to a control. In yet another aspect, the compounds inhibit expression of mRNA encoding B7-1 or B7-2 in 80% confluent HepG2 cells in culture at an optimum concentration by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50%, compared to a control. 
     The invention also relates to pharmaceutical compositions which comprise an antisense oligonucleotide to a B7 protein in combination with a second anti-inflammatory agent, such as a second antisense oligonucleotide to a protein which mediates intercellular interactions, e.g., an intercellular adhesion molecule (ICAM) protein. 
     Methods comprising contacting animals with oligonucleotides specifically hybridizable with nucleic acids encoding B7 proteins are herein provided. These methods are useful as tools, for example, in the detection and determination of the role of B7 protein expression in various cell functions and physiological processes and conditions, and for the diagnosis of conditions associated with such expression. Such methods can be used to detect the expression of B7 genes (i.e., B7-1 or B7-2) and are thus believed to be useful both therapeutically and diagnostically. Methods of modulating the expression of B7 proteins comprising contacting animals with oligonucleotides specifically hybridizable with a B7 gene are herein provided. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between B7 expression and T cell activation and proliferation. The present invention also comprises methods of inhibiting B7-associated activation of T cells using the oligonucleotides of the invention. Methods of treating conditions in which abnormal or excessive T cell activation and proliferation occurs are also provided. These methods employ the oligonucleotides of the invention and are believed to be useful both therapeutically and as clinical research and diagnostic tools. The oligonucleotides of the present invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides of the present invention may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art. 
     The methods disclosed herein are also useful, for example, as clinical research tools in the detection and determination of the role of B7-1 or B7-2 expression in various immune system functions and physiological processes and conditions, and for the diagnosis of conditions associated with their expression. The specific hybridization exhibited by the oligonucleotides of the present invention may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art. For example, because the oligonucleotides of this invention specifically hybridize to nucleic acids encoding B7 proteins, sandwich and other assays can easily be constructed to exploit this fact. Detection of specific hybridization of an oligonucleotide of the invention with a nucleic acid encoding a B7 protein present in a sample can routinely be accomplished. Such detection may include detectably labeling an oligonucleotide of the invention by enzyme conjugation, radiolabeling or any other suitable detection system. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue or cell sample with a detectably labeled oligonucleotide of the present invention under conditions selected to permit hybridization and measuring such hybridization by detection of the label, as is appreciated by those of ordinary skill in the art. 
     The present invention provides an antisense oligonucleotide which specifically hybridizes to a nucleic acid encoding human B7.2 protein, said antisense oligonucleotide comprising at least an 8 nucleobase portion of SEQ ID NO: 374, 391 or 440, wherein said antisense oligonucleotide inhibits expression of said human B7.2 protein. 
     In one aspect, the invention provides the antisense oligonucleotide of the invention, wherein said antisense oligonucleotide has the sequence shown in SEQ ID NO: 374, 391 or 440. 
     In another aspect, the antisense oligonucleotide of the invention has at least one modified internucleotide linkage. 
     In yet another aspect, the invention encompasses the antisense oligonucleotide of the invention wherein said modified linkage is a phosphorothioate. The antisense oligonucleotide of claim  2 , wherein all internucleotide linkages are phosphorothioate linkages. 
     In another aspect, the invention encompasses the antisense oligonucleotide of the invention having at least one 2′ sugar modification. The antisense oligonucleotide of claim  2 , wherein nucleotides 1-5 and 16-20 comprise 2′-MOE modifications. 
     In yet another aspect, the invention provides the antisense oligonucleotide of the invention wherein said 2′ sugar modification is a 2′-MOE. 
     In another aspect, the invention encompasses the antisense oligonucleotide of the invention having at least one base modification. 
     In another aspect, the invention provides the antisense oligonucleotide of the invention wherein said base modification is a 5-methylcytidine. The antisense oligonucleotide of claim  2 , wherein all cytidine residues are replaced with 5′methylcytidines. 
     In yet another aspect, the invention provides an antisense oligonucleotide having the sequence of SEQ ID NO: 374, 391 or 440, wherein all internucleotide linkages are phosphorothioate linkages, all cytidine residues are replaced with 5′methylcytidines and nucleotides 1-15 and 16-20 comprise 2′-MOE modifications. 
     In another aspect, the invention also provides a method of inhibiting expression of human B7.2 protein in cells or tissues comprising contacting said cells or tissues with the antisense oligonucleotide of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a bar graph showing the inhibitory effect of the indicated oligonucleotides on B7-1 protein expression in COS-7 cells. 
         FIG. 2  is a dose-response curve showing the inhibitory effect of oligonucleotides on cell surface expression of B7-1 protein. Solid line, ISIS 13812; dashed line, ISIS 13800; dotted line, ISIS 13805. 
         FIG. 3  is a bar graph showing the inhibitory effect of the indicated oligonucleotides on cell surface expression of B7-2 in COS-7 cells. 
         FIG. 4  is a bar graph showing the inhibitory effect of the indicated oligonucleotides, including ISIS 10373 (a 20-mer) and ISIS 10996 (a 15-mer) on cell surface expression of B7-2 in COS-7 cells. 
         FIG. 5  is a bar graph showing the specificity of inhibition of B7-1 or B7-2 protein expression by oligonucleotides. Cross-hatched bars, B7-1 levels; striped bars, B7-2 levels. 
         FIG. 6  is a dose-response curve showing the inhibitory effect of oligonucleotides having antisense sequences to ICAM-1 (ISIS 2302) or B7-2 (ISIS 10373) on cell surface expression of the ICAM-1 and B7-2 proteins. Solid line with X&#39;s, levels of B7-1 protein on cells treated with ISIS 10373; dashed line with asterisks, levels of ICAM-1 protein on cells treated with ISIS 10373; solid line with triangles, levels of B7-1 protein on cells treated with ISIS 2302; solid line with squares, levels of ICAM-1 protein on cells treated with ISIS 10373. 
         FIG. 7  is a bar graph showing the effect of the indicated oligonucleotides on T cell proliferation. 
         FIG. 8  is a dose-response curve showing the inhibitory effect of oligonucleotides on murine B7-2 protein expression in COS-7 cells. Solid line with asterisks, ISIS 11696; dashed line with triangles, ISIS 11866. 
         FIG. 9  is a bar graph showing the effect of oligonucleotides ISIS 11696 and ISIS 11866 on cell surface expression of murine B7-2 protein in IC-21 cells. Left (black) bars, no oligonucleotide; middle bars, 3 μM indicated oligonucleotide; right bars, 10 μM indicated oligonucleotide. 
         FIG. 10  is a graph showing the effect of ISIS 17456 on severity of EAE at various doses. 
         FIG. 11A-B  are graphs showing the detection of B7.2 mRNA ( FIG. 11A ) and B7.1 mRNA ( FIG. 11B ) during the development of ovalbumin-induced asthma in a mouse model. 
         FIG. 12  is a graph showing that intratracheal administration of ISIS 121874, an antisense oligonucleotide targeted to mouse B7.2, following allergen challenge, reduces the airway response to methacholine. 
         FIG. 13  is a graph showing the dose-dependent inhibition of the Penh response to 50 mg/ml methacholine by ISIS 121874. Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice (i.e., the sum of the airflow in the upper and lower respiratory tracts) during the respiratory cycle of the animal. The lower the Penh, the greater the airflow. The dose of ISIS 121874 is shown on the x-axis. 
         FIG. 14  is a graph showing the inhibition of allergen-induced eosinophilia by ISIS 121874. The dose of ISIS 121874 is shown on the x-axis. 
         FIG. 15  is a graph showing the lung concentration-dose relationship for ISIS 121874 delivered by intratracheal administration. 
         FIG. 16  is a graph showing the retention of ISIS 121874 in lung tissue following single dose (0.3 mg/kg) intratracheal instillation in the ovalbumin-induced mouse model of asthma. 
         FIG. 17  is a graph showing the effects of ISIS 121874, a 7 base pair mismatched control oligonucleotide (ISIS 131906) and a gap ablated control oligonucleotide which does not promote cleavage by RNase H (ISIS 306058). 
         FIGS. 18A-B  are graphs showing the effect of ISIS 121874 on B7.2 ( FIG. 18A ) and B7.1 ( FIG. 18B ) mRNA in lung tissue of ovalbumin-challenged mice. 
         FIGS. 19A-B  are graphs showing the effect of ISIS 121874 on B7.2 ( FIG. 19A ) and B7.1 ( FIG. 19B ) mRNA in draining lymph nodes of ovalbumin-challenged mice. 
         FIG. 20  is a graph showing that treatment with an antisense oligonucleotide targeted to B7.1 (ISIS 121844) reduces allergen-induced eosinophilia in the ovalbumin-induced mouse model of asthma. 
         FIGS. 21A-B  are graphs showing that treatment with ISIS 121844 reduces the levels of B7.1 mRNA ( FIG. 21A ) and B7.2 mRNA ( FIG. 21B ) in the mouse lung. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention employs oligonucleotides for use in antisense inhibition of the function of RNA and DNA encoding B7 proteins including B7-1 and B7-2. The present invention also employs oligonucleotides which are designed to be specifically hybridizable to DNA or messenger RNA (mRNA) encoding such proteins and ultimately to modulate the amount of such proteins transcribed from their respective genes. Such hybridization with mRNA interferes with the normal role of mRNA and causes a modulation of its function in cells. The functions of mRNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with mRNA function is modulation of the expression of a B7 protein, wherein “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a B7 protein. In the context of the present invention, inhibition is the preferred form of modulation of gene expression. 
     Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. Such oligonucleotides which specifically hybridize to a portion of the sense strand of a gene are commonly described as “antisense.” Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes, for example to distinguish between the functions of various members of a biological pathway. This specific inhibitory effect has, therefore, been harnessed by those skilled in the art for research uses. 
     “Hybridization”, in the context of this invention, means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. 
     It is understood in the art that the sequence of the oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the oligomeric compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). 
     In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which an oligomeric compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances and in the context of this invention; “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. 
     The specificity and sensitivity of oligonucleotides is also harnessed by those of skill in the art for therapeutic uses. For example, the following U.S. patents demonstrate palliative, therapeutic and other methods utilizing antisense oligonucleotides. U.S. Pat. No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression. U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions. U.S. Pat. No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenza virus. U.S. Pat. No. 4,806,463 provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementary base sequence to a portion of an oncogene. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells. U.S. Pat. No. 4,689,320 is directed to antisense oligonucleotides as antiviral agents specific to CMV. U.S. Pat. No. 5,098,890 provides oligonucleotides complementary to at least a portion of the mRNA transcript of the human c-myb gene. U.S. Pat. No. 5,242,906 provides antisense oligonucleotides useful in the treatment of latent EBV infections. 
     Oligonucleotides capable of modulating the expression of B7 proteins represent a novel therapeutic class of anti-inflammatory agents with activity towards a variety of inflammatory or autoimmune diseases, or disorders or diseases with an inflammatory component such as asthma, juvenile diabetes mellitus, myasthenia gravis, Graves&#39; disease, rheumatoid arthritis, allograft rejection, inflammatory bowel disease, multiple sclerosis, psoriasis, lupus erythematosus, systemic lupus erythematosus, diabetes, multiple sclerosis, contact dermatitis, eczema, atopic dermatitis, seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, rhinitis and various allergies. In addition, oligonucleotides capable of modulating the expression of B7 proteins provide a novel means of manipulating the ex vivo proliferation of T cells. 
     It is preferred to target specific genes for antisense attack. “Targeting” an oligonucleotide to the associated nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a cellular gene associated with several immune system disorders and diseases (such as inflammation and autoimmune diseases), as well as with ostensibly “normal” immune reactions (such as a host animal&#39;s rejection of transplanted tissue), for which modulation is desired in certain instances. The targeting process also includes determination of a region (or regions) within this gene for the oligonucleotide interaction to occur such that the desired effect, either detection or modulation of expression of the protein, will result. Once the target regions have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity to give the desired effect. 
     Generally, there are five regions of a gene that may be targeted for antisense modulation: the 5′ untranslated region (hereinafter, the “5′-UTR”), the translation initiation codon region (hereinafter, the “tIR”), the open reading frame (hereinafter, the “ORF”), the translation termination codon region (hereinafter, the “tTR”) and the 3′ untranslated region (hereinafter, the “3′-UTR”). As is known in the art, these regions are arranged in a typical messenger RNA molecule in the following order (left to right, 5′ to 3′): 5′-UTR, tIR, ORF, tTR, 3′-UTR. As is known in the art, although some eukaryotic transcripts are directly translated, many ORFs contain one or more sequences, known as “introns” which are excised from a transcript before it is translated; the expressed (unexcised) portions of the ORF are referred to as “exons” (Alberts et al., Molecular Biology of the Cell, 1983, Garland Publishing Inc., New York, pp. 411-415). Furthermore, because many eukaryotic ORFs are a thousand nucleotides or more in length, it is often convenient to subdivide the ORF into, e.g., the 5′ ORF region, the central ORF region, and the 3′ ORF region. In some instances, an ORF contains one or more sites that may be targeted due to some functional significance in vivo. Examples of the latter types of sites include intragenic stem-loop structures (see, e.g., U.S. Pat. No. 5,512,438) and, in unprocessed mRNA molecules, intron/exon splice sites. Within the context of the present invention, one preferred intragenic site is the region encompassing the translation initiation codon of the open reading frame (ORF) of the gene. Because, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Furthermore, 5′-UUU functions as a translation initiation codon in vitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:  Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology , Vol. 2, 1987, Neidhardt et al., eds., American Society for Microbiology, Washington, D.C., p. 1303). Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions, in order to generate related polypeptides having different amino terminal sequences (Markussen et al.,  Development,  1995, 121, 3723; Gao et al.,  Cancer Res.,  1995, 55, 743; McDermott et al,  Gene,  1992, 117, 193; Perri et al.,  J. Biol. Chem.,  1991, 266, 12536; French et al.,  J. Virol.,  1989, 63, 3270; Pushpa-Rekha et al.,  J. Biol. Chem.,  1995, 270, 26993; Monaco et al.,  J. Biol. Chem.,  1994, 269, 347; DeVirgilio et al.,  Yeast,  1992, 8, 1043; Kanagasundaram et al.,  Biochim. Biophys. Acta,  1992, 1171, 198; Olsen et al.,  Mol. Endocrinol.,  1991, 5, 1246; Saul et al.,  Appl. Environ. Microbiol.,  1990, 56, 3117; Yaoita et al.,  Proc. Natl. Acad. Sci. USA,  1990, 87, 7090; Rogers et al.,  EMBO J.,  1990, 9, 2273). In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a B7 protein, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. 
     In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases. 
     While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing. 
     The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode,  Caenorhabditis elegans  (Guo and Kempheus,  Cell,  1995, 81, 611-620). 
     Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al.,  Proc. Natl. Acad. Sci. USA,  1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in  Caenorhabditis elegans  resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al.,  Nature,  1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,  Science,  2002, 295, 694-697). 
     Oligomer and Monomer Modifications 
     As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside linkage or in conjunction with the sugar ring the backbone of the oligonucleotide. The normal internucleoside linkage that makes up the backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. 
     Modified Internucleoside Linkages 
     Specific examples of preferred antisense oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. 
     In the  C. elegans  system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that certain preferred oligomeric compounds of the invention can also have one or more modified internucleoside linkages. A preferred phosphorus containing modified internucleoside linkage is the phosphorothioate internucleoside linkage. 
     Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. 
     Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference. 
     In more preferred embodiments of the invention, oligomeric compounds have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH 2 —]. The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Preferred amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240. 
     Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2  component parts. 
     Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference. 
     Oligomer Mimetics 
     Another preferred group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA oligomeric compounds can be found in Nielsen et al.,  Science,  1991, 254, 1497-1500. 
     One oligonucleotide mimetic that has been reported to have excellent hybridization properties is peptide nucleic acids (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al.,  Science,  1991, 254, 1497-1500. 
     PNA has been modified to incorporate numerous modifications since the basic PNA structure was first prepared. The basic structure is shown below: 
     
       
         
         
             
             
         
       
     
     wherein 
     Bx is a heterocyclic base moiety; 
     T 4  is hydrogen, an amino protecting group, —C(O)R 5 , substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, a reporter group, a conjugate group, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; 
     T 5  is —OH, —N(Z 1 )Z 2 , R 5 , D or L α-amino acid linked via the α-amino group or optionally through the ω-amino group when the amino acid is lysine or ornithine or a peptide derived from D, L or mixed D and L amino acids linked through an amino group, a chemical functional group, a reporter group or a conjugate group; 
     Z 1  is hydrogen, C 1 -C 6  alkyl, or an amino protecting group; 
     Z 2  is hydrogen, C 1 -C 6  alkyl, an amino protecting group, —C(═O)—(CH 2 ) n -J-Z 3 , a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group; 
     Z 3  is hydrogen, an amino protecting group, —C 1 -C 6  alkyl, —C(═O)—CH 3 , benzyl, benzoyl, or —(CH 2 ) n —N(H)Z 1 ; 
     each J is O, S or NH; 
     R 5  is a carbonyl protecting group; and 
     n is from 2 to about 50. 
     Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. A preferred class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey,  Biochemistry,  2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits. 
     Morpholino nucleic acids have been prepared having a variety of different linking groups (L 2 ) joining the monomeric subunits. The basic formula is shown below: 
     
       
         
         
             
             
         
       
     
     wherein 
     T 1  is hydroxyl or a protected hydroxyl; 
     T 5  is hydrogen or a phosphate or phosphate derivative; 
     L 2  is a linking group; and 
     n is from 2 to about 50. 
     A further class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al.,  J. Am. Chem. Soc.,  2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate  E. Coli  RNase resulting in cleavage of the target RNA strand. 
     The general formula of CeNA is shown below: 
     
       
         
         
             
             
         
       
     
     wherein 
     each Bx is a heterocyclic base moiety; 
     T 1  is hydroxyl or a protected hydroxyl; and 
     T2 is hydroxyl or a protected hydroxyl. 
     Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid) can be prepared from one or more anhydrohexitol nucleosides (see, Wouters and Herdewijn,  Bioorg. Med. Chem. Lett.,  1999, 9, 1563-1566) and would have the general formula: 
     
       
         
         
             
             
         
       
     
     A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH 2 —) n  group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradation and good solubility properties. The basic structure of LNA showing the bicyclic ring system is shown below: 
     
       
         
         
             
             
         
       
     
     The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370). 
     LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex. 
     LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands. 
     Novel types of LNA-oligomeric compounds, as well as the LNAs, are useful in a wide range of diagnostic and therapeutic applications. Among these are antisense applications, PCR applications, strand-displacement oligomers, substrates for nucleic acid polymerases and generally as nucleotide based drugs. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in  Escherichia coli . Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished. 
     The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226. 
     The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., PCT International Application WO 98-DK393 19980914). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog with a handle has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA&#39;s have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported. 
     Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs having the formulas (amidite monomers shown): 
                         
(see Steffens et al.,  Helv. Chim. Acta,  1997, 80, 2426-2439; Steffens et al.,  J. Am. Chem. Soc.,  1999, 121, 3249-3255; and Renneberg et al.,  J. Am. Chem. Soc.,  2002, 124, 5993-6002). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm&#39;s) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.
 
     Another class of oligonucleotide mimetic referred to as phosphonomonoester nucleic acids incorporate a phosphorus group in the backbone. This class of oligonucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology. 
     The general formula (for definitions of Markush variables see: U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by reference in their entirety) is shown below. 
     
       
         
         
             
             
         
       
     
     Another oligonucleotide mimetic has been reported wherein the furanosyl ring has been replaced by a cyclobutyl moiety. 
     Modified Sugars 
     Oligomeric compounds of the invention may also contain one or more substituted sugar moieties. Preferred oligomeric compounds comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise a sugar substituent group selected from: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, amino alkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta,  1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 . 
     Other preferred sugar substituent groups include methoxy (—O—CH 3 ), aminopropoxy (—OCH 2 CH 2 CH 2 NH 2 ), allyl (—CH 2 —CH═CH 2 ), —O-allyl (—O—CH 2 —CH═CH 2 ) and fluoro (F). 2′-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. 
     Further representative sugar substituent groups include groups of formula I a  or II a : 
     
       
         
         
             
             
         
       
     
     wherein: 
     R b  is O, S or NH; 
     R d  is a single bond, O, S or C(═O); 
     R e  is C 1 -C 10  alkyl, N(R k )(R m ), N(R k )(R n ), N═C(R p )(R q ), N═C(R p )(R r ) or has formula III a ; 
     
       
         
         
             
             
         
       
     
     R p  and R q  are each independently hydrogen or C 1 -C 10  alkyl; 
     R r  is —R x —R y ; 
     each R s , R t , R u  and R v  is, independently, hydrogen, C(O)R w , substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; 
     or optionally, R u  and R v , together form a phthalimido moiety with the nitrogen atom to which they are attached; 
     each R w  is, independently, substituted or unsubstituted C 1 -C 10  alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl; 
     R k  is hydrogen, a nitrogen protecting group or —R x —R y ; 
     R p  is hydrogen, a nitrogen protecting group or —R x —R y ; 
     R x  is a bond or a linking moiety; 
     R y  is a chemical functional group, a conjugate group or a solid support medium; 
     each R m  and R n  is, independently, H, a nitrogen protecting group, substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted C 2 -C 10  alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH 3   + , N(R u )(R v ), guanidino and acyl where said acyl is an acid amide or an ester; 
     or R m  and R n , together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group; 
     R i  is OR z , SR z , or N(R z ) 2 ; 
     each R z  is, independently, H, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C(═NH)N(H)R u , C(═O)N(H)R u  or OC(═O)N(H)R u ; 
     R f , R g  and R h  comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic; 
     R j  is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R k )(R m )OR k , halo, SR k  or CN; 
     m a  is 1 to about 10; 
     each mb is, independently, 0 or 1; 
     mc is 0 or an integer from 1 to 10; 
     md is an integer from 1 to 10; 
     me is from 0, 1 or 2; and 
     provided that when mc is 0, md is greater than 1. 
     Representative substituents groups of Formula I are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety. 
     Representative cyclic substituent groups of Formula II are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNA Targeted 2′-Oligomeric compounds that are Conformationally Preorganized,” hereby incorporated by reference in its entirety. 
     Particularly preferred sugar substituent groups include O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. 
     Representative guanidino substituent groups that are shown in formula III and IV are disclosed in co-owned U.S. patent application Ser. No. 09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7, 1999, hereby incorporated by reference in its entirety. 
     Representative acetamido substituent groups are disclosed in U.S. Pat. No. 6,147,200 which is hereby incorporated by reference in its entirety. 
     Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled “2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6, 1999, hereby incorporated by reference in its entirety. 
     Modified Nucleobases/Naturally Occurring Nucleobases 
     Oligomeric compounds may also include nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. 
     Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in  The Concise Encyclopedia Of Polymer Science And Engineering , pages 858-859, Kroschwitz, J. I., ed. John Wiley &amp; Sons, 1990, those disclosed by Englisch et al.,  Angewandte Chemie , International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15 , Antisense Research and Applications , pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,  Antisense Research and Applications , CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. 
     In one aspect of the present invention oligomeric compounds are prepared having polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula: 
     
       
         
         
             
             
         
       
     
     Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (R 10 ═O, R 11 -R 14 ═H) [Kurchavov, et al.,  Nucleosides and Nucleotides,  1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R 10= S, R 11 -R 14 ═H), [Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R 10 ═O, R 11 -R 14 ═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388]. Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. patent application entitled “Modified Peptide Nucleic Acids” filed May 24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled “Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser. No. 10/013,295, both of which are commonly owned with this application and are herein incorporated by reference in their entirety). 
     Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (R 10 ═O, R 11 ═—O—(CH 2 ) 2 —NH 2 , R 12-14 ═H) [Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ΔT m  of up to 18° relative to 5-methyl cytosine (dC5 me ), which is the highest known affinity enhancement for a single modification, yet. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. The T m  data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5 me . It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding. 
     Further tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Pat. No. 6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992, which issued on Dec. 28, 1999, the contents of both are commonly assigned with this application and are incorporated herein in their entirety. 
     The enhanced binding affinity of the phenoxazine derivatives together with their uncompromised sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions are capable to activate RNaseH, enhance cellular uptake and exhibit an increased antisense activity [Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimize oligonucleotide design and to better understand the impact of these heterocyclic modifications on the biological activity, it is important to evaluate their effect on the nuclease stability of the oligomers. 
     Further modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patent application Ser. No. 09/996,292 filed Nov. 28, 2001, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference. 
     The oligonucleotides of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. Thus, a 20-mer may comprise 60 variations (20 positions×3 alternates at each position) in which the original nucleotide is substituted with any of the three alternate nucleotides. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of HCV mRNA and/or HCV replication. 
     Conjugates 
     A further preferred substitution that can be appended to the oligomeric compounds of the invention involves the linkage of one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligomeric compounds. In one embodiment such modified oligomeric compounds are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res.,  1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &amp;  Nucleotides,  1995, 14, 969-973), or adamantane acetic acid (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,  Biochim. Biophys. Acta,  1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al.,  J. Pharmacol. Exp. Ther.,  1996, 277, 923-937. 
     The oligomeric compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety. 
     Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference. 
     Chimeric Oligomeric Compounds 
     It is not necessary for all positions in an oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligomeric compound or even at a single monomeric subunit such as a nucleoside within an oligomeric compound. The present invention also includes oligomeric compounds which are chimeric oligomeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are oligomeric compounds that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid based oligomer. 
     Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligomeric compounds when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. 
     Chimeric oligomeric compounds of the invention may be formed as composite structures of two or more oligonucleotides, oligonucleotide analogs, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligomeric compounds have also been referred to in the art as hybrids hemimers, gapmers or inverted gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. 
     3′-Endo Modifications 
     In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry. There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appear efficient in triggering RNAi response in the  C. elegans  system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren&#39;t limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation. 
     
       
         
         
             
             
         
       
     
     Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as illustrated in  FIG. 2 , below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O&#39;kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation. Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &amp; Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modified nucleosides amenable to the present invention are shown below in Table I. These examples are meant to be representative and not exhaustive. 
     
