Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1992-07-06
2001-01-09
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024500, C514S04400A
Reexamination Certificate
active
06172208
ABSTRACT:
This invention relates to oligonucleotides which may bind to a DNA, and RNA, a protein, or a polypeptide, for use as a therapeutic agent or as a diagnostic probe. More particularly, this invention relates to oligonucleotides wherein at least one nucleotide unit of the oligonucleotide includes a conjugate moiety.
Oligonucleotides may be of value as therapeutic agents for the treatment of a wide variety of diseases. They offer the potential for a high degree of specificity by virtue of their capability for interaction with target macromolecules. Natural oligonucleotides, however, are relatively ineffective as therapeutic agents due to their poor penetrability into the cell, and their rapid degradation by enzymes. Therefore, relatively high concentrations of natural oligos are needed in order to achieve a therapeutic effect.
Applicants have found that the attachment of specific classes of conjugate groups to oligonucleotides improves their uptake into the cell, improves their stability, or both. Conjugate groups employed in the present invention, which include amino acids, dipeptide mimics, sugars, sugar phosphates, and neurotransmitters or analogues thereof, have been shown to be transported into the cell by specific transporter systems. These transporters can be used to improve the uptake of oligonucleotide conjugates into the cell, and, depending upon the site of attachment, can also prevent or reduce degradation.
Hydrophilic polymer conjugate groups which may be employed in the present invention, and which include poly-hydroxypropylmethacrylamide, dextrans, polymaleic anhydride, cyclodextrins, starches, and polyethyleneimine, may be used to reduce or prevent degradation of the oligonucleotide by blocking access of the conjugate to the enzymes which degrade oligonucleotides.
The scientific literature contains descriptions of certain conjugate groups attached to oligonucleotides.
Conjugates with polylysine have been described by Lemaitre, et al.,
Proc. Natl. Acad. Sci. USA,
Vol. 84, pgs. 648-562 (1987), and shown to be more active in cell culture than their unmodified counterparts. Polylysine, however, is not a preferred molecule for conjugation due to its relatively high toxicity. Amphiphilic oligonucleotide conjugates with polyethylene glycol have been described by Tullis in U.S. Pat. No. 4,904,582, and conjugates with cholesterol have been reported by Letsinger et al., (Abstracts, Conference on Nucleic Acid Therapeutics, Clearwater, Fl., (1991)). Many more conjugates have been synthesized for diagnostic applications. In these cases the molecule conjugated to the oligonucleotide acts as a reporter or signaling group, such as, for example, oligonucleotides attached to fluorescent groups which enable the duplex to be detached visually, biotin conjugates which can be detected after capture by streptavidin attached to a signaling group, or enzyme conjugates which can directly generate a signal upon addition of a suitable substrate. Several reviews on modified oligonucleotides, including conjugates have been published; see for example, Uhlmann and Peyman,
Chemical Reviews,
Vol. 90, pgs. 543-584 (1990), and Goodchild,
Bioconjugate Chemistry,
Vol. 1, pgs 165-187 (1990).
In accordance with an aspect of the present invention, there is provided an oligonucleotide wherein at least one nucleotide unit of the oligonucleotide is conjugated with a moiety selected from the group consisting of (a) amino acids; (b) dipeptide mimics; (c) sugars; (d) sugar phosphates; (e) neurotransmitters; (f) poly-hydroxypropylmethacrylamide; (g) dextrans; (h) polymaleic anhydride; (i) cyclodextrins; (j) starches; and (k) polyethyleneimine.
The term “oligonucleotide” as used herein means that the oligonucleotide may be a ribonucleotide, deoxyribonucleotide, or a mixed ribonucleotide/deoxyribo nucleotide; i.e., the oligonucleotide may include ribose or deoxyribose sugars or a mixture of both. Alternatively, the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example, arabiose, xylose, glucose, galactose, or deoxy derivative thereof or any mixture of sugars.
The phosphorus-containing moieties of the oligonucleotides of the present invention may be modified or unmodified. The pohosphorus-containing moiety may be, for example, a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, thiophosphonate, phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorothionate, phosphorothiolate, phosphoramidothiolate, and phosphorimidate. It is to be understood, however, that the scope of the present invention is not to be limited to any specific phosphorus moiety or moieties. Also, the phosphorus moiety may be modified with a cationic, anionic, or sqitterionic moiety. The oligonucleotides may also contain backbone linkages which do not contain phosphorus, such as carbonates, carboxymehtyl esters, acetamidates, carbamates, acetals, and the like.
The oligonucleotides also include any natural or unnatural, substituted or unsubstituted, purine or pyrimidine base. Such purine and pyrimidine bases include, but are not limited to, natural purines and pyrimidines such as adenine, cytosine, thymine, guanine, uracil, or other purines and pyrimidines, such as isocytosine, 6-methyluracil, 4,6-di-hydroxyprimidine, hypoxanthine, xanthine, 2,6-diaminopurine, 5-azactosine, 5-methyl cystosine, and the like.
In general, the oligonucleotide includes at least two, preferably at least 5, and most preferably from 5to 30 nucleotide units.
In one embodiment, the at least one nucleotide unit which includes the conjugate moiety is the 3′ terminal nucleotide unit. In another embodiment, the at lest one nucleotide unit is the 5′ terminal nucleotide unit.
Alternatively, the at least one nucleotide unit which includes a conjugate moiety as hereinabove described is one or more nucleotide units at the 3′ end and/or the 5′ end of the oligonucleotide. In yet another embodiment, the at least one nucleotide unit may alternate with nucleotide units which are unsubstituted (i.e., which do not include a conjugate moiety). In another embodiment, all of the nucleotide units include a conjugate moiety.
The conjugate moiety may be attached to the oligonucleotide at the purine or pyrimidine base, at the phosphate group, or to the sugar.
When the conjugate moiety is attached to the base, it is preferably attached at certain positions of the base, depending upon the base to which the moiety is attached. When the moiety is attached to adenine, it may be attached at the C2, N6, or C8 positions. When the moiety is attached to guanine, it may be attached at the N2 or C8 positions. When the moiety is attached to cytosine, it may be attached at the C5 or N4 positions. When the moiety is attached to thymine or uracil, it may be attached at the C5 position.
The moiety may be attached to a phosphate group at the 5′ end, at an internal position, or at the 3′ end of the oligonucleotide. The moiety may be attached to the 5′ end of the oligonucleotide via an —NH
2
—(CH
2
)
6
— linker (such as Aminolink II, for example), via a phosphodiester linkage, or via other linkers. A wide variety of linker groups may be employed, depending upon the nature of the nucleotide unit, the moiety, and whether the linker group is present during the synthesis of the oligonucleotide. The linker group may be a single atom, or a functional group. Examples of linkers include, but are not limited to —NH—, or amino groups, sulfur atoms, and polyvalent functional groups.
In another embodiment, the linking group is derived from a polyvalent functional group having at least one atom, and not more than about 60 atoms other than hydrogen, preferably not more than about 30 atoms other than hydrogen. The linker group in general has up to about 30 carbon atoms, preferably not more than about 20 carbon atoms, and up to about 10 heteroatoms, preferably up to about 6 heteroatoms, and in particular such heteroatoms may be oxygen, sulfur, nitrogen, or phosphorus. Representative examples of linker groups include, but are not lim
Genzyme Corporation
Horlick Kenneth R.
Lillie Raymond J.
Olstein Elliot M.
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