CN1242775A - G-rich oligo aptamers and methods of modulating immune response - Google Patents

Abstract

Aptamer oligonucleotides specifically bind to the DNA binding site of proteins such as Sp1 and Sp1-related proteins which regulate the genes which encode costimulatory molecules such as CD28 and cytokines such as IL-2 and GMCSF. The oligonucleotides compete with the DNA-binding sites of regulatory proteins which specifically regulate molecules to modulate T-cell activation. This serves to modulate gene expression by preventing transcription of the gene. Aptamers are administered to provide therapies for diseases which involve aberrant T-cell activation such as psoriasis, Type I (insulin-dependent) diabetes mellitus, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis), and septic shock.

Description

Be rich in the oligo aptamers of G and the method for adjustment immunne response

Invention field

Invention field is an immunology.

Background of invention

Be to reply by the abnormal immune due to the abnormal T cell-stimulating to cause much by T cell-mediated epiphytotics pathology and deterioration.It is believed that a lot of other diseases also are by due to the abnormal T cell-stimulating, described disease comprises I type (insulin-dependent) diabetes, thyroiditis, sarcoidosis, multiple sclerosis, autoimmunity uveitis, rheumatoid arthritis, systemic lupus erythematous, inflammatory bowel disease (CrohnShi and ulcerative colitis) and aplastic anemia.In addition, comprise that the cachectic multiple syndrome of septic shock and tumor inducing may relate to the increment generation of the lymphokine of t cell activation and genotoxic potential level.Normal t cell activation is also by providing the necessary signal of effective destruction " external source " donor tissue to mediate the repulsion of transplanted cells and organ.

Cause T cell proliferation and specific immunity to adjust independently signal of two of the genetic expression of cytokine and excretory T lymphocyte activator needs.First signal relates to by the antigen of the main histocompatibility complex molecular presentation on antigen presenting cell (APC) surface to be discerned by specific t-cell receptor/CD3 mixture.Antigen non-specific sexual cell interphase interaction between T cell and the APC provides second signal, and this signal can be used for regulating the T cell at antigenic reaction, described second or costimulatory signal determined the amount of T cell at antigenic reaction.Altogether irritation cell is by the level that increases the specific cell factor gene and transcribe with stablize selected mRNA and react.Lack activated T cell t cell responses that can lead to the failure or anergy when stimulating altogether.Main costimulatory signal provides (Linsley and Ledbetter (1993) Annu Rev Immunol 11:191-212) by the interaction of the B7 associated molecule on T cell surface receptor CD28 and the APC.CD28 is at 95%CD4 ⁺T cell (function of helper is provided for the generation of B cell antibody) and 50%CD8 ⁺The last constitutive expression of T cell (having the cell toxicant function) (Yamada etc. (1985), European Journal of Immunology, 15:1164-1168).After carrying out antigen or external short cell fission stimulation, further induced the CD28 of surface level and produced cytokine that some immunity is adjusted.Described cytokine comprises the interleukin II (IL-2) that T cell cycle progression is required, demonstrate the IFN-(IFN γ) of antiviral widely and antitumor action and be known as the interleukin 8 (IL-8) of neutrophil and lymphocytic strong chemokine.The CD28 approach of t cell activation demonstrates can regulate these cytokines (Fraser etc. (1991), science, 251:313-316, Seder etc. (1994), The Journal of Experimental Medicine, 179:299-304, Wechsler etc. (1994), Journal of Immunology, 153:2515-2523).IL-2, IFN γ and IL-8 promote that widely immunne response institute is requisite, demonstrated can be under the cell-mediated morbid state of a lot of T overexpression.

In psoriatic, the impaired T cell that is activated mainly discharges the Th1 cytokine, as IL-2 and IFN γ (Schlaak etc. (1994), chronic and refractory tetter magazine, 102:145-149).These are expressed and identical phenotype (HLA DR seen in the psoriatic infringement by the normal keratinocyte of excretory cytokine induction ⁺/ ICAM-1 ⁺) (Baadsgaard etc. (1990), chronic and refractory tetter magazine, 95:275-282).Because proinflammatory characteristic in the external and body of IL-8, again because the T cell that is activated of IL-8 and the keratinocyte of psoriatic infringement place secrete in a large number, therefore, IL-8 is considered to that pathology change seen in psoriatic's skin, as the major cause of the excessive propagation of keratinocyte.In addition, a member BB1 of acceptor B7 family (activated APC goes up the CD28 native ligand of finding) demonstrates can be at psoriatic's expression in vivo, but can form cell to skin keratin and impact (Nickoloff etc. (1993), American Journal of Pathology, 142:1029-1040), emphasized the importance of t cell activation in disease pathology.

In the cell-mediated tetter of other T, in allergic contact dermatitis and lichen planus, most of corium and epidermis CD3 ⁺In the T cell high level expression CD28, but in normal skin and rodent cancer (tetter of non-T cell mediation), CD28 only expresses in blood vessel week T cell.Similarly, in allergic contact dermatitis and lichen planus, the corium dendritic cell, observing B7 on corium APC and the keratinocyte expresses, and do not find that in normal skin and rodent cancer B7 expresses (Simon etc. (1994), chronic and refractory tetter magazine 103:539-543), therefore shows that the CD28/B7 approach is the cell-mediated dermopathic important amboceptor of T.

The principal character of the abnormal T cell-stimulating relevant with some autoimmune disease that is caused by the self tolerance forfeiture is CD28 ⁺The existence of T cell and part B7 thereof the expression on the specialized APC of activated (monocyte, scavenger cell or dendritic cell).Described disease comprises autoimmune Exophthalmus goiter (Garcia-Cozar etc. (1993), Immunologia 12 32), sarcoidosis (Vandenberghe etc. (1993) Int Lmmunol 5:317-321), rheumatoid arthritis (Verwilghen etc. (1994), Journal of Immunology, 153:1378-1385) and systemic lupus erythematous (Sfikakis etc. (1994), the clinical experiment immunology, 96:8-14).In mediation transplanted cells and organ rejection's normal t cell activation, CD28 in the TXi Baoshouti cohesive process by it suitable B7 part in conjunction with for for the suitable allotype reaction of the exogenous antigen on the donor tissue for example, being vital (Azuma etc. (1992), The Journal of Experimental Medicine, 175:353-360, Turka etc. (1992), Proc Nat Acad Sci USA, 89:11102-11105).

The traditional remedies of autoimmune disease does not stop t cell activation; Described activation is at the effect step in the autoreactivity immunne response of autoantigen.At present, use medicine to improve symptom, but these medicines can not stop advancing of disease as steroid and on-steroidal anti-inflammatory medicaments (NSAID).In addition, steroid may have side effect, as induces osteoporosis, organ toxicity and diabetes, and can quicken cartilage sex change process and cause the state of an illness aggravation of so-called injection back to reach 2 to 8 hours.NSAID has gastrointestinal side effect and can increase the risk of suffering from granulopenia and iatrogenic hepatitis.

Immunosuppressive drug also can be used as the therapy of another kind of form, and is particularly all the more so in stage terminal stage of a disease.Yet these medicines have suppressed whole immunity system, and regular treatment has severe side effect, comprise hypertension and pnehrotoxicity.Immunosuppressor of having set up such as S-Neoral and FK506 can not suppress CD28-dependent form t cell activation approach (June etc. (1987), molecular cytobiology, 7:4472-4481).