       
         
           
               
             
               
                 TABLE I 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     The preferred conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA like conformations, A-form duplex geometry in an oligomeric context, are selected for use in the modified oligonucleotides of the present invention. The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press., and the examples section below.) Nucleosides known to be inhibitors/substrates for RNA dependent RNA polymerases (for example HCV NS5B). 
     In one aspect, the present invention is directed to oligonucleotides that are prepared having enhanced properties compared to native RNA against nucleic acid targets. A target is identified and an oligonucleotide is selected having an effective length and sequence that is complementary to a portion of the target sequence. Each nucleoside of the selected sequence is scrutinized for possible enhancing modifications. A preferred modification would be the replacement of one or more RNA nucleosides with nucleosides that have the same 3′-endo conformational geometry. Such modifications can enhance chemical and nuclease stability relative to native RNA while at the same time being much cheaper and easier to synthesize and/or incorporate into an oligonucleotide. The selected sequence can be further divided into regions and the nucleosides of each region evaluated for enhancing modifications that can be the result of a chimeric configuration. Consideration is also given to the 5′ and 3′-termini as there are often advantageous modifications that can be made to one or more of the terminal nucleosides. The oligomeric compounds of the present invention include at least one 5′-modified phosphate group on a single strand or on at least one 5′-position of a double stranded sequence or sequences. Further modifications are also considered such as internucleoside linkages, conjugate groups, substitute sugars or bases, substitution of one or more nucleosides with nucleoside mimetics and any other modification that can enhance the selected sequence for its intended target. The terms used to describe the conformational geometry of homoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,  Biochem. Biophys. Res. Comm.,  1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm&#39;s) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2′ hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2′-endo pucker and O4′-endo pucker. This is consistent with Berger, et. al.,  Nucleic Acids Research,  1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4′-endo pucker contribution. 
     DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al.,  Nucleic Acids Res.,  1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al.,  Eur. J. Biochem.,  1993, 215, 297-306; Fedoroff et al.,  J. Mol. Biol.,  1993, 233, 509-523; Gonzalez et al.,  Biochemistry,  1995, 34, 4969-4982; Horton et al.,  J. Mol. Biol.,  1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as but not limited to antisense and RNA interference as these mechanisms require the binding of a synthetic oligonucleotide strand to an RNA target strand. In the case of antisense, effective inhibition of the mRNA requires that the antisense DNA have a very high binding affinity with the mRNA. Otherwise the desired interaction between the synthetic oligonucleotide strand and target mRNA strand will occur infrequently, resulting in decreased efficacy. 
     One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2′-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependant on the nature of the substituent at the 2′-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2′-halogens have been studied showing that the 2′-fluoro derivative exhibits the largest population (65%) of the C3′-endo form, and the 2′-iodo exhibits the lowest population (7%). The populations of adenosine (2′-OH) versus deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, the effect of the 2′-fluoro group of adenosine dimers (2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is further correlated to the stabilization of the stacked conformation. 
     As expected, the relative duplex stability can be enhanced by replacement of 2′-OH groups with 2′-F groups thereby increasing the C3′-endo population. It is assumed that the highly polar nature of the 2′-F bond and the extreme preference for C3′-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and  1 H NMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2′-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2′-substituent on the 3′-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2′-substituted adenosine diphosphates. It is not clear whether the 3′-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3′-endo conformation. 
     One synthetic 2′-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2-methoxyethoxy (2′-MOE, 2′-OCH 2 CH 2 OCH 3 ) side chain (Baker et al.,  J. Biol. Chem.,  1997, 272, 11944-12000). One of the immediate advantages of the 2′-MOE substitution is the improvement in binding affinity, which is greater than many similar 2′ modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P.,  Helv. Chim. Acta,  1995, 78, 486-504; Altmann et al.,  Chimia,  1996, 50, 168-176; Altmann et al.,  Biochem. Soc. Trans.,  1996, 24, 630-637; and Altmann et al.,  Nucleosides Nucleotides,  1997, 16, 917-926). Relative to DNA, the oligonucleotides having the 2′-MOE modification displayed improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides having 2′-MOE substituents in the wing nucleosides and an internal region of deoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotide or gapmer) have shown effective reduction in the growth of tumors in animal models at low doses. 2′-MOE substituted oligonucleotides have also shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis. 
     Chemistries Defined 
     Unless otherwise defined herein, alkyl means C 1 -C 12 , preferably C 1 -C 8 , and more preferably C 1 -C 6 , straight or (where possible) branched chain aliphatic hydrocarbyl. 
     Unless otherwise defined herein, heteroalkyl means C 1 -C 12 , preferably C 1 -C 8 , and more preferably C 1 -C 6 , straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, and preferably about 1 to about 3, hetero atoms in the chain, including the terminal portion of the chain. Preferred heteroatoms include N, O and S. 
     Unless otherwise defined herein, cycloalkyl means C 3 -C 12 , preferably C 3 -C 8 , and more preferably C 3 -C 6 , aliphatic hydrocarbyl ring. 
     Unless otherwise defined herein, alkenyl means C 2 -C 12 , preferably C 2 -C 8 , and more preferably C 2 -C 6  alkenyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon double bond. 
     Unless otherwise defined herein, alkynyl means C 2 -C 12 , preferably C 2 -C 8 , and more preferably C 2 -C 6  alkynyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon triple bond. 
     Unless otherwise defined herein, heterocycloalkyl means a ring moiety containing at least three ring members, at least one of which is carbon, and of which 1, 2 or three ring members are other than carbon. Preferably the number of carbon atoms varies from 1 to about 12, preferably 1 to about 6, and the total number of ring members varies from three to about 15, preferably from about 3 to about 8. Preferred ring heteroatoms are N, O and S. Preferred heterocycloalkyl groups include morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl. 
     Unless otherwise defined herein, aryl means any hydrocarbon ring structure containing at least one aryl ring. Preferred aryl rings have about 6 to about 20 ring carbons. Especially preferred aryl rings include phenyl, napthyl, anthracenyl, and phenanthrenyl. 
     Unless otherwise defined herein, hetaryl means a ring moiety containing at least one fully unsaturated ring, the ring consisting of carbon and non-carbon atoms. Preferably the ring system contains about 1 to about 4 rings. Preferably the number of carbon atoms varies from 1 to about 12, preferably 1 to about 6, and the total number of ring members varies from three to about 15, preferably from about 3 to about 8. Preferred ring heteroatoms are N, O and S. Preferred hetaryl moieties include pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl, etc. 
     Unless otherwise defined herein, where a moiety is defined as a compound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl and alkyl), etc., each of the sub-moieties is as defined herein. 
     Unless otherwise defined herein, an electron withdrawing group is a group, such as the cyano or isocyanato group that draws electronic charge away from the carbon to which it is attached. Other electron withdrawing groups of note include those whose electronegativities exceed that of carbon, for example halogen, nitro, or phenyl substituted in the ortho- or para-position with one or more cyano, isothiocyanato, nitro or halo groups. 
     Unless otherwise defined herein, the terms halogen and halo have their ordinary meanings. Preferred halo (halogen) substituents are Cl, Br, and I. 
     The aforementioned optional substituents are, unless otherwise herein defined, suitable substituents depending upon desired properties. Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties, NO 2 , NH 3  (substituted and unsubstituted), acid moieties (e.g. —CO 2 H, —OSO 3 H 2 , etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties, etc. 
     In all the preceding formulae, the squiggle (˜) indicates a bond to an oxygen or sulfur of the 5′-phosphate. 
     Phosphate protecting groups include those described in U.S. Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expressly incorporated herein by reference in its entirety. 
     The oligonucleotides in accordance with this invention (single stranded or double stranded) preferably comprise from about 8 to about 80 nucleotides, more preferably from about 12-50 nucleotides and most preferably from about 15 to 30 nucleotides. As is known in the art, a nucleotide is a base-sugar combination suitably bound to an adjacent nucleotide through a phosphodiester, phosphorothioate or other covalent linkage. 
     The oligonucleotides of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. Thus, a 20-mer may comprise 60 variations (20 positions×3 alternates at each position) in which the original nucleotide is substituted with any of the three alternate nucleotides. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of B7.1 or B7.2 mRNA. 
     The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. 
     The oligonucleotides of the present invention can be utilized as therapeutic compounds, diagnostic tools and as research reagents and kits. The term “therapeutic uses” is intended to encompass prophylactic, palliative and curative uses wherein the oligonucleotides of the invention are contacted with animal cells either in vivo or ex vivo. When contacted with animal cells ex vivo, a therapeutic use includes incorporating such cells into an animal after treatment with one or more oligonucleotides of the invention. While not intending to be bound to a particular utility, the ex vivo modulation of, e.g., T cell proliferation by the oligonucleotides of the invention can be employed in, for example, potential therapeutic modalities wherein it is desired to modulate the expression of a B7 protein in APCs. 
     As an example, oligonucleotides that inhibit the expression of B7-1 proteins are expected to enhance the availability of B7-2 proteins on the surface of APCs, thus increasing the costimulatory effect of B7-2 on T cells ex vivo (Levine et al.,  Science,  1996, 272, 1939). 
     For therapeutic uses, an animal suspected of having a disease or disorder which can be treated or prevented by modulating the expression or activity of a B7 protein is, for example, treated by administering oligonucleotides in accordance with this invention. The oligonucleotides of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide to a suitable pharmaceutically acceptable diluent or carrier. Workers in the field have identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Antisense oligonucleotides have been safely administered to humans and several clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic instrumentalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. 
     The oligonucleotides of the present invention can be further used to detect the presence of B7-specific nucleic acids in a cell or tissue sample. For example, radiolabeled oligonucleotides can be prepared by  32 P labeling at the 5′ end with polynucleotide kinase (Sambrook et al.,  Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 10.59). Radiolabeled oligonucleotides are then contacted with cell or tissue samples suspected of containing B7 message RNAs (and thus B7 proteins), and the samples are washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates the presence of bound oligonucleotide, which in turn indicates the presence of nucleic acids complementary to the oligonucleotide, and can be quantitated using a scintillation counter or other routine means. Expression of nucleic acids encoding these proteins is thus detected. 
     Radiolabeled oligonucleotides of the present invention can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of B7 proteins for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing a B7 gene. Quantitation of the silver grains permits detection of the expression of mRNA molecules encoding these proteins and permits targeting of oligonucleotides to these areas. 
     Analogous assays for fluorescent detection of expression of B7 nucleic acids can be developed using oligonucleotides of the present invention which are conjugated with fluorescein or other fluorescent tags instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently-labeled amidites or controlled pore glass (CPG) columns. Fluorescein-labeled amidites and CPG are available from, e.g., Glen Research, Sterling Va. 
     The present invention employs oligonucleotides targeted to nucleic acids encoding B7 proteins and oligonucleotides targeted to nucleic acids encoding such proteins. Kits for detecting the presence or absence of expression of a B7 protein may also be prepared. Such kits include an oligonucleotide targeted to an appropriate gene, i.e., a gene encoding a B7 protein. Appropriate kit and assay formats, such as, e.g., “sandwich” assays, are known in the art and can easily be adapted for use with the oligonucleotides of the invention. Hybridization of the oligonucleotides of the invention with a nucleic acid encoding a B7 protein can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonucleotide or any other suitable detection systems. Kits for detecting the presence or absence of a B7 protein may also be prepared. 
     In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a decrease or loss of function, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. 
     The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. In general, for therapeutics, a patient in need of such therapy is administered an oligonucleotide in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in doses ranging from 0.01 μg to 100 g per kg of body weight depending on the age of the patient and the severity of the disorder or disease state being treated. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease or disorder, its severity and the overall condition of the patient, and may extend from once daily to once every 20 years. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disorder or disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated. 
     In some cases, it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “a treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. In a preferred embodiment, the oligonucleotides of the invention are used in conjunction with an anti-inflammatory and/or immunosuppressive agent, preferably one or more antisense oligonucleotides targeted to an intercellular adhesion molecule (ICAM), preferably to ICAM-1. Other anti-inflammatory and/or immunosuppressive agents that may be used in combination with the oligonucleotides of the invention include, but are not limited to, soluble ICAM proteins (e.g., sICAM-1), antibody-toxin conjugates, prednisone, methylprednisolone, azathioprine, cyclophosphamide, cyclosporine, interferons, sympathomimetics, conventional antihistamines (histamine H 1  receptor antagonists, including, for example, brompheniramine maleate, chlorpheniramine maleate, dexchlorpheniramine maleate, tripolidine HCl, carbinoxamine maleate, clemastine fumarate, dimenhydrinate, diphenhydramine HCl, diphenylpyraline HCl, doxylamine succinate, tripelennamine citrate, tripelennamine HCl, cyclizine HCl, hydroxyzine HCl, meclizine HCl, methdilazine HCl, promethazine HCl, trimeprazine tartrate, azatadine maleate, cyproheptadine HCl, terfenadine, etc.), histamine H 2  receptor antagonists (e.g., ranitidine). See, generally,  The Merck Manual of Diagnosis and Therapy,  15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 302-336 and 2516-2522). When used with the compounds of the invention, such agents may be used individually, sequentially, or in combination with one or more other such agents. 
     In another preferred embodiment of the invention, an antisense oligonucleotide targeted to one B7 mRNA species (e.g., B7-1) is used in combination with an antisense oligonucleotide targeted to a second B7 mRNA species (e.g., B7-2) in order to inhibit the costimulatory effect of B7 molecules to a more extensive degree than can be achieved with either oligonucleotide used individually. In a related version of this embodiment, two or more oligonucleotides of the invention, each targeted to an alternatively spliced B7-1 or B7-2 mRNA, are combined with each other in order to inhibit expression of both forms of the alternatively spliced mRNAs. It is known in the art that, depending on the specificity of the modulating agent employed, inhibition of one form of an alternatively spliced mRNA may not result in a sufficient reduction of expression for a given condition to be manifest. Thus, such combinations may, in some instances, be desired to inhibit the expression of a particular B7 gene to an extent necessary to practice one of the methods of the invention. 
     Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. In the case of in individual known or suspected of being prone to an autoimmune or inflammatory condition, prophylactic effects may be achieved by administration of preventative doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. In like fashion, an individual may be made less susceptible to an inflammatory condition that is expected to occur as a result of some medical treatment, e.g., graft versus host disease resulting from the transplantation of cells, tissue or an organ into the individual. 
     The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer or metered dose inhaler; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. 
     Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. 
     Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Compositions for oral administration also include pulsatile delivery compositions and bioadhesive composition as described in copending U.S. patent application Ser. No. 09/944,493, filed Aug. 22, 2001, and 09/935,316, filed Aug. 22, 2001, the entire disclosures of which are incorporated herein by reference. 
     Compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. 
     Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. 
     The following examples illustrate the invention and are not intended to limit the same. Those skilled in the art will recognize, or be able to ascertain through routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of the present invention. 
     The following examples are provided for illustrative purposes only and are not intended to limit the invention. 
     EXAMPLES 
     Example 1 
     Synthesis of Nucleic Acids Oligonucleotides 
     Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry with oxidation using iodine. β-Cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step. 
     The 2′-fluoro phosphorothioate oligonucleotides of the invention were synthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared as disclosed in U.S. patent application Ser. No. 463,358, filed Jan. 11, 1990, and Ser. No. 566,977, filed Aug. 13, 1990, which are assigned to the same assignee as the instant application and which are incorporated by reference herein. The 2′-fluoro oligonucleotides were prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol: deprotection was effected using methanolic ammonia at room temperature. 
     The 2′-methoxy (2′-O-methyl)oligonucleotides of the invention were synthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base is increased to 360 seconds. Other 2′-alkoxy oligonucleotides are synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va. The 3′-base used to start the synthesis was a 2′-deoxyribonucleotide. The 2′-O-propyl oligonucleotides of the invention are prepared by a slight modification of this procedure. 
     The 2′ methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 ) oligonucleotides of the invention were synthesized according to the method of Martin,  Helv. Chim. Acta  1995, 78, 486. For ease of synthesis, the last nucleotide was a deoxynucleotide. All 2′-O—CH 2 CH 2 OCH 3 -cytosines were 5-methyl cytosines, which were synthesized according to the following procedures. 
     Synthesis of 5-Methyl Cytosine Monomers 
     2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine] 
     5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions. 
     2′-O-Methoxyethyl-5-methyluridine 
     2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH 2 Cl 2 /acetone/MeOH (20:5:3) containing 0.5% Et 3 NH. The residue was dissolved in CH 2 Cl 2  (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. 
     2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine 
     2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH 3 CN (200 mL). The residue was dissolved in CHCl 3  (1.5 L) and extracted with 2×500 mL of saturated NaHCO 3  and 2×500 mL of saturated NaCl. The organic phase was dried over Na 2 SO 4 , filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et 3 NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%). 
     3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine 
     2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl 3  (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). 
     3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine 
     A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl 3  was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added to the later solution dropwise, over a 45 minute period. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO 3  and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound. 
     2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine 
     A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH 4 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH 3  gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound. 
     N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine 
     2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl 3  (700 mL) and extracted with saturated NaHCO 3  (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO 4  and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et 3 NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound. 
     N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite 
     N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 Cl 2  (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO 3  (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH 2 Cl 2  (300 mL), and the extracts were combined, dried over MgSO 4  and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound. 
     2′-O-(Aminooxyethyl)nucleoside amidites and 2′-O-(dimethylaminooxyethyl)nucleoside amidites 
     2′-(Dimethylaminooxyethoxy)nucleoside amidites 
     2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl)nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine. 
     5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine 
     O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product. 
     5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine 
     In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure&lt;100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for are-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product. 
     2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine 
     5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%). 
     5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine 
     2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH 2 Cl 2  (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH 2 Cl 2  and the combined organic phase was washed with water, brine and dried over anhydrous Na 2 SO 4 . The solution was concentrated to get 2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%). 
     5′-O-tert-Butyldiphenylsilyl-2′-O—[N,N-dimethylaminooxyethyl]-5 
     5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na 2 SO 4 , evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO 3  (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na2SO4 and evaporated to dryness. The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH 2 Cl 2  to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%). 
     2′-O-(dimethylaminooxyethyl)-5-methyluridine 
     Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH 2 Cl 2  to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%). 
     5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine 
     2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 5  under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH 2 Cl 2  (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%). 
     5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P2O5 under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N1,N1-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). Ethyl acetate layer was dried over anhydrous Na2SO4 and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%). 
     2′-(Aminooxyethoxy)nucleoside amidites 
     2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl)nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly. 
     N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (PCT WO94/02501). Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]. 
     2′-dimethylaminoethoxyethoxy (2′-DMAEOE)nucleoside amidites 
     2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly. 
     2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine 
     2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O2-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155 C for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid. 
     5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine 
     To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers are washed with saturated NaHCO3 solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH2Cl2:Et3N (20:1, v/v, with 1% triethylamine) gives the title compound. 
     5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite 
     Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH 2 Cl 2  (20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound. 
     Purification: 
     After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides were purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0. Oligodeoxynucleotides and their phosphorothioate analogs were judged from electrophoresis to be greater than 80% full length material. 
     B7 Antisense Oligonucleotides 
     A series of oligonucleotides with sequences designed to hybridize to the published human B7-1 (hB7-1) and murine B7-1 (mB7-1) mRNA sequences (Freeman et al.,  J. Immunol.,  1989, 143, 2714, and Freeman et al.,  J. Exp. Med.,  1991, 174, 625 respectively). The sequences of and modifications to these oligonucleotides, and the location of each of their target sites on the hB7-1 mRNA, are given in Tables 1 and 2. Similarly, a series of oligonucleotides with sequences designed to hybridize to the human B7-2 (hB7-2) and murine B7-2 (mB7-2) mRNA published sequences (respectively, Azuma et al., Nature, 1993, 366, 76; Chen et al.,  J. Immunol.,  1994, 152, 4929) were synthesized. The sequences of and modifications to these oligonucleotides and the location of each of their target sites on the hB7-2 mRNA are described in Tables 3 and 4. Antisense oligonucleotides targeted to ICAM-1, including ISIS 2302 (SEQ ID NO: 17), have been described in U.S. Pat. No. 5,514,788, which issued May 7, 1996, hereby incorporated by reference. ISIS 1082 (SEQ ID NO: 102) and ISIS 3082 (SEQ ID NO: 101) have been previously described (Stepkowski et al.,  J. Immunol.,  1994, 153, 5336). 
     Subsequent to their initial cloning, alternative splicing events of B7 transcripts have been reported. The reported alternative splicing for B7-1 is relatively simple, in that it results in messages extended 5′ relative to the 5′ terminus of the human and murine B7-1 cDNA sequences originally reported (Borriello et al., J. Immunol., 1994, 153, 5038; Inobe et al., J. Immunol., 1996, 157, 588). In order to retain the numbering of the B7-1 sequences found in the references initially reporting B7-1 sequences, positions within these 5′ extensions of the initially reported sequences have been given negative numbers (beginning with position −1, the most 3′ base of the 5′ extension) in Tables 1 and 2. The processing of murine B7-2 transcripts is considerably more complex than that so far reported for B7-1; for example, at least five distinct murine B7-2 mRNAs, and at least two distinct human B7-2 mRNAs, can be produced by alternative splicing events (Borriello et al.,  J. Immunol.,  1995, 155, 5490; Freeman et al., WO 95/03408, published Feb. 2, 1995; see also Jellis et al., Immunogenet., 1995, 42, 85). The nature of these splicing events is such that different 5′ exons are used to produce distinct B7-2 mRNAs, each of which has a unique 5′ sequence but which share a 3′ portion consisting of some or all of the B7-2 sequence initially reported. As a result, positions within the 5′ extensions of B7-2 messages cannot be uniquely related to a position within the sequence initially reported. Accordingly, in Table 3, a different set of coordinates (corresponding to those of SEQ ID NO: 1 of WO 95/03408) and, in Table 4, the exon number (as given in Borriello et al., J. Immunol., 1995, 155, 5490) is used to specify the location of targeted sequences which are not included in the initially reported B7-2 sequence. Furthermore, although these 5′ extended messages contain potential in-frame start codons upstream from the ones indicated in the initially published sequences, for simplicity&#39;s sake, such additional potential start codons are not indicated in the description of target sites in Tables 1-4. 
     In Tables 1-4, the following abbreviations are used: UTR, untranslated region; ORF, open reading frame; tIR, translation initiation region; tTR, translation termination region; FITC, fluorescein isothiocyanate. Chemical modifications are indicated as follows. Residues having 2′ fluoro (2′F), 2′-methoxy (2′MO) or 2′-methoxyethoxy (2′ME) modification are emboldened, with the type of modification being indicated by the respective abbreviations. Unless otherwise indicated, interresidue linkages are phosphodiester linkages; phosphorothioate linkages are indicated by an “s” in the superscript position (e.g., T S A). Target positions are numbered according to Freeman et al.,  J. Immunol.,  1989, 143:2714 (human B7-1 cDNA sequence; Table 1), Freeman et al.,  J. Exp. Med.,  1991, 174, 625 (murine B7-1 cDNA sequence; Table 2), Azuma et al.,  Nature,  1993, 366:76 (human B7-2 cDNA sequence; Table 3) and Chen et al.,  J. Immunol.,  1994, 152:4929 (murine B7-2 cDNA sequence; Table 4). Nucleotide base codes are as given in 37 C.F.R. §1.822(b)(1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequences of Oligonucleotides Targeted to Human B7-1 mRNA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                 Target Position; Site 
                 Oligonucleotide Sequence (5′-&gt;3′) 
                 ID 
               
               
                 ISIS # 
                 (and/or Description) 
                 and Chemical Modifications 
                 NO: 
               
               
                   
               
               
                 13797 
                 0053-0072; 5′ UTR 
                 G S G S G S T S A S A S G S A S C S T S C S C S A S C S T S T S C S T S G S A 
                 22 
               
               
                   
               
               
                 13798 
                 0132-0151; 5′ UTR 
                 G S G S G S T S C S T S C S C S A S A S A S G S G S T S T S G S T S G S G S A 
                 23 
               
               
                   
               
               
                 13799 
                 0138-0157; 5′ UTR 
                 G S T S T S C S C S T S G S G S G S T S C S T S G S C S A S A S A S G S G S T 
                 24 
               
               
                   
               
               
                 13800 
                 0158-0177; 5′ UTR 
                 A S C S A S C S A S C S A S G S A S G S A S T S T S G S G S A S G S G S G S T 
                 25 
               
               
                   
               
               
                 13801 
                 0193-0212; 5′ UTR 
                 G S C S T S C S A S C S G S T S A S G S A S A S G S A S C S C S C S T S C S C 
                 26 
               
               
                   
               
               
                 13802 
                 0217-0236; 5′ UTR 
                 G S G S C S A S G S G S G S C S T S G S A S T S G S A S C S A S A S T S C S C 
                 27 
               
               
                   
               
               
                 13803 
                 0226-0245; 5′ UTR 
                 T S G S C S A S A S A S A S C S A S G S G S C S A S G S G S G S C S T S G S A 
                 28 
               
               
                   
               
               
                 13804 
                 0246-0265; 5′ UTR 
                 A S G S A S C S G S A S G S G S G S C S A S C S T S T S C S C S C S A S G S G 
                 29 
               
               
                   
               
               
                 13805 
                 0320-0339; tIR 
                 C S C S T S G S C S C S T S C S C S G S T S G S T S G S T S G S G S C S C S C 
                 30 
               
               
                   
               
               
                 13806 
                 0380-0399; 5′ ORF 
                 G S A S C S C S A S G S C S C S A S G S C S A S C S C S A S A S G S A S G S C 
                 31 
               
               
                   
               
               
                 13807 
                 0450-0469; 5′ ORF 
                 C S C S A S C S A S G S G S A S C S A S G S C S G S T S T S G S C S C S A S C 
                 32 
               
               
                   
               
               
                 13808 
                 0568-0587; 5′ ORF 
                 C S C S G S G S T S T S C S T S T S G S T S A S C S T S C S G S G S G S C S C 
                 33 
               
               
                   
               
               
                 13809 
                 0634-0653; central ORF 
                 G S C S C S C S T S C S G S T S C S A S G S A S T S G S G S G S C S G S C S A 
                 51 
               
               
                   
               
               
                 13810 
                 0829-0848; central ORF 
                 C S C S A S A S C S C S A S G S G S A S G S A S G S G S T S G S A S G S G S C 
                 34 
               
               
                   
               
               
                 13811 
                 1102-1121; 3′ ORF 
                 G S G S C S A S A S A S G S C S A S G S T S A S G S G S T S C S A S G S G S C 
                 35 
               
               
                   
               
               
                 13812 
                 1254-1273; 3′-UTR 
                 G S C S C S T S C S A S T S G S A S T S C S C S C S C S A S C S G S A S T S C 
                 36 
               
               
                   
               
               
                 13872 
                 (scrambled # 13812) 
                 A S G S T S C S C S T S A S C S T S A S C S C S A S G S C S C S G S C S C S T 
                 52 
               
               
                   
               
               
                 12361 
                 0056-0075; 5′ UTR 
                 T S C S A S G S G S G S T S A S A S G S A S C S T S C S C S A S C S T S T S C 
                 38 
               
               
                   
               
               
                 12348 
                 0056-0075; 5′ UTR 
                   T C A G G G   S T S A S A S G S A S C S T S   C   S   C A C T T C   
                 38 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12473 
                 0056-0075; 5′ UTR 
                 T S   C   S   A   S   G   S   G   S   G   S T S A S A S G S A S C S T S C S   C   S   A   S   C   S   T   S   T   S   C   
                 38 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12362 
                 0143-0162; 5′ UTR 
                 A S G S G S G S T S G S T S T S C S C S T S G S G S G S T S C S T S C S C S A 
                 39 
               
               
                   
               
               
                 12349 
                 0143-0162; 5′ UTR 
                   A G G G T G   S T S T S C S C S T S G S G S   G   S   T C T C C A   
                 39 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12474 
                 0143-0162; 5′ UTR 
                   A   S   G   S   G   S   G   S   T   S   G   S T S T S C S C S T S G S G S G S   T   S   C   S   T   S   C   S   C   S   A   
                 39 
               
               
                   
                   
                 (2′F1) 
                   
               
               
                   
               
               
                 12363 
                 0315-0334; tIR 
                 C S T S C S C S G S T S G S T S G S T S G S G S C S C S C S A S T S G S G S C 
                 40 
               
               
                   
               
               
                 12350 
                 0315-0334; tIR 
                   C T C C G T   S G S T S G S T S G S G S C S   C C A T G G C   
                 40 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12475 
                 0315-0334; tIR 
                   C   S   T   S   C   S   C   S   G   S   T   S G S T S G S T S G S G S C S C S   C   S   A   S   T   S   G   S   G   S   C   
                 40 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12364 
                 0334-0353; 5′ ORF 
                 G S G S A S T S G S G S T S G S A S T S G S T S T S C S C S C S T S G S C S C 
                 41 
               
               
                   
               
               
                 12351 
                 0334-0353; 5′ ORF 
                   G G A T G G   S T S G S A S T S G S T S T S   C C C T G C C   
                 41 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12476 
                 0334-0353; 5′ ORF 
                   G   S   G   S   A   S   T   S   G   S   G   S T S G S A S T S G S T S T S C S   C   S   C   S   T   S   G   S   C   S   C   
                 41 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12365 
                 0387-0406; 5′ ORF 
                 T S G S A S G S A S A S A S G S A S C S C S A S G S C S C S A S G S C S A S C 
                 42 
               
               
                   
               
               
                 12352 
                 0387-0406; 5′ ORF 
                   T G A G A A   S A S G S A S C S C S A S G S   C   S   C A G C A C   
                 42 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12477 
                 0387-0406; 5′ ORF 
                   T   S   G   S   A   S   G   S   A   S   A   S A S G S A S C S C S A S G S C S   C   S   A   S   G   S   C   S   A   S   C   
                 42 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12366 
                 0621-0640; central ORF 
                 G S G S G S C S G S C S A S G S A S G S C S C S A S G S G S A S T S C S A S C 
                 43 
               
               
                   
               
               
                 12353 
                 0621-0640; central ORF 
                   G G G C G C   S A S G S A S G S C S C S A S   G G A T C A C   
                 43 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12478 
                 0621-0640; central ORF 
                   G   S   G   S   G   S   C   S   G   S   C   S A S G S A S G S C S C S A S G S   G   S   A   S   T   S   C   S   A   S   C   
                 43 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12367 
                 1042-1061; 3′ ORF 
                 G S G S C S C S C S A S G S G S A S T S G S G S G S A S G S C S A S G S G S T 
                 44 
               
               
                   
               
               
                 12354 
                 1042-1061; 3′ ORF 
                   G G C C C A   S G S G S A S T S G S G S G S   A G C A G G T   
                 44 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12479 
                 1042-1061; 3′ ORF 
                   G   S   G   S   C   S   C   S   C   S   A   S G S G S A S T S G S G S G S A S   G   S   C   S   A   S   G   S   G   S   T   
                 44 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12368 
                 1069-1088; tTR 
                 A S G S G S G S C S G S T S A S C S A S C S T S T S T S C S C S C S T S T S C 
                 45 
               
