Oligo-2′-deoxynucleotides and their use as...

Details Oligo-2′-deoxynucleotides and their use as... Oligo-2′-deoxynucleotides and their use as...

1997-12-16

2001-12-11

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S023100, C536S024300, C536S024500, C536S025300, C536S025320, C536S025330

Reexamination Certificate

active

06329346

ABSTRACT:

Oligonucleotides whose sequences are complementary to the RNA or DNA of a viral sequence or to an oncogene are of potential interest for the therapy of viral infections since they can inhibit the expression of viral genes. The underlying method, denoted antisense principle, is described for example by Zamecnik, P. C. and Stephenson, M. L. (1978) in Proc. Natl. Acad. Sci. USA 75, 280.
However, it has turned out that when such antisense oligonucleotides are introduced into cells, preferentially intrinsic cell enzymes rapidly degrade these oligonucleotides by cleavage of phosphodiester bridges, and they thus become ineffective.
Therefore many attempts have been made to synthesize antisense oligonucleotides which are resistant to enzymatic degradation (Uhlmann, E. and Peyman, A. (1990) in “Antisense oligonucleotides: A new therap. Principle”, Chem. Rev. 90, 543-584). Up to now such modifications have been primarily carried out at the internucleotide bridges i.e. on the phosphorus atom. Thus for example oligonucleoside-phosphorothioates and -oligonucleoside-phosphorodithioates, as well as non-ionic oligonucleoside-methylphosphonates, oligonucleoside-methylphosphorothioates, oligonucleoside-alkylphosphotriesters and oligonucleoside-alkylphosphoramidates have been described which are resistant to enzymatic degradation. A disadvantage of these compounds is for example their chirality with regard to the phosphorus atom. This means that in each case there are two pairs of diastereoisomers. This non-uniformity, however, limits their pharmacological effectiveness or requires a complicated separation of the isomers before use as therapeutic agents.
A further known class of compounds which has been proposed for antisense therapy are oligonucleotides which contain intercalating or reactive ligands. Thus for example acridine-modified oligomers or oligonucleotides which can be cross-linked (psoralenazidoproflavin-substituted) have been described (Hélène, C. and Thuong, N. T. in “Antisense RNA and DNA”, Curr. Commun. Mol. Biol.; Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., 1987). The production of such oligonucleotides is, however, very complicated.
In addition it is known that those oligodeoxynucleotides which have a configuratively changed glyconic part (alpha DNA) can be used as antisense oligonucleotides. Such an alpha DNA which is comprised exclusively of purine-2′-deoxynucleosides and pyrimidine-2′-deoxynucleosides in the alpha-D configuration is not or only very slowly degraded by intrinsic cell enzymes and would therefore be suitable for antisense therapy. However, a disadvantage of these compounds is the very complicated and tedious synthesis (Cohen, J. S. in Topics in Molecular and Structural Biology, “Oligonucleotides: Antisense Inhibitors of Gene Expression”, MacMillan Press, Lt. 1989). In addition dinucleotide monophosphates are known which are resistant to nucleases due to a building block in the threo configuration (Sokolova et al.; Nucleosides and Nucleotides 9/4 (1990), 515-531). However, corresponding oligonucleotides are neither disclosed nor would it be possible to produce these without difficulty. In addition the dinucleotides are not suitable for hybridization and formation of duplexes.
The object of the present invention was therefore to provide oligonucleotides which are not enzymatically degraded in eukaryotic cells, which can be easily produced and are suitable as pharmaceutical antiviral agents based on the antisense principle.
The invention therefore concerns oligodeoxyribonucleotides in which at least two 2′-deoxy-&bgr;-D-erythro-pentofuranosyl groups are replaced by 2′-deoxy-&bgr;-D-threo-pentofuranosyl groups at both the 5′ and 3′ end and which are composed of 6 to 100 nucleotide building blocks.
