# Patent

## Title
Compositions and methods for the modulation of the expression of B7 protein

## Abstract

Antisense oligonucleotide compositions which specifically hybridize with nucleic acids encoding B7 proteins, and use of these compositions for inhibiting expression of B7 mRNA.

## Background
RELATED APPLICATIONS
This application is a U.S. National Phase application of PCT/US2004/015880 filed on May 19, 2004 which claims priority to four U.S. patent applications: Ser. No. 60/651,504, filed May 23, 2003; Ser. No. 60/510,614 filed, Oct. 10, 2003; Ser. No. 60/520,401, filed Nov. 13, 2003; and Ser. No. 60/537,291, filed Jan. 16, 2004, all of which are incorporated herein by reference in their entirety.


SEQUENCE LISTING
A sequence listing is filed herewith in accordance with CFR 1.821 and is hereby incorporated by reference.
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'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). CTLA4Ig, 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.
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. These oligonucleotides may be chemically modified or unmodified. 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 B7-2 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' 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.
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 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.
In one embodiment, there is provided a pharmaceutical composition comprising, as an active ingredient, a modified or unmodified antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients.
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.
In another embodiment, the invention provides methods of treating a condition associated with an increase in the expression of B7.1 or B7.2 protein comprising administering to an individual in need of such treatment an effective amount of an antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt thereof. That is, the present invention provides for the use of an antisense oligonucleotide of the present invention, or a pharmaceutical composition thereof, for the treatment of a disorder associated with increased expression of B7-1 or B7-2 proteins.
In another aspect, the present invention provides for the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the expression of B7-1 or B7-2. Thus, the present invention provides for the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disorder associated with expression of B7-1 or B7-2 by means of the methods described above.
In a preferred embodiment, the present invention provides a method for treating asthma comprising administering to a patient an individual in need of such treatment an effective amount of an antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt thereof.



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'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 ΦM indicated oligonucleotide; right bars, 10 ΦM 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 is a graph 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 is a graph 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.
FIG. 22 is a graph showing that aerosolized B7-2 antisense oligonucleotide (ISIS 121874), but not a mismatch control oligonucleotide (ISIS 131906), reduces the airway response to methacholine in an ovalbumin-induced mouse model of asthma.
FIG. 23 is a graph 'showing that aerosolized B7-2 antisense oligonucleotide (ISIS 121874), but not a mismatch-control oligonucleotide (ISIS 131906), reduces airway eosinophilia in ovalbumin-challenged mice.
FIG. 24 is a graph showing that aerosolized B7-2 antisense oligonucleotide (ISIS 121874), but not a mismatch control oligonucleotide (ISIS 131906), reduces mucus oveproduction in ovalbumin-challenged mice.



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.
Antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
While oligonucleotides are a preferred form of the antisense compounds of this invention, the present invention comprehends other families of antisense compounds as well, including but not limited to oligonucleotide analogs and mimetics such as locked nucleic acids (LNA), peptide nucleic acids (PNA), cyclohexynyl nucleic acids (CeNA) ethyloxy nucleic acids (ENA) which are described in more detail herein.
“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.
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' 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 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 Anormal@ immune reactions (such as a host animal'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 region 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 A5′-UTR@), the translation initiation codon region (hereinafter, the AtIR@), the open reading frame (hereinafter, the AORF@), the translation termination codon region (hereinafter, the AtTR@) and the 3′ untranslated region (hereinafter, the A3′-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 Aintrons,@ which are excised from a transcript before it is translated; the expressed (unexcised) portions of the ORF are referred to as Aexons@ (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 AAUG codon,@ the Astart codon@ or the AAUG 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 Atranslation initiation codon@ and Astart 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, Astart codon@ and Atranslation 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 Astop 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 Astart codon region@ and Atranslation 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 Astop codon region@ and Atranslation 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.
Antisense oligonucleotides of the present invention that further comprise modified internucleoside linkages are preferred, particularly one in which one of the oxygen atoms of the phosphate group in the phosphodiester bond is replaced with a sulfur atom to form a phosphorothioate linkage.
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, phosphoro-dithioates, 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 borano-phosphates 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 —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3) —CH2—, —CH2—N(CH3) —N(CH3) —CH2— and —O—N(CH3) —CH2—CH2— [wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2—]. 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 CH2 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.
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;
T4 is hydrogen, an amino protecting group, —C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 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;
T5 is —OH, —N(Z1)Z2, R5, 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;
Z1 is hydrogen, C1-C6 alkyl, or an amino protecting group;
Z2 is hydrogen, C1-C6 alkyl, an amino protecting group, —C(═O)—(CH2)n-J-Z3, 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;
Z3 is hydrogen, an amino protecting group, —C1-C6 alkyl, —C(═O)—CH3, benzyl, benzoyl, or —(CH2)n—N(H)Z1;
each J is O, S or NH;
R5 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 (L2) joining the monomeric subunits. The basic formula is shown below:




wherein
Bx is a heterocyclic base moiety;
T1 is hydroxyl or a protected hydroxyl;
T5 is hydrogen or a phosphate or phosphate derivative;
L2 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 an DNA/RNA molecule is replaced with a cyclohenyl 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;
T1 is hydroxyl or a protected hydroxyl;
T2 is hydroxyl or a protected hydroxyl;
L3 is a linking group; and
n is from 2 to about 50
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:




wherein

Bx is a heterocyclic base moiety;
T1 is a hydroxyl or a protected hydroxyl;
T2 is a hydroxyl or a protected hydroxyl;
L is a linking group; and
n is from 2 to about 50
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 (—CH2—)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:




wherein
Bx is a heterocyclic base moiety;
T1 is a hydroxyl or a protected hydroxyl;
T2 is a hydroxyl or a protected hydroxyl;
Z1 is a linking group; and
n is from 2 to about 50;
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 LINA 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'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):




DMT=dimethoxytrityl. (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'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 is referred to as phosphonomonoester nucleic acids incorporate a phosphorus group in the backbone. This class of olignucleotide 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 C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, 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—CH2CH2OCH3, 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. Especially preferred compounds of the invention comprise 2′-MOE modifications.
A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)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—CH2—O—CH2—N(CH3)2.
Other preferred sugar substituent groups include methoxy (—O—CH3), aminopropoxy (—OCH2CH2CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O—CH2—CH═CH2) 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 Ia or IIa:




wherein:
Rb is O, S or NH;
Rd is a single bond, O, S or C(═O);
Re is C1-C10 alkyl, N(Rk) (Rm), N(Rk) (Rn), N═C(Rp) (Rq), N═C(Rp) (Rr) or has formula IIIa;




Rp and Rq are each independently hydrogen or C1-C10 alkyl;
Rr is —Rx—Ry;
each Rs, Rt, Ru and Rv is, independently, hydrogen, C(O)Rw, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 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, Ru and Rv, together form a phthalimido moiety with the nitrogen atom to which they are attached;
each Rw is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
Rk is hydrogen, a nitrogen protecting group or —Rx—Ry;
Rp is hydrogen, a nitrogen protecting group or —Rx—Ry;
Rx is a bond or a linking moiety;
Ry is a chemical functional group, a conjugate group or a solid support medium;
each Rm and Rn is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH3+, N(Ru) (Rv), guanidino and acyl where said acyl is an acid amide or an ester;
or Rm and Rn 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;
Ri is ORz, SRz, or N(Rz)2;
each Rz is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(═NH)N(H)Ru, C(═O)N(H)Ru or OC(═O)N(H)Ru;
Rf, Rg and Rh 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;
Rj 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(Rk) (Rm) ORk, halo, SRk or CN;
ma 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. Pat. No. 6,172,209, hereby incorporated by reference in its entirety.
Representative cyclic substituent groups of Formula II are disclosed in United States Pat. No. 6,217,358, hereby incorporated by reference in its entirety.
Particularly preferred sugar substituent groups include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]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. Pat. No. 6,593,466, 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 Publication WO 00/08044, 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—CH3) 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. Particularly preferred base modifications of the present invention are 5-methyl cytosine modifications.
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 & 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.
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 (R10=O,R11-R14=H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R10=S, R11-R14=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 (R10=O, R11-R14=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 Publication No. US2003-0207804 entitled “Modified Peptide Nucleic Acids”; and Publication No. US2003-0185906 entitled “Nuclease Resistant Chimeric Oligonucleotides”, 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 (R10=O, R11=—O—(CH2)2—NH2, R12-14=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 ΔTm of up to 18° relative to 5-methyl cytosine (dC5me), 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 Tm data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5me. 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 20 mer 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 heterocycicic 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 Publication No. US 2003-0158403, 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 triethyl-ammonium 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 & 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. Pat. No. 6,656,730 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 a 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 appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren'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'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 & 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 oligoncleotides 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 oligonulceotide. 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'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 dependent 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 1H 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′-MOE modification (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′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Hel V. 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 on the market for the treatment of CMV retinitis.
Chemistries Defined
Unless otherwise defined herein, alkyl means C1-C12, preferably C1-C8, and more preferably C1-C6, straight or (where possible) branched chain aliphatic hydrocarbyl.
Unless otherwise defined herein, heteroalkyl means C1-C12, preferably C1-C8, and more preferably C1-C6, straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, and preferably about 1 to about 3, heteroatoms in the chain, including the terminal portion of the chain. Preferred heteroatoms include N, O and S.
Unless otherwise defined herein, cycloalkyl means C3-C12, preferably C3-C8, and more preferably C3-C6, aliphatic hydrocarbyl ring.
Unless otherwise defined herein, alkenyl means C2-C12, preferably C2-C8, and more preferably C2-C6 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 C2-C12, preferably C2-C8, and more preferably C2-C6 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, NO2, NH3 (substituted and unsubstituted), acid moieties (e.g. —CO2H, —OSO3H2, 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. Nos. 5,760,209, 5,614,621, 6,051,699, 6,020,475, 6,326,478, 6,169,177, 6,121,437, 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 8 to about 50, even 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 term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects thereto.
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). Sodium salts are especially preferred pharmaceutically acceptable salts of the compounds of the present invention.
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 Atherapeutic 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 32P 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, radiolabelling of the oligonucleotide or any other suitable detection systems. Kits for detecting the presence or absence of a B7 protein may also be prepared.
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 ug 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 Atreatment regimen@ is meant to encompass therapeutic, palliative and prophylactic modalities. In one 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) (E.g., 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 H1 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 H2 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 Φg 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 preventive doses, ranging from 0.01 Φg to 100 g per kg of body weight, once or more daily. 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, dry powder inhaler, or metered dose inhaler; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Pulmonary administration methods are described in more detail below.
Metered Dose Inhalers, MDI or pMDI.
This delivery system is the most widely used, and accounts for about 70-80% of the pulmonary delivery systems used worldwide and have been approved since the 1950's. MDI is based typically (except for QVARS which is a solution of beclomethasone DP in a mixture of ethanol and HFA 134a) on a suspension of a micronized drug in a CFC or HFA liquid under high pressure. The drug is metered through a small chamber in the valve (typically 25-150 μL, most common 50 μL) and the suspension/HFA is aerosolized from the valve upon actuation and delivered as a dry powder, due to the evaporation of the propellant. This device typically can have suspension concentrations of up to 2% solids in suspension, which will be able to deliver about 1 mg/puff of active to the airways.
Nebulization—Continuous Nebulizers (Jet and Ultrasonic)
This delivery system typically comprises a continuous generation of an aerosol cloud from a solution or a suspension in water by a stream of compressed air (jet nebulizers) or by a ultrasound energy (ultrasonic nebulizers). Dosing is based on the time that the individual is exposed to the aerosol cloud and on the concentration of the drug in the solution or suspension. The patient or clinician is responsible for the preparation of the unit before and after dosing. Nebulizers continuously release drug throughout the respiratory cycle, but output is increased during inspiration in response to the patient's breathing. Large amounts of drug can be delivered to the airways. Some nebulizera detect pressure changes during breathing and constantly adapt to the inspiratory and expiratory flow pattern of the patient.
Dry Powder Inhaler, DPI
Dry powder inhalers consist of a finely divided dry powder contained in a reservoir or in a metered unit dose that upon actuation (by inspiratory force—these devices are called passive DPI, or by energy provided by the device—these devices are called active DPI) will generate a plume of aerosolized powder particles that will be inhaled. The DPI available can deliver up to a dose of 20-30 mg of active to the airways (Nektar and Alkermes) per puff.
Single Dose Nebulizers.
These devices have the way of metering the dose or have the doses pre-metered in unit dosage packages. The aerosol is generated from an aqueous solution or suspension by energy provided by the device and some of them are breath actuated, which means that the dosing will only occur when the right inspiratory flow rate is achieve to ensure proper dosing. The unit doses are typically 50-150 μL per dose, so depending on the solution concentration a dose of 10 to 20 mg per puff to the airways based on a solution concentration of around 150 mg/mL and a efficiency of around 60%.
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′-MOE 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 Publication Numbers US2003-124196 and US2003-0083286, 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 EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug 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′-MOE 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′-MOE cytosines were 5-methyl cytosines, which were synthesized according to the following procedures.
Synthesis of 5-Methyl Cytosine Monomers:
2,2′-Anhydro[-(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 CH3CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH2Cl2/acetone/MeOH (20:5:3) containing 0.5% Et3NH. The residue was dissolved in CH2Cl2 (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 CH3CN (200 mL). The residue was dissolved in CHCl3 (1.5 L) and extracted with 2×500 mL of saturated NaHCO3 and 2×500 mL of saturated NaCl. The organic phase was dried over Na2SO4, 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% Et3NH. 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 CHCl3 (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 CHCl3. 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 CH3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added, to a solution of triazole (90 g, 1.3 M) in CH3CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl3 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 NaHCO3 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 NH4OH (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 NH3 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.
N4-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 CHCl3 (700 mL) and extracted with saturated NaHCO3 (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO4 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% Et3NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.
N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite
N4-Benzoy-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH2Cl2 (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 NaHCO3 (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH2Cl2 (300 mL), and the extracts were combined, dried over MgSO4 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
O2-2′-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-Butyldiphenylchlotosilane (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-O2-2′-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<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 P2O5 under high vacuum for two days at 40 EC. 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 CH2Cl2 (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10 EC to 0 EC. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH2Cl2 and the combined organic phase was washed with water, brine and dried over anhydrous Na2SO4. 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 EC under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10 EC. 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 CH2Cl2). Aqueous NaHCO3 solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na2SO4, 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 EC in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10 EC 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% NaHCO3 (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 CH2Cl2 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 CH2Cl2). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH2Cl2 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 P2O5 under high vacuum overnight at 40 EC. 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 CH2Cl2 (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 P205 under high vacuum overnight at 40 EC. 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 NaHCO3 (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—CH2—O—CH2—N(CH2)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 tetra-hydrofuran (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-dimethyl-aninoethoxy)ethyl)]-5-methyl uridine
To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)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 CH2Cl2 (2×200 mL). The combined CH2Cl2 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 CH2Cl2 (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 (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'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 AS@ in the superscript position (e.g., TsA) 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