               
                   
               
               
                 12355 
                 1069-1088; tTR 
                   A G G G C G   S T S A S C S A S C S T S T S   T C C C T T C   
                 45 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12480 
                 1069-1088; tTR 
                   A   S   G   S   G   S   G   S   C   S   G   S T S A S C S A S C S T S T S T S   C   S   C   S   C   S   T   S   T   S   C   
                 45 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12369 
                 1100-1209; tTR 
                 C S A S G S C S C S C S C S T S T S G S C S T S T S C S T S G S C S G S G S A 
                 46 
               
               
                   
               
               
                 12356 
                 1100-1209; tTR 
                   C A G C C C   S C S T S T S G S C S T S T S C S   T G C G G A   
                 46 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12481 
                 1100-1209; tTR 
                   C   S   A   S   G   S   C   S   C   S   C   S C S T S T S G S C S T S T S C S   T   S   G   S   C   S   G   S   G   S   A   
                 46 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12370 
                 1360-1380; 3′ UTR 
                 A S A S G S G S A S G S A S G S G S G S A S T S G S C S C S A S G S C S C S A 
                 47 
               
               
                   
               
               
                 12357 
                 1360-1380; 3′ UTR 
                   A A G G A G   S A S G S G S G S A S T S G S   C C A G C C A   
                 47 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 12482 
                 1360-1380; 3′ UTR 
                   A   S   A   S   G   S   G   S   A   S   G   S A S G S G S G S A S T S G S C S   C   S   A   S   G   S   C   S   C   S   A   
                 47 
               
               
                   
                   
                 (2′FI) 
                   
               
               
                   
               
               
                 12914 
                 (-0038 to -0059; 
                 
                   C 
                   S 
                   T 
                   S 
                   G 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   A 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   G 
                   S 
                   T 
                   S 
                   T 
                 
                 48 
               
               
                   
                 5′ UTR of 
                   S   T   S   G  (2′MO) 
                   
               
               
                   
                 alternative mRNA) 
                   
                   
               
               
                   
               
               
                 12915 
                 (-0035 to -0059; 
                 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   C 
                   S 
                   T 
                   S 
                   G 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   A 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                 
                 49 
               
               
                   
                 5′ UTR of 
                   S   G   S   T   S   T   S   T   S   G  (2′MO) 
                   
               
               
                   
                 alternative mRNA) 
                   
                   
               
               
                   
               
               
                 13498 
                 (-0038 to -0058; 
                 
                   C 
                   S 
                   T 
                   S 
                   G 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   A 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   G 
                   S 
                   T 
                   S 
                   T 
                 
                 50 
               
               
                   
                 5′ UTR of 
                   S   T (2′ME) 
                   
               
               
                   
                 alternative mRNA) 
                   
                   
               
               
                   
               
               
                 13499 
                 (-0038 to -0058; 
                   C   T   G   T   T   A   C   T   T   T   A   C   A   G   A   G   G   G   T   T   T   
                 50 
               
               
                   
                 5′ UTR of 
                 (2′ME) 
                   
               
               
                   
                 alternative mRNA) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Sequences of Oligonucleotides Targeted to Murine B7-1 mRNA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 Oligonucleotide Sequence (5′-&gt;3′) 
                 ID 
               
               
                 ISIS # 
                 Target Position; Site 
                 Chemical Modifications 
                 NO: 
               
               
                   
               
               
                 14419 
                 0009-0028; 5′ UTR 
                 A S G S T S A S A S G S A S G S T S C S T S A S T S T S G S A S G S G S T S A 
                 53 
               
               
                   
               
               
                 14420 
                 0041-0060; 5′ UTR 
                 G S G S T S T S G S A S G S T S T S T S C S A S C S A S A S C S C S T S G S A 
                 54 
               
               
                   
               
               
                 14421 
                 0071-0091; 5′ UTR 
                 G S T S C S C S A S G S A S G S A S A S T S G S G S A S A S C S A S G S A S G 
                 55 
               
               
                   
               
               
                 14422 
                 0109-0128; 5′ UTR 
                 G S G S C S A S T S C S C S A S C S C S C S G S G S C S A S G S A S T S G S C 
                 56 
               
               
                   
               
               
                 14423 
                 0114-0133; 5′ UTR 
                 T S G S G S A S T S G S G S C S A S T S C S C S A S C S C S C S G S G S C S A 
                 57 
               
               
                   
               
               
                 14424 
                 0168-0187; 5′ UTR 
                 A S G S G S C S A S C S C S T S C S C S T S A S G S G S C S T S C S A S C S A 
                 58 
               
               
                   
               
               
                 14425 
                 0181-0200; 5′ UTR 
                 G S C S C S A S A S T S G S G S A S G S C S T S T S A S G S G S C S A S C S C 
                 59 
               
               
                   
               
               
                 14426 
                 0208-0217; 5′ UTR 
                 C S A S T S G S A S T S G S G S G S G S A S A S A S G S C S C S A S G S G S A 
                 60 
               
               
                   
               
               
                 14427 
                 0242-0261; tIR 
                 A S A S T S T S G S C S A S A S G S C S C S A S T S A S G S C S T S T S C S A 
                 61 
               
               
                   
               
               
                 14428 
                 0393-0412; 5′ ORF 
                 C S G S G S C S A S A S G S G S C S A S G S C S A S A S T S A S C S C S T S T 
                 62 
               
               
                   
               
               
                 14909 
                 0478-0497; 5′ ORF 
                 C S C S C S A S G S C S A S A S T S G S A S C S A S G S A S C S A S G S C S A 
                 63 
               
               
                   
               
               
                 14910 
                 0569-0588; central ORF 
                 G S G S T S C S T S G S A S A S A S G S G S A S C S C S A S G S G S C S C S C 
                 64 
               
               
                   
               
               
                 14911 
                 0745-0764; central ORF 
                 T S G S G S G S A S A S A S C S C S C S C S C S G S G S A S A S G S C S A S A 
                 65 
               
               
                   
               
               
                 14912 
                 0750-0769; central ORF 
                 G S G S C S T S T S T S G S G S G S A S A S A S C S C S C S C S C S G S G S A 
                 66 
               
               
                   
               
               
                 14913 
                 0825-0844; 3′ ORF 
                 T S C S A S G S A S T S T S C S A S G S G S A S T S C S C S T S G S G S G S A 
                 67 
               
               
                   
               
               
                 14914 
                 0932-0951; 3′ ORF 
                 C S C S C S A S G S G S T S G S A S A S G S T S C S C S T S C S T S G S A S C 
                 68 
               
               
                   
               
               
                 14915 
                 1001-1020; 3′ ORF 
                 C S T S G S C S G S C S C S G S A S A S T S C S C S T S G S C S C S C S C S A 
                 69 
               
               
                   
               
               
                 14916 
                 1125-1144; tTR 
                 C S A S G S G S C S C S C S G S A S A S G S G S T S A S A S G S G S C S T S G 
                 70 
               
               
                   
               
               
                 14917 
                 1229-1248; 3′ UTR 
                 T S C S A S G S C S T S A S G S C S A S C S G S G S T S G S C S T S G S A S A 
                 71 
               
               
                   
               
               
                 14918 
                 1329-1348; 3′ UTR 
                 G S G S C S C S C S A S G S C S A S A S A S G S T S T S G S C S C S C S G S T 
                 72 
               
               
                   
               
               
                 14919 
                 1377-1393; 3′ UTR 
                 C S C S A S C S C S A S C S A S G S T S G S G S G S C S T S C S A S G S C S C 
                 73 
               
               
                   
               
               
                 12912 
                 -0067 to -0049; 5′ UTR 
                 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   C 
                   S 
                   A 
                   S 
                   T 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   A 
                   S 
                   A 
                   S 
                   T 
                   S 
                   C 
                   S 
                   T 
                   S 
                   A 
                   S 
                   A 
                 
                 74 
               
               
                   
                   
                 (2′MO) 
                   
               
               
                   
               
               
                 12913 
                 -0067 to -0047; 5′ UTR 
                 
                   G 
                   S 
                   T 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   C 
                   S 
                   A 
                   S 
                   T 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   A 
                   S 
                   A 
                   S 
                   T 
                   S 
                   C 
                   S 
                   T 
                   S 
                   A 
                   S 
                   A 
                 
                 75 
               
               
                   
                   
                 (2′MO) 
                   
               
               
                   
               
               
                 13496 
                 -0067 to -0047; 5′ UTR 
                 
                   G 
                   S 
                   T 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   C 
                   S 
                   A 
                   S 
                   T 
                   S 
                   G 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   A 
                   S 
                   A 
                   S 
                   T 
                   S 
                   C 
                   S 
                   T 
                   S 
                   A 
                   S 
                   A 
                 
                 75 
               
               
                   
                   
                 (2′ME) 
                   
               
               
                   
               
               
                 13497 
                 -0067 to -0047; 5′ UTR 
                   G   T   G   G   C   C   A   T   G   A   G   G   G   C   A   A   T   C   T   A   A   
                 75 
               
               
                   
                   
                 (2′ME) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Sequences of Oligonucleotides Targeted to Human B7-2 mRNA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                 ISIS # 
                 Target Position*; Site** 
                 Oligonucleotide Sequence (5′-&gt;3′) 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 9133 
                 1367-1386; 3′-UTR 
                 T S T S C S C S A S G S G S T S C S A S T S G S A S G S C S C S A S T S T S A 
                 3 
               
               
                 10715 
                 scrambled control of # 9133 
                 G S A S T S T S T S A S A S C S A S T S T S T S G S G S C S G S C S C S C S A 
                 76 
               
               
                 9134 
                 1333-1352; 3′-UTR 
                 C S A S T S A S A S G S G S T S G S T S G S C S T S C S T S G S A S A S G S T S G 
                 4 
               
               
                 9135 
                 1211-1230; 3′-UTR 
                 T S T S A S C S T S C S A S T S G S G S T S A S A S T S G S T S C S T S T S T S   
                 5 
               
               
                 9136 
                 1101-1120; tTR 
                 A S T S T S A S A S A S A S A S C S A S T S G S T S A S T S C S A S C S T S T S   
                 6 
               
               
                 10716 
                 (scrambled # 9136) 
                 A S A S A S G S T S T S A S C S A S A S C S A S T S T S A S T S A S T S C S T 
                 77 
               
               
                 9137 
                 0054-0074; 5′-UTR 
                 G S G S A S A S C S A S C S A S G S A S A S G S C S A S A S G S G S T S G S G S T 
                 7 
               
               
                 9138 
                 0001-0020; 5′-UTR 
                 C S C S G S T S A S C S C S T S C S C S T S A S A S G S G S C S T S C S C S T 
                 8 
               
               
                 9139 
                 0133-0152; tIR 
                 C S C S C S A S T S A S G S T S G S C S T S G S T S C S A S C S A S A S A S T 
                 9 
               
               
                 10877 
                 (scrambled # 9139) 
                 A S G S T S G S C S G S A S T S T S C S T S C S A S A S A S C S C S T S A S C 
                 78 
               
               
                 10367 
                 0073-0092; 5′-UTR 
                 G S C S A S C S A S G S C S A S G S C S A S T S T S C S C S C S A S A S G S G 
                 10 
               
               
                 10368 
                 0240-0259; 5′ ORF 
                 T S T S G S C S A S A S A S T S T S G S G S C S A S T S G S G S C S A S G S G 
                 11 
               
               
                 10369 
                 1122-1141; 3′-UTR 
                 T S G S G S T S A S T S G S G S G S C S T S T S T S A S C S T S C S T S T S T 
                 12 
               
               
                 10370 
                 1171-1190; 3′-UTR 
                 A S A S A S A S G S G S T S T S G S C S C S C S A S G S G S A S A S C S G S G 
                 13 
               
               
                 10371 
                 1233-1252; 3′-UTR 
                 G S G S G S A S G S T S C S C S T S G S G S A S G S C S C S C S C S C S T S T 
                 14 
               
               
                 10372 
                 1353-1372; 3′-UTR 
                 C S C S A S T S T S A S A S G S C S T S G S G S G S C S T S T S G S G S C S C 
                 15 
               
               
                 11149 
                 0019-0034; 5′-UTR 
                 T S A S T S T S T S G S C S G S A S G S C S T S C S C S C S C 
                 79 
               
               
                 11151 
                 0020-0034; 5′-UTR 
                 T S A S T S T S T S G S C S G S A S G S C S T S C S C S C 
                 80 
               
               
                 11150 
                 0021-0034; 5′-UTR 
                 T S A S T S T S T S G S C S G S A S G S C S T S C S C 
                 81 
               
               
                 10373 
                 0011-0030; 5′-UTR 
                 T S G S C S G S A S G S C S T S C S C S C S C S G S T S A S C S C S T S C S G 
                 16 
               
               
                 10721 
                 (scrambled # 10373) 
                 C S G S A S C S A S G S C S T S C S C S T S G S C S G S C S T S C S C S T S C 
                 82 
               
               
                 10729 
                 (5′FITC # 10373) 
                 T S G S C S G S A S G S C S T S C S C S C S C S G S T S A S C S C S T S C S C 
                 16 
               
               
                 10782 
                 (5′cholesterol # 10373) 
                 T S G S C S G S A S G S C S T S C S C S C S C S G S T S A S C S C S T S C S C 
                 16 
               
               
                   
               
               
                   
                 # 10373 Deletion Derivatives: 
                   
                   
               
               
                 10373 
                 0011-0030; 5′-UTR 
                 T S G S C S G S A S G S C S T S C S C S C S C S G S T S A S C S C S T S C S C 
                 16 
               
               
                 10888 
                 0011-0026; 5′-UTR 
                 A S G S C S T S C S C S C S C S G S T S A S C S C S T S C S C 
                 83 
               
               
                 10889 
                 0015-0030; 5′-UTR 
                 T S G S C S G S A S G S C S T S C S C S C S C S G S T S A S C 
                 84 
               
               
                 10991 
                 0015-0024; 5′-UTR 
                 C S T S C S C S C S C S G S T S A S C 
                 85 
               
               
                 10992 
                 0015-0025; 5′-UTR 
                 G S C S T S C S C S C S C S G S T S A S C 
                 86 
               
               
                 10993 
                 0015-0026; 5′-UTR 
                 A S G S C S T S C S C S C S C S G S T S A S C 
                 87 
               
               
                 10994 
                 0015-0027; 5′-UTR 
                 G S A S G S C S T S C S C S C S C S G S T S A S C 
                 88 
               
               
                 10995 
                 0015-0028; 5′-UTR 
                 C S G S A S G S C S T S C S C S C S C S G S T S A S C 
                 89 
               
               
                 10996 
                 0015-0029; 5′-UTR 
                 G S C S G S A S G S C S T S C S C S C S C S G S T S A S C 
                 90 
               
               
                 11232 
                 0017-0029; 5′ UTR 
                 G S C S G S A S G S C S T S C S C S C S C S G S T 
                 91 
               
               
                   
               
               
                   
                 # 10996 Derivatives: 
                   
                   
               
               
                 10996 
                 0015-0029; 5′-UTR 
                 G S C S G S A S G S C S T S C S C S C S C S G S T S A S C 
                 90 
               
               
                 11806 
                 (scrambled # 10996) 
                 G S C S C S G S C S C S G S C S C S A S A S G S T S C S T 
                 92 
               
               
                 11539 
                 (fully 2′MO # 10996) 
                   G   S   C   S   G   S   A   S   G   S   C   S   T   S   C   S   C   S   C   S   C   S   G   S   T   S   A   S C (2′MO) 
                 90 
               
               
                 11540 
                 (control for # 11539) 
                   G   S   C   S   C   S   G   S   C   S   C   S   G   S   C   S   C   S   A   S   A   S   G   S   T   S   C   S T (2′MO) 
                 92 
               
               
                 11541 
                 (# 10996 7-base “gapmer”) 
                   G   S   C   S   G   S   A   S G S C S T S C S C S C S C S   G   S   T   S   A   S   C  (2′MO) 
                 90 
               
               
                 11542 
                 (control for # 11541) 
                   G   S   C   S   C   S   G   S C S C S G S C S C S A S A S   G   S   T   S   C   S   T  (2′MO) 
                 92 
               
               
                 11543 
                 (# 10996 9-base “gapmer”) 
                   G   S   C   S   G   S A S G S C S T S C S C S C S C S G S   T   S   A   S   C  (2′MO) 
                 90 
               
               
                 11544 
                 (control for # 11543) 
                   G   S   C   S   C   S G S C S C S G S C S C S A S A S G S   T   S   C   S   T  (2′MO) 
                 92 
               
               
                 11545 
                 (# 10996 5′ “wingmer”) 
                   G   S   C   S   G   S   A   S   G   S   C   S   T   S   C   S C S C S C S G S T S A S C (2′MO) 
                 90 
               
               
                 11546 
                 (control for # 11545) 
                   G   S   C   S   C   S   G   S   C   S   C   S   G   S   C   S C S A S A S G S T S C S T (2′MO) 
                 92 
               
               
                 11547 
                 (# 10996 3′ “wingmer”) 
                 G S C S G S A S G S C S T S   C   S   C   S   C   S   C   S   G   S   T   S   A   S   C  (2′MO) 
                 90 
               
               
                 11548 
                 (control for # 11547) 
                 G S G S C S G S C S C S G S   C   S   C   S   A   S   A   S   G   S   T   S   C   S   T  (2′MO) 
                 92 
               
               
                 12496 
                 ((2′-5′)A 4  # 10996) 
                 GCGAGCTCCCCGTAC 
                 90 
               
               
                 13107 
                 ((2′-5′)A 4  # 10996) 
                 G S C S G S A S G S C S T S C S C S C S C S G S T S A S C 
                 90 
               
               
                 12492 
                 ((2′-5′)A 4  # 10996) 
                   G   S   C   S   G   S   A   S   G   S   C   S   T   S   C   S   C   S   C   S   C   S   G   S   T   S   A   S   C  (2′MO) 
                 90 
               
               
                 12495 
                 ((2′-5′)A 4  # 10996) 
                   G   S   C   S   G   S   A   S   G   S   C   S   T   S   C   S   C   S   C   S   C   S   G   S   T   S   A   S   C  (2′MO) 
                 90 
               
               
                 12887 
                 (1-24 of SEQ ID NO:1 of WO 
                   G   S   A   S   G   S   A   S   A   S   G   S   C   S   A   S   A   S   A   S   G   S   C   S   T   S   T   S   T   S   C   S   A   S   C   S   C   S   C - 
                 93 
               
               
                   
                 95/03408; alternative mRNA) 
                   S   T   S   G   S   T   S   G  (2′MO) 
                   
               
               
                 12888 
                 (1-22 of SEQ ID NO:1 of WO 
                 
                   G 
                   S 
                   A 
                   S 
                   A 
                   S 
                   G 
                   S 
                   C 
                   S 
                   A 
                   S 
                   A 
                   S 
                   A 
                   S 
                   G 
                   S 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   T 
                   S 
                   C 
                   S 
                   A 
                   S 
                   C 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   G 
                   S 
                   T 
                   S 
                 
                 94 
               
               
                   
                 95/03408; alternative mRNA) 
                   G  (2′MO) 
                   
               
               
                 12889 
                 (1-19 of SEQ ID NO:1 of WO 
                 
                   G 
                   S 
                   C 
                   S 
                   A 
                   S 
                   A 
                   S 
                   A 
                   S 
                   G 
                   S 
                   C 
                   S 
                   T 
                   S 
                   T 
                   S 
                   T 
                   S 
                   C 
                   S 
                   A 
                   S 
                   C 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   G 
                   S 
                   T 
                   S 
                   G 
                 
                 95 
               
               
                   
                 95/03408; alternative mRNA) 
                 (2′MO) 
                   
               
               
                 12890 
                 0001-0024 
                   C   S   T   S   C   S   C   S   C   S   C   S   G   S   T   S   A   S   C   S   C   S   T   S   C   S   C   S   T   S   A   S   A   S   G   S   G   S   C - 
                 96 
               
               
                   
                   
                   S   T   S   C   S   C   S   T  (2′MO) 
                   
               
               
                 12891 
                 0001-0022 
                 
                   C 
                   S 
                   C 
                   S 
                   C 
                   S 
                   C 
                   S 
                   G 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   A 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   T 
                   S 
                   C 
                   S 
                   C 
                   S 
                 
                 97 
               
               
                   
                   
                   T  (2′MO) 
                   
               
               
                 12892 
                 0001-0020 
                 
                   C 
                   S 
                   C 
                   S 
                   G 
                   S 
                   T 
                   S 
                   A 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   C 
                   S 
                   C 
                   S 
                   T 
                   S 
                   A 
                   S 
                   A 
                   S 
                   G 
                   S 
                   G 
                   S 
                   C 
                   S 
                   T 
                   S 
                   C 
                   S 
                   C 
                 
                 98 
               
               
                   
                   
                 (2′MO) 
               
               
                   
               
            
           
         
       
     
                     TABLE 4                  Sequences of Oligonucleotides Targeted to Murine B7-2 mRNA                                         SEQ                   ID       ISIS #   Target Position; Site   Oligonucleotide Sequence (5′-&gt;3′)   NO:               11347   1094-1113; 3′ UTR   A S G S A S A S T S T S C S C S A S A S T S C S A S G S C S T S G S A S G S A   121               11348   1062-1081; 3′ UTR   T S C S T S G S A S G S A S A S A S C S T S C S T S G S C S A S C S T S T S C   122               11349   1012-1031; 3′ UTR   T S C S C S T S C S A S G S G S C S T S C S T S C S A S C S T S G S C S C S T   123               11350   0019-1138; 5′ UTR   G S G S T S T S G S T S T S C S A S A S G S T S C S C S G S T S G S C S T S G   124               11351   0037-0056; 5′ UTR   A S C S A S C S G S T S C S T S A S C S A S G S G S A S G S T S C S T S G S G   103               11352   0089-0108; tIR   C S A S A S G S C S C S C S A S T S G S G S T S G S C S A S T S C S T S G S G   104               11353   0073-0092; tIR   C S T S G S G S G S G S T S C S C S A S T S C S G S T S G S G S G S T S G S C   105               11354   0007-0026; 5′ UTR   C S C S G S T S G S C S T S G S C S C S T S A S C S A S G S G S A S G S C S C   106               11695   0058-0077; 5′ UTR   G S G S T S G S C S T S T S C S C S G S T S A S A S G S T S T S C S T S G S G   107               11696   0096-0117; tIR   G S G S A S T S T S G S C S C S A S A S G S C S C S C S A S T S G S G S T S G   108               11866   (scrambled # 11696)   C S T S A S A S G S T S A S G S T S G S C S T S A S G S C S C S G S G S G S A   109               11697   0148-0167; 5′ ORF   T S G S C S G S T S C S T S C S C S A S C S G S G S A S A S A S C S A S G S C   110               11698   0319-0338; 5′ ORF   G S T S G S C S G S G S C S C S C S A S G S G S T S A S C S T S T S G S G S C   111               11699   0832-0851; 3′ ORF   A S C S A S A S G S G S A S G S G S A S G S G S G S C S C S A S C S A S G S T   112               11700   0753-0772; 3′ ORF   T S G S A S G S A S G S G S T S T S T S G S G S A S G S G S A S A S A S T S C   113               11701   0938-0957; 3′ ORF   G S A S T S A S G S T S C S T S C S T S C S T S G S T S C S A S G S C S G S T   114               11702   0890-0909; 3′ ORF   G S T S T S G S C S T S G S G S G S C S C S T S G S C S T S A S G S G S C S T   115               11865   (scrambled # 11702)   C S T S A S G S G S T S C S T S C S G S T S C S G S T S C S G S G S T S G S G   116               11703   1003-1022; tTR   T S C S T S C S A S C S T S G S C S C S T S T S C S A S C S T S C S T S G S C   117               13100   Exon 1 (Borriello et al.,     G   S   T   S   A   S   C   S   C   S   A   S   G   S   A   S   T   S   G   S   A   S   A   S   G   S   G   S   T   S   T   S   A   S   T   S   C   S   A     118           J. Immun., 1995, 155,     S   A (2′MO)               5490; 5′ UTR of alterna-                   tive mRNA)                       13101   Exon 4 (Borriello et al.;     C   S   T   S   T   S   T   S   G   S   G   S   A   S   G   S   A   S   T   S   T   S   A   S   T   S   T   S   C   S   G   S   A   S   G   S   T   S   T     119           5′ UTR of alternative mRNA)   (2′MO)                   13102   Exon 5 (Borriello et al.;     G   S   C   S   A   S   A   S   G   S   T   S   G   S   T   S   A   S   A   S   A   S   G   S   C   S   C   S   C   S   T   S   G   S   A   S   G   S   T     120           5′ UTR of alternative mRNA)   (2′MO)                    
cDNA Clones:
 
     A cDNA encoding the sequence for human B7-1 was isolated by using the reverse transcription/polymerase chain reaction (RT-PCR). Poly A+ RNA from Daudi cells (ATCC accession No. CCL 213) was reverse transcribed using oligo-dT primer under standard conditions. Following a 30 minute reaction at 42° C. and heat inactivation, the reaction mixture (20 μL) was brought to 100 μL with water. A 10 μL aliquot from the RT reaction was then amplified in a 50 μL PCR reaction using the 5′ primer, 
     
       
         
           
               
            
               
                 the 5′ primer, 
               
               
                 5′-GAT-CAG-GGT-ACC-CCA-AAG-AAA-AAG-TGA-TTT-GTC- 
               
               
                 ATT-GC-3′ (sense, SEQ ID NO: 20), and 
               
               
                   
               
               
                 the 3′ primer, 
               
               
                 5′-GAT-AGC-CTC-GAG-GAT-AAT-GAA-TTG-GCT-GAC-AAG- 
               
               
                 AC-3′ (antisense, SEQ ID NO: 21) 
               
            
           
         
       
     
     The primers included unique restriction sites for subcloning of the PCR product into the vector pcDNA-3 (Invitrogen, San Diego, Calif.). The 5′ primer was designed to have identity with bases 1 to 26 of the published human B7-1 sequence (Freeman et al.,  J. Immunol.,  1989, 143, 2714; positions 13-38 of the primer) and includes a Kpn I restriction site (positions 7-12 of the primer) for use in cloning. The 3′ primer was designed to be complementary to bases 1450 to 1471 of the published sequence for B7-1 (positions 14-35 of the primer) and includes a Xho I restriction site (positions 7-12 of the primer). Following PCR, the reaction was extracted with phenol and precipitated using ethanol. The product was digested with the appropriate restriction enzymes and the full-length fragment purified by agarose gel and ligated into the vector pcDNA-3 (Invitrogen, San Diego, Calif.) prepared by digesting with the same enzymes. The resultant construct, pcB7-1, was confirmed by restriction mapping and DNA sequence analysis using standard procedures. A mouse B7-1 clone, pcmB7-1, was isolated in a similar manner by RT-PCR of RNA isolated from a murine B-lymphocyte cell line, 70Z3. 
     A cDNA encoding the sequence for human B7-2, position 1 to 1391, was also isolated by RT-PCR. Poly A+ RNA from Daudi cells (ATCC accession No. CCL 213) was reverse transcribed using oligo-dT primer under standard conditions. Following a 30 minute reaction at 42° C. and heat inactivation, the reaction mixture (20 μL) was brought to 100 μL with water. A 10 μL aliquot from the RT reaction was then amplified in a 50 μL PCR reaction using 
     
       
         
           
               
            
               
                 the 5′ primer, 
               
               
                 5′-GAT-CAG-GGT-ACC-AGG-AGC-CTT-AGG-AGG-TAC-GG-3′ 
               
               
                 (sense, SEQ ID NO: 1), and  
               
               
                   
               
               
                 the 3′ primer, 
               
               
                 5′-GAT-AGC-CTC-GAG-TTA-TTT-CCA-GGT-CAT-GAG-CCA-3′ 
               
               
                 (antisense, SEQ ID NO: 2). 
               
