WO2004096117A2 - Drug delivery systems comprising transferrin-nucleic acid conjugates - Google Patents

Abstract

Compositions for delivering and expressing nucleic acids in a cell in a subject are provided. The compositions comprise a transferrin ligand and one or more other cell-specific ligands operatively linked to the transferrin ligand, and a nucleic acid that is operatively linked to the transferrin ligand. The nucleic acid comprises a coding sequence and a promoter for driving or promoting expression of the coding sequence. In certain embodiments, the compositions of the instant invention may comprise human vascular endothelial growth factor (VEGF), or human epidermal growth factor (EGF), or both human VEGF and human EGF, and at least one nucleic acid encoding a protein, polypeptide, or RNA of interest, each of which is operatively linked to a human transferrin ligand. Methods of using the present compositions to express nucleic acids in a cell in a subject, to confer immunity to an infectious agent on the subject, or to reduce levels of a disease or disorder causing agent in the subject are also provided.

Description

NUCLEIC ACID DELIVERY SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Patent Application Serial

Nos: 10/429,662 and 10/10/429,660, both of which were filed on May 2, 2003, and are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION [0002] The possibility of delivering foreign or heterologous nucleic acid molecules into cells raises hope for the development of many new therapeutics, particularly when such nucleic acids are expressed in vivo at therapeutic levels, i.e., levels that are beneficial to the host animal.

[0003] A number of methods have been developed to introduce nucleic acids into cells. All of these are based on contacting the nucleic acid with the target cells. Cells in vitro are easily physically accessible to direct contact through methods such as so-precipitation with calcium phosphate, electroporation, microinjection and particle bombardment ("gene gun"; firing of metallic microprojec tiles coated with nucleic acid at the target cells). Cells in vivo are often not easily physically accessible to the direct contact necessary for efficient uptake of nucleic acids. Thus, although the above-referenced techniques are effective for in vitro use, they are impractical and potentially damaging for use in vivo. [0004] Nucleic acids to be delivered to target cells in vivo are usually transported through the circulatory system where they are subject to damage and clearance from the body before ever reaching their target cells. Thus, carriers are often employed for transport of nucleic acid in vivo in order to provide protection from damage and prolong half-life. There are several broad categories of compositions and methods currently employed for in vivo delivery including; nucleic acid condensing agents, polymer systems, liposomes, viral vectors, and peptide-enhanced delivery methods.

[0005] For successful in vivo application it is important that nucleic acid delivery carriers be small enough to gain access to target cells by passage through cellular membranes. DNA can be condensed into small polyelectrolyte complexes by the addition of polycations, such as poly (L) lysine. These small complexes facilitate the transfer of DNA across the cell membrane for delivery to the nucleus. These polyelectrolyte complexes have shown limited usefulness due to rapid clearance from the circulation following intravenous injection. This rapid clearance results from destabilization of the polyelectrolyte complexes by serum proteins and degradation of the polyelectrolyte complexes by serum proteases. [0006] Polymer systems have also been used for the delivery of nucleic acids, for example, by complexing nucleic acid with silica or by complexing nucleic acid with polymeric cations such as natural proteins and starches. However, these systems are subject to degradation and rapid clearance from the body.

[0007] Liposomes are phospholipid bilayer vesicles that can be used to encapsulate and transport nucleic acids. The nucleic acid is trapped in the aqueous compartment of the liposome and enclosed by the completely sealed lipid bilayer. The membranes of most cells have a net negative charge and thus the cationic nature of liposomes allows for attraction of the liposome to the cellular membrane resulting in fusion of the liposome with the cell membrane and subsequent release of the nucleic acid contained within. However, several disadvantages are associated with use of liposomes for the delivery of nucleic acids. Liposomes are not effective for nucleic acid delivery to all cell types. Often high doses of nucleic acid are required for successful delivery. Additionally, liposomes lack selectivity and can bind plasma proteins and the extracellular matrix thus never reaching their target cells. [0008] Nucleic acids can also be integrated within a viral genome and delivered to target cells through viral infection. Adenovirus and retrovirus are commonly used for this purpose. The size of nucleic acid to be delivered by viral vector is limited to approximately 7- 8 kb. Additionally, viral genes are also delivered with the nucleic acid which can lead to undesirable side effects including; immunogenicity, fixation of complement, poor-target selectivity and potential toxicity. It is also difficult to produce virus in the quantities necessary for use with delivery methods.

[0009] Delivery can also be enhanced by conjugation of the nucleic acid with specific pep tides. Peptides containing aromatic amino acids (phenylalanine, tyrosine, tryptophan) are useful for this purpose. Additionally, viral peptides, such as fusogenic peptides derived from Staphylococcal Protein A enhance membrane fusion and thus can increase the efficiency of delivery. However, peptide-enhanced delivery is not effective for all types of cells. [0010] Since many disadvantages remain associated with all of the compositions and methods currently employed for in vivo delivery discussed in the above paragraphs, there is clearly a need for improved compositions and methods for delivery of nucleic acids to cells in vivo.

SUMMARY OF THE INVENTION [0011] The instant invention provides compositions capable of delivering to and enhancing the expression of heterologous nucleic acids within cells in a mammalian subject, more particularly a human subject. The compositions of the present invention comprise a transferrin ligand and one or more other cell-specific ligands operatively linked to the transferrin ligand, and a nucleic acid that is operatively linked to the transferrin ligand. The nucleic acid comprises a coding sequence that encodes an RNA, protein, or polypeptide of interest and a promoter for driving or promoting expression of the coding sequence. In certain embodiments, the compositions of the instant invention may comprise human vascular endothelial growth factor (VEGF), or human epidermal growth factor (EGF). or both human VEGF and human EGF, and at least one nucleic acid encoding a protein or RNA of interest, each of which is operatively linked to a human transferrin ligand. In one embodiment, the composition comprises human vascular endothelial growth factor (VEGF) and at least one nucleic acid each operatively linked to human transferrin wherein said human VEGF binds human VEGF receptors where expressed on cell surfaces and said human transferrin binds human transferrin receptors where expressed on cell surfaces. In another embodiment, the composition comprises human epidermal growth factor (EGF) and at least one nucleic acid encoding a protein, polypeptide or RNA of interest, each of which is operatively linked to human transferrin wherein said human EGF binds human EGF receptors where expressed on cell surfaces and said human transferrin binds human transferrin receptors where expressed on cell surfaces. In another embodiment, the composition comprises human vascular endothelial growth factor (VEGF), human epidermal growth factor (EGF) and at least one nucleic acid each operatively linked to human transferrin, wherein said human VEGF binds to human VEGF receptors where expressed on cell surfaces, said human EGF binds to human EGF receptors where expressed on cell surfaces and said human transferrin binds human transferrin receptors where expressed on cell surfaces. In certain embodiments the nucleic acid encodes an antibody, more particularly the light chain or heavy chain of an antibody, preferably a human antibody.