Target Position; Site
Oligonucleotide Sequence (5′ −> 3′) and
SEQ


ISIS #
(and/or Description)
Chemical Modifications
ID NO:





13797
0053-0072; 5′ UTR
GSGSGSTSASASGSASCSTSCSCSASCSTSTSCSTSGSA
22


 


13798
0132-0151; 5′ UTR
GSGSGSTSCSTSCSCSASASASGSGSTSTSGSTSGSGSA
23


 


13799
0138-0157; 5′ UTR
GSTSTSCSCSTSGSGSGSTSCSTSCSCSASASASGSGST
24


 


13800
0158-0177; 5′ UTR
ASCSASCSASCSASGSASGSASTSTSGSGSASGSGSGST
25


 


13801
0193-0212; 5′ UTR
GSCSTSCSASCSGSTSASGSASASGSASCSCSCSTSCSC
26


 


13802
0217-0236; 5′ UTR
GSGSCSASGSGSGSCSTSGSASTSGSASCSASASTSCSC
27


 


13803
0226-0245; 5′ UTR
TSGSCSASASASASCSASGSGSCSASGSGSGSCSTSGSA
28


 


13804
0246-0265; 5′ UTR
ASGSASCSCSASGSGSGSCSASCSTSTSCSCSCSASGSG
29


 


13805
0320-0339; tIR
CSCSTSGSCSCSTSCSCSGSTSGSTSGSTSGSGSCSCSC
30


 


13806
0380-0399; 5′ ORF
GSASCSCSASGSCSCSASGSCSASCSCSASASGSASGSC
31


 


13807
0450-0469; 5′ ORF
CSCSASCSASGSGSASCSASGSCSGSTSTSGSCSCSASC
32


 


13808
0568-0587; 5′ ORF
CSCSGSGSTSTSCSTSTSGSTSASCSTSCSGSGSGSCSC
33


 


13809
0634-0653; central ORF
GSCSCSCSTSCSGSTSCSASGSASTSGSGSGSCSGSCSA
51


 


13810
0829-0848; central ORF
CSCSASASCSCSASGSGSASGSASGSGSTSGSASGSGSC
34


 


13811
1102-1121; 3′ ORF
GSGSCSASASASGSCSASGSTSASGSGSTCASGSGSC
35


 


13812
1254-1273; 3′-UTR
GSCSCSTSCSASTSGSASTSCSCSCSCSASCSGSASTSC
36


 


13872
(scrambled # 13812)
ASGSTSCSCSTSASCSTSASCSCSASGSCSCSGSCSCST
52


 


12361
0056-0075; 5′ UTR
TSCSASGSGSGSTSASASGSASCSTSCSCSASCSTSTSC
38


 


12348
0056-0075; 5′ UTR
T C A G G GSTSASASGSASCSTSCSC A C T T C
38




(2′ME)



 


12473
0056-0075; 5′ UTR
TSCSASGSGSGSTSASASGSASCSTSCSCSASCSTSTSC
38




(2′F1)



 


12362
0143-0162; 5′ UTR
ASGSGSGSTSGSTSTSCSCSTSGSGSGSTSCSTSCSCSA
39


 


12349
0143-0162; 5′ UTR
A G G G T GSTSTSCSCSTSGSGSGST C T C C A
39




(2′ME)



 


12474
0143-0162; 5′ UTR
ASGSGSGSTSGSTSTSCSCSTSGSGSGSTSCSTSCSCSA
39




(2′F1)



 


12363
0315-0334; tIR
CSTSCSCSGSTSGSTSGSTSGSGSCSCSCSASTSGSGSC
40


 


12350
0315-0334; tIR
C T C C G TSGSTSGSTSGSGSCSC C A T G G C
40




(2′ME)



 


12475
0315-0334; tIR
CSTSCSCSGSTSGSTSGSTSGSGSCCSASTSGSGSC
40




(2′F1)



 


12364
0334-0353; 5′ ORF
GSGSASTSGSGSTSGSASTSGSTSTSCSCSCSTSGSCSC
41


 


12351
0334-0353; 5′ ORF
G G A T G GSTSGSASTSGSTSTSC C C T G C C
41




(2′ME)



 


12476
0334-0353; 5′ ORF
GSGSASTSGSGSTSGSASTSGSTSTSCSCSCSTSGSCSC
41




(2′F1)



 


12365
0387-0406; 5′ ORF
TSGSASGSASASASGSASCSCSASGSCSCSASGSCSASC
42


 


12352
0387-0406; 5′ ORF
T G A G A ASASGSASCSCSASGSCSC A G C A C
42




(2′ME)



 


12477
0387-0406; 5′ ORF
TSGSASGSASASASGSASCSCSASGSCSCSASGSCSASC
42




(2′F1)



 


12366
0621-0640; central ORF
GSGSGSCSGSCSASGSASGSCSCSASGSGSASTSCSASC
43


 


12353
0621-0640; central ORF
G G G C G CSASGSASGSCSASASG G A T C A C
43




(2′ME)



 


12478
0621-0640; central ORF
GSGSGSCSGSCSASGSASGSCSCSASGSGSASTSCSASC
43




(2′F1)



 


12367
1042-1061; 3′ ORF
GSGSCSCSCSASGSGSASTSGSGSGSASGSCSASGSGST
44


 


12354
1042-1061; 3′ ORF
G G C C C ASGSGSASTSGSGSGSA G C A G G T
44




(2′ME)



 


12479
1042-1061; 3′ ORF
GSGSCSCSCSASGSGSASTSGSGSGSASGSCSASGSGST
44




(2′F1)



 


12368
1069-1088; tTR
ASGSGSGSCSGSTSASCSASCSTSTSTSCSCSCSTSTSC
45


 


12355
1069-1088; tTR
A G G G C GSTSASCSASCSTSTST C C C T T C
45




(2′ME)



 


12480
1069-1088; tTR
ASGSGSGSCSGSTSASCSASCSTSTSTSCSCSCSTSTSC
45




(2′F1)



 


12369
1100-1209; tTR
CSASGSCSCSCSCSTSTSGSCSTSTSCSTSGSCSGSGSA
46


 


12356
1100-1209; tTR
C A G C C CSCSTSTSGSCSTSTSCST G C G G A
46




(2′ME)



 


12481
1100-1209; tTR
CSASGSCSCSCSCSTSTSGSCSTSTSCSTSGSCSGSGSA
46




(2′F1)



 


12370
1360-1380; 3′ UTR
ASASGSGSASGSASGSGSGSASTSGSCSCSASGSCSCSA
47


 


12357
1360-1380; 3′ UTR
A A G G A GSASGSGSGSASTSGSC C A G C C A
47




(2′ME)



 


12482
1360-1380; 3′ UTR
ASASGSGSASGSASGSGSGSASTSGSCSCSASGSCSCSA
47




(2′F1)



 


12914
(−0038 to −0059; 5′
CSTSGSTSTSASCSTSTSTSASCSASGSASGSGSGSTSTSTSG
48



UTR of alternative
(2'40 MO)




mRNA)




 


12915
(−0035 to −0059; 5′
CSTSTSCSTSGSTSTSASCSTSTSTSASCSASGSASGSGSGS
49



UTR of alternative
TSTSTSG




mRNA)
(2′MO)



 


13498
(−0038 to −0058; 5′
CSTSGSTSTSASCSTSTSTSASCSASGSASGSGSGSTSTST
50



UTR of alternative
(2′ME)




mrNA)




 


13499
(−0038 to −0058; 5′
C T G T T A C T T T A C A G A G G G T T T
50



UTR of alternative
(2′ME)




mRNA)
















TABLE 2







Sequences of Oligonucleotides Targeted to Murine B7-1 mRNA












Oligonucleotide Sequence (5′ −> 3′)
SEQ


ISIS #
Target Position; Site
and Chemical Modifications
ID NO:





14419
0009-0028; 5′ UTR
ASGSTSASASGSASGSTSCSTSASTSTSGSASGSGSTSA
53


 


14420
0041-0060; 5′ UTR
GSGSTSTSGSASGSTSTSTSCSASCSASASCSCSTSGSA
54


 


14421
0071-0091; 5′ UTR
GSTSCSCSASCSASGSASASTSGSGSASASCSASGSASG
55


 


14422
0109-0128; 5′ UTR
GSGSCSASTSCSCSASCSCSCSGSGSCSASGSASTSGSC
56


 


14423
0114-0133; 5′ UTR
TSGSGSASTSGSGSCSASTSCSCSASCSCSCSGSGSCSA
57


 


14424
0168-0187; 5′ UTR
ASGSGSCSASCSCSTSCSCSTSASGSGSCSTSCSASCSA
58


 


14425
0181-0200; 5′ UTR
GSCSCSASASTSGSGSASGSCSTSTSASGSGSCSASCSC
59


 


14426
0208-0217; 5′ UTR
CSASTSGSASTSGSGSGSGSASASASGSCSCSASGSGSA
60


 


14427
0242-0261; tIR
ASASTSTSGSCSASASGSCSCSASTSASGSCSTSTSCSA
61


 


14428
0393-0412; 5′ ORF
CSGSGSCSASASGSGSCSASGSCSASASTSASCSCSTST
62


 


14909
0478-0497; 5′ ORF
CSCSCSASGSCSASASTSGSASCSASGSASCSASGSCSA
63


 


14910
0569-0588; central ORF
GSGSTSCSTSGSASASASGSGSASCSCSASGSGSCSCSC
64


 


14911
0745-0764; central ORF
TSGSGSGSASASASCSCSCSCSCSGSGSASASGSCSASA
65


 


