WO2004015089A2

WO2004015089A2 - Polyionic organic acid formulations

Info

Publication number: WO2004015089A2
Application number: PCT/US2003/025419
Authority: WO
Inventors: Michael J. Bennett; Yen-Ju Chen; Edmund J. Niedzinski; Hsien Tseng; Sean Tucker
Original assignee: Genteric, Inc.
Priority date: 2002-08-12
Filing date: 2003-08-12
Publication date: 2004-02-19
Also published as: WO2004015089A3; AU2003265432A8; AU2003265432A1

Abstract

The present invention provides a nucleic acid transfection composition comprising a polyionic organic acid and a nucleic acid. Preferably, the polyionic organic acid is a dye. Efficient methods are also provided for administering the compositions, increasing the transfection efficiency in a cell (e.g., secretory gland cell), and using the compositions as nucleic acid stabilizers to prevent in vivo and in vitro nucleic acid degradation, thus increasing the half-life and shelf life of a nucleic acid.

Description

Polyionic Organic Acid Formulations

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] The present application claims priority to U.S. Provisional Patent Application Nos. 60/402,811, filed August 12, 2002, 60/453,999, filed March 11, 2003 and 60/476,145, filed June 4, 2003, the disclosures of which are hereby incoφorated by reference in their entirety for all puφoses.

FIELD OF THE INVENTION [02] The present invention relates to compositions for nucleic acid transfection comprising a polyionic organic acid and a nucleic acid. Methods are provided for administering the compositions to a cell with increased transfection efficiency. More particularly, the present invention is concerned with using the compositions as nucleic acid stabilizers to prevent nucleic acid degradation, and increasing the half-life and/or shelf life of a nucleic acid in vivo and in vitro, respectively. In other embodiments, the polyionic organic acids act directly as anti- viral compounds.

BACKGROUND OF THE INVENTION

[03] The ability to replace defective or absent genes has attracted wide attention as a method to treat a variety of human diseases (Crystal, Science 270:404 (1995), Lever et al.

Gene Therapy, Pearson Professional, New York p. 1-91 (1995), and Friedmann, Nature Med. 2: 144 (1996)). Gene-based therapy can be a useful means to supply exogenous gene products to the circulatory system for the treatment of a wide range of systemic disorders that involve deficiencies in circulating proteins, such as hormones, growth factors, clotting proteins, and immunoglobulins (Lever et al. (1995) and Buckel, TiPS 17:450 (1996)), as well as a means of administering other polypeptide drags. The success of this therapeutic application depends upon developing effective methods to deliver and express genes encoding proteins of interest in vivo. (Crystal (1995); Lever et al (1995)).

[04] The advent of recombinant DNA technology and genetic engineering has led to numerous efforts to develop methods that facilitate the transfection of therapeutic and other nucleic acid-based agents to specific cells and tissues. Known techniques provide for the delivery of such agents, including a variety of genes that are carried in recombinant expression constructs. Once the constracts enter the cell, they are capable of mediating expression of the genes. Such developments have been critical to many forms of molecular medicine, specifically gene therapy, whereby a missing or defective gene can be replaced by an exogenous copy of the functional gene. [05] Typically, nucleic acids are large, highly polar molecules. As such, nucleic acids face the impermeable barrier of the cellular membrane in eukaryotes and prokaryotes. The cell membrane acts to limit or prevent the entry of the nucleic acid into the cell. The development of various gene delivery methods has paralleled currently known gene therapy protocols. While much progress has been made in increasing the efficiency of gene delivery into cells, limited nucleic acid uptake or transfection remains a hindrance to the development of efficient gene therapy techniques.

[06] Common approaches for delivering a nucleic acid into a cell include ex vivo and in vivo strategies. In ex vivo gene therapy methods, the cells are removed from the host organism, such as a human, prior to experimental manipulation. These cells are then transfected with a nucleic acid in vitro using methods well known in the art. These genetically manipulated cells are then reintroduced into the host organism. Alternatively, in vivo gene therapy approaches do not require removal of the target cells from the host organism. Rather, the nucleic acid may be complexed with reagents, such as liposomes or retroviruses, and subsequently administered to target cells within the organism using known methods. See, e.g., Morgan et al., Science 237:1476, 1987; Gerrard et al., Nat. Genet. 3:180, 1993.

[07] Several different methods for transfecting cells can be used for either ex vivo or in vivo gene therapy approaches. Known transfection methods may be classified according to the agent used to deliver a select nucleic acid into the target cell. These transfection agents include viras dependent, lipid dependent, peptide dependent, and direct transfection ("naked DNA") approaches. Other approaches used for transfection include calcium co-precipitation and electroporation.

[08] There are currently two general methods for transferring exogenous genes into humans and other mammals: viral and non- viral. Both of these methods have their associated advantages and disadvantages involving either transfection efficiency or safety issues. For example, adenoviral vectors induce potent immune responses (Baum and O'Connell, Crit. Rev. Oral Biol. Med. 10(3):276 (1999)) and transfection efficiency is low when genes are transferred using non-viral delivery systems. Genetic manipulation of cells to express a protein for systemic delivery to the organism has been problematic. Thus, there is a need in the art to develop gene transfer techniques that enhance gene transfection efficiency in a safe manner.

[09] Viral approaches use a genetically engineered virus to infect a host cell, thereby "transfecting" the cell with an exogenous nucleic acid. Among known viral vectors are recombinant viruses, of which examples have been disclosed, including poxvirases, heφesvirases, adenoviruses, and retroviruses. Such recombinants can carry heterologous genes under the control of promoters or enhancer elements, and are able to cause their expression in vector-infected host cells. Recombinant viruses of the vaccinia and other types are reviewed by Mackett et al., J. Virol. 49:3, 1994; also see Kotani et al., Hum. Gene Ther. 5:19, 1994.

[10] However, viral transfection approaches carry a risk of mutagenicity due to possible viral integration into the cellular genome, or as a result of undesirable viral propagation. Many studies in vertebrate systems have established that insertion of retroviral DNA can result in inactivation or ectopic activation of cellular genes, thereby causing diseases. For a review, see Lee et al., J. Virol. 64:5958-5965, 1990. For example, one well known consequence of retroviral integration is activation of oncogenes. One study describes the activation of a human oncogene by insertion of HIV. Shiramizu et al., Cancer Res., 54:2069- 2072, 1994. Viral vectors also are susceptible to interference from the host immune system. [11] Non- viral vectors, such as liposomes, may also be used as vehicles for nucleic acid delivery in gene therapy. In comparison to viral vectors, liposomes are safer, have higher capacity, are less toxic, can deliver a variety of nucleic acid-based molecules, and are relatively nonimmunogenic. See Feigner, P. L. and Ringold, G. M., Nature 337, 387-388, 1989. Among these vectors, cationic liposomes are the most studied due to their effectiveness in mediating mammalian cell transfection in vitro. One technique, known as lipofection, uses a lipoplex made of a nucleic acid and a cationic lipid that facilitates transfection into cells. The lipid/nucleic acid complex fuses or otherwise disrupts the plasma or endosomal membranes and transfers the nucleic acid into cells. Lipofection is typically more efficient in introducing DNA into cells than calcium phosphate transfection methods. Chang et al., Focus 10:66, 1988. However, some of the lipid complexes commonly used with lipofection techniques are cytotoxic or have undesirable non-specific interactions with charged serum components, blood cells, and the extracellular matrix. Furthermore, these liposome complexes can promote excessive non-specific tissue uptake. [12] One known protein dependent approach involves the use of polylysine mixed with a nucleic acid. The polylysine/nucleic acid complex is then exposed to target cells for entry. See, e.g., Verma and Somia, Nature 389:239, 1997; Wolff et al., Science 247:1465, 1990. However, protein dependent approaches are disadvantageous because they are generally not effective and typically require chaotropic concentrations of polylysine. [13] "Naked" DNA transfection approaches involve methods where nucleic acids are administered directly in vivo. See U.S. Pat. No. 5,837,693 to German et al. Administration of the nucleic acid could be by injection into the interstitial space of tissues in organs, such as muscle or skin, introduction directly into the bloodstream, into desirable body cavities, or, alternatively, by inhalation. In these "Naked" DNA approaches, the nucleic acid is injected or otherwise contacted with the animal without any adjuvants, such as lipids or proteins, which typically results in only moderate levels of transfection, and the insufficient expression of the desired protein product. It has recently been reported that injection of free ("naked") plasmid DNA directly into body tissues, such as skeletal muscle or skin, can lead to protein expression, but also to the induction of cytotoxic T lymphocytes and antibodies against the encoded protein antigens contained in the plasmid. See Ulmer et al., Science, 259, 1993, 1745-1749; Wang et al., Proc. Nat. Acad. Sci. U.S.A. 90, 4157-4160, 1993; Raz et al., Proc. Nat. Acad. Sci. U.S.A. 91, 9519-9523, 1994.

