Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-05-06
2001-03-13
Schwartzman, Robert A. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S014800, C514S015800, C530S300000, C530S326000, C530S327000, C530S328000
Reexamination Certificate
active
06200956
ABSTRACT:
The present invention concerns the field of gene therapy and relates more particularly to the in vitro, ex vivo and/or in vivo transfer of genetic material. The invention proposes in particular a novel pharmaceutical composition which is useful for efficiently transfecting cells. The invention also relates to the uses of this composition.
Chromosomal deficiencies and/or anomalies (mutation, aberrant expression, and the like) are the cause of many diseases, of hereditary or non-hereditary nature. Conventional medicine has for a long time remained powerless as far as they are concerned. Nowadays, with the development of gene therapy, it is hoped to be able from now on to prevent or correct this type of chromosomal aberration. This novel medication consists in introducing genetic information into the affected cell or organ, for the purpose of correcting this deficiency or anomaly therein or alternatively for the purpose of expressing a protein of therapeutic value therein.
The main obstacle to the penetration of a nucleic acid into a cell or target organ lies in its size and the polyanionic nature of this nucleic acid, which oppose its passage across cell membranes.
In order to relieve this difficulty, various techniques are nowadays proposed including, more particularly, the transfection of naked DNA across the plasma membrane in vivo (WO90/11092) and the transfection of DNA via a transfection vector.
As regards the transfection of naked DNA, the efficacy of this still remains very low. Naked nucleic acids possess a short half-life in plasma on account of their degradation by enzymes and their removal via urinary routes.
Regarding the second technique, this also proposes two strategies:
The first uses natural transfection vectors, namely viruses. It is thus proposed to use adenoviruses, herpes-viruses, retroviruses and, more recently, adeno-associated viruses. Although these vectors prove to be of high performance as regards transfection, it is unfortunately not possible to exclude as far as they are concerned certain risks of pathogenicity, replication and/or immunogenicity, which are inherent to their viral nature.
The second strategy consists advantageously in using non-viral agents capable of promoting the transfer and expression of DNA in eukaryotic cells.
The subject of the present invention is directed more particularly towards this second strategy.
Chemical or biochemical vectors represent an advantageous alternative to natural viruses, in particular for this absence of viral recombination and/or immunological response. They have no pathogenic power, there is no risk of multiplication of the DNA within these vectors and there is no theoretical limit associated therewith as regards the size of the DNA to be transfected.
These synthetic vectors have two main functions, to condense the DNA to be transfected and to promote its cell binding as well as its passage across the plasma membrane and, where appropriate, the two nuclear membranes.
On account of its polyanionic nature, DNA naturally has no affinity for the plasma membrane of cells, which membrane is also polyanionic. In order to overcome this drawback, non-viral vectors generally all have polycationic charges.
Among the synthetic vectors developed, cationic polymers of polylysine and DE dextran type or alternatively cationic lipids or lipofectants are the most advantageous. They have the property of condensing DNA and of promoting its association with the cell membrane. More recently, the concept of targeted transfection mediated by a receptor has been developed. This technique exploits the principle of condensing DNA, by virtue of the cationic polymer, while at the same time directing the binding of the complex to the membrane using a chemical coupling between the cationic polymer and the ligand of a membrane receptor present at the surface of the cell type which it is desired to graft. Screenings of the receptor with transferrin and insulin, and screening of the hepatocyte asialoglycoprotein receptor have thus been described.
However, the synthetic vectors proposed to date are still far from giving as good performance as viral vectors. This could be the consequence of insufficient condensation of the DNA to be transfected and/or difficulties, encountered by the transfected DNA, of leaving the endosome and penetrating into the cell nucleus. Lastly, other drawbacks are directly associated with the nature of the cationic polymers of the lipofectants used.
The subject of the present invention is precisely to propose an advantageous solution to these problems.
More precisely, the present invention relates to a pharmaceutical composition which is useful for the transfection of a nucleic acid, characterized in that it contains, besides the said nucleic acid, at least one transfection agent and a compound involved in the condensation of the said nucleic acid; the said compound being derived, partly or totally, from a histone, a nucleoline, a protamine and/or one of the derivatives thereof.
The Applicant has discovered, unexpectedly, that the presence of such a compound within a transfecting composition based on a standard transfection agent made it possible to reduce considerably the amount of this agent, with the beneficial toxicological consequences stemming therefrom, without bringing any prejudice to bear on the transfecting activity of the said composition. On the contrary, this composition advantageously has a higher level of transfection.
In the sense of the invention, a compound involved in the condensation of the nucleic acid covers any compound which directly or indirectly compacts the nucleic acid. More precisely, this compound may either act directly on the nucleic acid to be transfected or may be involved at the level of an associated compound which itself is directly involved in the condensation of this nucleic acid.
Preferably, it acts directly on the nucleic acid.
According to a specific embodiment of the invention, the compound involved in the condensation of the nucleic acids consists, totally or partly, of peptide units (LysThrProLysLysAlaLysLysPro) SEQ ID No. 1 and/or (AlaThrProAlaLysLysAlaAla) SEQ ID No. 2 or one of their derivatives, it being possible for the number of units to range between 2 and 10. In the structure of the compound according to the invention, these units may be repeated continuously or non-continuously. Thus, they may be separated by connections of biochemical nature, for example one or more amino acids, or of chemical nature.
The particular choice, a compound according to the invention, of a peptide or pseudopeptide possessing a majority of amino acids with basic nature, such as lysine, histidine or arginine, is particularly advantageous in the context of the present invention. This compound may also possess a &bgr;-sheet conformational structure. Basic amino acids are, indeed, more specifically involved in peptide-nucleic acid bonding. They participate in the establishment of ionic hydrogen bonds between the two species, thus promoting the condensation of the nucleic acid. As regards the 8-sheet structure, this is characterized by better accessibility of the majority of the carbonyl bonds and of the hydrogen atoms which, on account of their respective acceptor and donor natures, also favour the formation of bonds with the nucleic acid to be compacted.
Such a compound is more preferably all or part of a histone, a nucleoline, protamine and/or one of the derivatives thereof.
Histones and protamines are cationic proteins which naturally compact DNA. They are thus responsible in vivo for the condensation of non-transcribed DNA and the DNA of certain viruses. As histones which may be used in the context of the present invention, mention may be made more particularly of histones H1, H2a, H3 and H4. As regards nucleoline, this is a nucleolar protein which would appear to possess a synergistic effect with respect to the histone H1 during the condensation of DNA by the latter. In the context of the present invention, the compound may advantageously be represented by a peptide sequence deri
Byk Gerardo
Scherman Daniel
Schwartz Bertrand
Aventis Pharma S.A.
Aventis Pharmaceuticals Products Inc.
Lacourciere Karen
Schwartzman Robert A.
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