WO2001011960A1 - A mucosal specific gene delivery system based on modified bacterial exotoxin - Google Patents

Abstract

A novel nucleic acid delivery system is described based on purified recombinant exotoxin protein variants. A purified exotoxin variant is described that includes a receptor targeting domain and a nucleic acid affinity domain operably linked to the exotoxin protein domain. For the transfer of nucleic acid material, the nucleic acid affinity domain is preferably a polycationic domain such as polylysine. The invention also describes methods for transferring nucleic acids material to tissues of interest, i.e., mucosal cells, using the delivery system of the invention and methods for therapeutic treatment using the delivery system.

Description

A MUCOSALSPECIFIC GENE DELIVERY SYSTEM BASED ON MODIFIED

BACTERIAL EXOTOXIN

FIELD OF THE INVENTION The invention consists of a novel nucleic acid delivery system that will specifically facilitate targeted delivery of nucleic acid. This novel system makes use of recombinant toxin protein components modified to contain a nucleic acid binding moiety resulting in the targeted delivery of nucleic acid to mucosal surfaces. The invention provides methods for therapeutic and prophylactic treatment using the delivery system.

BACKGROUND OF THE INVENTION

Foreign nucleic acids can be introduced into cells in vitro and in vivo by a variety of physical methods, including transfection, direct microinjection, electroporation, etc. However, most of these transfection or transduction techniques are non-specific and impractical for delivering nucleic acid to cells within intact animals. For this reason there is a great interest in developing nucleic acid delivery systems for use in gene therapy, antisense therapy and nucleic acid vaccine delivery. Exotoxins have been widely used as adjuvants and immunogens in the study of vaccine delivery. E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) are related members of the ADP-ribosylating exotoxin (bARE) family, which also include Bordetella pertussis-deήved pertussis toxin (PT), Pseudomonas aeruginosa exotoxin A (ETA), and Coryne bacterium diphtheria-deήved diphtheria toxin Krueger et al (1995). Both LT and CT have five identical B subunits that form a pentamer which facilitates binding to cell surface ganglioside receptors (GM1). LT exhibits a lectin- like binding capacity that results in the binding to a broader range of receptors on mammalian cells for LT than for CT, which binds only GM1 (Angstrom et al (1994); Clements et al (1980); Holgrem (1994)). Both CT and LT have an enzymatic active A subunit that enters the cell and catalyzes the ADP-ribosylation of guanine nucleotide binding proteins of the adenylate cyclase complex, resulting in activation of adenylate cyclase and increased intracellular cyclic AMP (cAMP). Both CTA and LTA are proteolytically cleaved into an enzymatically active Al subunit and an A2 linker fragment that is inserted into the central pore of the B pentamer The structure of these proteins has been characterized at the molecular level (Sixma et al (1993)

LT and CT are potent mucosal adjuvants (Williams et al (1999), Freytag et al (1999)) Some degree of A subunit enzyme activity is required for oral adjuvant function (Sixma et al (1993)) While ADP-ribosyltransferase activity enhances adjuvanticity, it is also responsible for toxicity Mutant LT and CT molecules have been constructed with altered A subunits, resulting in reduced ADP ribosylation activity and reduced toxicity, yet some maintained their adjuvant function The effect of CT and LT on the immune responses, include antigen presentation, cytokine production, with inhibitory as well as enhancing effects (Williams et al (1999), Matousek et al (1998)]

LT and CT have been engineered to carry protein or peptide molecules through chemical coupling or as chimeric fusion proteins (Williams et al (1999), Cardenas et al (1993), Lipscombe et al (1991), Loregian (1999), O'Doed et al (1999), Bagdasarian et al (1999)) Both CT and LT modified to remove their toxicity while maintaining their adjuvanticity have been demonstrated to enhance immune responses to a wide variety of co-administered antigens (Freytag et al (1999), Bagdasarian et al (1999))

For a review of synthetic DNA delivery systems please see Luo and Saltzman (Krueger et al (1995)) The use of a polylysine moiety to electrostatically couple naked DNA to a protein carrier has been demonstrated previously This method has been used to deliver DNA by receptor-mediated endocytosis via the transferrin receptor (Wagner et al (1990)) The major drawback of this system is the inability of the internalized DNA to escape the endosome, leading to its eventual degradation and resulting in poor transduction efficiency Subsequently, whole heat-inactivated adenovirus was added to the transferrin system to promote endosomolysis (Wagner et al (1990), Michael et al (1994), Tang et al (1997), Shi et al (1999))

Recently, researchers have reported that non-invasive direct application of vaccines onto the skin (Tang et al (1997), Glenn et al (1999)) results in immune responses In one example, application of recombinant adenovirus vectors (10⁸ PFU) to the skin resulted in an immune response (Tang et al (1997)) This non-invasive vaccination method was recently modified by crosslinking a DNA vector to the live

L recombinant adenovirus using polylysine (Shi et al (1999)) This combination was then administered to the shaved necks of mice by gluing a plastic cylinder to the neck of the mouse in order to allow the adenovirus-DNA complex to incubate with the skm for 1-18 hrs In another example, researchers demonstrated that the topical co- administration of cholera toxin with antigens of either tetanus or diphtheria toxoids to the skin of mice, resulted in immune responses (Glenn et al (1999)) These animals were protected against subsequent systemic tetanus toxin challenge These novel methods of administering vaccines is attractive due to their non-invasive approach, however, their method of delivery uses either recombinant live virus to administer DNA or the virulent cholera toxin to administer a protein antigen These safety issues limit their application and they do not target delivery to the mucosal surfaces

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 Benvemsty and Reshef, Proc Nat Acad Sci USA, 83 9551-55 (1986) injected calcium phosphate precipitated DNA lntrapeπtoneally into newborn rats and noted gene expression in the livers of the animals 48 hr 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 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 (naked) and relies on injection as a method of administration

Chemical based systems for the transfection of DNA include poly-L-lysine (PLL), DEAE-dextran, artifical lipids (1 e pofectamme), calcium phosphate, and controlled release polymers (1 e PLGA) Initial attempts to deliver naked DNA vaccines to mucosal surfaces failed, however, lipid-DNA complexes or encapsulated naked DNA in biodegradable poly (D,L-lactιde-co-glycohde (PLGA) microp articles have been successfully used for the in vivo delivery of DNA vaccines (Sagodira et al (1999A), Harpin et al (1999), Sagodira et al (1999B), Klavinskis et al (1999), Lunsford et al (2000), Walter et al (1999), Jones (1998), Jones (1997A), Jones (1997B)) However, these methods lack the specificity of targeting by receptor- mediated DNA delivery

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 polycatiomc protein (usually poly-L-lysine) containing a covalently attached hgand, 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-carπer complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier gand 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, polyormthine, histone, avidm, or protamine) and a tissue receptor- specific protein gand For targeting liver cells, Wu suggests "asialoglycoprotem (galactose-terminal) ligands" These may be formed, Wu says, either by desialation of appropriate glycoproteins, or by coupling lactose to non-galactose bearing proteins Low, et al , U S Pat No 5, 108,921, disclose binding biotin to DNA to transform a cell using receptor mediated endocytosis

Stomp, et al , U S Pat No 5, 122,466 and McCabe, et al , U S Pat No 5, 120,657 disclose attaching DNA to a metal pellet by covalently attaching polylysine to the material and then allowing DNA to be complexed to it The resulting product is then used for ballistic transformation of a cell See Stomp, et al , column 7, lines 29- 37 and McCabe, et al , column 7, lines 49-65 Wagner, et al., Proc. Natl Acad. Sci. USA, 88:4255-4259 (1991) 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.

All references cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert and applicants reserve the right to challenge the accuracy and pertinency of the cited documents.

SUMMARY OF THE INVENTION

The current invention describes an exotoxin engineered to contain a nucleic acid binding moiety such as polylysine or protamine for use as a nucleic acid carrier and delivery system. The current invention utilizes the general principle of coupling nucleic acid to a protein carrier to facilitate gene delivery, but in the described system the carrier protein will consist of recombinant exotoxin complexes engineered to carry the nucleic acid and target delivery to the mucosal surfaces. Such a carrier system for delivery of nucleic acid, makes use of exotoxin's specific ability to bind to a broad range of cells of the mucosal surfaces. The described invention is unique in that it does not use covalent linking to attach ligand to the poly cationic protein. The exotoxin variant of the present invention is capable of binding DNA while maintaining its receptor binding capabilities.

