Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-02-11
2004-12-14
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C514S001000, C514S04400A, C435S006120, C536S024300, C536S025300, C536S026600
Reexamination Certificate
active
06831166
ABSTRACT:
FIELD OF THE INVENTION
This application is directed to sequence specific oligonucleotides that include functionalized nucleosides having substituents such as steroids, reporter molecules, reporter enzymes, non-aromatic lipophilic molecules, peptides, or proteins attached via linking groups.
BACKGROUND OF THE INVENTION
Messenger RNA (mRNA) directs protein synthesis. Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding of oligonucleotides to RNA or single-stranded DNA via complementary Watson-Crick base pairs.
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in
Oligonucleotides: Antisense Inhibitors of Gene Expression
, CRC Press, Inc., Boca Raton, Fla. (1989), are thought to be of two types. The first, hybridization arrest, denotes a terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (see, e.g., Miller, et al.,
Anti
-
Cancer Drug Design
1987, 2, 117) and &agr;-anomer oligonucleotides, the two most extensively studied antisense agents, are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of targeted RNA by intracellular RNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA to form a duplex that activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides provide the most prominent example of antisense agents that operate by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research reagents, and therapeutics. At least for therapeutic purposes, the antisense oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express activity. One method for increasing membrane or cellular transport is by the attachment of a pendant lipophilic group.
Ramirez, et al.,
J. Am. Chem. Soc
. 1982, 104:, 5483, introduced the phospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3′ and 5′ positions. Subsequently Shea, et al.,
Nuc. Acids Res
. 1990, 18, 3777, disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain of the Shea, et. al. authors disclosed these and other compounds in patent application PCT/US90/01002. Another glucosyl phospholipid was disclosed by Guerra, et al.,
Tetrahedron Letters
1987, 28, 3581.
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3′ terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and by Letsinger, et al.,
Proc. Natl. Acad. Sci. USA
1989, 86, 6553. The aromatic intercalating agent anthraquinone was attached to the 2′ position of a sugar fragment of an oligonucleotide as reported by Yamana, et al.,
Bioconjugate Chem
. 1990, 1, 319.
Lemairte, et al.,
Proc. Natl. Acad. Sci. USA
1986, 84, 648 and Leonetti, et al.,
Bioconjugate Chem
. 1990, 1, 149, disclose modifying the 3′ terminus of an oligonucleotide to include a 3′-terminal ribose sugar moiety. Poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of this terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0, wherein the lysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′ terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3′ terminus of an oligonucleotide to link a peptide to the oligonucleotide, as described by Corey, et al.,
Science
1987, 238, 1401; Zuckermann, et al.,
J. Am. Chem. Soc
. 1988, 110, 1614; and Corey, et al.,
J. Am. Chem. Soc
. 1989, 111, 8524.
Nelson, et al.,
Nuc. Acids Res
. 1989, 17, 7187 describe a linking reagent for attaching biotin to the 3′-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol, is commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3′-Amine on and from Glen Research Corporation (Sterling, Va.) under the name 3′-Amino-Modifier. This reagent was also utilized to link a peptide to an oligonucleotide, as reported by Judy, et al.,
Tetrahedron Letters
1992, 32, 879. A similar commercial reagent (actually a series of linkers having various lengths of polymethylene connectors) for linking to the 5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6, also from Glen Research Corporation. These compounds or similar ones were utilized by Krieg, et al.,
Antisense Research and Development
1991, 1, 161 to link fluorescein to the 5′-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3′-terminus of an oligonucleotide. Asseline, et al.,
Proc. Natl. Acad. Sci. USA
1984, 81, 3297 described linking acridine on the 3′-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, et al.,
Tetrahedron Letters
1987, 28, 5199 reported building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3′hydroxyl group of the 3′terminal nucleotide of the oligonucleotide. Chollet,
Nucleosides & Nucleotides
1990, 9, 957 attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5′ terminal phosphate of an oligonucleotide. They then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
An EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer, et al.,
Proc. Natl. Acad. Sci. USA
1985, 82, 968. Fluorescein has been linked to an oligonucleotide in the same manner, as reported by Haralambidis, et al.,
Nucleic Acid Research
1987, 15, 4857 and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, et al.,
J. Am. Chem. Soc
. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT from Glen Research Corporation, can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Cholic acid linked to EDTA for use in radioscintigraphic imaging studies was reported by Betebenner, et al.,
Bioconjugate Chem
. 1991, 2, 117; however, it is not known to link cholic acid to nucleosides, nucleotides or oligonucleotides.
OBJECTS OF THE INVENTION
It is an object of this invention to provide sequence-specific oligonucleotides having improved transfer across cellular membranes.
It is a further object of this invention to provide improvements in research and diagnostic methods and materials for assaying bodily states in animals, especially disease states.
It is an additional object of this invention to provide therapeutic and research materials having improved transfer and uptake properties for the treatment of diseases through modulation of the activity of DNA or RNA.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with these and other objects evident from this specification, there are provided compounds that comprise a plurality of linked nucleosides wherein at least one of the nucleosides is functionalized at the 2′-position with a subst
Bennett Clarence Frank
Cook Phillip Dan
Manoharan Muthiah
ISIS Pharmaceuticals Inc.
Riley Jezia
Woodcock & Washburn LLP
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