[0012] The present invention also relates to a pharmaceutical composition comprising a pharmacologically effective amount of a carrier and a complex comprising a transferrin ligand, one or more other cell-specific ligands that are operatively linked to the transferrin ligand, and a nucleic acid operatively linked to the transferrin ligand. The nucleic acid encodes an RNA, a protein, or a polypeptide that directly or indirectly has a therapeutic effect on a disease or disorder of a mammalian subject, particularly a human subject. [0013] The present invention also relates to a method of delivering and expressing a nucleic acid that encodes an RNA, protein, or polypeptide in a human subject. The method comprises administering a complex comprising a transferrin molecule, the nucleic acid, and another ligand that binds to a receptor on within a cell in the subject. In certain preferred embodiments, the nucleic acid encodes an RNA protein or polypeptide has a direct or indirect therapeutic effect on a disease or disorder in a mammalian subject, preferably a human subject. The method comprises administering a pharmaceutical composition of the present invention to the subject.

[0014] The present invention also relates to a method of conferring immunity to an infectious agent to a mammalian subject, including a human subject. The method comprises administering a complex comprising a transferrin ligand, a nucleic acid comprising a coding sequence that encodes an antibody and a promoter that drives expression of the coding sequence, and another cell specific ligand. The nucleic acid and the cell-specific ligand are operatively linked to the transferrin ligand. In certain embodiments a single complex comprising a nucleic acid that encodes the heavy chain of the antibody, preferably a human antibody, and a nucleic acid that encodes the light chain of the antibody, preferably the human antibody, is administered to the subject. In other embodiments two complexes are administered to the subject, wherein one of the complexes comprises a nucleic acid that encodes the heavy chain of the antibody, preferably the heavy chain of a human antibody, and the other complex comprises a nucleic acid that encodes the light chain of the antibody, preferably the light chain of a human antibody.

[0015] The present invention also relates to a method of reducing levels of a disease or disorder causing agent in a subject. The method comprises administering a biologically effective amount of a composition comprising a transferrin ligand, a cell-specific ligand, and a nucleic acid comprising a coding sequence immunoreactive with the agent to the subject.

The cell-specific ligand and the nucleic acid, which also comprises a promoter for promoting expression of the coding sequence in the cell, are both operatively linked to the transferrin ligand. The disease causing agent may be a protein, polypeptide, polysaccharide or a lipopolysaccharide.

BRIEF DESCRIPTION OF THE FIGURES

[0016] FIGURE 1 shows a diagrammatic presentation of several embodiments of the compositions described herein.

[0017] FIGURE 2 shows a schematic presentation of the cloning steps necessary to produce the DNA vectors utilized in the experimental examples described herein.

[0018] FIGURE 3 shows a graphical presentation of the serum level of anti-digoxin antibody achieved in experimental SCID mice that have been injected with a composition comprising a transferrin ligand operatively linked to EGF and a vector comprising a sequence encoding the heavy chain of anti-digoxin antibody and a transferrin ligand operatively linked to EGF and a vector comprising a sequence encoding the light chain of anti-digoxin antibody. [0019] FIGURE 4 shows a graphical presentation of the serum level of anti- respiratory synctial virus (RSV) antibody achieved in experimental SCID mice that have been injected with a composition comprising a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the heavy chain of anti-RSV antibody and a composition comprising transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the light chain of anti-RSV antibody. Control animals have been injected with compositions in which the transferrin ligands are linked to expression vectors comprising a sequence encoding the heavy chain and light chain of anti- digoxin antibody.

[0020] FIGURE 5 is a bar graph showing the RSV titers in the lungs of SCID mice that were challenged with RSV following injection with a composition comprising a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the heavy chain of anti-RSV antibody and a composition comprising transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the light chain of anti-RSV antibody. Control animals were challenged with RSV following injection with compositions in which the transferrin ligands are operatively linked to EGF and expression vectors comprising the heavy and light chains of anti-digoxin antibody. [0021] FIGURE 6 is a graphical presentation showing the survival of SCID mice that have been infected with RSV following injection with a composition comprising a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the heavy chain of anti-digoxin antibody and a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the light chain of anti-RSV or injection with compositions in which the transferrin ligands are linked to expression vectors comprising a sequence encoding the heavy chain and light chain of anti-digoxin antibody (control animals).

[0022] FIGURE 7 shows a graphical presentation of the serum level of anti-TNF alpha antibody achieved in experimental SC D mice that have been injected with a composition comprising a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the heavy chain of anti-digoxin antibody and a transferrin ligand operatively linked to EGF and an expression vector comprising a sequence encoding the light chain of anti-TNF alpha antibody. Control animals have been injected with compositions in which the transferrin ligands are linked to expression vectors comprising a sequence encoding the heavy chain and light chain of anti-digoxin antibody.

[0023] The drawings constitute a part of this specification and include exemplary embodiments of the present invention and illustrate various objects and features thereof.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

[0024] The following list defines terms, phrases and abbreviations used throughout the instant specification. Although the terms, phrases and abbreviations are listed in the singular tense the definitions are intended to encompass all grammatical forms.

[0025] As used herein, the abbreviation "EGF" refers to epidermal growth factor.

[0026] As used herein, the abbreviation "EGFR" refers to epidermal growth factor receptor.

[0027] As used herein, the abbreviation "VEGF" refers to vascular endothelial growth factor.

[0028] As used herein, the abbreviation "VEGFR" refers to vascular endothelial growth factor receptor.

[0029] As used herein, the abbreviation "PEG" refers to polyethylene glygol.

[0030] As used herein, the abbreviation "TF" refers to transferrin.

[0031] As used herein, the abbreviation "SA" refers to streptavidin.

[0032] As used herein, the abbreviation "TF/SA" refers to a composition comprising transferrin linked to streptavidin.

[0033] As used herein, the abbreviation "MBS" refers to m-maleimidobenzoyl N- hydroxysuccinimide ester.

[0034] As used herein, the abbreviation "HPLC" refers to high performance liquid chromatography.

[0035] As used herein, the abbreviation "RP-HPLC" refers to reverse phase high performance liquid chromatography. As used herein, the abbreviation "NHS" refers to N hydroxysuccinimide.

[0036] As used herein, the abbreviation "TFA" refers to trifluoroacetic acid.

[0037] As used herein, the abbreviation "PBS" refers to phosphate buffered saline.

[0038] As used herein, the abbreviation "SCID" refers to a type of transgenic mouse that is severe combined immuno-deficient. [0039] As used herein, the term "selective delivery" is defined as delivery which is targeted to a specific cell type for the purpose of avoiding uniform or even delivery to all cell types.