The novel nucleic acid delivery system of the present invention will specifically facilitate delivery of nucleic acid by utilizing recombinant exotoxin protein components modified to contain a nucleic acid binding moiety resulting in the targeted delivery of genes to the specific cell target (i.e. mucosal membranes). This system consists of two primary components: 1) a modified recombinant exotoxin variant and 2) a nucleic acid containing the gene(s) to be delivered. The exotoxin is engineered to carry exogenous nucleic acid by addition of a nucleic acid affinity domain (NAAD) and/or condensing motif of polylysine, protamine or similar polycationic amino acid sequences, or nucleic acid binding motifs. These recombinant exotoxin proteins are engineered such that they retain their functional ability to self assemble and bind their natural receptor target (i.e. ganglioside receptor, GM1) The system thus utilizes components of exotoxins to enhance the specificity and efficiency of nucleic acid delivery, retaining some of the best properties of exotoxins as mucosal carriers, while avoiding problems inherent in biological agents and enabling pharmaceutical quality control over the final preparations. The system offers universal application for use in a variety of animal species including man, the receptor is found on cells in most animal species, as well as the system's added flexibility of complexing any combination or variety of nucleic acids carrying a gene of interest.

The manner and process for making and using the invention are described through practical examples, as shown below. However, the exact manner and process for making and using the invention is not limited to these practical examples described below. In particular, any exotoxin targeting moiety can be modified to bind and deliver the nucleic acid, including, but not limited to, members of the bacterial ADP-ribosylating exotoxin (bARE) family which include Cholera toxin (CT), E.coli heat-labile enterotoxin (LT), Bordetella pertussis-derived pertussis toxin (PT), Pseudomonas aeriiginosa exotoxin A (ETA) and Corynebacterium diphtheria-derived diphtheria toxin, etc. In particular, any nucleic acid binding moiety can be used to modify the exotoxin, including, but not limited to polylysine, protamine and other polycationic amino acid sequences, DNA binding motifs from transcription factors and other nucleoproteins, nucleic acid condensing molecules, and molecular conjugates. Variants of the nucleic acid delivery molecule enhanced for specific receptor targeted delivery and nucleic acid delivery can be generated by applying DNA shuffling techniques familiar to those in the art (for a review see Sedlack (2000); Licking (1999)).

This procedure may be applied to human gene therapy. The major advantages of this method are (i) the ease of preparation of the DNA complex; (ii) the ability to target genes to mucosal specific tissues ; and (iii) the relative safety of the complex, since it is devoid of infectious viral DNA.

This procedure has may also be applied to nucleic acid vaccine delivery. The major advantages of this method are (i) the ease of preparation of the DNA complex;

L (ii) the ability to target genes to mucosal specific tissues, the site of initial infection of most pathogens, and (iii) the relative safety of the complex, since it is devoid of infectious material

Utility of this invention includes, but is not limited to delivery of therapeutic or prophylactic genes, vaccines, or maker genes to cells and tissues by ex vivo or in cell culture This invention can be applied to induce an immune response to proteins encoded by the delivered genes, or the commercial production of proteins encoded by the delivered genes, or for the treatment of diseases such as cancer, infectious diseases, hereditary diseases, autoimmune diseases, allergic diseases etc , but its use is not limited to these diseases or to any specific disease state

This mucosal nucleic acid delivery system offers the following advantages over recombinant live vectors 1) capacity to transport nucleic acid, 2) the lack of size restriction and therefore the potential capacity to carry any size gene or combination of genes, 3) ease of constructing vectors carrying different antigenic genes, 4) the enhanced ability to enter cells and deliver nucleic acid, 5) elimination of costly production and purification of high titer recombinant viruses, 6) lack of viral genes and therefore virulence or toxicity associated with viral based systems, 7) elimination of potential interference with pre-existing immunity to the vaccine earner, 8) the elimination of problems associated with needle injections, and 9) the targeting of nucleic acid vaccine or gene delivery to the mucosal surfaces Advantages over naked DNA vaccines include the potential to eliminate problems associated with needle injections, facilitation of uptake via the mucosal surfaces, targeting vaccine or therapeutic gene delivery to the mucosal surfaces, efficient internalization, and gene expression The appended claims are hereby incorporated by reference into this description as a recitation of preferred embodiments of the invention

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 A and B show western blot and PAGE analysis of purified recombinant LTBpLh proteins expressed in E. coli Figure 1A shows a Coomassie stain of the PAGE analysis of LTBpLh fractions eluted from the Talon affinity column Lane 1 contains fraction 1, lane 2 contains fraction 2, and lane 3 contains

1 fraction 3 Figure IB shows reactivity of the LTBpLh with anti-his antibody Lane 1 contains pRSETB no insert cell lysate and lane 2 contains pRSET-LTBpLh cell lysate

Figure 2 shows PAGE analysis of recombinant LTBpLh and wild type LTB proteins Both are able to assemble into pentamer size complexes (Lanes 2 and 9, respectively) A monomer LTBpLh and wild type LTB proteins (wt LTB) show migration on denaturing SDS-PAGE corresponding to expected size lanes 1 and 8, respectively Lanes 3, 4, and 5, show the effect of DNA on pentamer formation of LTBpLh in the presence of 0 4, 3, or 12 micrograms of GFP plasmid DNA, respectively Lanes 6 and 7 show the effect of 50 μg of protamine sulfate on LTBpLh complex formation in the presence of 12 micrograms of DNA (lane 6) or absence of DNA (lane 7) Lane 9 shows pentamer formation by wt LTB in the absence of DNA, and lanes 10 and 1 1 in the presence of 12 micrograms of DNA Lanes 11 and 12 are in the presence of 50 micrograms of Protamine Sulfate M represents molecular weight marker lanes

Figure 3 shows PAGE analysis of recombinant LTBpLh treated with EKMax to remove vector derived sequences Lanes 1 and 2 represent boiled samples and unboiled samples of LTBpLh, respectively Lanes 3 and 4 represent boiled and unboiled LTBpLh-treated with enterokinase M represents molecular weight markers Figure 4 shows DNA binding analysis of recombinant LTBpLh and wild type

LTB by gel mobility shift analysis

Figure 5 shows quantitation of GM1 binding by ELISA analysis of recombinant LTBpLh and wild type LTB

Figures 6A-D show the effect of DNA (Fig 6 A), or Protamine Sulfate (Fig 6B) or varying concentrations of DNA, Protamine sulfate, and LTBpLh concentrations (Figs 6C and 6D) on GM1 bind by ELISA analysis of recombinant LTBpLh and wild type LTB

Figure 7 shows that recombinant LTBpLh enhances cellular DNA uptake and transfection of Yl cells The number of GFP fluorescent cells was assayed by UV microscopy

Figures 8A-D show that recombinant LTBpLh are able to target DNA delivery to Yl cells (Fig 8 A) whereas wt LTB did not (Fig 8B) Addition of protamine t sulfate further enhanced delivery by LTBpLh (Fig 8C) as compared to protamine sulfate alone (Fig 8D)

Figure 9 is the sequence of LTBpL (SEQ ID NO 9), an LTB fusion protein with a polylysine DNA binding moiety (bold) followed by a termination codon (asterisk)

Figure 10 is the sequence of LTBpLh (SEQ ID NO 10), a LTB fusion protein with a hinge region (boxed in capital letters) and a polylysine DNA binding moiety (bold), followed by a termination codon (asterisk)

Figure 11 is the sequence of LTB-P (SEQ ID NO 11), an LTB fusion protein with the DNA binding moiety protamine (bold) followed by a termination codon (asterisk)

Figure 12 is the sequence of LTB-Ph (SEQ ID NO 12), an LTB fusion protein with a hinge region (boxed in capital letters) and a DNA binding moiety protamine (bold) followed by a termination codon (asterisk)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Exotoxin "Exotoxins" which can be used to make the exotoxin variant of the present invention are preferably exotoxins which are ligands of receptors which are primarily found on mucosal cells, particularly if such receptors are substantially specific to mucosal cells The sub-category of enterotoxins falls within this definition Enterotoxins bind not only to intestinal cells, but usually bind to other mucosal cells as well, such as within the respiratory system Preferred examples of exotoxins in accordance with the present invention are members of the bacterial ADP-ribosylating exotoxin (bARE) family which include, but are not limited to, Cholera toxin (CT), E.coli heat-labile enterotoxin (LT), Bordetella pertussis-deήved pertussis toxin (PT), Pseudomonas aeruginosa exotoxin A (ETA) and Corynebacterium dψhtheria-deήved diphtheria toxin Thus, any known exotoxin family member may be used, particularly LT and CT "Mucosal cells" are cells which belong to tissues (including organs) of the organism, including cells belonging to (in the case of an animal) its digestive system (e g , the stomach and intestines), the respiratory system (e g , the nose and the lungs), the reproductive system, the endocrine system (the liver, spleen, thyroids, parathyroid), the skin, or any target cells expressing the exotoxin receptor

