Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-03-29
2002-03-19
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C514S04400A, C435S069100, C435S320100, C435S455000, C435S458000
Reexamination Certificate
active
06358524
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to target cell-specific non-viral vectors, to pharmaceutical compositions that comprise such vectors, and to the use of these vectors in gene therapy.
The aim of gene e therapy is to insert a foreign gene(s) into the cell s of an organism in order either to switch off defective genes, to replace a defective gene with an intact gene, or to enable these cells to form a protein that possesses a prophylactic or therapeutic effect.
Vectors for insertion of genes into eukaryotic cells, b based on viruses , are well known in the art. Viruses have developed a differentiated system by means of which they bind specifically to cells by means of coat proteins, and, after being endocytosed via endosomes, are able to penetrate the membrane of these endosomes and reach the interior of the host cells. Viruses have therefore been used as carriers for inserting foreign genes into the cell. This technology, in its different variations, and the viruses that are used for this purpose, have already been described in detail (see the reviews of Hodgson,
Bio/Technology
, 3: 222 (1995); and Jolly,
Cancer Gene Therapy
, 1: 51 (1994)).
The principle underlying this technology is that parts of the viral gene are replaced by the desired foreign gene so that a viral vector is produced. As a rule, viral vectors are no longer able to replicate, due to the manipulation. However, all the genes that encode the viral coat proteins and regulate the expression of these viral genes must be present to enable these viral vectors to replicate.
It has been found, however, that viral vectors can give rise to problems, particularly when being used in humans. There is the danger of recombination with wild-type viruses of the same species, as a result of which pathogenic viruses might be produced. Furthermore, viral coat proteins can trigger immune reactions in the recipient. As viral vectors take the same route of infection in the cell as do the corresponding wild viruses, there is the danger of the host genes being mutated as a result of the foreign genes being integrated into the host chromosomes (activation of quiescent genes, destruction of active genes).
A further disadvantage of viral vectors is that the geometry of the viruses restricts their ability to accommodate many foreign genes.
In view of these limitations and dangers of viral vectors, attempts have been made to find virus-independent methods of inserting genes into cells. The principle underlying one of these methods is fusing the negatively charged cell membrane with the negatively charged gene so that the gene is taken up by the cell, and penetrates into the cytoplasm through the endosomal membrane or the lysosomal membrane. Apart from developing physical (enclosure of gene particles, osmotic, thermal or electrical alterations to the cell membrane) or chemical (organic solvents, detergents, enzymes) methods for altering the cell membrane, gene carriers have been developed that mediate fusion of the genes with the cell membrane. These carriers include liposomes, cationic polypeptides, dendrimeric polymers or cationic amphiphilic substances (for reviews, see Behr,
Bioconjugate Chem
., 5: 382 (1994); Afione et al.,
Clin. Pharmakokinet
., 28: 181 (1995) and Felgner,
Adv. Drug Delivery Rev
., 5: 163 (1990)).
Synthetic cationic amphiphilic substances, such as dioleoyloxypropyltrimethylammonium bromide (DOTMA) in a mixture with dioleoylphosphatidylethanolamine (DOPE) or lipopolyamine (see Behr above), have gained considerable importance in this type of charged gene transfer. The mechanism of action of these cationic amphiphilic substances or substance mixtures is that, due to an excess of cationic charge, they both complex with the negatively charged genes and bind to the anionic cell surface. The amphiphilic character of these carriers leads to fusion with the cell membrane. However, the transfection rate which can be achieved is still markedly less than when using viral vectors. Furthermore, the excess cationic charge on the complexes composed of non-viral carriers and DNA is neutralized, after in-vivo administration, by anionic biological substances (proteins, heparins, etc.), thereby impairing binding to cells.
It, therefore, is an object of this invention to provide a means for inserting a foreign gene into a eukaryotic cell that avoids the drawbacks of prior art methods. This and related objects have been achieved by the invention described below.
SUMMARY OF THE INVENTION
The present invention is based in part on the concept that cells can take up genes through the process of endocytosis. The endocytosis process is normally followed by enzymic degradation of the foreign genes in the endosomes or lysosomes. Only those genes that can evade this enzymic degradation and can penetrate through the membrane of the endosomes/lysosomes into the cytoplasm and/or into the cell nucleus are able to be expressed by transcription. In the case of the novel target cell-specific vectors described herein, the local concentration of the vectors at the target cell is increased in vivo as the novel vectors are provided with target cell-specific ligands.
The present invention relates, therefore, to target cell-specific non-viral vectors for inserting at least one gene into cells of an organism, which vectors comprise the following components:
(a) a non-viral carrier for the gene to be inserted,
(b) a ligand which can bind specifically to the desired target cell,
(c) a fusion protein for the penetration of the vector into the cytoplasm of the target cell, and
(d) the gene to be introduced.
In the novel vectors, the individual components of the target cell-specific vector are bonded to each other covalently and/or by means of adsorptive bonding. The present invention also relates to a pharmaceutical composition comprising the above vector.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The non-viral carrier (a) (see description above) for the gene, that is used in accordance with the invention, is preferably a protein, polypeptide, polysaccharide, phospholipid, cationic lipid, glycoprotein, lipoprotein or lipopolyamine that can be cationized by introducing positively charged side groups, with the bonding between the non-viral carrier and a positively charged side chain being effected by adsorptive or covalent bonding. Furthermore, the carrier can be given amphiphilic properties by an additional adsorptive or covalent bonding-on of lipophilic side groups. In a particularly preferred embodiment, the non-viral carrier (a) is albumin or xylan.
The ligand (b) that is employed in accordance with the invention is selected on the basis of specific binding to the outer membrane of a particular animal or human cell. For binding specifically to endothelial cells, ligand (b) is preferably selected from the group consisting of a monoclonal antibody, or a fragment specific for endothelial cells, a glycoprotein that carries mannose terminally, glycolipid, polysaccharide, cytokine, growth factor, adhesion molecules and glycoproteins from the coats of viruses that possess a tropism for endothelial cells. The last-named is a particularly preferred embodiment.
In another preferred embodiment, ligand (b) that binds specifically to smooth muscle cells is selected from the group consisting of a monoclonal antibody, or its fragments thereof, that specifically bind to actin, cell membrane receptor, a growth factor, and a glycoprotein from the coats of viruses that possesses tropism for smooth muscle cells. The last-named is a particularly preferred embodiment.
In a further preferred embodiment, a ligand (b) that binds specifically to macrophages and/or lymphocytes is selected from the group consisting of a monoclonal antibody that is specific for a membrane antigen on macrophages or lymphocytes, an intact immunoglobulin or Fc fragments of polyclonal or monoclonal antibodies that are specific for membrane antigens on macrophages and lymphocytes, cytokine, growth factor, a peptide carrying mannose terminally, protein, lipid, polysaccharide a
Kissel Thomas
Klenk Hans-Dieter
Müller Rolf
Sedlacek Hans-Harald
Aventis Pharma Deutschland GmbH
Guzo David
Heller Ehrman White and McAuliffe
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