The up-to-date medicine that influences t cell activation comprises synthetic peptide, the t cell activation molecule of monoclonal antibody and soluble form.Do not identify as yet up to now at the t cell activation molecule, as CD28, the competitiveness of CD40L and CAM family adhesion molecule is synthesized peptide.Monoclonal antibody (mAb) demonstrates may have therapeutic action as psoriatic this T cell mediated diseases (anti--CD4 (Prinz etc. (1994), lancet, 338:320-321)), and the normal t cell activation of immunosuppression (resists-VCAM-1 and VLA-4 (Isobe etc. (1994) in allotransplantation, Journal of Immunology, 153:5810-5818)).Yet the host animal of accepting chronicity treatment can produce the antibody at monoclonal antibody, thereby has limited the purposes of monoclonal antibody.Develop " humanization " monoclonal antibody, it can obviously reduce the risk of inducing at the immunne response of these mAb, yet, these " humanization " monoclonal antibodies are still in the middle of development, in addition, these new mAb are larger protein, therefore are difficult to arrive its target site.Developed the t cell activation molecule of soluble form, as CTLA-4Ig, it contains the people CTLA-4 gene extracellular region territory (result and CD28 are relevant) of merging with people IgC γ chain.CTLA-4Ig demonstrates can be by preventing to occur xenogenesis (Lenschow etc. (1992) in the rat heart allotransplantation, science, 257:789-792) and allogeneic (Turka etc. (1992), Proc Nat AcadSci USA, 89:11102-11105) repel to come the normal t cell activation of specific inhibition, and to the abnormal T cell-stimulating, have therapeutic action (Nishikawa etc. (1994) as the t cell activation of in rat autoimmunity glomerulonephritis, finding, the Europe Journal of Immunology, 24:1249-1254).Yet except the height cost of its production, solubility CTLA-4Ig has the limitation similar to monoclonal antibody.In addition, the definite function of this CD28 quasi-molecule is still unknown, therefore, needs to measure the definite function of described molecule before any therapeutic action of assessment comprehensively.

The non-reacted prolongation or the disappearance that CD28 are caused activated T cell in the inhibition of cell surface expression.Inactivation can prevent that T cell proliferation and suppressor T cell from producing specific immunomodulating cytokines specifically, as interleukin II, and IFN-and interleukin 8.

Use the oligonucleotide with forming triplex of antisense can regulate CD28 genetic expression, described triplex is by (patent CD28 Tam) with DNA in oligodeoxyribonucleotide or oligoribonucleotide and CD28 gene or the promoter region or the formation of RNA sequence hybridization.Oligonucleotide has avoided being used to blocking a lot of danger that the up-to-date medicine of normal and the effect of abnormal T cell-stimulating is hidden.Yet, be designed to the Degradation sensitivity of these oligonucleotide of antisense strategy for the nuclease that exists in nuclease in the born of the same parents or the born of the same parents' external environment.

Proved before that DNA (or RNA) and combination of proteins were crucial approach, this approach may command genetic transcription.These regulate albumen or transcription factor is discerned the dna sequence dna with specific secondary structure, and consequential interaction can cause the plus or minus control of genetic expression.Regulon (aptamer) is the oligonucleotide sequence of the proteinic weak point of energy specificity binding specificity.Illustrated different regulon (aptameric) but the sequence specificity in conjunction with different protein, but as sequence GGNNGG specificity bind thrombin, N=guanosine (G) wherein, cytosine(Cyt) (C), adenosine (A) or thymidine (T) (Bock etc. (1992), nature, 355:564-566 and patent #5582981 (1996) Toole etc.).

Yet still unmanned the description overregulated subsequence, and the competitive inhibitor that described sequence can be used as the DNA binding site works, and described site is positioned on the adjusting albumen that is known as transcription factor.To be a class combine the protein of regulating described gene by the specificity adjusting sequence in main and the gene 5 ' upstream promoter zone to transcription factor, and this interaction causes transcription initiation.As Sp1, AP2, AP-1, some transcription factor of EGR-1 and NFkB is to activate T and bone-marrow-derived lymphocyte necessary (Skerka etc., biology and The Chemicals, 270:22500-22506, Jung etc. (1995), Ann NY Acad Sci 766:245-252).In some cases, these transcription factors are induced (Jung etc. (1995) by the signal that stimulates the back to start altogether, Ann N Y Acad Sci 766:245-252), therefore, still need to develop interactional medicine and the method that to disturb protein and specific DNA binding site, described interaction can cause some immunization route, comprises that common stimulation approach is suppressed.

The invention summary

According to the present invention, the regulon oligonucleotide is provided, described oligonucleotide be designed to specificity in conjunction with as the protein DNA binding site of Sp1 and Sp1 associated protein, described protein can be regulated and encode as the costimulatory molecules of CD28 with as the gene of the cytokine of IL-2 and GMCSF.

In preferred embodiments, oligonucleotide is designed to regulate protein binding with the specificity as Sp1 and Sp1 associated protein, and combines with the promoter region of the gene that is subjected to described transcription factor control with these transcription factor competitions.It adjusts genetic expression by stoping genetic transcription, and therefore, the regulon oligonucleotide can suppress the function of RNA or DNA, suppresses it and translates into protein, is transported in the kytoplasm or suppresses its all biological and learn necessary any other activity of function.RNA or DNA can not exercise its all or part of function and can cause the genome of part control t cell activation suitably not expressed, and therefore can adjust described metabolism.

Preferably with regulon nucleic acid phantom target as target with regulate proteic DNA binding site competition, but described adjusting albumen specificity is regulated the molecule that can adjust t cell activation.Found that CD28 albumen is particularly useful to this method.The inhibition of CD28 and CD28 related gene expression is expected to can be used for treating psoriatic and other tetter, abnormal T cell-stimulating syndrome, autoimmune disease and allograft rejection.

The present invention also provides the method for adjusting t cell activation, and described method comprises patient contact with oligonucleotide that described oligonucleotide is competed suppressing the known protein expression that is conditioned that can adjust t cell activation with the proteic DNA binding site of adjusting.Preferably with regulate the protein that CD28 and CD28 genes involved are transcribed, as Sp1 and Sp1 associated protein bonded oligonucleotide.

In another aspect of this invention, use regulon physics to be provided and for example to regulate normal t cell activation in the allograft rejection, described disease comprises the abnormal T cell-stimulating, as psoriatic, psoriatic and other tetter of AIDS-aggravation, I type (insulin-dependent) diabetes, thyroiditis, sarcoidosis, multiple sclerosis, the autoimmunity uveitis, rheumatoid arthritis, systemic lupus erythematous, inflammatory bowel disease (CrohnShi and ulcerative colitis), septic shock, the emaciation of tumor inducing and aplastic anemia.Synthetic and the expression that comprises the t cell activation molecule of CD28 and CD28 associated molecule by upset can be accomplished this point.

The present invention provides regulon on the other hand, described regulon can be regulated protein binding with the specificity as Sp1 and Sp1 associated protein, therefore can suppress gene transcription as CD28 and CD28 associated protein, CD28 and CD28 associated protein can b) can be adjusted t cell responses a) by described adjusting albumen normal regulating.

The accompanying drawing summary

Figure 1A and 1B illustrate respectively in extracellular fluid body and the Jurkat cell ³²The thiophosphoric acid of P-mark, the vitro stability of ICN 16064 (Seq#4).Fig. 1 C and 1D illustrate respectively in extracellular fluid body and the Jurkat cell ³²The thiophosphoric acid of P-mark, the vitro stability of ICN 16214 (Seq#21).

Fig. 1 E and 1F illustrate by electrophoresis on 20% polyacrylamide denaturant gel, then use PhosphorImager to observe each oligonucleotide ICN 16064 (Seq#4) and the ICN16214 (Seq#21) that assesses out, time-dependent degraded (0-96 hour) (2000cpm).With respect to t=0, mensuration is passed the 10000cpm born of the same parents outer (Fig. 1 E) of Nickspin post (Pharmacia) and the complete total length that cell (Fig. 1 F) elutriant keeps at each time point ³²P-RT03S (o) and ³²The per-cent of P-RTC06S ().While analyzing molecules amount standard (Std), ³²P-dNTP (N) and without ³²The ortho-phosphoric acid of P mark (P).