14912
0750-0769; central ORF
GSGSCSTSTSTSGSGSGSASASASCSCSCSCSCSGSGSA
66


 


14913
0825-0844; 3′ ORF
TSCSASGSASTSTSCSASGSGSASTSCSCSTSGSGSGSA
67


 


14914
0932-0951; 3′ ORF
CSCSCSASGSGSTSGSASASGSTSCSCSTSCSTSGSASC
68


 


14915
1001-1020; 3′ ORF
CSTSGSCSGSCSCSGSASASTSCSCSTSGSCSCSCSCSA
69


 


14916
1125-1144; tTR
CSASGSGSCSCSCSGSASASGSGSTSASASGSGSCSTSG
70


 


14917
1229-1248; 3′ UTR
TSCSASGSCSTSASGSCSASCSGSGSTSGSCSTSGSASA
71


 


14918
1329-1348; 3′ UTR
GSGSCSCSCSASGSCSASASASCSTSTSGSCSCSCSGST
72


 


14919
1377-1393; 3′ UTR
CSCSASCSCSASCSASGSTSGSGSGSCSTSCSASGSCSC
73


 


12912
−0067 to −0049; 5′ UTR
GSGSCSCSASTSGSASGSGSGSCSASASTSCSTSASA
74




(2′MO)



 


12913
−0067 to −0047; 5′ UTR
GSTSGSGSCSCSASTSGSASGSGSGSCSASASTSCSTSASA
75




(2′MO)



 


13496
−0067 to −0047; 5′ UTR
GSTSGSGSCSCSASTSGSASGSGSGSCSASASTSCSTSASA
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


ISIS #
Target Position*; Site**
Oligonucleotide Sequence (5′ −> 3′)
ID NO:













9133
1367-1386; 3′-UTR
TSTSCSCSASGSGSTSCSASTSGSASGSCSCSASTSTSA
3


 


10715
scrambled control of # 9133
GSASTSTSTSASASCSASTSTSTSGSGSCSGSCSCSCSA
76


 


9134
1333-1352; 3′-UTR
CSASTSASASGSGSTSGSTSGSCSTSCSTSGSASASGSTSG
4


 


9135
1211-1230; 3′-UTR
TSTSASCSTSCSASTSGSGSTSASASTSGSTSCSTSTSTS
5


 


9136
1101-1120; tTR
ASTSTSASASASASASCSASTSGSTSASTSCSASCSTSTS
6


 


10716
(scrambled # 9136)
ASASASGSTSTSASCSASASCSASTSTSASTSASTSCST
77


 


9137
0054-0074; 5′-UTR
GSGSASASCSASCSASGSASASGSCSASASGSGSTSGSGST
7


 


9138
0001-0020; 5′-UTR
CSCSGSTSASCSCSTSCSCSTSASASGSGSCSTSCSCST
8


 


9139
0133-0152; tIR
CSCSCSASTSASGSTSGSCSTSGSTSCSASCSASASAST
9


 


10877
(scrambled # 9139)
ASGSTSGSCSGSASTSTSCSTSCSASASASCSCSTSASC
78


 


10367
0073-0092; 5′-UTR
GSCSASCSASGSCSASGSCSASTSTSCSCSCSASASGSG
10


 


10368
0240-0259; 5′ ORF
TSTSGSCSASASASTSTSGSGSCSASTSGSGSCSASGSG
11


 


10369
1122-1141; 3′-UTR
TSGSGSTSASTSGSGSGSCSTSTSTSASCSTSCSTSTST
12


 


10370
1171-1190; 3′-UTR
ASASASASGSGSTSTSGSCSCSCSASGSGSASASCSGSG
13


 


10371
1233-1252; 3′-UTR
GSGSGSASGSTSCSCSTSGSGSASGSCSCSCSCSCSTST
14


 


10372
1353-1372; 3′-UTR
CSCSASTSTSASASGSCSTSGSGSGSCSTSTSGSGSCSC
15


 


11149
0019-0034; 5′-UTR
TSASTSTSTSGSCSGSASGSCSTSCSCSCSC
79


 


11151
0020-0034; 5′-UTR
TSASTSTSTSGSCSGSASGSCSTSCSCSC
80


 


11150
0021-0034; 5′-UTR
TSASTSTSTSGSCSGSASGSCSTSCSC
81


 


10373
0011-0030; 5′-UTR
TSGSCSGSASGSCSTSCSCSCSCSGSTSASCSCSTSCSC
16


 


10721
(scrambled # 10373)
CSGSASCSASGSCSTSCSCSTSGSCSGSCSTSCSCSTSC
82


 


10729
(5′FITC # 10373)
TSGSCSGSASGSCSTSCSCSCSCSGSTSASCSCSTSCSC
16


 


10782
(5′cholesterol # 10373)
TSGSCSGSASGSCSTSCSCSCSCSGSTSASCSCSTSCSC
16


 



#10373 Deletion Derivatives:




10373
0011-0030; 5′-UTR
TSGSCSGSASGSCSTSCSCSCSCSGSTSASCSCSTSCSC
16


 


10888
0011-0026; 5′-UTR
        ASGSCSTSCSCSCSCSGSTSASCSCSTSCSC
83


 


10889
0015-0030; 5′-UTR
TSGSCSGSASGSCSTSCSCSCSCSGSTSASC
84


 


10991
0015-0024; 5′-UTR
          CSTSCSCSCSCSGSTSASC
85


 


10992
0015-0025; 5′-UTR
        GSCSTSCSCSCSCSGSTSASC
86


 


10993
0015-0026; 5′-UTR
      ASGSCSTSCSCSCSCSGSTSASC
87


 


10994
0015-0027; 5′-UTR
    GSASGSCSTSCSCSCSCSGSTSASC
88


 


10995
0015-0028; 5′-UTR
  CSGSASGSCSTSCSCSCSCSGSTSASC
89


 


10996
0015-0029; 5′-UTR
GSCSGSASGSCSTSCSCSCSCSGSTSASC
90


 


11232
0017-0029; 5′ UTR
GSCSGSASGSCSTSCSCSCSCSGST
91


 



# 10996 Derivatives:




10996
0015-002 9; 5′-UTR
GSCSGSASGSCSTSCSCSCSCSGSTSASC
90


 


11806
(scrambled # 10996)
GSCSCSGSCSCSGSCSCSASASGSTSCST
92


 


11539
(fully 2′ MO # 10996)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


11540
(control for # 11539)
GSCSCSGSCSCSGSCSCSASASGSTSCST (2′ MO)
92


 


11541
(# 10996 7-base Agapmer@)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


11542
(control for # 11541)
GSCSCSGSCSCSGSCSCSASASGSTSCST (2′ MO)
92


 


11543
(# 109969-base Agapmer@)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


11544
(control for # 11543)
GSCSCSGSCSCSGSCSCSASASGSTSCST (2′ MO)
92


 


11545
(# 10996 5′ Awingmer@)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


11546
(control for # 11545)
GSCSCSGSCSCSGSCSCSASASGSTSCST (2′ MO)
92


 


11547
(# 10996 3′ Awingmer@)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


11548
(control for # 11547)
GSCSCSGSCSCSGSCSCSASASGSTSCST (2′ MO)
92


 


12496
((2′-5′)A4 # 10996)
G C G A G C T C C C C G T A C
90


 


13107
((2′-5′)A4 # 10996)
GSCSGSASGSCSTSCSCSCSCSGSTSASC
90


 


12492
((2′-5′)A4 # 10996)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


12495
((2′-5′)A4 # 10996)
GSCSGSASGSCSTSCSCSCSCSGSTSASC (2′ MO)
90


 


12887
(1-24 of SEQ ID NO: 1 of WO
GSASGSASASGSCSASASASGSCSTSTSTSCSASCSCSCS-
93



95/03408; alternative mrNA)
TSGSTSG (2′ MO)



 


12888
(1-22 of SEQ ID NO: 1 of WO
GSASASGSCSASASASGSCSTSTSTSCSASCSCSCSTSGSTSG
94



95/03408; alternative mRNA)
(2′ MO)



 


12889
(1-19 of SEQ ID NO: 1 of WO
GSCSASASASGSCSTSTSTSCSASCSCSCSTSGSTSG
95



95/03408; alternative mRNA)
(2′ MO)



 


12890
0001-0024
CSTSCSCSCSCSGSTSASCSCSTSCSCSTSASASGSGSCS-
96




TSCSCST (2′ MO)



 


12891
0001-0022
CSCSCSCSGSTSASCSCSTSCSCSTSASASGSGSCSTSCSCST
97




(2′ MO)



 


12892
0001-0020
CSCSGSTSASCSCSTSCSCSTSASASGSGSCSTSCSC
98




(2′ MO)
















TABLE 4







Sequences of Oligonucleotides Targeted to Murine B7-2 mRNA













SEQ


ISIS #
Target Position; Site
Oligonucleotide Sequence (5′ −> 3′)
ID NO:





11347
1094-1113; 3′ UTR
ASGSASASTSTSCSCSASASTSCSASGSCSTSGSASGSA
121


 


11348
1062-1081; 3′ UTR
TSCSTSGSASGSASASASCSTSCSTSGSCSASCSTSTSC
122


 


11349
1012-1031; 3′ UTR
TSCSCSTSCSASGSGSCSTSCSTSCSASCSTSGSCSCST
123


 


11350
0019-1138; 5′ UTR
GSGSTSTSGSTSTSCSASASGSTSCSCSGSTSGSCSTSG
124


 


11351
0037-0056; 5′ UTR
ASCSASCSGSTSCSTSASCSASGSGSASGSTSCSTSGSG
103


 


11352
0089-0108; tIR
CSASASGSCSCSCSASTSGSGSTSGSCSASTSCSTSGSG
104


 


11353
0073-0092; tIR
CSTSGSGSGSGSTSCSCSASTSCSGSTSGSGSGSTSGSC
105


 


11354
0007-0026; 5′ UTR
CSCSGSTSGSCSTSGSCSCSTSASCSASGSGSASGSCSC
106


 


11695
0058-0077; 5′ UTR
GSGSTSGSCSTSTSCSCSGSTSASASGSTSTSCSTSGSG
107


 


11696
0096-0117; tIR
GSGSASTSTSGSCSCSASASGSCSCSCSASTSGSGSTSG
108


 


11866
(Scrambled # 11696)
CSTSASASGSTSASGSTSGSCSTSASGSCSCSGSGSGSA
109


 


11697
0148-0167; 5′ ORF
TSGSCSGSTSCSTSCSCSASCSGSGSASASASCSASGSC
110


 


11698
0319-0338; 5′ ORF
GSTSGSCSGSGSCSCSCSASGSGSTSASCSTSTSGSGSC
111


 


11699
0832-0851; 3′ ORF
ASCSASASGSGSASGSGSASGSGSGSCSCSASCSASGST
112


 


11700
0753-0772; 3′ ORF
TSGSASGSASGSGSTSTSTSGSGSASGSGSASASASTSC
113


 


11701
0938-0957; 3′ ORF
GSASTSASGSTSCSTSCSTSCSTSGSTSCSASGSCSGST
114


 


11702
0890-0909; 3′ ORF
GSTSTSGSCSTSGSGSGSCSCSTSGSCSTSASGSGSCST
115


 


11865
(scrambled # 11702)
CSTSASGSGSTSCSTSCSGSTSCSGSTSCSGSGSTSGSG
116


 


11703
1003-1022; tTR
TSCSTSCSASCSTSGSCSCSTSTSCSASCSTSCSTSGSC
117


 


13100
Exon 1 (Borriello et al., J.
GSTSASCSCSASGSASTSGSASASGSGSTSTSASTSCSASA
118



Immun., 1995, 155, 5490;
(2′ MO)




5′ UTR of alternative mRNA).




