Method for preparing compositions for transferring nucleic acids

Details Method for preparing compositions for transferring nucleic acids Method for preparing compositions for transferring nucleic acids

1999-04-02

2000-12-05

Ketter, James

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

435458, 4353201, 536 231, A61K 9127

Patent

active

061563382

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a process for the preparation of compositions for the transfer of nucleic acids. The compositions obtained can be used for the transfer of nucleic acids into cells in vitro, ex vivo or in vivo.
The cellular penetration of a naked nucleic acid (generally of high molecular mass and negatively charged) is a rare phenomenon which, in general, only leads to the transfection of the cell with a very limited efficiency. For this reason, various types of vectors and techniques have been described in the prior art for carrying out this transfer. Two large families can be distinguished in this regard: viral vectors and physical techniques.
Among the viral vectors, there may be mentioned, for example, retroviruses, adenoviruses, AAVs, herpesviruses, baculoviruses and the like. The transfer efficiency obtained with viral vectors is generally very good. However, their construction and their production are difficult, the cloning capacity of these vectors is sometimes limited, and their use may, in some cases, exhibit certain disadvantages inherent in the use of viruses (spreading, pathogenicity and the like).
For these reasons, various physical techniques for the transfer of nucleic acids have been developed. There may be mentioned, for example, electroporation, coprecipitation or the use of particle guns.
Electroporation consists in applying an electric field to a cell suspension containing DNA. However, while this technique gives good results in some cases, it is difficult to optimize because of the risk of irreversible lesions of the cell membrane. In the particle gun technique, particles (gold, tungsten) are coated with nucleic acids and then discharged onto the cells. This method is however promising mainly for cells with a wall, such as plant cells. Moreover, the coprecipitation of DNA with certain polymers (DEAE-dextran) or calcium phosphate has the disadvantage of not being very reproducible and of being sometimes cytotoxic.
To overcome these disadvantages, synthetic transfer vectors have been developed. The role of these vectors is essentially to provide the nucleic acid in a form appropriate for cell penetration, to facilitate this penetration (into the cytoplasm and then into the nucleus) as well as to protect it from cytoplasmic nucleases.
Among these vectors, the cationic lipids possess advantageous properties. These vectors consist of a polar cationic part allowing the condensation of the nucleic acids and of a hydrophobic lipid part stabilizing the ionic interaction. An excess of lipid, because of the ionization of its polar part, would also promote the interaction with the cell membrane. Specific examples of cationic lipids are in particular lipopolylysine, monocationic lipids (DOTMA; Lipofectin.RTM.); certain cationic detergents (DDAB); lipospermines (DOGS, DPPES, and the like), lipothermines, and the like.
The use of this type of vector, optionally in combination with a fusogenic lipid (DOPE and the like), has shown that they possess good nucleic acid transfer properties in vitro, ex vivo and in vivo on numerous cell types. These vectors therefore constitute an advantageous alternative to the viral vectors and to the physical techniques for the transfer of nucleic acids. However, the exploitation of these vectors at the industrial level is currently limited. In particular, the methods of preparing these vectors which are described in the prior art are empirical, not very industrializable, not very reproducible and lead to poorly defined mixtures whose composition does not give optimum properties.
Accordingly, it is known to prepare compositions by mixing various lipid components in chloroform, drying in a stream of nitrogen for 20 minutes and then drying under vacuum. The lipid film thus obtained is taken up in deionized water [Hui 96], [Felgner 87]. The mixture is then vortexed in order to resuspend the lipid and sonicated in a sonication bath for 10 minutes until clarification of the suspension which can then be diluted. Compaction is immediately performed before addit

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