Fig. 2 illustrates gel mobility shift assay, this analysis is illustrated after HeLa nucleus extract is incubated, and contains band A that the oligonucleotide (swimming lane 5 and 11) of the 12 aggressiveness sequence motifs that are rich in G presents and moves different with electrophoresis with the observed B of being with of other phosphorothioate oligonucleotide.Band C only is ³²The P-oligonucleotide.

Fig. 3 illustrates the cell with plamid vector transfection Jurkat, then with and handle without phosphorothioate oligonucleotide ICN16064 and ICN16481 after the expression of E.C. 2.3.1.28 (CAT), described plasmid vector contains the 226bp of CD28 promoter region in CAT reporter gene upstream inset (residue-197 is to+28) (28b) or residue-51 to-22 mutant that replaced by table 1 Seq#3 (28h-1).

Fig. 4 illustrates the super displacement of gel and analyzes, and this analysis demonstrates combining of Sp1 and 28b, and the upstream region of CD28 gene-197 is to the+28th, and is specific.

Fig. 5 illustrate Sp1 with ³²The combination of the double-stranded few 28b of P-mark (derived from parental generation 28b-Seq#1 table 1) and the few 28b of cold two strands combine with the competitiveness of regulon oligonucleotide FIGURE 1A and 16481.

The detailed description of specific embodiments

With such as the DNA binding site specific binding of the adjusting albumen of Sp1 and Sp1 GAP-associated protein GAP Regulate sub-oligonucleotides and can stop the specific double-strand of regulating in albumen and the required gene promoter region DNA zone combination. Can stop genetic transcription by regulating sub-competitive binding, thereby suppress the heredity letter Breath flows to protein from DNA. Its characteristic that is specific to its target that makes of oligonucleotides also can be fit to Multiple use. Because oligonucleotides is the long-chain of 4 monomer unit, therefore be easy to for any target RNA sequence synthetic oligonucleotide.

In a lot of models and vitro system, prove by oligonucleotide mediated pressing down gene expression System, described inhibition is the (Uhlmann that have treatment to render a service as the New Policy of a lot of human diseases for the treatment of And Peyman (1990), chemistry comment, 90:544-584, Zon and Stec (1991), few nuclear Thuja acid and analog-method of operating: 87-108, Miller etc. (1981), biochemistry, 20:1874-1880, Orson etc. (1991), nucleic acids research, 19:3435-3441, Helene and Toulme (1990) Biochem Biophys Acta 1049:99-125, Thierry and Dritschilo (1992), nucleic acids research, 20:5691-5698). Because showing cellular uptake increases Strong, to the oligonucleotides that nuclease has resistance, comprise D2EHDTPA (Zon and Stec (1991), Oligonucleotides and analog-method of operating: 87-108) and D2EHDTPA-3 ' isopropanolamine (Tam etc. (1994), nucleic acids research, 22:977-986) synthetic latest developments can be considered the widow is examined at present Thuja acid is as the therapeutic agent of new model. Target is regulated the sub-oligonucleotides of the adjusting generation of protein binding site Show another kind of compound based on nucleic acid, they provide for the difficult problem that the method for prior art runs into Desirable solution. They participate in the adjustment of expression of specific gene directly, therefore cut off target Protein expression, but it does not participate in soluble recepter to the competitiveness inhibition of target protein, institute Stating interaction need to be to the fully understanding of binding mechanism and the affinity of receptor-ligand binding Power. Oligonucleotides is little molecule, therefore can not run into the space problem identical such as big molecule inhibitor. The description of target

The target of this paper expectation comprises and can be regulated by transcription factor, initial or keep in the immune response and rise The molecule of important function, these molecules comprise such as the costimulatory molecules of CD28 with such as IL-2, GM-CSF Cell factor with IFN γ.

As for therapeutic agent, can treat the disease of suffering under a cloud by using oligonucleotides of the present invention Animal, described disease can be by reducing the table such as the costimulatory molecules of CD28 or CD28 correlation molecule Reach to treat. Oligonucleotides can be formulated into pharmaceutical composition, except oligonucleotides, and described medicine Also can comprise carrier in the compositions, thickener, diluent, buffer, anticorrisive agent, live in the surface The property agent, liposome or liquid preparation etc. Except oligonucleotides, also can wrap in the pharmaceutical composition Draw together one or more active components, such as antimicrobial, antiphlogistic, anesthetic etc.

Pharmaceutical composition is administration in many ways, and this depends on needs part or whole body therapeutic, Zone with the need treatment. Administration can be local (comprise retina, vagina is in the rectum, nose), Oral, by suction, or parenterai administration, for example by intravenous drip, subcutaneous, in the peritonaeum Or intramuscular injection administration.

The preparation of topical can comprise ointment, washing lotion, missible oil, gel, drops, suppository, spray Mist agent, liquid and powder. Conventional pharmaceutical carrier, the aqueous solution, powder or oil base, thickener etc. Deng being essential or needing. It also is useful having by sheath or gloves.

Liquid preparations for oral administration comprises powder or particulate, water-soluble or suspension or solution, capsule, sachet or the tablet in the water-bearing media not.Thickening material, seasonings, thinner, emulsifying agent, dispersing auxiliary or wedding agent also are desirable.

The preparation that is used for parenterai administration comprises and contains damping fluid, the sterile aqueous solution of liposome thinner and other suitable additive.

Dosage depends on severity of disease and the reaction that needs treatment, but general every day potion or multi-agent, therapeutic process can extend to some months from several days or until curing or the morbid state disappearance.Those skilled in the art are easy to determine dose,optimum, method of administration and repetition rate.

In preferred whole body was used, used regulon once with the dosage intravenously of 5mg/kg every day.In preferred topical application, the regulon solution of using 1-5% every day once.In preferred lung was used, the regulon of using 5mg spraying dosage every day once.

The present invention uses the regulon oligonucleotide to be used to suppress to adjust the function of proteinic corresponding RNA of t cell activation and DNA.In the context of the present invention, term " oligonucleotide " refers to the oligomer or the polymer of Yeast Nucleic Acid or thymus nucleic acid.This term comprises by natural base, oligomer that sugar and a sugar (skeleton) Lian Jian form and the oligomer with intimate non-natural part.Compare with the oligonucleotide of natural form, the often more preferably this modified or oligonucleotide that replaces, for example cellular uptake strengthens and the characteristic of stability increase in the presence of nuclease because the latter has.

Oligonucleotide of the present invention preferably contains has an appointment 3 to about 50 nucleic acid base units, and more preferably this oligonucleotide contains has an appointment 8 to about 30 nucleic acid base units, and more preferably this oligonucleotide contains has an appointment 12 to about 22 nucleic acid base units.Should understand that nucleic acid base unit connects the suitable bonded base of key and adjacent nucleic acid base unit-sugar combination by phosphodiester bond or other.

Can be easily and prepare oligonucleotide used among the present invention routinely by well-known solid phase synthesis technique.The several manufacturers that comprise Applied Biosystems sell this synthesizer, also can use any other to be used for this synthetic device, yet the reality of oligonucleotide is synthetic to be well-known to those skilled in the art.Using similar techniques to prepare other oligonucleotide, also is well-known as thiophosphoric acid and 3 ' amine-thiophosphoric acid.

