WO1996024380A1 - Methods and compositions for regulation of cd28 expression - Google Patents
Methods and compositions for regulation of cd28 expression Download PDFInfo
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- WO1996024380A1 WO1996024380A1 PCT/US1996/001507 US9601507W WO9624380A1 WO 1996024380 A1 WO1996024380 A1 WO 1996024380A1 US 9601507 W US9601507 W US 9601507W WO 9624380 A1 WO9624380 A1 WO 9624380A1
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
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- C12N2310/315—Phosphorothioates
Definitions
- the invention is in the field of modulating gene expression tnrough the use of oligomers, particularly those oligomers
- the immune system plays a crucial role in protecting higher organisms against life-threatening infections, the immune system also plays a crucial part in the pathogenesis of numerous diseases.
- Those diseases in which the immune system plays a part include autoimmune diseases in which the immune system reacts against an autologous antigen, e.g., systemic lupus
- erythematosus or diseases associated with immunoregulation initiated by reaction to a foreign antigen, e . g . , graft vs. nest disease observed in transplantation rejection.
- T-cell mediated diseases result from an inappropriate immune response driven by abnormal T-cell activation.
- activate ⁇ T-cells have been reported in many T-cell mediated skin diseases (Simon et al . , (1994) J. Invest Derm., 103:539-543).
- psoriasis which afflicts 2_ of the Western population including four million Americans, is a skin disorder
- Th1 cytokines IL-2, interferon-gamma
- HLA DR+/ICAM-1+ phenotype of psoriasis lesions
- BBl tne natural ligands for CD28 found on activated APC
- T-cell activation A number of other diseases are thought to oe caused by aberrant T-cell activation, including Type I (insulin-dependent) diabetes mellitus, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus
- erythematosus erythematosus
- inflammatory bowel disease Crohn's and ulcerative colitis
- autoimmune hepatitis a variety of syn ⁇ romes including septic shock and tumor-induce ⁇ cacnexia may involve T-cell activation and augmented production of potentially toxic levels of lymphokines. Normal T-cell activation also me ⁇ iates the rejection of transplanted cells ana organs by providing the necessary signals for the effective destruction of the "foreign" donor tissue.
- the first signal involves the recognition, by specific T-cell receptor/CD3 complex, of antigen presented by major histocompati- bility complex molecules on the surface of antigen presenting cells (APCs).
- APCs antigen presenting cells
- Antigen-nonspecifIC intercellular interactions between T-cells and APCs provide the second signal that serves to regulate T-cell responses to antigen.
- Costimulated cells react by increasing the levels of specific cytokine gene transcription and by stabilizing selected mRNAs . In the absence of costimulation, T-cell activation results in an aborted or anergic T-cell response.
- One key costimulatory signal is provided by interaction of the T-cell surface receptor CD28 with B7-related molecules on APC (Linsley and Ledbetter (1993) Ann. Rev. Immunol., 11:191-212).
- CD28 is constitutiveiy expressed on 95% of CD4+ T-cells (whicn provide helper functions for B-cell antibody production) and 50% of CD8+ T-cells (which have cytotoxic functions) (Yamada et al . , (1985) Eur. J.
- cytokines include interleukin-2 (IL-2), required for cell cycle progression of T-cells, interferon-gamma, which displays a wide variety of anti-viral and anti-tumor effects and interleukin-8 (IL-8), known as a potent chemotactic factor for neutrophiis and lymphocytes.
- IL-2 interleukin-2
- interferon-gamma which displays a wide variety of anti-viral and anti-tumor effects
- IL-8 interleukin-8
- cytokines have oeen shown to be regulated by the CD28 pathway of T-cell activation (Fraser et al . , (1991) Science. 251:313-316, Seder et al . , (1994) J. Exp. M ed. , 179:299-304, Wechsler et al . , (1994) J. Immunol.,
- IL-2 interferon-gamma
- IL-8 are essential in promoting a wide range of immune responses and have been shown to be overexpressed in many T-cell mediated disease states.
- CD28 was expressed in high levels in the majority of dermal and epidermal CD3+ T-cells but in normal skin and basal cell carcinoma (a non T-cell mediated skin disease), CD28 was expressed only in perivascular T-ceils.
- B7 expression was found on dermal dendritic cells, ⁇ ermal APCs and on keratinocytes but not in normal skin and basal cell carcinoma (Simon et al . , (1994) J. Invest Derm., 103: 539-543). Therefore this suggests that the CD28/B7 pathway is an important mediator of T-cell-mediated skin diseases.
- autoimmune diseases caused by the loss of self-tolerance is predominantly characterized by the presence of CD28+ T-cells and expression of its ligand, B7 on activated professional APCs
- non-steroid anti-inflammatory drugs are currently used to ameliorate symptoms, but they do not prevent the progression of the disease.
- steroids can have side effects such as inducing osteoporosis, organ toxicity and diabetes, and can accelerate the cartilage degeneration process and cause so-called post-injection flares for up to 2 to 8 hours.
- NSAIDS can have gastrointestinal side effects and increase the risk of agranulocytosis and latrogenic hepatitis.
- Immunosuppressive drugs are also used as another form of therapy, especially in advanced disease stages. However, these ⁇ rugs suppress the entire immune system and often treatment has severe side effects including hypertension and nephrotoxicity.
- establisne ⁇ immunosuppressants such as cyclosporin and FK506 cannot inhibit the CD28-dependent T-cell activation pathway (June et al . , (1987) Mol. Cell. Biol., 7:4472-4481).
- the subject invention provides methods and compositions for the treatment of immune system-mediated diseases.
- the composxtions of the invention have the property of reducing the
- CD28 in cells of interest, which in turn moderate pathogenic effects of the immune system in an immune system-mediated disease.
- compositions of the invention include many different oligomers capable of reducing the expression of CD28.
- One aspect of the invention is to provide oligomers capable of reducing the expression of CD28 by interfering with the expression of CD28.
- the oligomers of the invention have nucleic acid base sequence homology to a CD28 gene or a CD28 gene
- oligomers of the invention may be DNA, RNA, or various synthetic analogs thereof.
- oligomers having 11 to 50 bases comprising at least two sequences of GGGG separated by 3 to 5 bases.
- Another aspect of the invention is to provide genetic engineering vectors for the intracellular expression of oligomers of the invention in cells of interest, preferably cells that naturally express CD28.
- Another aspect of the invention is to provide pharmaceutical formulations comprising one or more different oligomers of the invention.
- the pharmaceutical formulations may be adapted for various forms of aoministration to the body or administration to cells to be reintroduced into the body.
- Another aspect of the invention is to provide methods for the treatment of immune system-mediated diseases.
