Composition containing nucleic acids, preparation and uses

Details Composition containing nucleic acids, preparation and uses Composition containing nucleic acids, preparation and uses

1998-09-25

2001-01-09

Priebe, Scott D. (Department: 1633)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S320100, C435S325000, C435S455000, C435S458000, C536S023100, C536S024500, C424S450000

Reexamination Certificate

active

06172048

ABSTRACT:

The present invention relates to compositions based on nucleic acids, to their preparation and to their use. More particularly, it relates to compositions comprising at least one nucleic acid and one lipopolyamine and to their use in gene therapy, especially for the transfer of nucleic acids.
Gene therapy consists in correcting a deficiency or abnormality (mutation, aberrant expression, and the like) or in providing for the expression of a protein of therapeutic interest by introduction of genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue. Various techniques have been described for the transfer of this genetic information, including various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol., 1 (1967), 891), of DNA and nuclear proteins (Kaneda et al., Science, 243 (1989), 375), of DNA and lipids (Felgner et al., PNAS, 84 (1987), 7413) and of DNA and polylysine, the use of liposomes (Fraley et al., J. Biol. Chem., 255 (1980), 10431), and the like. More recently, the use of viruses as vectors for gene transfer has appeared as a promising alternative to these physicochemical transfection techniques. In this respect, various viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
However, the techniques developed until now do not make it possible to satisfactorily resolve the difficulties related to the transfer of genes into cells and/or the organism. In particular, the problems related to the penetration of nucleic acid into cells are not completely solved. In fact, the polyanionic nature of nucleic acids prevents their passage through cell membranes. While it has been shown that naked nucleic acids are capable of passing through the plasma membrane of certain cell types in vivo (see especially Application No. WO 90/11092), transfection efficiency remains fairly low. Moreover, naked nucleic acids have a short plasma half-life, due to their degradation by enzymes and their removal by urinary routes. Moreover, while recombinant viruses make it possible to improve the efficiency of transfer of nucleic acids, their use presents certain risks such as pathogenicity, transmission, replication, recombination, transformation, immunogenicity, and the like.
The present invention introduces an advantageous solution to these various problems. The Applicant has in fact shown that certain compositions comprising a nucleic acid and a lipopolyamine can make possible the in vivo transfer of the said nucleic acid into a cell and/or organ with high efficiency and without toxicity. The compositions of the invention also make it possible to avoid the disadvantages related to the use of viral vectors (potential dangers, limited size of transferred gene, high cost, and the like).
The use of certain lipopolyamines for the in vitro transfection of cell cultures has already been described in the prior art. Thus, Application EP 394,111 describes the use of certain lipopolyamines for the in vitro transfection of cell lines. The article by Demeneix et al. (Int. J. Dev. Biol., 35 (1991), 481) likewise describes the use of a lipopolyamine (dioctadecylamidoglycylspermine, DOGS) for the in ovo transfection of nucleic acids. According to these documents, the lipopolyamines must be used under conditions such that the positive charges of the lipopolyamine
egative charges of the nucleic acid ratio is between 2 and 5 and preferably equal to 3 or 4. However, as shown in Examples 8 and 9 of the present application, none of the conditions described in these documents, surprisingly, makes possible the in vivo transfection of nucleic acids. Due to interaction with anionic macromolecules or with the extracellular matrix of the tissues, the particles formed under these conditions are in fact incapable of diffusing out of the site of application and thus of transferring any nucleic acid in vivo. Moreover, the preparation conditions described in these documents are not applicable to the production of pharmaceutical compositions containing significant amounts of nucleic acids. The Applicant has now shown that, under certain conditions, lipopolyamines can be used for the in vivo transfection of nucleic acids. More particularly, the Applicant has found that compositions comprising a nucleic acid and a lipopolyamine under conditions such that the positive charges of the lipopolyamine
egative charges of the nucleic acid ratio is less than or equal to 2 surprisingly make possible the in vivo transfection of the said nucleic acid with high efficiency. Moreover, the Applicant has developed certain conditions making possible the preparation of these pharmaceutical compositions incorporating significant amounts of nucleic acid. The pharmaceutical compositions of the invention thus constitute particularly advantageous tools for the administration and transfer of nucleic acids in vivo.
A first subject of the invention thus lies in a composition comprising at least one nucleic acid and one lipopolyamine in which the ratio R=positive charges of the lipopolyamine
egative charges of the nucleic acid is less than or equal to 2.
Within the meaning of the present invention, the term lipopolyamine denotes any amphiphilic molecule comprising at least one hydrophilic polyamine region and one lipophilic region. The cationically charged polyamine region of the lipopolyamines is capable of combining reversibly with the negatively charged nucleic acid. This interaction strongly compacts the nucleic acid. The lipophilic region makes this ionic interaction insensitive to the external medium, by covering the nucleolipid particle formed with a lipid layer.
Advantageously, the polyamine region of the lipopolyamines used in the context of the invention corresponds to the general formula
H
2
N—(—(CH)
m
—NH—)
n
—H
in which m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, it being possible for m to vary between the different carbon groups included between two amines. Preferentially, m is between 2 and 6 inclusive and n is between 1 and 5 inclusive. Still more preferentially, the polyamine region is represented by spermine or an analogue of spermine which has retained its properties of binding to DNA.
The lipophilic region can be a saturated or unsaturated hydrocarbon chain, cholesterol, a natural lipid or a synthetic lipid capable of forming lamellar or hexagonal phases.
Use is advantageously made, in the context of the present invention, of the lipopolyamines as defined in Patent Application EP 394,111. This application also describes a process which can be used for the preparation of these lipopolyamines.
Use is particularly advantageously made, in the context of the invention, of dioctadecylamidoglycylspermine (DOGS) or of the 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES).
In order to obtain an optimum effect of the compositions of the invention, the respective proportions of the polyamine and of the nucleic acid are preferably determined so that the positive charges of the lipopolyamine
egative charges of the nucleic acid ratio R is between 0.1 and 1.9 and more preferentially between 0.5 and 1.5.
In the compositions of the present invention, the nucleic acid can be both a deoxyribonucleic acid and a ribonucleic acid. There can be sequences of natural or artificial origin and especially genomic DNA, cDNA, MRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences. These nucleic acids can be of human, animal, plant, bacterial or viral origin and the like. They can be obtained by any technique known to those skilled in the art and especially by screening banks, by chemical synthesis or alternatively by mixed methods including chemical or enzymatic modification of sequences obtained by

Affiliated with

Also associated with

Rating

Composition containing nucleic acids, preparation and uses does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Composition containing nucleic acids, preparation and uses, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Composition containing nucleic acids, preparation and uses will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2493508