[0040] As used herein, the term "selective concentration" is defined as concentrating a substance, such as nucleic acid, to a specific area for the purpose of avoiding uniform or even concentration of a substance in all areas.

[0041] As used herein, the term "nucleic acid" is meant to encompass both DNA and

RNA.

[0042] As used herein, the term "ligand" refers to a molecule that exhibits specific binding of high affinity for another molecule and upon binding with that molecule is internalized into the cellular interior. An illustrative, albeit non limiting example of how the term "ligand" is used in the context of the instant specification is a protein ligand binding to a cell surface receptor, such as EGF binding to the EGFR. As used herein the term "ligand" refers to a molecule that exhibits specific binding of high affinity for another molecule and upon binding with that molecule is internalized into the cellular interior. Any such ligand can be incorporated into the complexes of the present invention and used to selectively deliver nucleic acids to cells within a human subject. Illustrative, albeit non-limiting examples of ligands known and commonly used in the art are antibodies, cell adhesion molecules, hormones and cell-specific ligands. Researchers can select ligands and nucleic acid sequences to design a composition according to preferred use. Particularly preferred ligands for use are the cell-specific ligands, human epidermal growth factor (EGF), human vascular endothelial growth factor (VEGF) and human transferrin. The epidermal growth factor receptor (EGFR) is expressed by epithelial and epidermal cells. Cells expressing EGFR are found in human liver tissue. The vascular endothelial growth factor receptor (VEGFR) is expressed by non- resting endothelial cells of blood vessels. The transferrin receptor is ubiquitously expressed and shows an increase in expression on the cellular surface of actively proliferating cells. Accordingly, EGF functions as a vector for delivery of nucleic acids to cells expressing the EGFR, VEGF functions as a vector for delivery of nucleic acids to cells expressing the VEGFR and transferrin functions as a vector for delivery of nucleic acids to cells expressing the transferrin receptor.

[0043] As used herein, the term "receptor" refers to a molecule that exhibits specific binding of high affinity for its complementary ligand. An illustrative, albeit non-limiting example of how the term "receptor" is used in the context of the instant specification is a cell surface receptor binding to a ligand, such as the EGFR binding the EGF. [0044] As used herein, the term "complementary receptor" refers to the receptor a ligand specifically binds with high affinity, for example, the EGFR is the complementary receptor for EGF.

[0045] As used herein, the term "target" refers to a specific molecule expressed on the cellular surface such as a receptor to which a specific moiety can be directed, for example the

EGFR is a target for EGF. As used herein "target" can also refer to a cell or tissue, for example, a cell or tissue expressing the EGFR is a target for EGF.

[0046] As used herein, the term "targeting agent" refers to a specific molecule that binds to a complementary molecule expressed on the cellular surface such as a ligand, for example EGF is a targeting agent for the EGFR.

[0047] As used herein, the term "VEGF" refers to a glycosylated polypeptide that serves as a mitogen to stimulate vascular development. VEGF imparts activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors (VEGFR' s) which then activates signal transduction.

[0048] As used herein, the term "VEGFR" refers to a vascular endothelial cell plasma membrane-spanning tyrosine kinase receptor which binds VEGF thus exerting a mitogenic signal to stimulate vascularization of tissues.

[0049] As used herein, the term vascular endothelial growth factor (VEGF) encompasses VEGF and isolated peptide fragments or biologically active portions thereof, analogues of VEGF and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of VEGF.

[0050] As used herein, the term "EGF" refers to a mitogenic polypeptide that exhibits growth stimulatory effects for epidermal and epithelial cells. EGF imparts activity by binding to epidermal and/or epithelial cell plasma membrane spanning tyrosine kinase receptors

(EGFR's) which then activates signal transduction.

[0051] As used herein, the term "EGFR" refers to a epidermal and/or epithelial cell plasma membrane-spanning tyrosine kinase receptor which binds EGF thus exerting a mitogenic signal.

[0052] As used herein, the term epidermal growth factor (EGF) encompasses EGF and isolated peptide fragments or biologically active portions thereof, analogues of EGF and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of EGF.

[0053] As used herein, the term "transferrin" refers to a vertebrate glycoprotein that functions to bind and transport iron. [0054] As used herein, the term "transferrin receptor" refers to a receptor expressed on the surface of cells functioning to capture and bind iron saturated transferrin. Expression of the transferrin receptor is increased in cells which are actively proliferating.

[0055] As used herein, the term transferrin encompasses transferrin and isolated peptide fragments or biologically active portions thereof, analogues of transferrin and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of transferrin.

[0056] As used herein, the term "host" refers to any animal having cells to which nucleic acids can be delivered.

[0057] As used herein, the term "bioactivity" refers to the ability of a ligand to bind to its complementary receptor thus enabling internalization of the ligand into the cellular interior.

[0058] As used herein, the term "biologically active portion" refers to the portion of a ligand that has the ability to bind to its complementary receptor thus enabling internalization of the ligand into the cellular interior.

[0059] As used herein, the term "linker" refers to the molecules which join the ligands of the composition of the instant invention together to form a single compound; for example, EGF-PEG attached to biotin links streptavidin attached to transferrin.

[0060] As used herein, the phrase "operatively linked" means that the linkage does not destroy the functions of each of the separate elements of the composition of the instant invention, for example, when linked together by a linker to form the single compound of the instant invention the ligands retain the ability to bind their complementary receptors.

[0061] As used herein, the term "carrier" refers to a pharmaceutically inert substance that facilitates delivery of an active agent to a host, for example, as is shown in the experiments described herein, saline functions as a carrier for delivery of the compositions of the instant invention to the mouse host.

[0062] As used herein, the phrase "pharmacologically effective amount of a carrier" refers to an amount of a carrier that is sufficient to effectively deliver an active agent to a host.