[14] Electroporation is another transfection method. See U.S. Pat. No. 4,394,448 to Szoka, Jr., et al. and U.S. Pat. No. 4,619,794 to Hauser. The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA can enter directly into the cell cytoplasm either through these small pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores.

[15] A common disadvantage to known non- viral nucleic acid delivery techniques is that the amount of exogenous protein expression produced relative to the amount of exogenous nucleic acid administered remains too low for most diagnostic or therapeutic procedures.

Low levels of protein expression are often a result of a low rate of transfection of the nucleic acid or the instability of the nucleic acid.

[16] Nucleic acid degradation by the endonuclease family of DNases is thought to be a substantial barrier to efficient nucleic acid (e.g., plasmid DNA) transfection both in vitro and in vivo. For example, plasmid DNA is readily degraded in respiratory fluids by extracellular endonucleases (Glasspool-Malone et al., J. Gene Med. 4(3):323-332, 2002). Studies have also shown the potentially deleterious effects of intracellular endonucleases on transfection efficacy. See, e.g., Ross et al., Gene Ther. 5(9): 1244-1250, 1998; Pollard et al., J. Gene Med. 3(2):153-164, 2001. Besides confronting lysosomal endonucleases, a nucleic acid (e.g., plasmid DNA) may also encounter cytosolic endonucleases prior to nuclear uptake. Based on studies performed in HeLa and COS cells, the estimated half-life of plasmid DNA in cytosolic fluids is approximately 50-90 minutes (Lechardeur et al., Gene Ther. 6(4):482-497, 1999). In general, the presence of intracellular endonucleases may be responsible for an observation suggesting that only 1 out of 1000 plasmid DNA copies are effectively trafficked to the nucleus (Pollard et al., J. Gene Med. 3(2):153-164, 2001). Since the deleterious effect of endonucleases appears to be widespread, strategies directed at improving transfection efficacy by overcoming this barrier may have broad implications. [17] DNases such as endonucleases are phosphodiesterases capable of hydrolyzing polydeoxyribonucleic acid. It acts to extensively and non-specifically degrade DNA and in this regard it is distinguished from the relatively limited and sequence-specific restriction endonucleases. The two prominent non-specific endonucleases are DNase I and II. DNase I efficiently hydrolyzes single- or double-stranded DNA to a mixture of short 5'-phosphate- containing oligo- and mononucleotides. Cleavage preferentially occurs adjacent to pyrimidine residues, and the enzyme has a pH optimum near neutrality. In the presence of magnesium, cleavage of each strand of a double-stranded DNA substrate proceeds independently. In contrast, in the presence of manganese, DNase I cleaves both strands of DNA at approximately the same site, generating fragments with blunt ends or one or two base overhangs. DNase II exhibits an acid pH optimum, can be activated by divalent cations, and produces 3 '-phosphate oligonucleotides on hydrolysis of DNA using a nicking mechanism. DNase II primarily functions in DNA turnover, mitosis, anti- viral protection, and apoptotic or programmed cell death.

[18] Despite numerous research efforts directed at finding efficient methods for nucleic acid delivery, most known techniques fail to result in sufficient cell transfection and/or plasmid DNA stability to achieve the desired protein expression. There is still a need to develop a nucleic acid delivery method that efficiently introduces recombinant expression constructs encoding useful genes into cells, while minimizing undesirable effects. The present invention satisfies this and other needs.

SUMMARY OF THE INVENTION

[19] The present invention provides novel nucleic acid transfection compositions, efficient methods for administering them and/or localizing them in a cell (e.g., secretory gland cell), and methods for using the compositions as stabilizers to prevent nucleic acid degradation in vivo and in vitro. [20] Suφrisingly, the addition of certain polyionic organic acids (PODS) to nucleic acid (e.g., plasmid DNA) solutions results in an increase in in vivo salivary gland transfection efficiency. Specifically, PODS, such as Evans blue and Congo red, and the like, can be used to enhance in vivo gene delivery to the salivary glands resulting in high levels of secretion of exogenous protein (SEAP) into blood. In addition, mixtures of the PODS, such as aurintricarboxylic acid (ATA) and zinc chloride, can also be used to enhance in vivo salivary gland transfection efficiency.

[21] As such, in one embodiment, the present invention provides a nucleic acid transfection composition comprising a polyionic organic acid and a nucleic acid. In certain aspects, the composition also comprises a cationic lipid, a cationic polymer or cationic peptide. Preferably, the polyionic organic acid is a dye. In certain aspects, the dyes include, but are not limited to, Evans Blue, Congo Red, ponceau S, Congo corinth, Sirius red F3B, ponceau 6R, amido black 10B, biebrich scarlet and aurintricarboxylic acid. Preferably, the dye absorbs in the visible light spectrum. In certain other aspects, the polyionic organic acids of the present invention are not limited to dyes. For example, Suramin is a colorless dye and is suitable for the present invention. Suitable nucleic acid includes, but is not limited to, DNA, RNA, DNA/RNA hybrids, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, a plasmid DNA, nucleic acids encoding proteins, therapeutic proteins, antibodies, peptides, cyclic peptides, RNAi, antisense nucleic acids, and ribozymes. [22] In another embodiment, the present invention provides a method for administering a nucleic acid to a cell, such as a secretory gland cell. The method comprises contacting a cell (e.g., a secretory gland cell) with a nucleic acid transfection composition, the nucleic acid transfection composition comprising a polyionic organic acid and nucleic acid, thereby administering the nucleic acid to the cell. In a preferred embodiment, the cell is a secretory gland cell, such as a salivary gland cell, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, or a liver cell, hi a more preferred embodiment, the secretory gland cell is a salivary gland cell.

[23] In still yet another embodiment, the present invention provides a method for increasing nucleic acid transfection efficiency comprising contacting a cell with a nucleic acid transfection composition, the nucleic acid transfection composition comprising an polyionic organic acid and nucleic acid, thereby increasing the nucleic acid transfection efficiency.

[24] In a further embodiment, the present invention provides a method for stabilizing a nucleic acid, the method comprising: contacting the nucleic acid with a composition comprising a polyionic organic acid, thereby stabilizing the nucleic acid. In a preferred embodiment, the composition increases the half-life of a nucleic acid (in vivo and/or in vitro) by inhibiting DNase activity, achieving higher concentrations of plasmid DNA, enhanced transfection efficiency, increased levels of protein expression via plasmid DNA accumulation, and combinations thereof. In another preferred embodiment, the composition increases the in vitro shelf life of a nucleic acid by inhibiting DNase activity. [25] In yet a further embodiment, the present invention provides a method for neutralizing a viras, the method comprising: administering a polyionic organic acid optionally in combination with a nucleic acid to an animal infected with a virus, thereby neutralizing the virus. As used herein, "in combination" means prior to, concomitantly with, and/or subsequently to the administration of the polyionic organic acid. In a preferred embodiment, the method also comprises administering an ionizable or ionized transition metal enhancer in combination with a polyionic organic acid and optionally with a nucleic acid to neutralize the virus. Suitable ionizable or ionized transition metal enhancers include, but are not limited to, ZnCl₂, NiCl₂, CoCl₂, CuCl₂, A1C1₂, and GaCl₂. In another preferred embodiment, the method comprises administering to an animal infected with a viras a polyionic organic acid, thereby neutralizing the viras.

