Compositions and methods for highly efficient transfection

Details Compositions and methods for highly efficient transfection Compositions and methods for highly efficient transfection

1998-02-12

2002-11-12

Nguyen, Dave T. (Department: 1632)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S320100, C435S325000, C435S069100, C435S455000, C435S091400, C530S300000, C530S324000

Reexamination Certificate

active

06479464

ABSTRACT:

FIELD OF THE INVENTION
The invention relates in general to transfection of cells and to agents which condense nucleic acid.
BACKGROUND OF THE INVENTION
Cell transfection relies on efficient delivery of DNA to target cells, and expression of the delivered DNA in the nucleus of such cells.
Early experiments on introducing DNA into mammalian cells in vitro utilized DNA in precipitated form with low efficiency of transfection and required selectable marker genes (Wigler et al. (1977) Cell 16:777-85; Graham and Van der Eb (1979) Proc. Natl. Acad. Sci. USA 77:1373-76 and (1973) Virology 52:456)). Since this time molecular biologists have developed many other more efficient techniques for introducing DNA into cells, such as electroporation, complexation with asbestos, polybrene, DEAE, Dextran, liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984) Crit. Rev. Biochem. 16:349-79; Keown et al. (1990) Methods Enzymol. 185:527). Many of these methods are unsuitable for use clinically since they give highly variable and relatively poor levels of transfection. Another obstacle to the wider use of existing transfection complexes resides in their instability in vivo. It has been shown that particles of a similar size to the transfection complexes of the prior art are rapidly and efficiently removed from the blood by the reticuloendothelial system (Poste and Kirsch, Bio/Technology 1:869 (1984)).
Soluble DNA/polylysine complexes have been generated (Li et al., (1973) Biochem. J. 12:1763) and tagged with asialoglycoprotein to target DNA to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262:4429 (1987); U.S. Pat. No. 5,166,320). Lactosylated polylysine (Midoux et al. (1993) Nuc. Acids Res. 21:871-878) and galactosylated histones (Chen et al. (1994) Human Gene Therapy 5:429-435) have been used to target plasmid DNA to cells bearing lectin receptors, and insulin conjugated to polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 199:323-329) to cells bearing insulin receptors. However, Wagner et al. (supra) have shown that the latter approach is even less efficient than standard methods of transfection, and may therefore be considered unsuitable for pharmaceutical-development. Monoclonal antibodies have been used to target DNA to particular cell types (Machy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-8031; Trubetskoy et al. (1992) Bioconjugate Chem. 3:323-27 and WO 91/17773 and WO 92/19287).
Peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when coadministered with polylysine DNA complexes (Plank et al. (1994) J. Biol. Chem. 269:12918-24). Trubetskoy et al. (supra) and Mack et al. ((1994) Am. J. Med. Sci. 307: 138-143) suggest that cocondensation of polylysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency. WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.
Disulfide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al. (1992) Meth. Enzymol. 217:618-644); see also, Trubetskoy et al. (supra) However, the chemical modification of the various components, although group specific, is not regio-specific and leads to enormous molecular heterogeneity of the conjugated product. Disulfide bonds are also known to be unstable in biological fluids and thus limits the potency of such compounds in practice.
Similar heterogeneity is also produced by other standard conjugation methods such as carbodiimide coupling through side chain carboxyl groups (Wu et al. (1991) J. Biol. Chem. 266:14338-42). However, in addition to the above disadvantages, the resulting amide bond coupling the components is chemically stable within the cytosol and makes the components difficult to separate.
More specific coupling chemistry has been employed by Cotten et al. (supra). This method involves oxidation of the carbohydrate moieties using periodate, followed by subsequent reaction with polylysine. The Schiff base so formed was reduced with sodium cyanoborohydride to form a stable amide bond. However, due to the large number of available lysine residues, the resulting amide bond was linked at random to the polylysine component.
Trubetskoy (supra) observed increased efficiency of a conjugate made up of a heterogeneous polylysine moiety linked through the N-terminus non-specifically to amino functions on a monoclonal antibody.
Many prior art methods employ highly heterogeneous components linked by conjugation chemistry which itself leads to more heterogeneity. This heterogeneity leads to poor control during preparation and large batch-to-batch variability, low potency and poor solution stability.
Scale up and reproducible manufacture of the gene delivery vehicles described in the literature are problematic because of the extreme heterogeneity of the products and components of those systems. Key parameters such as quality control, process control and product identification are thus rendered imprecise. Therefore, an object of the invention is the development of a reproducible and scalable production process for pharmaceutical compositions which facilitate delivery of exogenous DNA to a target cell with high efficiency.
Another object of the invention is to provide an improved transfection complex having chemical components of defined stoichiometry and therefore reduced heterogeneity.
Yet another object of the invention is to provide pharmaceutical formulations for transfection which exhibit increased transfection efficiency.
SUMMARY OF THE INVENTION
The invention is based on the discovery of polypeptides which, when associated with a nucleic acid, confer a high efficiency upon host cell transfection.
The invention encompasses a polypeptide comprising or consisting of the following 29 amino acid composition: 6G's, 2F's, 2L's, 1W, 4R's, 2E's, 2N's, 3K's, 1T, 1S, 1A, 1Y, 1M, 1C, and 1I, and having additional cationic residues to provide a net number of positive charges of greater than 8. Preferably, the 29 amino acids are contiguous.
As used herein, “composition” refers to the amino acid content rather than an order of amino acids. “Cationic residue” refers to an amino acid or other molecule having a net positive charge, examples of which include but are not limited to lysine, ethyleneimine, arginine, methacrylate, amidoamine, protamine, spermine, and spermidine. “Cationic charge” refers to a net positive charge.
As the 29 amino acids specified above contained in the polypeptide contain 7 cationic residues and 2 anionic residues, and thus contain a net of 5 cationic charges, the additional cationic charges will number at least 3, preferably, 4 or 5 and more preferably number, for example, 6, 12, 18 or 24.
Polypeptides according to the invention will therefore contain the 29 amino acids specified above and additional cationic residues sufficient to net equal to or greater than 8 positively charged residues in the polypeptide, wherein the 29 amino acids specified above may be present in the polypeptide as (a) a block of: 29 contiguous amino acids and equal to or greater than (≧) 3 cationic residues (monomer) or (b) as two or more blocks of the 29+≧3 amino acids (dimer, trimer, etc., i.e., multimer). Where a 29+≧3 polypeptide is present in a multimeric form, several individual 29+≧3 polypeptides may be linked in conventional stable bonds (peptide, oxime, or thioether) or the polypeptides may be linked via labile bonds, e.g., disulfide bonds.
A cationic sequence useful herein may be a tract of contiguous cationic residues in the length range of 3 to 700, whereby the net cationic charge is at least 3. Alternatively, the tract of cationic sequences need not be contiguous, but may be dispersed among basic or neutral amino acids such that the net number of cationic charges is in the range of 3 to 700. Therefore, a polypeptide according to the invention may contain as few as (5+3=)8 net cationic ch

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