According to the present invention, it will be understood by those skilled in the art that:the messenger RNA (mRNA) that the open reading frame (ORF) by DNA is identified (transcribed obtain by above-mentioned DNA) not only comprises the information of ORF among the DNA, comprise that also forming above-noted persons is known as 5 ' untranslated, the relevant ribonucleotide in the zone in 3 ' untranslated zone and intervening sequence ribonucleotide.Therefore, can prepare oligonucleotide according to the present invention, described oligonucleotide is these relevant ribonucleotides of target and information ribonucleotide wholly or in part.In preferred embodiments, the regulon oligonucleotide with as the proteic DNA binding site of the adjusting of Sp1 and Spl-associated protein interact, thereby blocking-up is encoded and is related to the proteinic genetic expression of t cell activation.In preferred embodiments, the described protein of being regulated is CD28 and all homologues of CD28 molecule.The preferred contained sequence of oligonucleotide contains at least two zones of being rich in G, and the zone of being rich in G is meant the 4 Nucleotide zones of containing 3 guanosines (G) residue at least, as GGGG, and GNGG, GGNG, wherein N=A, C, G, U or T.By 6 residues at the most, the all or part of useful preferred sequence section of be preferably 4 or still less the zone of two this G of being rich in separating of residue be useful in the present invention. is: be accurate although sequence shown in 5 ' 3 ' the SEQ IDTTG GAG GGG GTG GTG GGG FIGURE 1AGGG GAG GAG GGG CTG GAA ICN 16481GGG GTG GTG GGG ICN 16525TTG GAG GGG GAG GAG GGG ICN 16475TTG GAG GGG GAG GTG GGG ICN 16479GGG TTG GAG GGG GTG GTG GGG ICN 16065 it is believed that, what the present invention relates to is to find wrong correct sequence. Can be used for oligonucleotide of the present invention and contain in these sequences one, or its part, therefore, shown in the preferred use above in these oligonucleotide any, or those skilled in the art adjust the t cell activation molecule according to being used to, and comprise any similar oligonucleotide that the relevant knowledge of CD28 and the preferred oligonucleotide target of CD28-associated molecule synthetic can prepare.The inhibition that expection CD28 and/or CD28-homologue produce or be adjusted at treatment disease aspect and have notable therapeutic effect.For the effectiveness of evaluation group compound, need test or a series of test.

The embodiment oligonucleotide

(394 types AppliedBiosystems) go up synthetic oligodeoxynucleotidecombination to the phosphoramidite chemistry of use standard at automatic dna synthesizer.β-cyanoethyl phosphoramidite, synthetic agent and CPG polystyrene columns available from Applied Biosystems (ABI, Foster City, CA), 3 ' Amino-Modifier C3 CPG post available from Glen Research (Sterling, VA).As for phosphorothioate oligonucleotide, the oxygen cylinder with tetraethylthiuram disulfide/acetonitrile replacement standard uses the ABI thiophosphoric acid program of standard progressively to add phosphorothioate bond.After cracking is got off from the controlled pore glass post,, oligonucleotide is handled 8 hours to remove blocking group with strong aqua by in 55 ℃.By using the anti-phase C8 post (ABI) that partly prepares to carry out HPLC and come purification of oligonucleotides.After the cracking of DMT blocking group, use 80% acetic acid treatment, again through ethanol sedimentation, (Beckman, Fullerton CA) carry out HPLC and assess degree of purity of production the C18 post by operational analysis.All oligonucleotide of purity＞90% are refrigerated to dried, with aseptic deionized water (ICN, Costa Mesa) reprovision oligonucleotide, with oligonucleotide concentration adjustment to 400 μ M, five equilibrium also is stored in-20C after OD260nm estimates before the experiment.In all cases, listed various oligonucleotide at least 3 batches of tables 1 have been used.The oligonucleotide in vitro stability study

(Tam etc. (1994), nucleic acids research 22:977-986) describedly carry out the analysis of oligonucleotide time stability to press document.Assess oligonucleotide degraded distribution plan and use the Nickspin post quantitative by electrophoresis.Clone and T cell purification

The blood of 60ml healthy donors is carried out after the Ficoll-Hypaque density gradient centrifugation, from yellowish chromatograph, separate and obtain peripheral blood lymphocytes (PBMC), use the Lymphokwik lymphocyte separation agent (LK-25T that is specific to the T cell then, One Lambda, Canoga Park CA) purifying T cell from PBMC, (contain 20mM HEPES damping fluid in 37 ℃ at 20-30ml RPMI-AP5 then, pH7.4,5% autologous plasma, the 1%L-glutamine, be 40-60 * 10 with mean yield in the RPMI-1640 substratum of 1% penicillin/streptomycin and 0.05%2-mercaptoethanol (ICN, Costa Mesa, CA)) ⁶Individual T cell incubated overnight is to remove the adhesive cell of any pollution.In all experiments, with RPMI-AP5 washing T cell, then with 2-3 * 10 ⁶The density of individual cell/ml is tiled on the 96 hole microtiter plates.

(contain 20mM HEPES damping fluid, pH7.4,10% foetal calf serum (FCS) (Hyclone at RPMI-10, Logan, UT), keep t cell lymphoma clone, Jurkat E6-1 (CD28 the RPMI-1640 substratum of 1%L-glutamine and 1% penicillin/streptomycin) ⁺/ CD4 ⁺) cell (152-TIB).Mitogen inductive t cell activation and oligonucleotide are handled

Adding human peripheral T cell or t cell lymphoma clone (0.2-0.3 * 10 ⁶) before, (San Diego CA) wraps by double 96 hole microtiter plates in advance for clone HIT3a, Pharmingen, and with cold phosphate buffered saline (PBS), pH7.4 (PBS) washed twice with the CD 3-resisting monoclonal antibody (mAb) (6.25-200ng/ hole) of purifying.By adding 2ng phorbol myristate acetate (PMA) (Calbiochem, La Jolla, CA) further activated T cell, and in 37 ℃ of insulations 48 hours.Handle anti--CD3/PMA-activated T cells with 1-20 μ M CD28-specific oligonucleotide and control oligonucleotide after activating at once, handle once more after 24 hours.Use the T cell in the revision test culture plate to carry out immunofluorescence analysis, 1A is used for cytokine research, and second culture plate is used for the T analysis of cell proliferation.Immunofluorescence research

After the activation, the 150 μ l cell conditioned medium liquid that derive from first revision test microtest plate are transferred to the production of cytokines that derives from cell in another microtest plate with analysis.Ooze salts solution with waiting, (Becton Dickinson, Mansfield MA) with all the other cell washings twice, and are suspended in 50 μ l etc. and ooze in the salts solution pH7.4 again, are divided into two duplicate samples again.With the PE-CI28/FITC-CD4 mAb sample aliquot that dyes altogether, assess non-specific fluorescence by using through second part of aliquots containig of isotype paired contrast monoclonal antibody dyeing of PE/FITC mark.All monoclonal antibodies through fluorescence-mark derive from Becton Dickinson (Son Jose, CA).In 4 ℃, use saturated mAb concentration insulation 45 minutes in the dark.Before FACScan flow cytometer (Becton Dickinson) analysis, use the PBS washing to remove uncorporated marker.Measure antigen density and be expressed as the average channel (MCF) of fluorescence in the viable cell indirect of gate.By from CD28 ^-CD4 ^-Deduct CD28 among the MCF of cell ⁺CD4 ⁺The MCF of cell can measure the painted CD4 through CD28 mAb ⁺The surface expression of cell subsets.In a plurality of donors, by all oligonucleotide are measured the undressed cells of contrast with vital dye iodate third ingot (final concentration is 5 μ g/ml) dyeing and in batches through the viability of the acid-treated cell of oligonucleoside.With dosage range is after whole oligonucleotide of all batches of 1-20 μ M are handled, and discharges the per-cent of the viable cell of iodate third ingot by cells were tested by flow cytometry, and described per-cent is＞90% (scope is 90-99%).Cytokine analysis