[0063] As used herein, the term "pharmaceutical composition" refers to the compositions of the instant invention combined with a pharmacologically effective amount of a carrier. COMPOSITIONS FOR DELIVERING NUCLEIC ACIDS INTO CELLS [0064] The present invention comprises composition for delivering nucleic acids encoding a protein, polypeptide or RNA of interest into a cell, particularly a cell within a mammalian subject. The composition comprises transferrin, the nucleic acid, and one or more other cell-specific ligands. The nucleic acid, which comprises a coding sequence for the protein, RNA, or polypeptide and a promoter for driving expression of the coding sequence, is operatively linked to the transferrin ligand. The one or more other cell-specific ligands are also operatively linked to the transferrin ligand. In certain preferred embodiments, one of the one or more cell-specific ligands is EGF. In other preferred embodiments, one of the one or more cell-specific ligands is VEGF. In other preferred embodiments, the composition comprises both EGF and VEGF. NUCLEIC ACID

[0065] The compositions of the present invention comprise a nucleic acid encoding an RNA, protein, or polypeptide of interest. The nucleic acid, which is operatively linked to the transferrin ligand, comprises a coding sequence and a promoter for driving or promoting expression of the coding sequence in cells of a mammalian subject, preferably a human subject. Such promoter can be constitutive (e.g., CMV enhancer/promoter from human cytomegalovirus) or inducible (e.g., MMTV enhancer/promoter from mouse mammary tumor virus). A variety of constitutive and inducible promoters and enhancers are known in the art. Other promoters that result in transcription of polynucleotide sequences in specific cell types, so-called "tissue-specific promoters," can also be used. A variety of promoters that are expressed in specific tissues exist and are known in the art. For example, promoters whose expression is specific to neural, liver, epithelial and other cells exist and are well known in the art. promoter. Useful promoters include the CMV promoter, the SV40 promoter, and the beta actin promoter. The nucleic acid coding sequence may be incorporated into a vector comprising the promoter, such as for example a plasmid. Plasmids comprising such promoters are available commercially. The vector may also include transcription initiation, termination and enhancer sequences, as well as sequences that enhance translation efficiency, such as the Kozak sequence. Optionally, the vector also includes a marker gene for selecting for the presence of the vector in the cell. In certain embodiments, the coding sequence encodes the heavy chain or light chain of an antibody, preferably a human antibody. Methods for making such DNA molecules (i.e., recombinant DNA methods) are well known to those skilled in the art. LINKERS

[0066] When assembling compositions from multiple elements, elements are either linked directly through chemical conjugation (for example through reaction with an amine or sulfhydryl group) or are linked indirectly through molecules termed linkers. When selecting a linker it is important to choose the appropriate length and flexibility of linker in order to reduce steric hindrance between the elements of the composition. For example, if an element of a composition is brought into close physical proximity of another element by linkage, the function of either or both elements can be affected. Each element of the composition must retain its bioactivity, for example in the instant invention, each ligand must retain its ability to bind to its complementary receptor after linkage with the other ligands of the composition. Illustrative, albeit non-limiting examples of linkers are glycols, alcohols and peptides. Particularly preferred linkers are PEG (polyethylene glycol) and the peptide linker shown as SEQ ID NO:8 (use of each of these linkers is illustrated in the examples described herein). PHARMACEUTICAL COMPOSITIONS

[0067] The present invention relates to a pharmaceutical composition comprising a carrier and a biologically effective amount of a composition of the present invention. The acceptable carrier is a physiologically acceptable diluent or adjuvant. The term physiologically acceptable means a non-toxic material that does not interfere with the effectiveness of the delivery composition or the anti-tumor agent. The characteristics of the carrier will depend on the route of administration and particular compound or combination of compounds in the composition. Illustrative, albeit non-limiting examples of carriers known in the art and suitable for use with the instant invention are water, saline solutions and dextrose solutions. A particularly preferred carrier is saline, the use of which is illustrated in the examples herein. Preparation of such formulations is within the level of skill in the art. The pharmaceutical composition may further comprise fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

METHODS OF DELIVERING NUCLEIC ACIDS INTO CELLS IN A MAMMALIAN SUBJECT

[0068] The present invention provides methods of delivering nucleic acids into cells in a mammalian subject, particularly a human subject. The methods comprise administering a composition of the present invention to the subject. In certain cases where the subject is a human that has been infected or may become infected, e.g., due to exposure, with an infectious agent, the composition may comprise a nucleic acid encoding the heavy chain of an antibody, preferably a human antibody, that is immunoreactive with the infectious agent or a component thereof (e.g. a protein) and a nucleic acid encoding the light chain of a human antibody that is immunoreactive with the infectious agent or a component thereof. Thus, the methods of the present invention can be used to confer immunity to the infectious agent to the subject. In other cases, the subject, particularly a human subject, may have elevated levels of an agent, e.g. a protein, peptide, lipopolysaccharide, or polysaccharide, that directly or indirectly causes a disease or a disorder in the subject. An example of such agent is TNF alpha which is associated with rheumatoid arthritis in humans. In such cases, the method may comprise administering a composition which comprises transferrin ligand, operatively linked to EGF, VEGF, or both ligands and a nucleic acid encoding the heavy chain of an anti- TNF alpha antibody and a nucleic acid encoding the light chain of an anti-TNF alpha antibody.

[0069] The compositions may, for example, be administered orally, intra-vascularly, intraperitoneally, intranasal, intrabronchial, subcutaneously, intramuscularly or topically (including aerosol). The pharmaceutical composition is administered once or repeatedly in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means the total amount of the composition that is sufficient to show a meaningful subject or patient benefit, e.g., production of antibodies sufficient to reduce the number of infectious agents or antigens that are immunoreactive with the antibody, in the subject. Initially, the attending physician will administer low doses of the composition and observe the patient's response. Larger doses of composition may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It may. be desirable to administer simultaneously or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to one individual as a single treatment episode. Ultimately, the attending physician will decide the amount of therapeutic composition with which to treat each individual patient.

EXAMPLES [0070] The invention may be better understood by reference to the following examples, which serve to illustrate but not to limit the present invention. EXPERIMENTAL PROCEDURES SEQUENCES

[0071] The following nucleic acid sequences and corresponding amino acid sequences were used to generate the DNA and polypeptides used in the experiments described herein. Homo sapiens (human) VEGF 165 (vascular endothelial growth factor isoform 165)nucleic acid sequence is disclosed as SEQ ID NO:l and translates into VEGF165 protein disclosed as amino acid sequence SEQ ID NO:2. Homo sapiens (human) transferrin nucleic acid sequence is disclosed as SEQ ID NO:3 and translates into transferrin protein disclosed as amino acid sequence SEQ ID NO:4. Homo sapiens (human) EGF (epidermal growth factor) nucleic acid sequence is disclosed as SEQ ED NO: 5 and translates into EGF protein disclosed as amino acid sequence SEQ ID NO:6. Homo sapiens (human) anti-digoxin antibody heavy chain nucleic acid sequence is disclosed as SEQ ID NO: 9 and translates into anti-digoxin antibody heavy chain protein disclosed as SEQ ID NO: 10. Homo sapiens (human) anti-digoxin antibody light chain nucleic acid sequence is disclosed as SEQ ID NO: 11 and translates into anti-digoxin antibody light chain protein disclosed as SEQ ID NO: 12. Sequences that encode Homo sapiens (human) respiratory synctial virus (RSV) antibody heavy chain and homo sapiens (human) anti-RSV antibody light chain nucleic acid sequence are known in the art. Similarly, sequences that encode homo sapiens (human) anti- TNFα antibody heavy chain and homo sapiens (human) anti- TNF antibody light chain are also know in the art.