The "Mucosal Cell Binding Moiety (MCBM)" is a moiety of the exotoxin which binds specifically to an accessible structure (the "receptor") of the intended mucosal cells It is not necessary that it be absolutely specific for those cells, however, it must be sufficiently specific for the conjugate to be therapeutically effective There is no absolute minimum affinity which the MCBM must have for an accessible structure of the mucosal cell, however, its cross-reactivity with other cells should be minimum The MCBM may interact with a lectin, for which there is a cognate carbohydrate structure on the cell surface The MCBM may be a hgand which is specifically bound by any of the known receptors of the exotoxin bARE family or those yet to be defined carried by the mucosal cells One class of ligands of interest are carbohydrates, especially mono- and oligosaccharides Suitable ligands include galactose, lactose and mannose For example it has been shown that LTB binding capacity for ganghoside GMl receptor, D-galactose and addition receptor molecules with lectin-like properties that have yet to be identified

Exotoxin Variants Exotoxin variants of the present invention are preferably derived from exotoxin proteins as defined above, or at least the MCBM thereof, through the fusion of a nucleic acid affinity domain to the precursor polypeptide The nucleic acid affinity domain can be operably linked to the precursor polypeptide, such as LTB, at the amino-terminus, the carboxy-terminus or at any suitable position within the precursor polypeptide sequence

The nucleic acid affinity domain (NADD) is chosen such that it posses an affinity for the nucleic acid to be delivered in the sub-millimolar range One or more nucleic acid affinity domains can be present on a single exotoxin variant The choice of nucleic acid affinity domain can be determined by one skilled in the art

Any substance which binds reversibly to a nucleic acid may serve as a nucleic acid affinity domain (NAAD) provided that it binds sufficiently strongly and specifically to the nucleic acid to retain it until the conjugate reaches and enters the target cell, and does not, through its binding, substantially damage or alter the nucleic acid The ultimate criterion is one of therapeutic effectiveness of the conjugate Preferably, the NAAD is a polycation. Its positively charged groups bind ionically to the negatively charged DNA, and the resulting charge neutralization reduces nucleic acid-solvent interactions. Examples of NAAD include, but are not limited to polycationic domains such as polylysine, polyhistidine, polyarginine, other mixed sequences composed primarily of Arg-Lys-His mixed polymers, polyornithine, histones, avidin, and protamines. NAAD also include domains with homology to know NAAD including helix-turn-helix, leucine zipper, zinc finger, helix-loop-helix, single stranded DNA binding motifs, and RNA binding motifs. Other domains know to bind nucleic acids or those yet to be found are also included. Nucleic acid affinity domains can be readily screened by one skilled in the art for their ability to bind nucleic acids through an electrophoretic gel mobility shift assay or nucleic acid filter binding assays.

A second desired feature for the addition of a nucleic acid affinity domain to the exotoxin is that the resulting exotoxin variant must remain structurally functional. For example, it must remain competent for receptor binding and entry into the cell. Variants with suitable NAAD, can be screened for their structural and functional integrity, for example, by screening for pentamer formation of the LTB using native polyacrylamide gel electrophoresis as outlined in Example 3, or any other technique known in the art, including sedimentation studies, by screening for receptor binding activity as described Example 3.

Exotoxin variants include any derivatives of the variant modified by mutation for enhancement of functional activity. For example variants can be modified through mutation for enhancement of receptor binding and targeting and or enhanced for nucleic acid binding and delivery. Techniques used to introduce such mutations are known to those skilled in the art for examples, the use of polymerase chain reaction or random mutagenesis to introduce mutations. Examples include point mutations that result in altered codon usage, amino acid substitutions, additions, and or deletions such that the alteration results in a variants of the exotoxin with enhanced or altered structural or functional properties. An example of such mutations includes the substitution of various receptor binding domains.

Nucleic Acid Material

\ \ The "nucleic acid material" which may be delivered by the exotoxin variant of the present invention may be any molecule containing nucleic acid. The nucleic acid may be a DNA, RNA, or a DNA or RNA derivative such as a derivative resistant to degradation in vivo, as discussed below. Within this specification, references to DNA apply, mutatis mutandis, to other nucleic acids as well, unless clearly forbidden by the context. The nucleic acid may be single or double stranded. The bases may be the "normal" bases adenine (A), guanine (G), thymidine (T), cytosine (C) and uracil (U), or abnormal bases such as those listed in 37 CFR §1.822 (p) (1). The nucleic acid molecule can be synthetic, cDNA, or of genomic origin, or a combination thereof. The gene may be one which occurs in nature, a non-naturally occurring gene which nonetheless encodes a naturally occurring polypeptide, or a gene which encodes a recognizable mutant of such a polypeptide. It may also encode an mRNA which will be "antisense" to a DNA found or an mRNA normally transcribed in the host cell, but which antisense RNA is not itself translatable into a functional protein. DNA can include non-transcribed and transcribed regions (such as 5' and 3' non-coding regions, introns and exons) or cDNA and mRNA molecules contain sequences corresponding to transcribed regions. The genetic material may also be a hybrid of the above types of material, or a hybrid with a protein or proteins.

The "nucleic acid material" may be prepared by any desired procedure. For example, DNA may be produced by amplification reaction (such as polymerase chain reaction (PCR)), or DNA or RNA may be produced by oligonucleotide synthesis and or ligation of smaller fragments.

In a preferred embodiment, the nucleic acid material comprises an expressible gene which is functional in the target cell. For example, the gene may encode vaccine antigens or the genes may encode enzymes or factors involved in specific metabolic defects, receptors, or membrane transporters.

For the gene to be expressible, the coding sequence must be operably linked to a promoter sequence functional in the target cell. Two DNA sequences (such as a promoter region sequence and a coding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation in the region sequence to direct the transcription of the desired gene sequence, or (2) interfere with the ability of the gene sequence to be transcribed by the promoter region sequence A promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a mRNA if it contains nucleotide sequences which contain transcπptional regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the RNA The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but in general include a promoter which directs the initiation of RNA transcription Such regions may include those 5'-non-codιng sequences involved with initiation of transcription such as the TATA box

If desired, the non-coding region 3' to the gene sequence coding for the desired RNA product may be obtained This region may be retained for its transcπptional termination regulatory sequences, such as those which provide for termination and polyadenylation Thus, by retaining the 3 '-region naturally contiguous to the coding sequence, the transcπptional termination signals may be provided Where the transcπptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted

The promoter may be an "ubiquitous" promoter active in essentially all cells of the host orgamsm, e g , for mammals, the beta-actin promoter, or it may be a promoter whose expression is more or less specific to the target cell or a promoter native to a gene which is naturally expressed in the target cell may be used for this purpose Examples of promoters include albumin, metallothionem, surfactant, apoE, pyruvate kinase, LDL receptor HMG CoA reductase or any promoter which has been isolated, cloned and shown to have an appropriate pattern of tissue specific expression and regulation by factors (hormones, diet, heavy metals, etc ) required to control the transcription of the gene in the target tissue In addition, a broad variety of viral promoters can be used, these include MMTV, SV-40 and CMV An "expression vector" is a vector which (due to the presence of appropriate transcπptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing an RNA or protein product Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences

In addition to or instead of an expressible gene, the nucleic acid may comprise sequences homologous to genetic material of the target cell, whereby it may insert itself ("integrate") into the genome by homologous recombination, thereby displacing a coding or control sequence of a gene, or deleting a gene altogether

In another embodiment, the nucleic acid molecule is "antisense" to a genomic or other DNA sequence of the target organism (including viruses and other pathogens) or to a messenger RNA transcribed in cells of the organisms, which hybridizes sufficiently thereto to inhibit the transcription of the target genomic DNA or the translation of the target messenger RNA The efficiency of such hybridization is a function of the length and structure of the hybridizing sequences The longer the sequence and the closer the complementarily to perfection, the stronger the interaction As the number of base pair mismatches increases, the hybridization efficiency will fall off Furthermore, the GC content of the packaging sequence DNA or the antisense RNA will also affect the hybridization efficiency due to the additional hydrogen bond present in a GC base pair compared to an AT (or AU) base pair Thus, a target sequence richer in GC content is preferable as a target It is desirable to avoid antisense sequences which would form secondary structure due to intramolecular hybridization, since this would render the antisense nucleic acid less active or inactive for its intended purpose One of ordinary skill in the art will readily appreciate whether a sequence has a tendency to form a secondary structure An oligonucleotide complementary to the target sequence may be synthesized from natural mononucleosides or, alternatively, from mononucleosides having substitutions at the non-bridging phosphorous bound oxygens A preferred analogue is a methylphosphonate analogue of the naturally occurring mononucleosides More generally, the mononucleoside analogue is any analogue whose use results in oligonucleotides which have the advantages of (a) an improved ability to diffuse through cell membranes and/or (b) resistance to nuclease digestion within the body of a subject (Miller, P S. et al , Biochemistry 20 1874-1880 (1981)) Such nucleoside analogues are well-known in the art The nucleic acid molecule may be an analogue of DNA or RNA The present invention is not limited to use of any particular DNA or RNA analogue, provided it is capable of fulfilling its therapeutic purpose, has adequate resistance to nucleases, and adequate bioavailabi ty and cell take-up DNA or RNA may be made more resistant to in vivo degradation by enzymes, e g , nucleases, by modifying internucleoside linkages (e g , methylphosphonates or phosphorothioates) or by incorporating modified nucleosides (e g , 2'0-methylπbose or 1 '-alpha-anomers)