 


13101
Exon 4 (Borriello et al.;
CSTSTSTSGSGSASGSASTSTSASTSTSCSGSASGSTST
119



5′ UTR of alternative mRNA)
(2′ MO)



 


13102
Exon 5 (Borriello et al.;
GSCSASASGSTSGSTSASASASGSCSCSCSTSGSASGST
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 uL) was brought to 100 uL with water. A 10 uL aliquot from the RT reaction was then amplified in a 50 uL PCR reaction using 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 uL) was brought to 100 uL with water. A 10 uL aliquot from the RT reaction was then amplified in a 50 uL 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,









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). OptiMEMTM (GIBCO-BRL, Gaithersburg, Md.) medium was added along with 15 Φg/mL of LipofectinTM (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 ΦL of staining buffer (PBS, 0.2% BSA, 0.1% azide) with 5 Φ L 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 ΦL 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 IC50 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&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 RNATM 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 a-32P-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 IITM kit (Ambion) according to the manufacturer's directions. Total RNA (5 Φg) was hybridized overnight, at 42° C., with 105 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 Φ L 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'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 by 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 10996 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 10889, 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′ methoxethoxy substitutions were prepared, including a fully substituted derivative (ISIS 11539), Agapmers@ (ISIS 11541 and 11543) and Awingmers@ (ISIS 11545 and 11547). As explained in Example 5, the 2′-MOE 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 “caps” 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 “caps” 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′-MOE modifications in order to enhance their affinity for their target molecules, and 2′-MOE 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 PolymorphprepTM (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 LipofectinTM (8 ug/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&D Systems, Minneapolis, Minn.) (66 ng/mL) and granulocyte-macrophage colony-stimulating factor (GM-CSF; R&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 IC50 of approximately 250 nM. ISIS 10373 had only a slight effect on ICAM-1 expression even at a dose of 1 uM. 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 IC50 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 [3H] 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 NH4Cl lysis. T cells were purified using a T cell enrichment column (R&D Systems, Minneapolis, Minn.) essentially according to the manufacturer'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's directions. T cells were determined to be >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 ug/mL) for 1 hour then washed 3 times with PBS. APCs (105 cells) were then combined with 4×104 CD4+T cells in 350 uL of culture media. Where indicated, purified CD3 mAb was also added at a concentration of 1 ug/mL. During the last 6 hours of the 48 hour incubation period, proliferation was measured by determining uptake of 1.5 uCi of [3H]-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 IC50 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 uL of OptiMEMTM (GIBCO-BRL, Gaithersburg, Md.) supplemented with 15 ug/mL of LipofectinTM (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 ug/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 LipofectinTM (GIBCO-BRL, Gaithersburg, Md.). The results are shown in FIG. 9. At a concentration of 10 uM, 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 uM, levels of induced expression were greatly reduced by ISIS 11696, while ISIS 11866 had little effect.
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 oligqnucleotide 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′-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)













SEQ
TARGET GENE
SEQ


ISIS
NUCLEOTIDE SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
REGION














22313
AGACTCCACTTCTGAGATGT
126
0048-0067
5′-UTR


 


22314
TGAAGAAAAATTCCACTTTT
127
0094-0113
5′-UTR


 


22315
TTTAGTTTCACAGCTTGCTG
128
0112-0129
5′-UTR


 


22316
GCTCACGTAGAAGACCCTCC
26
0193-0212
5′-UTR


 


22317
TCCCAGGTGCAAAACAGGCA
129
0233-0252
5′-UTR


 


22318
GTGAAAGCCAACAATTTGGA
130
0274-0293
5′-UTR


 


22319
CATGGCTTCAGATGCTTAGG
131
0301-0320
AUG


 


22320
TTGAGGTATGGACACTTGGA
132
0351-0370
coding


 


22321
GACCAGCCAGCACCAAGAGC
31
0380-0399
coding


 


22322
GCGTTGCCACTTCTTTCACT
133
0440-0459
coding


 


22323
TTTTGCCAGTAGATGCGAGT
134
0501-0520
coding


 


22324
GGCCATATATTCATGTCCCC
135
0552-0571
coding


 


22325
GCCAGGATCACAATGGAGAG
136
0612-0631
coding


 


22326
GTATGTGCCCTCGTCAGATG
137
0640-0659
coding


 


22327
TTCAGCCAGGTGTTCCCGCT
138
0697-0716
coding


 


22328
GGAAGTCAGCTTTGACTGAT
139
0725-0744
coding


 


22329
CCTCCAGAGGTTGAGCAAAT
140
0798-0817
coding


 


22330
CCAACCAGGAGAGGTGAGGC
141
0827-0846
coding


 


22331
GAAGCTGTGGTTGGTTGTCA
142
Q94Q-Q959
coding


 


22332
TTGAAGGTCTGATTCACTCT
143
0987-1006
coding


 


22333
AAGGTAATGGCCCAGGTTGG
144
1050-1069
coding


 


22334
AAGCAGTAGGTCAGGCAGCA
145
1098-1117
coding


 


22335
CCTTGCTTCTGCGGACACTG
146
1185-1204
3′-UTR


 


22336
AGCCCCTTGCTTCTGCGGAC
147
1189-1208
3′-UTR


 


22337
TGACGGAGGCTACCTTCAGA
148
1216-1235
3′-UTR


 


22338
GCCTCATGATCCCCACGATC
36
1254-1273
3′-UTR


 


22339
GTAAAACAGCTTAAATTTGT
149
1286-1305
3′-UTR


 


22340
AGAAGAGGTTACATTAAGCA
150
1398-1417
3′-UTR


 


22341
AGATAATGAATTGGCTGACA
151
1454-1473
3′-UTR


 


24733
GCGTCATCATCCGCACCATC
152
control



 


24734
CGTTGCTTGTGCCGACAGTG
153
control



 


24735
GCTCACGAAGAACACCTTCC
154
control





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2Co-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 oligonucleodides 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














ASO Gene

% mRNA
% mRNA


ISIS #
SEQ ID 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′-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
SEQ


ISIS
NUCLEOTIDE SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
REGION





18104
AGAGAAACTAGTAAGAGTCT
155
0018-0037
5′-UTR


 


18105
TGGCATCCACCCGGCAGATG
156
0110-0129
5′-UTR


 


18106
TCGAGAAACAGAGATGTAGA
157
0144-0163
5′-UTR


 


18107
TGGAGCTTAGGCACCTCCTA
158
0176-0195
5′-UTR


 


18108
TGGGGAAAGCCAGGAATCTA
159
0203-0222
5′-UTR


 


18109
CAGCACAAAGAGAAGAATGA
160
0310-0329
coding


 


18110
ATGAGGAGAGTTGTAACGGC
161
0409-0428
coding


 


18111
AAGTCCGGTTCTTATACTCG
162
0515-0534
coding


 


18112
GCAGGTAATCCTTTTAGTGT
163
0724-0743
coding


 


18113
GTGAAGTCCTCTGACACGTG
164
0927-0946
coding


 


18114
CGAATCCTGCCCCAAAGAGC
165
0995-1014
coding


 


18115
ACTGCGCCGAATCCTGCCCC
166
1002-1021
coding


 


18116
TTGATGATGACAACGATGAC
167
1035-1054
coding


 


18117
CTGTTGTTTGTTTCTCTGCT
168
1098-1117
coding


 


18118
TGTTCAGCTAATGCTTCTTC
169
1134-1153
coding


 


18119
GTTAACTCTATCTTGTGTCA
170
1263-1282
3′-UTR


 


18120
TCCACTTCAGTCATCAAGCA
171
1355-1374
3′-UTR


 


18121
TGCTCAATACTCTCTTTTTA
172
1680-1699
3′-UTR


 


18122
AGGCCCAGCAAACTTGCCCG
173
1330-1349
3′-UTR


 


18123
AACGGCAAGGCAGCAATACC
174
0395-0414
coding





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2Co-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′-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













SEQ
TARGET GENE
GENE


ISIS
NUCLEOTIDE SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
REGION





22284
TGCGAGCTCCCCGTACCTCC
 16
0011-0030
5′-UTR


 


22285
CAGAAGCAAGGTGGTAAGAA
175
0049-0068
5′-UTR


 


22286
GCCTGTCCACTGTAGCTCCA
176
0113-0132
5′-UTR


 


22287
AGAATGTTACTCAGTCCCAT
177
0148-0167
AUG


 


22288
TCAGAGGAGCAGCACCAGAG
178
0189-0208
coding


 


22289
TGGCATGGCAGGTCTGCAGT
179
0232-0251
coding


 


22290
AGCTCACTCAGGCTTTGGTT
180
0268-0287
coding


 


22291
TGCCTAAGTATACCTCATTC
181
0324-0343
coding


 


22292
CTGTCAAATTTCTCTTTGCC
182
0340-0359
coding


 


22293
CATATACTTGGAATGAACAC
183
0359-0378
coding


 


22294
GGTCCAACTGTCCGAATCAA
184
0392-0411
coding


 


22295
TGATCTGAAGATTGTGAAGT
185
0417-0436
coding


 


22296
AAGCCCTTGTCCTTGATCTG
186
0430-0449
coding


 


22297
TGTGATGGATGATACATTGA
187
0453-0472
coding


 


22298
TCAGGTTGACTGAAGTTAGC
188
0529-0548
coding


 


22299
GTGTATAGATGAGCAGGTCA
189
0593-0612
coding


 


22300
TCTGTGACATTATCTTGAGA
190
0694-0713
coding


 


22301
AAGATAAAAGCCGCGTCTTG
191
0798-0817
coding


 


22302
AGAAAACCATCACACATATA
192
0900-0919
coding


 


22303
AGAGTTGCGAGGCCGCTTCT
193
0947-0968
coding


 


22304
TCCCTCTCCATTGTGTTGGT
194
0979-0998
coding


 


22305
CATCAGATCTTTCAGGTATA
195
1035-1054
coding


 


22306
GGCTTTACTCTTTAATTAAA
196
1115-1134
stop


 


22307
GAAATCAAAAAGGTTGCCCA
197
1178-1197
3′-UTR


 


22308
GGAGTCCTGGAGCCCCCTTA
198
1231-1250
3′-UTR


 


22309
TTGGCATACGGAGCAGAGCT
199
1281-1300
3′-UTR


 


22310
TGTGCTCTGAAGTGAAAAGA
200
1327-1346
3′-UTR


 


22311
GGCTTGGCCCATAAGTGTGC
201
1342-1361
3′-UTR


 


22312
CCTAAATTTTATTTCCAGGT
202
1379-1398
3′-UTR


 


24736
GCTCCAAGTGTCCCAATGAA
203
control



 


24737
AGTATGTTTCTCACTCCGAT
204
control



 


24738
TGCCAGCACCCGGTACGTCC
205
control





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2Co-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 wee 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














ASO Gene

% mRNA
% mRNA


ISIS #
SEQ ID 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′-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













SEQ
TARGET GENE
GENE


ISIS
NUCLEOTIDE SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
REGION





18084
GCTGCCTACAGGAGCCACTC
206
0003-0022
5′-UTR


 


18085
TCAAGTCCGTGCTGCCTACA
207
0013-0032
5′-UTR


 


18086
GTCTACAGGAGTCTGGTTGT
208
0033-0052
5′-UTR


 


18087
AGCTTGCGTCTCCACGGAAA
209
0152-0171
coding


 


18088
TCACACTATCAAGTTTCTCT
210
0297-0316
coding


 


18089
GTCAAAGCTCGTGCGGCCCA
211
0329-0348
coding


 


18090
GTGAAGTCGTAGAGTCCAGT
212
0356-0375
coding


 


18091
GTGACCTTGCTTAGACGTGC
213
0551-0570
coding


 


18092
CATCTTCTTAGGTTTCGGGT
214
0569-0588
coding


 


18093
GGCTGTTGGAGATACTGAAC
215
0663-0682
coding


 


18094
GGGAATGAAAGAGAGAGGCT
216
0679-0698
coding


 


18095
ACATACAATGATGAGCAGCA
217
0854-0873
coding


 


18096
GTCTCTCTGTCAGCGTTACT
218
0934-0953
coding


 


18097
TGCCAAGCCCATGGTGCATC
219
0092-0111
AUG


 


18098
GGATTGCCAAGCCCATGGTG
108
0096-0115
AUG


 


18099
GCAATTTGGGGTTCAAGTTC
220
0967-0986
coding


 


18100
CAATCAGCTGAGAACATTTT
221
1087-1106
3′-UTR


 


18101
TTTTGTATAAAACAATCATA
222
0403-0422
coding


 


18102
CCTTCACTCTGCATTTGGTT
223
0995-1014
stop


 


18103
TGCATGTTATCACCATACTC
224
0616-0635
coding





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2Co-ordinates from Genbank Accession No. S70108 locus name “S70108”.