- the methods of the invention involve modulating CD28 expression by administering an effective amount of the oligomers of the invention.
- the methods of the invention include methods of treating autoimmune disease, methods of reducing inflammation, response, methods of reducing the production of selected cytokines, methods of inactivating T cells, and methods of immunosuppressing a
- Figure 1 is the sequence of the 5' untranslated region of the CD28 gene (1A) and the mRNA sequence of human CD28 (1B, 1C). Figures 1B and 1C represent different contiguous portions of a polynucleotide sequence.
- Figure 2 is a graphical representation of the percentage of viable (live) T-cells following treatment with various CD28- specific and control phosphorothioate ana pnosphorotnioate- 3'hy ⁇ roxypropylamine oligonucleotides.
- Figure 3 is a graphical representation of anti-CD3
- Figure 4 is a graphical representation of A) the in ⁇ uction of T-cell proliferation by mitogens in human T-cells from donor KS006 and B) the effect of CD28-specific and control
- Figure 5 is a graphical representation of the in ⁇ uction of interleukin-2 (IL-2) production by anti-CD3 monoclonal antibody and PMA in human T-cells (A) and the effect of CD28-specific and control phosphorothioate (B) phosphorothioate- 3'hydroxypropylamine (C) oligonucleotides on anti-CD3 monoclonal antibo ⁇ y/PMA-induced IL-2 production in human peripheral T-cells.
- IL-2 interleukin-2
- Figure 6 is a graphical representation of the in ⁇ uction of interferon-gamma (IFN ⁇ ) production by anti-CD3 monoclonal antibody and PMA in human T-cells (A) and the effect of
- CD28-specific and control phosphorothioate B
- phosphorothioate-3'hydroxypropylamine C
- oligonucleotides on anti-CD3 monoclonal antibody/PMA-induced interferon-gamma production in human peripheral T-cells B
- Figure 7 is a graphical representation of the induction of interleukin-8 (IL-8) production by anti-CD3 monoclonal antibody and PMA in human T-cells (A) and the effect of CD28-specific ana control phosphorothioate (B) phosphorothioate-3'hydroxypropylamine (C) oligonucleotides on anti-CD3 monoclonal antibody/PMA-induced IL-8 production in human peripneral T-cells.
- IL-8 interleukin-8
- Figure 8 is a graphical representation of the induction of interieukin-2 receptor (IL-2R, otherwise known as CD25) (A) and intracellular adhesion molecule-1 (ICAM-1 otherwise Known as CD54) (B) expression by anti-CD3 monoclonal antibody and PMA in human peripheral T-cells treated with and without CD28-specific and control phosphorothioate 3'hydroxypropylamine oligonucleotides.
- IL-2R interieukin-2 receptor
- IAM-1 intracellular adhesion molecule-1 otherwise Known as CD54
- Figure 9 is a graphical representation of CD28 expression in HUT 78 (A) and Jurkat (B) human T-cell lines before and after anti-CD3 monoclonal antibody and PMA treatment, and the effect of CD28-specific phosphorothioate oligonucleotides in anti-CD3 monoclonal antibody and PMA-treated Jurkat cells (C).
- Figure 10 is a graphical representation of the effect of CD28-specific phosphorothioate oligonucleotides on interleukin-2 production in anti-CD3 monoclonal antibody and PMA-treated HUT 78 (A) and Jurkat (B) human T-cell lines.
- Figure 11 is a graphical representation of the effect of phosphorothioate oligonucleotides on surface expression of accessory molecules and on cytokine secretion in activated T cells.
- Figure 12 is a graphical representation of the effect cf phosphorothioate oligonucleotides on CD28 and CD25 mRNA levels.
- Figure 13 is a graphical representation of the specificity of oligonucleotides RT03S (SEQ ID NO: 44) and RT04S (SEQ ID NO: 45) with respect to inhibitory effect on functional CD28
- Figure 14 is a graphical representation of the tolerance induction in vi tro by the CD28-specific oligonucleotides, RT03S (SEQ ID NO: 44) and RT04S (SEQ ID NO: 45).
- Figure 15 is a graphical representation of the in vi tro stability of 32 P-labeled phosphorothioates, RT03S (SEQ ID NO: 44) and RTC06S (SEQ ID NO: 48) in extracellular supernatants (top panel) and Jurkat cell lysates (bottom panel).
- RT03S SEQ ID NO: 44
- RTC06S SEQ ID NO: 48
- Described herein are methods and compositions for treating immune system-mediated diseases, wherein the desired therapeutic effect is achieved by decreasing the expression of CD28, thereby abrogating activated CD28 + T cell function and decreasing
- the inventor has discovered that antigen-dependent T cell activation may be inhibited by decreasing the expression of CD28 in CD28 + T cells.
- the invention provides numerous compounds that may be used to decrease the expression of CD28 in T cells.
- the invention described herein involves the discovery that decreasing CD28 expression in T cells can interfere with the antigen-specific activation of T cells.
- the discovery may be used to provide numerous methods of treating immune system-mediated diseases with oligomers targeted to CD28 and with non-oligomer compounds that decrease CD28 expression.
- numerous methods of treating immune system-mediated diseases are provided, such methods may employ non-oligomer compounds that have not yet been synthesized or purified.
- One aspect of the invention is to provide for oligomers that can be used to inhibit gene expression of certain genes is an established technique frequently referred to as the use of "anti-sense” oligonucleotides or "anti-sense therapy.”
- anti-sense refers to oligomers (including oligo- nucleotides) capable of forming either double-stranded or triple- stranded (triplex) helices with poiynucleotides so as to
- the oligomers of the invention are capable of modulating the expression of the CD28 gene.
- the oligomers of the invention include those oligomers that have the property of being able to form either a double-stranded polynucleotide helix by hybridizing with CD28 transcripts (or portions thereof), or a double-stranded polynucleotide helix by hybridizing with a portion or portions of a CD28 gene, wherein the helix formation may occur under
- the oligomers of the invention also include those oligomers that are capable of affecting the
- oligomers of the invention include those oligomers that are capable of forming a triple-stranded polynucleotide helix with a portion or portions of a CD28 gene, wherein the helix formation may occur under intracellular conditions.
- double-stranded helix and triple-stranded helix base pairing relationships between nucleic acid bases are known to the person o f ordinary s ki ll in the art and may be employed in the design of the
- Regions of the CD28 gene or CD28 gene transcript at which double-stranded helix or triple-stranded helix formation can occur with a given oligomer of the invention are said to be "targeted” by that oligomer.
