Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-09-30
2001-04-10
Yucel, Remy (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S091310, C435S320100, C435S375000, C435S459000, C536S023100, C536S024500, C424S001290, C424S461000, C424S009350, C424S452000, C424S458000, C424S001210
Reexamination Certificate
active
06214621
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention pertains to conjugates which make it possible to address oligonucleotides, antisense, and ribozymes, in cells. The process for their preparation, and pharmaceutical compositions containing them are also disclosed.
Oligonucleotides constitute a particularly interesting class of molecules because of their ability to form, by hybridization, specific complexes with complementary nucleic acid sequences. These complexes may include duplexes resulting from the hybridization of oligonucleotides with single-strand DNA sequences or with RNA sequences such as mRNA, and triplexes formed by hybridization with double-strand DNA molecules [1-6].
The properties cited below confer on the oligonucleotides remarkable possibilities for the study of genes, and in the area of therapeutic treatment [17]. Thus, the invention particularly pertains to antisense oligonucleotides and ribozymes, whose capacity to inhibit specifically the expression of genes on in vitro models [16,18] and the proliferation of cells in vivo [19,20], as well as the RNAse activity of the ribozymes, have already been studied.
The fixation of oligonucleotides and ribozymes on mRNA leads to the inhibition of translation of mRNA according to two general processes; on the one hand, degradation of this mRNA or the RNA/DNA duplexes by the cellular ribonuclease H (RNase H), or by the catalytic activity of the ribozymes, and on the other hand the steric blockage of the cellular machinery [1-9].
The use of chemically modified oligonucleotides makes it possible to improve their incorporation by the cell and their resistance to nucleases [10], particularly the 3′-exonucleases. Among the chemical modifications of oligonucleotides proposed in the prior art, the most promising seems to be the use of structural analogs of the phosphodiester oligonucleotides like the phosphorothioate oligonucleotides. The latter are resistant to cleavage by nucleases, and do not inhibit degradation by Rnase H [28]. These advantages have led to several cellular and pharmacokinetic studies [18,29,30], as well as to clinical tests of these compounds as antitumor and antiviral agents (31). However, the discovery of nonspecific effects has considerably limited the enthusiasm for the therapeutic use of antisense oligonucleotides (17,32,33).
In addition, a major constraint on the use of these modified oligonucleotides is that the hybridization complexes between the RNA and the oligonucleotide should be sufficiently stable so they are not dissociated by the cellular machinery. Thus, when the antisense oligonucleotides or the ribozymes are directed toward the coding region, they are separated from their target by the translation ribozymes (11-15). This dissociation can be avoided by combining the oligonucleotides with reagents that can react spontaneously, or after irradiation, with the target RNA (3-6,10,11).
For example, international patent application number WO 90/12020, proposes coupling furocoumarin to an oligonucleotide by means of a ribose or deoxyribose sugar. European Patent Application No. 316,016, International Application No. WO 89/06702, and German Patent Application No. 3,928,900 describe the use of conjugates of psoralen and oligonucleotides to block genetic expression. French Patent Application No. 2,568,254 describes the application of oligonucleotide compounds linked to an intercalating agent for the selective blockage of a nucleic acid sequence. More specifically, the application of these compounds to the selective blockage in vivo of the expression of a gene or a sequence involved in the initiation, the propagation, or the termination of the replication of a nucleic acid, the transcription of one or more genes, and/or their translation is disclosed in this French patent application.
However, these chemical methods present disadvantages because the induction of bridging by chemical agents is often accompanied by nonspecific reactions, and photochemical activation difficult to implement in vivo.
The effectiveness of the antisense oligonucleotides and ribozymes is also limited, on the one hand by their polyanionic nature, which leads to nonspecific interactions with extracellular cationic molecules [21,22] and, on the other hand, because of their weak diffusion through the plasma membrane [23-25]. To remedy these disadvantages, the prior art has proposed using, as indicated above, chemically modified oligonucleotides or transport and delivery systems [27].
The strategies of encapsulation of the oligonucleotides and ribozymes seem to constitute a better approach than the chemical modifications for favoring both transport and stability of unmodified oligonucleotides, while preserving their specificity of hybridization. Thus, the prior art conducted encapsulation of oligonucleotides in liposomes, in immunoliposomes (34,35), or pH-sensitive liposomes (36). It has been shown that encapsulation permits relative protection of the oligonucleotides against the nucleases and increases their delivery into the cells. In spite of these advantages, the encapsulation of oligonucleotides in liposomes is not entirely satisfactory, particularly because of problems in the encapsulation yield. The prior art also considers that the interaction between oligonucleotides which are conjugated to cholesterol with natural LDLs makes it possible to prolong the plasma half-life of the oligonucleotides [38] from 1 to 10 minutes, and to increase in the in vitro efficiency of antisense oligonucleotides [39]. However, the preparation of LDL from human plasma and the weak stability of the associated oligonucleotides remain major obstacles to their therapeutic use.
Cationic lipids, such as DOTMA or DOTAP, already known for DNA transfection, could also constitute transporters of oligonucleotides [40,41] and ribozymes. Their effectiveness has been demonstrated, particularly the oligonucleotide complexes and DOTAP, allowing for an increase in transport and a decrease in intra- and extracellular degradation of the oligonucleotides [41]. However, the cellular toxicity of these complexes limits their use in in vitro experiments [27,42] or for local administration [43].
More recently, the adsorption of oligonucleotides on nanoparticles of polyalkylcyanoacrylate made it possible to reinforce the protection against degradation by nucleases [44]. The inhibition of neoplastic growth in nude mice has been observed with a concentration of oligonucleotides adsorbed on these nanoparticles, 100 times weaker than with the free oligonucleotides [45]. But the possibilities for systemic use of these vehicles have not been demonstrated to date.
Therefore, there is currently no system of transport, addressing, and effective protection of the oligonucleotides, antisense and ribozymes, which would allow them to be used therapeutically.
SUMMARY OF INVENTION
The applicants have developed their expertise in the preparation of synthetic particle vectors, designated by the general term of Supramolecular Biovector nanoparticules (SMBV), or biovectors, which can fix and advantageously target different substances or active ingredients, and are therefore useful for the manufacture of pharmaceutical compositions.
A first type of supramolecular biovector, and the process for its preparation, has been described in European Patent No. 344,040.
These particles include:
a central nonliquid hydrophilic nucleus made up of a polysaccharide or oligosaccharide nucleus, naturally or chemically crosslinked, which can be modified by various ionic groupings;
a layer of fatty acids grafted by covalent bonds to the nucleus; and
one or more lipid layers, made up specifically of phospholipids.
The developments of this first generation of Supramolecular Biovector nanoparticule shave led the applicants to design and prepare new Supramolecular Biovector nanoparticules with improved properties, particula
Betbeder Didier
De Miguel Ignacio
Jarvis Thale
Kravtzoff Roger
Pavco Pamela
Biovector Therapeutics S.A.
Feit Irving N.
Hoffmann & Baron , LLP
Yucel Remy
Zara Jane
LandOfFree
Conjugates of a particle vector and oligonucleotides,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Conjugates of a particle vector and oligonucleotides,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Conjugates of a particle vector and oligonucleotides,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2552226