TABLE 15







Inhibition of Mouse B7-2 mRNA Expression by Chimeric (deoxy


gapped) Phosphorothioate Oligodeoxynucleotides












SEQ
GENE




ISIS
ID
TARGET
% mRNA
% mRNA


No:
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 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 2. 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 B7-2 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%
100%


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%
100%


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














ASO Gene

% Surface
% Surface


ISIS #
SEQ ID 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














ASO Gene

% Surface
% Surface


ISIS #
SEQ ID 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'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














Dose
%




ISIS # (s)
SEQ ID NO
mg/kg
Incidence
Peak day1
Severity2





control

—
  70%
 6.7 ± 2.9
3.2 ± 1.1


17456 (B7-
173
10
  50%
12.1 ± 4.6
2.7 ± 1.3


1)







11696 (B7-
108
10
37.5%
11.6 ± 4.5
3.4 ± 1.8


2)







17456/11696

10
  30%
 1.0 ± 0.6
0.7 ± 0.4


18110 (B7-
161
10
55.6%
 2.0 ± 0.8
2.0 ± 1.3


1)







18089 (B7-
211
10
44.4%
 6.8 ± 2.2
2.3 ± 1.3


2)







18110/18089

10
  60%
11.6 ± 0.7
4.5 ± 1.7





1Peak day is the day from onset of maximum swelling for each joint measure.


2Severity 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'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′-MOE nucleotides at the wings and a central region of ten deoxynucleotides. All cytidines shown in Table 21 are 5-methyl cytidines. 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 1×107 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 Φg/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, 113507, 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













SEQ
TARGET GENE
GENE


ISIS
NUCLEOTIDE SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
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





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosine residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2Co-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′-MOE nucleotides at the wings and a central region of ten deoxynucleotides. All cytidnes shown in Table 23 are 5-methyl cytidines. 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 1×107 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 ΦM) 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 SEQUENCE1
ID
NUCLEOTIDE
TARGET


NO.
(5′ −> 3′)
NO:
CO-ORDINATES2
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





1Emboldened residues are 2′-MOE residues (others are 2′-deoxy). All 2′-MOE cytosines and 2′-deoxy cytosine residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.


2For 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 “HSU04343”.













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
3′ UTR
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, 2=-MOE modifications at nucleotides 1-5 and 16-20 and all cytidines were 5-methyl cytidines.
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 are 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<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>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 et al., Proc. Natl. Acad. Sci. U.S.A. 94:1350-1355, 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 & 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-(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's tongue was extended with forceps and 25 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, and all cytidines are 5-methyl cytidines. 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 (>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 (>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:









forward:



5′-GGCCCTCCTCCTTGTGATG-3′ (SEQ ID NO: 288)


 



probe:



5′-/56-FAM/TGCTCATCATTGTATGTCACAAGAAGCCG/36-



TAMTph/-3′ (SEQ ID NO: 289)


 



reverse:



5′-CTGGGCCTGCTAGGCTGAT3′ (SEQ ID NO: 290)


 



B7-1:



forward:



5′-CAGGAAGCTACGGGCAAGTT3′ (SEQ ID NO: 291)


 



probe:



5′-/56-FAM/TGGGCCTTTGATTGCTTGATGACTGAA/36-



TAMTph/-3′ (SEQ ID NO: 292)


 



reverse:



5′-GTGGGCTCAGCCTTTCCA3′ (SEQ ID NO: 293)






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 bty 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 naive 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. Treatment with a mismatch control oligonucleotide did not reduce airway hyperresponsiveness. 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). There was no increase in inflammatory infiltrates at pharmacologically relevant doses. No increases in spleen weight or AST/ALT levels were observed at dosages up to 10 mg/kg. The observed pharmacology is compatible with daily or weekly dosing in humans.
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 naive mice at 0.001 mg/kg dose (estimated inhaled dose=1 μg/kg). No gross adverse effects were seen.
In another aerosolized ISIS 121874 study, mice were treated with 0.01, 0.3 or 10.0 μg/kg estimated inhaled dose (n=10 mice per group), four times per day, or with a 7 base pair mismatched control oligonucleotide. The airway response to methacholine (100 mg/ml) was reduced to the level seen in naive mice at 0.01-0.3 μg/kg dose (FIG. 22). A mismatch control oligonucleotide had no effect on the airway response: to methacholine. In addition, ISIS 121874 reduced airway eosinophilia in a dose-dependent manner (FIG. 23). The mismatch control oligonucleotide had no effect. In addition, aerosolized ISIS 121874, but not a mismatch control oligonucleotide, reduced the percentage,of eosinophils/macrophages in lungs from ovalbumin-challenged mice. Lastly, aerosolized ISIS 121874, but not a mismatched control oligonucleotide, reduced mucus overproduction in a dose-dependent manner in ova-challenged mice (FIG. 24). Thus, both intratracheal and aerosol administration of B7.2 antisense oligonucleotides effectively reduce airway hyperresponsiveness and pulmonary inflammation in a mouse model of allergen-induced asthma, and represents a therapeutically useful treatment of human pulmonary inflammatory disorders, including asthma.
In addition, a dose-dependent increase in lung concentration of ISIS 121874 was observed at 24 hours following single (0.06 mg/kg) or multiple (0.00006, 0.006, 0.005 or 0.06 mg/kg, q2d×5).
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 (SingulairTM), albuterol, beclomethasone dipropionate, triamcinolone acetonide, ipratropium bromide (AtroventTM), flunisolide, fluticasone propionate (FloventTM) 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.
The tolerability of a 2′-MOE modified antisense oligonucleotide was investigated in an inhalation toxicity study in mice. CD1 mice were exposed to nine lung delivered doses of 1, 3 and 10 mg/kg over 18 days. No effect was seen on survival, clinical signs of toxicity, body weight or clinical pathology parameters. Lung morphology was unaffected except for dose-dependent changes in alveolar macrophages. The treatment doses tested were three orders of magnitude greater than doses with which in vivo efficacy was observed.
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 and having a two-nucleobase overhang of deoxythymidine (dT) would have the following structure:




In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG may be prepared with blunt ends (no single stranded overhang) as shown:




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 uM. Once diluted, 30 uL of each strand is combined with 15 uL 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 uL. 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 uM. 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′-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 phosphorothioate


oligonucleotides having 2′-MOE wings and a deoxy gap













Isis

SEQ
%
Genbank
Target



Number
Sequence 5′ to 3′
ID NO:
INHIB
Accesion Target
Site
Region
















322216
ACCAAAAGGAGTATTTGCGA
298
N.D.
U04343.1
26
5′UTR


 


322217
CATTCCCAAGGAACACAGAA
299
N.D.
U04343.1
64
5′UTR


 


322218
ACTGTAGTTCCAAAAAGAGA
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
TCCCTGTTACAGCAGCAGAG
304
N.D.
BC040261.1
32
5′UTR


 


322223
ATCTGGAAATGACCCCACTC
305
N.D.
BC040261.1
71
5′UTR


 


322224
GTGACCTAATATCTGGAAAT
306
N.D.
BC040261.1
81
5′UTR


 


322225
CATTTTGGCTGCTTCTGCTG
307
N.D.
EC040261.1
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
TGGAACTCTTGCCTTTCGGA
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
genomic


 


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
GCCTAAGTATACTTCATTCA
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
CAAGCCCTTGTCCTTTGATC
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
ACTGTTATATTAGAAATTGG
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
TTGGTTWCCACATTATAAGA
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









Example 28
Real-time Quantitative PCR Analysis of B7.2 mRNA Levels
Quantitation of B7.2 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5× PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
Probes and primers to human B7.2 were designed to hybridize to a human B7.2 sequence, using published sequence information (GenBank accession number U04343.1.1, incorporated herein as SEQ ID NO:295). For human B7.2 the PCR primers were:

    forward primer: TGTTTCATTCCCTGATGTTACGA (SEQ ID NO: 445)
    reverse primer: AAAAGCCGCGTCTTGTCAGT (SEQ ID NO: 446) and the
    PCR probe was: FAM-CAATATGACCATCTTCTGTATTCTGGA-TAMRA (SEQ ID NO: 447) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:
    forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:448)
    reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:449) and the
    PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 450) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.


Example 29
Oligonucleotides Targeted to Human B7.2
In accordance with the present invention, an additional series of antisense compounds was designed to target different regions of a 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, GenBank accession number BI824940.1, which is incorporated herein as SEQ ID NO: 451). The compounds are shown in Table 26. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. ISIS 331931 through ISIS 332004 (SEQ ID NO: 453 through SEQ ID NO: 526), ISIS 335973 (SEQ ID NO: 544), ISIS 335978 (SEQ ID NO: 549), ISIS 335993 (SEQ ID NO: 723) and ISIS 336004 (SEQ ID NO: 574), in additional to targeting human CD86, are exact matches to CD86 sequences from various other primate species, including Cynomolgous monkey and African Green Monkey.
ISIS 331993 through 332004 (SEQ ID NOS: 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526) are chimeric oligonucleotides 14-20 nucleotides in length, composed of varying combinations of 2′-deoxynucleotides and 2′-MOE nucleotides. The 2′-MOE modifications are at nucleobases 11-18 in 331993; 13-18 in 331994 and 331997; 1-4 and 15-18 in 331995 and 331996; 13-16 in 331998; 1-3 and 14-16 in 331999 and 332000; 1-2 and 13-14 in 332001 and 332002; and 11-20 in 332003 and 332004. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
ISIS 336088 through 336096 (SEQ ID NOs: 655, 656, 657, 658, 659, 660, 661, 662 and 663) are 20 nucleotides in length, composed of 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
ISIS 336098, 336101 and 336104 (SEQ ID NOs: 664, 665 and 666, respectively) are 20 to 21 nucleotides in length, composed of 2′deoxynucleotides. The internucleoside (backbone) linkages are phosphorophosphate throughout the nucleotdide. All cytidine residues are 5-methylcytidines.
The remaining compounds in Table 26 and ISIS 129686 (CGTTATTAACCTCCGTTGAA, SEQ ID NO: 452) 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′-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. THP-1 cells were electroporated in 1 mm cuvettes at a density of 1×107 cells/mL, a voltage of 75V and pulse length of 6 ms, in the presense of 10 ΦM oligonucleotide. Cells were immediately treated with 1 Φg/ml LPS and 0.5 mM dbcAMP, as described in other examples herein. Target mRNA was measured by quantitative real-time PCR as described in other examples herein. ISIS 129686 (SEQ ID NO: 452) was used as a scrambled control oligonucleotide. Data are averages from two experiments and are shown in Table 26. If present, “N.D.” indicates “no data”.







TABLE 26







Inhibition of human B7.2 mRNA levels by oligonucleotides













ISIS

SEQ
%
GenBank
Target



#
Sequence
ID NO
Inhib
Accession Target
Site
Region
















331931
CAGTCCCATAGTGCACTGGG
453
77
BC040261.1
124
Coding


 


331932
ACTCAGTCCCATAGTGCACT
454
70
BC040261.1
127
Coding


 


331933
TTGAAATAAGCTTGAATCTT
455
37
BC040261.1
195
Coding


 


331934
CATTGAAATAAGCTTGAATC
456
46
BC040261.1
197
Coding


 


331935
CTCATTGAAATAAGCTTGAA
457
56
BC040261.1
199
Coding


 


331936
TGCAGTCTCATTGAAATAAG
458
60
BC040261.1
205
Coding


 


331937
TCTGCAGTCTCATTGAAATA
459
32
BC040261.1
207
Coding


 


331938
GGCAGGTCTGCATTCTCATT
460
83
BC040261.1
213
Coding


 


331939
CATGGCAGGTCTGCAGTCTC
461
74
BC040261.1
216
Coding


 


331940
AATTGGCATGGCAGGTCTGC
462
82
BC040261.1
222
Coding


 


331941
GCAAATTGGCATGGCAGGTC
463
89
BC040261.1
225
Coding


 


331942
TTTGCAAATTGGCATGGCAG
464
60
BC040261.1
228
Coding


 


331943
GAGTTTGCAAATTGGCATGG
465
66
BC040261.1
231
Coding


 


331944
TGAGAGTTTGCAAATTGGCA
466
77
BC040261.1
234
Coding


 


331945
TTTTGAGAGTTTGCAAATTG
467
32
BC040261.1
237
Coding


 


331946
GGTTTTGAGAGTTTGCAAAT
468
63
BC040261.1
239
Coding


 


331947
CCAAAATACTACTAGCTCAC
469
75
BC040261.1
268
Coding


 