- Human CD28 is a 90-kDa homodimeric transmemorane glycoprotein present on the surface of a subset of T ceils. CD23 is present on most CD4 + T cells and about 50% of CD8 + T cells. The DNA sequence encoding human CD28 has been resolved as can be found, among other places, in Lee et al . Journal of Immune logy. 145:344-352 (1990) and on publicly accessible gene ⁇ atabases such as GenBank.
- the human CD28 gene comprises four exons, each defining a functional domain of the predicted protein.
- compositions and methods of the invention are provided.
- sequence of the CD28 gene may be obtained by, among other methods, using previously identified CD28 gene sequences from humans (or other mammals) as gene library hybridization probes and/or PCR (polymerase chain
- amplification primers While the published nucleotide sequences of tne CD28 gene are believed to be accurate, the subject invention may be practiced by the person of ordinary skill in the art even if the published nucleotide base sequence of CD28 contains sequencing errors. The proper nucleotide base sequence errors in published sequences may be detected by, among other means, re-sequencing regions of the CD28 gene (or CD28 gene transcripts) targeted by the oligomers of the invention. Resequencing may be performed by means of conventional DNA
- the oligomers of the invention preferably comprise from about 11 to aoout 50 nucleic acid base units. It will be readily appreciated by the person of ordinary skill in the art that oligomers of the invention may be significantly longer than 50 nucieic acid oase units. In a more preferred embodiment cf tne invention, the oligomers comprise from about 8 to about 25 nucleic acid base units; more preferably from about 14 nucleic acid base units to about 22 nucleic base units.
- the preferred size limitations for the oligomers of the invention pertain only to those oligomers that are to be administered extracellulariy to a cell and are not applicable to intracellularly produced CD28 specific oligomers, e.g., as produced from vectors for the genetic manipulation of target host ceils.
- the oligomers of the invention may have numerous different nucleic acid base sequences.
- the oligomers of the invention may be selected to reduce expression of CD28 by hybridizing (througn nucleic acid - nucieic acid interaction) to virtually any region of a CD28 transcript of CD28 gene in order to reduce expression of CD28, or by hybridizing (through nucleic acid - protein interaction) to non-nucleic acid molecules that recognize untranslated sequences of the CD28 gene.
- oligomers of the invention may be selected so as to be able to hybridize to translated regions of a CD28 transcript, untranslated regions of a CD28 transcript, unspliced regions of a CD28 transcript, CD28 gene introns, CD28 promoter sequences, and CD28 regulatory sequence, the 5' cap region of a CD28 transcript, CD28 gene coding regions, and the like (including combinations of various distinct regions).
- Preferred embodiments of the CD28 gene and CD28 gene transcripts by the oligomers of the invention are in the translational and/or transcriptional initiation regions of the CD28 gene (and transcripts thereof).
- the potency of the oligomer i.e., the amount required to produce the desired biological effect will be varied.
- Preferred embodiments of the oligomers of the invention have the highest possible potency.
- the potency of different oligomers of the invention may be measured by various in vitro assays known to the person of ordinary skill in the art.
- the subject oligomers exhibit perfect nucleic acid base complementarity to the selected target sequence, i . e . , every nucleic acid base in the oligomer may enter into a base pairing relationship with a second (or third) nucleic acid base on another strand of a double (or triple) helix.
- every nucleic acid base in the oligomer may enter into a base pairing relationship with a second (or third) nucleic acid base on another strand of a double (or triple) helix.
- oligomers specific for a CD28 gene target and/or capable of inhibiting CD28 expression may have nucleotide base sequences that lack perfect hybridization to the CD28 gene (either strand), CD28 gene transcripts, or CD28-specific regulatory proteins.
- the oligomers having the nucleotide base sequences indicated in RT01, RT02, RT03, and RT04 are phosphorothioates.
- Particularly preferred oligomers are phosphorothioate-3'hydroxypropylamine, as described in Tam et al . , Nucl. Acid. Res. 22:977-966 (1994).
- Oligomers of the invention may be designed so as to decrease tne expression of CD28 in T cells that have internalized
- oligomers of the invention may be designed so as to decrease expression of CD28 when the oligomers are produced intracellularly through the use of genetic expression vectors. Inhibition of CD28 expression may be effected through (I) interference with CD28 gene transcription, (ii) interference with the transcription of CD28 gene transcripts, (iii) interference with the processing of CD28 gene transcripts, or any combination of (I), (ii), and (iii).
- the precise degree and mechanism of the interference of CD28 expression will depend on factors such as the structure of the particular oligomer, the nucleotide base sequence of the oligomer, the dosage of oligomer, the means of administering the subject oligomer, and the like.
- oligomer refers to both naturally occurring polynucleotides, e.g., DNA, RNA, and to various
- oligomers that are artificial analogs of naturally occurring polynucleotides have properties that make them superior to DNA or RNA for use in the methods of the invention. These properties include higher affinity for DNA/RNA, endonuclease resistance, exonuclease resistance, lipid solubility, RNAse H activation, and the like.
- enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phosphate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleotide or alkylphosphotriester oligonucleotide.
- Non-ionic oligomers such as these are
- Methylphosphonate (and other alkyl phosphonate) oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Fiftina, Nucl. Acids Res., 3:109-3024 (1979); Miller et al . . Biochemistry, 18:5134-5142 (1979); Miller et al . , J. Biol. Chem., 255:9659-9665 (1980); Miller e t al . , Nucl. Acids Res., 11:5189-5204 (1983); Miller et al . , Nucl. Acids Res., 11:6225-6242 (1983); Miller et al . ,
- polynucleotide analogs of interest include compounds having acetals or thioacetals in the backbone structure. Examples of how to make and use such compounds can be found, among other places in, Gao et al . , Biochemistry 31:6223-6236 (1992), Quaedflieg et al . , Tetrahedron Lett. 33(21):3081-7034 (1992), Jones et al . , J. Org. Chem. 58:2983-2991 (1993).
- polynucleotide analogs of interest include compounds having silyl and siloxy bridges in the backbone structure.
- polynucleotide analogs of interest include compounds having silyl and acetamidate bridges in the backbone structure. Examples of how to make and use such compounds can be found, among other places in Gait et al . , J. Chem. Soc., Perkin Trans. 1:1684 (1974), Mungall and Kaiser, J. Org. Chem. 42(4):703-706 (1977), and Coull et al . , and Tetrahedron Lett. 28(7):745-748 (1987).
- PNAs peptide nucieic acids
- PNAs peptide nucieic acids
- a description of how to make and use peptide nucleic acids can be found in, among other places, Buchardt et al . , Trends in
- oligomers may be further modified so as to increase the stability of duplexes and/or increase cellular uptake.
