Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-05-19
2004-06-15
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S456000, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000, C536S023100
Reexamination Certificate
active
06750058
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a transfecting peptide vector, to a composition containing the said vector as well as to their applications in the treatment (medicaments) and the prevention (vaccines) of human and animal diseases. The said vector is in particular capable of dispensing to suitable target cells nucleic sequences, proteins, peptides and chemical substances of interest.
In the field of gene therapy, many compositions useful for efficiently transfecting eukaryotic cells with a selected genetic material have been described.
There are essentially two main types of transfection vectors:
the natural transfection vectors, such as viruses or modified viruses, which are efficient but which have limits to their use: tissue nonspecificity, necessity to obtain constructs for each gene of interest and potential risks for the environment which lead to the setting in place of costly and constraining clinical infrastructures for the patient and staff;
nonviral agents (synthetic vectors), capable of promoting the transfer and the expression of chemical substances such as DNA into eukaryotic cells. The latter strategy represents an alternative to viral vectors.
These synthetic vectors must essentially have two functions: to condense the DNA to be transfected and to promote its cellular attachment as well as its passage across the plasma membrane and possibly the nuclear membranes; such vectors must therefore mimic the functioning of viruses in order to be efficient; however, it appears that the different vectors provided in the prior art do not exhibit these two functions in an optimum manner and may, in addition, depending on the cases, be toxic for the cells.
Among these nonviral agents, there may be mentioned first of all the cationic polymers and the cationic lipids. The former generally consist of polylysine, whereas a wide variety of cationic lipids (liposomes or pseudoliposomes) exist, each giving transfection efficiencies which vary according to the cell types (DOTMA, and the like).
The lipid portion which interacts with and/or destabilizes the membranes allows the fusion and the entry of the DNA/liposome complex.
However, the transfection of DNA by liposomes, although less immunogenic than that performed with the aid of cationic polymers, is in fact a relatively inefficient method.
The major mechanism of entry of the DNA/liposome complexes is, it seems, endocytosis; consequently, the transfected DNA is trapped in the intracellular vesicles and destroyed by the lysosomal enzymes.
Even if a portion of the transfected DNA is released into the cytoplasm by a mass action effect, only a small fraction of this DNA is effectively present in the nucleus.
Agents capable of increasing the release of the DNA from the endosomal vesicles and its passage into the nucleus can increase the gene transfer rate.
Among these agents, there may be distinguished:
those which target the complex towards another point of entry: the targeting is obtained, for example, by coupling ligands with the polylysine polymers; the targeting can also occur after internalization, by directing the complexes to the nucleus (PCT International Application WO 95/31557), and
those which avoid endosomal degradation; to escape endosomal degradation, it has been proposed to incorporate an endosomolytic agent into the complex, such as adenoviral particles (PCT International Application WO 93/07283) or more recently synthetic peptides with endosomolytic activity, which increase the release of the DNA into the cytoplasm.
Taking into account the preceding text, various types of complexes have been provided; there may be mentioned complexes combining liposomes and peptides, such as those described in:
International Application WO 96/25508, which describes compositions comprising (i) the nucleic acid to be transfected, (ii) a transfection agent, such as a cationic polymer and/or a lipofectant, (iii) a peptide compound involved at the level of the condensation of the nucleic acid, consisting as a whole or in part of peptide motifs possessing a majority of amino acids with a basic character, such as lysine, histidine, arginine (histones, nucleolin, protamine or derivatives thereof) and optionally (iv) a targeting element which makes it possible to orient the transfer of the nucleic acid, such as a ligand of the intracellular type such as a nuclear localization signal sequence (NLS) which favours the accumulation of the transfected DNA inside the nucleus and which may be combined with the peptide compound to form a chimeric peptide comprising a protein fragment (histone or protamine or nucleolin) and an NLS sequence. However, this system requires the presence of a cationic polymer and/or a lipofectant, which have the disadvantage of being toxic and/or costly,
International Application WO 97/30170 which also describes compositions for transfecting eukaryotic cells, which comprise the nucleic acid to be transfected, at least one cationic lipid at a suboptimal concentration and at least one acidic peptide (active on the membrane) which destabilizes the endosomal membrane and thus increases the transfection efficiency. The positive charge
egative charge ratio is between 0 and 3. The selected peptides are derived from the influenza virus, so as to induce effective rupture of the endosomes. The presence of the lipids is necessary in this composition because of the fact that the selected peptide does not allow passage across the first cell membrane.
Such complexes do not therefore make it possible to avoid the disadvantages linked to the use of liposomes.
That is undoubtedly the reason why complexes using only peptides have been provided:
European Patent Application 0,544,292, which describes a complex for transfecting nucleic acid which comprises a fusion protein consisting of a cellular factor (growth factor, viral antigen, toxin, integrin or lipoprotein) and a basic polycationic peptide comprising arginine and/or lysine residues,
International Application WO 94/23751, which describes a transfer peptide which comprises three parts: (1) a ligand L1 (peptide of 2 to 100 amino acids), capable of binding to a binding site at the surface of eukaryotic cells (membrane receptor) (example: peptide RGD, domain for binding of growth factors, hormones, viral antigens or lipoproteins), (2) a ligand L2 similar to L1 (peptide of 2 to 20 amino acids), which binds to the outer nuclear membrane of eukaryotic cells, such as an NLS sequence and (3) a ligand L3 corresponding to a basic peptide (3 to 100 amino acids) (histone fragment H1 or H2B, for example). The transfer peptides described in this Application therefore have a general ligand structure for a membrane receptor-ligand for the outer nuclear membrane-basic peptide. Such a structure was proposed in order to improve the specificity of the complex towards target cells, but has a toxicity level of the same order as that of the liposomes; in addition, the construct should be adapted as a function of the target cells (presence of specific receptors on the target cells), and
International Application WO 95/31557 which describes a transfection vector comprising a synthetic peptide and the nucleic acid to be transfected. The synthetic peptide comprises a polymeric chain of basic amino acids, preferably at the C-terminal position (10-50 amino acids, such as lysine, arginine and ornithine), an NLS peptide (6-15 amino acids, such as the NLS sequence of the SV40 T antigen, the NLS sequence of the polyoma T antigen, the NLS sequence of adenovirus E1a or the NLS sequence of adenovirus E1b, preferably at the N-terminal position and a hinge region of neutral amino acids (6-50 amino acids selected from glycine, alanine, leucine and isoleucine), between the polymeric chain and the NLS peptide. The preferred NLS sequence is the sequence of the SV40 virus T antigen (small sequence of basic amino acids: PKKKRKV SEQ ID NO: 25), which is efficient in mammalian cells or a short hydrophobic sequence which contains one or more basic amino acids (KIPIK SEQ ID NO:43). The hinge sequence com
Chroboczek Jadwiga
Fender Pascal
Alston & Bird LLP
Commissariat a l'Energie Atomique
Guzo David
LandOfFree
Transfecting peptide vector, composition containing same and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Transfecting peptide vector, composition containing same and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Transfecting peptide vector, composition containing same and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3349564