[26] In still yet another preferred embodiment, the present invention provides a method for determining whether a polyionic organic acid directly neutralizes a virus, the method comprising: (i) administering a polyionic organic acid and a nucleic acid to an animal; (ii) isolating plasma from the animal; (iii) removing the antibodies from the plasma; (iv) testing the ability of the antibody-depleted plasma to neutralize the viras; and (v) determining the IC₅o of the polyionic organic acid to eliminate the viras. [27] Viruses that can be neutralized according to these methods include, but are not limited to, HIV, Epstein Barr viras, heφes simplex viras, hepatitis A, hepatitis B, hepatitis C, hepatitis E, mumps, measles, polio, and chicken pox. Preferably, the viras to be neutralized is HIV. In certain aspects, the polyionic organic acid is a dye. Preferably, the dye is Congo Red or aurintricarboxylic acid. Suitable nucleic acids include, but are not limited to, DNA and plasmid DNA encoding a viral protein. Preferably, the nucleic acid encodes a viral envelope protein.

[28] Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description and figures which follows. BRIEF DESCRIPTION OF THE DRAWINGS [29] Figure 1 shows several preferred embodiments of a polyionic organic acid of the present invention.

[30] Figure 2 shows the effect of the polyionic organic acid Evans Blue on salivary gland transfection efficiency.

[31] Figure 3 shows the effect of the concentration of the polyionic organic acid Evans Blue on salivary gland transfection efficiency.

[32] Figure 4 shows the effect of the polyionic organic acid Evans Blue in combination with calcium on the levels of observed human growth hormone protein in plasma.

[33] Figure 5 compares the concentration effect of the polyionic organic acids Evans Blue and Congo Red on salivary gland transfection efficiency.

[34] Figure 6 illustrates the accumulation of plasmid DNA in the submandibular gland for a polyionic organic acid such as Evans Blue and Congo Red, as well as a metal such as zinc.

[35] Figure 7 illustrates an effect of a composition of the present invention on DNase type I.

[36] Figure 8 illustrates an effect of a composition of the present invention on DNase type II. [37] Figure 9 shows the effect of the polyionic organic acid ATA in combination with the metal zinc on observed transgene expression after non- viral salivary gland transfection.

[38] Figure 10 shows the effect of plasmid DNA dose on salivary gland transfections containing both the polyionic organic acid ATA and the metal zinc. [39] Figure 11 shows the effect of the concentration of the polyionic organic acid

ATA in combination with the metal zinc on salivary gland transfection efficiency.

[40] Figure 12 illustrates the accumulation of plasmid DNA in the submandibular gland for the polyionic organic acid ATA in combination with the metal zinc.

[41] Figure 13 illustrates an inhibitory effect of the polyionic organic acid ATA in combination with the metal zinc on DNase types I and II.

[42] Figure 14 shows the effect of the polyionic organic acid ATA in combination with the metal zinc on salivary gland histology after non-viral transfection.

[43] Figure 15 shows a comparison of SEAP plasma concentrations after congo red- and suramin/zinc-mediated transfection in the rat submandibular gland. [44] Figure 16 shows SEAP protein in rat salivary gland tissues observed after suramin/zinc-mediated transfection.

[45] Figure 17 shows sample data demonstrating the effects of direct Congo Red viral neutralization. [46] Figure 18 shows a time course comparing anti-gp 120 plasma IgG titers from retroductal introduction of formulations with DNA encoding gpl20 with or without a polyionic organic acid into the salivary gland of rats.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[47] As used herein, the following terms have the meanings ascribed to them below unless otherwise specified.

[48] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleotides may be referred to by their commonly accepted single-letter codes. These are A, adenine; C, cytosine; G, guanine; and T, fhymine (DNA), or U, uracil (RNA). [49] The term "codon" refers to a sequence of nucleotide bases that specifies an amino acid or represents a signal to initiate or stop a function. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605 (1985); Rossolini et al, Mol. Cell. Probes 8:91 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. [50] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers, as well as, amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. [51] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified through post translational modification, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refers to compounds that have the same fundamental chemical stracture as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical stracture as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a stracture that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by. the IUPAC-IUB Biochemical Nomenclature Commission. [52] The term "recombinant" when used with reference, e.g. , to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[53] The terms "promoter" and "expression control sequence" are used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. In some embodiments, the nucleic acid is linked to a tissue specific expression control sequence. In other embodiments, the tissue is intestinal epithelium, liver, lung, pancreas, breast, brain, or muscle. In one embodiment, the tissue is intestinal epithelium. [54] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g. , a fusion protein).

[55] An "expression vector" is a nucleic acid constract, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, viras, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

[56] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985)). Techniques for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).

[57] A "salivary gland" is a gland of the oral cavity which secretes saliva, including the glandulae salivariae majores of the oral cavity (the parotid, sublingual, and submandibular glands) and the glandulae salivariae minores of the tongue, lips, cheeks, and palate (labial, buccal, molar, palatine, lingual, and anterior lingual glands). [58] Recently, salivary glands have been the target of gene transfer experiments aimed at developing clinical applications to treat salivary gland disorders or diseases involving systemic protein deficiencies (see, U.S. Provisional Patent No. 60/458,793, filed 03/26/2003, U.S. Patent No. 6,255,289, U.S. Patent No. 6,004,944, U.S. Patent No. 5,885,971, U.S. Patent No. 5,827,693, Baccaglini et al, J. Gene. Med. 3:82 (2001), Baum et al, Int. J. Oral

Maxillofac. Surg. 29:163 (2000), Baum and O'Connell, Crit. Rev. Oral Biol. Med. 10(3):276 (1999), He et al, Gene Therapy 5:537 (1998), and Masfrangeli et al, Am. J. Physiol. 266:G1146 (1994)). Salivary glands are a good target for in vivo gene transfer because of their exocrine gland characteristics. The main secretory duct can conveniently be used to easily access the major salivary glands. The majority of salivary parenchymal cells can be transfected this way, thus these glands are capable of producing and secreting therapeutic proteins by both exocrine and endocrine secretory pathways. Moreover, expression of therapeutic proteins can be regulated physiologically in response to meal, chewing or different neural and hormonal stimulation. (See, e.g., U.S. Patent No. 5,837,693). Expressed therapeutic proteins may be secreted into the saliva or into the bloodstream. (See, id.). [59] A nucleic acid may be administered to the salivary gland with or without a "formulant," i.e., a substance that enhances transfection efficiency. Suitable formulants include, for example, divalent transition metals. "Divalent transitions metal compounds" refer to compounds comprising a divalent transition metal, such as, for example, zinc, copper, cobalt, or nickel.

[60] A nucleic acid administered to the salivary gland may be encapsulated in, for example, a liposome (or other cationic, anionic, or neutral polymer) formulation. [61] A "therapeutic protein" or "therapeutic nucleic acid" is any protein or nucleic acid that provides a therapeutic or prophylactic effect. A therapeutic protein may be naturally occurring or produced by recombinant means. A "therapeutically effective amount" of a nucleic acid or protein is an amount of nucleic acid or protein sufficient to provide a therapeutic or prophylactic effect in a subject. Such therapeutic or prophylactic effects may be local or systemic. Therapeutic and prophylactic effects include, for example, eliciting or modulating an immune response. Selby et al. (2000) J. Biotechnol 83(1-2): 147-52. In some embodiments, the therapeutic protein is expressed in an intestinal epithelial cell.

