United States Patent:  6,077,834

Appl. No.:  790290

Disclosed is a delivery system for biologically active molecules or agents which must enter cells to exert their effect. The delivery system comprises a mixture of cationic lipid in combination with a receptor ligand and is particularly suited for intracellular delivery of polynucleotides.

SUMMARY OF THE INVENTION

This invention employs a delivery system for biologically active molecules which must enter cells to exert their biological effect. Such molecules include but are not limited to peptides, proteins, or polynucleotides such as DNA or RNA. In a preferred embodiment the molecule is a gene for transfection in gene therapy protocal. The delivery system is a mixture composed of a cationic lipid which is preferably in a liposome formulation and a receptor ligand, such as transferrin, wherein the ligand is not covalently bound to a liposome component. The receptor ligand is first added to the cationic liposome formulation and incubated. The biologically active agent to be delivered to cells is then added to the resulting ligand-liposome combination and the mixture is again incubated. The order in which the components are combined, i.e. ligand with liposome formulation followed by nucleic acid, is critical to obtaining high efficiency transfection.

Generally, the methods of this invention can employ any ligand having an affinity for a cell surface receptor. The term ligand is used broadly herein and includes receptor ligands such as transferrin, insulin, cholera toxin, adenovirus fiber KNOB peptide, as well as antibodies and antibody fragments (e.g. Fab fragments) to receptors, such as anti-secretory components, and peptides and proteins, such as epidermal growth factor and viral proteins, particularly those viral proteins which the receptor-mediated endocytosis mechanism stimulates. The ligand employed can be one, such as transferrin or insulin, that targets a wide range of cell types or one that targets a specific cell type.

Generally, the methods of this invention can employ any mono-or polycationic lipid and neutral lipid in a cationic liposome formulation. Cationic lipids including DOTMA, DDAB, DOSPA, DORI, DORI-ester, DORI-ether, DMRIE, DOTAP, TM-TPS and cationic lipids structurally related thereto. [Definitions for each of these acronyms are provided herein and are well-known in the art.]

More specifically, this invention provides transfection agents in which the ligand is transferrin, insulin, cholera toxin or adenovirus fiber KNOB peptide, and in which the cationic liposome formulation comprises the cationic lipids DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl), or DDAB (dimethyl dioctadecylammonimum bromide) and an appropriate neutral lipid. Useful cationic liposome formulations include those in which the neutral lipid is DOPE (dioleoyl phosphatidylethanolamine). Preferred cationic liposome formulations are "lipofectin" (Trademark) which is a commercially available 1:1 liposome formulation of DOTMA and DOPE and "lipofectace" (Trademark) which is a commercially available 1:2.5 liposome formulation of DDAB and DOPE.

Certain fractions and/or components of serum have been found to enhance transfection efficiency of cationic liposomes. These fractions or components, when used in place of receptor ligands in the protocols of this invention, enhance liposome transfection efficiency.

The method of this invention is a high-efficiency gene transfer method which employs transfection reagents that are easy to prepare. In exemplified embodiments the reagent ingredients are commercially available. Further, receptor ligands prepared from one animal source may be employed for gene therapy in the same animal species, thus mitigating or preventing host inflammatory and immune responses which have been a major drawback for human gene therapy employing adenoviral vectors. Liposomes appear to cause little or no apparent host inflammatory and immune response. Utilization of human transferrin or other human ligands in combination with cationic liposomes can circumvent the host immune response while achieving high gene transfer efficiency in humans.

The methods described herein with "lipofectin" and transferrin can yield 100% transfection efficiency in HeLa cells. The "lipofectin"-transferrin transfection protocol of this invention can also be used to correct the chloride conductance abnormality in immortalized CF airway epithelial cells (CFT1) by delivery of CFTR CDNA. Transferrin also significantly enhances transfection by "lipofectace" of HeLa cells.

The transfection agents and methods of this invention are useful in in vitro and in vivo transfection applications. The simplicity of the formulation and high transfection efficiency by these reagents facilitate the development of suitable transfection reagents for human gene therapy.

Claim 1 of 19 Claims

1. A method for intracellular delivery of a polynucleotide comprising:

(a) first combining a non-viral receptor ligand and a cationic lipid to form a mixture, so that said ligand and lipid become associated although not covalently bound; and thereafter

(b) adding to said mixture a polynucleotide, so that said polynucleotide becomes associated with said lipid to form a molecular mixture; and

(c) introducing said molecular mixture to a cell, wherein said molecular mixture enhances the delivery of said polynucleotide to said cell.

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