Mensuration derives from the human cell factor concentration that derives from cell in the cell conditioned medium liquid of first revision test microtest plate.Use commercially available ELISA test kit (R﹠amp; D systems Quantikine test kit, Minneapolis, MN) mensuration is through the variation of mitogen inductive interleukin II (IL-2) level.All ELISA results represent with pg/ml.Electrophoretic mobility shift assay (EMSA)

According to the scheme of manufacturer use the T4 polynucleotide kinase (Gibco BRL, Gaithersburg, MD), with [γ- ³²P]-(CA) mark is tried 5 ' end of oligonucleotide to ATP for ICN, Costa Mesa.Under the room temperature, with about 80,000cpm is incubated 20 minutes together through the oligonucleotide of mark with 10 μ g HeLa nucleus extracts (Promega).The association reaction mixture contains 10mM Tris-HCl (pH7.5), 50mM NaCl, 0.5mM DTT, 0.5mM EDTA, 1mM MgCl ₂, 4% glycerine and 0.5 μ g poly (dI.dC).In 100V, about 3 hours of electrophoresis is to differentiate the DNA-protein complex on 4% polyacrylamide gel that contains 0.5 * tbe buffer liquid (50mM Tris, 45mM boric acid, 0.5mM EDTA).Desiccant gel also uses PhosphorImager (Biorad, Richmond CA) carries out radioactive automatic developing.Be DNA enzyme footprint and gel displacement test preparation cDNA

Protein-DNA bonded the cDNA (about 300 base pairs) that is used for DNA enzyme footprint and gel displacement test separates the 28b from plasmid pCAT3e, pCAT3e 28h or pCAT3e28h-1.Digest the various plasmids of 60 μ g with BglII, some of them are at first placed on the sepharose to check linearity, then remaining being carried out phenol/sevag extracts, ethanol sedimentation, again suspend in water again and with SacI digestion, again sub-fraction is placed on the gel electrophoresis to check whether plasmid is cut (should occur the band of 2 bands: 4kb and the band of 300bp at present), remaining is carried out phenol/sevag extract ethanol sedimentation.In order to carry out following dephosphorylation, the DNA throw out is suspended in the less water (62 μ l) again, (BoehringerMannheim, Indianapolis is IN) with 7 μ l, 10 * reaction buffer to add 1 μ l 20U/ μ l alkaline phosphatase.In 37 ℃ with reaction mixture insulation 1 hour after, add 7 μ l pH8.0,0.2M EGTA, in 65 ℃ whole test tube was heated 10 minutes, (Santa Clarita, Qiaquick gel extraction kit CA) places on 1% sepharose band with purifying 300bp with the dephosphorylized DNA of whole 77 μ l available from Qiagen in use.The cumulative volume of purified 300bp band is 70 μ l, its concentration calculated as described below: the μ g of 60 μ g * (300bp/4300bp)=4.2, suppose that the rate of recovery after all these operations is 50%:2.1 μ g/70 μ l=30ng/ μ l.For each warp ³²The 300bp DNA of 5 μ l to 7 μ l purifying has been used in the end-labelled reaction of P (having used kinases).Be gel displacement test preparation polyacrylamide gel

According to 4% non-denaturing polyacrylamide gel solution (4% acrylamide, 0.05% bisacrylamide, 2.5% glycerine, 0.5 * TBE) among Promega gel displacement pilot system technical bulletin preparation 0.5 * TBE.The liquid storage of preparation 250ml above-mentioned gelating soln filters and places 4 ℃.During each the use, in every 25ml 4% gel storage liquid, add 12.5 μ l TEMED and 187.5 μ l, 10% ammonium persulphate, pour in the sheet glass of 16.5cm * 16.5cm * 0.75mm.Always allow gel polymerisation spend the night to reach best effect.Before the last sample, in making gel prerunning 30 minutes in 0.5 * tbe buffer liquid under the 100V.The formation of double chain oligonucleotide and purifying

Method used herein is referring to Jacob Joseph etc., nucleic acids research, 1997, the 25th volume, o. 11th, 2182-2188, " antiparallel poly-purine phosphorothioate oligonucleotide and rat α (I) glue protogene promotor form stable triplex and suppress to transcribe in the rat fibroblast of cultivating ".In 80 ℃, in 0.25M NaCl,, then slowly cool to room temperature with the complementary strand heating of equivalent 5 minutes.Use Sambrook, Fritsch﹠amp; Maniatis, the method described in " molecular cloning, laboratory manual ", by electrophoresis on the polyacrylamide gel of 6% non-sex change (29: 1) with purifying annealed double chain oligonucleotide, downcut described oligonucleotide band then, adhesive tape is broken into pieces and soaked, with ethanol sedimentation it.In each labeled reactant, use about 20ng double chain oligonucleotide.The end of DNA ³²The P mark

In 37 ℃, in 10 μ l volumes with the cDNA of 150 to 200ng 300bp or 20ng double chain oligonucleotide and 10 μ Ci[γ- ³²P] (Irvine CA) is incubated 1 hour with 10U kinases and 1 * kinase buffer liquid (all deriving from Promega) to ATP together for 4500Ci/mmole, ICN, and (Princeton Separation, Adelphia NJ) go up purifying at the CentriSpin-10 post.In each gel sigmatropic reaction, use about 80,000-100, the DNA that 000cpm handles through kinases.Gel displacement test

Adding is before the DNA that kinases is handled, (Promega, Madison WI) are incubated 5-10 minute together with 1 * gel displacement damping fluid with protein (transcription factor of nuclear extract or purifying) under room temperature, add after the above-mentioned DNA, under room temperature, be incubated 20-30 minute again.Then sample is splined on 4% native gel that electrophoresis is crossed in advance, in 100V, in 0.5 * TBE after the about 3-4 of electrophoresis hour, desiccant gel and being exposed in phosphorus-visual instrument spends the night on 2 Whatman filter paper.The super displacement test of antibody gel

Add ³²Before the cDNA or oligonucleotide of P mark, will (Santa Cruz be CA) with Sp1 (Promega) pre-incubation of purifying 1 hour for clone 1C6, Santa Cruz Biotechnologies at the antibody of Sp1.Competitive gel displacement test

Add ³²Before the DNA of P mark, the oligonucleotide (or strand or two strands) of un-marked that will about 70-100 molar excess under room temperature about 30 minutes with the protein pre-incubation.PCAT3e28b, pCAT3e28h, the structure of pCAT3e28h-1

Use the total RNA of Jurkat to be template, produce CD28 upstream cDNA (197 to+28) by RT PCR.At first this section cDNA is cloned into TA cloning vector PCR2.1 (Invitrogen, Carlsbad, CA) in, then by insert the XhoI-SacI site with identical cDNA subclone to pCAT3e (Promega).PCAT3e28h and pCAT3e28h-1 are the mutant of pCAT3e28b, wherein-51 replace to-22 sequence deletions and by 15 other Nucleotide.Preceding 1 day of transfection (transient expression) transfection, in 2 or 3 T150, the cell preparation Jurkat cell that is paved with by 80-90% with the extent of dilution of 1: 4 or 1: 5.Before the transfection, all cells collect in the culturing bottle and counting (concentration should be 40 * 10 ⁴About/ml).In the 50ml conical tube centrifugal 10 transfections reaction used 11 * 4 * 10 ⁶Individual cell.The PBS that with volume is original half is suspended in cell washing 1 time in the fresh Jurkat substratum (90%RPMI 1640,10%FBS, 1%L-glutamine, 1% penicillin/streptomycin) of the pre-temperature of 44ml, so final concentration is 1 * 10 then again ⁶Ml.The 4ml cell is moved in each hole of 6 well culture plates, the 2mg/ml plasmid (pCAT3e series) of 2.5 μ l is moved in the 1.5ml pipe, add 147.5 μ l RPMI, 1640 substratum (serum-frees, antibiotic-free), in plasmid/culture medium solution, add the Superfact reagent that 20 μ l derive from Qiagen then, by drawing 5 times up and down, placed 5 to 10 minutes under the room temperature to mix.In each cell hole, drip transfection mixture, slowly rotate culture plate to mix.In 37 ℃, 5%CO ₂Incubation cell in the incubator, harvested cell is to carry out the CAT test after 24 hours.If add oligonucleotide after the transfection, then add in the cell setting constantly the 400 μ l oligonucleotide that (after the transfection 1 hour) store 50 μ l, send cell back to incubator again.The CAT test

