Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Virus or bacteriophage
Reexamination Certificate
1998-02-24
2001-07-17
Hauda, Karen M. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Virus or bacteriophage
C435S320100, C435S455000, C514S04400A
Reexamination Certificate
active
06261554
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of providing cells with additional genetic information through recombinant DNA techniques. It especially relates to methods and means of providing specific groups of cells with additional genetic information, in particular in the context of gene therapy.
Gene therapy is a recently developed concept which has a very broad range of applications, ranging from supplementing deficiencies in a mammals' set of proteins, usually resulting from genetic disorders, to the treatment of cancer, (auto-)immune diseases or (viral) infections, usually by eliminating or suppressing the responsible set of cells or organisms.
One of the main problems in gene therapy is delivering the genetic material to the target cells and not to other cells. Another problem in gene therapy is that certain cell types are extremely refractory to current gene transfer techniques.
The present invention relates generally to gene delivery vehicles and their use in gene therapy, and more particularly, to recombinant viruses which can be targeted to susceptible target cells.
Retroviruses are RNA viruses which efficiently integrate their genetic information into the genomic DNA of infected cells via a reverse-transcribed DNA intermediate. This property of their life-cycle and the fact that parts of their genetic material can be replaced by foreign DNA sequences make retroviruses one of the most promising vectors for the delivery of genes in human gene therapy procedures, most notably for gene therapies which rely on gene transfer into dividing tissues. Most retroviral vector systems are based on mouse retroviruses and consist of two components, i.e. (i) the recombinant retroviral vector carrying the foreign sequences of interest, and (ii) so-called packaging cells expressing the structural viral proteins of which the encoding sequences are lacking in the retroviral vector. Expression of (i) in (ii) results in the production of recombinant retroviral particles capable of transducing susceptible target cells.
The infectivity and host cell range of the retrovirus particle is conferred by an envelope glycoprotein which specifically binds to a receptor molecule on the target cell membrane. The envelope glycoprotein of all known retroviruses consists of two associated peptides, which are derived by proteolytic cleavage from the same precursor protein encoded by the retroviral env gene (Dickson et al. in Weiss et al. (ed.) Molecular biology of tumor viruses (1984), Cold Spring Harbor Press, pp. 513-648). The amino terminal peptide encompasses the specific binding site for its receptor on the target cell membrane, thus determining the virus host range (Hunter and Swanstrom, Curr. Top. Microbiol. Immunol. 157(1990): 187). The carboxy terminal peptide, which contains trans-membrane anchor sequences, is assumed to account for the selective uptake of the envelope glycoprotein in the virus particle and to mediate fusion between the virus membrane and—depending on the type of virus—the plasma membrane or intracellular vesicle membrane of the target cell.
Several envelope glycoprotein variants with different infection spectra for mammalian cells have been identified. All known env variants have a rather broad infection spectrum in common. Here lies one of the major shortcomings of current recombinant retrovirus technology. In numerous gene therapy applications targeted delivery of genes into defined cells would be desired, most notably in the case of in vitro gene transfer into cell types present with low abundance in cell mixtures and in approaches for in vivo gene transfer into cells in a living mammalian body. Conventional gene transfer techniques have the disadvantage in such applications of low efficiency of gene transfer to desired target cells, because the gene transfer vehicles are taken up by other cells as well. In addition, the cell mixture or living mammalian body may contain cells to which gene transfer is absolutely undesired. E.g., genes providing protection against chemotherapeutic drugs should not be transferred into malignant cells. Therefore, increasing attention is focused on devising procedures to limit the retrovirus infection spectrum. By employing particular env variants the transduction spectrum can be limited to some extend, but true specificity for most target cells of interest can not be obtained this way. On the other hand, on the surface of some specific cell types the expression of receptors for retroviral entry is extremely low. An important example of a cell type with low retrovirus receptor expression is the pluripotent stem cell of the hematopoietic system (Orlic et al., Blood 86 suppl 1 (1995): 628a). A preferred strategy to accomplish targeted delivery is to direct the retrovirus particle to cell membrane molecules differing from the natural envelope glycoprotein receptor, said molecule being specifically expressed on the membrane of the desired target cell. Present ideas about how this could be done include:
1. direct chemical coupling of a ligand for a target cell molecule to the viral envelope glycoprotein (Neda et al, J. Biol. Chem. 266(1991): 14143),
2. bridging the viral envelope glycoprotein to a molecule on the target cell through a complex of two antibodies, one directed against the viral envelope glycoprotein and the other against the molecule on the target cell (Goud et al., virology 163(1988): 251; Roux et al., Proc. Natl. Acad. Sci. USA 86(1989): 9079; Etienne-Julan et al., J. Gen. Virol. 73(1992): 3251),
3. bridging the viral envelope glycoprotein to a molecule on the target cell through a complex of an antibody directed against the viral envelope glycoprotein and a peptide ligand for the molecule on the target cell (Etienne-Julan et al., J. Gen. Virol. 73(1992): 3251)
4. replacing the specific binding site of the viral envelope glycoprotein for its receptor by a peptide ligand for a target cell surface molecule,
5. co-expression on the virus membrane of the natural viral envelope glycoprotein and a ligand for a target cell surface molecule (Young et al., Science 250(1990): 1421), and
6. co-expression on the virus membrane of the natural viral envelope glycoprotein and an altered viral envelope glycoprotein in which the specific binding site for its receptor has been replaced by a peptide ligand for a target cell surface molecule (Young et al., Science 250(1990): 1421; Russell et al., Nucl. Acid Res. 5(1993): 1081; Chu et al., Gene Ther. 1(1994): 292; Kasahara et al., Science 266(1994): 1373; Chu and Dornburg, J. Virol. 69(1995): 2659).
Monoclonal antibodies or fragments thereof, exhibiting high specificity and affinity for the target cell specific molecule, are amongst the preferred ligands for targeted delivery. Approaches nos. 2 and 3 mentioned above rely on antibodies and promising tools for use in the approaches nos. 4 and 6 are chimaeric molecules between viral envelope glycoproteins and single-chain antibody fragments of the variable antigen-binding domain of immunoglobulins (scFv) (Russell et al., Nucl. Acid Res. 5(1993): 1081; Chu et al., Gene Ther. 1(1994): 292; Chu and Dornburg, J. Virol. 69(1995): 2659). A chimaeric molecule of such an scFv fragment and a different membrane anchoring protein than the viral envelope glycoprotein could be used for approach no. 5.
An important limitation of all these previously invented approaches is that a new virus with a specific targeting ligand (chemically or genetically modified envelope glycoprotein, or co-expressed ligand) or a new specific dual-antibody complex has to be made for each target cell type. The present invention bypasses this limitation.
Adenovirus capsids are regular icosahedrons composed of 252 subunits, of which 240 are so-called hexons and 12 are so-called pentons. The pentons are located at the vertices of the icosahedron. They contain a penton base on the surface of the capsid which is composed of five molecules of a 85 kD polypeptide. A fiber composed of a homotrimer of 62 kD polypeptides projects from the penton base outward. The fiber protein
Valerio Domenico
van Beusechem Victor Willem
Baker Anne-Marie
Hauda Karen M.
IntroGene B.V.
TraskBritt
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