CROSSLINKING OF VEGF (AND EGF) TO A BIOTINYLATED-POLYLINKER [0072] EGF and VEGF are crosslinked to a biotinylated polylinker by carrying out the following protocol. The polylinker used consists of 15 amino acid residues shown as SEQ ID NO:8. The cDNA sequence encoding this polylinker is shown as SEQ ID NO:7. The first glycine residue at the N- terminal was biotinylated. EDC (l-Ethyl-3- (3-Dimethylaminopropyl)carbodiimide Hydrochloride) and NHS (N-Hydroxysuccinimide) were equilibrated to room temperature. 0.4 mg of EDC and 0.6 mg of NHS were added to 1 mg/ml of the polylinker peptide solution (in activation buffer: 0.1 M MES (2-[N- morpholino] ethane sulfonic acid), 0.5 M NaCl, pH 6.0) to a final concentration of EDC and NHS of 2 mM and 5 mM respectively. The reaction mixture was then held for 15 minutes at room temperature. 1.4 μl of 2-mercaptoethanol was then added (to a final concentration of 20 mM). The reaction mixture was then run through P2 gel filtration mini-column and eluted by the activation buffer. Fractions containing the protein were then pooled together. Equal mole:mole ratios of either VEGF or EGF protein were added to the pooled fractions and reacted for 2 hours at room temperature. Hydroxylamine was added to a final concentration of 10 mM and the VEGF- linker or EGF-linker was purified by P2 gel filtration mini- column. SYNTHESIS OF TF/SA COMPOSITION

[0073] 8.84 mg of transferrin (TF) was thiolated by adding a 5-fold molar excess of

2-Iminothiolane hydrochloride (Traut's reagent) in pH 8.0, 0.16 M borate. Following 90 minutes at room temperature, the thiolated TF was desalted and concentrated by Centricon microconcentrators. Ellman's reagent (Pierce) was then used to demonstrate that a single thiol group was inserted on the surface of TF. 7 mg of streptavidin (SA) (in PBS) was activated by adding to a 20:1 molar ratio of m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS)(stock at lmg/ml in dimethylformamide). After 20 minutes, the activated SA was desalted on a microconcentrator and immediately, the activated S A was added to a 10 molar excess of thiolated TF. They were mixed and then incubated at room temperature for 3 hours. Purification of the TF/SA composition was done by HPLC using TSK-G3000 column. The number of biotin binding sites per TF/SA composition was determined with ³H-biotin binding assay.

CONJUGATION OF VEGF-LINKER-BIOTIN (AND EGF-LINKER-BIOTIN) TO TF- SA

[0074] VEGF-Linker-Biotin and EGF-Linker-Biotin are added to TF/SA by carrying out the following protocol. The composition of VEGF-Linker-biotin (or EGF-Linker-biotin) and TF/SA was prepared by mixing 5 nmol of VEGF-Linker-biotin (or 5 nmol of EGF- Linker-biotin) with 8 nmol of TF/SA (1 :1.6 molar ratio). HPLC was then used to purify the VEGF-Linker-biotin-TF-SA composition (or EGF-Linker-biotin-TF-SA composition). CONJUGATION OF VEGF (AND EGF) TO PEG3400-BIOTIN

[0075] Alternatively to linkage with a peptide linker, VEGF and EGF can also be linked to transferrin using PEG by carrying out the following protocol. NHS-PEG3400-biotin was obtained from Shearwater Polymers (Huntsville, AL), where NHS = N-hydroxysuccinimide and PEG3400 = poly(ethylene glycol) of 3400 Da molecular mass. NHS-PEG3400-biotin (20 nmol in 310 μl of 0.05 M NaHCO3) was added in a 1:1 molar ratio to either VEGF or EGF (16 nmol in 250 μl of 0.05 M NaHCO3) followed by incubation at room temperature for 60 minutes. The mixture was then applied to two Sepharose 12 HR 10/30 FPLC columns in series, followed by the elution in 0.01 M NaH2PO4/0.15 M NaCl/pH 7.5 at a flow rate of 0.7 mL/minute for 120 minutes. Fraction(s) that contained VEGF or EGF bound to PEG3400-biotin moiety were pooled together. CONJUGATION OF VEGF-PEG3400-BIOTIN (AND EGF-PEG3400-BIOTIN) TO TF-SA

[0076] Following reaction of EGF and/or VEGF with NHS-PEG3400-biotin and transferrin with streptavidin, both compositions were purified by HPLC. The EGF (and/or VEGF)-NHS-PEG3400-biotin and TF/SA compositions were then mixed (1 :1.6 molar ratio). HPLC was then used to purify the EGF (and/or VEGF)-NHS-PEG3400-biotin-TF-SA compositions.

SYNTHESIS OF BIOTINYLATED POLYLYSINE FOR NUCLEIC ACID CARRIER AND ATTACHMENT TO VEGF-TF (AND/OR EGF-TF)

[0077] After attachment of the ligands(such as, EGF and VEGF) to the transferrin ligand, the nucleic acid was condensed and attached to the transferrin ligand according to the following protocol. Two polylysine (PLL) peptides were used for the DNA molecule to form a complex: Cys-Trp-Lysl9 (Kl9, BioPeptide, San Diego, CA) and Lysl50 (K150, average molecular weight of 20,000, Sigma, St Louis, MO). Biotin reagents for peptide modification were purchased from Pierce (Rockford, IL). Peptide Kl9 was modified with a biotin group through the terminal cysteine residue by reaction of the sulfhydryl group with the iodoacetyl group of the biotinylation reagent, EZ-link-PEO-iodoacetyl-biotin. The peptide Kl9 (10 mg) was dissolved in 90 μl of buffer (50 mM Tris, 5 mM EDTA, pH = 8.3) that was previously bubbled with nitrogen gas. The EZ-link-PEO-iodoacetyl-biotin (48 mg) was also dissolved in 200 μl of buffer (0.1 M sodium phosphate, 5 mM EDTA, pH = 6.0). The biotin solution was added dropwise to the peptide solution, mixed gently and incubated for 90 minutes. The starting peptide solution and the reaction mixture were analyzed by HPLC to determine if the reaction had gone to completion. The starting peptide solution and the reaction mixture were resolved by injecting 50 μg through a C18 RP-HPLC column eluted with water (0.1% trifluoroacetic acid) and an acetonitrile gradient (0.1% TFA, 0 to 95% over 50 minutes at 60°C) while detecting the absorbance at 260 nm. For purification, sephadex (G15) was equilibrated in deionized water for 30 minutes prior to packing in a glass column (2 cm diameter x 12 cm height). The reaction mixture was passed though the column using deionized water. Thirty fractions were collected, and the presence of the tryptophan side chain was examined by measuring the absorbance at 260 nm (Beckman Instruments Inc., Fullerton, CA). The fractions with the greatest absorbance at 260 nm were lyophilized. The purified biotinylated peptide (K.19-B) was stored as a powder at -20°C. [0078] The peptide K.150 was biotinylated using succinimide ester (NHS)/amine chemistry. K150 (10 mg) was dissolved in 1 mL of phosphate buffered saline (PBS, pH = 7) and EZ-link-Sulfo-NHS-LC-biotin (2.8 mg) was added directly to the solution, mixed gently and incubated for 2 hours at 4°C. The reaction mixture was purified using dialysis cassettes immersed in deionized water. The dialyzed product was further purified using a monomeric avidin column to separate the biotinylated components from nonbiotinylated species. The biotinylated product was eluted with 10 mL of a 10 mM biotin solution and dialyzed to remove the unconjugated biotin. The purified biotinylated peptide (K150-B) was then lyophilized and stored as a powder at -20°C.