[62] An "immunogenic peptide or protein" is one that elicits or modulates an immune response. Preferably, the peptide or protein induces or enhances an immune response in response to a particular antigen. Immune responses include humoral immune responses and cell-mediated immune responses. An immunogenic peptide or protein can be used therapeutically or prophylactically to treat or prevent disease at any stage. [63] "Retroductally introducing" refers to introduction of a composition through a duct in a salivary gland, wherein the composition flows through the salivary gland duct in a retrograde manner. Suitable ducts include all major and minor salivary gland ducts. For example, the Wharton's duct or the Stenson's duct is suitable.

[64] "Electroporation" involves contacting cells, tissues, glands, or organs with electrodes and "pulsing" the cells, tissues, glands, or organs, i.e., passing an electric signal through the tissues, glands, or organs via the electrode. One preferred embodiment of the present invention comprises contacting a salivary gland with an electrode and "pulsing" the salivary gland. After contacting and pulsing the salivary gland, electrodes may be "repositioned" to come into contact with the same or different position on the salivary gland. After repositioning of the electrode, the salivary gland may be pulsed again. "Electrodes" that can be used to contact the cells, tissues, glands, or organs, include needles, laparoscopic needles, probes, needles with paddles, and needles with flat plates or calipers. Electrodes may comprise individual needles, laparoscopic needles, probes, needles with paddles, and flat plates or may comprise an array of multiple needles, e.g. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 needles, laparoscopic needles, probes, needles with paddles, and needles with flat plates or calipers. "Contacting" includes, placing the electrodes at or near the cells, tissues, glands, or organs; touching the cells, tissues, glands, or organs with the electrodes, or penetrating the tissues, glands or organs with the electrodes.

[65] The term "cationic lipid" refers to any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-N,N- dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride ("DOTAP"); 3 -(N-rN'.N'-dimethylaminoethane)- carbamoyl)cholesterol ("DC-Choi") and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide ("DMRIE"). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTD ^® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-SH-3-phosphoethanolamine ("DOPE"), from GIBCO/BRL, Grand Island, New York, USA); LfPOFECTAMINE^® (commercially available cationic liposomes comprising N-(l-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)efhyl)-N,N- dimethylammonium trifluoroacetate ("DOSPA") and("DOPE"), from GIBCO/BRL); and TRANSFECTAM^® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine ("DOGS") in ethanol from Promega Coφ., Madison, Wisconsin, USA). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA and the like.

[66] A "polyionic organic acid' (POD) as used herein, is preferably a polyprotic polyaromatic organic compound wherein the compound contains at least two aromatic components. "Polyionic" compounds refer to compounds comprising one or more ionizable units, either as in the protonated form or as the conjugate salt. In certain embodiments, the POD has associated therewith, such as complexed with, a transition metal enhancer of the type described below. In certain other embodiments, the POD acts directly as an antiviral compound.

[67] The term "in vivo" refers to being within a living organism such as a plant or animal, and includes, but is not limited to, cells, tissues, glands, organs, and the like, in the living organism. The term "in vitro" refers to an artificial environment outside a living organism, and includes, but is not limited to, cells, tissues, glands, organs, and the like, outside of the living organism.

[68] The term "nuclease" refers to an enzyme which cuts, digests, or degrades DNA, RNA, plasmid DNA, or any other nucleic acid as described herein. The terms "DNase" and "deoxyribonuclease" are used interchangeably herein to refer to an enzyme which cuts, digests, or degrades DNA or plasmid DNA in either single- or double-stranded form by cleaving phosphodiester linkages. The terms encompass DNases that act as non-specific endonucleases, restriction endonucleases, or exonucleases. Preferably, the DNase is a nonspecific endonuclease including, but not limited to, DNase I and DNase II. In certain embodiments, the PODS as described herein act to inhibit DNases, preferably DNase I and II. In further embodiments, the PODS as described herein act together with a transition metal enhancer to inhibit DNases, preferably DNase I and II. In yet further embodiments, the PODS as described herein act with or without a transition metal enhancer together with a known DNase inhibitor of the type described below to inhibit DNases, preferably DNase I and II. In particularly preferred embodiments, the various combinations of PODS, transition metal enhancers, and known DNase inhibitors are effective at inhibiting DNase activities in vitro and in vivo.

[69] The term "half-life" refers to the time required for half the quantity of a compound deposited in a living organism or a cell outside a living organism to be metabolized, eliminated, or degraded by normal biological processes. The term "shelf life" refers to the length of time a compound may be stored without becoming unsuitable for use or consumption. In certain aspects, both the half-life and shelf life of a compound, preferably a nucleic acid, is a measure of its stability. In preferred embodiments, the present invention provides compositions and methods for increasing the half-life of nucleic acids in vivo and in vitro, and for increasing the shelf life of nucleic acids in vitro.

[70] The term "neutralize" refers to blocking the ability of a virus to bind to a target cell, or inactivating, inhibiting, making ineffective, or counteracting the activity or effect of a viras. Viruses that can be neutralized include, but are not limited to, HIV, Epstein Barr viras, heφes simplex viras, hepatitis A, hepatitis B, hepatitis C, hepatitis E, mumps, measles, polio, and chicken pox. Preferably, the virus to be neutralized is HIV.

[71] The term "IC₅₀" refers to the concentration of a compound required to reduce viral infectivity by 50% The compound includes, but is not limited to, a polyionic organic acid, an antibody, a peptide, a protein, or a nucleic acid. Preferably, the IC₅₀ refers to the concentration of a polyionic organic acid required to neutralize 50% of a viras. The IC₅₀ is also referred to as the median inhibitory concentration. The ICso can be a measure of the binding affinity of a compound to a target. The target includes, but is not limited to, a viras, an antigen, a cell, a peptide, a protein, or a nucleic acid. In a preferred aspect, the IC₅₀ is a measure of the binding affinity of a polyionic organic acid to a viras. For example, a low IC₅₀ for a polyionic organic acid could correspond to a strong binding affinity, or interaction, of the polyionic organic acid to the virus.

[72] The term "affinity" refers to the strength of non-covalent chemical binding between a compound and a target and can be measured by the association constant (K_A) or dissociation constant (K_D) of the complex. The compound includes, but is not limited to, a polyionic organic acid, an antibody, a peptide, a protein, or a nucleic acid. The target includes, but is not limited to, a viras, an antigen, a cell, a peptide, a protein, or a nucleic acid. In certain aspects, the affinity is the strength of binding between an antibody and an antigen, such as a virus. In other aspects, the affinity is the strength of binding between a polyionic organic acid and a viras. An "increase in affinity" refers to a higher K_A or lower K_D between the compound and the target as compared to control samples. In particular, the increase in affinity corresponds to a K_A that is preferably 1 to 2-fold, or more preferably at least 2-fold higher than that observed for control samples. The increase in affinity also corresponds to a K_D that is preferably 1- to 2-fold, or more preferably at least 2-fold lower than that observed for control samples. For example, the increase in affinity (i.e., higher K_A or lower K_D) can be determined for an antibody and a viras with or without (control sample) the addition of a polyionic organic acid.

II. General

[73] Prior to the advent of the present invention, commonly used chemical adjuvants that enhance in vivo gene transfer efficiency included cationic liposomes, cationic polymers, divalent cationic metals, and cationic polypeptides.

[74] The present invention provides compositions to enhance in vivo secretory gland transfection efficiency. Efficient transfection of, for example, the human salivary gland tissues can be used to treat a variety of diseases including: diabetes, hemophilia, cancer, ionizing radiation, an autoimmune disorder, Sjδgren's syndrome, graft- versus-host-disease, systemic lupus erythematosis, rheumatoid arthritis, H1N-1 infection, ageing, treatment with medications/drags, autonomic dysfunction, conditions affecting the CΝS, psychogenic disorder, trauma, or decrease in mastication and the like. The disclosed compositions are effective at enhancing transfection efficiency in a variety of other target tissues and cells in vivo, in vitro and ex vivo.