- modifications can be fcun ⁇ in PCT publication WO 93/24507 entitled "Conformationally Restrained Oligomers Containing Amide or Carbamate Linkages for Sequence-Specific Binding," Nielsen et al .
- the oligomers of the invention comprise various nucleic acid bases. In addition to nucleic acid bases found to occur
- oligomers of the invention naturally in DNA or RNA, e.g., cytosine, adenine, guanine, thymidine, uracil, and hypoxanthine, the oligomers of the
- inventions may comprise one or more nucleic acid bases that are synthetic analogs of naturally occurring acid bases.
- nonnaturally occurring heterocyclic bases include, but are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine analogs as well as other heterocyclic base analogs, wherein one or more of the carbon and nitrogen atoms of the purine and pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.
- Preferred base moieties are those bases that may be incorporated into one strand of double-stranded polynucleotides so as to maintain a base pairing structural relationship with a naturally occurring base on the complementary strand at the double-stranded polynucleotide .
- the invention provides many methods of treating a variety of immune disorders.
- treatment or “treating” as used herein with reference to a disease refers both to prophylaxis ana to the amelioration of symptoms already present in an individual. It will be appreciated by the person of ordinary skill in the art that a treatment need not be completely effective in preventing the onset of a disease or in reducing the symptoms associated with the disease. Any reduction of the severity of symptoms, delay in the onset of symptoms, or delay in the progression of severity of symptoms is desirable to a patient.
- Immune disorders that can be treated by the methods of the invention include the diseases in which CD28 expressing T cells mediate or contribute to an undesired ldiopathic effect.
- the methods of the invention include, but are not limited to, methods of treating diseases in which pathogenesis is mediated through interleukin-2, interferon-gamma, interleukin-8, or a combination thereof, whereby a T cell mediated immune response is interrupted or reduced.
- diseases in which pathogenesis is mediated through interleukin-2, interferon-gamma, interleukin-8, or a combination thereof whereby a T cell mediated immune response is interrupted or reduced.
- immune disorders that may oe treated by administering the subject oligomers to a patient include organ transplantation rejection, septic shock, tumor-induced cachexia, and numerous auto-immune diseases.
- Autoimmune diseases that may be treated by the subject methods include diseases that are mediated by aberrant T cell activation
- Type I diabetes insulin-dependent diabetes, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, ulcerative colitis, aplastic anemia, systemic lupus erythematosus,
- compositions of the invention provide for the treatment of a variety of syndromes, including septic shock and tumor-induced cachexia, in which the pathogenic effects are mediated, at least in part, by the lymphokine secreted from activated CD28 + T cells.
- the disease treatment methods of the invention comprise the steps of administering an effective amount of the subject oligomers to a patient.
- the precise dosage, i . e . , effective amount, of CD28-specific oligomer to be administered to a patient will vary with numerous factors such as the specific disease to be treated, the precise oligomer (or oligomers) in the
- the invention contemplates methods of treatment in which CD28' cells (or ceils having the potential to express CD28) are removed from a patient (with or without other cells) and transformed with one or more different oligomers of the invention. Transformation may be by any of a variety of means well known to the person skilled in the art, e.g.,
- Transformed cells may then be reintroduced into the body.
- Another aspect of the invention is to provide methods of treating immune disorder by means of administering CD28-specific oligomers, wherein the oligomers are produced intracellularly through recombinant polynucleotide expression vectors.
- Intracellularly-produced CD28-specific oligomers are necessarily RNA or D ⁇ A molecules.
- Recombinant polynucleotide vectors for the expression of polynucleotide sequences of interest are well known to the person of ordinary skill in the art of molecular biology. Detailed descriptions of recombinant vectors for the expression of polynucleotides of interest can be found in, among other places, "Somatic Gene Therapy," ed. P. L. Chang, CRC Press, Boca Raton (1995), R. C. Mulligan, Science. 260:926-932 (1993), F. W. Anderson, Science, 256:808-873 (1992), Culver et al . , Hum. Gene Ther., 2:107-109 (1991), and the like. Suitable recombinant vectors for use in the subject methods of treating immune
- CD28-specific oligomers for use in
- intracellular administration are preferably significantly longer than CD28-specific oligomers for extracellular administration.
- the CD28-specific oligomer is complementary to one or more entire CD28 transcripts or the entire CD28 gene; however, suitable intracellularly-produced CD28-specific
- CD28-specific oligomers may be considerably shorter in length. Unlike extra-cellularly-administered CD28-specific oligomers, CD28-specific oligomers do not present problems with intracellular uptake or hydrolysis by extracellular enzymes.
- the subject methods of using intracellular CD28-specific oligomers may involve
- CD28-specific oligomer-encoding recombinant vectors directly to a patient.
- CD28-specific oligomer-producing vectors may be administered directly to cells that have been removed from a patient (i.e., stem cells, T cells, whole blood, marrow, etc. ) , whereby transformed cells are produced. The transformed cells may be subsequently be reintroduced into a patient .
- the invention also specifically provides for expression vectors capable of expressing one or more of the oligomers of the invention.
- expression vectors comprise, in operable combination, a promoter and a polynucleotide sequence encoding an oligomer capable of inhibiting the expression of CD28 in a T cell.
- promoters may be used in the vectors of the invention, preferred promoters are capable of driving the high level expression in T cells.
- the expression vectors of the invention may also comprise various regulatory sequences. Currently available expression vectors, especially those vectors explicitly designed for gene therapy, may readily be adapted for the expression of CD28-targeted oligomers of the invention.
- the vectors may be adapted for the expression of CD28-targeted oligomers using conventional genetic engineering techniques such as those described in Sambrook et al . , Molecular Cloning, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (1989).
- Another aspect of the invention is to provide pharmaceutical formulations for the administration of the oligomers of the invention so as to effect the treatment of immune system-mediated diseases. These pharmaceutical formulations may be reaoily produced by the person of ordinary skill in the art of
- Such formulations comprise one or more of the oligomers of the invention; however, in embodiments of the invention comprising more than one different types of oligomers, the oligomers are preferably selected so as to not be able to hybridize with on. another.
- the pharmaceutical formulations of the invention may be adapted for administration to the body in a number of ways suitable for the selected method of
- the subject pharmaceutical formulations may comprise one or more non-biologically active compounds, i . e . , excipients, such as
- stabilizers to promote long term storage
- emulsifiers to promote long term storage
- Dinding agents to promote long term storage
- thickening agents to promote long term storage
- salts to promote long term storage
- preservatives to promote long term storage
- Formulations for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives.