            
           
         
       
     
     The 5′ primer was designed to have identity with bases 1-20 of the published B7-2 sequence (Azuma et al.,  Nature,  1993, 366, 76 and Genbank Accession No. L25259; positions 13-32 of the primer) and includes a Kpn I site (positions 7-12 of the primer) for use in cloning. The 3′ primer was designed to have complementarity to bases 1370-1391 of the published sequence for B7-2 (positions 13-33 of the primer) and includes an Xho I restriction site (positions 7-12 of the primer). Following PCR, the reaction was extracted with phenol and precipitated using ethanol. The product was digested with Xho I and Kpn I, and the full-length fragment purified by agarose gel and ligated into the vector pcDNA-3 (Invitrogen, San Diego, Calif.) prepared by digesting with the same enzymes. The resultant construct, pcB7-2, was confirmed by restriction mapping and DNA sequence analysis using standard procedures. 
     A mouse B7-2 clone, pcmB7-2, was isolated in a similar manner by RT-PCR of RNA isolated from P388D1 cells using the 5′ primer, 
     
       
         
           
               
               
            
               
                   
                 the 5′ primer, 
               
               
                   
                 5′-GAT-CAG-GGT-ACC-AAG-AGT-GGC-TCC-TGT-AGG-CA  
               
               
                   
                 (sense, SEQ ID NO: 99), and   
               
               
                   
                   
               
               
                   
                 the 3′ primer, 
               
               
                   
                 5′-GAT-AGC-CTC-GAG-GTA-GAA-TTC-CAA-TCA-GCT-GA  
               
               
                   
                 (antisense, SEQ ID NO: 100). 
               
            
           
         
       
     
     The 5′ primer has identity with bases 1-20, whereas the 3′ primer is complementary to bases 1096-1115, of the published murine B7-2 sequence (Chen et al.,  J. Immun.,  1994, 152, 4929). Both primers incorporate the respective restriction enzyme sites found in the other 5′ and 3′ primers used to prepare cDNA clones. The RT-PCR product was restricted with Xho I and Kpn I and ligated into pcDNA-3 (Invitrogen, Carlsbad, Calif.). 
     Other cDNA clones, corresponding to mRNAs resulting from alternative splicing events, are cloned in like fashion, using primers containing the appropriate restriction sites and having identity with (5′ primers), or complementarity to (3′ primers), the selected B7 mRNA. 
     Example 2 
     Modulation of hB7-1 Expression by Oligonucleotides 
     The ability of oligonucleotides to inhibit B7-1 expression was evaluated by measuring the cell surface expression of B7-1 in transfected COS-7 cells by flow cytometry. 
     Methods: 
     A T-175 flask was seeded at 75% confluency with COS-7 cells (ATCC accession No. CRL 1651). The plasmid pcB7-1 was introduced into cells by standard calcium phosphate transfection. Following a 4 hour transfection, the cells were trypsinized and seeded in 12-well dishes at 80% confluency. The cells were allowed to adhere to the plastic for 1 hour and were then washed with phosphate-buffered saline (PBS). OptiMEM™ (GIBCO-BRL, Gaithersburg, Md.) medium was added along with 15 μg/mL of Lipofectin™ (GIBCO-BRL, Gaithersburg, Md.) and oligonucleotide at the indicated concentrations. After four additional hours, the cells were washed with phosphate buffered saline (PBS) and incubated with fresh oligonucleotide at the same concentration in DMEM (Dulbecco et al.,  Virol.,  1959, 8, 396; Smith et al.,  Virol.,  1960, 12, 185) with 10% fetal calf sera (FCS). 
     In order to monitor the effects of oligonucleotides on cell surface expression of B7-1, treated COS-7 cells were harvested by brief trypsinization 24-48 hours after oligonucleotide treatment. The cells were washed with PBS, then resuspended in 100 μL of staining buffer (PBS, 0.2% BSA, 0.1% azide) with 5 μL conjugated anti-B7-1-antibody (i.e., anti-hCD80-FITC, Ancell, Bayport, Minn.; FITC: fluorescein isothiocyanate). The cells were stained for 30 minutes at 4° C., washed with PBS, resuspended in 300 μL containing 0.5% paraformaldehyde. Cells were harvested and the fluorescence profiles were determined using a flow cytometer. 
     Results: 
     The oligonucleotides shown in Table 1 were evaluated, in COS-7 cells transiently expressing B7-1 cDNA, for their ability to inhibit B7-1 expression. The results ( FIG. 1 ) identified ISIS 13805, targeted to the translation initiation codon region, and ISIS 13812, targeted to the 3′ untranslated region (UTR), as the most active oligonucleotides with greater than 50% inhibition of B7-1 expression. These oligonucleotides are thus highly preferred. ISIS 13799 (targeted to the 5′ untranslated region), ISIS 13802 (targeted to the 5′ untranslated region), ISIS 13806 and 13807 (both targeted to the 5′ region of the ORF), and ISIS 13810 (targeted to the central portion of the ORF) demonstrated 35% to 50% inhibition of B7-1 expression. These sequences are therefore also preferred. Oligonucleotide ISIS 13800, which showed essentially no inhibition of B7-1 expression in the flow cytometry assay, and ISIS Nos. 13805 and 13812 were then evaluated for their ability to inhibit cell surface expression of B7-1 at various concentrations of oligonucleotide. The results of these assays are shown in  FIG. 2 . ISIS 13812 was a superior inhibitor of B7-1 expression with an IC 50  of approximately 150 nM. ISIS 13800, targeted to the 5′ UTR, was essentially inactive. 
     Example 3 
     Modulation of hB7-2 Protein by Oligonucleotides 
     In an initial screen, the ability of hB7-2 oligonucleotides to inhibit B7-2 expression was evaluated by measuring the cell surface expression of B7-2 in transfected COS-7 cells by flow cytometry. The methods used were similar to those given in Example 2, with the exceptions that (1) COS-7 cells were transfected with the plasmids pbcB7-2 or BBG-58, a human ICAM-1 (CD54) expression vector (R&amp;D Systems, Minneapolis, Minn.) introduced into cells by standard calcium phosphate transfection, (2) the oligonucleotides used were those described in Table 2, and (3) a conjugated anti-B7-2 antibody (i.e., anti-hCD86-FITC or anti-CD86-PE, PharMingen, San Diego, Calif.; PE: phycoerythrin) was used during flow cytometry. 
     Results: 
     The results are shown in  FIG. 3 . At a concentration of 200 nM, ISIS 9133, ISIS 9139 and ISIS 10373 exhibited inhibitory activity of 50% or better and are therefore highly preferred. These oligonucleotides are targeted to the 3′ untranslated region (ISIS 9133), the translation initiation codon region (ISIS 9139) and the 5′ untranslated region (ISIS 10373). At the same concentration, ISIS 10715, ISIS 10716 and ISIS 10721, which are scrambled controls for ISIS 9133, ISIS 9139 and ISIS 10373, respectively, showed no inhibitory activity. Treatment with ISIS 10367 and ISIS 10369 resulted in greater than 25% inhibition, and these oligonucleotides are thus also preferred. These oligonucleotides are targeted to the 5′ (ISIS 10367) and 3′(ISIS 10369) untranslated regions. 
     Example 4 
     Modulation of hB7-2 mRNA by Oligonucleotides 
     Methods: 
     For ribonuclease protection assays, cells were harvested 18 hours after completion of oligonucleotide treatment using a Totally RNA™ kit (Ambion, Austin, Tex.). The probes for the assay were generated from plasmids pcB7-2 (linearized by digestion with Bgl II) and pTRI-b-actin (Ambion Inc., Austin, Tex.). In vitro transcription of the linearized plasmid from the SP6 promoter was performed in the presence of α- 32 P-UTP (800 Ci/mmole) yielding an antisense RNA complementary to the 3′ end of B7-2 (position 1044-1391). The probe was gel-purified after treatment with DNase I to remove DNA template. Ribonuclease protection assays were carried out using an RPA II™ kit (Ambion) according to the manufacturer&#39;s directions. Total RNA (5 μg) was hybridized overnight, at 42° C., with 10 5  cpm of the B7-2 probe or a control beta-actin probe. The hybridization reaction was then treated, at 37° C. for 30 minutes, with 0.4 units of RNase A and 2 units of RNase T1. Protected RNA was precipitated, resuspended in 10 μL of gel loading buffer and electrophoresed on a 6% acrylamide gel with 50% w/v urea at 20 W. The gel was then exposed and the lanes quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) essentially according to the manufacturer&#39;s instructions. 
     Results: 
     The extent of oligonucleotide-mediated hB7-2 mRNA modulation generally paralleled the effects seen for hB7-2 protein (Table 5). As with the protein expression (flow cytometry) assays, the most active oligonucleotides were ISIS 9133, ISIS 9139 and 10373. None of the oligonucleotides tested had an inhibitory effect on the expression of b-actin mRNA in the same cells. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Activities of Oligonucleotides Targeted to hB7-2 mRNA 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 % Control 
                 % Control RNA 
               
               
                   
                 ISIS NO. 
                 SEQ ID NO. 
                 Protein 
                 Expression 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 9133 
                 3 
                 70.2 
                 46.0 
               
               
                   
                 9134 
                 4 
                 88.8 
                 94.5 
               
               
                   
                 9135 
                 5 
                 98.2 
                 83.4 
               
               
                   
                 9136 
                 6 
                 97.1 
                 103.1 
               
               
                   
                 9137 
                 7 
                 80.5 
                 78.1 
               
               
                   
                 9138 
                 8 
                 86.4 
                 65.9 
               
               
                   
                 9139 
                 9 
                 47.9 
                 32.6 
               
               
                   
                 10367 
                 10 
                 71.3 
                 52.5 
               
               
                   
                 10368 
                 11 
                 81.0 
                 84.5 
               
               
                   
                 10369 
                 12 
                 71.3 
                 81.5 
               
               
                   
                 10370 
                 13 
                 84.3 
                 83.2 
               
               
                   
                 10371 
                 14 
                 97.3 
                 92.9 
               
               
                   
                 10372 
                 15 
                 101.7 
                 82.5 
               
               
                   
                 10373 
                 16 
                 43.5 
                 32.7 
               
               
                   
                   
               
            
           
         
       
     
     Example 5 
     Additional hB7-1 and hB7-2 Oligonucleotides 
     Oligonucleotides having structures and/or sequences that were modified relative to the oligonucleotides identified during the initial screening were prepared. These oligonucleotides were evaluated for their ability to modulate human B7-2 expression using the methods described in the previous examples. ISIS 10996, an oligonucleotide having a 15 nucleotide sequence derived from the 20 nucleotide sequence of ISIS 10373, was also prepared and evaluated. ISIS 10996 comprises 15 nucleotides, 5′-GCG-AGC-TCC-CCG-TAC (SEQ ID NO: 90) contained within the sequence of ISIS 10373. Both ISIS 10373 and 10996 overlap a potential stem-loop structure located within the B7-2 message comprising bases 1-67 of the sequence of hB7-2 presented by Azuma et al. ( Nature,  1993, 366, 76). While not intending to be bound be any particular theory regarding their mode(s) of action, ISIS 10373 and ISIS 10996 have the potential to bind as loop 1 pseudo-half-knots at a secondary structure within the target RNA. U.S. Pat. No. 5,5152,438, the contents of which are hereby incorporated by reference, describes methods for modulating gene expression by the formation of pseudo-half-knots. Regardless of their mode(s) of action, despite having a shorter length than ISIS 10373, the 15-mer ISIS 10886 is as (or more) active in the B7-2 protein expression assay than the 20-mer from which it is derived ( FIG. 4 ; ISIS 10721 is a scrambled control for ISIS 10373). A related 16-mer, ISIS 10998, was also active in the B7-2 protein expression assay. However, a structurally related 14-mer (ISIS 10995), 13-mer (ISIS 10994), 12-mer (ISIS 10993), 11-mer (ISIS 10992) and 10-mer (ISIS 10991) exhibited little or no activity in this assay. ISIS 10996 was further derivatized in the following ways. 
     ISIS 10996 derivatives having 2′ methoxyethoxy substitutions were prepared, including a fully substituted derivative (ISIS 11539), “gapmers” (ISIS 11541 and 11543) and “wingmers” (ISIS 11545 and 11547). As explained in Example 5, the 2′ methoxyethoxy substitution prevents the action of some nucleases (e.g., RNase H) but enhances the affinity of the modified oligonucleotide for its target RNA molecule. These oligonucleotides are tested for their ability to modulate hB7-2 message or function according to the methods of Examples 3, 4, 7 and 8. 
     ISIS 10996 derivatives were prepared in order to be evaluated for their ability to recruit RNase L to a target RNA molecule, e.g., hB7-2 message. RNase L binds to, and is activated by, (2′-5′)(A) n , which is in turn produced from ATP by (2′-5′)(A) n  synthetase upon activation by, e.g., interferon. RNase L has been implicated in antiviral mechanisms and in the regulation of cell growth as well (Sawai,  Chemica Scripta,  1986, 21, 169; Charachon et al.,  Biochemistry,  1990, 29, 2550). The combination of anti-B7 oligonucleotides conjugated to (2′-5′)(A) n , is expected to result in the activation of RNase L and its targeting to the B7 message complementary to the oligonucleotide sequence. The following oligonucleotides have identical sequences (i.e., that of ISIS 10996) and identical (2′-5′)(A) 4  “caps” on their 5′ termini: ISIS 12492, 12495, 12496 and 13107. The adenosyl residues have 3′ hydroxyl groups and are linked to each other by phosphorothioate linkages. The (3′-5′) portion of the oligonucleotide, which has a sequence complementary to a portion of the human B7-2 RNA, is conjugated to the (2′-5′)(A) 4  “cap” via a phosphorothioate linkage from the 5′ residue of the (3′-5′) portion of the oligonucleotide to an n-aminohexyl linker which is bonded to the “cap” via another phosphorothioate linkage. In order to test a variety of chemically diverse oligonucleotides of this type for their ability to recruit RNase L to a specific message, different chemical modifications were made to this set of four oligonucleotides as follows. ISIS 12496 consists of unmodified oligonucleotides in the (3′-5′) portion of the oligonucleotide. In ISIS 13107, phosphorothioate linkages replace the phosphate linkages found in naturally occurring nucleic acids. Phosphorothioate linkages are also employed in ISIS 12492 and 12495, which additionally have 2′-methoxyethoxy substitutions. These oligonucleotides are tested for their ability to modulate hB7-2 message or function according to the methods of Examples 3, 4, 7 and 8. 
     Derivatives of ISIS 10996 having modifications at the 2′ position were prepared and evaluated. The modified oligonucleotides included ISIS 11539 (fully 2′-O-methyl), ISIS 11541 (having 2′-O-methyl wings and a central 7-base gap), ISIS 11543 (2′-O-methyl wings with a 9-base gap), ISIS 11545 (having a 5′2′-O-methyl wing) and ISIS 11547 (having a 3′2′-O-methyl wing). The results of assays of 2′-O-methyl oligonucleotides were as follows. ISIS 11539, the fully 2′O-methyl version of ISIS 10996, was not active at all in the protein expression assay. The gapped and winged oligonucleotides (ISIS 11541, 11543, 11545 and 11547) each showed some activity at 200 nM (i.e., from 60 to 70% expression relative to untreated cells), but less than that demonstrated by the parent compound, ISIS 10996 (i.e., about 50% expression). Similar results were seen in RNA expression assays. 
     ISIS 10782, a derivative of ISIS 10373 to which cholesterol has been conjugated via a 5′ n-aminohexyl linker, was prepared. Lipophilic moieties such as cholesterol have been reported to enhance the uptake by cells of oligonucleotides in some instances, although the extent to which uptake is enhanced, if any, remains unpredictable. ISIS 10782, and other oligonucleotides comprising lipophilic moieties, are tested for their ability to modulate B7-2 message or function according to the methods of Examples 3, 4, 7 and 8. 
     A series of 2′-methoxyethoxy (herein, “2′ME”) and 2′-fluoride (herein, “2′F”) “gapmer” derivatives of the hB7-1 oligonucleotides ISIS 12361 (ISIS Nos. 12348 and 12473, respectively), ISIS 12362 (ISIS Nos. 12349 and 12474), ISIS 12363 (ISIS Nos. 12350 and 12475), ISIS 12364 (ISIS Nos. 12351 and 12476), ISIS 12365 (ISIS Nos. 12352 and 12477), ISIS 12366 (ISIS Nos. 12353 and 12478), ISIS 12367 (ISIS Nos. 12354 and 12479), ISIS 12368 (ISIS Nos. 12355 and 12480), ISIS 12369 (ISIS Nos. 12356 and 12481) and ISIS 12370 (ISIS Nos. 12357 and 12482) were prepared. The central, non-2′-modified portions (“gaps”) of these derivatives support RNase H activity when the oligonucleotide is bound to its target RNA, even though the 2′-modified portions do not. However, the 2′-modified “wings” of these oligonucleotides enhance their affinity to their target RNA molecules (Cook, Chapter 9  In: Antisense Research and Applications , Crooke et al., eds., CRC Press, Boca Raton, 1993, pp. 171-172). 
     Another 2′ modification is the introduction of a methoxy (MO) group at this position. Like 2′ME- and 2′F-modified oligonucleotides, this modification prevents the action of RNase H on duplexes formed from such oligonucleotides and their target RNA molecules, but enhances the affinity of an oligonucleotide for its target RNA molecule. ISIS 12914 and 12915 comprise sequences complementary to the 5′ untranslated region of alternative hB7-1 mRNA molecules, which arise from alternative splicing events of the primary hB7-1 transcript. These oligonucleotides include 2′ methoxy modifications, and the enhanced target affinity resulting therefrom may allow for greater activity against alternatively spliced B7-1 mRNA molecules which may be present in low abundance in some tissues (Inobe et al.,  J. Immun.,  1996, 157, 582). Similarly, ISIS 13498 and 13499, which comprise antisense sequences to other alternative hB7-1 mRNAs, include 2′ methoxyethoxy modifications in order to enhance their affinity for their target molecules, and 2′ methoxyethoxy or 2′methoxy substitutions are incorporated into the hB7-2 oligonucleotides ISIS 12912, 12913, 13496 and 13497. These oligonucleotides are tested for their ability to modulate hB7-1 essentially according to the methods of Example 2 or hB7-2 according to the methods of Examples 3, 4, 7 and 8, with the exception that, when necessary, the target cells are transfected with a cDNA clone corresponding to the appropriate alternatively spliced B7 transcript. 
     Example 6 
     Specificity of Antisense Modulation 
     Several oligonucleotides of the invention were evaluated in a cell surface expression flow cytometry assay to determine the specificity of the oligonucleotides for B7-1 as contrasted with activity against B7-2. The oligonucleotides tested in this assay included ISIS 13812, an inhibitor of B7-1 expression ( FIG. 1 ; Example 2) and ISIS 10373, an inhibitor of B7-2 expression ( FIG. 3 ; Example 3). The results of this assay are shown in  FIG. 5 . ISIS 13812 inhibits B7-1 expression with little or no effect on B7-2 expression. As is also seen in  FIG. 5 , ISIS 10373 inhibits B7-2 expression with little or no effect on B7-1 expression. ISIS 13872 (SEQ ID NO: 37, AGT-CCT-ACT-ACC-AGC-CGC-CT), a scrambled control of ISIS 13812, and ISIS 13809 (SEQ ID NO: 51) were included in these assays and demonstrated essentially no activity against either B7-1 or B7-2. 
     Example 7 
     Modulation of hB7-2 Expression by Oligonucleotides in Antigen Presenting Cells 
     The ability of ISIS 10373 to inhibit expression from the native B7-2 gene in antigen presenting cells (APCs) was evaluated as follows. 
     Methods: 
     Monocytes were cultured and treated with oligonucleotides as follows. For dendritic cells, EDTA-treated blood was layered onto Polymorphprep™ (1.113 g/mL; Nycomed, Oslo, Norway) and sedimented at 500×g for 30 minutes at 20° C. Mononuclear cells were harvested from the interface. Cells were washed with PBS, with serum-free RPMI media (Moore et al.,  N.Y. J. Med.,  1968, 68, 2054) and then with RPMI containing 5% fetal bovine serum (FBS). Monocytes were selected by adherence to plastic cell culture cell culture dishes for 1 h at 37° C. After adherence, cells were treated with oligonucleotides in serum-free RPMI containing Lipofectin™ (8 μg/mL). After 4 hours, the cells were washed. Then RPMI containing 5% FBS and oligonucleotide was added to cells along with interleukin-4 (IL-4; R&amp;D Systems, Minneapolis, Minn.) (66 ng/mL) and granulocyte-macrophage colony-stimulating factor (GM-CSF; R&amp;D Systems, Minneapolis, Minn.) (66 ng/mL) to stimulate differentiation (Romani et al.,  J. Exp. Med.,  1994, 180, 83, 1994). Cells were incubated for 48 hours, after which cell surface expression of various molecules was measured by flow cytometry. 
     Mononuclear cells isolated from fresh blood were treated with oligonucleotide in the presence of cationic lipid to promote cellular uptake. As a control oligonucleotide, ISIS 2302 (an inhibitor of ICAM-1 expression; SEQ ID NO: 17) was also administered to the cells. Expression of B7-2 protein was measured by flow cytometry according to the methods of Example 2. Monoclonal antibodies not described in the previous Examples included anti-hCD3 (Ancell, Bayport, Minn.) and anti-HLA-DR (Becton Dickinson, San Jose, Calif.). 
     Results: 
     As shown in  FIG. 6 , ISIS 10373 has a significant inhibitory effect on B7-2 expression with an IC 50  of approximately 250 nM. ISIS 10373 had only a slight effect on ICAM-1 expression even at a dose of 1 μM. ISIS 2302 (SEQ ID NO: 17), a control oligonucleotide which has been shown to inhibit ICAM-1 expression, had no effect on B7-2 expression, but significantly decreased ICAM-1 levels with an IC 50  of approximately 250 nM. Under similar conditions, ISIS 10373 did not affect the cell surface expression of B7-1, HLA-DR or CD3 as measured by flow cytometry. 
     Example 8 
     Modulation of T Cell Proliferation by Oligonucleotides 
     The ability of ISIS 2302 and ISIS 10373 to inhibit T cell proliferation was evaluated as follows. Monocytes treated with oligonucleotide and cytokines (as in Example 6) were used as antigen presenting cells in a T cell proliferation assay. The differentiated monocytes were combined with CD4+ T cells from a separate donor. After 48 hours, proliferation was measured by [ 3 H]thymidine incorporation. 
     Methods: 
     For T cell proliferation assays, cells were isolated from EDTA-treated whole blood as described above, except that a faster migrating band containing the lymphocytes was harvested from just below the interface. Cells were washed as described in Example 6 after which erythrocytes were removed by NH 4 Cl lysis. T cells were purified using a T cell enrichment column (R&amp;D Systems, Minneapolis, Minn.) essentially according to the manufacturer&#39;s directions. CD4+ T cells were further enriched from the entire T cell population by depletion of CD8+ cells with anti-CD8-conjugated magnetic beads (AMAC, Inc., Westbrook, Me.) according to the manufacturer&#39;s directions. T cells were determined to be &gt;80% CD4+ by flow cytometry using Cy-chrome-conjugated anti-CD4 mAb (PharMingen, San Diego, Calif.). 
     Antigen presenting cells (APCs) were isolated as described in Example 6 and treated with mitomycin C (25 μg/mL) for 1 hour then washed 3 times with PBS. APCs (10 5  cells) were then combined with 4×10 4  CD4+ T cells in 350 μL of culture media. Where indicated, purified CD3 mAb was also added at a concentration of 1 μg/mL. During the last 6 hours of the 48 hour incubation period, proliferation was measured by determining uptake of 1.5 μCi of [ 3 H]-thymidine per well. The cells were harvested onto filters and the radioactivity measured by scintillation counting. 
     Results: 
     As shown in  FIG. 7 , mononuclear cells which were not cytokine-treated slightly induced T cell proliferation, presumably due to low levels of costimulatory molecules expressed on the cells. However, when the cells were treated with cytokines and induced to differentiate to dendritic-like cells, expression of both ICAM-1 and B7-2 was strongly upregulated. This resulted in a strong T cell proliferative response which could be blocked with either anti-ICAM-1 (ISIS 2302) or anti-B7-2 (ISIS 10373) oligonucleotides prior to induction of the mononuclear cells. The control oligonucleotide (ISIS 10721) had an insignificant effect on T cell proliferation. A combination treatment with both the anti-ICAM-1 (ISIS 2302) and anti-B7-2 (ISIS 10373) oligonucleotides resulted in a further decrease in T cell response. 
     Example 9 
     Modulation of Murine B7 Genes by Oligonucleotides 
     Oligonucleotides (see Table 4) capable of inhibiting expression of murine B7-2 transiently expressed in COS-7 cells were identified in the following manner. A series of phosphorothioate oligonucleotides complementary to murine B7-2 (mB7-2) cDNA were screened for their ability to reduce mB7-2 levels (measured by flow cytometry as in Example 2, except that a conjugated anti-mB7-2 antibody (i.e., anti-mCD86-PE, PharMingen, San Diego, Calif.) in COS-7 cells transfected with an mB7-2 cDNA clone. Anti-mB7-2 antibody may also be obtained from the hybridoma deposited at the ATCC under accession No. HB-253. Oligonucleotides (see Table 2) capable of modulating murine B7-1 expression are isolated in like fashion, except that a conjugated anti-mB7-1 antibody is used in conjunction with COS-7 cells transfected with an mB7-1 cDNA clone. 
     For murine B7-2, the most active oligonucleotide identified was ISIS 11696 (GGA-TTG-CCA-AGC-CCA-TGG-TG, SEQ ID NO: 18), which is complementary to position 96-115 of the cDNA, a site which includes the translation initiation (AUG) codon.  FIG. 8  shows a dose-response curve for ISIS 11696 and a scrambled control, ISIS 11866 (CTA-AGT-AGT-GCT-AGC-CGG-GA, SEQ ID NO: 19). ISIS 11696 inhibited cell surface expression of B7-2 in COS-7 cells with an IC 50  in the range of 200-300 nM, while ISIS 11866 exhibited less than 20% inhibition at the highest concentration tested (1000 nM). 
     In order to further evaluate the murine B7-2 antisense oligonucleotides, the IC-21 cell line was used. IC-21 monocyte/macrophage cell line expresses both B7-1 and murine B7-2 (mB7-2) constitutively. A 2-fold induction of expression can be achieved by incubating the cells in the presence of lipopolysaccharide (LPS; GIBCO-BRL, Gaithersburg, Md.) (Hathcock et al.,  Science,  1993, 262, 905). 
     IC-21 cells (ATCC; accession No. TIB 186) were seeded at 80% confluency in 12-well plates in DMEM media with 10% FCS. The cells were allowed to adhere to the plate overnight. The following day, the medium was removed and the cells were washed with PBS. Then 500 μL of OptiMEM™ (GIBCO-BRL, Gaithersburg, Md.) supplemented with 15 μg/mL of Lipofectin™ (GIBCO-BRL, Gaithersburg, Md.) was added to each well. Oligonucleotides were then added directly to the medium at the indicated concentrations. After incubation for 4 hours, the cells were washed with PBS and incubated overnight in culture medium supplemented with 15 μg/mL of LPS. The following day, cells were harvested by scraping, then analyzed for cell surface expression by flow cytometry. 
     ISIS 11696 and ISIS 11866 were administered to IC-21 cells in the presence of Lipofectin™ (GIBCO-BRL, Gaithersburg, Md.). The results are shown in  FIG. 9 . At a concentration of 10 μM, ISIS 11696 inhibited mB7-2 expression completely (and decreased mB7-2 levels below the constitutive level of expression), while the scrambled control oligonucleotide, ISIS 11866, produced only a 40% reduction in the level of induced expression. At a concentration of 3 μM, levels of induced expression were greatly reduced by ISIS 11696, while ISIS 11866 had little effect. 
     Modified oligonucleotides, comprising 2′ substitutions (e.g., 2′ methoxy, 2′ methoxyethoxy) and targeted to alternative transcripts of murine B7-1 (ISIS 12914, 12915, 13498, 13499) or murine B7-2 (ISIS 13100, 13100 and 13102) were prepared. These oligonucleotides are tested for their ability to modulate murine B7 essentially according to the above methods using IC-21 cells or COS-7 transfected with a cDNA clone corresponding to the appropriate alternatively spliced B7 transcript. 
     Example 10 
     Modulation of Allograft Rejection by Oligonucleotides 
     A murine model for evaluating compounds for their ability to inhibit heart allograft rejection has been previously described (Stepkowski et al., J. Immunol., 1994, 153, 5336). This model was used to evaluate the immunosuppressive capacity of antisense oligonucleotides to B7 proteins alone or in combination with antisense oligonucleotides to intercellular adhesion molecule-1 (ICAM-1). 
     Methods: 
     Heart allograft rejection studies and oligonucleotide treatments of BALB/c mice were performed essentially as previously described (Stepkowski et al., J. Immunol., 1994, 153, 5336). Antisense oligonucleotides used included ISIS 11696, ISIS 3082 (targeted to ICAM-1) and ISIS 1082 (a control oligonucleotide targeted to the herpes virus UL-13 gene sequence). Dosages used were 1, 2, 2.5, 5 or 10 mg/kg of individual oligonucleotide (as indicated below); when combinations of oligonucleotides were administered, each oligonucleotide was given at a dosage of 1, 5 or 10 mg/kg (total oligonucleotide dosages of 2, 10 and 20 mg/kg, respectively). The survival times of the transplanted hearts and their hosts were monitored and recorded. 
     Results: 
     The mean survival time for untreated mice was 8.2±0.8 days (7, 8, 8, 8, 9, 9 days). Treatment of the mice for 7 days with ISIS 1082 (SEQ ID NO: 125, unrelated control oligonucleotide) slightly reduced the mean survival times to 7.1±0.7 days (5 mg/kg/day; 6, 7, 7, 7, 8, 8) or 7.0±0.8 days (10 mg/kg/day; 6, 7, 7, 8). Treatment of the mice for seven days with the murine B7-2 oligonucleotide ISIS 11696 (SEQ ID NO: 108) increased the mean survival time to 9.3 days at two doses (2 mg/kg/day, 9.3±0.6 days, 9, 9, 10; 10 mg/kg/day, 9.3±1.3 days, 8, 9, 9, 11). Treatment of mice for seven days with an ICAM-1 oligonucleotide, ISIS 3082, also increased the mean survival of the mice over several doses. Specifically, at 1 mg/kg/day, the mean survival time (MSD) was 11.0±0.0 (11, 11, 11); at 2.5 mg/kg/day, the MSD was 12.0±2.7 (10, 12, 13, 16); at 5 mg/kg/day, the MSD was 14.1±2.7 (10, 12, 12, 13, 16, 16, 17, 17); and, at 10 mg/kg/day, the MSD was 15.3±5.8 (12, 12, 13, 24). Some synergistic effect was seen when the mice were treated for seven days with 1 mg/kg/day each of ISIS 3082 and 11696: the MSD was 13.8±1.0 (13, 13, 14, 15). 
     Example 11 
     Detection of Nucleic Acids Encoding B7 Proteins 
     Oligonucleotides are radiolabeled after synthesis by  32 P-labeling at the 5′ end with polynucleotide kinase. Sambrook et al., “Molecular Cloning. A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 11.31. Radiolabeled oligonucleotide capable of hybridizing to a nucleic acid encoding a B7 protein is contacted with a tissue or cell sample suspected of B7 protein expression under conditions in which specific hybridization can occur, and the sample is washed to remove unbound oligonucleotide. A similar control is maintained wherein the radiolabeled oligonucleotide is contacted with a normal tissue or cell sample under conditions that allow specific hybridization, and the sample is washed to remove unbound oligonucleotide. Radioactivity remaining in the samples indicates bound oligonucleotide and is quantitated using a scintillation counter or other routine means. A greater amount of radioactivity remaining in the samples, as compared to control tissues or cells, indicates increased expression of a B7 gene, whereas a lesser amount of radioactivity in the samples relative to the controls indicates decreased expression of a B7 gene. 
     Radiolabeled oligonucleotides of the invention are also useful in autoradiography. A section of tissues suspected of expressing a B7 gene is treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to standard autoradiography procedures. A control of a normal tissue section is also maintained. The emulsion, when developed, yields an image of silver grains over the regions expressing a B7 gene, which is quantitated. The extent of B7 expression is determined by comparison of the silver grains observed with control and test samples. 
     Analogous assays for fluorescent detection of expression of a B7 gene use oligonucleotides of the invention which are labeled with fluorescein or other fluorescent tags. Labeled oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard phosphoramidite chemistry. b-Cyanoethyldiisopropyl phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). Fluorescein-labeled amidites are purchased from Glen Research (Sterling, Va.). Incubation of oligonucleotide and biological sample is carried out as described above for radiolabeled oligonucleotides except that, instead of a scintillation counter, a fluorescence microscope is used to detect the fluorescence. A greater amount of fluorescence in the samples, as compared to control tissues or cells, indicates increased expression of a B7 gene, whereas a lesser amount of fluorescence in the samples relative to the controls indicates decreased expression of a B7 gene. 
     Example 12 
     Chimeric (Deoxy Gapped) Human B7-1 Antisense Oligonucleotides 
     Additional oligonucleotides targeting human B7-1 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 6. 
     Oligonucleotides were screened as described in Example 4. Results are shown in Table 7. 
     Oligonucleotides 22315 (SEQ ID NO: 128), 22316 (SEQ ID NO: 26), 22317 (SEQ ID NO: 129), 22320 (SEQ ID NO: 132), 22324 (SEQ ID NO: 135), 22325 (SEQ ID NO: 136), 22334 (SEQ ID NO: 145), 22335 (SEQ ID NO: 146), 22337 (SEQ ID NO: 148), and 22338 (SEQ ID NO: 36) resulted in 50% or greater inhibition of B7-1 mRNA in this assay. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Human B7-1 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                 TARGET GENE 
                 GENE 
               
