Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2001-01-26
2004-11-09
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S375000, C435S455000, C536S023100
Reexamination Certificate
active
06815429
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the therapeutic use of oligonucleotides, both in the antisense approach, and as immunostimulatory agents.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modem 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.
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.
Both of these modem 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. Antl. Acad. Sci. USA
85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
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.
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 focussed 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: 1216-124 (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.
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
The invention provides methods for modulating the immune response caused by CpG-containing oligonucleotides. 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. Thus, the invention further provides oligonucleotides having optimal levels of immunostimulatory effect for either application and methods for making and using such oligonucleotides.
The present inventor has surprisingly discovered that positional modification of CpG-containing oligonucleotides dramatically affects their immunostimulatory capabilities. In particular, 3′ alkylation or alkoxylation of oligonucleotides, or introduction of an uncharged internucleoside linkage, at particular positions 5′ or 3′ to the CpG dinucleotide either enhances or reduces their immunostimulatory effect in a reproducible and predictable manner.
In a first aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing a 3′ substituted nucleoside into the oligognucleotide at a position adjacent to, and on the 5′ and/or 3′ side of the CpG dinucleotide. In preferred embodiments, this method includes creating a 2′-5′ linkage between the 2′ position of a 3′ substituted nucleoside and the 5′ position of another nucleoside, which may or may not be a 3′ substituted nucleoside.
In a second aspect, the invention provides a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 3′ substituted nucleoside at a position adjacent to, and on the 5′ and/or 3′ side of the CpG dinucleotide. In preferred embodiments, CpG-containing oligonucleotides according to this aspect of the invention include a 2′-5′ linkage between the 2′ position of a 3′ substituted nucleoside and the 5′ position of another nucleoside, which may or may not be a 3′ substituted nucleoside.
In a third aspect, the invention provides a method for obtaining an antisense-specific reduction in the expression of a gene in a mammal, the method comprising administering to the mammal a CpG-containing oligonucleotide having a reduced immunostimulatory effect, wherein the oligonucleotide comprises a 3′ substituted nucleoside at a position adjacent to, and on the 5′ and/or 3′ side of the CpG dinucleotide. In preferred embodiments, CpG-containing oligonucleotides used in this aspect of the invention include a 2′-5′ linkage between the 2′ position of a 3′ substituted nucleoside and the 5′ position of another nucleoside, which may or may not be a 3′ substituted nucleoside.
In a fourth aspect, the invention provides a method for reducing the immunostimulatory effect of a CpG-containing oligonucleotide. The method according to this aspect of the invention comprises introducing an uncharged internucleoside linkage into the oligonucleotide at a position adjacent to, and on the 5&pr
Hybridon, Inc.
Keown & Associates
LeGuyader John L.
Zara Jane
LandOfFree
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