- formulations of the invention may be designed to promote the cellular uptake of the oligomers in the composition, e.g., the oligomers may be encapsulated in suitable liposomes.
- compositions for topical administration are especially useful for localized treatment.
- Formulations for topical treatment included ointments, sprays, gels, suspensions, lotions, creams, and the like.
- administration may include, in addition to the subject oligomers, known carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, etc.
- carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, etc.
- the pharmaceutically acceptable carrier may also include a known chemical absorption promoter. Examples of absorption promoters are e.g., dimethylacetamide (United States Patent No. 3,472,931),
- the oligomers may also be used as an analytical laboratory tool for the study of T cell activation.
- T cells nave several surface receptors in addition to CD28 and the antigen specific T cell receptors. Difficulties arise in studying the individual biological properties of selected receptors because of potential and actual interactions between multiple receptor-mediated pathways.
- the oligomers and methods of the invention al so provide useful laboratory methods for studying T cell behavior independently of the CD28 activation pathway.
- Oligodeoxynucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry. ⁇ -cyanoethylphosphoramidites, synthesis reagents and CPG polystyrene columns were purchased from Applied Biosystems (ABI, Foster City, CA) . 3'-AminoModifler C3 CPG columns were purchased from Glen Research (Sterling, VA). For pnospnorothioate oligonucleotides, the standard oxidation bottle was replaced with tetraethylthiuram
- PBMCs Peripheral blood mononuclear cells
- T-cell isolation reagent specific for T-cells (LK-25T, One Lambda, Canoga Park CA) .
- An average yield of 40 - 60 ⁇ 10 6 T-cells were then incubated overnight at 37°C in 20 - 30 ml RPMI-AP5
- RPMI-1640 medium (ICN, Costa Mesa, CA) containing 20 ⁇ M HEPES buffer, pH 7.4, 5% autologous plasma, 1 % L-glutamine, 1 % penicillin/streptomycin and 0.05% 2-mercaptoethanol) to remove any contaminating adherent cells.
- T-cells were washed with RPMI-AP5 and then plated on 96-well microtitre plates at a cell concentration of 2 - 3 ⁇ 10 6 cells/ml.
- T-cell lymphoma cell lines Jurkat E6-1 (CD28+/CD4+ . cells (152-TIB) and HUT 78 (CD28-/CD4+) cells (TIB-161) ⁇ ATCC, Rockville, MD) were maintained in RPMI-10 (RPMI-1640 medium containing 20 ⁇ M HEPES buffer, pH 7.4, 10 % fetal calf serum (FCS) (Hyclone, Logan, UT), 1 % L-glutamine and 1 % penicillin/streptomycin).
- FCS fetal calf serum
- microtitre plates were pre-coated with purified anti-CD3
- mAb monoclonal antibody
- Anti-CD3 mAb-treated T-cells were further activated by the addition of 2 ng phorbol 12-myristate 13-acetate (PMA) (Calbiochem, La Jolla, CA) and incubated for 48 h at 37°C.
- PMA phorbol 12-myristate 13-acetate
- Anti-CD3/PMA-activated T-cells were treated with 1 - 20 ⁇ M CD28-specific and control oligonucleotides immediately following activation and re-treated 24 h later.
- T-cells from one duplicate plate was used for immunofluorescence analysis and the supernatants used for cytokine studies and the second plate was used for T-cell proliferation analysis.
- fluorescence-labeled monoclonal antibodies were obtained from Bec.on Dickinson (San Jose, CA). Incubations were performed in the dark at 4°C for 45 min using saturating mAb concentrations. Unincorporated label was removed by washing in PBS prior to the analysis with a FACScan flow cytometer (Becton Dickinson).
- Antigen density was indirectly determined in gated live cells and expressed as the mean channel of fluorescence (MCF).
- MCF mean channel of fluorescence
- CD54, CD25 surface expression of specific antigens (CD54, CD25) was represented as the mean channel shift (MCS) obtained by subtracting the MCF of FITC- or PE-labeled isotype-matched (IgGl) control mAb-stained cells from the MCF of FITC- or PE-labeled antigen-specific mAb stained cells.
- MCS mean channel shift
- CD4 + -subset of cells stained with CD28 mAb was determined by subtracting the MCF of CD28' CD4 + from the MCF of CD28- CD4- cells.
- the viability of control untreated and oligonucleotide-treated cells were determined in each batch of all oligonucleotides in multiple donors by staining with the vital dye, propidium iodide (5 ⁇ g/ml final concentration).
- the percentage of live cells which excluded propidium iodide was determined by flow cytometry and was > 90 % (range 90 - 99 %) following treatment with all batches of all oligonucleotides at a dose range of 1 - 20 ⁇ M ( Figure 2) .
- Cell-derived human cytokine concentrations were determined in cell supernatants from the first duplicate microplate.
- IL-2 interleukin-2
- IL-2-dependent cell line CTLL-2 (ATCC, Rockville, MD)
- Mi togen-induced changes in interferon-gamma and interieukin-8 (IL-9) levels were determined by ELISA using kits from Endogen (Cambridge, MA) and R & D systems (Quantikine kit, Minneapolis, MN) respectively. All ELISA results were expressed as pg/ml and the CTLL-2 bioassay as counts per minute representing the
- the second duplicate microplate in all experiments were analyzed for changes in mitogen-induced T-cell proliferation. 72h following anti-CD3/PMA activation and in the absence or presence of oligonucleotides, cells were pulsed with 1 ⁇ Ci
- Anti-CD3/PMA treatment of human T-cells increased the surface expression of CD28 (using immunofluorescence analysis) from a MCS of 122 ⁇ 7.74 in resting T-cells to a MCS of 150 ⁇ 9.27
- anti-CD3/PMA-activated T-cells with phosphorothioate (denoted as S-oligomers, Figure 3B) and phosphorothioate-3' amine (denoted as A-oligomers, Figure 3C) forms of CD28-specific and control oligonucleotides in 2 donors and with 2 separate batches of each oligonucleotide.
- both phosphorothioate and phosphorothioate-3' amine forms cf RT03 and RT04 were the most active inhibitors of mitogen-induced CD28 expression, both inhibiting induced CD28 expression by greater than 50% (IC 50 ) at 5 ⁇ M or less.
- IC 50 50%
- the effect of anti-CD3 and PMA on T-cell proliferation are synergistic as shown in Figure 4A.
- Figure 4B shows a representative experiment of the effect of CD28-specific and control phosphorothioate oligonucleotides on
- Activated T-cells produce a variety of immunomodulatory cytokines including IL-2, interferon-gamma and IL-8.
