Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1993-11-23
2002-04-09
Martinell, James (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C536S023100
Reexamination Certificate
active
06369038
ABSTRACT:
The present invention relates to compounds of the oligonucleotide type, as well as to their applications.
Antisense oligonucleotides are short synthetic DNA or RNA molecules of sequence complementary to a target sequence belonging to a gene or to a messenger RNA whose expression it is desired to block specifically. Antisense oligonucleotides may be directed towards a messenger RNA sequence, or alternatively towards a DNA sequence. Antisense oligonucleotides hybridize with the sequence to which they are complementary and can thus block the expression of the messenger RNA carrying this sequence.
The term “oligonucleotide” is used in a general manner to denote a polynucleotide of ribo- or deoxyribo-series. Where the issue of a particular property linked to the use of a deoxyribo-series or a ribo-series is involved, the complete name oligodeoxyribonucleotide or oligoribonucleotide may be used. An oligonucleotide can be single-stranded, that is to say contain only one line of nucleotides which are not paired with another chain, or can alternatively be double-stranded, that is to say contain nucleotides paired with another polynucleotide chain. Two complementary oligonucleotides form a double-stranded structure. A single-stranded oligonucleotide can, however, possess double-stranded regions by intra-chain pairings between complementary sequences carried on the same strand.
The term hybridization used here means the formation of hydrogen bonds between pairs of complementary bases, guanine and cytosine forming three hydrogen bonds and adenine and thymine forming two.
Antisense oligonucleotides are synthesized chemically, and frequently contain modifications which change the actual skeleton of the molecule or carry additional reactive groups localized at their ends. The objectives of these modifications introduced into anti-sense oligonucleotides are either to enhance the resistance of these molecules to nucleolytic degradation, or to promote their interactions with their targets, or to permit specific degradation/modification reactions of the RNA or DNA targets, or to increase their intracellular penetration.
Antisense oligonucleotides are sensitive to nuclease degradation, and mainly to the action of exonucleases. Nucleases occur in all compartments—cellular and extracellular, especially in the serum—and cause a rapid degradation of these molecules. A pharmacological use of antisense molecules involves solving these problems of degradation in order to achieve satisfactory pharmacokinetics and hence an adequate perpetuation of the effects of these molecules. Many chemical modifications enable antisense oligonucleotides to become nuclease-resistant. Some modifications directly affect the structure or nature of the phosphodiester bond (methylphosphonates, phosphorothioates, alpha-oligonucleotides, phosphoramidates, to mention a few examples), other [sic] consist in adding blocking groups to the 3′ and 5′ ends of the molecules (Perbost et al., 1989; Bertrand et al., 1989; Bazile et al., 1989; Andrus et al., 1989; Cazenave et al., 1989; Zon, 1988; Maher and Dolnick, 1988; Gagnor et al., 1987; Markus-Sekura, 1987).
To increase the efficacy of the interactions between an oligonucleotide and its target, an inter-calating group (acridines for example) may be added to one end of the antisense oligonucleotide. Lastly, re-active groups (alkylating agents, psoralens, Fe-EDTA for example) capable of causing cleavages or permanent chemical changes in the target may be added to the antisense oligonucleotides (Sun et al., 1989; Helene, 1989; Durand et al., 1989; Sun et al., 1988; Helene and Thuong, 1988; Verspieren et al., 1987; Sun et al., 1987; Cazenave et al., 1988, 1987; Le Doan et al., 1987; Toulme et al., 1986; Vlassov et al., 1986).
The last type of conventional modification of antisense oligonucleotides consists in adding groups which modify the charge and/or hydrophilicity of the molecules in order to facilitate their passage through the membrane (Kabanov et al., 1990; Degols et al., 1989; Stevenson et al., 1989; Leonetti et al., 1988).
All these modifications can obviously be combined with one another.
Not all the regions of a messenger RNA are sensitive in a like manner to the effects of an antisense oligonucleotide. A messenger RNA is not a set linear molecule but, on the contrary, a molecule possessing many secondary structural features (complex intramolecular hybridizations) and tertiary structural features (refoldings and particular conformations, pseudo-nodes), and which interacts with structural and functional nucleo-proteins (basic proteins, splicing, polyadenylation and capping complexes, translation complex for example). The effective availability and accessibility of the different regions of a messenger RNA will depend on their engagement in these structural features. Correspondingly, the efficacy of an inhibitory agent which interacts with this or that sequence will also depend on the engagement of this sequences [sic] in a particular function. The target regions for antisense molecules must be accessible to the oligonucleotide.
The use of software for prediction of secondary structures enables theoretical degrees of accessibility to be predicted, and hence the choice of targets for antisense oligonucleotides to be guided. As a whole, the regions most widely used as targets are translation initiation sites (AUG initiation region) and also splicing sites (SD/SA junctions). Many other sequences not having particular functional properties and not engaged in intramolecular pairing have also proved effective as a target for antisense oligonucleotides (see the examples mentioned later).
Antisense oligodeoxyribonucleotides may also be directed towards certain regions of double-stranded DNA (homopurine/homopyrimidine sequences or purine/pyrimidine-rich sequences), and can thus form triple helices (Perroualt et al., 1990; François et al.(A), 1989; François et al.(B), 1989; François et al.(C), 1989; Wang et al., 1989; Maher et al., 1989; Sun et al., 1989; Boidot-Forget et al., 1988; Moser and Dervan, 1987; Dervan, 1986). Oligonucleotides directed in this manner towards DNA have been termed “anti-gene” or alternatively “anti-code”. The formation of a triple helix at a particular sequence can block the binding of proteins involved in the expression of a gene and/or permit the introduction of irreversible damage into the DNA if the oligonucleotide in question possesses a particular reactive group. Such antisense oligonucleotides can become true artificial restriction endonucleases, directed on request towards specific sequences.
The hybridization between an antisense oligonucleotide and a target messenger RNA can block expression of the latter in several ways, either sterically or pseudocatalytically (Gagnor et al., 1989; Jesus et al., 1988; Markus-Sekura, 1987):
the interaction between the messenger RNA and a complementary antisense oligonucleotide can create a physical barrier preventing the binding and/or progression of proteins or protein complexes needed for translation, maturation, stabilization or transport of the messenger RNA. This physical blockade will lead finally to an inhibition of the expression of the target messenger RNA.
the hybridization between a messenger RNA and an antisense oligodeoxyribonucleotide will create a substrate for RNase H, an enzyme present in all eukaryotic cells. RNase H is an enzyme which specifically degrades RNA when it is hybridized with DNA. The hybridization of an antisense oligonucleotide with a target RNA will hence lead to cleavage of this target RNA at the location of this hybridization, and hence to its permanent inactivation.
moreover, as stated above, antisense oligonucleotides can contain reactive groups capable of directly producing irreversible damage in the target RNA molecule.
As regards antisense oligonucleotides directed towards DNA, these can act either by inhibiting the binding of a regulatory protein essential to expression of the target gene (transcription factor for example), or by producing irreve
Blumenfeld Marta
Brandys Pascal
d'Auriol Luc
Vasseur Marc
Genset
Martinell James
Saliwanchik Lloyd & Saliwanchik
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