After the incubation 24 hours,, guarantee with the cell culture medium wash-out hole not stay any cell with collecting cell by the cell in each hole being moved in the conical test tube of 15ml.Under the room temperature, with 2,000rpm eccentric cell 5 minutes is removed substratum, with 2ml PBS each cell precipitation thing is washed (add PBS, vortex is handled, and is centrifugal, removes substratum) 3 times.Remove final PBS washing lotion as much as possible with moving the drop head, in each cell transfer pipet, add 400 μ l, 1 * Reporter lysis buffers (Promega CAT Enzyme Assay System), and be transferred in the 1.5ml test tube.Under the room temperature, in lysis buffer, the cell precipitation thing is incubated 30 minutes, turn once in a while.Be incubated after 30 minutes, in 60 ℃ these test tubes heated 10 minutes, then at room temperature with 12, centrifugal 2 minutes of 000rpm moves into supernatant liquor (lysate) in the fresh 1.5ml test tube.100 μ l lysates are used in each CAT test reaction, with all the other lysates place-80 ℃ freezing.By each CAT of following setting test: in the 1.5ml test tube with 18.5 μ l water and 100 μ l lysates, positive butyryl CoA of 5 μ l5mg/ml (Promega) and 1.5 μ l, 0.1 μ Ci/ μ l paraxin- ¹⁴C (ICN) mixes (cumulative volume is 125 μ l), and in 37 ℃ of insulations 1 hour.When insulation finishes, in each test tube, add 300 μ l dimethylbenzene (ICN), the violent rotation for 5 seconds, full speed is centrifugal 3 minutes under the room temperature, 280 μ l upper stratas (dimethylbenzene) are moved in the fresh test tube mutually, add 100 μ l0.25M Tris at 280 μ l dimethylbenzene in mutually, pH8.0 is by above-mentioned rotation and centrifugal.200 μ l upper stratas are moved in the scintillation vial mutually, add the 5ml scintillation solution, reversing mixes, and counts sample on scintillometer.Oligonucleotide expands to the biologic activity of phosphorothioate oligonucleotide in external stability

Can give the nuclease resistance with the company's key modified oligonucleotide between thiophosphoric acid Nucleotide, therefore (the Stein (1993) that the external biological activity expanded to 24 hours from 1-2 hour, science, 261:1004-1012), this paper has illustrated the oligonucleotide FIGURE 1A (Seq#4) that is rich in G and has had higher vitro stability than the thiophosphoric acid FIGURE 1B (Seq#21) that is not rich in G.In Figure 1A, electrophorogram clearly illustrates that: for extracellular 1A (S) and cell 1B (L), after Jurkat cell insulation 96 hours, residual is complete ³²The FIGURE 1A (Seq#4) of P mark is more much more than FIGURE 1B (Seq#21), also observes Nickspin post data (Figure 1B) simultaneously.Herein, the complete oligonucleotide per-cent that reclaims from FIGURF 1A (Seq#4) after 96 hours is 54% (S) and 59% (L), and that reclaim from FIGURE 1B (Seq#21) is 10% (S) and 34% (L).These data show that stronger nuclease resistance is given by the existence of being rich in the zone of G among the FIGURE 1A (Seq#4) purely, infer that this is relevant with the ability that this special oligonucleotide forms folding secondary structure.Be conditioned that functional CD28 among the sub-oligonucleotide activated human T-cell expresses and the inhibition of CD28 specificity IL-2 generation depends on the primitive of the specific G of being rich in

Phosphorothioate oligonucleotide sequence #4 to 21 in the table 1 (5 μ M) expresses the relative restraining effect that produces with CD28 specificity IL-2 to CD28 and is shown in table 2.We have tested the bioactive accurate sequence demand of these regulon oligonucleotide herein.Table 2 shows that it is sequence-dependent suppressing activity, specifically depends on exist (Seq#5-8) of the primitive that contains two GGGG that separated by 4 bases.These data show that the oligonucleotide as FIGURE 1A (Seq#4) depends on and similar accurate conformation demand seen in oligonucleotide-protein interaction with its interaction of inferring target, rather than nucleic acid: nucleic acid hybridization demand (seen in antisense and the anti-genetic model).The oligonucleotide that contains specificity 12 aggressiveness sequence motifs forms the specific protein oligonucleotide complex

Fig. 2 demonstrates the warp with the pre-incubation of HeLa cell extract ³²The electrophoretic mobility displacement of the oligonucleotide of P mark is analyzed.Oligonucleotide row in the table 3 comprise that two oligonucleotide [FIGURE 1A (Seq#4) and ICN 16481 (Seq#5)] that contain 12 aggressiveness primitives, described primitive carry the G tetrad that two covers are separated by 4 Nucleotide.The oligonucleotide (swimming lane 5 and 11) that contains described primitive is the only oligonucleotide that tried that demonstrates this oligonucleotide-protein displacement (A band) that is different from other phosphorothioate oligonucleotide.These data show that the oligonucleotide that contains 12 aggressiveness primitives can cause specific protein-oligonucleotide and interact.It is relevant with the existence of specific oligonucleotide-protein complex to be conditioned the inhibition that functional CD28 expresses among the sub-oligonucleotide activated human T-cell

Table 4 has compared the inhibition effect of some phosphorothioate oligonucleotide of 5 μ M to mitogen-inductive CD28 expression and IL-2 generation, described oligonucleotide has the regulon ability of formation specific oligonucleotide-protein complex when being incubated with the HeLa nucleus extract that is rich in transcription factor.These data clearly show that the oligonucleotide that contains primitive is expressed CD28 and the IL-2 excretory suppresses relation between the formation of activity and specificity gel displacement band.Replacement in 2 G tetrads causes the forfeiture of function and causes the disappearance of oligonucleotide-protein complex.CD28 upstream promoter zone-197 combines with Sp1 to+28 (28b)

Will through ³²The CD28 promoter region-197 of P mark is to+28, perhaps also can be referred to as 28b is incubated with Sp1 albumen with the Sp1 antibody of the initial continuous 3 times of dilutions of 0.5 μ g, carry out the super displacement test of gel, tell DNA-protein-antibody complex behind the electrophoresis, data are shown in Fig. 4.Data show that Sp1 combines with the 28b zone of CD28 promotor, and this interaction is specific, because with after specific Sp1 antibody serial dilution to the 0.00617 μ g, and Sp1/ ³²P-28b/Sp1 antibody band (band B) disappears, and only stays 28b/Sp1 band (band A).This shows 28b specifically in conjunction with Sp1, without ³²The 28b of P mark is band C.Derive from CD28 upstream promoter zone-197 to+28 (28b) and the oligonucleotide-51 that contains the 12 aggressiveness primitives that are rich in G to-22 also can be in conjunction with Sp1