- CD28 immunomodulatory cytokines
- CD28-specific oligonucleotides RT03 (SEQ ID NO: 3) and RT04 (SEQ ID NO: 4) but not the control oligonucleotides, RTC01 (SEQ ID NO: 5)- RTC06 (SEQ ID NO: 10) (data not shown)), inhibited
- the specificity of the CD28-specific oligonucleotides, RT03 and RT04 was evaluated by three methods.
- the first method was to determine whether these CD28- specific oligonucleotides were able to inhibit the expression of other human T-cell activation markers which act independently of the CD28 costimulatory pathway. Activation of resting T-cells significantly increases the expression of both the IL-2 receptor (CD25) and the intracellular adhesion molecule, ICAM-1 (CD54). However, both these accessory molecules are regulated
- Figure 8 shows the effect of CD28-specific and control oligonucleotides on CD25 ( Figure 8A) and CD54 ( Figure 8B) expression in mitogen- activated T-cells. No significant decrease in the activated T-cell expression of both CD25 and CD54 were observed following treatment with all CD28- specific and control oligonucleotides in the dose range 2 - 10 ⁇ M. This clearly
- the second method was to demonstrate that the CD28 pathway was really the target for CD28-specific oligonucleotides by comparing mitogen-induced IL-2 production in a CD28+, T-cell leukemia cell line, Jurkat E6-1 and a CD28-, T-cell lymphoma cell line, HUT 78.
- Figure 9A confirms the absence of CD28 expression in both resting and activated HUT 78 cells whereas constitutive levels of CD28 increases upon activation in Jurkat E6-1 cell s ( Figure 9B).
- CD28-specific but not control oligonucleotides were able to inhibit mitogen-induced CD28 expression (Figure 9C) and also mitogen-induced IL-2
- the third method was to activate resting T-cells specifically via the CD28 pathway using anti-CD28 monoclonal antibody in combination with mitogens (anti-CD3/PMA) using identical protocols to those used in activating T-cells with mitogens alone.
- Anti-CD28 mAb in combination with PMA or anti-CD3 mAb has been previously shown to provide the costimulatory signal to resting T-cells and promote only CD28-dependent and not
- CD28-specific but not control oligonucleotides were able to inhibit CD28-dependent activation of IL-2, IL-8 and interferon-gamma production and T-cell proliferation in
- Oligonucleotides were synthesized with an Applied Biosystems 394 DNA synthesizer. Phosphorothioate linkages were introduced after the standard oxidation bottle was replaced with tetraethyl- chiuram disulfide/acetonitrile. The purity of the oligonucleotides was assessed by analytical HPLC. All oligonucleotides of >90 % purity were lyophilized to dryness and reconstituted in water (400 ⁇ M). At least three batches of each oligomer listed in Tables 3 and 5 were used. 5' labeling of oligonucleotides was achieved using T4 polynucleotide kinase and 32 P- ⁇ ATP.
- PBMCs Peripheral blood mononuclear cells
- T cells Contaminating monocytes were removed by adherence to plastic.
- Purified T cells were > 99% CD2 + , ⁇ 1% HLA-DR + and ⁇ 5 % CD25 + .
- Jurkat E6-1 (CD237CD4 + ) T cells and HUT 78 (CD28VCD4 + ) T cells and the monocytic cell line, THP were obtained from ATCC.
- Cells were cultured at a concentration of 0.2 - 0.3 ⁇ 10 6 /well and activated with plate-immobilized anti-CD3 monoclonal antibody (mAb) (HIT3A 0.25 ⁇ g/ml) (Pharmingen) and 2 ng phorbol
- mAb plate-immobilized anti-CD3 monoclonal antibody
- PE-CD54/FITC-CD25 mAb or with PE/FITC-labeled isotype-matched controls (Becton Dickinson).
- Cell surface antigen density (CD28, CD54, CD25) was confirmed by flow cytometry (FACScan, Becton Dickinson).
- Viability was assessed by propidium iodide (5 ⁇ g/ml) exclusion in control untreated and oligo-treated CD4 + cells from multiple donors and was typically > 90 % (range 90 - 99 %) following 48h incubation with 1 - 10 ⁇ M of each batch of all oligonucleotides .
- the cDNA synthesis reaction (Promega) was performed using oligomer (dT) 15 primer and AMV reverse transcriptase (H. C).
- the PCR reaction (GeneAmp PCR kit, Perkin-Elmer Cetus) consisted of 50 ⁇ l mixture containing 3 ⁇ l of cDNA, dNTPs (each at 200 ⁇ M), 0.5 ⁇ M of each primer and 1 unit of Taq polymerase.
- the primers used were as follows :CD28, 5'-CTGCTCTTGGCTCTCAACTT-3' (sense) and 5' AAGCTATAGCAA GCCAGGAC- 3' (antisense), inter-leukin-2 receptor p55 alpha chain primers (Stratagene) and pHE7 ribosomal gene. Kao, H.-T., Nevins, J. R. (1983) Mol. Cell.
- PBMCs were cultured (1:1) with mitomycin C-treated (50 ⁇ g/ml) PBMCs from a HLA-disparate individual.
- mitomycin C-treated PBMCs 50 ⁇ g/ml PBMCs from a HLA-disparate individual.
- alloantigen-specific T cell assays T cells isolated from PBMCs of tetanus-toxoid-primed healthy donors were cultured (1:1) with autologous mitomycin C-treated PBMCs in the presence of tetanus toxoid (2 ⁇ g/ml. List Biologicals). In both assays, 2 ⁇ 10 5 cells/well were cultured for 6 days at 37°C prior to further analysis.
- Inhibition of CD28 expression by phosphorothioate oligonucleotides is specific and affects activated T cell function.
- Figure 11 summarizes the effect of the phosphorothioate oligonucleotides from Table 3 on surface expression of accessory molecules and on cytokine secretion in activated T cells.
- the oligomers used were designed to hybridize to the 5' untranslated region (UT) of the CD28 gene, and were either antisense (AS) or G-rich sequences.
- Control oligomers were either sense strand ( SS ) or complementary strand (CS) sequences.
- oligomers (Table 3) can specifically block activation-induced CD28 expression in CD4 + T cells.
- Figure 12 shows the effect of phosphorothioate oligonucleotides at 10 ⁇ M on CD28 and CD25 mRNA levels. Resting (lane 1) and anti-CD3/PMA-activated (6h) levels of CD28 and CD25 mRNA, in the absence (lane 2) or presence of the oligonucleotides, RT03S (SEQ ID NO: 44) (lane 4), RTCO6S (SEQ ID NO: 64) (lane 5) and RT03D (SEQ ID NO: 49) (lane 6) were detected following RT-PCR of total cellular RNA and Southern analysis using specific, radiolabeled probes.