III. Compositions

[75] The present invention provides compositions useful for gene delivery. The present compositions comprise a polyionic organic acid and a nucleic acid. In vivo salivary gland transfection experiments comparing the use of the present compositions, such as Evans Blue, Congo Red, and aurintricarboxylic acid/zinc chloride complexes, with "state of the art" non- viral gene delivery methods reveal that the compositions of the present invention containing DΝA solutions are at least 1000-fold more efficacious. As such, the compositions of the present invention have widespread applicability in the fields of cell biology and gene therapy. These applications may also include regulating enzymes that require the interaction between proteins and nucleic acids. One example involves the inhibition of certain DΝA binding enzymes, such as deoxyribonucleases, ribonucleases, isomerases, integrases, polymerases, transcriptases. More specifically, this may include the inhibition of DΝase I and II. A. Polyionic Organic Acids

[76] In one embodiment, the polyionic organic acid can be a polyprotic polyaromatic organic compound wherein the compound is multivalent with at least two aromatic components. As used herein, the term "multivalent" means having the ability to adopt multiple valence configurations. Figure 1 and Table I set forth polyionic organic acids that increase in vivo gene transfer efficacy in the rat submandibular gland. In the case of aurintricarboxylic acid, a divalent metal chloride (zinc chloride) is added to obtain an optimal effect. [77] One example of a polyionic organic acid is a dye. As used herein, a dye is a compound that absorbs radiation in the ultraviolet, visible and/or infrared regions of the electromagnetic spectrum. These regions of the electromagnetic spectrum correspond to radiation having wavelengths of 10^"9 to 4xl0^"7, 4 - 7xl0^'7 and 7xl0^"7 to 10^"4 meters, respectively. Dyes which are useful in the present invention include, but are not limited to, an acid dye, a disperse dye, a direct dye and a reactive dye. In a preferred embodiment, an acid dye is used. Suitable acid dyes include, but are not limited to, direct red dye, direct blue dye, acid black dye, an acid blue dye, an acid orange dye, an acid red dye, an acid violet dye, and an acid yellow dye. In certain other preferred embodiments, suitable acid dyes include, but are not limited to, Evans Blue, Congo Red, ponceau S, Congo corinth, Sirius red F3B, ponceau 6R, amido black 10B, biebrich scarlet and aurintricarboxylic acid. In yet another preferred embodiment, a direct dye is used. Prefened direct dyes include direct red, direct blue, direct yellow and direct green. More preferably, direct blue 15 (Light Blue), direct red 28 (Congo Red) and direct blue 53 (Evans Blue) are used. Preferably, the dye absorbs in the visible light spectrum, between about 400 nm to 700 nm. [78] The PODS of the present invention are not limited to dyes. For example, Suramin (Figure 1) is a colorless dye and is suitable for the present invention.

[79] In another preferred embodiment, the polyionic organic acid and nucleic acid composition is an aqueous solution having a physiological acceptable pH. [80] In a preferred embodiment, the polyionic organic acid is comprised of a complex formed between ATA and/or suramin and a transitional metal enhancer, such as a divalent metal halide such as zinc chloride. Suφrisingly, the addition of a divalent metal to a formulation with ATA and DNA results in a synergistic increase in gene expression (see Figure 9). [81] In certain aspects, the in vitro concentration of PODS used herein are 10 to about 0.0001 mg/mL. Preferably, the concentration of PODS used herein is 1 to about 0.001 mg/mL. More preferably, the concentration of PODS used herein is 0.1 to about 0.01 mg/mL.

[82] In certain aspects, it is possible to determine whether a candidate compound is a polyionic organic acid. For example, an assay can be performed wherein a candidate polyionic organic acid is tested using routine procedures for its ability to disrupt, inhibit, and/or prevent the interaction between a protein and a nucleic acid such that they are no longer bound or complexed to one another. The assay can be performed in vitro or in vivo. In certain instances, the protein is an enzyme that binds nucleic acid. In preferred instances, the enzyme is a nuclease or reverse transcriptase. The effect of the polyionic organic acid on a protein/nucleic acid complex is measured using standard assay protocols, including, but not limited to, light scattering, circular dichroism techniques, DNA/protein crosslinking, DNA footprinting, electromobility shift, and competitive DNA/nuclease binding assays. Those of skill in the art will know of other techniques useful for determining whether a candidate compound is a polyionic organic acid.

[83] Suitable polyionic organic acids for use in the present invention are listed in Figure 1 and Table I.

Table I

4-hydroxy-5-[[4- phenylamino]5-sulfo-l- naphthalenyl]azo]-2,7- naphthalenedisulfonic acid trisodium salt

8-(phenylamino)-5-[[4-(3- sulphonatophenyl)azo]-l - naphthalenyl)azo]-l- naphthalenesulfonic acid disodium salt

4-[(2-hydroxy-l- naphthalenyl)-azo]- benzenesulfonic acid monosodium salt

4-[4,5-dihydro-3-oxo-4)- (4-methyl-3-phenylamino) sulfonyl(phenyl)-azo)-(₅- ethyl- 1 H-pyrazol- 1 -yl)- benzenesulfonic acid monosodium salt

4-hydroxy-3 -(phenylazo)-

₅-(methoxymethylamin)-

2,7-naphthalenedisulfonic acid disodium salt

4-hydroxy-3,4'-azo-di-l- naphthalene-disulfonic acid disodium salt

4-[(2,4-dimethylphenyl)- azo]-3-hydroxy-2,7- naphthalene-disulfonic acid disodium salt

7-hydroxy-8-[[4-

(phenylazo))-phenyl]-azo]-

1 ,3-naphthalene-disulfonic acid disodium salt

8-((3,3'-dimethyl-4'-((4-

(((4-methylphenyl)- sulfonyl)-oxy)-phenyl)- azo)(l ,1 '-biphenyl)-4-yl)- azo)-7-hydroxy-l ,3- naphthalene-disulfonic

acid disodium salt

4-amino-3-[[4'-](2,4- diaminophenyl)-azo]-

[1,1 'biphenyl]-4-yl-[azo]-

₅-hydroxy-6-(phenylazo)-

2,7-naphthalene-disulfonic acid disodium salt

3,3'-[(3,3'-dimethoxy-4,4'- biphenylene)-bis-(azo)]- bis-[₅-amino-4-hydroxy-

6,8-naphthalene-disulfonic acid] tetrasodiu salt

3,3'-((4,4'-biphenylylene)- bis(azo))-bis-(5-amino-4- hydroxy-2,7-naphthalene- disulfonic acid) tetrasodium salt

3,3-[3,3-dimethyl-[[l,l- biphenyl] -4,4-diyl)-bis-

(azo)]-bis-[5-amino-4- hydroxy-2,7-naphthalene- disulfonic acid] tetrasodium salt

3,3-[3,3-dimethoxy[[l,l- biphenyl]-4,4-diyl]-bis- azo]-bis (5-amino-4- hydroxy-2,7- naphthalenedisulfonic acid) tetrasodium salt

Direct Blue 78

l,l-((l,l-biphenyl)-4,4- diyl-bis(azo))-(4-hydroxy- 3-carboxylate)-(2-hydroxy- 3-azo)-2-hydroxy-4- benzenesulfonic acid disodium salt

Direct Red 79

Red

-

(2- phenyl

- acid

acid

salt

,6-

acid

1 - H-

3H-

83 Hoechst 33342

84 Direct Violet ₅1

85 Direct Red 2₅

86 Direct Red 23

87 N,N'-(phenyl)-bis-(4-

(phenyl)azo)-3-hydroxy-2- naphthalenecarboxamide

88 4-[(2,5-dichlorophenyl)- azo]-N-(2,3-dihydro-2- oxo-1 H-benzimidazol-5- yl)-3-hydroxy-2- naphthalenecarboxamide

89 1- (2,4-dinitrophenyl)-azo - 2-naphthalenol,

90 4,4*- (3,3'-dichloro l,r- biphenyl -4,4'-diyl)bis(azo) bis 2,4-dihydro-5-methyl- 2-phenyl-3H-pyrazol-3-one