Production Methods Exotoxin variants can be prepared by a number of known techniques, including peptide synthesis, chemical or enzymatic gation of peptides, and preferably by recombinant expression from host cells

To produce the exotoxin variants of the present invention by recombinant expression from host cells, host cells containing expression vectors with nucleic acid molecules encoding the variants of the present invention must be used Thus, the present invention includes nucleic acid molecules encoding the exotoxin protein variants of the invention, vectors, host cells, and production methods

Basic procedures for constructing recombinant DNA and RNA molecules in accordance with the present invention are disclosed by Sambrook, J et al , In Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N Y (1989) A description of suitable nucleic acids is described in U S patent 6,077,835, which reference is herein incorporated by reference

A nucleic acid molecule according to the invention encodes, or is complementary to, an exotoxin protein variant of the invention A complementary nucleotide sequence is capable of forming Watson-Cπck bonds with its complement, in which adenine pairs with thymine or uracil and guamne pairs with cytosine A double-stranded nucleic acid molecule encodes one of the exotoxin protein vaπants, whereas a single-stranded DNA or RNA molecule is either the coding (sense) or the noncoding (anti-sense) strand

Because of the redundancy of the genetic code, there are a large number of possible nucleic acid molecules related to each exotoxin protein vaπant More specifically, because several different codons encode the same ammo acid, a large number of different nucleic acid molecules encode (or are complementary to a nucleic acid encoding) the same exotoxin protein vaπant In some instances it may be advantageous to change one or more codons in a nucleic acid molecule without altering the encoded amino acid Examples of such "silent mutations" within the scope of the present invention include, e g , mutations that create or destroy restriction endonuclease sites to facilitate construction of a desired vector and mutations that enhance expression of the encoded exotoxin protein variant Examples of the latter include nucleotide substitutions designed to reduce formation of 5' stem and loop structures in transcribed mRNA or to provide codons that are more readily transcribed by the selected host (e g , the well-known preference codons for E coli or yeast expression)

A nucleic acid molecule of the present invention can be incorporated into a vector for propagation and/or expression in a host cell Such vectors typically contain a replication sequence capable of effecting replication of the vector in a suitable host cell (l e , an origin of replication) as well as sequences encoding a selectable marker, such as antibiotic resistance gene Upon transformation of a suitable host, the vector can replicate and function independently of the host genome or integrate into the host genome Vector design depends, among other things, on the intended use and host cell for the vector, and the design of a vector of the invention for a particular use and host cell is within the level of skill in the art If the vector is intended for expression of exotoxin variant, the vector includes one or more control sequences capable of effecting and/or enhancing expression of an operably linked exotoxin variant coding sequence Control sequences that are suitable for expression in prokaryotes, for example, include a promoter sequence, an operator sequence, and a πbosome binding site Control sequences for the expression in eukaryotic cells include a promoter, an enhancer, and a transcription termination sequence (l e , a polyadenylation signal)

An exotoxin variant expression vector can also include other sequences, for example, nucleic acid sequences encoding a signal sequence or an amp fiable gene A signal sequence directs the secretion of a polypeptide fused thereto from a cell expressing the protein In the expression vector, nucleic acid encoding a signal sequence is linked to the exotoxin vanant coding sequence so as to preserve the reading frame of exotoxin vaπant coding sequence The vectors of the invention most commonly encode a purification domain that is operably linked to the expressed protein, resulting in the expression of a fusion protein. Suitable purification domains are hexahistidine sequences that bind metal affinity columns; glutathione-S-transferase, that binds to glutathione; protein A (or derivative or fragments thereof), that binds IgG molecules; maltose binding protein, that binds maltose or any variety of other protein or protein domains that can bind to an affinity support with an association constant (Ka) of >10⁵ M^"1 . The purification domain is most frequently linked to the exotoxin variant through a linker region. The linker region most often encodes a substrate sequence for a sequence specific protease to allow the elution of the fusion protein during purification. The linker sequence may also contain any other cleavable peptide sequence as would be known by one skilled in the art, including sequences susceptible to chemically induced cleavage.

A vector of the present invention is produced by linking desired elements by ligation at convenient restriction site. If such sites do not exist, suitable sites can be introduced by standard mutagenesis (e.g., site-directed or cassette mutagenesis) or synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.

The present invention also provides a host cell containing a vector for this invention. A wide variety of host cells are available for propagation and/or expression. Examples include prokaryotic cells (such as E. coli and strains of Bacillus, Pseudomonas, and other bacteria), yeast or fungal cells (including S. cerevesiae and P. pastoris), insect cells, plant cells, and phage, as well as higher eukaryotic cells (such as human embryonic kidney cells and other mammalian cells). Host cells according to the invention include cells in culture and cells present in living organisms, such as transgenic plants and animals. A preferred embodiment of the present invention utilizes E. coli as a host cell for expression of the exotoxin protein variant.

A vector of the present invention is introduced into a cell by any convenient method, which will vary depending on the vector-host system employed. Generally, a vector is introduced into a host cell by transformation (also known as transfection) or infection with a virus (i.e., phage) bearing the vector. Known methods, suitable for the host-vector system employed, acceptable for the use in the invention include:

\ 1 infection, chemical transformation, (i e, polyethylene glycol treatment, calcium phosphate precipitation, DEA-dextran, Lipids, PLL-based, PEI), mechanical transformation, (i e , low-voltage electrophoration, bombardment, pressure), electrical transformation, protoplast fusion and direct microinjection To produce exotoxin variants recombinantly, host cells containing the exotoxin variant expression vector are prepared and cultured under conditions suitable for cell growth and for expression of the exotoxin variant In particular, the culture media contains appropriate nutrients and growth factors for the host cell employed The nutrients and growthfactors are, in many cases, well known or can be readily determined empirically by those skilled in the art

In addition , the culture conditions should allow transcription, translation, and protein transport between cellular components Factors that affect these processes are well-known and include DNA/RNA copy number, factors that stabilize DNA, nutrients, supplements, and transcriptional inducers or repressors present in the culture medium, temperature, pH and osmolality of the culture, and cell density The adjustment of these factors to promote expression in a particular vector-host cell system is within the level of skill in the art

The cell culture procedure employed in the production of the exotoxin variant of the present invention can be any of a number of well known procedures for large- or small-scale production of proteins These include, but are not limited to, the use of a shaker flask, fermentor, a fluidized bed bioreactor, a roller bottle culture system, and a stirred tank bioreactor system The protein can be produced in a batch, fed-batch, or continuous mode

Methods for recovery of the recombinant exotoxin proteins produced as described above are well-known and vary depending on the expression system used For example, if the exotoxin variant contains a signal sequence the recombinant protein is recovered from the culture media or the periplasm The recombinant exotoxin protein is secreted into the periplasmic space as a mature protein Alternatively, the recombinant exotoxin protein can also be expressed intracellularly and recovered from cell lysates

The recombinant exotoxin can be purified from the culture media or a cell lysate by any method capable of separating the recombinant exotoxin protein from

I* components of the host cell or culture media. Typically, the exotoxin variant is separated from the host cell and /or culture media components that would interfere with the intended use of the exotoxin variant. As a first step, the culture medium or cell lysate is usually centrifuged or filtered to remove cellular debris. The supernatant is then typically concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification.

The exotoxin variant is further purified using well-known techniques. The techniques chosen will vary depending on the properties of the exotoxin variant. If, for example, the exotoxin variant is expressed as a fusion protein containing an affinity domain, purification typically includes the use of an affinity column containing the cognate binding partner. For instance, the exotoxin variant fused with the hexahistidine or similar metal affinity tags can be purified by fractionation on an immobilized metal affinity column.

The following exemplary procedures can be used or adapted for purifying the exotoxin variant of the invention: fractionation on an immuno affinity column, fractionation on an ion-exchange column, fractionation on a immobilized D-galactose column, ammonium sulfate or ethanol precipitation, reverse phase HPLC chromatography on silica, isoelectric focusing, SDS-PAGE or gel filtration.

Exotoxin Delivery System

With targeting Hgand sequences and a material conjugating moiety engineered into the exotoxin variant, this recombinant variant represents the minimal components necessary to achieve efficient targeted delivery of exogenous material to cells expressing the specific receptor that binds the ligand. The delivery system is assembled from the exotoxin variant and the nucleic acid to be delivered. The components are mixed to form functional complexes. It is preferable to mix the components at low temperature with the therapeutic material of interest.

