Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1995-06-07
2001-02-20
Schwartzman, Robert A. (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C536S023100
Reexamination Certificate
active
06191257
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the use of DNA binding proteins to effect or enhance entry of DNA into cells for gene transfer or gene therapy. It more particularly relates to the production of chimeric, recombinant, or synthetic proteins containing DNA binding elements for the purposes of effecting or enhancing gene transfer or gene therapy. It also relates to the use of lactoferrin for enhancing entry of DNA into cells.
BACKGROUND OF INVENTION
The essential process of somatic gene therapy is the ability to perform gene transfer. In this process, recombinant genes are introduced into selected somatic cells. In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell. This process is called microinjection (1). Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription, and a gene product will be expressed. The utility of microinjection for gene therapy is obviously limited by the number of cells which can be injected. Thus, more efficient methods have been developed for introducing DNA into larger numbers of cells. These methods include: transfection, where DNA is precipitated with CaPO
4
and taken into cells by pinocytosis (2); electroporation, where cells are exposed to large voltage pulses to introduce holes into the membrane (3); lipofection/liposome fusion, where DNA is packaged into lipophilic vesicles which fuse with a target cell (4); and particle bombardment using DNA bound to small projectiles (5). Another important method for introducing DNA into cells is to couple the DNA to chemically modified proteins. These modified proteins have the ability to bind DNA through a chemically attached synthetic polylysine peptide, and bind to specific receptors on target cells. After these complexes are taken up by a specific receptor mediated endocytosis, the genes encoded by the DNA can be expressed by the target cell. Experiments have been performed with transferrin/polylysine/DNA complexes (6-11) as well as with asialoglycoprotein/polylysine/DNA complexes (12-25). The covalently, chemically combined natural ligands are used: (1) to specifically targeted DNA to different tissues; (2) to provide more efficient uptake process. These methods are limited because they require in vitro modification of the ligands using chemical or enzymatic methods in order to create a compound capable of binding DNA.
An important advance in methods of gene transfer was the demonstration that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to adenoviral particles by polylysine covalently attached to the adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (6). Further studies have also suggested that proteins purified from other pathogenic bacteria, viruses or parasites may have similar properties.
In vivo studies with asialo-orosomucoid/polylysine/DNA complexes have shown that it is possible to achieve short term expression (several days) of recombinant genes in organs such as the liver (12-15). For this expression to be useful, however, it will be necessary to administer the complex repetitively either to treat intermittent symptoms of disease, or to establish chronic, steady-state levels of the gene product. One problem with repetitive administration using the asialo-orosomucoid/polylysine/DNA complex method, however, is that this complex is highly antigenic and that anaphylaxis can result from repetitive administration. Significantly, antibodies are formed against both the covalently modified orosomucoid as well as polylysine. Presumably, the natural protein (orosomucoid) which is not normally antigenic, is rendered antigenic by the covalent addition of polylysine. (It is less likely that removal of sialic acid makes this protein antigenic since this is a normal intermediate in synthesis and degradation of the protein.)
The present invention describes an improved method for gene transfer which enables specific targeting of DNA, enhanced uptake in various cell types and endosomal destabilization without the need to produce and purify viral proteins and without the need for covalent, chemical or enzymatic modifications to couple the DNA to the ligand. The present invention thus provides a method for gene transfer which will be easier and safer than currently available methods.
LITERATURE CITED IN TEXT
(1) Capecchi M R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22:479-88 (1980).
(2) Chen C. and Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7:2745-52 (1987).
(3) Chu G., Hayakawa H. and Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 15:1311-26 (1987).
(4) Felgner P L., Gadek T R., Holm M., et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA. 84:7413-7 (1987).
(5) Yang N S., Burkholder J., Roberts B, Martinell B. and McCabe D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc. Natl. Acad. Sci. 87:9568-72 (1990).
(6) Curiel D T., Agarwal S., Romer M U., Wagner E., Cotten M., Birnstiel M L. and Boucher R C. Gene transfer to respiratory epithelial cells via the receptor-mediated endocytosis pathway. Am. J. Respir. Cell. Mol. Biol. 6:247-52 (1992).
(7) Wagner E., Cotten M., Mechtler K., Kirlappos H. and Birnstiel M L. DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety. Bioconjug-Chem. 2:226-31 (1991).
(8) Wagner E., Cotten M., Foisner R. and Birnstiel M L. Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. USA. 88:4255-9 (1991).
(9) Wagner E., Zenke M., Cotten M., Beug H. and Birnstiel M L. Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA. 87:3410-4 (1990).
(10) Zenke M., Steinlein P., Wagner E., Cotten M., Beug H. Birnstiel M L. Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. USA. 87:3655-9 (1990).
(11) Cotten M. Langle-Rouault F., Kirlappos H., Wagner E., Mechtler K., Zenke M., Beug H. and Birnstiel M L. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. USA. 87:4033-7 (1990).
(12) Wilson J M., Grossman M., Wu C H., Chowdhury N R., Wu G Y. and Chowdhury J R. Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J. Biol. Chem. 267:963-7 (1992).
(13) Wilson J M., Grossman M., Cabrera J A., Wu C H., Wu G Y. A novel mechanism for achieving transgene persistence in vivo after somatic gene transfer into hepatocytes. J. Biol. Chem. 267: 11483-9 (1992).
(14) Wu G Y., Wilson J M., Shalaby F., Grossman M., Shafritz D A. and Wu C H. Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats. J. Biol. Chem. 266:14338-42 (1991).
(15) Wu C H., Wilson J M. and Wu G Y. Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J. Biol. Chem. 264: 16985-7 (1989).
(16) Wu G Y. and Wu C H. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262:4429-32 (1987).
(17) Wu G Y. and Wu C H. Targeted delivery and expression of foreign genes in hepatocytes. Targeted Diagn. Ther. 4:127-49 (1991).
(18) Wu G Y. and Wu C H. Delivery systems for gene therapy. Biother
Ledley Fred D.
Stankovics Jozsef
Baylor College of Medicine
Lyon & Lyon LLP
Schwartzman Robert A.
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