               
                 ISIS 
                 NUCLEOTIDE SEQUENCE 1   
                 ID 
                 NUCLEOTIDE 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO: 
                 COORDINATES 2   
                 REGION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 22313 
                   AGACT CCACTTCTGA GATGT   
                 126 
                 0048-0067 
                 5′-UTR 
               
               
                   
               
               
                 22314 
                   TGAAG AAAAATTCCA CTTTT   
                 127 
                 0094-0113 
                 5′-UTR 
               
               
                   
               
               
                 22315 
                   TTTAG TTTCACAGCT TGCTG   
                 128 
                 0112-0129 
                 5′-UTR 
               
               
                   
               
               
                 22316 
                   GCTCA CGTAGAAGAC CCTCC   
                 26 
                 0193-0212 
                 5′-UTR 
               
               
                   
               
               
                 22317 
                   TCCCA GGTGCAAAAC AGGCA   
                 129 
                 0233-0252 
                 5′-UTR 
               
               
                   
               
               
                 22318 
                   GTGAA AGCCAACAAT TTGGA   
                 130 
                 0274-0293 
                 5′-UTR 
               
               
                   
               
               
                 22319 
                   CATGG CTTCAGATGC TTAGG   
                 131 
                 0301-0320 
                 AUG 
               
               
                   
               
               
                 22320 
                   TTGAG GTATGGACAC TTGGA   
                 132 
                 0351-0370 
                 coding 
               
               
                   
               
               
                 22321 
                   GACCA GCCAGCACCA AGAGC   
                 31 
                 0380-0399 
                 coding 
               
               
                   
               
               
                 22322 
                   GCGTT GCCACTTCTT TCACT   
                 133 
                 0440-0459 
                 coding 
               
               
                   
               
               
                 22323 
                   TTTTG CCAGTAGATG CGAGT   
                 134 
                 0501-0520 
                 coding 
               
               
                   
               
               
                 22324 
                   GGCCA TATATTCATG TCCCC   
                 135 
                 0552-0571 
                 coding 
               
               
                   
               
               
                 22325 
                   GCCAG GATCACAATG GAGAG   
                 136 
                 0612-0631 
                 coding 
               
               
                   
               
               
                 22326 
                   GTATG TGCCCTCGTC AGATG   
                 137 
                 0640-0659 
                 coding 
               
               
                   
               
               
                 22327 
                   TTCAG CCAGGTGTTC CCGCT   
                 138 
                 0697-0716 
                 coding 
               
               
                   
               
               
                 22328 
                   GGAAG TCAGCTTTGA CTGAT   
                 139 
                 0725-0744 
                 coding 
               
               
                   
               
               
                 22329 
                   CCTCC AGAGGTTGAG CAAAT   
                 140 
                 0798-0817 
                 coding 
               
               
                   
               
               
                 22330 
                   CCAAC CAGGAGAGGT GAGGC   
                 141 
                 0827-0846 
                 coding 
               
               
                   
               
               
                 22331 
                   GAAGC TGTGGTTGGT TGTCA   
                 142 
                 0940-0959 
                 coding 
               
               
                   
               
               
                 22332 
                   TTGAA GGTCTGATTC ACTCT   
                 143 
                 0987-1006 
                 coding 
               
               
                   
               
               
                 22333 
                   AAGGT AATGGCCCAG GATGG   
                 144 
                 1050-1069 
                 coding 
               
               
                   
               
               
                 22334 
                   AAGCA GTAGGTCAGG CAGCA   
                 145 
                 1098-1117 
                 coding 
               
               
                   
               
               
                 22335 
                   CCTTG CTTCTGCGGA CACTG   
                 146 
                 1185-1204 
                 3′-UTR 
               
               
                   
               
               
                 22336 
                   AGCCC CTTGCTTCTG CGGAC   
                 147 
                 1189-1208 
                 3′-UTR 
               
               
                   
               
               
                 22337 
                   TGACG GAGGCTACCT TCAGA   
                 148 
                 1216-1235 
                 3′-UTR 
               
               
                   
               
               
                 22338 
                   GCCTC ATGATCCCCA CGATC   
                 36 
                 1254-1273 
                 3′-UTR 
               
               
                   
               
               
                 22339 
                   GTAAA ACAGCTTAAA TTTGT   
                 149 
                 1286-1305 
                 3′-UTR 
               
               
                   
               
               
                 22340 
                   AGAAG AGGTTACATT AAGCA   
                 150 
                 1398-1417 
                 3′-UTR 
               
               
                   
               
               
                 22341 
                   AGATA ATGAATTGGC TGACA   
                 151 
                 1454-1473 
                 3′-UTR 
               
               
                   
               
               
                 24733 
                   GCGTC ATCATCCGCA CCATC   
                 152 
                 control 
                   
               
               
                   
               
               
                 24734 
                   CGTTG CTTGTGCCGA CAGTG   
                 153 
                 control 
                   
               
               
                   
               
               
                 24735 
                   GCTCA CGAAGAACAC CTTCC   
                 154 
                 control 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 Co-ordinates from Genbank Accession No. M27533, locus name “HUMIGB7”. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Inhibition of Human B7-1 mRNA Expression by 
               
               
                 Chimeric (deoxy gapped) Phosphorothioate 
               
               
                 Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ 
                 GENE 
                   
                   
               
               
                 ISIS 
                 ID 
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
               
                 basal 
                 — 
                 — 
                 100% 
                 — 
               
               
                   
               
               
                 13805 
                 30 
                 AUG 
                 46% 
                 54% 
               
               
                   
               
               
                 13812 
                 36 
                 3′-UTR 
                 22% 
                 78% 
               
               
                   
               
               
                 22313 
                 126 
                 5′-UTR 
                 75% 
                 25% 
               
               
                   
               
               
                 22314 
                 127 
                 5′-UTR 
                 69% 
                 31% 
               
               
                   
               
               
                 22315 
                 128 
                 5′-UTR 
                 49% 
                 51% 
               
               
                   
               
               
                 22316 
                 26 
                 5′-UTR 
                 42% 
                 58% 
               
               
                   
               
               
                 22317 
                 129 
                 5′-UTR 
                 43% 
                 57% 
               
               
                   
               
               
                 22318 
                 130 
                 5′-UTR 
                 63% 
                 37% 
               
               
                   
               
               
                 22319 
                 131 
                 AUG 
                 68% 
                 32% 
               
               
                   
               
               
                 22320 
                 132 
                 coding 
                 45% 
                 55% 
               
               
                   
               
               
                 22321 
                 31 
                 coding 
                 57% 
                 43% 
               
               
                   
               
               
                 22324 
                 135 
                 coding 
                 46% 
                 54% 
               
               
                   
               
               
                 22325 
                 136 
                 coding 
                 46% 
                 54% 
               
               
                   
               
               
                 22326 
                 137 
                 coding 
                 62% 
                 38% 
               
               
                   
               
               
                 22328 
                 139 
                 coding 
                 64% 
                 36% 
               
               
                   
               
               
                 22329 
                 140 
                 coding 
                 59% 
                 41% 
               
               
                   
               
               
                 22330 
                 141 
                 coding 
                 54% 
                 46% 
               
               
                   
               
               
                 22331 
                 142 
                 coding 
                 62% 
                 38% 
               
               
                   
               
               
                 22332 
                 143 
                 coding 
                 67% 
                 33% 
               
               
                   
               
               
                 22333 
                 144 
                 coding 
                 73% 
                 27% 
               
               
                   
               
               
                 22334 
                 145 
                 coding 
                 43% 
                 57% 
               
               
                   
               
               
                 22335 
                 146 
                 3′-UTR 
                 43% 
                 57% 
               
               
                   
               
               
                 22336 
                 147 
                 3′-UTR 
                 55% 
                 45% 
               
               
                   
               
               
                 22337 
                 148 
                 3′-UTR 
                 42% 
                 58% 
               
               
                   
               
               
                 22338 
                 36 
                 3′-UTR 
                 40% 
                 60% 
               
               
                   
               
               
                 22339 
                 149 
                 3′-UTR 
                 69% 
                 31% 
               
               
                   
               
               
                 22340 
                 150 
                 3′-UTR 
                 71% 
                 29% 
               
               
                   
               
               
                 22341 
                 151 
                 3′-UTR 
                 59% 
                 41% 
               
               
                   
               
            
           
         
       
     
     Dose response experiments were performed on several of the more active oligonucleotides. The oligonucleotides were screened as described in Example 4 except that the concentration of oligonucleotide was varied as shown in Table 8. Mismatch control oligonucleotides were included. Results are shown in Table 8. 
     All antisense oligonucleotides tested showed a dose response effect with inhibition of mRNA approximately 60% or greater. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Dose Response of COS-7 Cells to B7-1 Chimeric 
               
               
                 (deoxy gapped) Antisense Oligonucleotides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
                   
                   
               
               
                   
                 ID 
                 Gene 
                   
                 % mRNA 
                 % mRNA 
               
               
                 ISIS # 
                 NO: 
                 Target 
                 Dose 
                 Expression 
                 Inhibition 
               
               
                   
               
               
                 basal 
                 — 
                 — 
                 — 
                 100% 
                 — 
               
               
                   
               
               
                 22316 
                 26 
                 5′-UTR 
                  10 nM 
                 99% 
                 1% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 73% 
                 27% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 58% 
                 42% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 33% 
                 67% 
               
               
                   
               
               
                 24735 
                 154 
                 control 
                  10 nM 
                 100% 
                 — 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 95% 
                 5% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 81% 
                 19% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 75% 
                 25% 
               
               
                   
               
               
                 22335 
                 146 
                 3′-UTR 
                  10 nM 
                 81% 
                 19% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 63% 
                 37% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 43% 
                 57% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 35% 
                 65% 
               
               
                   
               
               
                 24734 
                 153 
                 control 
                  10 nM 
                 94% 
                 6% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 96% 
                 4% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 94% 
                 6% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 84% 
                 16% 
               
               
                   
               
               
                 22338 
                 36 
                 3′-UTR 
                  10 nM 
                 68% 
                 32% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 60% 
                 40% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 53% 
                 47% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 41% 
                 59% 
               
               
                   
               
               
                 24733 
                 152 
                 control 
                  10 nM 
                 90% 
                 10% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 91% 
                 9% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 90% 
                 10% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 80% 
                 20% 
               
               
                   
               
            
           
         
       
     
     Example 13 
     Chimeric (Deoxy Gapped) Mouse B7-1 Antisense Oligonucleotides 
     Additional oligonucleotides targeting mouse B7-1 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 9. 
     Oligonucleotides were screened as described in Example 4. Results are shown in Table 10. Oligonucleotides 18105 (SEQ ID NO: 156), 18106 (SEQ ID NO: 157), 18109 (SEQ ID NO: 160), 18110 (SEQ ID NO: 161), 18111 (SEQ ID NO: 162), 18112 (SEQ ID NO: 163), 18113 (SEQ ID NO: 164), 18114 (SEQ ID NO: 165), 18115 (SEQ ID NO: 166), 18117 (SEQ ID NO: 168), 18118 (SEQ ID NO: 169), 18119 (SEQ ID NO: 170), 18120 (SEQ ID NO: 171), 18122 (SEQ ID NO: 173), and 18123 (SEQ ID NO: 174) resulted in greater than approximately 50% inhibition of B7-1 mRNA in this assay. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Mouse B7-1 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                 TARGET GENE 
                 GENE 
               
               
                 ISIS 
                 NUCLEOTIDE SEQUENCE 1   
                 ID 
                 NUCLEOTIDE 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO: 
                 CO-ORDINATES 2   
                 REGION 
               
               
                   
               
               
                 18104 
                   AGAGA AACTAGTAAG AGTCT   
                 155 
                 0018-0037 
                 5′-UTR 
               
               
                   
               
               
                 18105 
                   TGGCA TCCACCCGGC AGATG   
                 156 
                 0110-0129 
                 5′-UTR 
               
               
                   
               
               
                 18106 
                   TCGAG AAACAGAGA TGTAGA   
                 157 
                 0144-0163 
                 5′-UTR 
               
               
                   
               
               
                 18107 
                   TGGAG CTTAGGCACC TCCTA   
                 158 
                 0176-0195 
                 5′-UTR 
               
               
                   
               
               
                 18108 
                   TGGGG AAAGCCAGGA ATCTA   
                 159 
                 0203-0222 
                 5′-UTR 
               
               
                   
               
               
                 18109 
                   CAGCA CAAAGAGAAG AATGA   
                 160 
                 0310-0329 
                 coding 
               
               
                   
               
               
                 18110 
                   ATGAG GAGAGTTGTA ACGGC   
                 161 
                 0409-0428 
                 coding 
               
               
                   
               
               
                 18111 
                   AAGTC CGGTTCTTAT ACTCG   
                 162 
                 0515-0534 
                 coding 
               
               
                   
               
               
                 18112 
                   GCAGG TAATCCTTTT AGTGT   
                 163 
                 0724-0743 
                 coding 
               
               
                   
               
               
                 18113 
                   GTGAA GTCCTCTGAC ACGTG   
                 164 
                 0927-0946 
                 coding 
               
               
                   
               
               
                 18114 
                   CGAAT CCTGCCCCAA AGAGC   
                 165 
                 0995-1014 
                 coding 
               
               
                   
               
               
                 18115 
                   ACTGC GCCGAATCCT GCCCC   
                 166 
                 1002-1021 
                 coding 
               
               
                   
               
               
                 18116 
                   TTGAT GATGACAACG ATGAC   
                 167 
                 1035-1054 
                 coding 
               
               
                   
               
               
                 18117 
                   CTGTT GTTTGTTTCT CTGCT   
                 168 
                 1098-1117 
                 coding 
               
               
                   
               
               
                 18118 
                   TGTTC AGCTAATGCT TCTTC   
                 169 
                 1134-1153 
                 coding 
               
               
                   
               
               
                 18119 
                   GTTAA CTCTATCTTG TGTCA   
                 170 
                 1263-1282 
                 3′-UTR 
               
               
                   
               
               
                 18120 
                   TCCAC TTCAGTCATC AAGCA   
                 171 
                 1355-1374 
                 3′-UTR 
               
               
                   
               
               
                 18121 
                   TGCTC AATACTCTCT TTTTA   
                 172 
                 1680-1699 
                 3′-UTR 
               
               
                   
               
               
                 18122 
                   AGGCC CAGCAAACTT GCCCG   
                 173 
                 1330-1349 
                 3′-UTR 
               
               
                   
               
               
                 18123 
                   AACGG CAAGGCAGCA ATACC   
                 174 
                 0395-0414 
                 coding 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 Co-ordinates from Genbank Accession No. X60958, locus name “MMB7BLAA”. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Inhibition of Mouse B7-1 mRNA Expression by 
               
               
                 Chimeric (deoxy gapped) Phosphorothioate 
               
               
                 Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 GENE 
                   
                   
               
               
                   
                 ISIS 
                 SEQ ID 
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                   
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 basal 
                 — 
                 — 
                 100.0% 
                 — 
               
               
                   
               
               
                   
                 18104 
                 155 
                 5′-UTR 
                 60.0% 
                 40.0% 
               
               
                   
               
               
                   
                 18105 
                 156 
                 5′-UTR 
                 32.0% 
                 68.0% 
               
               
                   
               
               
                   
                 18106 
                 157 
                 5′-UTR 
                 51.0% 
                 49.0% 
               
               
                   
               
               
                   
                 18107 
                 158 
                 5′-UTR 
                 58.0% 
                 42.0% 
               
               
                   
               
               
                   
                 18108 
                 159 
                 5′-UTR 
                 82.0% 
                 18.0% 
               
               
                   
               
               
                   
                 18109 
                 160 
                 coding 
                 45.5% 
                 54.5% 
               
               
                   
               
               
                   
                 18110 
                 161 
                 coding 
                 21.0% 
                 79.0% 
               
               
                   
               
               
                   
                 18111 
                 162 
                 coding 
                 38.0% 
                 62.0% 
               
               
                   
               
               
                   
                 18112 
                 163 
                 coding 
                 42.0% 
                 58.0% 
               
               
                   
               
               
                   
                 18113 
                 164 
                 coding 
                 24.6% 
                 75.4% 
               
               
                   
               
               
                   
                 18114 
                 165 
                 coding 
                 25.6% 
                 74.4% 
               
               
                   
               
               
                   
                 18115 
                 166 
                 coding 
                 33.5% 
                 66.5% 
               
               
                   
               
               
                   
                 18116 
                 167 
                 coding 
                 65.6% 
                 34.4% 
               
               
                   
               
               
                   
                 18117 
                 168 
                 coding 
                 46.7% 
                 53.3% 
               
               
                   
               
               
                   
                 18118 
                 169 
                 coding 
                 31.7% 
                 68.3% 
               
               
                   
               
               
                   
                 18119 
                 170 
                 3′-UTR 
                 24.0% 
                 76.0% 
               
               
                   
               
               
                   
                 18120 
                 171 
                 3′-UTR 
                 26.7% 
                 73.3% 
               
               
                   
               
               
                   
                 18121 
                 172 
                 3′-UTR 
                 114.0% 
                 — 
               
               
                   
               
               
                   
                 18122 
                 173 
                 3′-UTR 
                 42.0% 
                 58.0% 
               
               
                   
               
               
                   
                 18123 
                 174 
                 coding 
                 42.0% 
                 58.0% 
               
               
                   
               
            
           
         
       
     
     Example 14 
     Chimeric (Deoxy Gapped) Human B7-2 Antisense Oligonucleotides 
     Additional oligonucleotides targeting human B7-2 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 11. 
     Oligonucleotides were screened as described in Example 4. Results are shown in Table 12. Oligonucleotides 22284 (SEQ ID NO: 16), 22286 (SEQ ID NO: 176), 22287 (SEQ ID NO: 177), 22288 (SEQ ID NO: 178), 22289 (SEQ ID NO: 179), 22290 (SEQ ID NO: 180), 22291 (SEQ ID NO: 181), 22292 (SEQ ID NO: 182), 22293 (SEQ ID NO: 183), 22294 (SEQ ID NO: 184), 22296 (SEQ ID NO: 186), 22299 (SEQ ID NO: 189), 22300 (SEQ ID NO: 190), 22301 (SEQ ID NO: 191), 22302 (SEQ ID NO: 192), 22303 (SEQ ID NO: 193), 22304 (SEQ ID NO: 194), 22306 (SEQ ID NO: 196), 22307 (SEQ ID NO: 197), 22308 (SEQ ID NO: 198), 22309 (SEQ ID NO: 199), 22310 (SEQ ID NO: 200), and 22311 (SEQ ID NO: 201) resulted in greater than 50% inhibition of B7-2 mRNA in this assay. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Human B7-2 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 TAGET GENE 
                   
               
               
                   
                   
                 SEQ 
                 NUCLEOTIDE 
                 GENE 
               
               
                 ISIS 
                 NUCLEOTIDE SEQUENCE 1   
                 ID 
                 CO- 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO: 
                 ORDINATES 2   
                 REGION 
               
               
                   
               
               
                 22284 
                   TGCGA GCTCCCCGTA CCTCC   
                  16 
                 0011-0030 
                 5′-UTR 
               
               
                   
               
               
                 22285 
                   CAGAA GCAAGGTGGT AAGAA   
                 175 
                 0049-0068 
                 5′-UTR 
               
               
                   
               
               
                 22286 
                   GCCTG TCCACTGTAG CTCCA   
                 176 
                 0113-0132 
                 5′-UTR 
               
               
                   
               
               
                 22287 
                   AGAAT GTTACTCAGT CCCAT   
                 177 
                 0148-0167 
                 AUG 
               
               
                   
               
               
                 22288 
                   TCAGA GGAGCAGCAC CAGAG   
                 178 
                 0189-0208 
                 coding 
               
               
                   
               
               
                 22289 
                   TGGCA TGGCAGGTCT GCAGT   
                 179 
                 0232-0251 
                 coding 
               
               
                   
               
               
                 22290 
                   AGCTC ACTCAGGCTT TGGTT   
                 180 
                 0268-0287 
                 coding 
               
               
                   
               
               
                 22291 
                   TGCCT AAGTATACCT CATTC   
                 181 
                 0324-0343 
                 coding 
               
               
                   
               
               
                 22292 
                   CTGTC AAATTTCTCT TTGCC   
                 182 
                 0340-0359 
                 coding 
               
               
                   
               
               
                 22293 
                   CATAT ACTTGGAATG AACAC   
                 183 
                 0359-0378 
                 coding 
               
               
                   
               
               
                 22294 
                   GGTCC AACTGTCCGA ATCAA   
                 184 
                 0392-0411 
                 coding 
               
               
                   
               
               
                 22295 
                   TGATC TGAAGATTGT GAAGT   
                 185 
                 0417-0436 
                 coding 
               
               
                   
               
               
                 22296 
                   AAGCC CTTGTCCTTG ATCTG   
                 186 
                 0430-0449 
                 coding 
               
               
                   
               
               
                 22297 
                   TGTGA TGGATGATAC ATTGA   
                 187 
                 0453-0472 
                 coding 
               
               
                   
               
               
                 22298 
                   TCAGG TTGACTGAAG TTAGC   
                 188 
                 0529-0548 
                 coding 
               
               
                   
               
               
                 22299 
                   GTGTA TAGATGAGCA GGTCA   
                 189 
                 0593-0612 
                 coding 
               
               
                   
               
               
                 22300 
                   TCTGT GACATTATCT TGAGA   
                 190 
                 0694-0713 
                 coding 
               
               
                   
               
               
                 22301 
                   AAGAT AAAAGCCGCG TCTTG   
                 191 
                 0798-0817 
                 coding 
               
               
                   
               
               
                 22302 
                   AGAAA ACCATCACAC ATATA   
                 192 
                 0900-0919 
                 coding 
               
               
                   
               
               
                 22303 
                   AGAGT TGCGAGGCCG CTTCT   
                 193 
                 0947-0968 
                 coding 
               
               
                   
               
               
                 22304 
                   TCCCT CTCCATTGTG TTGGT   
                 194 
                 0979-0998 
                 coding 
               
               
                   
               
               
                 22305 
                   CATCA GATCTTTCAG GTATA   
                 195 
                 1035-1054 
                 coding 
               
               
                   
               
               
                 22306 
                   GGCTT TACTCTTTAA TTAAA   
                 196 
                 1115-1134 
                 stop 
               
               
                   
               
               
                 22307 
                   GAAAT CAAAAAGGTT GCCCA   
                 197 
                 1178-1197 
                 3′-UTR 
               
               
                   
               
               
                 22308 
                   GGAGT CCTGGAGCCC CCTTA   
                 198 
                 1231-1250 
                 3′-UTR 
               
               
                   
               
               
                 22309 
                   TTGGC ATACGGAGCA GAGCT   
                 199 
                 1281-1300 
                 3′-UTR 
               
               
                   
               
               
                 22310 
                   TGTGC TCTGAAGTGA AAAGA   
                 200 
                 1327-1346 
                 3′-UTR 
               
               
                   
               
               
                 22311 
                   GGCTT GGCCCATAAG TGTGC   
                 201 
                 1342-1361 
                 3′-UTR 
               
               
                   
               
               
                 22312 
                   CCTAA ATTTTATTTC CAGGT   
                 202 
                 1379-1398 
                 3′-UTR 
               
               
                   
               
               
                 24736 
                   GCTCC AAGTGTCCCA ATGAA   
                 203 
                 control 
                   
               
               
                   
               
               
                 24737 
                   AGTAT GTTTCTCACT CCGAT   
                 204 
                 control 
                   
               
               
                   
               