91 N,N'-(3,3-dichloro-l,l'- biphenyl-4,4'-diyl)-bis(4-

(2-chlorophenyl)azo)-3- hydroxy-2- naphthalenecarboxamide

92 4,4^,- (3,3'-dichloro l,l'- biphenyl -4,4'-diyl)bis(azo) bis 2,4-dihydro-5-methyl- 2-(4-methylphenyl)- 3H- pyrazol-3-one

93 2-((4-chloro-2- nitrophenyl)azo)-N-(2,3- dihydro-2-oxo- 1 H- benzimidazol-5-yl)-3-oxo- butana ide

94 N-(4-(acetylamino)phenyl

-4-((5-(aminocarbonyl)-2- chlorophenyl)azo)-3- hydroxy-2- naphthalenecarboxamide

95 1 -((4-nitrophenyl)azo)-2- naphthalenol

96 l-((4-methyl-2- nitrophenyl)azo)-2- naphthalenol

97 N-(5-chloro-2,4- dimethoxyphenyl)-4-[[5- [(diethylamino)sulfonyl] - 2-methoxyphenyl] azo] -3- hydroxy-2- naphthalenecarboxamide

98 N-(4-chlorophenyl)-3- hydroxy-4-((2-methyl-5- nitrophenyl)azo)-2- naphthalenecarboxamide

99 4-((2,5-dichlorophenyl)- azo) -3-hydroxy-N-(2- methoxyphenyl)- 2- naphthalenecarboxamide

100 3-hydroxy-4-[(2-methyl-4- nitrophenyl)azo]-N-(2- methylphenyl)-2- naphthalenecarboxamide

101 3-hydroxy-4-[(2-methyl-5- nitrophenyl)azo]-N- phenyl-2- naphthalenecarboxamide

102 4-[(₅-chloro-4-methyl-2- sulfophenyl)azo]-3- hydroxy-2-naphthalene carboxylic acid

103 2-[(2-hydroxy-l- naphthalenyl)azo]-l- naphthalenesulfonic acid, monosodium salt

104 4-[(4-chloro-5-methyl-2- sulfophenyl)azo]-3- hydroxy-2- naphthalenecarboxylic acid, disodium salt

105 5-chloro-2-[(2-hydroxy-l - naphthalenyl)azo]-4- methyl-benzenesulfonic acid, monosodium salt

106 3-hydroxy-4-[(4-methyl-2- sulfophenyl)azo]-2- naphthalenecarboxylic acid, disodium salt

B. Nucleic Acids

[84] Suitable nucleic acids useful in the present invention include, for example, DNA, RNA, a DNA/RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, a plasmid DNA, viral vector DNA, nucleic acids encoding proteins, therapeutic proteins, antibodies, peptides, cyclic peptides, RNAi, antisense nucleic acids, and ribozymes. [85] In a typical embodiment, nucleic acids that can be used in the present invention are those encoding therapeutic proteins that may be useful for treating or preventing a disease or disorder in a subject. Nucleic acids administered according to the compositions and methods of the present invention may encode proteins that have local or systemic effects. Proteins encoded by nucleic acids administered according to the methods of the present invention can be used, for example, to treat or prevent any disorder amenable to treatment or prevention by expression of a therapeutic protein into the blood stream, by secretion of a therapeutic protein to the gastrointestinal tract (e.g. by secretion of the protein into the saliva), or by expression of the therapeutic protein by the transfected cell, tissue, gland, or organ. The subject can be a mammal such as, for example, a mouse, a rat, a guinea pig, a cat, a dog, a sheep, a goat, a cow, a horse, a non-human primate, or a human; or a non-mammal, such as, for example, a frog, a toad, a lizard, a snake, a turtle, a tortoise, or a salamander. [86] Suitable therapeutic proteins encoded by nucleic acids according to the compositions of the present invention include, for example, growth hormones, clotting factors such as Factor XIII, Factor IX, Factor X, and the like, lysosomal enzymes, plasma proteins, plasma protease inhibitors, proteases, protease inhibitors, hormones, pituitary hormones, growth factors, somatomedins, gonadofrophins, apolipoproteins, insulinotrophic hormones, immunoglobulins, chemotactins, chemokines, interleukins, interferons, cytokines, fusion proteins, and immunogenic peptides or proteins.

[87] Nucleic acids encoding suitable immunogenic peptides or proteins include, for example, antigens such as cancer antigens, bacterial antigens, viral antigens, fungal antigens, parasitic antigens, and antigens overexpressed on neoplastic cells. Cancer antigens include, for example, antigens expressed, for example, in colon cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, prostate cancer, breast cancer, skin cancer (e.g., melanoma), leukemia, lymphoma, or myeloma. Exemplary cancer antigens include, for example, HPV LI, HPV L2, HPV El, HPV E2, PSA, placental alkaline phosphatase, AFP, BRCA1, Her2/neu, CA 15-3, CA 19-9, CA-125, CEA, hCG, urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor and MAGE-1. Bacterial antigens may be derived from, for example, Staphylococcus aureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetella pertussis, Yersiniapestis, Neisseria gonorrhoeae, Treponema pallidum, Mycoplasm sp., Neisseria meningitidis, Legionella pneumophila, Rickettsia typhi, Chlamydia trachomatis, and Shigella dysenteriae. In certain aspects, the bacterial antigen is, for example, anthrax protective antigen. Viral antigens may be derived from, for example, human immunodeficiency viras, human papilloma virus, Epstein Barr virus, heφes simplex viras, human heφes viras, rhinoviruses, cocksackievirases, enterovirases, hepatitis A, hepatitis B, hepatitis C, and hepatitis E, rotavirases, mumps viras, rubella virus, measles viras, polioviras, smallpox viras, influenza viras, rabies viras, and Variella-zoster viras. In certain aspects, the viral antigen is, for example, HIV envelope protein or a portion thereof (e.g. , gp 160 or a portion thereof, gp 120 or a portion thereof, or gp41 or a portion thereof). Fungal antigens maybe derived from, for example, Tinea pedis, Tinea corporus, Tinea cruris, Tinea unguium, Cladosporium carionii, Coccidioides immitis, Candida sp., Aspergillus fumigatus, and Pneumocystis carinii. Parasitic antigens may be derived from, for example, Giardia lamblia, Leishmania sp., Trypanosoma sp., Trichomonas sp., Plasmodium sp., and Schistosoma sp.

[88] Exemplary proteins encoded by nucleic acids suitable for use according to the compositions of the present invention include, for example, insulin, insulintropin, glucagon, glucagon-like peptide (GLP), human growth hormone (hGH), bovine growth hormone (bGH), factor VIII and factor IX, erythropoietin (EPO), antithrombin III, thrombopoietin (TPO), calcitonin, α-galactosidase, α-glucosidase, glucocerebrosidase, /3-glucuronidase, parathyroid like hormone (PTH), fibroblast growth factor (FGF), insulin-like growth factor (IGF), neurite growth factor (NGF), epidermal growth factor (EGF), transforming growth factor (TGF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon, γ-interferon, /5-interferon, IL-1, IL-1 RA, IL-2, IL-4, IL-5, IL-10, IL-12, phenylalanine ammonia lyase, arginase, L-asparaginase, uricase, platelet derived growth factor (PDGF), brain derived neurite factor (BDNF), purine nucleotide phosphorylase, tumor necrosis factor (TNF), lipid-binding proteins (lbp), α-1- antitrypsin, apolipoprotein B-48, apolipoprotein Al₂, tissue plasminogen activator (fPA), urokinase, streptokinase, superoxide dismutase (SOD), catalase, adenosine deamidase, cholecystokinin (cck), ob gene product, vasoactive intestinal peptide (VD?), gastric inhibitory peptide (GIP), somatostatin, pepsin, trypsin, chymotrypsin, elastase, carboxypeptidase, lactase, sucrase, intrinsic factor, and zsig63. The zsig63 polypeptide is described in U.S. Patent Application No. 2002/0173027A1, published November 21, 2002, and incoφorated herein by reference.