Accurate Sp1 land is limited in the effort of being rich in the G primitive in the CD28 promoter region-197 to+28 in 28b, we have synthesized two strands (ds) the 30 aggressiveness oligonucleotide that are called as 28b oligonucleotide (table 1Seq#1), the sequence of described oligonucleotide contains 12 aggressiveness GGGGAGGAGGGG, the Sp1 binding site in the CD28 promoter region of inferring that Here it is.Warp ³²Behind the P mark, the 28b oligonucleotide is incubated with the Sp1 extract, in fact they interosculate (Fig. 5, the band A, swimming lane 2 and 3).Can cause being with A to disappear with the ds 28b competitive oligonucleotide of un-marked, this in fact description taken in conjunction be (swimming lane 4) that is specific to Sp1.Surprisingly, the strand thiophosphoric acid is rich in combining of the oligonucleotide FIGURE 1A (swimming lane 5) of G and 16481 (swimming lane 6) (all containing the primitive that is rich in G) rather than control oligonucleotide ICN 16476 (swimming lane 7) competition and Sp1.This data declaration in fact thiophosphoric acid is rich in the DNA binding site bonded regulon that the oligonucleotide FIGURE 1A of G and ICN 16481 can be used as with Sp1 and works.This results of interaction is to stop Sp1 to combine with the Sp1 site at-51 to-22 places in the promoter region, thereby suppresses the CD28 genetic transcription of Sp1 mediation and reduce the proteic expression of sophisticated CD28.

Therefore, the invention discloses regulon and utilize this regulon to adjust the method for immunne response.Although herein disclosed is specific embodiment, except the soluble the present invention of appended claims, scope of the present invention is also unrestricted.

Sequence table (1) physical data: (i) applicant: Robert Tam is the invention exercise question (ii): the method that is rich in the oligo aptamers of G and adjusts immunne response is sequence number (iii): (iv) address:

(A) addressee: Crockett ﹠amp; Fish

(B) street: 1440 N.Harbor Blvd., Suite 706

(C) city: Fullerton

(D) state: California

(E) country: the U.S.

(F) postcode: 92835 (v) computer-reader form:

(A) media type: floppy disk

(B) computer: but IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: WordPerfect 6.1 (vi) present application materials:

(A) application number: uncertain

(B) applying date: November 21 nineteen ninety-five

(C) classification number: uncertain (viii) proxy/act on behalf of data:

(A) name: Fish, Robert D.

(B) registration number: 33,880

(C) data/number of documents: 213/015 (ix) communications data:

(A) phone: 714-525-3433

(B) fax: 714-525-3303

(C) telegram: the data of (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:1:GGGTTCCTCG GGGAGGAGGGG GCTGGAACCC (3) SEQ ID NO:2:

(i) sequence signature:

(A) length: 15 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:2:GGAGCACAGG GTGCT (4) SEQ ID NO:3:

(i) sequence signature:

(A) length: 15 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:3:TCATCACAGG GTGCT (5) SEQ ID NO:4:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:4:TTGGAGGGGG TGGTGGGG (6) SEQ ID NO:5:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:5:GGGGAGGAGG GGCTGGAA (7) SEQ ID NO:6:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:6:GGGTTGGAGG GGGTGGTGGG G (8) SEQ ID NO:7:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:7:TTGGAGGGGG AGGAGGGG (9) SEQ ID NO:8:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:8:TTGGAGGGGG AGGTGGGG (10) SEQ ID NO:9:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:9:TTGGAGGCGG TGGTGGCG (11) SEQ ID NO:10:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown is molecule type (ii): DNA (genomic) is sequence description (xi): the data of SEQ ID NO:10:TTGGAGCCGG TGGTGGCC (12) SEQ ID NO:11: (i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown is molecule type (ii): DNA (genomic) is sequence description (xi): the data of SEQ ID NO:11:TTGGAGGGGC TCCTCGGG (13) SEQ ID NO:12: (i) sequence signature:

(A) length: 16 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown is molecule type (ii): DNA (genomic) is sequence description (xi): the data of SEQ ID NO:12:TTGGAGCCGG TGGTGG (14) SEQ ID NO:13: (i) sequence signature:

(A) length: 12 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown is molecule type (ii): DNA (genomic) is sequence description (xi): the data of SEQ ID NO:13:GGGGTGGTGG GG (15) SEQ ID NO:14:

(i) sequence signature:

(A) length: 10 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:14:GGGGTTGGGG (16) SEQ ID NO:15:

(i) sequence signature:

(A) length: 5 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:15:TGGGG (17) SEQ ID NO:16:

(i) sequence signature:

(A) length: 4 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:16:GGGG (18) SEQ ID NO:17:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:17:CACTGCGGGG AGGGCTGGGG (19) SEQ ID NO:18:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:18:ATGGGGTGCA CAAACTGGGG (20) SEQ ID NO:19:

(i) sequence signature:

(A) length: 15 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:19:AACGTTGAGG GGCAT (21) SEQ ID NO:20:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:20:TTCCAGCCCC TCCTCCCC (22) SEQ ID NO:21:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:21:AACCTCCCCC ACCACCCC (23) SEQ ID NO:22:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:22:ATTCGATCGG GGCGGGGCGA GC (24) SEQ ID NO:23:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:23:CGCTTGATGA GTCAGCCGGA A (25) SEQ ID NO:24:

(i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:24:GATCGAACTG ACCGCCCGCG GCCCCT (26) SEQ ID NO:25:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:25:AGTTGAGGGG ACTTTCCCAG GC (27) SEQ ID NO:26:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:26:TGTCGAATGC AAATCACTAG AA (28) SEQ ID NO:27:

(i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: the data of SEQ ID NO:27:AGAGATTGCC TGACGTCAGA GAGCTAG (29) SEQ ID NO:28:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ii) molecule type: DNA (genomic)

(xi) sequence description: SEQ ID NO:28:GCAGAGCATA TAAGGTGAGG TAGGA table 1. sequence number oligonucleotide identification number sequence 1. 28b 5 ' GGG TTC CTC GGG GAG GAG GGG CTG GAA CCC

3’?CCC?AAG?GAG?CCC?CTC?CTC?CCC?GAC?CTT?GGG?2. 28h 5’?GGA?GCA?CAG?GGT?GCT

3’?CCT?CGT?GTC?CCA?CGA?3. 28h-1 5’?TCA?TCA?CAG?GGT?GCT

3’?AGT?AGT?GTC?CCA?CGA?4 ICN?16064 5’?TTG?GAG?GGG?GTG?GTG?GGG?5 ICN?16481 5’?GGG?GAG?GAG?GGG?CTG?GAA?6 ICN?16065 5’?GGG?TTG?GAG?GGG?GTG?GTG?GGG?7 ICN?16475 5’?TTG?GAG?GGG?GAG?GAG?GGG?8 ICN?16479 5’?TTG?GAG?GGG?GAG?GTG?GGG?9 ICN?16480 5’?TTG?GAG?GCG?GTG?GTG?GCG10 ICN?16538 5’?TTG?GAG?CCG?GTG?GTG?GCC11 ICN?16539 5’?TTG?GAG?GGG?CTC?CTC?GGG12 ICN?16523 5’?TTG?GAG?CCG?GTG?GTG?G13 ICN?16525 5’?GGG?GTG?GTG?GGG14 ICN?16526 5’?G?GGG?TTG?GGG15 ICN?16483 5’?TG?GGG16 ICN?16482 5’?G?GGG17 ICN?16527 5’?CAC?TGC?GGG?GAG?GGC?TGG?GG18 ICN?16528 5’?ATG?GGG?TGC?ACA?AAC?TGG?GG19 ICN?16487 5’?AAC?GTT?GAG?GGG?CAT20 ICN?16476 5’?TTC?CAG?CCC?CTC?CTC?CCC21 ICN?16214 5’?AAC?CTC?CCC?CAC?CAC?CCC22 SP1 5’?ATT?CGA?TCG?GGG?CGG?GGC?GAG?C