               
                 24738 
                   TGCCA GCACCCGGTA CGTCC   
                 205 
                 control 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 Co-ordinates from Genbank Accession No. U04343 locus name “HSU04343”. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Inhibition of Human B7-2 mRNA Expression by 
               
               
                 Chimeric (deoxy gapped) Phosphorothioate 
               
               
                 Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 GENE 
                   
                   
               
               
                 ISIS 
                 SEQ ID 
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
               
                 basal 
                 — 
                 — 
                 100% 
                 0% 
               
               
                   
               
               
                 10373 
                  16 
                 5′-UTR 
                 24% 
                 76% 
               
               
                   
               
               
                 22284 
                  16 
                 5′-UTR 
                 30% 
                 70% 
               
               
                   
               
               
                 22285 
                 175 
                 5′-UTR 
                 74% 
                 26% 
               
               
                   
               
               
                 22286 
                 176 
                 5′-UTR 
                 39% 
                 61% 
               
               
                   
               
               
                 22287 
                 177 
                 AUG 
                 27% 
                 73% 
               
               
                   
               
               
                 22288 
                 178 
                 coding 
                 38% 
                 62% 
               
               
                   
               
               
                 22289 
                 179 
                 coding 
                 41% 
                 59% 
               
               
                   
               
               
                 22290 
                 180 
                 coding 
                 42% 
                 58% 
               
               
                   
               
               
                 22291 
                 181 
                 coding 
                 41% 
                 59% 
               
               
                   
               
               
                 22292 
                 182 
                 coding 
                 39% 
                 61% 
               
               
                   
               
               
                 22293 
                 183 
                 coding 
                 43% 
                 57% 
               
               
                   
               
               
                 22294 
                 184 
                 coding 
                 21% 
                 79% 
               
               
                   
               
               
                 22295 
                 185 
                 coding 
                 66% 
                 34% 
               
               
                   
               
               
                 22296 
                 186 
                 coding 
                 42% 
                 58% 
               
               
                   
               
               
                 22297 
                 187 
                 coding 
                 54% 
                 46% 
               
               
                   
               
               
                 22298 
                 188 
                 coding 
                 53% 
                 47% 
               
               
                   
               
               
                 22299 
                 189 
                 coding 
                 46% 
                 54% 
               
               
                   
               
               
                 22300 
                 190 
                 coding 
                 39% 
                 61% 
               
               
                   
               
               
                 22301 
                 191 
                 coding 
                 51% 
                 49% 
               
               
                   
               
               
                 22302 
                 192 
                 coding 
                 41% 
                 59% 
               
               
                   
               
               
                 22303 
                 193 
                 coding 
                 46% 
                 54% 
               
               
                   
               
               
                 22304 
                 194 
                 coding 
                 41% 
                 59% 
               
               
                   
               
               
                 22305 
                 195 
                 coding 
                 57% 
                 43% 
               
               
                   
               
               
                 22306 
                 196 
                 stop 
                 44% 
                 56% 
               
               
                   
               
               
                 22307 
                 197 
                 3′-UTR 
                 45% 
                 55% 
               
               
                   
               
               
                 22308 
                 198 
                 3′-UTR 
                 40% 
                 60% 
               
               
                   
               
               
                 22309 
                 199 
                 3′-UTR 
                 42% 
                 58% 
               
               
                   
               
               
                 22310 
                 200 
                 3′-UTR 
                 41% 
                 59% 
               
               
                   
               
               
                 22311 
                 201 
                 3′-UTR 
                 49% 
                 51% 
               
               
                   
               
               
                 22312 
                 202 
                 3′-UTR 
                 83% 
                 17% 
               
               
                   
               
            
           
         
       
     
     Dose response experiments were performed on several of the more active oligonucleotides. The oligonucleotides were screened as described in Example 4 except that the concentration of oligonucleotide was varied as shown in Table 13. Mismatch control oligonucleotides were included. Results are shown in Table 13. 
     All antisense oligonucleotides tested showed a dose response effect with maximum inhibition of mRNA approximately 50% or greater. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Dose Response of COS-7 Cells to B7-2 
               
               
                 Chimeric (deoxy gapped) Antisense 
               
               
                 Oligonucleotides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ ID 
                 ASO Gene 
                   
                 % mRNA 
                 % mRNA 
               
               
                 ISIS # 
                 NO: 
                 Target 
                 Dose 
                 Expression 
                 Inhibition 
               
               
                   
               
               
                 basal 
                 — 
                 — 
                 — 
                 100% 
                 — 
               
               
                   
               
               
                 22284 
                  16 
                 5′-UTR 
                  10 nM 
                 92% 
                 8% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 72% 
                 28% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 59% 
                 41% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 48% 
                 52% 
               
               
                   
               
               
                 24738 
                 205 
                 control 
                  10 nM 
                 81% 
                 19% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 92% 
                 8% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 101% 
                 — 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 124% 
                 — 
               
               
                   
               
               
                 22287 
                 177 
                 AUG 
                  10 nM 
                 93% 
                 7% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 79% 
                 21% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 66% 
                 34% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 45% 
                 55% 
               
               
                   
               
               
                 24737 
                 204 
                 control 
                  10 nM 
                 85% 
                 15% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 95% 
                 5% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 87% 
                 13% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 99% 
                 1% 
               
               
                   
               
               
                 22294 
                 184 
                 coding 
                  10 nM 
                 93% 
                 7% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 95% 
                 5% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 58% 
                 42% 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 45% 
                 55% 
               
               
                   
               
               
                 24736 
                 203 
                 control 
                  10 nM 
                 102% 
                 — 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                  30 nM 
                 101% 
                 — 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM 
                 100% 
                 — 
               
               
                   
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM 
                 107% 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 15 
     Chimeric (Deoxy Gapped) Mouse B7-2 Antisense Oligonucleotides 
     Additional oligonucleotides targeting mouse B7-2 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 14. 
     Oligonucleotides were screened as described in Example 4. Results are shown in Table 15. 
     Oligonucleotides 18084 (SEQ ID NO: 206), 18085 (SEQ ID NO: 207), 18086 (SEQ ID NO: 208), 18087 (SEQ ID NO: 209), 18089 (SEQ ID NO: 211), 18090 (SEQ ID NO: 212), 18091 (SEQ ID NO: 213), 18093 (SEQ ID NO: 215), 18095 (SEQ ID NO: 217), 18096 (SEQ ID NO: 218), 18097 (SEQ ID NO: 219), 18098 (SEQ ID NO: 108), 18102 (SEQ ID NO: 223), and 18103 (SEQ ID NO: 224) resulted in 50% or greater inhibition of B7-2 mRNA expression in this assay. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Mouse B7-2 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 TARGET GENE 
                   
               
               
                   
                 NUCLEOTIDE 
                 SEQ 
                 NUCLEOTIDE 
                 GENE 
               
               
                 ISIS 
                 SEQUENCE 1   
                 ID 
                 CO- 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO: 
                 ORDINATES 2   
                 REGION 
               
               
                   
               
               
                 18084 
                   GCTGC CTACAGGAGC CACTC   
                 206 
                 0003-0022 
                 5′-UTR 
               
               
                   
               
               
                 18085 
                   TCAAG TCCGTGCTGC CTACA   
                 207 
                 0013-0032 
                 5′-UTR 
               
               
                   
               
               
                 18086 
                   GTCTA CAGGAGTCTG GTTGT   
                 208 
                 0033-0052 
                 5′-UTR 
               
               
                   
               
               
                 18087 
                   AGCTT GCGTCTCCAC GGAAA   
                 209 
                 0152-0171 
                 coding 
               
               
                   
               
               
                 18088 
                   TCACA CTATCAAGTT TCTCT   
                 210 
                 0297-0316 
                 coding 
               
               
                   
               
               
                 18089 
                   GTCAA AGCTCGTGCG GCCCA   
                 211 
                 0329-0348 
                 coding 
               
               
                   
               
               
                 18090 
                   GTGAA GTCGTAGAGT CCAGT   
                 212 
                 0356-0375 
                 coding 
               
               
                   
               
               
                 18091 
                   GTGAC CTTGCTTAGA CGTGC   
                 213 
                 0551-0570 
                 coding 
               
               
                   
               
               
                 18092 
                   CATCT TCTTAGGTTT CGGGT   
                 214 
                 0569-0588 
                 coding 
               
               
                   
               
               
                 18093 
                   GGCTG TTGGAGATAC TGAAC   
                 215 
                 0663-0682 
                 coding 
               
               
                   
               
               
                 18094 
                   GGGAA TGAAAGAGAG AGGCT   
                 216 
                 0679-0698 
                 coding 
               
               
                   
               
               
                 18095 
                   ACATA CAATGATGAG CAGCA   
                 217 
                 0854-0873 
                 coding 
               
               
                   
               
               
                 18096 
                   GTCTC TCTGTCAGCG TTACT   
                 218 
                 0934-0953 
                 coding 
               
               
                   
               
               
                 18097 
                   TGCCA AGCCCATGGT GCATC   
                 219 
                 0092-0111 
                 AUG 
               
               
                   
               
               
                 18098 
                   GGATT GCCAAGCCCA TGGTG   
                 108 
                 0096-0115 
                 AUG 
               
               
                   
               
               
                 18099 
                   GCAAT TTGGGGTTCA AGTTC   
                 220 
                 0967-0986 
                 coding 
               
               
                   
               
               
                 18100 
                   CAATC AGCTGAGAAC ATTTT   
                 221 
                 1087-1106 
                 3′-UTR 
               
               
                   
               
               
                 18101 
                   TTTTG TATAAAACAA TCATA   
                 222 
                 0403-0422 
                 coding 
               
               
                   
               
               
                 18102 
                   CCTTC ACTCTGCATT TGGTT   
                 223 
                 0995-1014 
                 stop 
               
               
                   
               
               
                 18103 
                   TGCAT GTTATCACCA TACTC   
                 224 
                 0616-0635 
                 coding 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 Co-ordinates from Genbank Accession No. S70108 locus name “S70108”. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Inhibition of Mouse B7-2 mRNA Expression by Chimeric (deoxy gapped) 
               
               
                 Phosphorothioate Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 GENE 
                   
                   
               
               
                 ISIS 
                   
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                 No: 
                 SEQ ID NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 basal 
                 — 
                 — 
                 100.0% 
                 0.0% 
               
               
                 18084 
                 206 
                 5′-UTR 
                 36.4% 
                 63.6% 
               
               
                 18085 
                 207 
                 5′-UTR 
                 35.0% 
                 65.0% 
               
               
                 18086 
                 208 
                 5′-UTR 
                 40.1% 
                 59.9% 
               
               
                 18087 
                 209 
                 coding 
                 42.1% 
                 57.9% 
               
               
                 18088 
                 210 
                 coding 
                 52.3% 
                 47.7% 
               
               
                 18089 
                 211 
                 coding 
                 20.9% 
                 79.1% 
               
               
                 18090 
                 212 
                 coding 
                 36.6% 
                 63.4% 
               
               
                 18091 
                 213 
                 coding 
                 37.1% 
                 62.9% 
               
               
                 18092 
                 214 
                 coding 
                 58.9% 
                 41.1% 
               
               
                 18093 
                 215 
                 coding 
                 32.7% 
                 67.3% 
               
               
                 18094 
                 216 
                 coding 
                 63.8% 
                 36.2% 
               
               
                 18095 
                 217 
                 coding 
                 34.3% 
                 65.7% 
               
               
                 18096 
                 218 
                 coding 
                 32.3% 
                 67.7% 
               
               
                 18097 
                 219 
                 AUG 
                 24.5% 
                 75.5% 
               
               
                 18098 
                 108 
                 AUG 
                 32.2% 
                 67.8% 
               
               
                 18099 
                 220 
                 coding 
                 66.8% 
                 33.2% 
               
               
                 18100 
                 221 
                 3′-UTR 
                 67.2% 
                 32.8% 
               
               
                 18101 
                 222 
                 coding 
                 88.9% 
                 11.1% 
               
               
                 18102 
                 223 
                 stop 
                 33.8% 
                 66.2% 
               
               
                 18103 
                 224 
                 coding 
                 30.2% 
                 69.8% 
               
               
                   
               
            
           
         
       
     
     Example 16 
     Effect of B7 Antisense Oligonucleotides on Cell Surface Expression 
     B7 antisense oligonucleotides were tested for their effect on cell surface expression of both B7-1 and B7-2. Cell surface expression was measured as described in Example 1. Experiments were done for both human B7 and mouse B7. Results for human B7 are shown in Table 16. Results for mouse B7 are shown in Table 17. 
     In both species, B7-1 antisense oligonucleotides were able to specifically reduce the cell surface expression of B7-1. B7-2 antisense oligonucleotides were specific for the B-7-2 family member. These oligonucleotides were also specific for their effect on B7-1 and B7-2 mRNA levels. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Inhibition of Human B7 Cell Surface Expression by Chimeric 
               
               
                 (deoxy gapped) Phosphorothioate Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
                   
               
               
                 ISIS 
                 ID 
                 GENE 
                 % B7-1 
                 % B7-2 
               
               
                 No: 
                 NO: 
                 TARGET 
                 EXPRESSION 
                 EXPRESSION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 basal 
                 — 
                 — 
                 100% 
                 0% 
               
               
                 22316 
                 26 
                 B7-1 
                 31% 
                 100% 
               
               
                 22317 
                 129 
                 B7-1 
                 28% 
                 91% 
               
               
                 22320 
                 132 
                 B7-1 
                 37% 
                 86% 
               
               
                 22324 
                 135 
                 B7-1 
                 37% 
                 91% 
               
               
                 22325 
                 136 
                 B7-1 
                 32% 
                 89% 
               
               
                 22334 
                 145 
                 B7-1 
                 28% 
                 92% 
               
               
                 22335 
                 146 
                 B7-1 
                 23% 
                 95% 
               
               
                 22337 
                 148 
                 B7-1 
                 48% 
                 101% 
               
               
                 22338 
                 36 
                 B7-1 
                 22% 
                 96% 
               
               
                 22284 
                 16 
                 B7-2 
                 88% 
                 32% 
               
               
                 22287 
                 177 
                 B7-2 
                 92% 
                 35% 
               
               
                 22294 
                 184 
                 B7-2 
                 77% 
                 28% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Inhibition of Mouse B7 Cell Surface Expression by Chimeric 
               
               
                 (deoxy gapped) Phosphorothioate Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ 
                 GENE 
                   
                   
               
               
                 ISIS 
                 ID 
                 TARGET 
                 % B7-1 
                 % B7-2 
               
               
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 EXPRESSION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 basal 
                 — 
                 — 
                 100% 
                 0% 
               
               
                 18089 
                 211 
                 B7-2 
                 85% 
                 36% 
               
               
                 18097 
                 219 
                 B7-2 
                 87% 
                 28% 
               
               
                 18110 
                 161 
                 B7-1 
                 31% 
                 93% 
               
               
                 18113 
                 164 
                 B7-1 
                 25% 
                 91% 
               
               
                 18119 
                 170 
                 B7-1 
                 27% 
                 98% 
               
               
                   
               
            
           
         
       
     
     Dose response experiments were performed on several of the more active human B7-1 antisense oligonucleotides. The oligonucleotides were screened as described in Example 2 except that the concentration of oligonucleotide was varied as shown in Table 18. Results are shown in Table 18. 
     All antisense oligonucleotides tested showed a dose response effect with inhibition of cell surface expression approximately 60% or greater. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Dose Response of COS-7 Cells to B7-1 
               
               
                 Chimeric (deoxy gapped) Antisense Oligonucleotides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ ID 
                 ASO Gene 
                   
                 % Surface 
                 % Surface 
               
               
                 ISIS # 
                 NO: 
                 Target 
                 Dose 
                 Expression 
                 Inhibition 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 basal 
                 — 
                 — 
                 — 
                 100% 
                 — 
               
               
                 22316 
                 26 
                 5′-UTR 
                 10 nM 
                 74% 
                 26% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 74% 
                 26% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 47% 
                 53% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 34% 
                 66% 
               
               
                 22335 
                 146  
                 3′-UTR 
                 10 nM 
                 81% 
                 19% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 69% 
                 31% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 47% 
                 53% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 38% 
                 62% 
               
               
                 22338 
                 36 
                 3′-UTR 
                 10 nM 
                 78% 
                 22% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 65% 
                 35% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 50% 
                 50% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 40% 
                 60% 
               
               
                   
               
            
           
         
       
     
     Dose response experiments were performed on several of the more active human B7-2 antisense oligonucleotides. The oligonucleotides were screened as described in Example 2 except that the concentration of oligonucleotide was varied as shown in Table 19. Results are shown in Table 19. 
     All antisense oligonucleotides tested showed a dose response effect with maximum inhibition of cell surface expression 85% or greater. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Dose Response of COS-7 Cells to B7-2 
               
               
                 Chimeric (deoxy gapped) Antisense Oligonucleotides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ ID 
                 ASO Gene 
                   
                 % Surface 
                 % Surface 
               
               
                 ISIS # 
                 NO: 
                 Target 
                 Dose 
                 Expression 
                 Inhibition 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 basal 
                 — 
                 — 
                 — 
                 100% 
                 — 
               
               
                 22284 
                  16 
                 5′-UTR 
                 10 nM 
                 63% 
                 37% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 60% 
                 40% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 37% 
                 63% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 15% 
                 85% 
               
               
                 22287 
                 177 
                 AUG 
                 10 nM 
                 93% 
                 7% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 60% 
                 40% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 32% 
                 68% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 15% 
                 85% 
               
               
                 22294 
                 184 
                 coding 
                 10 nM 
                 89% 
                 11% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 30 nM 
                 62% 
                 38% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 100 nM  
                 29% 
                 71% 
               
               
                 ″ 
                 ″ 
                 ″ 
                 300 nM  
                 12% 
                 88% 
               
               
                   
               
            
           
         
       
     
     Example 17 
     Effect of B7-1 Antisense Oligonucleotides in a Murine Model for Rheumatoid Arthritis 
     Collagen-induced arthritis (CIA) was used as a murine model for arthritis (Mussener, A., et al., Clin. Exp. Immunol., 1997, 107, 485-493). Female DBA/1LacJ mice (Jackson Laboratories, Bar Harbor, Me.) between the ages of 6 and 8 weeks were used to assess the activity of B7-1 antisense oligonucleotides. 
     On day 0, the mice were immunized at the base of the tail with 100 μg of bovine type II collagen which is emulsified in Complete Freund&#39;s Adjuvant (CFA). On day 7, a second booster dose of collagen was administered by the same route. On day 14, the mice were injected subcutaneously with 100 μg of LPS. Oligonucleotide was administered intraperitoneally daily (10 mg/kg bolus) starting on day −3 (three days before day 0) and continuing for the duration of the study. Oligonucleotide 17456 (SEQ ID NO. 173) is a fully phosphorothioated analog of 18122. 
     Weights were recorded weekly. Mice were inspected daily for the onset of CIA. Paw widths are rear ankle widths of affected and unaffected joints were measured three times a week using a constant tension caliper. Limbs were clinically evaluated and graded on a scale from 0-4 (with 4 being the highest). 
     Results are shown in Table 20. Treatment with B7-1 and B7-2 antisense oligonucleotides was able to reduce the incidence of the disease, but had modest effects on severity. The combination of 17456 (SEQ ID NO. 173) and 11696 (SEQ ID NO. 108) was able to significantly reduce the incidence of the disease and its severity. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Effect of B7 antisense oligonucleotide on CIA 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ 
                 Dose 
                 % 
                   
                   
               
               
                 ISIS #(s) 
                 ID NO 
                 mg/kg 
                 Inci-dence 
                 Peak day 1   
                 Severity 2   
               
               
                   
               
               
                 control 
                   
                 — 
                 70% 
                 6.7 ± 2.9 
                 3.2 ± 1.1 
               
               
                 17456 (B7-1) 
                 173 
                 10 
                 50% 
                 12.1 ± 4.6  
                 2.7 ± 1.3 
               
               
                 11696 (B7-2) 
                 108 
                 10 
                 37.5%   
                 11.6 ± 4.5  
                 3.4 ± 1.8 
               
               
                 17456/11696 
                   
                 10 
                 30% 
                 1.0 ± 0.6 
                 0.7 ± 0.4 
               
               
                 18110 (B7-1) 
                 161 
                 10 
                 55.6%   
                 2.0 ± 0.8 
                 2.0 ± 1.3 
               
               
                 18089 (B7-2) 
                 211 
                 10 
                 44.4%   
                 6.8 ± 2.2 
                 2.3 ± 1.3 
               
               
                 18110/18089 
                   
                 10 
                 60% 
                 11.6 ± 0.7  
                 4.5 ± 1.7 
               
               
                   
               
               
                   1 Peak day is the day from onset of maximum swelling for each joint measure. 
               
               
                   2 Severity is the total clinical score divided by the total number of mice in the group. 
               
            
           
         
       
     
     Example 18 
     Effect of B7-1 Antisense Oligonucleotides in a Murine Model for Multiple Sclerosis 
     Experimental autoimmune encephalomyelitis (EAE) is a commonly accepted murine model for multiple sclerosis (Myers, K. J., et al., J. Neuroimmunol., 1992, 41, 1-8). SJL/H, PL/J, (SJLxPL/J)F1, (SJLxBalb/c)F1 and Balb/c female mice between the ages of 6 and 12 weeks are used to test the activity of a B7-1 antisense oligonucleotide. 
     The mice are immunized in the two rear foot pads and base of the tail with an emulsion consisting of encephalitogenic protein or peptide (according to Myers, K. J., et al., J. of Immunol., 1993, 151, 2252-2260) in Complete Freund&#39;s Adjuvant supplemented with heat killed  Mycobacterium tuberculosis . Two days later, the mice receive an intravenous injection of 500 ng  Bordetella pertussis  toxin and additional adjuvant. 
     Alternatively, the disease may also be induced by the adoptive transfer of T-cells. T-cells are obtained from the draining of the lymph nodes of mice immunized with encephalitogenic protein or peptide in CFA. The T cells are grown in tissue culture for several days and then injected intravenously into naive syngeneic recipients. 
     Mice are monitored and scored daily on a 0-5 scale for signals of the disease, including loss of tail muscle tone, wobbly gait, and various degrees of paralysis. 
     Oligonucleotide 17456 (SEQ ID NO. 173), a fully phosphorothioated analog of 18122, was compared to a saline control and a fully phosphorothioated oligonucleotide of random sequence (Oligonucleotide 17460). Results of this experiment are shown in  FIG. 11 . 
     As shown in  FIG. 11 , for all doses of oligonucleotide 17456 tested, there is a protective effect, i.e. a reduction of disease severity. At 0.2 mg/kg, this protective effect is greatly reduced after day 20, but at the higher doses tested, the protective effect remains throughout the course of the experiment (day 40). The control oligonucleotide gave results similar to that obtained with the saline control. 
     Example 19 
     Additional Antisense Oligonucleotides Targeted to Human B7-1 
     Additional oligonucleotides targeting human B7-1 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region often deoxynucleotides. Oligonucleotide sequences are shown in Table 21. 
     The human promonocytic leukaemia cell line, THP-1 (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 growth media supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, Md.). A total of 1×10 7  cells were electroporated at an oligonucleotide concentration of 10 micromolar in 2 mm cuvettes, using an Electrocell Manipulator 600 instrument (Biotechnologies and Experimental Research, Inc.) employing 200 V, 1000 μF. Electroporated cells were then transferred to petri dishes and allowed to recover for 16 hrs. Cells were then induced with LPS at a final concentration of 1 μg/ml for 16 hours. RNA was isolated and processed as described in previous examples. Results are shown in Table 22. 
     Oligonucleotides 113492, 113495, 113498, 113499, 113501, 113502, 113504, 113505, 1113507, 113510, 113511, 113513 and 113514 (SEQ ID NO: 228, 231, 234, 235, 237, 238, 240, 241, 243, 246, 247, 249 and 250) resulted in 50% or greater inhibition of B7-1 mRNA expression in this assay. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Human B7-1 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                 NUCLEOTIDE 
                 SEQ 
                 TARGET GENE 
                 GENE 
               
               
                 ISIS 
                 SEQUENCE 1   
                 ID 
                 NUCLEOTIDE 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO. 
                 CO-ORDINATES 2   
                 REGION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 113489 
                 CCCTCCAGTGATGTTTACAA 
                 225 
                 179 
                 5′UTR 
               
               
                   
               
               
                 113490 
                 GAAGACCCTCCAGTGATGTT 
                 226 
                 184 
                 5′UTR 
               
               
                   
               
               
                 113491 
                 CGTAGAAGACCCTCCAGTGA 
                 227 
                 188 
                 5′UTR 
               
               
                   
               
               
                 113492 
                 TTCCCAGGTGCAAAACAGGC 
                 228 
                 234 
                 5′UTR 
               
               
                   
               
               
                 113493 
                 TGGCTTCAGATGCTTAGGGT 
                 229 
                 299 
                 5′UTR 
               
               
                   
               
               
                 113494 
                 CCTCCGTGTGTGGCCCATGG 
                 230 
                 316 
                 AUG 
               
               
                   
               
               
                 113495 
                 GGTGATGTTCCCTGCCTCCG 
                 231 
                 330 
                 Coding 
               
               
                   
               
               
                 113496 
                 GATGGTGATGTTCCCTGCCT 
                 232 
                 333 
                 Coding 
               
               
                   
               
               
                 113497 
                 AGGTATGGACACTTGGATGG 
                 233 
                 348 
                 Coding 
               
               
                   
               
               
                 113498 
                 GAAAGACCAGCCAGCACCAA 
                 234 
                 384 
                 Coding 
               
               
                   
               
               
                 113499 
                 CAGCGTTGCCACTTCTTTCA 
                 235 
                 442 
                 Coding 
               
               
                   
               
               
                 113500 
                 GTGACCACAGGACAGCGTTG 
                 236 
                 454 
                 Coding 
               
               
                   
               
               
                 113501 
                 AGATGCGAGTTTGTGCCAGC 
                 237 
                 491 
                 Coding 
               
               
                   
               
               
                 113502 
                 CCTTTTGCCAGTAGATGCGA 
                 238 
                 503 
                 Coding 
               
               
                   
               
               
                 113503 
                 CGGTTCTTGTACTCGGGCCA 
                 239 
                 567 
                 Coding 
               
               
                   
               
               
                 113504 
                 CGCAGAGCCAGGATCACAAT 
                 240 
                 618 
                 Coding 
               
               
                   
               
               
                 113505 
                 CTTCAGCCAGGTGTTCCCGC 
                 241 
                 698 
                 Coding 
               
               
                   
               
               
                 113506 
                 TAACGTCACTTCAGCCAGGT 
                 242 
                 706 
                 Coding 
               
               
                   
               
               
                 113507 
                 TTCTCCATTTTCCAACCAGG 
                 243 
                 838 
                 Coding 
               
               
                   
               
               
                 113508 
                 CTGTTGTGTTGATGGCATTT 
                 244 
                 863 
                 Coding 
               
               
                   
               
               
                 113509 
                 CATGAAGCTGTGGTTGGTTG 
                 245 
                 943 
                 Coding 
               
               
                   
               
               
                 113510 
                 AGGAAAATGCTCTTGCTTGG 
                 246 
                 1018 
                 Coding 
               
               
                   
               
               
                 113511 
                 TGGGAGCAGGTTATCAGGAA 
                 247 
                 1033 
                 Coding 
               
               
                   
               
               
                 113512 
                 TAAGGTAATGGCCCAGGATG 
                 248 
                 1051 
                 Coding 
               
               
                   
               
               
                 113513 
                 GGTCAGGCAGCATATCACAA 
                 249 
                 1090 
                 Coding 
               
               
                   
               
               
                 113514 
                 GCCCCTTGCTTCTGCGGACA 
                 250 
                 1188 
                 3′UTR 
               
               
                   
               
               
                 113515 
                 AGATCTTTTCAGCCCCTTGC 
                 251 
                 1199 
                 3′UTR 
               
               
                   
               
               
                 113516 
                 TTTGTTAAGGGAAGAATGCC 
                 252 
                 1271 
                 3′UTR 
               
               
                   
               
               
                 113517 
                 AAAGGAGAGGGATGCCAGCC 
                 253 
                 1362 
                 3′UTR 
               
               
                   
               
               
                 113518 
                 CAAGACAATTCAAGATGGCA 
                 254 
                 1436 
                 3′UTR 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 Co-ordinates from Genbank Accession No. M27533 to which the oligonucleotides are targeted. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Inhibition of Human B7-1 mRNA Expression by Chimeric (deoxy gapped) 
               
               
                 Phosphorothioate Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 GENE 
                   
                   
               
