2′ Modified oligonucleotides

Details 2′ Modified oligonucleotides 2′ Modified oligonucleotides

1998-08-10

2002-11-05

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S016800, C536S026260, C536S026700, C536S026800, C536S027210, C536S028100, C536S022100, C536S025300, C536S025320, C536S025340

Reexamination Certificate

active

06476205

ABSTRACT:

TECHNICAL FIELD
The invention relates to modified oligonucleotides useful in technologies which rely on complementarity or specificity of oligomer sequences for drug delivery or for direct interference with nucleic acid activity. More specifically, the invention concerns oligomers derivatized at the 2′ position, which are stable to nuclease activity.
BACKGROUND ART
There has been considerable activity in recent years concerning the design of nucleic acids as diagnostic and therapeutic tools. One aspect of this design relies on the specific attraction of certain oligomer sequences for nucleic acid materials in vivo which mediate disease or tumors. This general approach has often been referred to as “anti-sense” technology. An oversimplified statement of the general premise is that the administered oligomer is complementary to the DNA or RNA which is associated with, and critical to, the propagation of an infectious organism or a cellular condition such as malignancy. The premise is that the complementarity will permit binding of the oligomer to the target nucleic acid, thus inactivating it from whatever its ordinary function might have been.
A simple illustration would be the administration of a DNA oligomer complementary to an mRNA which encodes a protein necessary to the progress of infection. This administered DNA would inactivate the translation of the mRNA and thus prevent the formation of the protein. Presumably the DNA could be directly administered, or could be used to generate an mRNA complement to the target mRNA in situ. There is by now extensive literature concerned with this general approach, and the methods of utilizing oligomers of this type which are complementary to target RNA or DNA sequences are set forth, for example, in van der Krol, A. R., et al.,
Biotechniques
(1988) 6:958-976; Stein, C. A., et al.,
Cancer Research
(1988) 48:2659-2668; Izant, J. G., et al.,
Science
(1985) 229:345-352; and Zon, G.,
Pharmaceutical Research
(1988) 5:539-549, all incorporated herein by reference. In addition, a bibliography of citations relating to anti-sense oligonucleotides has been prepared by Dr. Leo Lee at the Frederick Cancer Research Facility in Frederick, Md.
There are two conceptual additions to the general idea of using complementarity to interfere with nucleic acid functionality in vitro. The first of these is that strict complementarity in the classical base-pairing sense can be supplemented by the specific ability of certain oligonucleotide sequences to recognize and bind sequences in double-helical DNA and to insert itself into the major groove of this complex. A fairly recent but reasonably definitive series of papers has elucidated the current rules for such specificity. These papers take account of very early work by, for example, Arnott, S., et al.,
J Mol Biol
(1974) 88:509-521, which indicates the general principle of binding as triplexes poly-dT/poly-dA/poly-dT, and the corresponding analogous triplex involving poly-dC as summarized by Moser, H. E., et al.,
Science
(1987) 238:645-650. More recent studies show that the earlier rule (which was that recognition could be achieved by a homopyrimidine oligomer to homopurine/homopyrimidine stretches in the duplex) could be extended to patterns whereby mixed sequences can also be recognized (Griffin, L. C., et al.,
Science
(1989) 245:907-971. Further summaries of these phenomena are given, for example, in a review article by Maher III, L. J., et al.,
Science
(1989) 245:725-730. Additional related disclosures of triple-helix formation are those by Cooney, M., et al.,
Science
(1988) 241:456-459; Francois, J.-C.,
Nucleic Acids Res
(1988) 16:11431-11440; and Strobel, S. A., et al.,
J Am Chem Soc
(1988) 110:7927-7929. While further details are needed to provide exact sequence specificity studies in this context, it is clear that the rules for “complementarity” in this sense of specific embedding into the major groove of the double-helix are rapidly emerging.
The second aspect of anti-sense technology which deviates from the simple concept of base-pair complementarity in native oligonucleotides results from the early recognition that oligonucleotides, especially RNAs, are highly susceptible to nuclease cleavage in biological systems. In order for these materials to remain active drugs, it would be necessary to stabilize the administered oligonucleotides against this degradation. The approach that has so far been used has been to modify the phosphodiester linkages so as to be resistant to attack by these enzymes. In particular, the phosphodiester linkage has been replaced by phosphoramidate linkages, methylphosphonate linkages, and phosphorothioate linkages. These approaches have certain results with regard to stereoisomerism and its associated impact on hybridization to the target sequences that make them less than completely satisfactory. An alternate approach has been to modify the nucleosides by using 2′-O-methyl ribose or the alpha-anomers of the conventional nucleoside residues. In addition, oligomers containing 2′ amino groups have been prepared via their triphosphate analogs and enzyme-catalyzed polymerization by Hobbs, J., et al.,
Biochemistry
(1973) 12:5138-5145. Some of these approaches have been summarized in the Zon review cited in the previous paragraph.
The present invention provides additional 2′-substituted pentose moieties for inclusion in the oligomers useful in this technology which are resistant to nuclease activity, and may optionally be combined with additional modifications such as those set forth above.
DISCLOSURE OF THE INVENTION
The invention is directed to nucleosides and nucleotides of the formula:
wherein
B is a purine or pyrimidine residue or analog thereof;
W
1
is H, (PO
3
)
m
−2
wherein m is an integer of 1-3; a protecting group, or a group reactive to link hydroxyl groups;
W
2
is H, PO
3
−2
, a protecting group, or a group reactive to link hydroxyl groups;
X is O, S, NR or CR
2
wherein each R is independently H or alkyl (1-6 C);
Y is a linker moiety, a drug residue optionally attached through a linker moiety, a label optionally attached through a linker moiety, or a property-affecting residue optionally attached through a linker moiety, wherein said X—Y substituent renders an oligomer in which said nucleoside or nucleotide of formula (1) is included more stable to treatment with nuclease than said oligomer which incorporates a corresponding nucleotide having —H
2
or —HOH at the 2-position.
These materials are useful as intermediates in the synthesis of the oligomers of the invention, which are oligomers of the formula:
wherein each B is independently a purine or pyrimidine residue or analog thereof;
W
3
and W
4
are each independently H, PO
3
−2
, a protecting group, or a group reactive to link hydroxyl groups;
n is an integer of 1-200;
each Z is independently a nucleotide linking residue covalently conjugating the hydroxyl groups of sequential nucleotide residues;
each A is independently selected from the group consisting of H, OH, OH derivatized to a protecting group, and X—Y wherein
X is O, S, NR, or CR
2
wherein each R is independently H or alkyl (1-6 C); and
Y is a linker moiety, a drug residue optionally attached through a linker moiety, a label optionally attached through a linker moiety, or a property-affecting group optionally attached through a linker moiety;
wherein at least one A is X—Y; and
wherein the oligomer is more stable to nuclease than the Corresponding oligomer wherein all A are H or OH.
The oligomeric materials of formula (2) are useful as therapeutic or prophylactic agents in protocols which are directed against infectious disease or malignancy by targeting specific DNA and/or RNA sequences associated with the condition, as well as in diagnostic applications.
MODES OF CARRYING OUT THE INVENTION
A. Definitions
The oligomers of the invention contain the residue of at least one nucleotide of formula (1). In this formula, and in the oligomers, B represents a conventional purine or pyrimidine ba

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