The material to be delivered can be any nucleic acid material that would be desired to be delivered to the target cell. Any nucleic acid of interest can be chosen for inclusion in the nucleic acid delivery system of the invention. Nucleic acid includes those described under "Nucleic Acid" section of this specification. For example, the nucleic acid may be a marker gene which includes but is not limited to β-lactamase, green fluorescent protein, luciferase and selectable markers such as the neomycin resistance gene, the β-galactosidase (lacZ) gene, and the chloramphenicol transferase (CAT) gene The nucleic acid material of interest can also include therapeutic genes If the targeted cell or tissue-type has a defective gene, for instance as a result of a hereditary condition, a functional copy of the gene may be included in the nucleic acid material Suitable genes for this approach include, but are not limited to, tumor necrosis factor (TNF) genes, such as TNF-alpha, genes encoding interferons such as Interferon-alpha, Interferon-beta, Interferon-gamma, genes encoding interleukins such as IL-1, IL-lβ, and Interleukins 2 throughl5, genes encoding G-CSF, M-CSF, and GM-CSF, genes encoding adenosme deaminase, or ADA, Zap70 kinase gene, genes encoding cellular growth factors, such as lymphokines, which are growth factors for lymphocytes, the glucocerebrosidase gene, genes encoding epidermal growth factor (EGF), and keratinocyte growth factor (KGF), genes encoding soluble CD4, the beta-globm gene, Factor VIII, Factor IX, T-cell receptors, the alpha-iduronidase gene, the LDL receptor, ApoE, ApoC, ApoAI and other genes involved in cholesterol transport and metabolism, the alpha- 1 antitrypsin (alpha 1 AT) gene, the ormthine transcarbamylase (OTC) gene, the CFTR gene, the insulin gene, suicide genes such as, for example, viral thymidine kinase genes, such as the Herpes Simplex Virus thymidine kinase gene, the cytomegalovirus thymidine kinase gene, and the varicella-zoster virus thymidine kinase gene, Fc receptors for antigen-binding domains of antibodies, antisense sequences which inhibit viral replication, such as antisense sequences which inhibit replication of hepatitis B or hepatitis non-A non-B virus, antisense c-myb o gonucleotides, multidrug resistance genes such as the MDR-1 gene, and antioxidants such as, but not limited to, manganese superoxide dismutase (Mn-SOD), catalase, copper-zinc superoxide dismutase (CuZn-SOD), extracellular superoxide dismutase (EC-SOD), and glutathione reductase Genes with function impaired as a result of somatic mutations in cancer can also be included Such genes include tumor suppressors such as p53 The nucleic acid material can also include nucleic acids or nucleic acid analogs designed to bind to a target mRNA to provide antisense inhibition of the gene, or nucleic acids or nucleic acid analogs that bind to other cellular components Furthermore, the nucleic acid material can comprise genes encoding toxic proteins In the treatment of tumors, for instance, tissue-specific gene transfer of a gene encoding a toxic protein could be used to kill tumor cells Likewise, delivery directed to virally infected cells may be used to kill such infected cells

The nucleic acid material can also encode proteins that catalyze the conversion of prodrugs with reduced toxicity to cytotoxic drugs within a cell or tissue-type of interest An example of such an enzyme is the herpes simplex virus thymidine kinase Cells expressing this enzyme are susceptible to the drug ganciclovir

The nucleic acid material can also encode proteins with systemic or long range effects, such as hormones An example is endostatin, which inhibits angiogenesis and has proven effective for tumor regression The nucleic acid material can also encode proteins that stimulate an immune response Such genes could be used as prophylactic or therapeutic vaccines They include nucleic acid vaccines that are capable of inducing an appropriate immune response in a host subject such that administration results in amelioration or prevention of the disease condition Examples include for prevention or treatment of infectious diseases or for the prevention or treatment of autoimmune or allergy diseases It is particularly useful to produce such vaccines in mucosal cells Protection against disease includes amelioration to the symptoms of the disease, decrease in mortality and morbidity, or decrease in susceptibility to the infectious organism An example would be genes encoding pathogen derived proteins that are capable of inducing an appropriate immune response in a host subject such that results in reducing or preventing infection Such genes can be derived from bacteria, viruses, fungi, and parasites or infectious organisms The methods and compositions of the nucleic acid vaccine are intended for use both in immature and mature vertebrates, in particular birds, mammals, and humans Useful antigens, as examples and not by way of limitation, would include genes encoding antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumomae, Neisseria gonorrheae, Neisseria menmgitidis, Corynebacterium dψhtheriae, Clostridium botulmum, Clostridium perf ingens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus a reus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tar da, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shi gel la dysenteriae, Shigellaflexneri, Shigella sonnei, Salmonella typhimuήum, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Bnicella abortus, Bnicella suis, Bnicella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsiitsiigumushi, Chlamydia spp.), Helicobacter pylori; pathogenic fungi (Coccidioides immitis, Aspergil us fiimigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsitlatum); protozoa (Apicomplexia, Coccidea Eimeria, Isospora, Cyclospora, Cryptosporidium, Besnoitia, Caryospora, Frenkelia, Hammondia, Neospora, Sarcocystis, Toxoplasma, Entomoeba histolytica, Trichomonas tenas, Trichomor as hominis, Tήchomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms); pathogenic viruses (as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non- B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus). Further examples of relevant vaccines include, but are not limited to, influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid combined with pertussis vaccine, hepatitis A vaccines hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles

2Z vaccines rubella vaccine, mumps vaccine, mixed vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, malaria vaccine, varicella vaccine, gonorrhea vaccine, HIV vaccines schistosomiasis vaccine, rota vaccine, mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine, and others Influenza vaccine a vaccine comprising genes encoding the whole or part of hemagglutmm, neuramimdase, nucleoprotein and matrix protein which are obtainable from purified Influenza virus by genetic engineering techniques or chemical synthesis, Pertussis vaccine a vaccine comprising genes encoding the whole or a part of pertussis toxin, hemagglutmm and K-agglutin which are obtained from bacterial cells of Bordetella pertussis by genetic engineering techniques or chemical synthesis, Diphtheria and tetanus toxoid combined with pertussis vaccine a vaccine mixed with genes encoding pertussis vaccine, diphtheria and tetanus toxoid antigens, Japanese encephalitis vaccine a vaccine comprising the genes encoding whole or part of an antigenic protein which are obtained from the purified virus or by genetic engineering techniques or chemical synthesis, Hepatitis B vaccine a vaccine comprising genes encoding the whole or part of an S antigen protein which is obtained from hepatitis virus or by genetic engineering techniques or by chemical synthesis, Measles vaccine a vaccine comprising the genes encoding whole or part of antigenic proteins which are obtained from the purified virus or by genetic engineering techniques or chemical synthesis, Rubella vaccine a vaccine comprising the genes encoding whole or part of antigenic proteins which are obtained from the purified virus or by genetic engineering techniques or chemical synthesis, Mumps vaccine a vaccine comprising the genes encoding whole or part of antigemc proteins which are obtained from the purified virus or by genetic engineering techmques or chemical synthesis, Mixed vaccine of measles, rubella and mumps a vaccine produced by mixing measles, rubella and mumps vaccines Rota vaccine a vaccine comprising the genes encoding whole or part of antigenic proteins which are obtained from the purified virus or by genetic engineering techniques or chemical synthesis, Mycoplasma vaccine a vaccine comprising the genes encoding whole or part of antigenic proteins which are obtained from the purified bacteria or by genetic engineering techniques or chemical synthesis Additional list of vaccine candidates would include genes isolated from transmissible diseases which are classified by the Institute for International Cooperation in Animal Bιologιcs(IICAB) and OIE Collaborating Centre for the Diagnosis of Animal Diseases and Vaccine Evaluation in the Americas as are described in both OIE List A Diseases and both OIE List B Diseases, which are hereby incorporated by reference Those conditions for which effective prevention may be achieved by the present method will be obvious to the skilled artisan

Another example is the use as a therapeutic vaccine which would include genes encoding autoimmune antigens or allergens that are capable of inducing an appropriate immune response in a host subject such that administration results in amelioration or prevention of the disease condition Autoimmune antigens include antigens specific for autoimmune diseases whereas allergen antigens, include antigens involved in eliciting specific allergic reactions Examples include genes which encode proteins that are known to protect against autoimmune diseases, to treat autoimmune diseases, or to prevent or treat allergic reactions Protection against disease includes amelioration to the symptoms of the autoimmune disease, decrease in mortality and morbidity, or decrease in sensitivity to the antigen Example of such genes include but is not limited to genes which encode insulin or glutamate decarboxylase (GAD) or heat shock protein (HSP) for the treatment of diabetes mellitus type I, genes which encode myehn basic protein (MBP) or myelin proteo pid protein (PLP) or mye n-oligodendrocyte glycoprotein (MOG) for the treatment of multiple sclerosis, genes which encode type II collagen for the treatment of rheumatoid arthritis, genes which encode acetylcho ne receptor (AChR) for the treatment of myasthenia gravis, genes which encode interphotoreceptor retinoid- bindmg protein (IRBP) or S-antigen for the treatment of uvoretimtis, genes which encode thyroglobu n for the treatment of thyroiditis, genes which encode MHC alloantigen HLA protein allele B7(MHC) for the prevention of transplant rejection, genes which encode allergens for the prevention of allergic reactions or hypersensitivity to the allergens, which include but are not limited to the genes which encode feline domesticus major allergen (Fel dl), bee venom honeybee phospho pase A2 (PLA-2), house dust mite allergens (Der p I) or (Der f I), and genes which encode plant pollens including grass or tree pollens