               
                 ISIS 
                 SEQ ID 
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 113489 
                 225 
                 5′ UTR 
                 122 
                 — 
               
               
                 113490 
                 226 
                 5′ UTR 
                 183 
                 — 
               
               
                 113491 
                 227 
                 5′ UTR 
                 179 
                 — 
               
               
                 113492 
                 228 
                 5′ UTR 
                 27 
                 73 
               
               
                 113493 
                 229 
                 5′ UTR 
                 488 
                 — 
               
               
                 113494 
                 230 
                 AUG 
                 77 
                 23 
               
               
                 113495 
                 231 
                 Coding 
                 43 
                 57 
               
               
                 113496 
                 232 
                 Coding 
                 71 
                 29 
               
               
                 113497 
                 233 
                 Coding 
                 78 
                 22 
               
               
                 113498 
                 234 
                 Coding 
                 37 
                 63 
               
               
                 113499 
                 235 
                 Coding 
                 25 
                 75 
               
               
                 113500 
                 236 
                 Coding 
                 83 
                 17 
               
               
                 113501 
                 237 
                 Coding 
                 36 
                 64 
               
               
                 113502 
                 238 
                 Coding 
                 26 
                 74 
               
               
                 113503 
                 239 
                 Coding 
                 65 
                 35 
               
               
                 113504 
                 240 
                 Coding 
                 46 
                 54 
               
               
                 113505 
                 241 
                 Coding 
                 40 
                 60 
               
               
                 113506 
                 242 
                 Coding 
                 105 
                 — 
               
               
                 113507 
                 243 
                 Coding 
                 36 
                 64 
               
               
                 113508 
                 244 
                 Coding 
                 117 
                 — 
               
               
                 113509 
                 245 
                 Coding 
                 62 
                 38 
               
               
                 113510 
                 246 
                 Coding 
                 43 
                 57 
               
               
                 113511 
                 247 
                 Coding 
                 48 
                 52 
               
               
                 113512 
                 248 
                 Coding 
                 73 
                 27 
               
               
                 113513 
                 249 
                 Coding 
                 48 
                 52 
               
               
                 113514 
                 250 
                 3′ UTR 
                 35 
                 65 
               
               
                 113515 
                 251 
                 3′ UTR 
                 184 
                 — 
               
               
                 113516 
                 252 
                 3′ UTR 
                 83 
                 17 
               
               
                 113517 
                 253 
                 3′ UTR 
                 201 
                 — 
               
               
                 113518 
                 254 
                 3′ UTR 
                 97 
                 03 
               
               
                   
               
            
           
         
       
     
     Example 20 
     Additional Antisense Oligonucleotides Targeted to Human B7-2 
     Additional oligonucleotides targeting human B7-2 were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 23. 
     The human promonocytic leukaemia cell line, THP-1 (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 growth media supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, Md.). A total of 1×10 7  cells were electroporated at an oligonucleotide concentration of 10 micromolar in 2 mm cuvettes, using an Electrocell Manipulator 600 instrument (Biotechnologies and Experimental Research, Inc.) employing 200 V, 1000 μF. Electroporated cells were then transferred to petri dishes and allowed to recover for 16 hrs Cells were then induced with LPS and dibutyryl cAMP (500 μM) for 16 hours. RNA was isolated and processed as described in previous examples. Results are shown in Table 24. 
     Oligonucleotides ISIS 113131, 113132, 113134, 113138, 113142, 113144, 113145, 113146, 113147, 113148, 113149, 113150, 113153, 113155, 113157, 113158, 113159 and 113160 (SEQ ID NO: 255, 256, 258, 262, 266, 268, 269, 270, 271, 272, 273, 274, 277, 279, 281, 282, 283 and 284) resulted in 50% or greater inhibition of B7-2 mRNA expression in this assay. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Human B7-2 Chimeric 
               
               
                 (deoxy gapped) Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                 TARGET GENE 
                 GENE 
               
               
                 ISIS 
                 NUCLEOTIDE SEQUENCE 1   
                 ID 
                 NUCLEOTIDE 
                 TARGET 
               
               
                 NO. 
                 (5′-&gt;3′) 
                 NO: 
                 CO-ORDINATES 2   
                 REGION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 113131 
                 CGTGTGTCTGTGCTAGTCCC 
                 255 
                 38 
                 5′UTR 
               
               
                   
               
               
                 113132 
                 GCTGCTTCTGCTGTGACCTA 
                 256 
                 83 
                 5′UTR 
               
               
                   
               
               
                 113133 
                 TATTTGCGAGCTCCCCGTAC 
                 257 
                 15 
                 5′UTR 
               
               
                   
               
               
                 113134 
                 GCATAAGCACAGCAGCATTC 
                 258 
                 79 
                 5′UTR 
               
               
                   
               
               
                 113135 
                 TCCAAAAAGAGACCAGATGC 
                 259 
                 97 
                 5′UTR 
               
               
                   
               
               
                 113136 
                 AAATGCCTGTCCACTGTAGC 
                 260 
                 117 
                 5′UTR 
               
               
                   
               
               
                 113137 
                 CTTCAGAGGAGCAGCACCAG 
                 261 
                 191 
                 Coding 
               
               
                   
               
               
                 113138 
                 GAATCTTCAGAGGAGCAGCA 
                 262 
                 195 
                 Coding 
               
               
                   
               
               
                 113139 
                 CAAATTGGCATGGCAGGTCT 
                 263 
                 237 
                 Coding 
               
               
                   
               
               
                 113140 
                 GCTTTGGTTTTGAGAGTTTG 
                 264 
                 257 
                 Coding 
               
               
                   
               
               
                 113141 
                 AGGCTTTGGTTTTGAGAGTT 
                 265 
                 259 
                 Coding 
               
               
                   
               
               
                 113142 
                 GCTCACTCAGGCTTTGGTTT 
                 266 
                 267 
                 Coding 
               
               
                   
               
               
                 113143 
                 GGTCCTGCCAAAATACTACT 
                 267 
                 288 
                 Coding 
               
               
                   
               
               
                 113144 
                 AGCCCTTGTCCTTGATCTGA 
                 268 
                 429 
                 Coding 
               
               
                   
               
               
                 113145 
                 TGTGGGCTTTTTGTGATGGA 
                 269 
                 464 
                 Coding 
               
               
                   
               
               
                 113146 
                 AATCATTCCTGTGGGCTTTT 
                 270 
                 473 
                 Coding 
               
               
                   
               
               
                 113147 
                 CCGTGTATAGATGAGCAGGT 
                 271 
                 595 
                 Coding 
               
               
                   
               
               
                 113148 
                 ACCGTGTATAGATGAGCAGG 
                 272 
                 596 
                 Coding 
               
               
                   
               
               
                 113149 
                 TCATCTTCTTAGGTTCTGGG 
                 273 
                 618 
                 Coding 
               
               
                   
               
               
                 113150 
                 ACAAGCTGATGGAAACGTCG 
                 274 
                 720 
                 Coding 
               
               
                   
               
               
                 113151 
                 TGCTCGTAACATCAGGGAAT 
                 275 
                 747 
                 Coding 
               
               
                   
               
               
                 113152 
                 AAGATGGTCATATTGCTCGT 
                 276 
                 760 
                 Coding 
               
               
                   
               
               
                 113153 
                 CGCGTCTTGTCAGTTTCCAG 
                 277 
                 787 
                 Coding 
               
               
                   
               
               
                 113154 
                 CAGCTGTAATCCAAGGAATG 
                 278 
                 864 
                 Coding 
               
               
                   
               
               
                 113155 
                 GGGCTTCATCAGATCTTTCA 
                 279 
                 1041 
                 Coding 
               
               
                   
               
               
                 113156 
                 CATGTATCACTTTTGTCGCA 
                 280 
                 1093 
                 Coding 
               
               
                   
               
               
                 113157 
                 AGCCCCCTTATTACTCATGG 
                 281 
                 1221 
                 3′UTR 
               
               
                   
               
               
                 113158 
                 GGAGTTACAGGGAGGCTATT 
                 282 
                 1261 
                 3′UTR 
               
               
                   
               
               
                 113159 
                 AGTCTCCTCTTGGCATACGG 
                 283 
                 1290 
                 3′UTR 
               
               
                   
               
               
                 113160 
                 CCCATAAGTGTGCTCTGAAG 
                 284 
                 1335 
                 3′UTR 
               
               
                   
               
               
                   1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages. 
               
               
                   2 For ISIS #113131 and 113132, co-ordinates are from Genbank Accession No. L25259, locus name “HUMB72A”. For remaining oigonucleotides, co-ordinates are from Genbank Accession No. U04343, locus name “HSUU04343”. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Inhibition of Human B7-2 mRNA Expression by 
               
               
                 Chimeric (deoxy gapped) Phosphorothioate 
               
               
                 Oligodeoxynucleotides 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 GENE 
                   
                   
               
               
                 ISIS 
                 SEQ ID 
                 TARGET 
                 % mRNA 
                 % mRNA 
               
               
                 No: 
                 NO: 
                 REGION 
                 EXPRESSION 
                 INHIBITION 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 113131 
                 255 
                 5′UTR 
                 13 
                 87 
               
               
                   
               
               
                 113132 
                 256 
                 5′UTR 
                 17 
                 83 
               
               
                   
               
               
                 113133 
                 257 
                 5′UTR 
                 214 
                 — 
               
               
                   
               
               
                 113134 
                 258 
                 5′UTR 
                 27 
                 73 
               
               
                   
               
               
                 113135 
                 259 
                 5′UTR 
                 66 
                 34 
               
               
                   
               
               
                 113136 
                 260 
                 5′UTR 
                 81 
                 19 
               
               
                   
               
               
                 113137 
                 261 
                 Coding 
                 57 
                 43 
               
               
                   
               
               
                 113138 
                 262 
                 Coding 
                 12 
                 88 
               
               
                   
               
               
                 113140 
                 264 
                 Coding 
                 214 
                 — 
               
               
                   
               
               
                 113141 
                 265 
                 Coding 
                 126 
                 — 
               
               
                   
               
               
                 113142 
                 266 
                 Coding 
                 35 
                 65 
               
               
                   
               
               
                 113143 
                 267 
                 Coding 
                 118 
                 — 
               
               
                   
               
               
                 113144 
                 268 
                 Coding 
                 41 
                 59 
               
               
                   
               
               
                 113145 
                 269 
                 Coding 
                 46 
                 54 
               
               
                   
               
               
                 113146 
                 270 
                 Coding 
                 32 
                 68 
               
               
                   
               
               
                 113147 
                 271 
                 Coding 
                 35 
                 65 
               
               
                   
               
               
                 113148 
                 272 
                 Coding 
                 23 
                 77 
               
               
                   
               
               
                 113149 
                 273 
                 Coding 
                 29 
                 71 
               
               
                   
               
               
                 113150 
                 274 
                 Coding 
                 19 
                 81 
               
               
                   
               
               
                 113151 
                 275 
                 Coding 
                 208 
                 — 
               
               
                   
               
               
                 113152 
                 276 
                 Coding 
                 89 
                 11 
               
               
                   
               
               
                 113153 
                 277 
                 Coding 
                 19 
                 81 
               
               
                   
               
               
                 113154 
                 278 
                 Coding 
                 63 
                 37 
               
               
                   
               
               
                 113155 
                 279 
                 Coding 
                 13 
                 87 
               
               
                   
               
               
                 113156 
                 280 
                 Coding 
                 83 
                 17 
               
               
                   
               
               
                 113157 
                 281 
                 3tUTR 
                 13 
                 87 
               
               
                   
               
               
                 113158 
                 282 
                 3′UTR 
                 20 
                 80 
               
               
                   
               
               
                 113159 
                 283 
                 3′UTR 
                 43 
                 57 
               
               
                   
               
               
                 113160 
                 284 
                 3′UTR 
                 09 
                 91 
               
               
                   
               
            
           
         
       
     
     Example 21 
     Human Skin Psoriasis Model 
     Animal models of psoriasis based on xenotransplantation of human skin from psoriatic patients are advantageous because they involve the direct study of affected human tissue. Psoriasis is solely a disease of the skin and consequently, engraftment of human psoriatic skin onto SCID mice allows psoriasis to be created with a high degree of fidelity in mice. 
     BALB/cByJSmn-Prkdcscid/J SCID mice (4-6 weeks old) of either sex (Jackson Laboratory, Bar Harbor, Me.) were maintained in a pathogen free environment. At 6-8 weeks of age, mice were anesthetized by intraperitoneal injection of 30 mg/kg body weight ketamine-HCl and 1 mg/kg body weight acepromazine. After anesthesia, mice were prepared for transplantation by shaving the hair from the dorsal skin, 2 cm away from the head. The area was then sterilized and cleaned with povidone iodide and alcohol. Graft beds of about 1 cm×1 cm were created on the shaved areas by removing full thickness skin down to the fascia. Partial thickness human skin was then orthotopically transferred onto the graft bed. The transplants were held in place by gluing the human skin to mouse-to-mouse skin with Nexband liquid, a veterinary bandage (Veterinary Products Laboratories, Phoenix, Ariz.). Finally, the transplant and the wounds were covered with a thick layer of antibiotic ointment. After 4 weeks of transplantation, a 2 mm punch biopsy was obtained to confirm the acceptance of the graft and the origin of the skin in the transplant area. Only mice whose grafts did not show signs of infection were used for the study. Normal human skin was obtained from elective plastic surgeries and psoriatic plaques were obtained from shave biopsies from psoriatic volunteers. Partial thickness skin was prepared by dermatome shaving of the skin and transplanted to the mouse as described above for the psoriatic skin. 
     Animals (n=5) were topically treated with 2.5% (w/w) of each antisense oligonucleotide in a cream formulation comprising 10% isopropyl myristate, 10% glyceryl monooleate, 3% cetostearyl alcohol, 10% polyoxyl-20-cetyl ether, 6% poloxamer 407, 2.5% phenoxyethanol, 0.5% methylparaben, 0.5% propylparaben and water (final pH about 7.5). 
     The following oligonucleotides were used: human B7-1 (5′-TTCCCAGGTGCAAAACAGGC-3′; SEQ ID NO: 228) (ISIS 113492) and human B7-2 (5′-CGTGTGTCTGTGCTAGTCCC-3′; SEQ ID NO: 255) (ISIS 113131). Both sequences contained only phosphorothioate linkages and had 2′-MOE modifications at nucleotides 1-5 and 16-20. 
     Plaques from the same patients were also transplanted onto control mice (n=5) and treated only with the vehicle of the active cream preparation. Both groups received the topical preparation twice a day for 4 weeks. Within 3-4 weeks the animals were sacrificed and 4 mm punch biopsies were taken from each xenograft. Biopsies were fixed in formalin for paraffin embedding and/or transferred to cryotubes and snap-frozen in liquid nitrogen and stored at −80° C. 
     Significant histological improvement marked by reduction of hyperkeratosis, acanthosis and lymphonuclear cellular infiltrates was observed in mice treated with the antisense oligonucleotides. Rete pegs, finger-like projections of the epidermis into the dermis, were also measured. These are phenotypic markers for psoriasis which lengthen as the disease progresses. The shortening of these rete pegs is a good measure of anti-psoriatic activity. In mice treated with the active agent, the rete pegs changed from 238.56±98.3 μm to 168.4±96.62 μm (p&lt;0.05), whereas in the control group the rete pegs before and after treatment were 279.93±40.56 μm and 294.65±45.64 μm, respectively (p&gt;0.1). HLA-DR positive lymphocytic infiltrates and intraepidermal CD8 positive lymphocytes were significantly reduced in the transplanted plaques treated with the antisense oligonucleotide cream. These results show that antisense oligonucleotides to B7 inhibit psoriasis-induced inflammation and have therapeutic efficacy in the treatment of psoriasis. 
     Example 22 
     Mouse Model of Allergic Inflammation 
     In the mouse model of allergic inflammation, mice were sensitized and challenged with aerosolized chicken ovalbumin (OVA). Airway responsiveness was assessed by inducing airflow obstruction with a methacholine aerosol using a noninvasive method. This methodology utilized unrestrained conscious mice that are placed into the main chamber of a plthysmograph (Buxco Electronics, Inc., Troy, N.Y.). Pressure differences between this chamber and a reference chamber were used to extrapolate minute volume, breathing frequency and enhanced pause (Penh). Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice (i.e., the sum of the airflow in the upper and lower respiratory tracts) during the respiratory cycle of the animal. The lower the Penh, the greater the airflow. This parameter closely correlates with lung resistance as measured by traditional invasive techniques using ventilated animals (Hamelmann . . . Gelfand, 1997). Dose-response data were plotted as raw Penh values to increasing concentrations of methacholine. This system was used to test the efficacy of antisense oligonucleotides targeted to human B7-1 and B7-2. 
     There are several important features common to human asthma and the mouse model of allergic inflammation. One of these is pulmonary inflammation, in which cytokine expression and Th2 profile is dominant. Another is goblet cell hyperplasia with increased mucus production. Lastly, airway hyperresponsiveness (AHR) occurs resulting in increased sensitivity to cholinergic receptor agonists such as acetylcholine or methacholine. The compositions and methods of the present invention may be used to treat AHR and pulmonary inflammation. 
     Ovalbumin-Induced Allergic Inflammation 
     Female Balb/c mice (Charles Rivers Laboratory, Taconic Farms, N.Y.) were maintained in micro-isolator cages housed in a specific pathogen-free (SPF) facility. The sentinel cages within the animal colony surveyed negative for viral antibodies and the presence of known mouse pathogens. Mice were sensitized and challenged with aerosolized chicken OVA. Briefly, 20 μg alum-precipitated OVA was injected intraperitoneally on days 0 and 14. On day 24, 25 and 26, the animals were exposed for 20 minutes to 1.0% OVA (in saline) by nebulization. The challenge was conducted using an ultrasonic nebulizer (PulmoSonic, The DeVilbiss Co., Somerset, Pa.). Animals were analyzed about 24 hours following the last nebulization using the Buxco electronics Biosystem. Lung function (Penh), lung histology (cell infiltration and mucus production), target mRNA reduction in the lung, inflammation (BAL cell type &amp; number, cytokine levels), spleen weight and serum AST/ALT were determined. 
     This method has been used to show that prophylactic treatment with an anti-B7.2 monoclonal antibody continued throughout the sensitization and challenge periods decreases OVA-specific serum IgE and IgE levels, IL-4 and IFN-γ levels in bronchoalveolar lavage (BAL) fluid, airway eosinophilia and airway hyperresponsiveness (Haczku et al.,  Am. J. Respir. Crit. Care Med.  159:1638-1643, 1999). Treatment during antigen challenge with both anti-B7.1 and anti-B7.2 mAbs is effective; however, either mAb alone is only partially active (Mathur et al., 21:498-509, 1999). However, the anti-B7.2 mAb had no activity when administered after the OVA challenge. The anti-B7.1 monoclonal antibody had no effect, either prophylactically or post-antigen challenge. Thus, there is a need for an effective B7 inhibitor which can be administered after antigen challenge, and which will reduce airway hyperresponsiveness and pulmonary inflammation. As described below, the antisense oligonucleotides of the present inventors fit this description. 
     Oligonucleotide Administration 
     Antisense oligonucleotides (ASOs) were dissolved in saline and used to intratracheally dose mice every day, four times per day, from days 15-26 of the OVA sensitization and challenge protocol. Specifically, the mice were anesthetized with isofluorane and placed on a board with the front teeth hung from a line. The nose was covered and the animal&#39;s tongue was extended with forceps and 251 μl of various doses of ASO, or an equivalent volume of saline (control) was placed at the back of the tongue until inhaled into the lung. The deposition pattern of an ASO in the lung, ISIS 13920 (5′-TCCGTCATCGCTCCTCAGGG-3′; SEQ ID NO:285) was also examined by immunohistochemical staining using a monoclonal antibody to the oligonucleotide, and showed that the ASO is taken up throughout the lung, most strongly by antigen presenting cells (APCs) and alveolar epithelium. 
     The B7 oligonucleotides used were: 
     
       
         
           
               
               
               
            
               
                 B7-1: 
                 5′-GCTCAGCCTTTCCACTTCAG-3′ 
                 (ISIS 121844; 
               
               
                   
                   
                 SEQ ID NO: 286) 
               
               
                   
               
               
                 B7-2: 
                 5′-GCTCAGCCTTTCCACTTCAG-3′ 
                 (ISIS 121874; 
               
               
                   
                   
                 SEQ ID NO: 287) 
               
            
           
         
       
     
     Both of these oligonucleotides are phosphorothioates with 2′-MOE modifications on nucleotides 1-5 and 16-20, and 2′-deoxy at positions 6-15. These ASOs were identified by mouse-targeted ASO screening by target mRNA reduction in mouse cell lines. For B7-2, 19 mouse-targeted ASOs were screened by target mRNA reduction (Northern analysis) in IC-21 macrophages. Dose-response confirmation led to selection of ISIS 121874 (&gt;70% reduction at 25 nM). For B7-1, 22 mouse-targeted ASOs were screened by target mRNA reduction (RT-PCR) in L-929 fibroblasts. Dose-response confirmation led to selection of ISIS 121844 (&gt;70% reduction at 100 nM). No cross hybridization was predicted, and no cross-target reduction was detected in transfected cells. 
     RT-PCR Analysis 
     RNA was harvested from experimental lungs removed on day 28 of the OVA protocol. B7.2 and B7.1 levels were measured by quantitative RT-PCR using the Applied Biosystems PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.). Primers and probes used for these studies were synthesized by Operon Technologies (Alameda, Calif.). The primer and probe sequences were as follows: 
                            B7-2:           (SEQ ID NO: 288)           forward: 5′-GGCCCTCCTCCTTGTGATG-3′                       (SEQ ID NO: 289)           probe:           5′-/56-FAM/TGCTCATCATTGTATGTCACAAGAAGCCG/36-           TAMTph/-3′                       (SEQ ID NO: 290)           reverse: 5′-CTGGGCCTGCTAGGCTGAT-3′                                   B7-1:           (SEQ ID NO: 291)           forward: 5′-CAGGAAGCTACGGGCAAGTT-3′                                   (SEQ ID NO: 292)           probe:           5′-/56-FAM/TGGGCCTTTGATTGCTTGATGACTGAA/36-           TAMTph/-3′                       (SEQ ID NO: 293)           reverse: 5′-GTGGGCTCAGCCTTTCCA-3′            
Collection of Bronchial Alveolar Lavage (BAL) Fluid and Blood Serum for the Determination of Cytokine and Chemokine Levels
 
     Animals were injected with a lethal dose of ketamine, the trachea was exposed and a cannula was inserted and secured by sutures. The lungs were lavaged twice with 0.5 ml aliquots of ice cold PBS with 0.2% FCS. The recovered BAL fluid was centrifuged at 1,000 rpm for 10 min at 4° C., frozen on dry ice and stored at −80° C. until used. Luminex was used to measure cytokine levels in BAL fluid and serum. 
     BAL Cell Counts and Differentials 
     Cytospins of cells recovered from BAL fluid were prepared using a Shandon Cytospin 3 (Shandon Scientific LTD, Cheshire, England). Cell differentials were performed from slides stained with Leukostat (Fisher Scientific, Pittsburgh, Pa.). Total cell counts were quantified by hemocytometer and, together with the percent type by differential, were used to calculate specific cell number. 
     Tissue Histology 
     Before resection, lungs were inflated with 0.5 ml of 10% phosphate-buffered formalin and fixed overnight at 4° C. The lung samples were washed free of formalin with 1×PBS and subsequently dehydrated through an ethanol series prior to equilibration in xylene and embedded in paraffin. Sections (6μ) were mounted on slides and stained with hematoxylin/eosin, massons trichome and periodic acid-schiff (PAS) reagent. Parasagittal sections were analyzed by bright-field microscopy. Mucus cell content was assessed as the airway epithelium staining with PAS. Relative comparisons of mucus content were made between cohorts of animals by counting the number of PAS-positive airways. 
     As shown in  FIGS. 11A-11B , B7.2 mRNA ( FIG. 11A ) and B7.1 mRNA ( FIG. 11B ) were detected in mouse lung and lymph node during the development of ovalbumin-induced asthma. Treatment with ISIS 121874 following allergen challenge reduces the airway response to methacholine ( FIG. 12 ). The Penh value in B7.2 ASO-treated mice was about 40% lower than vehicle-treated mice, and was statistically the same as naïve mice which were not sensitized with the allergen or treated with the ASO. This shows that B7.2 ASO-treated mice had significantly better airflow, and less inflammation, than mice which were not treated with the ASO. The dose-dependent inhibition of the Penh response to methacholine by ISIS 121874 is shown in  FIG. 13 . The inhibition of allergen-induced eosinophilia by ISIS 121874 is shown in  FIG. 14 . ISIS 121874 at 0.3 mg/kg reduced the total number of eosinophils by about 75% compared to vehicle-treated mice. Since increased numbers of eosinophils result from inflammation, this provides further support for the anti-inflammatory properties of the B7.2 ASO. In addition, daily intratracheal delivery of ISIS 121874 does not induce splenomegaly, the concentration of ISIS 121874 achieved in lung tissue via daily intratracheal administration is proportional to the dose delivered ( FIG. 15 ) and ISIS 121874 is retained in lung tissue for at least one week following single dose (0.3 mg/kg) intratracheal administration as determined by capillary gel electrophoresis (CGE) analysis ( FIG. 16 ). 
     Example 23 
     Support for an Antisense Mechanism of Action for ISIS 121874 
     Two variants of ISIS 121874 were synthesized: a 7 base mismatch 5′-TCAAGTCCTTCCACACCCAA-3′ (ISIS 306058; SEQ ID NO: 294) and a gap ablated oligonucleotide ISIS 306058 having the same sequence as ISIS 121874, but with 2′-MOE modifications at nucleotides 1, 2, 3, 6, 9, 13, 16, 18, 19 and 20. Because of the presence of 2′-MOE in the gap, this oligonucleotide is no longer an RNase H substrate and will not recruit RNase H to the RNA-DNA hybrid which is formed. 
     The results ( FIG. 17 ) show that at 0.3 mg/kg, only ISIS 121874, and not the mismatch and gap ablated controls, significantly lowered Penh, which supports that ISIS 121874 is working by an antisense mechanism. 
     The effects of ISIS 121874 and the control oligonucleotides on airway mucus production in the ovalbumin-induced model were also tested. The results ( FIG. 18 ) show that only ISIS 121874 significantly inhibited mucus production. 
     The effect of ISIS 121874 on B7.2 and B7.1 mRNA in lung tissue of allergen-challenged mice is shown in  FIGS. 19A and 19B , respectively. The effect of ISIS 121874 on B7.2 and B7.1 mRNA in draining lymph nodes of allergen-challenged mice is shown in  FIGS. 20A and 20B , respectively. This shows that ISIS 121874 reduces both B7.2 and B7.1 mRNA (greater in lung vs. node). 
     In summary, ISIS 121874 resulted in a dose-dependent inhibition of airway hypersensitivity, inhibited eosinophilia and reduced B7.1 and B7.2 expression in the lung and lymph nodes. In addition, ISIS 121874 reduced levels of the following inflammatory molecules: IgE mRNA in the lung and IgE protein in the serum; reduced IL-5 mRNA in the lung and IL-5 protein in the BAL fluid; and reduced the serum level of macrophage chemokine (KC). 
     In the aerosolized ISIS 121874 study, treatment with 0.001, 0.01, 0.1 or 1.0 mg/kg estimated inhaled dose was delivered by nose-only inhalation of an aerosol solution, four times per day, on days 15-26 (n=8 mice per group). The airway response to methacholine was reduced to the level seen in naïve mice at 0.001 mg/kg dose (estimated inhaled dose=1 μg/kg). No gross adverse effects were seen. 
     Example 24 
     B7.1 ASO in Ovalbumin Model of Asthma 
     The same protocols described above for the B7.2 ASOs were used to test the effect of the B7.1 ASO ISIS 121844 (SEQ ID NO: 286). In contrast to the B7.2 ASO, ISIS 121844 had no effect on the Penh response in mice challenged with methacholine. Although there was no effect on Penh, ISIS 121844 reduced allergen-induced airway eosinophilia ( FIG. 21 ) and reduced the levels of B7.1 and B7.2 in the mouse lung. ( FIGS. 22A-B ). Thus, treatment with B7.1 ASO produced anti-inflammatory effects, but did not prevent airway hyper-responsiveness. There was no effect of ISIS 121844 on the Penh response despite achieving an 80% reduction of B7.2 mRNA in the lung ( FIG. 21B ). Treatment with ISIS 121844 reduced eosinophil and PMN numbers in BAL fluid. This effect was associated with a reduction in lung B7.2 (not B7.1) mRNA. 
     The combined use of B7.1 or B7.2 with one or more conventional asthma medications including, but not limited to, montelukast sodium (Singulair™), albuterol, beclomethasone dipropionate, triamcinolone acetonide, ipratropium bromide (Atrovent™), flunisolide, fluticasone propionate (Flovent™) and other steroids is also contemplated. The combined use of oligonucleotides which target both B7.1 and B7.2 for the treatment of asthma is also within the scope of the present invention. B7.1 and B7.2 may also be combined with one or more conventional asthma medications as described above for B7.1 or B7.2 alone. 
     Example 25 
     Design and Screening of Duplexed Antisense Compounds Targeting B7.1 or B7.2 
     In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target B7.1 or B7.2. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide to B7.1 or B7.2 as described herein. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 445) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure: 
     
