Nucleic acid transfer system

Details Nucleic acid transfer system Nucleic acid transfer system

1997-04-25

2002-12-24

McKelvey, Terry (Department: 1636)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Reexamination Certificate

active

06498233

ABSTRACT:

The invention pertains to a nucleic acid transfer system suitable for targeting a nucleic acid, e.g. a gene, to a specific cell, and obtaining expression of said nucleic acid. The nucleic acid transfer system of the invention comprises a multidomain protein component and a nucleic acid component. Furthermore, the present invention relates to the multidomain protein, a nucleic acid encoding said protein, suitable amplification and expression systems for said nucleic acid, and processes for the preparation and uses of the above subject matters.
Gene transfer to eukaryotic cells may be accomplished using viral vectors, such as recombinant adenoviruses, or non-viral gene transfer vectors. Owing to several disadvantages, e.g. constraints in the size of the DNA to be delivered, incapability of transducing terminally differentiated cells, potential safety hazards and insufficient targetability, such viral DNA transfer systems seem to be of limited use in gene therapy strategies. As an alternative to viral systems, ligand-mediated approaches via molecular conjugate vectors have been developed. Such molecular conjugate vectors comprise the DNA molecule to be transferred and a target cell-specific ligand which is chemically coupled to a polycation, particularly a polyamine (for review, see e.g. Michael & Curiel, Gene Therapy 1: 223, 1994). The polycation binds to the DNA through electrostatic forces, thus acting to tie up the ligand with the gene to be delivered. For example, human transferrin or chicken conalbumin were covalently linked to poly-L-lysine or protamine through a disulfide linkage. Complexes of protein-polycation—conjugate and a bacterial plasmid containing a luciferase encoding gene were supplied to eukaryotic cells, resulting in expression of the luciferase gene (Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410, 1990). To achieve higher levels of gene expression, adenovirus particles were chemically coupled to the complex (see e.g. Curiel et al., Proc. Natl. Acad. Sci. USA 88: 8850, 1991; Christiano et al., Proc. Natl. Acad. Sci. USA 90: 11548, 1993). However, molecular conjugate vectors also have limitations, including large size, inhomogeneity, lack of specificity pertaining to the binding of the DNA component, and non-specific binding due to electrostatic interactions between the polycation and the cell membrane, which may at least partially neutralize the targetability imposed by the ligand.
Thus there is still a need for a simple, efficient nucleic acid transfer system which allows e.g. the target cell-specific introduction of nucleic acids to be expressed, but lacks the disadvantages of the prior art concepts.
It is the object of the present invention to provide such a system. The nucleic acid transfer system according to the invention is characterized by the following two components:
1) a multi-domain protein comprising several functional domains including a nucleic acid binding domain
2) an effector nucleic acid, particularly a DNA, comprising the nucleic acid, e.g. the gene, to be delivered to and expressed in a selected target cell, and a cognate structure recognizable by the nucleic acid binding domain of the protein.
The multi-domain protein component combines in a single molecule a target cell recognition function, also referred to as ligand domain, an endosome escape function and a nucleic acid binding function, particularly a DNA binding function. Such a protein does not occur in nature. The nucleic acid binding function serves to mediate the specific, high affinity and non-covalent interaction of the protein component with the effector nucleic acid component. Unlike the above described molecular conjugate vector of the prior art, the protein
ucleic acid complex of the present invention is formed by specific interaction of the nucleic acid binding domain with its cognate structure on the effector nucleic acid. Advantageously, the binding affinity of the proteinaceous nucleic acid binding domain for its cognate structure on the effector nucleic acid surpasses the affinity of the proteinaceous target cell recognition function for its cognate molecular structure on the target cell. Within the nucleic acid transfer system of the present invention the effector nucleic acid component may be e.g. a complete or partial plasmid carrying the nucleic acid to be expressed in the target cell. The nucleic acid delivery system of the invention is designed such that the rate of nucleic acid transfer is optimized.
Advantageously, the present system makes use of physiological target-cell inherent mechanisms of macromolecular transport involving endosomes, particularly receptor-mediated endocytosis. The protein
ucleic acid complex according to the invention is targetable in that it may be efficiently internalized only by a predetermined cell-type or cell population carrying a molecular structure, e.g. a receptor, which specifically interacts with the target cell recognition function of said complex. After entering the cell, the protein
ucleic acid complex of the invention becomes localized in endosomes from where it is released into the cytoplasm. Owing to the selective internalization of the protein
ucleic acid complex, expression of the particular nucleic acid(s) to be delivered by the complex of the invention occurs in a way that distinguishes (transfected) target cells from (non-transfected) non-target cells, e.g expression is essentially confined to the predetermined target cell. The nucleic acid to be transported to and expressed in the target cell may be therapeutically active or encode a therapeutically active product, e.g. tumor cells may be transfected to introduce a gene coding for a therapeutically active protein.
More specifically, the present invention provides a two-component system for the target cell-specific delivery and uptake of a non-covalently linked protein
ucleic acid complex leading to the expression in said target cells of one or more nucleic acids comprised by the transferred effector nucleic acid. Preferentially, such system of the invention essentially consists of a protein
ucleic acid complex containing two components:
a polypeptide chain containing several different functional, domains of eukaryotic, prokaryotic or synthetic origin, and
an effector nucleic acid.
Advantageously, the protein
ucleic acid complex is sufficiently stable in physiological fluids to enable its application in vivo. The complex of the invention is a molecular complex, whose stochiometry is essentially determined by the number of cognate structures of the protein nucleic acid binding domain on the effector nucleic acid. For example, the cognate structure of the yeast GAL4 binding domain is thought to bind a protein dimer. Accordingly, the ratio of multidomain protein to effector nucleic acid in the complex of the invention is 2:1 by using one nucleic acid binding domain. However, it is preferred to use nucleic acids which contain multiple sequences (preferably 2-8 which recognize the nucleic acid binding domain).
Successful transfer and expression of the desired nucleic acid depends on the specific interaction of the protein
ucleic acid complex with the target cell and on the efficient transfer of the nucleic acid of interest across systemic or subcellular barriers. To examine whether the complex of the invention is transported into or within the target cell, the complex may be suitably labeled and its accumulation on and in cells determined, e.g. by fluorescence imaging. For example, the complex may be fluoresence-labeled and its cellular localization be visualized, e.g. by video-enhanced microscopy and quantitative confocal laser scanning. Other assays suitable for determining the functionality of the nucleic acid transfer system of the invention, such as an assay for the expression of a delivered reporter gene, are described in the Examples. Further assays are known in the art and evident to the skilled person.
The nucleic acid delivery system of the invention provides for e.g. for efficient gene transfer in that it enables e.g. transit of said gene through

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