Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Patent
1996-08-26
1999-01-05
Guzo, David
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
536 231, 536 245, 435 6, 435 691, 435325, C07K 500, C07H 2104, C12Q 168, C12N 1585
Patent
active
058564357
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/FR95/00098, filed Jan. 27, 1995.
The present invention relates to compositions based on nucleic acids, to their preparation and to their use. More especially, it relates to compositions comprising nucleic acids and oligopeptides and their use in gene therapy, in particular for the transfer of nucleic acids.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introducing genetic information into the cell or organic affected. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. Different techniques have been described for the transfer of this genetic information, including various techniques of transfection 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), and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), 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 been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
However, the techniques developed hitherto do not enable the difficulties associated with the transfer of genes into cells and/or the body to be solved satisfactorily. In particular, the problems associated with the entry of the nucleic acid into the cells have not been solved completely. In effect, the polyanionic nature of nucleic acids prevents them from passing through the cell membranes. While it has been shown that naked nucleic acids are capable of passing through the plasma membrane ex vivo (see, in particular, Application No. WO90/11092), the efficiency of transfection remains fairly low. Furthermore, naked nucleic acids have a short plasma half-time on account of their degradation by enzymes and their elimination via the urine. Moreover, while recombinant viruses enable the efficiency of transfer of nucleic acids to be improved, their use incurs some risks, such as pathogenicity, transmission, replication, recombination, transformation, and the like.
The present invention provides an advantageous solution to these different problems. The Applicant has, in effect, shown that it is possible to form pairs of ions between particular cationic oligopeptides and the phosphate groups of nucleic acids, and that the complexes thus formed are stable and are capable of entering cells or of being encapsulated in transfer vectors such as liposomes, nanoparticles or low density lipoproteins (LDL), with high yields.
Hence a first subject of the invention lies in a composition comprising a nucleic acid and a cationic oligopeptide capable of forming secondary structures. The term secondary structure denotes peptides capable of adopting a particular spatial conformation under physiological conditions, as opposed to peptides not displaying any particular organization of their primary structure. The secondary structure may arise either in some solvents, or in aqueous solution, or after complexing with the nucleic acid.
More especially, the oligopeptides used in the context of the invention are capable of forming .alpha. helices or .beta. sheets.
The complexing of a nucleic acid with a polylysine has already been described in the prior art. However, the degree of complexing and the stability of the complex formed with polylysine are relatively low, and these complexes cannot be encapsulated satisfactorily in transfer vectors (see Examples). In contrast, the complexes according to the invention, which involve cationic oligopeptides capable of forming secondary structures (.alpha.helices, .beta. sheet
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Bazile Didier
Emile Carole
Helene Claude
Spenlehauer Gilles
Guzo David
Hansen Christine M.
Rhone-Poulenc Rorer SA
Wang Andrew
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