Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2000-07-06
2003-02-18
Ketter, James (Department: 1636)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C435S458000, C514S04400A, C536S023100
Reexamination Certificate
active
06521252
ABSTRACT:
The present invention relates to a new agent for transferring nucleic acids into cells. This transfer agent is more particularly characterized in that it contains one or more disulphide bridges which are sensitive to reducing conditions. This new agent can be used to transfer nucleic acids of interest into different cell types either in vitro, in vivo or ex vivo.
With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It involves the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labelling studies or also therapeutic approaches. It may also be the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.
The various synthetic vectors developed so far in order to improve the transfer of nucleic acids into cells possess a considerable structural diversity which reflects the observation of the fact that their efficiency is different depending on the desired application and the intended cell types. This efficiency is largely dependent on their structure.
Among the synthetic vectors developed hitherto, cationic lipids have an important place. These vectors consist of a cationic polar part which interacts with the nucleic acids, and a hydrophobic lipid part which enables the complex formed to be protected from the external medium. The following may be mentioned by way of example: the monocationic lipids (DOTMA: Lipofectin®); lipopolyamines, in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxy-spermylamide of palmitoylphosphatidylethanolamine (DPPES), whose preparation has been described, for example, in Patent Application EP 394 111; or else the cationic lipids cited in Applications WO 96/17823 and WO 97/18185 (incorporated into the present by way of reference).
Many studies have clearly indicated that cationic lipids possess properties which make it possible to promote transfection. However, it now appears necessary to develop cationic lipids having novel structures which make it possible to provide additional beneficial properties. Thus, there is a need for cationic lipids which would be more particularly suitable for crossing membrane barriers. Indeed, numerous obstacles prevent a real transfection efficiency, among which the difficulty for the nucleic acid to cross biological membranes and to penetrate into the cellular compartments (“
Cellular and Molecular Barriers to Gene Transfer by a Cationic Lipid
”, Zabner, J. et al., J. Biol. Chem., 1995, No. 32, 18997-19007). It is this technical difficulty which the present invention proposes to solve.
Thus, the present invention relates to novel nucleic acid transfer agents which comprise at least one cationic hydrophilic region capable of noncovalently combining with nucleic acids and at least one lipophilic region, these regions being connected to each other through a so-called “spacer” arm, and comprising, in addition, at least one disulphide bridge positioned such that its reduction causes partial degradation of the lipophilic region, or alternatively positioned such that its reduction causes separation of the said transfer agent when it is symmetrical.
These transfer agents are capable of efficiently complexing nucleic acids by virtue of their cationic hydrophilic parts, this interaction strongly compacting the said nucleic acid, and the lipophilic region makes this ionic interaction insensitive to the external medium by covering the particle formed with a lipid film.
However, in addition to these properties which are desired for vectorization, the transfer agents according to the invention possess an extremely advantageous detergent property, by generating, at the level of the reducing cellular medium, because of the presence of the disulphide bridge(s), molecules of the polyaminated alkyl chain type which are membrane destabilizers. Indeed, the disulphide bridges are capable of constituting stable covalent bonds in oxidizing medium, and of breaking in reducing medium, according to the following scheme:
X—S—S—Y→X—SH+HS—Y
This type of structure is present, for example, in certain proteins possessing cysteine amino acids, and contributes to their three-dimensional structure and therefore to their biological activity. Disulphide bridges have, moreover, already been introduced into certain chimeric proteins, and in particular into immunotoxins, in order to connect the targeting domain to the active domain.
“Reducing medium” is understood to mean, for the purposes of the invention, a natural reducing medium, for example the intracellular medium, in particular the cytoplasm and in particular the endosomes. An artificial reducing medium representative of natural conditions is for example a medium comprising 0.1% to 20% of dithiotreitol (DTT).
By contrast, “oxidizing medium” is understood to mean any medium which is in contact with atmospheric oxygen and which contains no reducing agent, in particular the extracellular medium. A representative oxidizing medium for example consists of a 150 mM isotonic solution of sodium chloride, or of a solution containing 5% glucose.
The Applicant has thus demonstrated, quite unexpectedly, that one of the disulphide bridges could be introduced into a synthetic vector for the transfer of nucleic acids, in particular of the cationic lipid type, and that this did not affect its capacity to complex the nucleic acids in a non-reducing medium. It also shows that the nucleic acid transfer properties of these agents are preserved, or even improved. Moreover, the complexes formed are degraded in reducing medium, and therefore in particular in the cell, which makes it possible to generate detergent molecules, thus making a larger quantity of nucleic acid accessible to the cellular transcription machinery.
“Detergent” is understood to mean, for the purposes of the invention, any amphiphilic molecule having the property of being inserted into biological membranes and destabilizing them. This results from the capacity of detergents amphiphilic molecules to rupture the membranes by becoming inserted into the phospholipid double layers and by solubilizing the lipids and the proteins (
La Cellule
, Ed. Vigot and Décarie, 1988, pp. 581-583).
Another advantage of the transfer agents according to the invention also consists in their reduced intrinsic toxicity. Indeed, the transfer agent being degraded in the cell at the level of the disulphide bridges which are sensitive to reducing conditions, it does not exert the toxic effect observed for conventional transfer vectors. Furthermore, the improvement of the passage across the membranes allows the use of smaller doses of nucleic acid/transfer agent complex, with the beneficial consequences resulting therefrom on toxicity.
Finally, the Applicant has also demonstrated that the transfer properties are significantly improved when the lipophilicity of the transfer agents is sufficient and when they are used in adequate quantity. More particularly, it has been shown that one of the major advantages of increasing the lipophilicity of these agents, or of introducing a chain derived from a steroid, is the induction of improved resistance to serum.
The transfer agents according to the invention can have two types of structure, without this having an influence on their technical effect. In the first case, this structure can be represented in the following manner:
cationic hydrophilic region-spacer-lipophilic region
In such a structure, the disulphide bridge(s) are positioned in the lipophilic region so as to generate a detergent amphiphilic molecule when
Byk Gerardo
Dubertret Catherine
Pitard Bruno
Scherman Daniel
Aventis Pharma S.A.
Ketter James
Newman Irving
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