2H Method of Treating a Subject The present invention also provides for methods of treating patients with exotoxin-nucleic acid complexes (ENAC) that provides protection against cancer, hereditary diseases, infectious diseases, autoimmune disease and allergy disease, comprising administering ENAC composition including a nucleic acid encoding a relevant gene having an amount of ENAC sufficient to protect the patient against the specific disease to which the nucleic acid is directed

The methods and compositions are directed towards treating and protecting humans as well as animals, including domestic animals Indeed, any multicellular organism into which it may be desirable to introduce exogenous nucleic acid is a potential subject for the present invention The multicellular organism may be a plant or an animal, preferably the latter The animal is preferably a vertebrate animal, and more preferably a higher vertebrate, l e , a mammal or bird, the former being especially preferred Among mammals, preferred subjects are human and other primates, laboratory animals such as mice, rats, rabbits and hamsters, pet animals such as dogs and cats, and farm animals such as horses, cows, goats, pigs and sheep It will be noted that these animals come from four orders of class Mammalia Pπmata, Rodenta, Carnivora and Artiodactyla

The mode of administration may be, by way of example and not by way of limitation, by an intradermal, intramuscular, lntrapeπtoneal, intravenous, subcutaneous, transdermal, epidural, pulmonary, oral, nasal, gastric, intestinal, rectal, vaginal, or urethral route Preferably, the route of administration is a mucosal route of administration, I e , through a mucosal membrane or surface, such as an oral, nasal, gastric, intestinal, rectal, vaginal or urethral route More preferably, the mucosal route of admimstration is through oral or nasal membrane or any other means of administration capable of delivering the ENAC to the target cell of interest can be used The route and site of administration may be chosen to enhance targeting For example, to target cells in the respiratory system, the DNA-complex may be administered m aerosol form Another example to target cells in the digestive tract involves administering the DNA-complex by feeding Polymers of positively charged amino acids are known to act as nuclear localization signals (NLS) in many nuclear proteins Treatment of patients with the ENAC produced according to the present invention results in the amelioration of symptoms, decrease in morbidity, and prevention of further disease development As the genes for treatment of the diseases are well known, the nucleic acid can readily be generated according to the present invention and used to treat patients suffering from these diseases

'Targeting" is the administration of the nucleic acid in such a manner that it enters the target cells, i e , the mucosal cells, in amounts effective to achieve the clinical purpose The amount of nucleic acid administered is such that it is effective to achieve the clinical purpose In this regard, it should be noted that DNA and RNA are capable of replication in the nucleus of the target cell, and in consequence the ultimate level of the nucleic acid in the cell may increase after uptake Moreover, if the clinical effect is mediated by a protein expressed by the nucleic acid, it should be noted that the nucleic acid acts as a template, and thus high levels of protein expression can be achieved, even if the number of copies of the nucleic acid in the cell is low Nonetheless, it is desirable to deliver high concentrations of DNA to increase the number of target cells which take up the DNA and the number of DNA molecules taken up by each cell

The invention is illustrated, but not limited by the following examples

EXAMPLE 1 Cloning of Enterotoxin proteins in bacterial expression plasmids.

Sequences encoding the E. coli heat-labile enterotoxin (eltB) were amplified from NADC LT(+) strain of E. coli carrying the porcine LT enterotoxin [Dallas,W S and Falkow,S 1980, Nature 288 (5790), 499-501, GenBank accession number Ml 7873] by polymerase chain reaction(PCR) and subcloned into the bacterial expression plasmid pRSET-B (Invitrogen) PCR amplification condition consisted of one cycle for one minute at 95°C followed by 32 cycles of one minute at 95°C,one minute at 52°C, and one minute at 72°C followed by one cycle often minutes at 72°C PCR was performed using the high fidelity DNA polymerase Pfu (Stratagene) which is much less prone to misincorporation of nucleotides and mutagenesis than standard Taq polymerase and the following primers which were designed for direct insertion of the PCR product in-frame into BamHI and Xhol sites of the pRSET B vector (Invitrogen) 5' Primer-SEQ ID 1 [5' GAC GAA TTC GGA TCC GAT GAA TAA AGT AAA ATG TTA GT 3'] and 3' Primer-SEQ ID 2 [5' CGG CCG CTC GAG CTA CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CGA GCTCGG TAC CCG GGG ATCATG GTT TTT CAT ACT GAT TGC CGC 3 '] The resulting construct was designated LTBpLh and has the sequence shown in Fig 10 In order to minimize any potential structural disruption of LTB pentamer formation or binding and maintain biological activity of the LTB portions of the chimeric fusion proteins, the 3' Primer-SEQ ID 2 was designed to contain 24 nucleotides which encode a hinge or spacer region (HDPRVPSS) (SEQ ID NO 6) that was fused in frame to the carboxy terminus of the eltB followed in frame by the NAAD The 3 ' Primer SEQ ID 2 contains the NAAD of 30 nucleotides which encodes a 10 amino acid polylysine repeat (underlined) fused in frame to the spacer region (bold) followed by a termination codon The resulting construct was designated LTBpLh and has the sequence shown in Fig 10 Examples of amino acid sequences of hinge regions used to make LTB fusion proteins are HDPRVPSS (SEQ ID NO 6) or GPGPE (SEQ ID NO 7) (described in Cardenas et al (1993), Lipscombe et al ( 1991 ), Clements ( 1990)) The isolated PCR fragment was digested with BamHI and Xhol restriction enzymes and cloned into the BamHI/XhoI site of pRSETB Positive clones were confirmed by DNA sequencing

Another 3' Primer SEQ ID 3 was designed containing an alternate NAAD 5' [CGG CCG CTC GAG CTA ACG CCT CCT GCG GCC TCC TCT CCT GCG ACG CCG GGA CAC CCT GGG GCG ACG GCG CCT GCG GAC ACG GCG GCT GGC TCT GCG TCT TCT GGG CAT CGA GCTCGG TAC CCG GGG ATCATG GTT TTT CAT ACT GAT TGC CGC 3'] This primer contains the identical sequence as SEQ ID 2 desrcibed above with the following exception, CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT (residues 16-45 of SEQ ID NO 2) was replaced by 93 nucleotides which encode a 31 amino acid nucleoprotamine (MPRRRRASRRVRRRRRPRVSRRRRRGGRRRR) (SEQ ID NO 8) which was fused in frame to the 3' terminus of the 24 nucleotides which encode hinge region (HDPRVPSS) (SEQ ID NO 6) followed by a termination codon. An example of the amino acid sequence of a fish protamine (GenBank Accession number K03051 ) The resulting construct was designated LTB-Ph and has the sequence shown in Fig 12

zn Alternatively the chimeπc LTB proteins can be derived using 3' primers that contain no spacer (hinge) regions which have the following sequences 3' Primer SEQ ID 4 [5' CGG CCG CTC GAG CTA CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT GTT TTT CAT ACT GAT TGC CGC 3'] which is identical to SEQ ID 2 with the exception that the 24 nucleotides which encode hinge region (HDPRVPSS) (SEQ ID NO 6) have been deleted which results in the 30 nucleotides encoding the 10 polylysine NAAD sequence being fused in frame to the 3' end of the eltB gene sequence (the resulting construct being designated LTBpL and having the sequence shown in Fig 9), or 3' Primer SEQ ID 5 [5' CGG CCG CTC GAG CTA ACG CCT CCT GCG GCC TCC TCT CCT GCG ACG CCG GGA CAC CCT GGG GCG ACG GCG CCT GCG GAC ACG GCG GCT GGC TCT GCG TCT TCT which is identical to SEQ ID 3 with the exception that the 24 nucleotides which encode hinge region (HDPRVPSS) (SEQ ID NO 6) have been deleted which results in the 93 nucleotides which encodes the 31 amino acid nucleoprotamine NAAD (MPRRRRASRRVRRRRRPRVSRRRRRGGRRRR) (SEQ ID NO 8) sequence being fused in frame to the 3' end of the eltB gene sequence (the resulting construct being designated LTB-Ph and having the sequence shown in Fig 1 1)

Researchers have reported that genetic fusions to the enterotoxins can result in decreased expression of the modified enterotoxin in bacteria There are a number of various expression vectors known to those skilled in the art Therefore, the choice of expression vectors for the recombinant LTB chimeπc proteins is not limited to those described In order to facilitate high level expression and purification of the chimeπc LTB proteins, the following can be subcloned into secretory baculovirus expression vector pAcGP67 (PharMingen) or into the secretory yeast expression vector PICZα (Invitrogen) Both of these expression vectors are designed for expression and secretion into the media The media is then concentrated and processed for isolation of the modified proteins All constructs are confirmed by DNA sequencing after cloning

