Stabilized oligonucleotides and their use

Details Stabilized oligonucleotides and their use Stabilized oligonucleotides and their use

1994-11-10

2002-02-19

Martinell, James (Department: 1633)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S022100, C536S023100, C536S025300, C514S04400A

Reexamination Certificate

active

06348312

ABSTRACT:

The invention relates to novel stabilized oligonucleotides in which at least one non-terminal pyrimidine nucleoside is modified, and to their use as a diagnostic or pharmaceutical for the treatment of viral infections, cancer or diseases in which integrins or cell-cell adhesion receptors are active.
Antisense oligonucleotides (AO) and triple-helix-forming oligonucleotides (TFO) have proved to be specific gene expression inhibitors in a large number of systems, both in vitro and in vivo [Uhlmann & Peyman, Chem. Rev. 1990, 90, 543; Milligan et al., J. Med. Chem. 1993, 36, 1923; Stein & Cheng, Science 1993, 261, 1004].
One of the main problems when using naturally occurring phosphodiesters (PO) oligonucleotides is their rapid degradation by a range of nucleolytic activities both in cells and in the cell culture medium. A range of chemical modifications was used to stabilize oligonucleotides. A review of the prior art is given, for example, by Milligan et al., supra, and Uhlmann & Peyman, supra. Stabilization against nucleolytic degradation can be effected by modifying or replacing the phosphate bridge, the sugar unit, the nucleic base, or by replacing the sugar-phosphate backbone of the oligonucleotides. Since the phosphate bridge is the center of nucleolytic attack, a large number of modifications of the internucleoside bridge were described, in particular. The most frequently used nuclease-resistant internucleoside bridges are phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PA) bridges.
It must be borne in mind that the introduction of modifications alters not only the stability to nucleases, but simultaneously a large number of characteristics of the antisense oligonucleotides or triple-helix-forming oligonucleotides, such as, for example, their ability to enter cells, activation of RNase H, their specificity and their ability to hybridize with RNA (in the case of AO) or DNA (in the case of TFO) and the like. Moreover, there are indications that the stability of the serum, which is frequently used as a criterion for stability to nucleases, does not always reflect the intracellular activity [P. D. Cook in “Antisense Research and Applications”, Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chapter 9, pp. 149 et sec.]. This is why, in addition to the resistance to nucleases, the biological activity of antisense oligonucleotides or triple-helix-forming oligonucleotides gives information about the quality of such modifications.
As regards the question of the positions in the oligonucleotide at which such modifications are ideally to be effected, the following strategies have been developed [P. D. Cook (supra); Uhlmann & Peyman (supra); Milligan et al. (supra)]:
I) Exchange of All Internucleoside Bridges, for Example to Produce All-PS Oligonucleotides
This exchange gives oligonucleotides which are extremely stable to nucleases. For example, degradation by endo-nucleases (S1 nucleases) and by endo/exo nuclease P1 in an all-PS oligonucleotide is slowed down by a factor of 2-45 relative to a PO oligonucleotide [Stein et al., Nucl. Acids Res. 1988, 16, 1763]. All-PS oligonucleotides are also resistant in intact cells. 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 three hours under the same conditions [Woolf et al., Nucl. Acids Res. 1990, 18, 1763]. All-MeP oligonucleotides are also extremely resistant to nucleases.
The disadvantage of all-PS, or all-MeP, oligonucleotides compared with the PO oligonucleotides is that their capability of forming stable hybrids with the target RNA is reduced. A further disadvantage of the all-PS oligonucleotides are the unspecific (“non-antisense”) effects, which are frequently observed in this class of compounds [Milligan et al., supra; Stein & Cheng, supra].
Other uniformly modified derivatives for example all-2′-O-methyl-derivatives or all-&agr;-2′-deoxyribo derivatives, are generally also characterized by having lost the capability of activating RNase H.
II) Copolymers of Modified and Unmodified Phosphodiester Bridges
Ghosh et al. [Anti-Cancer Drug Design 1993, 8, 15] describe a phosphorothioate-phosphodiester oligonucleotide containing various percentages of PS bridges. Their construction follows, for example, the pattern —(PS—PO—PO—PO)
n
, (PS—PO—PO)
n
, (PS—PO)
n
, ((PO)
2
—(PS)
2
)
n
, or (PO—PS—PS)
n
. More specifically,
Ghosh
et al. disclose the following constructions: (PS—PO—PO—PO)
n
and ((PO)
2
—(PS)
2
)
n
, wherein n=4, and (PS—PO)
n
and (PO—PS)
n
wherein n=8. They teach that a PS bridge content of at least 50% is required for selective translation inhibition and that the activity drops drastically when this content is less. The present invention shows that these results are incorrect and that a PS bond content of far less than 50% is sufficient for selective inhibition if the modifications are positioned correctly (see below). Ghosh et al., furthermore teach that good results are achieved using the end capping/gap technique described under III.
The alternating exchange of every other internucleoside bridge, for example for MeP bridges (Furdan et al., Nucl. Acids Res. 1989, 17, 9193), brings no advantage in comparison with uniformly modified MeP oligonucleotides.
For example, alternatingly MeP-modified oligonucleotides equally do not activate RNase H. A comparison has shown that oligonucleotides with alternating phosphate-O-ethyl or phosphate-O-isopropyl esters and alternating MeP oligonucleotides are also less active than all-MeP or all-PS oligonucleotides [Marcus-Secura et al., Nucl. Acids Res. 1987, 15, 5749].
III) The exchange of one, two or three internucleoside bridges on the 5′ or the 3′ end of the oligonucleotides (end capping) and the exchange of one, two or three internucleoside bridges on the 5′ and the 3′ end of the oligonucleotides (gap technique).
As regards the efficacy of end capping, the results are contradictory in some cases. In particular 3′ end capping by means of PS, PA or MeP bridges is described as a protection against nucleases [P. D. Cook, supra, Milligan et al., supra]. A protection by means of 3′ end capping was also achieved by a series of other modifications. 3′-3′ end capping was described by various authors as a protection against nucleolytic degradation [Shaw et al., Nucl. Acids Res. 1991, 19, 747; Seliger et al., Mucleoside & Nucleotides 1991, 10, 469]. A further variant of 3′ end capping is the introduction of conjugate molecules on the 3′ end, which also increases stability to nucleases, such as, for example, 3′-dodecanol or 3′-acridine [P. D. Cook, supra], or 3-amino-2,3-propanediol [WO92/20697]. The gap technique, ie. the exchange of one, two or three internucleoside bridges on the 5′ and the 3′ end of the oligonucleotides, has proved particularly advantageous since, apart from PS oligonucleotides, most uniform modifications entail a lose of the capability to activate RNase H and thus a severe loss of activity. Again, a wide range of derivatives, modified phosphodiester bridges, modified sugars, modified bases, such as, for example, MeP-, PS-, PA-, 2′-O-alkyl- or 2′-F-derivatized oligonucleotides, were employed for stabilization purposes. These results are compiled in P. D. Cook supra. Within the gap, a sequence of two to four PO bonds will then suffice to activate RNase H.
Giles et al. [Anti-cancer Drug Design 1993, 8, 33] describe chimeric methylphosphonate-phosphodiester oligonucleotides in which the gap of unmodified PO bridges was reduced continuously from eight to two bridges. While a tendency was found that a reduced gap improved uptake into the cell, the oligonucleotides were not examined for their antisense activity.
An interesting comparison between various strategies can be found

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