[0079] The composition of polylysine-biotin and TF/SA was prepared by mixing polylysine-biotin with TF/SA (1 :1.6 molar ratio). HPLC was then used to purify the polylysine-biotin-TF-SA composition. The reaction mixture was applied to a TSK-gel G3000 SWXL HPLC gel filtration column, followed by elution in 0.01 M Na2HPO4/0.15 M NaCl pH 7.4/0.05% Tween-20 at a flow rate of 0.5 mL/min for 40 minutes, and 0.5 mL fractions were collected and lyophilized and stored as a powder at -20°C. EXAMPLE 1 : Composition Comprising Transferrin Ligand Operatively Linked to Nucleic Acids Encoding the Heavy Chain and Light Chain of an Anti-Digoxin Antibody [0080] Digoxin is a cardiac glycoside found in the leaves of the foxglove plant group.

Digoxin is used for long-term treatment of chronic heart weakness and defective heart valves. However, if administered in incorrect dosages, digoxin can be highly toxic. Anti-digoxin antibodies are often used to counter these toxic effects, (see Concise Encyclopedia Biochemistry and Molecular Biology, Third Edition, revised and expanded by Thomas A. Scott and E. Ian Mercer, Walter de Gruyter publisher, Berlin and New York, 1997, page 173, for a discussion of digoxin).

[0081] In order to test the efficacy of the compositions of the instant invention, nucleic acid encoding the human anti-digoxin antibody (IgG) was selected for delivery to SCID mice. Nucleic acid sequence SEQ ID NO: 9 encodes the heavy chain (including constant and variable regions) of the human anti-digoxin antibody and nucleic acid sequence SEQ ID NO: 11 encodes the light chain of the human anti-digoxin antibody (including constant and variable regions). Figure 2 shows a schematic presentation of the cloning steps used to generate the DNA vectors containing SEQ ED NO:9 and SEQ ID NO: 11 which were used in the experiments illustrated herein. SEQ ID NO:9 was inserted into the multiple cloning site of the pVAXl plasmid to form the first vector (heavy chain) and SEQ ED NO: 11 was inserted into the multiple cloning site of the pVAXl plasmid to form the second vector (light chain). Symbols and abbreviations shown in Figure 2 are defined as follows: IgH, refers to cDNA encoding the heavy chain of the immunoglobulin antibody against digoxin (SEQ ED NO:9); IgL, refers to cDNA encoding the light chain of the immunoglobulin antibody against digoxin (SEQ ID NO:l 1); T7, refers to the T7 promoter useful for achieving efficient transcription; CMV, refers to the human cytomegalovirus immediate-early promoter/enhancer useful for achieving efficient high-level expression of the sequences encoded in the multiple cloning site of the plasmids; BGH, refers to the bovine growth hormone polyadenylation (pA) signal useful for achieving efficient transcription termination and polyadenlylation of mRNA; kanamycin, refers to the gene encoding kanamycin resistance useful as a selection marker in E. coli to select cells expressing the cloned sequences and pUC ori, refers to the pUC-derived origin of replication useful for achieving high-copy number plasmid replication and growth in E. coli.

[0082] Following preparation of the vectors, 50 μg of each DNA vector (250 μg/ml) was mixed with polylysine-biotin-TF-SA to provide the composition.

EXAMPLE 2: Method of Using the the Composition of Example 1 to Protect

Animals from Digoxin EXPERIMENTAL MICE

[0083] Severe combined immuno-deficient C.B.-17 scid/scid (SCID) mice were bred and maintained according to the protocol of Sandhu et al. (Critical Reviews in Biotechnology 16(1):95-118 1996). Mice were used when 6-8 weeks old and were pre-treated with a dose of 3 Gy γ radiation administered from a ¹³⁷CS source (Gamacell, Atomic Energy of Canada Ltd.Commercial Products). The irradiated SCID mice receive intraperitoneal injection of 20 μl ASGM1 sera diluted to 100 μl with saline, 4 hours pre- bone transplantation and every 7 days thereafter for the duration of the experiments. ANIMAL STUDIES

[0084] The following experiment was undertaken to demonstrate that capable of efficient delivery of nucleic acids to cells in vivo.

[0085] An EGF-PEG3400-biotin-TF-SA-DNA composition was prepared as described above, particularly in Example 1.. The composition (250μl 1) was injected intravenously into two groups of SCID mice; group one were normal SCED mice (non-tumor bearing) and group two were SCED mice implanted with JJ5 tumors (tumor-bearing). The control mice (also in two groups of tumor-bearing and non-tumor bearing) were administered the same amount of a similar composition, however the nucleic acid contained in this control composition did not encode for human anti-digoxin antibody. SCDD mice serum was analyzed every two weeks using ELISA (Fishwild et al. Nature Biotechnology 14(7):845-851 1996) to determine the serum levels of human anti-digoxin antibody. Serum samples were exposed to plate-adsorbed digoxin-BSA, and biotinylated goat anti-mouse and anti-human heavy chain secondary antibodies were used to detect the hapeten-bound antibody. A strepavi din-alkaline phosphatase conjugate was used to quantify anti-digoxin antibodies through hydrolysis of p-nitrophenylphosphate. Figure 3 shows that the experimental group of mice produced the antibody for at least 112 days, and in contrast very little antibody was detected in the serum of the control group of mice.