EXAMPLE 2

Bacterial production and purification of recombinant LTBpLh proteins

IX LTBpLh protein expression was IPTG induced in E. coli BL21 cells (Novagen) and the modified recombinant enterotoxin protein was purified by Talon (Clonetech) column affinity chromatography per manufacture's protocol However, alternate methods of purification will be apparent to those skilled in the art Coomassie and Western analysis of whole cell lysates (using anti-HIS) indicated LTBpLh was expressed as an apparent molecular weight of 13 kDa band (predicted size 20 kDa) Initial attempts to purify LTBpLh using the Talon system indicated the protein seemed to be insoluble as it remained with the membrane pellet after sonication of the cells as analyzed by PAGE and Coomassie staining indicating that the protein appeared to form inclusion bodies Others have reported that alteration of the carboxyl-terminus of LTB changes assembly and secretion properties in E. coli (Sandkvist et al (1987)) Therefore, the membrane pellet was denatured with 6 M Guanidine HC1 and the protein was purified in a denatured form using the Talon system from Clonetech The eluted protein fractions were dialyzed in PBS to renature the protein and analyzed by Coomassie and Western analysis using either goat α-LTB polyclonal antibody (gift from Dr John Clements, Tulane University) and α-goat- HRP polyclonal antibody (Sigma Chemical) or α-His monoclonal antibody (Clonetech) and α-mouse-HRP (Sigma Chemical) Coomassie and Western analysis confirmed the expression and isolation of chimeric LTBpLh The results are shown in Figures 1 A and IB

EXAMPLE 3 Characterization of the functional properties of recombinant enterotoxins Demonstration of the functional properties of recombinant enterotoxins 1) Assembly of recombinant enterotoxin proteins The wild type LTB and recombinant LTBpLh proteins were tested for their ability to assemble properly into pentamers by non-denaturing polyacrylamide gel electrophoresis The wild type LTB monomers have a predicted molecular weight around 14,124 Da whereas the predicted molecular weight of the wild type LTB pentameric complex is 70,620 Da The predicted molecular weights of the chimeric LTBpLh monomer is 20,018 Da and that of the LTBpLh pentameric complex is 100,090 Da The wild type LTB proteins showed proper assembly into a 70 kDa complex (Figure 2, lane 9) and the addition of DNA or protamine sulfate did not appear interfere with the formation of this complex (Figure 2, lanes 10,11, and 12). Although the highly charged polylysine tracts in the recombinant LTBpLh might theoretically inhibit pentamer formation, this was not the case, as LTBpLh was able to form the predicted 100 kDa complex (Figure 2, lane 2), whereas the addition of DNA did not appear to interfere with the complex formation,it did appear to further shift the complex to a higher molecular weight (Figure 2, lanes 3-5) as indicated by material remaining in the stacking gel well as compared to wild type LTB mixed with DNA which showed no difference(Figure 2, lane 10). This observation would indicate that DNA is interacting with the LTBpLh pentamers to form larger complexes. The addition of protamine sulfate also did not interfere with the LTBpLh complex formation (Figure 2, lanes 6, 7, 11 and 12).

Expression of gene constructs in the pRSET plasmid produces proteins with a polyhistidine moiety at the N-terminus, enabling affinity purification. After purification and elution from the column, this moiety may be removed by treatment with a recombinant preparation of the catalytic subunit of bovine enterokinase

(EKMax, Invitrogen),which cleaves the fusion protein at a cleavage site two amino acids upstream of the LTB sequence. In order to determine if amino terminal sequences derived from the pRSET expression vector interfered with LTBpLh complex formation, after purification and elution from the Talon column, this moiety was removed by treatment with EKMax (Invitrogen) per manufacture's protocol. The removal of the vector derived sequences by EKMax treatment did not appear to alter the activity the LTBpLh as analyzed by non-denaturing polyacrylamide gel electrophoresis (boiled vs. unboiled) and Coomassie staining, Western blot or GMl ELISA analysis. The EK-Max treated sample had several bands of similar intensity with apparent molecular weights of 14, 10, 8 kDa in both the boiled and unboiled fractions representing the cleavage products of the vector derived sequence and the LTBpLh and an additional lower smear implying some degradation was occurring. The unboiled EKMax treated LTBpLh sample was able to form the predicted pentamer complex (Figure 3 lane 4). Nucleic acid gel mobility shift experiment 2) Binding of DNA

o The wild type LTB or recombinant LTBpLh were incubated with either a Emerald GFP plasmid DNA ( approximately 4 5 kb) or with Gibco DNA ladder molecular weight marker and examined by gel shift assay to determine if the addition of polylysine tract had conferred the ability to bind DNA thereby confirming the previous observation of the higher complex formation of LTBpLh in the presence of DNA The given protein concentration of 1, 10 ,25, or 50 micrograms of protein was mixed with 1 microgram of either DNA ladder or plasmid DNA in a 50 microliter reaction and incubated for 30 minutes at room temperature Five micro liters of lOx DNA loading dye was added and the samples were loaded on 0 8% agarose gel run in lx TBE buffer at 80 volts for approximately 1 5 hours Gels were visualized by ethidium bromide staining As shown in Figure 4, only LTBpLh bound DNA fragments, as demonstrated by formation of the LTBpLh-DNA complexes (Figure 4, lane 2) as compared to the absence of complex formation in the wild type LTB-DNA sample (Figure 4, lane 6) LTBpLh was also capable of binding plasmid DNA as demonstrated in Figure 4, lanes 8-1 1 This demonstrates the versatility of the recombinant LTBpLh system in its ability to bind a variety of multiple nucleic acid fragments or molecules which could represent multiple gene delivery targets or vaccine combination targets 3) Receptor binding to ganghosides Receptor binding of recombinant enterotoxin proteins was quantitated by

ELISA to determine if they retain their ability to bind their natural receptor GMl ganghosides as described in PCT WO 98/32461 and Guidry et al (1997) The amount of LT binding is quantitated by subsequent detection with affinity-purified goat antiserum against LT followed by rabbit antiserum against goat IgG conjugated to horse radish peroxidase (Sigma Chemical) Substrate reactions were stopped by the addition of 1 N NH2SO4 and optical densities at 450 nm were determined using a Bio-Tek microtiter reader Titers are compared to wild type LTB to determine binding efficiency Briefly, 96 well microtiter plates were coated with 1 5 μg/well of GMl ganghosides (Sigma Chemical) in carbonate buffer Plates were incubated overnight and stored at 4°C Prior to use plates were washed three times with carbonate buffer followed by two washes with PBS-0 05% Tween and tapped dry after the last wash on a paper towel Optimal dilution of either wild type LTB or LTBpLh protein was determined by two-fold serial dilutions in 100 μl of PBS-T starting at 50 micrograms of protein/well across the plate using a multichannel pipet Plates were incubate at 37°C for 1 5 hours and rinsed three times with PBS-T and tapped dry after the last wash on a paper towel LTB binding was detected by the addition of lOOμl/well of goat α-LTB antibody diluted 1 500 in PBS-T Plates were incubate at 37°C for 1 hour and then rinse three times with PBS-T as before Followed by the addition of lOOμl/well of secondary antibody α-goat-HRP (Sigma Chemical) diluted 1 40,000 in PBS-T Plates were incubate at 37°C for 1 hour and then rinse three times with PBS-T as before Followed by the addition of 200 μl/well of TMB ELISA substrate (Gibco) Development was stopped by the addition of

1NH2SO4 Plates were lightly vortex to assure mixing of reagent and read at an O D of 450 nm on a Bio-Tek microtiter reader

LTBpLh had a higher binding reactivity than wild type LTB proteins at concentration between 50 to 0 78 micrograms of protein However, this diminished more sharply as compared to the wild type LTB when tittered below 0 2 micrograms or less as shown in Figure 5

Effect of DNA and Protamine sulfate on GMl binding by LTB or LTBpLh were analyzed by the GMl ELISA Various concentration of plasmid DNA between 0 0078 μg and 3 μg encoding the Green Fluorescent Protein marker gene (pGFP) under the control of a viral promoter (for example CMV) was conjugated to varying concentrations of either wild type LTB or LTBpLh between 0 195 μg and 50 μg in duplicate samples and incubated at room temperature for 30 minutes In order to neutralize the charge of the DNA and increase the efficiency, the enterotoxin-DNA complex can be mixed with free polylysine or protamine to increase the condensing of the DNA This may also result in the protection of the DNA from degradation After incubation of the enterotoxin-DNA complex, varying concentration of protamine sulfate(PS) between 0 488 μg and 50 μg (Sigma Chemical) were added to one set of samples and incubated an additional 15 minutes The various complex formation samples were then added to GMl coated microtiter plates for GMl ELISA analysis ELISA results are shown in Figures 6A-D