The invention in addition concerns oligodeoxyribonucleotides in which at least 20% of the 2′-deoxy-&bgr;-D-erythro-pentofuranosyl groups are replaced by 2′-deoxy-&bgr;-D-threo-pentofuranosyl groups in consecutive nucleotide building blocks and which are composed of 6-100 nucleotide building blocks.
Surprisingly such oligonucleotides (also denoted oligonucleotides in the following) are resistant or substantially resistant to nucleolytic degradation by cellular enzymes such as for example phosphodiesterases, exonucleases and endonucleases. In addition they form stable double-stranded hybrid structures with natural 2′-deoxyribonucleotides under the natural conditions in eukaryotic cells although they presumably at least in part have left-helical DNA structures as demonstrated by their CD spectra. The oligonucleotides according to the present invention are suitable for inhibiting the expression of viral genes and oncogenes in eukaryotic cells and can therefore be used therapeutically as antisense oligonucleotides.
30% and particularly preferably all of the 2′-deoxy-&bgr;-D-erythro-pentofuranosyl groups are replaced by 2′-deoxy-&bgr;-D-threo-pentofuranosyl groups.
If all the 2′-deoxy-&bgr;-D-erythro-pentofuranosyl groups are replaced by 2′-deoxy-&bgr;-D-threo-pentofuranosyl groups in an oligonucleotide, it is expedient that this oligonucleotide be designated an oligodeoxyxylonucleotide.
The structure of such an oligonucleotide according to the present invention is shown schematically in
FIG. 1
(section from an oligodeoxyxylonucleotide).
In the following the nucleotides which contain 2′-deoxy-&bgr;-D-erythro-pentofuranosyl groups are designated 2′-deoxyribonucleotides and the nucleotides which contain 2′-deoxy-&bgr;-D-threo-pentofuranosyl groups are designated 2′-deoxyxylonucleotides or building blocks.
In addition 2′-deoxyribonucleotide building blocks are referred to as dB (e.g. dA, dT, dC, dG) and 2′-deoxyxylonucleotide building blocks are referred to as dxB (e.g. dxA, dxT, dxC, dxG).
It is preferred that the dxBs and dBs occur consecutively in blocks in the oligonucleotides according to the present invention.
Accordingly preferred oligonucleotides according to the present invention are:
d {xBcB(B)
n
xBxB}
d {(B)
m
(xB)
n
(B)
o
}
d {(xB)
m
(B)
n
(xB)
o
}
in which n, m and o is at least 4, provided that the total length of the oligonucleotides according to the present invention does not exceed 100 nucleotide building blocks.
In an equally preferred embodiment, one or several nucleotide building blocks dB can be replaced by dxB at specific positions on the oligonucleotide (e.g. recognition sequences of endonucleases) in order to prevent cleavage by endonucleases.
All natural or modified nucleobases are suitable as bases. Particularly preferred modified bases are 5-methylcytosine or deazapurine such as 1-deazaadenine, 3-deazaadenine, 7-deazaadenine, 1-deazaguanine, 3-deazaguanine, 7-deazaguanine, 1-deazahypoxanthine, 3-deazahypoxanthine, 7-deazahypoxanthine and those bases which are substituted at the C-5 of pyrimidines, at the C-7 in the case of 7-deazapurines or at the C-8 of purines.
In addition the oligonucleotides according to the present invention can contain modifications at the internucleotide bridges in which case the modifications are preferably present at all internucleotide bridges of the oligonucleotide according to the present invention. In this connection phosphorothioates, methylphosphonates and phosphoroamidates are preferred.
The 3′ and/or 5′ ends the oligonucleotides according to the present invention can contain all suitable terminal groups known to a person skilled in the art. Hydrogen, mono-, di- or triphosphate, a reporter group or an intercalator group is preferred for the 3′ end and for the 5′ end. The other nucleotide building blocks can also be modified by reporter groups or intercalator groups.
A reporter group within the meaning of the invention is understood as a hapten such as e.g. biotin or digoxigenin or a fluorescent dye residue. Suitable intercalator groups are described by Hélène, C., loc. cit. and are preferably phenanthroline, acridine, actinomycin or its chromo

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