Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1997-02-12
1999-03-02
Ketter, James
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C12N 1511
Patent
active
058773029
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the compaction of nucleic acids and the delivery of compacted exogenous nucleic acids to cells of multicellular organisms, in vivo.
2. Description of the Background Art
Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes to cells within intact animals.
Receptor-Mediated Uncompacted DNA Delivery In Vivo. Receptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells.
Wu, et al., U.S. Pat. No. 5,166,320, discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands". These may be formed, Wu says, either by desialation of appropriate glycoproteins, or by coupling lactose to non-galactose bearing proteins. The molar ratio of polynucleic acid to conjugate is in the range 1:10 to 10:1, more typically 1:5 to 5:1, more preferably 1:2 to 3:1. While not stated by Wu et al., in our hands, Wu's method resulted in structures with a diameter of at least 80 nm.
Low, et al., U.S. Pat. No. 5,108,921, disclose binding biotin to DNA to transform a cell using receptor mediated endocytosis.
Stomp, et al., U.S. Pat. No. 5,122,466 and McCabe, et al., U.S. Pat. No. 5,120,657 disclose attaching DNA to a metal pellet by covalently attaching polylysine to the material and then allowing DNA to be complexed to it. The resulting product is then used for ballistic transformation of a cell. See Stomp, et al., column 7, lines 29-37and McCabe, et al., column 7, lines 49-65.
Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event. Wagner et al. do not recognize the value of attaining smaller diameter structures or teach how to obtain a greater degree of compaction. It is believed that Wagner et al's structures are multimolecular complexes, which have the disadvantage that they are more vulnerable to macrophage phagocytosis and less amenable to uptake by target tissues.
Direct injection of Naked, Uncompacted DNA. The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al, Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal. Benvenisty and Reshef, Proc. Nat. Aca
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Ferkol Thomas W.
Hanson Richard W.
Perales Jose C.
Case Western Reserve University
Cooper Iver
Ketter James
Ohio University
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