[0086] To examine if the anti-digoxin antibody seen in the serum was biologically active, mice survival studies using digoxin were done on all control and all experimental mice at 7 weeks post administration of the EGF-PEG3400-biotin-TF-SA-DNA composition. A general anesthetic [intramuscular administration of Xylazine (4 μl /20g mouse), and Ketamine (4 μl /20g mouse) in 40 μl of 0.9% sodium chloride] was administered to the mice under sterile conditions. The anesthetized mice were injected intravenously with 20 mg kg-1 of digoxin [purchased from Sigma USA] and animals were monitored continuously for 5 hours following digoxin administration. All the control mice (showing no serum anti-digoxin antibody) died from the digoxin overdose. In contrast the mice showing serum concentration of the anti-digoxin antibody survived the digoxin administration. These studies clearly demonstrate that the EGF-PEG3400-biotin-TF-SA-DNA composition of the instant invention is capable of efficient delivery of nucleic acids to cells in vivo and that the resulting protein produced in vivo is biologically active.

[0087] At the end of the digoxin-dosing experiments, DNA was isolated from the following tissues of the mice; lungs, liver, tumor, brain, kidney, blood, heart and muscle. Polymerase chain reaction was performed according to standard molecular biology protocols found in Molecular Cloning: A Laboratory Manual Authors: Joseph Sambrook and David W. Russell third edition 2001 on the isolated DNA for the presence of DNA coding for the anti- digoxin antibody. The antibody sequences were detected in the liver and the tumors in the tumor-bearing mice and only in the liver in the non-tumor bearing mice. This data suggests that the DNA is targeted to the liver by the VEGF, EGF and transferrin in the non-tumor bearing mice. In the tumor-bearing mice the VEGF, transferrin and EGF can target both the liver and tumor.

[0088] Antibodies of appropriate affinity and specificity have been shown capable of reversing advanced cardiac glycoside toxicity due to digoxin overdose. Without being bound by any particular theory, the reversal of digoxin effects by the digoxin antibody in the mice may be mediated by at least two mechanisms. First, the antibody may simply bind to cardiac glycoside and decrease the concentration of free digoxin in the serum, or second, the antibody may dislodge digoxin from its receptor.

EXAMPLE 2: Production of Anti- RSV Antibody in Animals Injected with Compositions Comprising Transferrin, EGF, and Nucleic Acids Encoding the Heavy Chain and Light Chain of Anti-RSV Antibody

[0089] A composition comprising transferrin operatively linked to EGF and a nucleic acid sequence encoding human anti-RSV heavy chain antibody and a composition comprising transferrin operatively linked to EGF and a nucleic acid sequence encoding human anti-RSV light chain antibody was prepared as described above for the anti-digoxin antibody. The composition were administered to experimental SCDD mice (n=10) and compositions comprising DNA encoding the heavy and light chains of anti-digoxin antibody were administered to control animals (n=10) by intravenous injection. Two weeks after administration of the test and control composition, all the SCID mice were challenged intranasally with 10⁶ plaque forming units (p.f.u) of RSV every week for the duration of the experiments.

[0090] Tissue Culture. HEp-2 cells (CCL 23; American Type Culture Collection

[ATCC], Rockville, MD) were used to grow and test for the presence of RSV. This cell line was originally obtained from the ATCC and was serially passaged in MEM supplemented with 10% fetal calf serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 2 mM L-glutamine, and 0.2% sodium bicarbonate whenever they became confluent. MEM supplemented with 2% FCS was used to maintain HEp-2 cell cultures and in all virus and antibody assays. P815 (H-2d), a mouse mastocytoma line, and EL-4 (H-2b), a mouse lymphoma line, were maintained using standard cell culture procedures. [0091] Virus RSV A2, an A subtype RSV, was obtained from the ATCC (catalog no. VR1302). Challenge pools of this virus were prepared by infecting monolayers of HEp-2 cells. When the infected monolayers exhibited approximately 90% syncytia formation, the cells and medium from the monolayers were collected, pooled, and clarified by centrifiigation (450g). The resulting supernatant fluids were passed through a 0.45 μm filter, portioned, and stored at -70°C until required. RSV long strain A2 were diluted in 0.2 ml of test medium 199 (medium 199 with 1 2% FCS). The culture medium was then replaced by the test medium containing RSV and incubated for 1 h at 37 C. [0092] The test medium containing RSV was replaced by 1 ml of overlay medium

(medium 199 with 1 2% FCS and 1% methylcellulose) and the plate was incubated for 6 days. The overlay medium was discarded and cells were fixed with 1 ml of ice-cold 80% methanol for 30 min at 48°C and blocked for 1 h with blotto. 18B2 antibody (0.2 ml of 0.5 mg/ml) in blotto was added into each well and incubated for 1 h with shaking. HRP- conjugated anti-mouse IgG antibody (0.2 ml of 1 :2000 dilution) was added into each well and incubated for 1 h. HRP substrate (0.2 ml; 0.6 mg/ml of 3,3- diaminobenzidine tetrahydrochloride 10 '03% of NiC12- 1 1 ml/ ml of 30% H₂O₂) was added into each well and incubated for 20 min. Plaques appeared as dark blue spots. RSV Titers in SCID Mouse Lung.

[0093] Experimental animals (n=10) were administered compositions comprising

DNA coding for anti-RSV antibody and control animals were administered compositions comprising DNA coding for anti-digoxin antibody. Two weeks after administration of the test and control compositions, all the SCID mice were infected intranasally (i.n.) under anesthesia with 20 μ\ of MEM containing 10⁶ p.f.u of RSV every week for the duration of the experiments. The mice were anesthetized by intramuscular (i.m.) injection of 2.5 ml/kg of a 4/1 mixture (v/v) of ketamine (Imalge'ne 500) (Rhone Merieux, Lyon, France) and xylazine (Rompun at 2%) (Bayer, Puteaux, France).

[0094] Three weeks post RSV challenge mice were killed by cervical dislocation.

Lungs were removed, placed into centrifuge tubes containing 3 ml of cold MEM-10% FBS, and sonicated on ice for 30 s. Tissue suspensions were clarified for 10 min (2,000 x g, 10 min, 4°C), and supernatants either were tested immediately by plaque assay or were frozen at - 80°C for subsequent testing. Virus was quantitated by plaque assay in Vero cell monolayers grown in 24-well polystyrene tissue culture plates. Serial 10-fold dilutions of virus were adsorbed to the cells for 1 h at 37°C, and then the cells were overlaid with 1 ml of MEM-10%) FBS containing 0.75% agarose. After 4 days, a second 1-ml overlay containing 0.01% neutral red was added. Plaques were counted 6 to 8 h later. Average tissue weights of 0.16 g for lungs were used to calculate the number of PFU per gram of tissue The significance of differences between groups was determined by a two-tailed t test. As shown in Figure 5, RSV titers were much lower in animals injected with test compositions comprising the transferrin ligand, EGF and nucleic acids encoding the heavy chain and light chain of anti- RSV antibody than in to animals injected with the control composition. As shown in Figure 4, test animals produced anti-RSV antibody for at least 42 days after injection of the test composition. In contrast very little, anti-RSV antibody was detected in the serum of the control group of mice As shown in Figure 6, test animals were able to survive infection with

RSV, as compared to animals injected with the control composition ^• .