331948
TAAGTATACCTCATTCAGAA
470
41
BC040261.1
307
Coding


 


331949
TTTGCCTAAGTATACCTCAT
471
85
BC040261.1
313
Coding


 


331950
AATTTCTCTTTGCCTAAGTA
472
47
BC040261.1
321
Coding


 


331951
TCAAATTTCTCTTTGCCTAA
473
45
BC040261.1
324
Coding


 


331952
GTGCGGCCCATATACTTGGA
474
77
BC040261.1
354
Coding


 


331953
AAACTTGTGCGGCCCATATA
475
61
BC040261.1
360
Coding







 













331954
ATGATACATTGATACAAGCC
476
71
BC040261.1
432
Coding







 













331955
AGTTAGCAAGCACTGACAGT
477
36
BC040261.1
503
Coding


 


331956
AGGTTGACTGAAGTTAGCAA
478
56
BC040261.1
514
Coding







 













331957
TTTCAGGTTGACTGAAGTTA
479
54
BC040261.1
518
Coding







 













331958
CGTGTATAGATGAGCAGGTC
480
64
BC040261.1
581
Coding







 













331959
TAACCGTGTATAGATGAGCA
481
63
BC040261.1
585
Coding







 













331960
TCTGGGTAACCGTGTATAGA
482
75
BC040261.1
591
Coding







 













331961
TTGGTTCTTAGCAAAACACT
483
56
BC040261.1
624
Coding







 













331962
TGAATTCTTGGTTCTTAGCA
484
57
BC040261.1
631
Coding







 













331963
CTCGATAGTTGAATTCTTGG
485
37
BC040261.1
640
Coding







 













331964
ATACTCGATAGTTGAATTCT
486
33
BC040261.1
643
Coding







 













331965
ATCATACTCGATAGTTGAAT
487
72
BC040261.1
646
Coding







 













331966
GTGACATTATCTTGAGATTT
488
70
BC040261.1
678
Coding







 













331967
GGTCCTCAAGCTCTATAGAG
489
25
BC040261.1
812
Coding







 













331968
GAGGGTCCTCAAGCTCTATA
490
74
BC040261.1
815
Coding







 













331969
CTGAGGGTCCTCAAGCTCTA
491
66
BC040261.1
817
Coding







 













331970
GGCTGAGGGTCCTCAAGCTC
492
71
BC040261.1
819
Coding







 













331971
TTCCATAGAATTAGACAGAA
493
55
BC040261.1
903
Coding







 













331972
ATTTCCATAGAATTAGACAG
494
31
BC040261.1
905
Coding







 













331973
TCTTCCATTTCCATAGAATT
495
42
BC040261.1
911
Coding







 













331974
CACATTTATAAGAGTTGCGA
496
28
BC040261.1
944
Coding







 













331975
TCCACATTTATAAGAGTTGC
497
67
BC040261.1
946
Coding







 













331976
GTTCCACATTTATAAGAGTT
498
36
BC040261.1
948
Coding







 













331977
GTGTTGGTTCCACATTTATA
499
22
BC040261.1
954
Coding







 













331978
CATTGTGTTGGTTCCACATT
500
38
BC040261.1
958
Coding







 













331979
TCCATTGTGTTGGTTCCACA
501
76
BC040261.1
960
Coding







 













331980
TGTTCACTCTCTTCCCTCTC
502
70
BC040261.1
978
Coding







 













331981
TGAATACTTGTATGGGCTTT
503
62
BC040261.1
1116
3′ UTR







 













331982
GCAGCACCAGAGAGCAGGAA
504
48
BC040261.1
168
Coding







 













331983
GCTTCTTCTTCTTCCATTTC
505
32
BC040261.1
920
Coding







 













331984
ATAGTGCACTGGGGATCCAT
506
38
BC040261.1
117
Start Codon







 













331985
GCCCATATACTTGGAATGAA
507
84
BC040261.1
349
Coding







 













331986
GGCCCATATACTTGGAATGA
508
61
BC040261.1
350
Coding







 













331987
CGGCCCATATACTTGGAATG
509
69
BC040261.1
351
Coding







 













331988
GCGGCCCATATACTTGGAAT
510
49
BC040261.1
352
Coding







 













331989
TGCGGCCCATATACTTGGAA
511
74
BC040261.1
353
Coding







 













331990
TATCACTTTTGTCGCATGAA
512
71
BC040261.1
1076
Coding







 













331991
GTATCACTTTTGTCGCATGA
513
38
BC040261.1
1077
Coding







 













331992
TGTATCACTTTTGTCGCATG
514
87
BC040261.1
1078
Coding







 













331993
 CGGCCCATATACTTGGAA
515
52
5C040261.1
353
Coding







 













331994
 TATCACTTTTGTCGCATG
516
80
BC040261.1
1078
Coding







 













331995
 CCATATACTTGGAATGAA
517
75
BC040261.1
349
Coding







 













331996
 TCACTTTTGTCGCATGAA
518
90
BC040261.1
1076
Coding







 













331997
  GCCCATATACTTGGAA
519
46
BC040261.1
353
Coding







 













331998
  TCACTTTTGTCGCATG
520
46
BC040261.1
1078
Coding







 













331999
  CATATACTTGGAATGA
521
10
BC040261.1
350
Coding







 













332000
  CACTTTTGTCGCATGA
522
80
BC040261.1
1077
Coding







 













332001
   ATATACTTGGAATG
523
 0
BC040261.1
351
Coding







 













332002
   ACTTTTGTCGCATG
524
33
BC040261.1
1078
Coding







 













332003
TGCGGCCCATATACTTGGAA
525
43
BC040261.1
353
Coding







 













332004
TGTATCACTTTTGTCGCATG
526
74
BC040261.1
1078
Coding







 













335956
CAAAAGGAGTATTTGCGAGC
527
20
U04343.1
24
5′ UTR







 













335957
AACCAAAAGGAGTATTTGCG
528
14
U04343.1
27
5′ UTR







 













335958
CAAGGTGGTAAGAATAAACC
529
15
U04343.1
43
5′ UTR







 













335959
GAACACAGAAGCAAGGTGGT
530
26
U04343.1
54
5′ UTR







 













335960
AGCATTCCCAAGGAACACAG
531
17
U04343.1
66
5′ UTR







 













335961
CACAGCAGCATTCCCAAGGA
532
25
U04343.1
72
5′ UTR







 













335962
AAGAGACCAGATGCATAAGC
533
10
U04343.1
91
5′ UTR







 













335963
AGAAGCAAAGCTTTCACCCT
534
51
BC040261.1
14
5′ UTR







 













335964
CAGCAGAGAAGCAAAGCTTT
535
46
BC040261.1
20
5′ UTR







 













335965
TTACAGCAGCAGAGAAGCAA
536
20
BC040261.1
26
5′ UTR







 













335966
ACCCCACTCATCCGTGTGTC
537
86
BC040261.1
60
5′ UTR







 













335967
AATGACCCCACTCATCCGTG
538
32
BC040261.1
64
5′ UTR







 













335968
GGAAATGACCCCACTCATCC
539
45
BC040261.1
67
5′ UTR







 













335969
TCTGGAAATGACCCCACTCA
540
32
BC040261.1
70
5′ UTR







 













335970
ATATCTGGAAATGACCCCAC
541
71
BC040261.1
73
5′ UTR







 













335971
GACCTAATATCTGGAAATGA
542
28
BC040261.1
79
5′ UTR







 













335972
GGGATCCAITFTGGCTGCTT
543
27
BC040261.1
106
Start Codon







 













335973
AGGCCATCACAAAGAGAATG
544
18
BC040261.1
149
Coding







 













335974
ATCTTCAGAGGAGCAGCACC
545
36
BC040261.1
180
Coding







 













335975
CTTGAATCITCAGAGGAGCA
546
38
BC040261.1
185
Coding







 













335976
AAGCTTGAATCTTCAGAGGA
547
50
BC040261.1
188
Coding







 













335977
ATATACTTGGAATGAACACT
548
19
BC040261.1
345
Coding







 













335978
TTTTGTGATGGATGATACAT
549
17
BC040261.1
443
Coding







 













335979
GCTTTTTGTGATGGATGATA
550
41
BC040261.1
446
Coding







 













335980
AAATTTATGTACACATTTTC
551
58
BC040261.1
561
Coding







 













335981
CTTCTTAGGTTCTGGGTAAC
552
35
BC040261.1
601
Coding







 













335982
AAAACACTCATCTTCTTAGG
553
25
BC040261.1
612
Coding







 













335983
GCATAATACCATCATACTCG
554
43
BC040261.1
656
Coding







 













335984
TCTGCATAATACCATCATAC
555
66
BC040261.1
659
Coding







 













335985
GATTTTCTGCATAATACCAT
556
26
BC040261.1
664
Coding







 













335986
CTATAGAGAAAGGTGAAGAT
557
21
BC040261.1
800
Coding







 













335987
TCTATAGAGAAAGGTGAAGA
558
10
BC040261.1
801
Coding







 













335988
GTAATCCAAGGAATGTGGTC
559
52
BC040261.1
846
Coding







 













335989
GTTGCGAGGCCGCTTCTTCT
560
35
BC040261.1
931
Coding







 













335990
TAAGAGTTGCGAGGCCGCIT
561
42
BC040261.1
936
Coding







 













335991
AGATCTTTCAGGTATATGGA
562
24
BC040261.1
1018
Coding







 













335992
ATCAGATCTTTCAGGTATAT
563
29
BC040261.1
1021
Coding







 













335993
GGGTTTCATCAGATCTTTCA
723
56
BC040261.1
1028
Coding







 













335994
CACGCTGGGTTTCATCAGAT
564
43
BC040261.1
1034
Coding







 













335995
AAACACGCTGGGTTTCATCA
565
23
BC040261.1
1037
Coding







 













335996
CGAACTTTTAAAAACACGCT
566
62
BC040261.1
1048
Coding







 













335997
GTCTTCGAACTTTTAAAAAC
567
79
BC040261.1
1053
Coding







 













335998
AGATGTCTTCGAACTTTTAA
568
21
BC040261.1
1057
Coding







 













335999
CGCATGAAGATGTCTTCGAA
569
47
BC040261.1
1064
Coding







 













336000
TTGTCGCATGAAGATGTCTT
570
29
BC040261.1
1068
Coding







 













336001
AACATGTATCACTTTTGTCG
571
22
BC040261.1
1082
Coding







 













336002
AAAAACATGTATCACTTTTG
572
24
BC040261.1
1085
Coding







 













336003
CTCTTTAATTAAAAACATGT
573
21
BC040261.1
1095
Stop Codon







 













336004
ACTTACAAAGGAAAGGGTAG
574
16
BC040261.1
1142
3′ UTR







 













336005
TTGCCCAGGAACTTACAAAG
575
31
BC040261.1
1152
3′ UTR







 













336006
GCCTTCTGGAAGAAATCAAA
576
66
BC040261.1
1176
3′ UTR







 













336007
TGGGCTTGGCCCATAAGTGT
577
26
BC040261.1
1331
3′ UTR







 













336008
AGCCATTAAGCTGGGCTTGG
578
30
BC040261.1
1342
3′ UTR







 













336009
TTATTTCCAGGTCATGAGCC
579
69
BC040261.1
1358
3′ UTR







 













336010
AAGAATCTGATCTGGAGGAG
580
52
BC040261.1
1394
3′ UTR







 













336011
GATAAAAGGCAGTTTTCCAG
581
22
BC040261.1
1462
3′ UTR







 













336012
TGGGCAGATAAAAGGCAGTT
582
39
BC040261.1
1468
3′ UTR







 













336013
GCACCAGCTTAGAATTCTGG
583
48
BC040261.1
1485
3′ UTR







 













336014
CCCACCCAGACTGAGGAGGT
584
44
BC040261.1
1540
3′ UTR







 













336015
TATTAACACTATAAGGTCAT
585
 2
BC040261.1
1572
3′ UTR







 













336016
TAGATCTCTATGTTTCAAGA
586
38
BC040261.1
1592
3′ UTR







 













336017
ACTATTACAGTACATAGATC
587
47
BC040261.1
1606
3′ UTR







 













336018
AGAGCATAGTAATCACACTA
588
43
BC040261.1
1622
3′ UTR







 