[89] In certain embodiments, the protein is selected from the group of gamma glutamyl transpeptidase, manganese superoxide dismutase, metallothionein, glutathione peroxidase (GPx)-4, catalase, IFN-alpha, IL-10, sTNFR, TGF-/3, IL-4, VIP, anti-TNF antibody, ILl-RA, other antibodies to proinflammatory cytokines, soluble gp39, soluble CD40, aquaporin-1, aquaporin-5, and zsig63.

[90] In certain aspects, the gene therapy methods of the present invention involve an in vivo method that provides a polynucleotide encoding a protein capable of neutralizing or eliminating a toxic free radical, superoxide anion and/or heavy metal cation, wherein the protein is transiently expressed in the individual. The transgenes of the present invention encode protein(s), such as metallothionein, superoxide dismutase or gamma glutamyl transpeptidase, that scavenge a toxic free radical, superoxide anion and/or heavy metal cation. [91] γ-Glutamyltranspeptidase (γ-GTP) is a plasma membrane-associated ectoenzyme that catalyzes the transpeptidation of extracellular glutathione into amino acid intermediates, which are then transported across the cell membrane and used to resynthesize glutathione de novo. Glutathione (GSH) detoxifies free-radicals. Cells generally synthesize GSH de novo from the constituent amino acids. A cell's sensitivity to radiation is directly correlated with its ability to transpeptidate extracellular glutathione via γ -GTP. Cell lines with high γ.-GTP activity are more resistant to the effects of radiation and are more capable of repairing damage induced by low doses of γ-irradiation than cell lines with low γ -GTP activity. [92] Protection against superoxide radicals requires antioxidants, such as GSH, and the radical scavenging enzyme superoxide dismutase (SOD). There are various forms of SODs: copper-zinc (CuZnSOD), manganese (MnSOD), iron (FeSOD) and EC (SOD) see, for example, U.S. Patent Nos. 5,472,681, 5,788,961, 5,366,729, 5,248,603, and 5,130,245. Although CuZnSOD and FeSOD are made constituitively, MnSOD synthesis is inducible. Induction of MnSOD activity has been shown to follow X-irradiation of heart tissue. [93] The nucleic acid comprises a promoter to facilitate expression of the nucleic acid within a salivary gland cell, more preferably a parotid gland cell, even more preferably a submandibular salivary gland cell. Suitable promoters include strong, eukaryotic promoter such as, for example promoters from cytomegalovirus (CMV), mouse mammary tumor viras (MMTV), Rous sarcoma viras (RSV), and adenovirus. More specifically, suitable promoters include the promoter from the immediate early gene of human CMV (Boshart et al., Cell 41:521 (1985)) and the promoter from the long terminal repeat (LTR) of RSN (Gorman et al,

Proc. Natl. Acad. Sci. USA 19:6111 (1982)).

[94] Salivary gland specific promoters may also be used in accordance with the present invention and include, for example, salivary α-amylase promoters and mumps viral gene promoters which are specifically expressed in salivary gland cells. Multiple salivary α- amylase genes, have been identified and characterized in both mice and humans (see, for example, Jones et al, Nucleic Acids Res., 17(16):6613 (1989); Pittet et al, J. Mol. Biol.

182:359 (1985); Hagenbuchle et al, J. Mol. Biol, 185:285 (1985); Schibler et al, Oxf. Surv.

Eukaiyot. Genes 3:210 (1986); and Sierra et al, Mol. Cell. Biol., 6:4067-(1986) for murine salivary α-amylase genes and promoters; Samuelson et al, Nucleic Acids Res., 16:8261

(1988); Groot et al, Genomics, 5:29 (1989); and Tomita et al, Gene, 76:11 (1989) for human salivary α-amylase genes and their promoters). The promoters of these α-amylase genes direct salivary gland specific expression of their corresponding α-amylase encoding DΝAs.

These promoters may thus be used in the constracts of the invention to achieve salivary gland-specific expression of a nucleic acid of interest. Sequences which enhance salivary gland specific expression are also well known in the art (see, for example, Robins et al,

Genetica 86:191 (1992)).

C. Transition Metal Enhancers

[95] In certain other embodiments, the compositions of the present invention further comprise an ionizable or ionized fransition metal enhancer, including, but not limited to, a complex, an adduct, a cluster or a salt of an element including, but not limited to, a d-block element, a first row f-block element, aluminum and gallium. In a preferred embodiment, the ionizable or ionized transition metal enhancer is a complex, an adduct, a cluster or a salt of an element including, but not limited to, zinc, nickel, cobalt, copper, aluminum and gallium. More preferably, the ionizable or ionized transition metal enhancer includes, but is not limited to, zinc sulfate, zinc acetate, nickel sulfate, nickel acetate, cobalt sulfate, cobalt acetate, copper sulfate and copper acetate. In another preferred embodiment, the ionizable or ionized transition metal enhancer includes, but is not limited to, zinc acetate or zinc sulfate. [96] In other embodiments, the ionizable or ionized transition metal enhancer is a metal halide including, but not limited to, zinc halide, nickel halide, cobalt halide, copper halide, aluminum halide and gallium halide. In certain preferred embodiments, the ionizable or ionized transition metal enhancer includes, but is not limited to, ZnCl , NiCl₂, CoCl₂, CuCl₂, A1C1₂ and GaCl₂.

D. DNase Inhibitors

[97] In certain aspects, the compositions of the present invention further include a DNase inhibitor, specifically, an inhibitor of endonuclease activity. In a preferred embodiment, such endonuclease inhibitors act together with one or more polyionic organic acids with or without a transition metal to enhance the transfection efficiency of a nucleic acid (e.g., plasmid DNA). In another preferred embodiment, such endonuclease inhibitors act together with one or more polyionic organic acids with or without a transition metal to stabilize DNA and prevent nucleic acid degradation in vitro and in vivo, thus increasing the half-life and shelf life of a nucleic acid.

[98] Endonuclease activity is thought to be a significant barrier to effective nucleic acid (e.g., plasmid DNA) delivery into cells in vitro and tissues in vivo. Both extracellular and intracellular endonucleases have the potential to degrade the administered nucleic acid (e.g. , plasmid DNA) prior to its entry into the nucleus. DNases have been well-characterized with respect to their roles in apoptosis, and they can be classified into three groups: 1) the Ca²⁺/Mg²⁺-dependent endonucleases; 2) the Mg²⁺-dependent endonucleases; and 3) the acid or cation-independent endonucleases. The first group includes DNase I, DNase gamma, and other DNases. The caspase-3-activated DNase, or CAD/DFF40, belongs to the Mg²⁺- dependent family of endonucleases. The third group includes DNase H, in addition to other endonucleases.

[99] Various DNase inhibitors known in the art can be used in combination with one or more polyionic organic acids with or without a transition metal to enhance the transfection efficiency of a nucleic acid (e.g. , plasmid DNA) and to promote nucleic acid stability.

Suitable endonuclease inhibitors include, but are not limited to, inhibitors of DNase I and II, and may act either directly or indirectly on the endonuclease to inhibit its activity. For example, the 12 amino acid ID2 peptides were shown to be potent inhibitors of DNase II (Sperinde et al., J. Gene Med. 3(2):101-108, 2001). DMI-2, a polyketide metabolite of Streptomyces sp. strain 560, also inhibits DNase II (Ross et al., Gene Ther. 5(9): 1244-1250, 1998). The metal zinc is particularly effective at inhibiting DNase I and other Ca²⁺/Mg²⁺- dependent endonucleases. Preferably, divalent cation chelators that act as endonuclease inhibitors, such as EDTA, EGTA, DTP A, and the like, can be included in the compositions of the present invention.

