Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-21
2001-03-13
LeGuyader, John L. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C530S300000, C530S324000, C530S326000, C530S330000, C435S455000
Reexamination Certificate
active
06200801
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the in vivo delivery of exogenous nucleic acids to cells of multicellular organisms. In particular, the present invention relates to the delivery of exogenous nucleic acids to cells having a serpin enzyme complex receptor on their surface.
BACKGROUND
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, polyomithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests “asialoglycoprotein (galactose-terminal) ligands”.
Wagner, et al.,
Proc. Natl. Acad. Sci
., 88:4255-4259 (1991) and U.S. Pat. No. 5,354,844 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.
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. Acad. Sci. USA
, 83:9551-55 (1986) injected calcium phosphate precipitated DNA intraperitoneally into newborn rats and noted gene expression in the livers of the animals 48 hours after transfection. In 1990, Wolff et al.,
Science
, 247:1456-68 (1990), reported that the direct injection of DNA or RNA expression vectors into the muscle of mice resulted in the detectable expression of the genes for periods for up to 2 months. This technique has been extended by Acsadi et al.,
New Biologist
, 3:71-81 (1991) to include direct injection of naked DNA into rat hearts; the injected genes were expressed in the heart of the animals for up to 25 days. Other genes, including the gene for dystrophin have been injected into the muscle of mice using this technique. This procedure forms the base of a broad approach for the generation of immune response in an animal by the administration of a gene by direct injection into the target tissue. The gene is transiently expressed, producing a specific antigen. (See Donnelly et al.,
The Immunologist
, 21, pp. 20-26 (1994) for a recent review). However, the DNA used in these experiments has not been modified or compacted to improve its survival in the cell, its uptake into the nucleus or its rate of transcription in the nucleus of the target cells.
SUMMARY OF THE INVENTION
The present invention relates to the delivery of exogenous nucleic acids to cells, including but not limited to the cells of multicellular organisms. When the nucleic acid includes an expressible gene, that gene can be expressed in the cell. In some embodiments, a tissue-specific carrier molecule is prepared, which is a bifunctional molecule having a nucleic acid-binding moiety and a target tissue-binding moiety.
The nucleic acid can be compacted at high concentrations with the carrier molecule at a critical salt concentration. The nucleic acid-loaded carrier molecule is then administered to the organism.
In one embodiment, the present invention contemplates a method for delivering an oligonucleotide to a mammalian cell, comprising the steps of: a) providing: i) a target binding moiety capable of binding to a serpin enzyme complex receptor; ii) a nucleic acid binding moiety; iii) an expression vector comprising an oligonucleotide encoding one or more gene products; iv) a mammalian cell having on its exterior surface a serpin enzyme complex receptor; b) conjugating the target binding moiety to the nucleic acid binding moiety to form a carrier; c) coupling the expression vector with the carrier to form a pharmaceutical composition; and d) contacting the mammalian cell with the pharmaceutical composition under conditions such that the pharmaceutical composition binds to the receptor and results in delivery of the pharmaceutical composition to the interior of the mammalian cell. It is preferred that the expression vector (i.e., the nucleic acid or oligonucleotide encoding one or more gene products) is compacted. The compaction of nucleic acids (e.g., expression vectors) associated with a carrier comprising a conjugate between a TBM and a NABM is described in detail herein. Preferably, the pharmaceutical compound comprising the carrier and the expression vector are compacted to a diameter of less than 100 nm, preferably less than 80 nm and most preferably having a diameter of about 10 to 25 nm, with a diameter of about 15 to 25 nm being particularly preferred.
As used herein, a “pharmaceutical composition” is a composition comprising an aggregate (i.e., a complex) between an expression vector (i.e., a nucleic acid molecule) and a carrier comprising a target binding moiety conjugated to a nucleic acid binding moiety. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The terms “pharmaceutical composition” and therapeutic composition” are used herein interchangeably. It is not intended that the pharmaceutical compositions be limited to any particular expression vector, carrier or exciepient.
In a preferred embodiment, the expression vector further comprises a promoter sequence operably linked to the oligonucleotide encoding one or more gene products. The present invention is not limited by the nature of the promoter sequence employed. Any promoter sequence which is functional in the target cell (i.e., the cell expressing a serpin enzyme complex (SEC) receptor on its exterior surface), may be employed to achieve expression of the gene(s) of interest. The promoter sequence may be from a mammalian gene, including but not limited to the gene encoded by the expression vector (i.e., the gene(s) of interest present on the expression vector may be under the transcriptional control of their native or endogenous promoter).
The promoter sequence may be derived (i.e., obtained or isolated) from a gene expressed in all mammalian cells (i.e., a constitutive or ubiquitous promoter) such as &bgr;-actin, human elongation factor 1&agr; gene, etc. Alternatively, the promoter may be derived from a gene which is expressed in a tissue-specific manner so long as the promoter is active in the target cell. For example, when liver cells are the target cells, promoters derived from genes expressed in the live
Davis Pamela B.
Ferkol, Jr. Thomas W.
Ziady Assem-Galal
Banner & Witcoff , Ltd.
Case Western Reserve University
LeGuyader John L.
Nguyen Dave Trong
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