- RT03S SEQ ID NO: 44
- RTCO6S SEQ ID NO: 64
- RT03D SEQ ID NO: 49
- the CD28 probe was derived from exon 2 (5'-ACGGGGTTC AACTGTGATGGGAAATTGGGCAA-3' ) and for IL-2 receptor, the probe was generated from the original primer mix. Equivalent loading was assessed following hybridization with a probe generated from pHE7 sense primer. RNA from CD28-deficient HUT (7) and THP (8) cell lines were used as controls. The data shown are representative of 3 separate experiments.
- active oligomers abrogated activation-in ⁇ uced T cell function, as RT03S (SEQ ID NO: 44) and RT04S (SEQ ID NO: 45) but not RT01S (SEQ ID NO: 42) or RT02S (SEQ ID NO: 43) or the control oligomers, RTC01S (SEQ ID NO: 46), RTC02S (SEQ ID NO: 47) and RTC06S (SEQ ID NO: 48), markedly inhibited anti-CD3/PMA-induced synthesis of the cytokines, IL-2, IFN ⁇ and IL-8 by activated T cells (IC 50 ⁇ 5 ⁇ M, range 2 - 10 ⁇ M) ( Figure 11B).
- lymphokine synthesis in activated T cells (Damle, N. K.,
- CD28-independent IL-2 secretion in activated HUT 78 cells (D, right). The data shown are representative of three separate experiments.
- T cell activation (expression of CD28, IL-2, IL-8 and IFN ⁇ ) directed by a specific anti-CD28 mAb in combination with anti-CD3, was blocked by biologically active phosphorothioate oligomers (data not shown) .
- Direct crosslinking of CD28 molecules has been previously shown to promote only
- RT03S SEQ ID NO: 44
- RT04S SEQ ID NO: 45
- resting (A, B) and activated (E, F) levels of CD28 are indicated for tetanus toxoid-specific T cell assay (top panel, A and B) and mixed lymphocyte reaction (bottom panel, E and F).
- the percentage of CD4 + , CD28-hi T cells is shown in the right-hand marker for A, B, E and F.
- Oligomer activity was assessed by the potential of two additions (0 and 96h) of 1 ⁇ M ( ) 2 ⁇ M
- Results are expressed as the difference in surface antigen expression of activated T cells (MCF A ) and oligonucleotide-treated activated T cells (MCF x ).
- CD28 expression in resting T cells on day 2 to 4 was in the range 119 - 121. "ND" represents no distinguishable difference.
- Figure 15 summarizes test results on the in vi tro stability of 32 P-labeled phosphorothioates, RT03S (SEQ ID NO: 44) and RTC06S (SEQ ID NO: 48) in extracellular supernatants (top panel) and Jurkat cell lysates (bottom panel).
- RT03D had little bioactivity (Table 5) and from in vi tro stability studies, only had a half-life of 24h (data not shown).
- IL-2 production The values for the phosphodiester form of RT03D (SEQ ID NO: 49) are in parentheses.
- RT03S Active phosphorothioate, RT03S (SEQ ID NO: 44), is an 18 mer originally designed to hybridize to the 5' untranslated region of the human CD28 gene, and has a sequence containing two sets of contiguous four G's. To identify the sequence-related factors critical for inhibition of activation-induced CD28 expression in human T cells and CD28-dependent IL-2 production in Jurkat T cells, bases were selectively added, deleted or substituted from RT03S (SEQ ID NO: 44) and activity assessed relative to the parent oligomer (Table 5).
- deletion or replacement of one or more G's by cytosine (C) within both sets of four G's resulted in a marked loss of activity relative to RT03S (SEQ ID NO: 44).
- RT19S SEQ ID NO: 58
- R20S SEQ ID NO: 61
- RT21S SEQ ID NO: 62
- TGGGG, GGGG or sequences containing 4 consecutive G's such as RT15S (SEQ ID NO: 63 had little or no inhibitory activity relative to RT03S (SEQ ID NO: 44).
- oligo-mediated effects on IL-2 receptor and ICAM-1 expression two accessory molecules known to be regulated independently of the CD28 pathway (Damle, N. K., et al . , (1992) J. Immunol. 148, 1985-1992; June, CH. et al . , (1987) Mol. Cell Biol. 7, 4472-4481; Stein, C. A., et al .
- CD28-specific oligomers must abrogate this function.
- Administration of active oligomers resulted in concomitant modulation of activation-induced IL-2, IFN ⁇ and IL-8 production.
- oligomers mediated alloantigen-specific tolerance in vi tro provide a promising alternative to the ligand capture strategy for inducing T cell hyporesponsiveness such as seen with CTLA 4 Ig, a high affinity B7 binder (Tan, P., Anasetti, C, Hansen, J. A., Melrose, J., Brunvand, M., Bradshaw, J., Ledbetter, J. A., Linsley, P.S. (1993) J. EXP. Med. 177, 165-173.).
- RT03S SEQ ID NO: 44
- RTC06S SEQ ID NO: 48
- our in vi tro stability studies showed that secondary structure, mediated by the G-rich sequence in RT03S (SEQ ID NO: 44), increased two- to fourfold the nuclease resistance typically associated with phosphorothioates (Stein, C. A., Cheng, Y. C. (1993) Science 261, 1004-1012.).
- the extended half life (96h) of 32 P-RT03S (SEQ ID NO: 44) correlated with its duration of bioactivity.
- RT03D the phosphodiester counterpart of RT03S (SEQ ID NO: 44)
- nuclease stability granted by both phosphorothioate modification and secondary structure may account for the prolonged inhibitory activity of RT03S (SEQ ID NO: 44).
- 3202-3209 demonstrated that activity of their phosphorothioate oligomers was based on possible G-quartet formation in sequences containing two sets of three or more consecutive G and this suggested that oligo-mediated regulation of human phospholipase A, was through specific nucleic acid-protein interaction. Specific protein recognition by a range of G-quartet structures have been demonstrated in telomeres, centromeres
- oligomers capable of forming an intermolecular four stranded G-quartet structure from a set of four contiguous G's such as those in telomeres (Smith, F. W., Feigon, J. (1992) Nature 356, 164-167), weakly inhibited CD28 expression.
- RT15S SEQ ID NO: 63, whose G-rich sequence was previously shown by others to inhibit c-myc expression (sequence 14 in Burgess, T. L., Fisher, E. F., Ross, S.
- RT03S (SEQ ID NO: 44) arises from an alternate G-quartet structure.
