Compositions containing at least one nucleic acid

Details Compositions containing at least one nucleic acid Compositions containing at least one nucleic acid

1999-01-06

2002-05-14

Ketter, James (Department: 1632)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C424S001210, C424S417000, C424S420000, C435S458000

Reexamination Certificate

active

06387396

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to new compositions containing at least one nucleic acid and their applications in the biomedical field, particularly in gene therapy.
Up to the end of the Eighties, the use of liposomes in view of biomedical applications and, more specifically, in gene therapy did not seem very promising. The results of transfection (translation and expression of a gene of prokaryotic or eukaryotic cells by eukaryotic cells), essentially transient, were relatively mediocre. The recourse to external virus envelopes has very significantly added to the results of gene transfers in a multitude of cell lines in vitro. On the contrary, it is certain that their use in human therapy and animal therapy gives rise to problems given the possible iatrogenic dangers that they represent. Towards the end of the last decade, it appeared that the use of cationic surfactants very clearly improved the results of transfection. Since then, despite certain problems linked to the use of cationic vectors, many formulations have been proposed some of which are patented and marketed.
In general, three sorts of non-viral vectors are used:
cationic polymers,
biochemical vectors constituted of a cationic protein combined with a cell receptor,
cationic lipids associated or non-associated to liposomes.
All these vectors contain cationic molecules.
The use of such vectors is notably found described in the following publications:
J. P. Behr et al. Proc. Nat. Ac. Scienc. 86, 6982, 1989
M. Cotten and Wagner Curr. Opin. Cell Biol. 4, 705, 1993
J. Haensler and F. C. Szoka Bioconjug. Chem. 4, 372, 1993
C. P. Hodgson Bio Technology 13, 222, 1995
F. D. Ledley Hum. Gene Ther. 6, 1129, 1995
J. S. Remy et al. Proc. Nat. Ac. Scienc. 92, 1744, 1995
V. S. Trubettskoy et al. Biochem. Biophys, Acta 1131, 311, 1992
Two essential problems arise from the use of cationic vectors
1—these surfactants are generally cytotoxic and even if certain specific molecules have been developed in order to reduce the toxicity thereof, the problem is not entirely solved,
2—their application cannot be envisaged in vivo since these vectors, due to their charge, interact very strongly with proteins, for example, with those present in the serum, as well as with the cell walls. They therefore stick rapidly onto the cells neighbouring the site of injection, reducing the systemic diffusion thereof.
This is the reason why they find their preferential use in vitro for cells in culture, in the transfer of genes.
It is well known that liposomes are colloidal structures which comprise an aqueous core separated from the external medium by one or more bi-layers of phospholipid molecules. Their application in cosmetics and in pharmacology has been the subject of a large number of patents which describe many uses of these vectors since their discovery in the 1960's.
From the point of view of applications, liposomes composed of one sole bi-layer, liposomes composed of several bi-layers often designated by the abbreviation MLV which corresponds to <<Multi-layered Vesicles>>, have been very rapidly distinguished. The multi-layered vesicles have a size which is badly controlled and is generally far greater than a micrometer. Amongst the uni-lamellar vesicles, small vesicles, often designated as the abbreviation SUV corresponding to <<Small uni-lamellar vesicles>> the size of which does not exceed 100 to 300 nm, have been distinguished from large vesicles often designated by the abbreviation LUV corresponding to <<Large uni-lamellar vesicles>> which can attain several tens of micrometers in size.
From the fact of their significant size, the multilamellar vesicles (MLVs) are only of little use in medical applications. In fact, risks of embolism that the introduction of particles of size of greater than a micrometer into the blood circulation brings about render their use impossible by intra-venous injection. Moreover, too great a size hinders the vesicles from passing through the tissue barriers, and this enables finding them in -the blood when they are injected intramuscularly or sub-cutaneously. Finally, really efficient processes do not exist for producing MLVs in a perfectly controlled way. The major part of the applications have been developed with SUVs which fulfil the criteria of safety and control necessary for a biomedical use.
The encapsulation of nucleic acids in vesicles based on surfactants, generally lipidic surfactants, has also been described in the literature. All the vesicles described to date have in common the fact of having several layers of surfactants surrounding a liquid core. Such vesicles are described, in particular, in the patents U.S. Pat. Nos. 4,394,448 and WO-95/16437.
One of the practical problems of the use of liposomes for the vectorisation of medicaments or their use in gene therapy is the low yield due to the fact that the percentage of the starting aqueous solution effectively encapsulated rarely exceeds 30%. Moreover, the method generally followed calls for steps of evaporation of organic solvents which intervene in the method of preparation. Finally, the experimental results have revealed to be very deceiving.
In fact, the methods commonly accepted for the incorporation of lipidic vesicles, or cationic complexes which contain DNA, in the cells, pass via a mechanism of endocytosis. In the cytoplasm, the object undergoing penetration is included in an endosome which contains enzymes, in particular DNAses which are capable of destroying any intruding genetic material. The liposomes, due to their low number of lipidic membranes which surround the aqueous core, do not provide sufficient protection for resisting the destructive action of the endosomal nucleases and proteases. From this, even if they are efficient in penetrating the cytoplasm of the cell, the liposomes enable the DNA to reach the nucleus with only a low yield.
It is therefore crucial to develop synthetic vectors which are compatible with an in vivo use and this all the more so since there does not exist at present an efficient vector for molecules which can be applied in human and/or animal gene therapy. Alone, the viral envelopes show an efficiency in transfection which is compatible with a human or animal application.
The vesicles of the liposome type or paucilamellar vesicles have in common to be constituted of one or more lamellar layers surrounding an aqueous core. Apart from this type of vesicles, multi-lamellar vesicles are also known which are structurally different from the preceding ones by the fact that they have a structure known as an <<onion>> structure and are constituted, from their centre through to their periphery, of a succession of lamellar layers which are separated by a liquid medium. These vesicles may be obtained by a method which comprises the preparation of a liquid crystal lamellar phase and its transformation by the application of a shearing. Such a method is described in particular in the by patent WO 93/19735 originating from French patent FR-2 689 418 or WO 95/18601 introduced herein by reference.
According to French patent FR-2 689 418, this transformation may be made during a homogenous shearing step of the liquid crystal phase, and this leads to vesicles which are known as microcapsules of controlled size. However, in adjusting the formulation of the liquid crystal lamellar phase, in particular in adjusting the nature of the surfactants entering in its composition, the transformation of this liquid crystal phase into vesicles can be obtained by a simple mechanical manipulation, in particular during the mixing of the constituents.
SUMMARY OF THE INVENTION
The research conducted by the inventors have lead them to discover that the use of the technologies described above allowed developing new multi-lamellar vectors of small size, which are non-cationic, and which enable encapsulating, protecting, and delivering DNA into cells; and all this with a high encapsulation efficiency as well as with a great ease of preparation.
Another advantage is that all

Affiliated with

Also associated with

Rating

Compositions containing at least one nucleic acid does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Compositions containing at least one nucleic acid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions containing at least one nucleic acid will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2848189