3’?TAA?GCT?AGC?CCC?GCC?CCG?CTC?G23 AP1?(c-jun) 5’?CGC?TTG?ATG?AGT?CAG?CCG?GAA

3’?GCG?AAC?TAC?TCA?GTC?GGC?CTT24 AP2 5’?GAT?CGA?ACT?GAC?CGC?CCG?CGG?CCC?CT

3’?CTA?GCT?TGA?CTG?GCG?GGC?GCC?GGG?GA25 NF-KB 5’?AGT?TGA?GGG?GAC?TTT?CCC?AGG?C

3’?TCA?ACT?CCC?CTG?AAA?GGG?TCC?G26 OCT1 5’?TGT?CGA?ATG?CAA?ATC?ACT?AGA?A

3’?ACA?GCT?TAC?GTT?TAG?TGA?TCT?T27 CREB 5’?AGA?GAT?TGC?CTG?ACG?TCA?GAG?AGC?TAG

3’?TCT?CTA?ACG?GAC?TGC?AGT?CTC?TCG?ATC28 TFIID 5’?GCA?GAG?CAT?ATA?AGG?TGA?GGT?AGG?A

3 ' CGT CTC GTA TAT TCC ACT CCA TCC T table 2. suppresses the oligonucleotide sequence evaluation that CD28 expressed and depended on the IL-2 generation of CD28

CD28 IL-2ICN 16064 TTG GAG GGGGTG GTGGGG 100 100ICN 16481 GGGGAG GAGGGGCTG GAA 100 100ICN 16065 GGG TTG GAG GGGGTG GTGGGG 100 100ICN 16475 TTG GAG GGGGAG GAGGGG 100 100ICN 16479 TTG GAG GGGGAG GTGGGG 100 100ICN 16480 TTG GAG GCGGTG GTGGCG 31 38ICN 16538 TTG GAG CCGGTG GTGGC C 40 57ICN 16539 TTG GAG GGGCTC CTC GGG 44 25ICN 16523 TTG GAG CCGGTG GTG G 38 57ICN 16525 GGGGTG GTGGGG 100 120ICN 16526 GGGG TTGGGG 30 39ICN 16483 TGGGG 2 2ICN 16482 GGGG 2 2ICN 16527 CAC TGC GGGGAG GGC TGGGG 58 76ICN 16528 ATGGGG TGC ACA AAC TGGGG 51 63ICN 16487 AAC GTT GAGGGG CAT 26 52ICN 16476 TTC CAG CCC CTC CTC CCC 29 22ICN 16214 AAC CTC CCC CAC CAC CCC 4 242GGGG12, ( ) CD3/PMA-TICN 16064CD28Jurkat TIL-2ICN 16064。^*With respect to activity (100%) ecbatic of 5um ICN 16064, the restraining effect that it is expressed CD28 in 7 experiments is 52-79%, and the restraining effect that IL-2 is produced is 76-89%.

Table 3: the nuclear extract protein of phosphorothioate oligonucleotide-in conjunction with distribution plan (see figure 2) oligonucleotide sequence swimming lane ICN 16064 TTG GAG GGG GTG GTG GGG 11,12ICN 16481 GGG GAG GAG GGG CTG GAA 5,6ICN 16480 TTG GAG GCG GTG GTG GCG 7,8ICN 16538 TTG GAG CCG GTG GTG GCC 1,2ICN 16485 GTT GGA GAC CGG GGT TGG 3,4ICN 16476 TTC CAG CCC CTC CTC CCC 9,10

Table 4: the restraining effect that functional CD28 is expressed is relevant with the existence of specific oligonucleotide-protein complex

To express relative inhibition (%) protein complex oligonucleotide sequence CD28 IL-2 oligonucleotides/protein complex ICN 16064 TTG GAG GGG GTG GTG GGG 100 100 are that ICN 16481 GGG GAG GAG GGG CTG GAA 100 100 are that ICN 16480 TTG GAG GCG GTG GTG GCG 31 38 no ICN 16538 TTG GAG CCG GTG GTG GCC 40 57 no ICN 16485 GTT GGA GAC CGG GGT TGG 11 15 no ICN 16476 TTC CAG CCC CTC CTC CCC 29 22 no ICN 16214 AAC CTC CCC CAC CAC CCC 42 are no

Claims (25)

1. have the regulon of the sequence that comprises at least two zones of being rich in G, described zone is selected from GGnG, GGGG, and GnGG and GGG, wherein G is a guanosine, n is any Nucleotide.

2. the regulon of claim 1, wherein at least two at least two zones are less than 7 Nucleotide separately.

3. the regulon of claim 1, wherein at least two at least two zones are comprised that by 3 to 6 Nucleotide 3 and 6 Nucleotide separately.

4. the regulon of claim 1, wherein at least two at least two zones by 4 Nucleotide separately.

5. the regulon of claim 1, the nucleic acid binding site of described regulon competition immune modulator.

6. the regulon of claim 2, wherein immune modulator is selected from SP1, NFKB, EGR1 and AP2.

7. the regulon of claim 1, the nucleic acid binding site of described regulon competition immune modulator, wherein at least one at least two zones of being rich in G contains GGnG, and at least two at least two zones are less than 7 Nucleotide separately.

8. the regulon of claim 1, the nucleic acid binding site of described regulon competition immune modulator, wherein at least one at least two zones of being rich in G contains GGGG, and at least two at least two zones are less than 7 Nucleotide separately.

9. the regulon of claim 1, the nucleic acid binding site of described regulon competition immune modulator, wherein at least one at least two zones of being rich in G contains GnGG, and at least two at least two zones are less than 7 Nucleotide separately.

10. the regulon of claim 1, the nucleic acid binding site of described regulon competition immune modulator, wherein at least one at least two zones of being rich in G contains GGG, and at least two at least two zones are less than 7 Nucleotide separately.

11. the regulon of claim 1, described regulon contain sequence 5 ' TTG GAG GGG GTG GTGGGG3 ' (Seq.Id.No.4).

12. the regulon of claim 1, described regulon contain sequence 5 ' GGG GAG GAG GGG CTGGAA3 ' (Seq.Id.No.5).

13. the regulon of claim 1, described regulon contain sequence 5 ' GGG GTG GTG GGG3 ' (Seq.Id.No.13).

14. the regulon of claim 1, described regulon contain sequence 5 ' TTG GAG GGG GAG GAGGGG3 ' (Seq.Id.No.7).

15. the regulon of claim 1, described regulon contain sequence 5 ' TTG GAG GGG GAG GTGGGG3 ' (Seq.Id.No.8).

16. the regulon of claim 1, described regulon contain sequence 5 ' GGG TTG GAG GGG GTGGTG GGG3 ' (Seq.Id.No.6).

17. adjust the method that patient's immunity system is replied, described method comprises to patient to be used according to each regulon of claim 1 to 16.

18. treatment suffers from patient's the method for the disease that is characterised in that inappropriate immunity system is replied, described method comprises to patient to be used according to each regulon of claim 1 to 16.

19. the method for claim 18, wherein disease comprises graft-vs-host reaction.

20. the method for claim 18, wherein disease comprises autoimmune disease.

21. the method for claim 20, wherein disease comprises rheumatoid arthritis.

22. the method for claim 20, wherein disease comprises multiple sclerosis.

23. the method for claim 20, wherein disease comprises lupus erythematosus.

24. the method for claim 20, wherein disease comprises insulin-dependent diabetes mellitus.

25. the method for claim 20, wherein disease comprises psoriatic.

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