EXAMPLE 2: Production of Anti- TNFα Antibody in Animals Injected with Compositions Comprising Transferrin, EGF, and Nucleic Acids Encoding the Heavy Chain and Light Chain of Anti-TNFα Antibody

[0095] A composition comprising transferrin operatively linked to EGF and a nucleic acid sequence encoding human anti-TNFα heavy chain antibody and a composition comprising transferrin operatively linked to EGF and a nucleic acid sequence encoding human anti-TNFα light chain antibody (test composition) was prepared as described above for the anti-digoxin antibody. The test compositions were administered to experimental SCID mice (n=10) and compositions comprising DNA encoding the heavy and light chains of anti-digoxin antibody were administered to control animals (n=10) by intravenous injection. As shown in Figure 7, test animals produced anti- TNFα antibody for at least 46 days after injection of the test composition. In contrast very little, anti-TNFα antibody was detected in the serum of the control group of mice.

[0096] In summary, the compositions of the instant invention enable delivery of nucleic acids to at least two appropriate target cells in vivo to exclusion of all other cell types with the resulting production of a biologically active protein in the target cells. The compositions of the instant invention are amenable for use with any nucleic acid sequence of interest and permit the introduction of these sequences into a variety of cells and tissues. However, these compositions are particularly useful for introduction of such therapeutic molecules such as antibodies. As is evidenced by the experimental examples described and shown herein, the instant invention provides compositions capable of increasing the selectivity of nucleic acid delivery by specifically targeting multiple cell types using multiple ligands, thereby simultaneously increasing the efficiency of nucleic acid transport and the resulting gene expression.

[0097] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the instant invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0098] It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement of parts herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification.

[0099] One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The oligonucleotides, peptides, polypeptides, biologically related compounds, methods,procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMS I claim:

1. A composition for delivering and expressing nucleic acids in a subject, comprising: a transferrin ligand operatively linked to i.) one or more other ligands that bind to a molecule on or within a cell of the subject; and ii) a nucleic acid comprising a coding sequence for a protein, polypeptide or RNA of interest, and a promoter for promoting expression of the coding sequence in the cell.

2. The composition of claim 1, wherein the cell is a liver cell.

3. The composition of claim 1, wherein at least one of said one or more other ligands is epidermal growth factor (EGF).

4. The composition of claim 1, wherein at least one of said one or more other ligands is vascular endothelial growth factor (VEGF).

5. The composition of claim 1, wherein the a coding sequence encodes the heavy chain of an antibody.

6. The composition of claim 1, wherein the coding sequence encodes the light chain of an antibody.

7. The composition of claim 5, wherein the antibody is a human antibody.

8. The composition of claim 6, wherein the antibody is a human antibody.

9. The composition of claim 1, wherein the nucleic acid comprises a coding sequence that encodes the heavy chain of an antibody and a coding sequence that encodes the light chain of an antibody.

10. The composition of claim 1, wherein the other ligands are linked to the tranferrin ligand indirectly through linkers.

11. The composition of claim 10, wherein the linker is a glycol, alcohol or peptide.

12. A pharmacological composition comprising: a pharmacologically effective amount of a carrier and a composition comprising a transferrin ligand operative linked to i) a nucleic acid comprising a coding sequence for a protein, polypeptide, or

RNA that directly or indirectly has an effect on a disease or disorder in a subject, and a promoter sequence for promoting expression of the coding sequence in a cell of the subject, and ii) one or more other ligands, wherein at least one of said one or more other ligands is VEGF or EGF.

13. The pharmacological composition of claim 12, wherein both EGF and VEGF are operatively linked to the transferrin ligand.

14. A method of delivering and expressing a nucleic acid in a subject, comprising: administering to the subject a composition comprising a transferrin ligand operatively linked to i) one or more other ligands that bind to a molecule on or within a cell of the subject, and ii) a nucleic acid comprising a coding sequence for a protein, polypeptide or RNA of interest, and a promoter for driving expression of the coding sequence in the cell.

13. The method of claim 12, wherein at least one of said one or more other ligands is epidermal growth factor (EGF).

14. The method of claim 12, wherein at least one of said one or more other ligands is vascular endothelial growth factor (VEGF).

17. The method of claim 12, wherein the composition is administered orally, intra- vascularly, intraperitoneally, intranasally, intrabronchially, subcutaneously, intramuscularly or topically.

18. The method of claim 12, wherein the composition is administered repeatedly over time.

19. A method of producing antibodies immunoreactive with an antigen in a subject comprising: administering to the subject one or more compositions comprising a transferrin ligand operatively linked to i) one or more other ligands that bind to a molecule on or within a cell of the subject; and ii) an expression vector comprising a coding sequence for an antibody immunoreactive with the antigen, and promoter sequence for promoting expression of the antibody in the cell.

20. The method of claim 19, wherein a single composition is administered to the subject, and wherein the expression vector comprises a coding sequence for the heavy chain of the antibody and a coding sequence for the light chain of the antibody.

21. The method of claim 19, wherein a composition comprising a vector comprising a coding sequence for the heavy chain of the antibody and a composition comprising a vector comprising a coding sequence of the light chain of the antibody are administered to the subject.

22. The method of claim 19, wherein the antigen is a component of an infectious agent.

23. The method of claim 19, wherein the coding sequences encode the heavy chain of a human antibody and the light chain of a human antibody.

24. The method of claim 19, wherein the composition is administered to a subject that has been exposed or may be exposed to an infectious agent, and the composition or compositions comprise nucleic acids encoding the light chain and the heavy chain of an antibody immunoreactive with the infectious agent or an anti genie component thereof.

25. A method of reducing levels of a disease or disorder causing agent in a subject, comprising: administering to the subject a biologically effective amount of a composition comprising a transferrin ligand operatively linked to i) one or more other ligands that bind to a protein on the surface of a cell within the subject; and ii) an expression vector comprising a coding sequence for an antibody immunoreactive with the agent and a promoter sequence for promoting expression of the antibody in the cell.

26. The method of claim 25, wherein the agent is a protein, polypeptide, polysaccharide, or a lipopolysaccharide.

27. The method of claim 25, wherein a single composition is administered to the subject, and wherein the expression vector comprises a coding sequence for the heavy chain of the antibody and a coding sequence for the light chain of the antibody.

28. The method of claim 25, wherein a composition comprising a vector comprising a coding sequence for the heavy chain of the antibody and a composition comprising a vector comprising a coding sequence of the light chain of the antibody are administered to the subject.