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  Modulation of oligonucleotide cpg-mediated immune stimulation by positional modification of nucleosidesUSPTO Application #: 20060142556Title: Modulation of oligonucleotide cpg-mediated immune stimulation by positional modification of nucleosides Abstract: The invention provides methods for modulating the immune response caused by CpG dinucleotide-containing compounds. The methods according to the invention enables both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. (end of abstract)
Agent: Joseph C. Zucchero - Woburn, MA, US Inventor: Sudhir Agrawal USPTO Applicaton #: 20060142556 - Class: 536023100 (USPTO) Related Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060142556. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09/845,623, filed on Apr. 30, 2001, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/201,578, filed on May 1, 2000. Each of the patent applications listed above is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates to the therapeutic use of oligonucleotides, both in the antisense approach, and as immunostimulatory agents. [0004] 2. Summary of the Related Art [0005] Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides. [0006] The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach. [0007] Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Anti. Acad. Sci. USA.about.: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry. [0008] More recently, several researchers have demonstrated the validity of the antisense approach to therapeutic treatment of disease. Crooke, Antisense Nucleic Acid Drug Dev. 8: vii-viii, discloses the successful marketing approval of a phosphorothioate oligonucleotide for the treatment of human cytomegalovirus induced retinitis. Unfortunately, the use of phosphorothioate oligonucleotides has become more complex than originally expected. Certain effects caused by phosphorothioate oligonucleotides could not be explained by the expected antisense mechanism. For example, McIntyre et al., Antisense Res. Dev. 3: 309-322 (1993) teaches that a "sense" phosphorothioate oligonucleotide causes specific immune stimulation. This and other side effects have complicated the picture for phosphorothioate oligonucleotides. [0009] On the other hand, the observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in developing this side effect as a therapeutic tool. These efforts have focused on phosphorothioate oligonucleotides containing the dinucleotide CpG. Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) teaches that phosphodiester oligonucleotides containing a palindrome that includes a CpG dinucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al., Nature 371: 546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al., Vaccine 16: 1216-1224 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, The Journal of Immunology 161: 4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent adjuvants, enhancing immune response against hepatitis B surface antigen. [0010] These reports make clear that there is a need to be able to modulate the immune response caused by CpG-containing oligonucleotides. Ideally, such modulation should include decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. BRIEF SUMMARY OF THE INVENTION [0011] The invention provides methods for modulating the immune response caused by CpG dinucleotide-containing compounds. The methods according to the invention enable both decreasing the immunostimulatory effect for antisense applications, as well as increasing the immunostimulatory effect for immunotherapy applications. Thus, the invention further provides compounds having optimal levels of immunostimulatory effect for either application and methods for making and using such oligonucleotides. [0012] The present inventor has surprisingly discovered that positional modification of CpG-containing oligonucleotides dramatically affects their immunostimulatory capabilities. In particular, 2' or 3' sugar or base modifications of oligonucleotides, or introduction of a modified internucleoside linkage, at particular positions 5' or 3', including 5' or 3' end modifications, to the CpG dinucleotide either enhances or reduces their immunostimulatory effect in a reproducible and predictable manner. [0013] In a first aspect, the invention provides a method for modulating the immunostimulatory effect of a CpG dinucleotide containing compound by introducing an immunomodulatory moiety at a position either 5' to or 3' to the CpG dinucleotide. [0014] In a second aspect, the invention provides compounds having increased or reduced immunostimulatory effect, the compounds comprising a CpG dinucleotide and an immunomodulatory moiety, wherein the increased or reduced immunomodulatory effect is relative to a similar compound lacking the immunomodulatory moiety. [0015] In a third aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, including a human, the method comprising administering to the mammal an oligonucleotide that is complementary to the gene and which comprises a CpG dinucleotide and an immunomodulatory moiety, wherein the oligonucleotide has less immunostimulatory effect than a similar oligonucleotide lacking the immunomodulatory moiety. [0016] In a fourth aspect, the invention provides a method for inducing an immune response in a mammal, including a human, the method comprising administering to the mammal a compound comprising a CpG dinucleotide and an immunomodulatory moiety, wherein the compound has greater immunostimulatory effect than a similar compound lacking the immunomodulatory moiety. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1 shows preferred embodiments of immunomodulatory moieties according to the invention. Note that the Figures use X3X4 for the 3' side and X1X2 for the 5' side. This use is illustrative for this figure only and should not be used to interpret the claims, which use the Y and X designations taught in this specification. [0018] FIG. 2 (SEQ ID NOs 1-3, respectively, in order of appearance) shows a modified compound according to the invention and results of spleen assays using this compound. [0019] FIG. 3 (SEQ ID NOs 4-6, respectively, in order of appearance) shows another modified compound according to the invention and results of spleen assays using this compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Continue reading... Full patent description for Modulation of oligonucleotide cpg-mediated immune stimulation by positional modification of nucleosides Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Modulation of oligonucleotide cpg-mediated immune stimulation by positional modification of nucleosides patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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