336019
TCTCTAGAGCATAGTAATCA
589
39
BC040261.1
1627
3′ UTR







 













336020
TGACAGAGGGATGAGAACTC
590
43
BC040261.1
1666
3′ UTR







 













336021
TTTCCTTACTGACCCTGACA
591
34
BC040261.1
1681
3′ UTR







 













336022
AGGCCACCGTTTTCCTTACT
592
65
BC040261.1
1691
3′ UTR







 













336023
TTGGTCTGCTCATTGTTGCC
593
48
BC040261.1
1719
3′ UTR







 













336024
CAGCCCAGATAGAAGTGGCT
594
39
BC040261.1
1781
3′ UTR







 













336025
AGCAACAGCCCAGATAGAAG
595
17
BC040261.1
1786
3′ UTR







 













336026
AGTTCTTCAAGAGAAACAGG
596
 1
BC040261.1
1886
3′ UTR







 













336027
TAGTCAGTTCTTCAAGAGAA
597
 3
BC040261.1
1891
3′ UTR







 













336028
TGTTTCAAGACTTGTTGATG
598
 0
BC040261.1
2034
3′ UTR







 













336029
GTTGCTTGTTTCAAGACTTG
599
27
BC040261.1
2040
3′ UTR







 













336030
CTTGGGTATCACCAAAACCC
600
39
BC040261.1
2121
3′ UTR







 













336031
CCCAGCTCTGCTTCCAGGAA
601
45
BC040261.1
2157
3′ UTR







 













336032
CTCTCAGCCAGGAGTGGAAA
602
 0
BC040261.1
2228
3′ UTR







 













336033
AATTTGCTAGCCCTATGCTG
603
34
BC040261.1
2340
3′ UTR







 













336034
GAGCCAGGTTTTCAAAGTCA
604
59
BC040261.1
2433
3′ UTR







 













336035
GATCCCACCTTAGAGCCAGG
605
73
BC040261.1
2445
3′ UTR







 













336036
CCTTAGTGATCCCACCTTAG
606
49
BC040261.1
2452
3′ UTR







 













336037
CATCCCTTAGTGATCCCACC
607
61
BC040261.1
2456
3′ UTR







 













336038
ATGGGAATATTACATGGACA
608
39
BC040261.1
2587
3′ UTR







 













336039
CTAGCCAGGTGCTAGGAGTC
609
50
BC040261.1
2634
3′ UTR







 













336040
AAACTAGCCAGGTGCTAGGA
610
53
BC040261.1
2637
3′ UTR







 













336041
TTAGAAACTAGCCAGGTGCT
611
50
BC040261.1
2642
3′ UTR







 













336042
AACATGTTAGAAACTAGCCA
612
49
BC040261.1
2647
3′ UTR







 













336043
CCAGACAAATCATTTTGAAC
613
 0
NT_005543.12
245
intron







 













336044
CAGAGAAGCAAAGCTTTCAC
614
49
NT_005543.12
1017
Coding







 













336045
AGCAGCAGAGAAGCAAAGCT
615
23
NT_005543.12
1022
Coding







 













336046
AAGAATTATTACTCACCACT
616
33
NT_005543.12
1127
exon:intron







 













335047
CCCTCTATGGTACCACAAAA
617
64
NT_005543.12
2463
intron







 













336048
GAACAGACATTGTTTAATGG
618
31
NT_005543.12
2610
intron







 













336049
TCTTAGCAAGATAGAGGGTT
619
22
NT_005543.12
2730
intron







 













336050
AGCACAACAATTGTCTTGGA
620
61
NT_005543.12
5446
intron







 













336051
GGTTGTGAGGAAAGAATCTG
621
9
NT_005543.12
5561
intron







 













336052
TCTGGTCTGGATGGTGCAGA
622
30
NT_005543.12
9759
intron







 













336053
TCAATCAAGAGGTAACTACT
623
26
NT_005543.12
9923
intron







 













336054
CCTGCTCAGCAGAGCAATGT
624
15
NT_005543.12
10002
intron







 













336055
TCTTGAAATATAAGACTTGG
625
 4
NT_005543.12
10252
intron







 













336056
CTGAACCTCTGATGCATTAA
626
 0
NT_005543.12
10379
intron







 













336057
TCGCTCTGACTCACTAAAGC
627
30
NT_005543.12
10455
intron







 













336058
AAAGCTCTTAGATTCAAGCA
628
27
NT_005543.12
11268
intron







 













336059
AATCTCAGAACTTCTTCTGA
629
38
NT_005543.12
14386
intron







 













336060
TAAGTATAGCCTTCAGATGT
630
 0
NT_005543.12
15560
intron







 













336061
ATTTGCTTATATTAAAAGGT
631
23
NT_005543.12
15656
intron







 













33T062
GATTCTACAAGTGAATGCAG
632
13
NT_005543.12
15756
intron







 













336063
GTTCCACAAAGGGAGTAGAT
633
57
NT_005543.12
16658
intron







 













336064
CACAGTTCCTGAATTATTCT
634
42
NT_005543.12
19375
intron







 













336065
TGCAGGAAAGGAAGGAGACT
635
34
NT_005543.12
19577
intron







 













336066
AAAGCCCATGGAAGAGATGA
636
29
NT_005543.12
19972
intron







 













336067
TACCAGAACATATTATTTTA
637
22
NT_005543.12
21030
intron







 













336068
AGGCCATGTGCTTCACTTCT
638
29
NT_005543.12
23119
intron







 













336069
CCTTCCCTGACAGATTCCCA
639
11
NT_005543.12
23275
intron







 













336070
TGCACCAGTCCTACCCTGCT
640
42
NT_005543.12
25661
intron







 













336071
GTGCTAAGTAGGTGAAGGGC
641
27
NT_005543.12
29098
intron







 













336072
CTATCTCATACATTTGAAAA
642
 0
NT_005543.12
33910
intron







 













336073
ATAGATCATGGTACTAAATG
643
 0
NT_005543.12
35788
intron







 













336074
GATCTGGCTGCTAAAGTCTC
644
24
NT_005543.12
36943
intron







 













336075
CCCATAGTGCTAGAGTCGAG
645
55
NT_005543.12
37217
intron:exon







 













336076
TATGATGATTATACAATGAC
646
 0
NT_005543.12
39043
intron







 













336077
AGTTGTGCGGACTGGTGACA
647
 0
NT_005543.12
45144
intron







 













336078
AGTAATCATGCTGAAGAATA
648
 5
NT_005543.12
49023
intron







 













336079
ATGTGGGTGTCAAGGTCAGC
649
27
NT_005543.12
53243
intron







 













336080
AAAGTCCCAGGCTCAGGGCC
650
25
NT_005543.12
54727
intron







 













336081
ATGTCGCCTTTCTTTGAAGT
651
29
NT_005543.12
55223
intron







 













336082
TGTGTTGGTTCCTGAAAGAC
652
 6
NT_005543.12
63666
intron:exon







 













336083
TGGTCTCTGCTCTTCATAGG
653
53
NT_005543.12
64347
intron







 













336084
AAGCCTTCCATCCCATACCT
654
 0
NT_005543.12
20
intron







 













336088
TATTACTCACCACTGGGGAT
655
N.D
NT_005543.12
1121
exon:intron







 













336089
ACAAACATACCTGTCACAAA
656
N.D
NT_005543.12
23663
exon:intron







 













336090
CCGTACCTCCTAAGGCTCCT
657
N.D
U04343.1
1
5′ UTR







 













336091
TACGGGGGCGTACAGCCTGC
658
N.D
B1824940.1
1
5′ UTR







 













336092
TTGGCTGCTTCTGCTGTGAC
659
N.D
BC040261.1
96
5′ UTR







 













336093
TGTCCACTGTAGCTCCAAAA
660
N.D
U04343.1
110
5′ UTR







 













336094
TCACCCTGTGCAAGCCTTCC
661
N.D
BC040261.1
1
5′ UTR







 













336095
CCTAAATTTTATTTCCAGGT
662
N.D
U04343.1
1379
3′ UTR







 













336096
ACAAGCATTTATTAAAAACT
663
N.D
BC040261.1
2678
3′ UTR







 













336098
GACCCCACTCATCCGTGTGT
664
N.D
BC040261.1
61
5′ UTR







 













336101
GCATAAGCACAGCAGCATTCC
665
N.D
U04343.1
78
5′ UTR







 













336104
ACCAGAGAGCAGGAAGGCC
666
N.D
U04343.1
177
Coding









In one embodiment, the antisense compound is targeted to nucleobases 195-537 of a coding region of a nucleic acid encoding human B7.2 (SEQ ID NO: 296).
Example 30
Antisense Inhibition of Human B7.2 Expression: Dose Response
In accordance with the present invention, several oligonucleotides were tested in a dose response study. ISIS 323624 (SEQ ID NO: 374), ISIS 323641 (SEQ ID NO: 391), ISIS 323690 (SEQ ID NO: 440), ISIS 331941 (SEQ ID NO: 463), ISIS 331949 (SEQ ID NO: 471), ISIS 331985 (SEQ ID NO: 507), ISIS 331992 (SEQ ID NO: 514), ISIS 331996 (SEQ ID NO: 518) were identified as oligonucleotides with good activity based on the results in Examples 27 and 29.
ISIS 13650 (TCCCGCCTGTGACATGCATT, SEQ ID NO: 667) and ISIS 129700 (TAGTGCGGACCTACCCACGA, SEQ ID NO: 668), used as control oligonucleotides, 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′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
Cells were electroporated in 1 mm cuvettes at a density of 1×107 cells/ml, a voltage of 75 V and a pulse length of 6 ms with an oligonucleotide dose of 2.5, 5, 10 or 20 uM. After electroporation, cells wer treated with 1 ug/ml LPS and 0.5 mM dbcAMP for 16 hours to induce B7-2 expression. Control cells were stimulated with 1 ug/ml LPS and 0.5 mM dbcAMP, but not treated with oligonucleotides.
Target mRNA levels were determined by quantitative real-described time PCR as described in other examples herein, using two different primer-probe sets to confirm the results of the antisense igonucleotide treatment. The first primer probe set was 1608, consisting of a forward primer (SEQ ID NO: 445), reverse primer (SEQ ID NO: 446) and probe (SEQ ID NO: 447) described in Example 28. The second primer probe set was #1770, consisting of CGCATCCACCAGATGAATTCT (forward primer, SEQ ID NO: 669), AATTGGTACTATTTCAGGTTGACTGAAG (reverse primer, SEQ ID NO: 670) and AACTGTCAGTGCTTGCT (probe, SEQ ID NO: 671). The data are the average of two experiments and are presented in Table 27. The percent inhibition is expressed relative to the LPS/dbcAMP-stimulated cells that were not exposed to oligonucleotides.
The results demonstrate that the oligonucleotides of the present invention, unlike the control oligonucleotides, inhibit the expression of B7-2 in a dose-dependent manner.