EXAMPLE 4 Cell binding and gene delivery by recombinant enterotoxins in vitro

Various concentration of plasmid DNA or a PCR dervived DNA between 0 0078 μg and 3 μg encoding the Green Fluorescent Protein marker gene (pGFP) under the control of a viral promoter (for example CMV) was conjugated to varying concentrations of either wild type LTB or LTBpLh between 0 195 μg and 50 μg in duplicate samples and incubated at room temperature for 30 minutes In order to neutralize the charge of the DNA and increase the efficiency, the enterotoxin-DNA complex can be mixed with free polylysine or protamine to increase the condensing of the DNA This may also result in the protection of the DNA from degradation After incubation of the enterotoxin-DNA complex, varying concentration of protamine sulfate (PS) between 0 488 μg and 50 μg (Sigma Chemical) were added to one set of samples and incubated an additional 15 minutes The various complex formation samples were layered on to a monolayer of either Caco-2 cells, COS cell or Yl adrenal cells that had previously been rinsed with serum free media However, other cell types are also available to those skilled in the art The preferable cell line is one that expresses the receptor (in this case the GMl receptor) The levels of DNA delivery are compared between the wild type LTB-DNA GFP conjugate, the modified LTBpLh-DNA/GFP conjugate, LTB-DNA/GFP conjugate with PS, the modified LTBpLh-DNA/GFP conjugate with PS and DNA/GFP alone by comparing the number of fluorescing cells by UV light microscopy Initial transfection of CaCo 2A cells and COS cells did not result in positive fluorescence However, the transfection of confluent Yl cells with the modified LTBpLh-DNA/GFP conjugate did result in positive fluorescence which was enhanced by the addition of PS to the LTBpLh- DNA/GFP conjugate as summarized Figure 7 The wild-type LTB-DNA/GFP did not result in any positive fluorescence as shown in Figures 7 and 8B This is due to the binding of the DNA to the polylysine tracts of the recombinant LTBpLh , which can then mediate internalization by binding the GMl receptor The number of fluorescent colonies obtained was significantly lower than the positive control lipofectamine transfected cells This may represent the non-targeted and non specific transformation of the lipofectamine samples as compared to the receptor mediated transfection by the modified LTBpLh-DNA/GFP conjugate as shown in Figures 8A- D The anticipated efficiency of transfection with a receptor mediated DNA transfection would correlate to receptor expression and availability in the cell culture system. Methods to establish reproducible receptor expression levels can be obtained by varying growth conditions of the cells and by monitoring receptor expression (i.e. L A of cultures for GMl).

EXAMPLE 5 Testing the ability of recombinant enterotoxins to target delivery of nucleic acids in vivo. Testing the ability of recombinant enterotoxin-DNA complexes for targeting and immunogenicity using DNA encoding a sporozoite attachment factors (pBKCMV- SAP (United States patent 5,861, 160).) or GP900 (United States patent 6,015,882). We have previously shown that the intramuscular immunization of both pigs and mice with DNA encoding a coccidiosis sporozoite attachment factor (SAP) results in immune response to the SAP. By using this animal model we are able to compare the immune response obtained by intramuscular administration of naked DNA to immune responses in administration of targeted enterotoxin-nucleic acid complexes. Animals will be divided into six groups representing five different routes of vaccine administration, either oral; nasal; transcutaneous; intraperitoneal; or intramuscular, and a control group receiving no treatment. Each group will be subdivided into three groups. These subdivided groups represent the three different vaccine combinations to be administered consisting of either DNA alone, DNA conjugated to the modified LTBpLh, DNA conjugated to the modified LTBpLh plus protamine sulfate. Optimum complex formation conditions as defined by the Yl cell transformation protocol are used to form the enterotoxin nucleic acid complexes. Vaccine will be administered orally and or intranasally (10 μl total dose starting at 10 μg of LTBpLh:5 μg of DNA with or without 32 μg protamine sulfate) four times at one week intervals. Serum samples will be collected prior to vaccination and on days 7, 14, 21 and 35 (end of test). Sera samples are tested for reactivity with the sporozoite attachment factors and LTB protein by ELISA and/or western blot analysis. Western blot analysis is described in the previous example. ELISA titers are determined as follows, the amount of anti-LT or anti-SAP antibody is quantitated by analyzing two-fold serial dilutions of serum in 96-well microtiter plates coated with either purified recombinant LTB or SAP proteins followed by subsequent detection with anti-mouse conjugated to horseradish peroxidase (Sigma Chemical) Substrate reactions are stopped by the addition of 1 N H₂SO and optical densities at 450 nm are determined using a Bio-Tek microtiter reader End point titers (reciprocal dilution of O D > 0 2) are statistically compared between the vaccinated groups Briefly, 96 well microtiter plates are coated with 1 tol 5 μg/well of recombinant protein for LTB or SAP in carbonate buffer Plates are incubated overnight and stored at 4°C Prior to use, plates are washed three times with carbonate buffer followed by two washes with PBS-0 05% Tween and tapped dry after the last wash on a paper towel Plates are blocked by the addition of 200 μl/well of 1% ova or bovine albumin in PBS and incubate for 30 minutes at room temperature followed by rinsing as previously described Two-fold serial dilutions of the serum collected on days 0, 7, 14, 21 or 35 are made in 100 μl of PBS-T starting at 1 2 dilution of serum/well across the plate using a multichannel pipet Plates are incubated at room temperature for 1 5 to 2 hours and rinsed three times with PBS-T and tapped dry after the last wash on a paper towel Antibody binding is detected by the addition of lOOμl/well of α-mouse- HRP conjugated antibody (Sigma Chemical) diluted 1 60,000 in PBS-T Plates are incubated at room temperature for 1 hour and then rinsed three times with PBS-T as before, followed by the addition of 200 μl/well of TMB ELISA substrate (Gibco) Development is stopped by the addition of IN H₂SO Plates are lightly vortexed to assure mixing of reagent and read at an O D of 450 nm on a Bio-Tek microtiter reader

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention Thus the expressions "means to " and

3tT "means for...", or any method step language, as may be found in the specification above and/or in the claims below, followed by a functional statement, are intended to define and cover whatever structural, physical, chemical or electrical element or structure, or whatever method step, which may now or in the future exist which carries out the recited function, whether or not precisely equivalent to the embodiment or embodiments disclosed in the specification above, i.e., other means or steps for carrying out the same functions can be used; and it is intended that such expressions be given their broadest interpretation.

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3*

Claims

CLAIMS What is claimed is:

1. An exotoxin protein variant consisting of a sequence comprising the sequence of a mucosal cell binding moiety of an exotoxin and the sequence of a nucleic acid affinity domain.

2. The exotoxin protein variant of claim 1, wherein said mucosal cell binding moiety is an enterotoxin receptor binding domain.

3. The exotoxin protein variant of claim 1 , wherein said nucleic acid affinity domain is a polycationic domain.

4. The exotoxin protein variant of Claim 3, wherein said nucleic acid affinity domain is a polylysine, polyhistidine, polyarginine, other mixed sequences composed primarily of Arg-Lys-His mixed polymers, polyornithine, histones, avidin, or protamines.

5. A delivery system for specific delivery of nucleic acid, comprising an exotoxin protein variant in accordance with claim 1 complexed at the nucleic acid affinity domain thereof with a nucleic acid.

6. The delivery system of claim 5, wherein said nucleic acid encodes prophylactic or therapeutic proteins derived from bacteria, viruses, parasites, or animals including humans.

7. A method for selectively delivering nucleic acid to mucosal cells, comprising administering the delivery system of claim 5.

8. A method for inducing an immune response, comprising administering a delivery system in accordance with claim 5, wherein said nucleic acid encodes an antigen to which said immune response is desired.

9. A method for selectively delivering a gene to a mucosal cell, comprising administering a delivery system in accordance with claim 5, wherein said nucleic acid is a gene which is desired to be incorporated into mucosal cells.

10. A method of achieving expression of a protein in a subject, comprising administering to a subject a composition comprising a delivery system in accordance with claim 5, wherein said nucleic acid encodes a protein which is expressed in the subject.

5^ The method of claim 10, wherein the delivery system is associated with pid molecules The method of claim 10, wherein the composition is administered by means of a route selected from the group consisting of subcutaneous, intrapulmonary, and oral administration The method of claim 10, wherein the composition is administered by means of topical admimstration The method of claim 10, wherein the composition is administered by means of inhalation of an aerosolized composition A DNA molecule comprising a sequence encoding an exotoxin protein variant in accordance with claim 1 An expression vector comprising a DNA molecule in accordance with claim 15 A host cell comprising a vector in accordance with claim 16 A method of production of an exotoxin protein variant, comprising cultuπng a host cell in accordance with claim 17 and isolating the exotoxin protein variant produced thereby An exotoxin protein variant in accordance with claim 1, wherein the nucleic acid is an antisense nucleic acid