- RT03S (SEQ ID NO: 44) indeed has a similar 12 mer sequence to a motif predicted by others (Smith, F. W., Feigon, J. (1993) Biociemistry 32, 8682-8692) to be essential for dimeric G-quartet formation.
- Dimeric G-quartets can arise from two strands of DNA, alternately parallel and antiparallel .
- adjacent strands contribute four G' s to form four stacked G-quartets.
- a motif on each strand consisting of twelve residues with four bases separating two sets of contiguous four G's, was associated with formation and stability.
- RT18S SEQ ID NO: 57
- G to C substitutions R10S (SEQ ID NO: 53), RT23S (SEQ ID NO: 56), RT24S (SEQ ID NO: 54), RT25S (SEQ ID NO: 55)
- R10S SEQ ID NO: 53
- RT23S SEQ ID NO: 56
- RT24S SEQ ID NO: 54
- RT25S SEQ ID NO: 55
- Double stranded oligomers can act as decoys for the transcription factor, E2F (Morishita, R., Gibbons, G. H., Horuchi, M., Ellison, K. E., Nakajima, M., Zhang, L., Kaneda, Y., Ogihara, T., Dzau, V. J. (1995) Proc. Natl. Acad. Sci. USA 92, 5855-5859). 6) G-rich oligomers have been shown to mediate the induction of Spl transcription factor (Perez, J. R., Li, Y., Stein, C. A.,
Abstract
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EP96909477A EP0810882A4 (en) | 1995-02-09 | 1996-02-05 | Methods and compositions for regulation of cd28 expression |
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Cited By (8)
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WO1997020924A1 (en) * | 1995-12-04 | 1997-06-12 | Saicom S.R.L | A class of oligonucleotides, therapeutically useful as antitumoural agents |
EP0968226A1 (en) * | 1996-12-27 | 2000-01-05 | ICN Pharmaceuticals, Inc. | G-rich oligo aptamers and methods of modulating an immune response |
WO2000014217A2 (en) * | 1998-09-03 | 2000-03-16 | Coley Pharmaceutical Gmbh | G-motif oligonucleotides and uses thereof |
US6943240B2 (en) | 2000-09-15 | 2005-09-13 | Coley Pharmaceuticals Gmbh | Nucleic acids for high throughput screening of CpG-based immuno-agonist/antagonist |
US7723500B2 (en) | 1994-07-15 | 2010-05-25 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
WO2014090985A1 (en) * | 2012-12-13 | 2014-06-19 | Universität Leipzig | T-cell modulation by exon skipping |
US20150267204A1 (en) * | 2009-07-24 | 2015-09-24 | Curna, Inc. | Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt) |
US11390868B2 (en) | 2009-09-25 | 2022-07-19 | Curna, Inc. | Treatment of filaggrin (FLG) related diseases by modulation of FLG expression and activity |
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WO1992015671A1 (en) * | 1991-03-08 | 1992-09-17 | Cytomed, Inc. | Soluble cd28 proteins and methods of treatment therewith |
WO1994028123A1 (en) * | 1993-05-26 | 1994-12-08 | Ontario Cancer Institute | Mammals lacking expression of cd28 transgenic |
-
1996
- 1996-02-05 PL PL96321711A patent/PL321711A1/en unknown
- 1996-02-05 EP EP96909477A patent/EP0810882A4/en not_active Withdrawn
- 1996-02-05 WO PCT/US1996/001507 patent/WO1996024380A1/en not_active Application Discontinuation
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WO1992015671A1 (en) * | 1991-03-08 | 1992-09-17 | Cytomed, Inc. | Soluble cd28 proteins and methods of treatment therewith |
WO1994028123A1 (en) * | 1993-05-26 | 1994-12-08 | Ontario Cancer Institute | Mammals lacking expression of cd28 transgenic |
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See also references of EP0810882A4 * |
THE JOURNAL OF IMMUNOLOGY, Volume 145, Number 1, issued 01 July 1990, LEE et al., "The Genomic Organization of the CD28 Gene: Implications for the Regulation of CD28 mRNA Expression and Heterogeneity", pages 344-352. * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
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US7723500B2 (en) | 1994-07-15 | 2010-05-25 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
WO1997020924A1 (en) * | 1995-12-04 | 1997-06-12 | Saicom S.R.L | A class of oligonucleotides, therapeutically useful as antitumoural agents |
EP0968226A1 (en) * | 1996-12-27 | 2000-01-05 | ICN Pharmaceuticals, Inc. | G-rich oligo aptamers and methods of modulating an immune response |
EP0968226A4 (en) * | 1996-12-27 | 2001-02-14 | Icn Pharmaceuticals | G-rich oligo aptamers and methods of modulating an immune response |
US6994959B1 (en) * | 1996-12-27 | 2006-02-07 | Valeant Research & Development | G-rich oligo aptamers and methods of modulating an immune response |
WO2000014217A2 (en) * | 1998-09-03 | 2000-03-16 | Coley Pharmaceutical Gmbh | G-motif oligonucleotides and uses thereof |
WO2000014217A3 (en) * | 1998-09-03 | 2000-07-13 | Cpg Immunopharmaceuticals Gmbh | G-motif oligonucleotides and uses thereof |
AU777225B2 (en) * | 1998-09-03 | 2004-10-07 | Coley Pharmaceutical Gmbh | G-motif oligonucleotides and uses thereof |
US6943240B2 (en) | 2000-09-15 | 2005-09-13 | Coley Pharmaceuticals Gmbh | Nucleic acids for high throughput screening of CpG-based immuno-agonist/antagonist |
US7820379B2 (en) | 2000-09-15 | 2010-10-26 | Coley Pharmaceutical Gmbh | Process for high throughput screening of CpG-based immuno-agonist/antagonist |
US20150267204A1 (en) * | 2009-07-24 | 2015-09-24 | Curna, Inc. | Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt) |
US10563202B2 (en) * | 2009-07-24 | 2020-02-18 | GuRNA, Inc. | Treatment of Sirtuin (SIRT) related diseases by inhibition of natural antisense transcript to a Sirtuin (SIRT) |
US11390868B2 (en) | 2009-09-25 | 2022-07-19 | Curna, Inc. | Treatment of filaggrin (FLG) related diseases by modulation of FLG expression and activity |
WO2014090985A1 (en) * | 2012-12-13 | 2014-06-19 | Universität Leipzig | T-cell modulation by exon skipping |
Also Published As
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EP0810882A4 (en) | 1999-05-19 |
HUP9801629A3 (en) | 2000-10-30 |
HUP9801629A2 (en) | 1998-10-28 |
PL321711A1 (en) | 1997-12-22 |
EP0810882A1 (en) | 1997-12-10 |
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