TABLE 27







Antisense inhibition of B7-2 mRNA expression: dose response









% Inhibition of B7-2 mRNA



Primer-Probe set










1680
1770









Dose of oligonucleotide















ISIS #
2.5 uM
5 uM
10 uM
20 uM
2.5 uM
5 uM
10 uM
20 uM


















323624
5
17
41
63
9
22
47
62


323641
23
55
74
89
31
59
73
88


323690
38
55
71
84
35
58
71
84


331941
63
75
87
87
63
76
86
87


331949
27
41
63
77
26
45
65
76


331985
26
46
62
82
28
49
64
82


331992
48
62
79
88
50
65
79
87


331996
44
57
80
88
44
59
80
88


13650
0
4
4
3
0
8
8
7


129700
0
9
0
13
0
1
0
3









Example 31
Inhibition of B7-2 Protein Expression by Antisense Oligonucleotide Treatment: Dose Response
In accordance with the present invention, the effects of antisense oligonucleotide treatment on cell surface expression of B7-2 was evaluated in THP-1 cells. The cell surface expression of B7-1 was also measured as a test of the specificity of the B7-2 antisense oligonucleotides.
The oligonucleotides used for this study were ISIS 22300 (SEQ ID NO: 190), ISIS 113131 (SEQ ID NO: 255), ISIS 323641 (SEQ ID NO: 391), ISIS 323643 (SEQ ID NO: 393), ISIS 323653 (SEQ ID NO: 403), ISIS 323660 (SEQ ID NO: 410), ISIS 323661 (SEQ ID NO: 411), ISIS 323675 (SEQ ID NO: 425), ISIS 323690 (SEQ ID NO: 440). ISIS 115675 (SEQ ID NO: 672) and ISIS 129690 (SEQ ID NO: 673), used as control oligonucleotides, 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′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
THP-1 cells were electroporated in the presence of 2.5, 10, or 20 uM of antisense or control oligonucleotides. The cells were immediately treated with 1 ug/ml LPS and 0.5 mM dbcAMP for 42 hours to induce B7-2 expression. Control cells were stimulated with 1 ug/ml LPS and dbcAMP, but not exposed to oligonucleotides. Cells were harvested by brief trpsinization, washed with PBS, the resuspended in 100 uL of staining buffer (PBS, 0.2% BSA) containing both 10 uL of FITC-conjugated anti-B7-2 antibody (FITC-anti-hCD86; FITC:fluorescien isothiocyanate; BD Biosciences, San Jose, Calif.) and 10 ul of PE-conjugated anti-B7-1 antibody (PE:phycoerythrin; PE-anti-hCD80, BD Biosciences, San Jose, Calif.). The cells were stained for 30 minutes at 4° C., washed with PBS, resuspended in 300 uL PBS containing 0.5% paraformaldehyde. Measurements of mean fluorescence activity were made by flow cytometry using the FL-1 and FL-2 channels of a BD Bioscience FACScan (BD Biosciences, San Jose, Calif.). With this method, both B7-2 and B7-1 protein expression on the surface of the same cell was measured. The data are the average of two electroporation experiments and are presented in Table 28. The dat are expressed as percent inhibition of protein expression relative to LPS/dbcAMP-stimulated cells that were not exposed to oligonucleotides. The data illustrate that the oligonucleotides of the present invention can reduce cell surface expression of B7-2 protein in a dose dependent manner. Furthermore, the antisense inhibition is specific to B7-2, as the cell surface expression of B7-1 is not reduced.







TABLE 28







Inhibition of cell surface B7-2 protein expression with


antisense oligonucleotide treatment: dose response










% Inhibition of B7-2
% Inhibition of B7-1



expression
expression









Dose of oligonucleotide













ISIS #
2.5 uM
10 uM
20 uM
2.5 uM
10 uM
20 uM
















22300
2
46
63
0
0
0


113131
31
73
83
0
0
0


323641
53
85
92
0
0
0


323643
16
79
90
0
0
0


323653
29
62
83
0
0
0


323660
25
74
84
18
0
0


323661
38
72
80
9
0
0


323675
9
46
72
0
0
0


323690
46
85
92
0
0
0


115675
0
2
29
0
0
0


129690
0
15
0
0
0
0









Example 32
Double Stranded RNAs Targeted to B7.2
In a further embodiment of the present invention, a series of blunt-ended nucleic acid duplexes comprising the antisense compounds of the present invention and their complements was designed to target human B7-2 as described in Example 25, 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—005612.13, a portion of which is incorporated herein as SEQ ID NO: 674, GenBank accession number L25259.1, which is incorporated herein as SEQ ID NO: 675). The sequences of the antisense strands are listed in Table 29 The sense strand of the dsRNA was designed and synthesized as the complement of the antisense strand, making both strands of the dsRNA duplex complementary over the entire length of the oligomeric compound. Target sites are indicated by the first (5′ most) nucleotide number, as given in the sequence source reference (indicated by GenBank accession number) to which the oligonucleotide binds. All compounds in Table 29 are oligoribonucleotides, 20 nucleotides in length with phosphodiester internucleoside linkages (backbones) throughout.
RNA strands of the duplex are 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 uM. Once diluted, 30 uL of each strand is combined with 15 uL 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 uL. 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 uM. 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 B7-2 expression. When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium. (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.







TABLE 29







Design of double stranded oligomeric compounds targeting human B7-2












ISIS

SEQ
GenBank
Target



#
SEQUENCE
ID NO
Accession Number
Site
Region















341416
UUGUCGCAUGAAGAUGUCUU
570
BC040261.1
1068
Coding


 


341417
UUGGCAUACGGAGCAGAGCU
199
U04343.1
1281
3′ UTR


 


341418
GCUUGGCCCAUAAGUGUGCU
676
NT_005612.13
65758
3′ UTR


 


341419
GUCAUGAGCCAUUAAGCUGG
677
NT_005612.13
65778
3′ UTR


 


341420
CUGAUCUGGAGGAGGUAUUG
678
NT_005612.13
65818
3′ UTR


 


341421
UCCAGAAAUUGAGAGGUCUA
679
NT_005612.13
65878
3′ UTR


 


341422
GAGGUAGUUGUUUAGUCACA
680
NT_005612.13
65958
3′ UTR


 


341423
ACUAUAAGGUCAUAAAUACA
681
NT_005612.13
65998
3′ UTR


 


341424
CUAUGUUUCAAGAUAUUAAC
682
NT_005612.13
66018
3′ UTR


 


341425
CUAUUACAGUACAUAGAUCU
683
NT_005612.13
66038
3′ UTR


 


341426
UCUAGAGCAUAGUAAUCACA
684
NT_005612.13
66058
3′ UTR


 


341427
GACAGAGGGAUGAGAACUCC
685
NT_005612.13
66098
3′ UTR


 


341428
GUUGCCUGUACCCUAGGCCA
686
NT_005612.13
66138
3′ UTR


 


341429
UUUAGGUUGGUCUGCUCAUU
687
NT_005612.13
66158
3′ UTR


 


341430
GGCUCCAGGUUCUAUCUCUG
688
NT_005612.13
66198
3′ UTR


 


341431
GCAACAGCCCAGAUAGAAGU
689
NT_005612.13
66218
3′ UTR


 


341432
GGCAAGCCUCCUCAAUAUUA
690
NT_005612.13
66238
3′ UTR


 


341433
CACUAUGGCUUGUUGGGUGG
691
NT_005612.13
66258
3′ UTR


 


341434
CCUGUUUAUUCAGUUCUCUC
692
NT_005612.13
66278
3′ UTR


 


341435
CCAGGCCAUCUCACUAGUCA
693
NT_005612.13
66338
3′ UTR


 


341436
UUUGAGUAUUGUGAUCUCUU
694
NT_005612.13
66378
3′ UTR


 


341437
UCUGUGGAUCAAGAUCUCUC
695
NT_005612.13
66418
3′ UTR


 


341438
GACCAGACAUUUCAUGUAUU
696
NT_005612.13
66438
3′ UTR


 


341439
CAUCUGUUGCUUGUUUCAAG
697
NT_005612.13
66478
3′ UTR


 


341440
GUCCAUUUGGACAGACUAUC
698
NT_005612.13
66498
3′ UTR


 


341441
GAAACUGCUGUCUGUCUUAU
699
NT_005612.13
66518
3′ UTR


 


341442
AUAACUUGGGUAUCACCAAA
700
NT_005612.13
66558
3′ UTR


 


341443
UCCAGGAAGAUGACAUCCCA
701
NT_005612.13
66578
3′ UTR


 


341444
CCCAUUAUCAAGGUGAUGGC
702
NT_005612.13
66618
3′ UTR


 


341445
CUAAGCCUCCUUCCAUUCAU
703
NT_005612.13
66638
3′ UTR


 


341446
UCAGCCAGGAGUGGAUAGUC
704
NT_005612.13
66658
3′ UTR


 


341447
GUGUCUUGGUCUUCCUAAUU
705
NT_005612.13
66698
3′ UTR


 


341448
CCGUAGGACAUCUGUAGGCU
706
NT_005612.13
66738
3′ UTR


 


341449
ACUCAAAUUUGCUAGCCCUA
707
NT_005612.13
66778
3′ UTR


 


341450
CAAGUUUCCUCUGGUUGCCU
708
NT_005612.13
66818
3′ UTR


 


341451
GAUCCCACCUUAGAGCCAGG
605
BC040261.1
2445
3′ UTR


 


341452
AUAACCCUUGAGGGUUUACA
709
NT_005612.13
66978
3′ UTR


 


341453
CUUGUCUAGCAUGGCAGUCU
710
NT_005612.13
66998
3′ UTR


 


341454
UGCUAGGAGUCUAAUCAAGG
711
NT_005612.13
67058
3′ UTR


 


341494
UCACCCUGUGCAAGCCUUCC
661
BC040261.1
1
5′ UTR


 


341495
GCAGCAGAGAAGCAAAGCUU
712
NT_005612.13
1396
5′ UTR


 


341496
CUGUGCUAGUCCCUGUUACA
713
NT_005612.13
1416
5′ UTR


 


341497
GACCCCACUCAUCCGUGUGU
664
BC040261.1
61
5′ UTR


 


341498
GUGACCUAAUAUCUGGAAAU
306
BC040261.1
81
5′ UTR


 


341499
CCAUUUUGGCUGCUUCUGCU
714
NT_005612.13
1476
5′ UTR


 


341500
UCCCAUAGUGCACUGGGGAU
715
L25259.1
111
Coding


 


341501
AUUUGCGAGCUCCCCGUACC
716
NT_005612.13
23918
5′ UTR


 


341502
AAGGAGUAUUUGCGAGCUCC
717
NT_005612.13
23925
5′ UTR


 


341503
CACAGAAGCAAGGUGGUAAG
718
NT_005612.13
23955
5′ UTR


 


341504
GAACACAGAAGCAAGGUGGU
719
U04343.1
54
5′ UTR


 


341505
AGCACAGCAGCAUUCCCAAG
530
NT_005612.13
23978
5′ UTR


 


341506
AUGCAUAAGCACAGCAGCAU
720
NT_005612.13
23985
5′ UTR


 


341507
UGCCUGUCCACUGUAGCUCC
721
NT_005612.13
24018
5′ UTR


 


341508
UCCCAUAGUGCUGUCACAAA
722
U04343.1
134
Start Codon

## Claims

1. A compound comprising a chimeric gapmer oligonucleotide consisting of 20 to 50 linked nucleosides and having a nucleobase sequence comprising SEQ ID NO:463, wherein the chimeric gapmer oligonucleotide is a single-stranded oligonucleotide, and wherein said chimeric gapmer oligonucleotide is at least 90% complementary to SEQ ID NO:296 as measured over the entirety of said chimeric gapmer oligonucleotide, and wherein said chimeric gapmer oligonucleotide inhibits the expression of human B7-2 mRNA.

2. A compound comprising a chimeric gapmer oligonucleotide consisting of 20 linked nucleosides having SEQ ID NO:463, wherein the chimeric gapmer oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, and wherein each internucleoside linkage of said chimeric gapmer oligonucleotide is a phosphorothioate linkage, and wherein said chimeric gapmer oligonucleotide inhibits the expression of human B7-2 mRNA.

3. The compound of claim 1, wherein at least one internucleoside linkage is a modified internucleoside linkage.

4. The compound of claim 3, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

5. The compound of claim 1, wherein at least one nucleoside comprises a modified sugar, and wherein at least one modified sugar is a bicyclic sugar.

6. The compound of claim 1, wherein at least one nucleoside comprises a modified sugar, and wherein at least one modified sugar comprises a 2′-O-methoxyethyl or a 4′-(CH2)n-O-2′ bridge, wherein n is 1 or 2.

7. The compound of claim 1, wherein at least one nucleoside comprises a modified nucleobase.

8. The compound of claim 7, wherein the modified nucleobase is a 5-methylcytosine.

9. The compound of claim 1, wherein each cytosine of said chimeric gapmer oligonucleotide is a 5-methylcytosine.

10. The compound of claim 1, wherein said chimeric gapmer oligonucleotide is at least 95% complementary to SEQ ID NO:296 as measured over the entirety of said chimeric oligonucleotide.

11. The compound of claim 1, wherein said chimeric gapmer oligonucleotide is 100% complementary to SEQ ID NO:296 as measured over the entirety of said chimeric gapmer oligonucleotide.

12. The compound of claim 2, wherein at least one nucleoside comprises a modified nucleobase.

13. The compound of claim 12, wherein the modified nucleobase is a 5-methylcytosine.

14. The compound of claim 2, wherein each cytosine of said chimeric gapmer oligonucleotide is a 5-methylcytosine.

15. The compound of claim 2, wherein the chimeric gapmer oligonucleotide is a single-stranded oligonucleotide.