       
         
         
             
             
         
       
     
     RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 μL of each strand is combined with 15 μL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 μL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 μM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times. 
     Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate B7.1 or B7.2 expression according to the protocols described herein. 
     Example 26 
     Design of Phenotypic Assays and In Vivo Studies for The Use of B7.1 or B7.2 Inhibitors 
     Phenotypic Assays 
     Once B7.1 or B7.2 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. 
     Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of B7.1 or B7.2 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.). 
     In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with B7.1 or B7.2 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints. 
     Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest. 
     Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the B7.1 or B7.2 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells. 
     Example 27 
     Antisense Inhibition of Human B7.2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap 
     In accordance with the present invention, an additional series of antisense compounds were designed to target different regions of the human B7.2 RNA, using published sequences (GenBank accession number U04343.1, incorporated herein as SEQ ID NO: 295, GenBank accession number BC040261.1, incorporated herein as SEQ ID NO: 296 and GenBank accession number NT — 005543.12, a portion of which is incorporated herein as SEQ ID NO: 297). The compounds are shown in Table 25. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 25 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human B7.2 mRNA levels in THP-1 cells by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, “N.D.” indicates “no data”. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Inhibition of human B7.2 mRNA levels by chimeric phos- 
               
               
                 phorothioate oligonucleotides having 2′-MOE wings and a 
               
               
                 deoxy gap 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                 Genbank 
                   
                   
               
               
                 Isis 
                 Sequence 
                 ID 
                   
                 Accesion 
                 Target 
                   
               
               
                 Number 
                 5′ to 3′ 
                 NO: 
                 % INHIB 
                 Target 
                 Site 
                 Region 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 322216 
                 ACCAAAAGGAGTATTTGCGA 
                 298 
                 N.D. 
                 U04343.1 
                 26 
                 5′UTR 
               
               
                   
               
               
                 322217 
                 CATTCCCAAGGAACACAGAA 
                 299 
                 N.D. 
                 U04343.1 
                 64 
                 5′UTR 
               
               
                   
               
               
                 322218 
                 ACTGTAGCTCCAAAAAGAGA 
                 300 
                 N.D. 
                 U04343.1 
                 105 
                 5′UTR 
               
               
                   
               
               
                 322219 
                 CTGTCACAAATGCCTGTCCA 
                 301 
                 N.D. 
                 U04343.1 
                 124 
                 5′UTR 
               
               
                   
               
               
                 322220 
                 TCAGTCCCATAGTGCTGTCA 
                 302 
                 N.D. 
                 U04343.1 
                 138 
                 START 
               
               
                   
               
               
                 322221 
                 CTGTTACAGCAGCAGAGAAG 
                 303 
                 N.D. 
                 BC040261.1 
                 29 
                 5′UTR 
               
               
                   
               
               
                 322222 
                 TCCCTGTTACAGCAGCACTC 
                 304 
                 N.D. 
                 BC040261.1 
                 32 
                 5′UTR 
               
               
                   
               
               
                 322223 
                 ATCTGGAAATGACCCCAAAT 
                 305 
                 N.D. 
                 BC040261.1 
                 71 
                 5′UTR 
               
               
                   
               
               
                 322224 
                 GTGACCTAATATCTGGACTG 
                 306 
                 N.D. 
                 BC040261.1 
                 81 
                 5′UTR 
               
               
                   
               
               
                 322225 
                 CATTTTGGCTGCTTCTGCTG 
                 307 
                 N.D. 
                 BC040261.1 
                 100 
                 START 
               
               
                   
               
               
                 322226 
                 GGAACTTACAAAGGAAAGGG 
                 308 
                 N.D. 
                 BC040261.1 
                 1145 
                 3′UTR 
               
               
                   
               
               
                 322227 
                 AAAAAGGTTGCCCAGGAACT 
                 309 
                 N.D. 
                 BC040261.1 
                 1159 
                 3′UTR 
               
               
                   
               
               
                 322228 
                 TGCCTTCTGGAAGAAATCAA 
                 310 
                 N.D. 
                 BC040261.1 
                 1177 
                 3′UTR 
               
               
                   
               
               
                 322229 
                 TTTTTGCCTTCTGGAAGAAA 
                 311 
                 N.D. 
                 BC040261.1 
                 1181 
                 3′UTR 
               
               
                   
               
               
                 322230 
                 CTATTCCACTTAGAGGGAGT 
                 312 
                 N.D. 
                 BC040261.1 
                 1233 
                 3′UTR 
               
               
                   
               
               
                 322231 
                 TCTGATCTGGAGGAGGTATT 
                 313 
                 N.D. 
                 BC040261.1 
                 1389 
                 3′UTR 
               
               
                   
               
               
                 322232 
                 AGAAATTGAGAGGTCTATTT 
                 314 
                 N.D. 
                 BC040261.1 
                 1444 
                 3′UTR 
               
               
                   
               
               
                 322233 
                 CACCAGCTTAGAATTCTGGG 
                 315 
                 N.D. 
                 BC040261.1 
                 1484 
                 3′UTR 
               
               
                   
               
               
                 322234 
                 AGGTAGTTGTTTAGTCACAG 
                 316 
                 N.D. 
                 BC040261.1 
                 1524 
                 3′UTR 
               
               
                   
               
               
                 322235 
                 CCAGACTGAGGAGGTAGTTG 
                 317 
                 N.D. 
                 BC040261.1 
                 1535 
                 3′UTR 
               
               
                   
               
               
                 322236 
                 CAGTACATAGATCTCTATGT 
                 318 
                 N.D. 
                 BC040261.1 
                 1599 
                 3′UTR 
               
               
                   
               
               
                 322237 
                 TTACAGTACATAGATCTCTA 
                 319 
                 N.D. 
                 BC040261.1 
                 1602 
                 3′UTR 
               
               
                   
               
               
                 322238 
                 GATGAGAACTCCTTAGCAGG 
                 320 
                 N.D. 
                 BC040261.1 
                 1657 
                 3′UTR 
               
               
                   
               
               
                 322239 
                 TAGCAACAGCCCAGATAGAA 
                 321 
                 N.D. 
                 BC040261.1 
                 1787 
                 3′UTR 
               
               
                   
               
               
                 322240 
                 TCTGTTGCTTGTTTCAAGAC 
                 322 
                 N.D. 
                 BC040261.1 
                 2043 
                 3′UTR 
               
               
                   
               
               
                 322241 
                 TCCATTTGGACAGACTATCC 
                 323 
                 N.D. 
                 BC040261.1 
                 2064 
                 3′UTR 
               
               
                   
               
               
                 322242 
                 GGGAAACTGCTGTCTGTCTT 
                 324 
                 N.D. 
                 BC040261.1 
                 2087 
                 3′UTR 
               
               
                   
               
               
                 322243 
                 TGCTTCCAGGAAGATGACAT 
                 325 
                 N.D. 
                 BC040261.1 
                 2149 
                 3′UTR 
               
               
                   
               
               
                 322244 
                 ATTCATCCCATTATCAAGGT 
                 326 
                 N.D. 
                 BC040261.1 
                 2191 
                 3′UTR 
               
               
                   
               
               
                 322245 
                 AGCCAGGAGTGGAAAGTCCT 
                 327 
                 N.D. 
                 BC040261.1 
                 2223 
                 3′UTR 
               
               
                   
               
               
                 322246 
                 CTTCCTAATTCCGTTGCAGC 
                 328 
                 N.D. 
                 BC040261.1 
                 2255 
                 3′UTR 
               
               
                   
               
               
                 322247 
                 CATCTGTAGGCTAAGTAAGC 
                 329 
                 N.D. 
                 BC040261.1 
                 2297 
                 3′UTR 
               
               
                   
               
               
                 322248 
                 CCCGTAGGACATCTGTAGGC 
                 330 
                 N.D. 
                 BC040261.1 
                 2306 
                 3′UTR 
               
               
                   
               
               
                 322249 
                 GCCCTATGCTGGGCCAGCCC 
                 331 
                 N.D. 
                 BC040261.1 
                 2331 
                 3′UTR 
               
               
                   
               
               
                 322250 
                 GTCTCTGTATGCAAGTTTCC 
                 332 
                 N.D. 
                 BC040261.1 
                 2396 
                 3′UTR 
               
               
                   
               
               
                 322251 
                 CCAGTATATCTGTCTCTGTA 
                 333 
                 N.D. 
                 BC040261.1 
                 2407 
                 3′UTR 
               
               
                   
               
               
                 322252 
                 CCAGGTTTTCAAAGTCATTT 
                 334 
                 N.D. 
                 BC040261.1 
                 2430 
                 3′UTR 
               
               
                   
               
               
                 322253 
                 AGCCAGGTTTTCAAAGTCAT 
                 335 
                 N.D. 
                 BC040261.1 
                 2432 
                 3′UTR 
               
               
                   
               
               
                 322254 
                 CCCTTAGTGATCCCACCTTA 
                 336 
                 N.D. 
                 BC040261.1 
                 2453 
                 3′UTR 
               
               
                   
               
               
                 322255 
                 CTGCCCCATCCCTTAGTGAT 
                 337 
                 N.D. 
                 BC040261.1 
                 2462 
                 3′UTR 
               
               
                   
               
               
                 322256 
                 TTTATGTTTGGGCAGAGACT 
                 338 
                 N.D. 
                 BC040261.1 
                 2480 
                 3′UTR 
               
               
                   
               
               
                 322257 
                 CATGGCAGTCTATAACCCTT 
                 339 
                 N.D. 
                 BC040261.1 
                 2556 
                 3′UTR 
               
               
                   
               
               
                 322258 
                 TAGCATGGCAGTCTATAACC 
                 340 
                 N.D. 
                 BC040261.1 
                 2559 
                 3′UTR 
               
               
                   
               
               
                 322259 
                 TCTAGCATGGCAGTCTATAA 
                 341 
                 N.D. 
                 BC040261.1 
                 2561 
                 3′UTR 
               
               
                   
               
               
                 322260 
                 TTGTCTAGCATGGCAGTCTA 
                 342 
                 N.D. 
                 BC040261.1 
                 2564 
                 3′UTR 
               
               
                   
               
               
                 322261 
                 AAGCTTGTCTAGCATGGCAG 
                 343 
                 N.D. 
                 BC040261.1 
                 2568 
                 3′UTR 
               
               
                   
               
               
                 322262 
                 ACATGGACAAGCTTGTCTAG 
                 344 
                 N.D. 
                 BC040261.1 
                 2576 
                 3′UTR 
               
               
                   
               
               
                 322263 
                 TTACATGGACAAGCTTGTCT 
                 345 
                 N.D. 
                 BC040261.1 
                 2578 
                 3′UTR 
               
               
                   
               
               
                 322264 
                 GAATATTACATGGACAAGCT 
                 346 
                 N.D. 
                 BC040261.1 
                 2583 
                 3′UTR 
               
               
                   
               
               
                 322265 
                 AACTAGCCAGGTGCTAGGAG 
                 347 
                 N.D. 
                 BC040261.1 
                 2636 
                 3′UTR 
               
               
                   
               
               
                 322266 
                 AATTATTACTCACCACTGGG 
                 348 
                 N.D. 
                 NT_005543.12 
                 1124 
                 genomic 
               
               
                   
               
               
                 322267 
                 TAATATTTAGGGAAGCATGA 
                 349 
                 N.D. 
                 NT_005543.12 
                 13890 
                 genomic 
               
               
                   
               
               
                 322268 
                 GGACCCTGGGCCAGTTATTG 
                 350 
                 N.D. 
                 NT_005543.12 
                 22504 
                 genomic 
               
               
                   
               
               
                 322269 
                 CAAACATACCTGTCACAAAT 
                 351 
                 N.D. 
                 NT_005543.12 
                 23662 
                 genomic 
               
               
                   
               
               
                 322270 
                 GTGATATCAATTGATGGCAT 
                 352 
                 N.D. 
                 NT_005543.12 
                 29265 
                 genomic 
               
               
                   
               
               
                 322271 
                 TGCTACATCTACTCAGTGTC 
                 353 
                 N.D. 
                 NT_005543.12 
                 31796 
                 genomic 
               
               
                   
               
               
                 322272 
                 TGGAAACTCTTGCCTTCGGA 
                 354 
                 N.D. 
                 NT_005543.12 
                 32971 
                 genomic 
               
               
                   
               
               
                 322273 
                 CCATCCACATTGTAGCATGT 
                 355 
                 N.D. 
                 NT_005543.12 
                 34646 
                 genomic 
               
               
                   
               
               
                 322274 
                 TCAGGATGGTATGGCCATAC 
                 356 
                 N.D. 
                 NT_005543.12 
                 36251 
                 genomic 
               
               
                   
               
               
                 322275 
                 TCCCATAGTGCTAGAGTCGA 
                 357 
                 N.D. 
                 NT_005543.12 
                 37218 
                 genomic 
               
               
                   
               
               
                 322276 
                 AGGTTCTTACCAGAGAGCAG 
                 358 
                 N.D. 
                 NT_005543.12 
                 37268 
                 genomic 
               
               
                   
               
               
                 322277 
                 CAGAGGAGCAGCACCTAAAA 
                 359 
                 N.D. 
                 NT_005543.12 
                 49133 
                 genomic 
               
               
                   
               
               
                 322278 
                 GACCACATACCAAGCACTGA 
                 360 
                 N.D. 
                 NT_005543.12 
                 49465 
                 genomic 
               
               
                   
               
               
                 322279 
                 ATCTTTCAGAAACCCAAGCA 
                 361 
                 N.D. 
                 NT_005543.12 
                 51347 
                 genomic 
               
               
                   
               
               
                 322280 
                 GAGTCACCAAAGATTTACAA 
                 362 
                 N.D. 
                 NT_005543.12 
                 51542 
                 genomic 
               
               
                   
               
               
                 322281 
                 CTGAAGTTAGCTGAAAGCAG 
                 363 
                 N.D. 
                 NT_005543.12 
                 51815 
                 genomic 
               
               
                   
               
               
                 322282 
                 ACAGCTTTACCTATAGAGAA 
                 364 
                 N.D. 
                 NT_005543.12 
                 52118 
                 genonic 
               
               
                   
               
               
                 322283 
                 TCCTCAAGCTCTACAAATGA 
                 365 
                 N.D. 
                 NT_005543.12 
                 54882 
                 genomic 
               
               
                   
               
               
                 322284 
                 GACTCACTCACCACATTTAT 
                 366 
                 N.D. 
                 NT_005543.12 
                 55027 
                 genomic 
               
               
                   
               
               
                 322285 
                 AGTGATAGCAAGGCTTCTCT 
                 367 
                 N.D. 
                 NT_005543.12 
                 56816 
                 genomic 
               
               
                   
               
               
                 322286 
                 CTTGGAGAGAATGGTTATCT 
                 368 
                 N.D. 
                 NT_005543.12 
                 61044 
                 genomic 
               
               
                   
               
               
                 322287 
                 GAAGATGTTGATGCCTAAAT 
                 369 
                 N.D. 
                 NT_005543.12 
                 63271 
                 genomic 
               
               
                   
               
               
                 322288 
                 GTGTTGGTTCCTGAAAGACA 
                 370 
                 N.D. 
                 NT_005543.12 
                 63665 
                 genomic 
               
               
                   
               
               
                 322289 
                 CAGGATTTACCTTTTCTTGG 
                 371 
                 N.D. 
                 NT_005543.12 
                 63711 
                 genomic 
               
               
                   
               
               
                 322290 
                 AGGGCAGAATAGAGGTTGCC 
                 372 
                 N.D. 
                 NT_005543.12 
                 64973 
                 Genomic 
               
               
                   
               
               
                 322291 
                 TTTTTCTCTGGAGAAATAGA 
                 373 
                 N.D. 
                 NT_005543.12 
                 65052 
                 genomic 
               
               
                   
               
               
                 323624 
                 GTTACTCAGTCCCATAGTGC 
                 374 
                 59 
                 U04343.1 
                 143 
                 START 
               
               
                   
               
               
                 323625 
                 CAAAGAGAATGTTACTCAGT 
                 375 
                 21 
                 U04343.1 
                 153 
                 Coding 
               
               
                   
               
               
                 323626 
                 CCATCACAAAGAGAATGTTA 
                 376 
                 32 
                 U04343.1 
                 159 
                 Coding 
               
               
                   
               
               
                 323627 
                 GGAAGGCCATCACAAAGAGA 
                 377 
                 54 
                 U04343.1 
                 165 
                 Coding 
               
               
                   
               
               
                 323628 
                 GAGCAGGAAGGCCATCACAA 
                 378 
                 44 
                 U04343.1 
                 170 
                 Coding 
               
               
                   
               
               
                 323629 
                 CCAGAGAGCAGGAAGGCCAT 
                 379 
                 36 
                 U04343.1 
                 175 
                 Coding 
               
               
                   
               
               
                 323630 
                 AAATAAGCTTGAATCTTCAG 
                 380 
                 22 
                 U04343.1 
                 205 
                 Coding 
               
               
                   
               
               
                 323631 
                 AGTCTCATTGAAATAAGCTT 
                 381 
                 56 
                 U04343.1 
                 215 
                 Coding 
               
               
                   
               
               
                 323632 
                 AGGTCTGCAGTCTCATTGAA 
                 382 
                 41 
                 U04343.1 
                 223 
                 Coding 
               
               
                   
               
               
                 323633 
                 CTACTAGCTCACTCAGGCTT 
                 383 
                 50 
                 U04343.1 
                 273 
                 Coding 
               
               
                   
               
               
                 323634 
                 AAATACTACTAGCTCACTCA 
                 384 
                 30 
                 U04343.1 
                 278 
                 Coding 
               
               
                   
               
               
                 323635 
                 CTGCCAAAATACTACTAGCT 
                 385 
                 24 
                 U04343.1 
                 284 
                 Coding 
               
               
                   
               
               
                 323636 
                 TTCAGAACCAAGTTTTCCTG 
                 386 
                 23 
                 U04343.1 
                 307 
                 Coding 
               
               
                   
               
               
                 323637 
                 CCTCATTCAGAACCAAGTTT 
                 387 
                 19 
                 U04343.1 
                 312 
                 Coding 
               
               
                   
               
               
                 323638 
                 GTATACCTCATTCAGAACCA 
                 388 
                 20 
                 U04343.1 
                 317 
                 Coding 
               
               
                   
               
               
                 323639 
                 GCCTAAGTATACCTCATTCA 
                 389 
                 55 
                 U04343.1 
                 323 
                 Coding 
               
               
                   
               
               
                 323640 
                 CTCTTTGCCTAAGTATACCT 
                 390 
                 28 
                 U04343.1 
                 329 
                 Coding 
               
               
                   
               
               
                 323641 
                 CCCATATACTTGGAATGAAC 
                 391 
                 88 
                 U04343.1 
                 361 
                 Coding 
               
               
                   
               
               
                 323642 
                 CTTGTGCGGCCCATATACTT 
                 392 
                 27 
                 U04343.1 
                 370 
                 Coding 
               
               
                   
               
               
                 323643 
                 ATCAAAACTTGTGCGGCCCA 
                 393 
                 80 
                 U04343.1 
                 377 
                 Coding 
               
               
                   
               
               
                 323644 
                 CCCTTGTCCTTGATCTGAAG 
                 394 
                 71 
                 U04343.1 
                 427 
                 Coding 
               
               
                   
               
               
                 323645 
                 ACAAGCCCTTGTCCTTGATC 
                 395 
                 56 
                 U04343.1 
                 432 
                 Coding 
               
               
                   
               
               
                 323646 
                 TTGATACAAGCCCTTGTCCT 
                 396 
                 33 
                 U04343.1 
                 437 
                 Coding 
               
               
                   
               
               
                 323647 
                 ATACATTGATACAAGCCCTT 
                 397 
                 41 
                 U04343.1 
                 442 
                 Coding 
               
               
                   
               
               
                 323648 
                 TGGATGATACATTGATACAA 
                 398 
                 31 
                 U04343.1 
                 448 
                 Coding 
               
               
                   
               
               
                 323649 
                 GAATTCATCTGGTGGATGCG 
                 399 
                 81 
                 U04343.1 
                 493 
                 Coding 
               
               
                   
               
               
                 323650 
                 GTTCAGAATTCATCTGGTGG 
                 400 
                 92 
                 U04343.1 
                 498 
                 Coding 
               
               
                   
               
               
                 323651 
                 TGACAGTTCAGAATTCATCT 
                 401 
                 64 
                 U04343.1 
                 503 
                 Coding 
               
               
                   
               
               
                 323652 
                 AGCACTGACAGTTCAGAATT 
                 402 
                 87 
                 U04343.1 
                 508 
                 Coding 
               
               
                   
               
               
                 323653 
                 TAGCAAGCACTGACAGTTCA 
                 403 
                 96 
                 U04343.1 
                 513 
                 Coding 
               
               
                   
               
               
                 323654 
                 TGAAGTTAGCAAGCACTGAC 
                 404 
                 87 
                 U04343.1 
                 519 
                 Coding 
               
               
                   
               
               
                 323655 
                 TTGACTGAAGTTAGCAAGCA 
                 405 
                 65 
                 U04343.1 
                 524 
                 Coding 
               
               
                   
               
               
                 323656 
                 CTATTTCAGGTTGACTGAAG 
                 406 
                 76 
                 U04343.1 
                 534 
                 Coding 
               
               
                   
               
               
                 323657 
                 TCTGTTATATTAGAAATTGG 
                 407 
                 43 
                 U04343.1 
                 556 
                 Coding 
               
               
                   
               
               
                 323658 
                 GCAGGTCAAATTTATGTACA 
                 408 
                 36 
                 U04343.1 
                 581 
                 Coding 
               
               
                   
               
               
                 323659 
                 GTATAGATGAGCAGGTCAAA 
                 409 
                 56 
                 U04343.1 
                 591 
                 Coding 
               
               
                   
               
               
                 323660 
                 GGGTAACCGTGTATAGATGA 
                 410 
                 71 
                 U04343.1 
                 601 
                 Coding 
               
               
                   
               
               
                 323661 
                 AGGTTCTGGGTAACCGTGTA 
                 411 
                 68 
                 U04343.1 
                 608 
                 Coding 
               
               
                   
               
               
                 323662 
                 TAGCAAAACACTCATCTTCT 
                 412 
                 22 
                 U04343.1 
                 629 
                 Coding 
               
               
                   
               
               
                 323663 
                 GTTCTTAGCAAAACACTCAT 
                 413 
                 23 
                 U04343.1 
                 634 
                 Coding 
               
               
                   
               
               
                 323664 
                 ATTCTTGGTTCTTAGCAAAA 
                 414 
                 35 
                 U04343.1 
                 641 
                 Coding 
               
               
                   
               
               
                 323665 
                 GATAGTTGAATTCTTGGTTC 
                 415 
                 43 
                 U04343.1 
                 650 
                 Coding 
               
               
                   
               
               
                 323666 
                 ACCATCATACTCGATAGTTG 
                 416 
                 71 
                 U04343.1 
                 662 
                 Coding 
               
               
                   
               
               
                 323667 
                 ATCTTGAGATTTCTGCATAA 
                 417 
                 52 
                 U04343.1 
                 683 
                 Coding 
               
               
                   
               
               
                 323668 
                 ACATTATCTTGAGATTTCTG 
                 418 
                 39 
                 U04343.1 
                 688 
                 Coding 
               
               
                   
               
               
                 323669 
                 CGTACAGTTCTGTGACATTA 
                 419 
                 68 
                 U04343.1 
                 702 
                 Coding 
               
               
                   
               
               
                 323670 
                 AGACAAGCTGATGGAAACGT 
                 420 
                 19 
                 U04343.1 
                 722 
                 Coding 
               
               
                   
               
               
                 323671 
                 GAAACAGACAAGCTGATGGA 
                 421 
                 26 
                 U04343.1 
                 727 
                 Coding 
               
               
                   
               
               
                 323672 
                 GGAATGAAACAGACAAGCTG 
                 422 
                 33 
                 U04343.1 
                 732 
                 Coding 
               
               
                   
               
               
                 323673 
                 CATCAGGGAATGAAACAGAC 
                 423 
                 38 
                 U04343.1 
                 738 
                 Coding 
               
               
                   
               
               
                 323674 
                 CGTAACATCAGGGAATGAAA 
                 424 
                 47 
                 U04343.1 
                 743 
                 Coding 
               
               
                   
               
               
                 323675 
                 AGCTCTATAGAGAAAGGTGA 
                 425 
                 77 
                 U04343.1 
                 817 
                 Coding 
               
               
                   
               
               
                 323676 
                 CCTCAAGCTCTATAGAGAAA 
                 426 
                 24 
                 U04343.1 
                 822 
                 Coding 
               
               
                   
               
               
                 323677 
                 GGAGGCTGAGGGTCCTCAAG 
                 427 
                 55 
                 U04343.1 
                 835 
                 Coding 
               
               
                   
               
               
                 323678 
                 AGTACAGCTGTAATCCAAGG 
                 428 
                 23 
                 U04343.1 
                 868 
                 Coding 
               
               
                   
               
               
                 323679 
                 TTGGAAGTACAGCTGTAATC 
                 429 
                 60 
                 U04343.1 
                 873 
                 Coding 
               
               
                   
               
               
                 323680 
                 ATAATAACTGTTGGAAGTAC 
                 430 
                 51 
                 U04343.1 
                 883 
                 Coding 
               
               
                   
               
               
                 323681 
                 CATCACACATATAATAACTG 
                 431 
                 8 
                 U04343.1 
                 893 
                 Coding 
               
               
                   
               
               
                 323682 
                 TCCATTTCCATAGAATTAGA 
                 432 
                 35 
                 U04343.1 
                 921 
                 Coding 
               
               
                   
               
               
                 323683 
                 TCTTCTTCCATTTCCATAGA 
                 433 
                 16 
                 U04343.1 
                 927 
                 Coding 
               
               
                   
               
               
                 323684 
                 ATTTATAAGAGTTGCGAGGC 
                 434 
                 32 
                 U04343.1 
                 954 
                 Coding 
               
               
                   
               
               
                 323685 
                 TTGGTTCCACATTTATAAGA 
                 435 
                 18 
                 U04343.1 
                 964 
                 Coding 
               
               
                   
               
               
                 323686 
                 CTCTCCATTGTGTTGGTTCC 
                 436 
                 53 
                 U04343.1 
                 976 
                 Coding 
               
               
                   
               
               
                 323687 
                 CTTCCCTCTCCATTGTGTTG 
                 437 
                 19 
                 U04343.1 
                 981 
                 Coding 
               
               
                   
               
               
                 323688 
                 TGGTCTGTTCACTCTCTTCC 
                 438 
                 58 
                 U04343.1 
                 996 
                 Coding 
               
               
                   
               
               
                 323689 
                 TTCATCAGATCTTTCAGGTA 
                 439 
                 43 
                 U04343.1 
                 1037 
                 Coding 
               
               
                   
               
               
                 323690 
                 ATCACTTTTGTCGCATGAAG 
                 440 
                 82 
                 U04343.1 
                 1088 
                 Coding 
               
               
                   
               
               
                 323691 
                 GCTTTACTCTTTAATTAAAA 
                 441 
                 40 
                 U04343.1 
                 1114 
                 STOP 
               
               
                   
               
               
                 323692 
                 GTATGGGCTTTACTCTTTAA 
                 442 
                 57 
                 U04343.1 
                 1120 
                 3′UTR 
               
               
                   
               
               
                 323693 
                 ATACTTGTATGGGCTTTACT 
                 443 
                 62 
                 U04343.1 
                 1126 
                 3′UTR 
               
               
                   
               
               
                 323694 
                 AATGAATACTTGTATGGGCT 
                 444 
                 71 
                 U04343.1 
                 1131 
                 3′UTR