Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-02-17
2004-04-20
Lacourciere, Karen A. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S091100, C435S325000, C435S375000, C435S006120, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06723706
ABSTRACT:
This invention relates to a specific modified oligonucleotide complementary to a section of the human Ha-ras gene and mRNA, and its use to specifically regulate, modulate or inhibit expression of the Ha-ras gene, and its use as a pharmaceutical for the treatment of conditions arising from abnormal expression of the Ha-ras gene.
Antisense oligonucleotides (AO) have proven to be specific inhibitors of gene expression in a large number of systems, both in vivo and in vitro. (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
One of the major problems encountered when using unmodified oligonucleotides containing only phosphodiester internucleoside linkages (PO-oligonucleotides) is the rapid degradation of this type of oligonucleotide in cells and biological fluids, such as, for example, serum and cerebrospinal fluid, by a range of nucleolytic activities. A wide range of chemical modifications to oligonucleotides have been carried out in order to improve their nucleolytic stability (Uhlmann and Peyman, Chem. Res. 1990, 90, 543). These modifications include the modification or replacement of the phosphodiester internucleoside linkage, the sugar unit, the nucleobase; or the sugar-phosphate backbone of the oligonucleotides. The most thoroughly investigated type of modification is alteration of the internucleoside linkage, including phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PSS) linkages. It should be stressed that modification of the oligonucleotide alters not only its nuclease stability but also other characteristics of the oligonucleotide, such as, for example, their cellular uptake, RNaseH activation, and the strength and specificity of binding to their target nucleic acid, and the like. It should be borne in mind that the stability of the modified oligonucleotide in serum, frequently used to determine the nuclease stability of the oligonucleotide, is not the sole determinant of intracellular activity (P. D. Cook in “Antisense Research and Applications”, Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chap.9, p149 et seq.).
The phosphorothioate (PS) modified oligonucleotides are the most widely used type of modified oligonucleotide. The following strategies have been developed for the positioning of PS linkages in antisense oligonucleotides:
(1) Replacement of all phosphodiester internucleoside linkages with phosphorothioate linkages.
The resulting all-phosphorothioate oligonucleotides are much more stable to nucleases than PO-oligonucleotides (Monia et al. J. Biol. Chem. 1996, 271, 14533). For example, degradation of all-PS oligonucleotides by endonucleases is slowed down by a factor of 2-45 relative to a PO oligonucleotide (Stein et al. Nucleic Acids Res. 1988, 16, 3209). In Xenopus oocytes or embryos, the degradation of microinjected PO oligonucleotides proceeds with a half-life of 30 minutes, while all-PS oligonucleotides have a half-life of over 3 hours under the same conditions (Woolf et al. Nucleic Acids Res. 1990, 18, 1763). All-PS oligonucleotides retain their ability to activate RNaseH. The major disadvantages of all-PS oligonucleotides are that their ability to form stable hybrids with their target nucleic acid is reduced, and that they frequently give rise to unspecific “non-antisense” effects (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(2) Oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages.
In an effort to overcome the non-antisense effects observed with all-PS oligonucleotides, oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages have been synthesized and tested for stability and biological activity.
Ghosh et al. (Anticancer Drug Design 1993, 8, 15) describe a PS—PO oligonucleotide containing various percentages of PS linkages. Their construction follows, for example, the pattern (PS—PO—PO—PO)
n
, (PO—PO—PS)
n
, (PS—PO)
n
, [(PO)
2
—(PS)
2
]
n
, [PO—PS—PS]
n
. They teach that a PS linkage content of at least 50% is required for selective translation inhibition in vitro and that activity drops drastically when the PS content is less than 50%. More recently it has been demonstrated that an oligonucleotide containing 50% PS-linkages arranged in the pattern (PS—PO)
n
showed no biological activity in an assay system where an all-PS oligonucleotide and “end-capped” PO—PS oligonucleotides (see below) of the same sequence were highly active (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(3) “End-capped” Oligonucleotides, where one, two or three internucleoside bridges on the 5′ and/or the 3′ end of the oligonucleotide are phosphorothioate modified. (also known as the “gap technique”)
This type of modification is designed primarily to protect the oligonucleotide from degradation by exonucleases. In particular modifications at the 3′-end of the oligonucleotide are desirable as they offer protection from 3′-exonucleases, which are the most abundant nucleases in serum (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
An interesting comparison of strategies is found in Hoke et al. (Nucleic Acids Res. 1991, 19, 5743). The authors compare the activity of a range of antisense PS-oligonucleotides against HSV-1 in cell culture. Their findings confirm that 3′, or 3′+5′, end-capped oligonucleotides (the first three internucleoside linkages being modified in each case), similarly to all-PS oligonucleotides are sufficiently protected against degradation by nucleases in serum. In contrast internally modified (three PS bridges) oligonucleotides and oligonucleotides in which only the 5′-end has been capped (again, the first three internucleoside linkages being modified) are degraded rapidly. In contrast, the authors found that neither 5′ nor 3′ end capping nor both are sufficient for activity within the cell, and they drew the conclusion that a uniform modification (all-PS) is required to achieve sufficient stability to nucleases in cells.
More recently it has been discovered that pyrimidine nucleosides are the most nuclease susceptible points in oligonucleotides (Peyman, A. and Uhlmann, E., Biol. Chem. Hoppe-Seyler 1996, 377, 67; EP 0 653 439 A2). It was found that a combination of end-capping and PS protection of the pyrimidine positions of oligonucleotides (the so called “minimal modification” approach) is sufficient to make them highly resistant to nuclease degradation. The biological activity (against Herpes simplex virus) of an oligonucleotide with this type of PS modification pattern was comparible to that of an all-PS oligonucleotide.
One of the major problems encountered when using AOs, whether or not they are stabilized against degradation, is their poor cellular uptake. Many approaches have been tried to attempt to ameliorate this problem. Most of these approaches involve the attachment of a variety of substances to the oligonucleotide. Modifications include: the attachment of peptides to oligonucleotides (Lemaitre, M. et al. Proc. Natl. Acad. Sci. USA 1987, 84, 648) and the attachment of lipophilic residues, such as alkyl chains or cholesterol, to oligonucleotides (Saison-Behmoaras, T. et al. EMBO J. 1991, 10, 1111-1118; Will, D. W. and Brown, T. Tetrahedron Lett. 1992, 33, 2729). It has been found, however, that in many cases the introduction of a lipophilic group causes biological effects which are independent of the sequence of the oligonucleotide. Non-specific effects have been reported for cholesterol-oligonucleotide conjugates (Henderson, G. B. and Stein, C. A. Nucleic Acids Res. 1995, 23, 3726.), and for oligonucleotides attached to alkyl chains (Shea, R. G. et al. Nucleic Acids Res. 1990, 18, 3777). Saison-Behmoaras et al (EMBO J. 1991, 10, 1111-1118; WO 96/34008) have reported that a 9mer all-PO oligonucleotide derivatized with a 3′-dodecanol moiety and a 5′-acridine crosslinking agent, and antisense to mutated Ha-ras inhibited T24 human bladder carcinoma cell proliferation. No comparison of the antiproliferative activity of this oligonucleotide with that o
Chang Esther
Peyman Anuschirwan
Pirollo Kathleen
Rait Antonina
Uhlmann Eugen
Aventis Pharma Deutschland GmbH
Lacourciere Karen A.
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