[100] Other DNase inhibitors include polymers such as polyvinylpyrrolidone (PVP), for example, Plasdone-C^®15, MW 10,000 and Plasdone-C^®30, MW 50,000, polyvinyl alcohol, polyethyleneimine, polyamidomine, polyethylene oxide, polyethylene glycol, and polyethylene glycol-polyethyleneimine-transferrin complexes that coat the DNA and protect it from DNases; DNA binding agents such as histones or intercalaters that protect the DNA from DNases; DNA nicking inhibitors such as carbohydrates, disaccharides, or higher molecular weight saccharides, including, but not limited to, fructose, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gluose, idose, galactose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, fractofuranose, ribofuranose, ribose, deoxyribose, mannitol, sialic acid, sucrose, lactose, maltose, cellobiose, trehalose, lactulose, starch, glycogen, cellulose, chondroitin, keratin, haparin, dermatan, haluronic acid; detergents such as anionic detergents, cationic detergents, or nonionic detergents, including, but not limited to, sodium dodecyl sulfate, ethylene oxide condensation products such as ethoxylated fatty acid esters of polyhydric alcohols, and polyoxyethylene sorbitan monolaurate (TWEEN 20^®); chaotropic salts such as urea, formaldehyde, guanidinium isothiocyanate, guanidinium hydrochloride, formamide, dimethylsulfoxide, ethylene glycol, and tetrafluoroacetate; and amino acids such as L-lysine.

E. Cationic Lipids

[101] In certain aspects, the compositions of the present invention further include a cationic lipid. Suitable cationic lipids, include, for example, DOTAP, DOTMA, DDAB, L-PE, and the like. Liposomes containing a cationic lipid, such as {N(l-2-3-dioleyloxy) propyl}-N,N,N- triethylammonium} (DOTMA), dimethyl dioctadecyl ammonium bromide (DDAB), or 1, 2- dioleoyloxy-3-(trimethylammonio) propane (DOTAP) or lysinylphosphatidylethanolamine (L-PE) and a second lipid, such as distearoylphosphatidylethanolamine (DOPE) or cholesterol (Choi). DOTMA synthesis is described in Feigner, et al, Proc. Nat. Acad. Sciences USA 84:7413 (1987). DOTAP synthesis is described in Stamatatos, et al, Biochemistry 27:3917 (1988). DOTMA:DOPE liposomes is commercially available from, for example, BRL. DOTAP :DOPE liposomes is commercially available from Boehringer Mannheim. Cholesterol and DDAB are commercially available from Sigma Coφoration. DOPE is commercially available from Avanti Polar Lipids. DDAB:DOPE is commercially available from Promega.

[102] Additionally, complexing the cationic lipid with a second lipid, primarily either cholesterol or DOPE can maximize transgene expression in vivo. For example, mixing cholesterol instead of DOPE with DOTAP, DOTMA, or DDAB may substantially increase transgene expression in vivo.

[103] Anionic and neutral liposomes are commercially available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using commercially available materials, such as, for example, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphoshatidylethanolamine (DOPE). Methods for making liposomes using these materials are well known in the art.

F. Mechanisms

[104] Without being bound to any particular theory, it is believed that the compositions and methods of the present invention employ a variety of mechanisms of action. These include, for example, DNase inhibition, cell membrane disruption, and dipole-dipole interactions which is related to the intra-molecular octanol- water partition distribution of the polyionic organic acid compounds. Other possibilities also exist.

[105] For example, the polyionic organic compounds may act as DNase inhibitors by directly or indirectly inhibiting DNase. Alternatively, the polyionic organic compounds may act by perturbing the cell membrane. This may occur as a result of the polyionic organic acid perturbing the packing of the lipid bilayer (e.g., intercalating into the lipid bilayer).

[106] Disruption of the lipid bilayer of the cell membrane may be a result, in part, of the hydrophobicity of the polyionic organic acid. The degree of hydrophobicity of the polyionic organic acid can be inferred from the Hansch-Leo estimate of the octanol- water partitioning coefficient (P) for an organic molecule. The Hansch-Leo partitioning coefficient is calculated by the following formula:

Log P = a_nf_n + Σb_mF_m where the f_n values are the fragmental constants for the different groups in the molecule, the a_n values are the number of any type of group in the molecule, the F_m values are factors for certain molecular features such as single bonds or double bonds, and the b_m values are the number of any such molecular feature. [107] Those of skill in the art will recognize that the Hansch-Leo approach to estimating partition constants, in which approach the Hansch-Leo fragmental constants are applied, does not yield precisely the empirical partition constant. See Hansch and Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, 1979; James, Solubility and Related Properties, Marcel Dekker, New York, 1986, pp. 320-325. However, the approach is precise enough to define the hydrophobicity features of the transfection delivery vehicles, specifically the polyionic organic acids of the present invention. [108] For example, active agents such as nucleic acid, which has a very negative log P, (P being the partition coefficient between octanol and water), are very large in size, and are hydrophilic, thereby associating closely with lipid membranes. These molecules diffuse very slowly into a cell, and can be administered prior to or substantially simultaneous with the electric pulse during elecfroporation (described in detail below). In addition, certain agents may require modification in order to allow more efficient entry into the cell. Elecfroporation facilitates entry of nucleic acids into a cell by creating pores in the cell membrane.

IV. Methods

[109] According to the present invention, the nucleic acid can be administered according to any means known in the art. Nucleic acids of the present invention can be administered to a subject prior to, simultaneous with, or subsequent to, for example, elecfroporation. Suitable methods of administration of the nucleic acid to the cells, tissues, glands, or organs include, for example, cannulation or injection of the nucleic acid into the cells, tissues, glands, or organs using a syringe, cannula, catheter, or shunt. The type of syringe used is not a critical part of the invention. One of skill in the art will appreciate that multiple types of syringes may be used to administer nucleic acids according to the present invention. Suitable types of syringes include, for example, an aspirating syringe, a removable needle syringe, a modified microliter syringe, a microliter syringe, a gastight syringe, a sample lock syringe, a threaded plunger syringe. [110] The present invention also relates to a process for administering a nucleic acid to a secretory gland cell, comprising contacting a cell with a nucleic acid transfection composition, the nucleic acid transfection composition comprising a polyionic organic acid and nucleic acid, thereby administering the nucleic acid to the secretory gland cell. Suitable secretory gland cells include, but are not limited to, a salivary gland cell, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, and a liver cell. It is particularly preferred that the secretory gland cell is a salivary gland cell. [Ill] The nucleic acid composition can be delivered to the secretory gland cell of the mammal by intraductal delivery or direct administration. A preferred method of delivery is by elecfroporation administration. Preferably, the process for administering a nucleic acid composition to a secretory gland cell includes an elecfroporation administration that comprises contacting the salivary gland with an electrode comprising at least two needles and pulsing the salivary gland. [112] According to the methods of the present invention, the nucleic acid composition can be retroductally introduced into the lumen of a salivary or lacrimal gland duct.

"Retroductally introduced" refers to introduction of a composition through a duct in a salivary gland, wherein the composition flows through the salivary gland duct in a retrograde manner. Suitable ducts include all major and minor salivary gland ducts. For example, the Wharton's duct or the Stenson's duct is suitable. The composition may be introduced alone or with an adjuvant. In some embodiments of the present invention, the adjuvant is administered at the same time as the composition. In other with embodiments of the present invention, the adjuvant is administered after the composition, e.g., 6, 12, 18, 24, 36, 48, 60, or 72 hours after administration of the composition. [113] Suitable methods of retroductal introduction of the composition to the salivary or lacrimal gland duct include, for example, cannulation or injection of the composition into the salivary gland duct using a syringe, cannula, catheter, or shunt. The type of syringe, cannula, catheter, or shunt used is not a critical part of the invention. One of skill in the art will appreciate that multiple types of syringes, cannulas, catheters, or shunts may be used to administer compositions according to the methods of the present invention. [114] Delivery of the nucleic acid, e.g. retroductal delivery, may be via gravity or an assisted delivery system. Suitable assisted delivery systems include controlled release pumps, time release pumps, osmotic pumps, and infusion pumps. The particular delivery system or device is not a critical aspect of the invention. One of skill in the art will appreciate that multiple types of assisted delivery systems may be used to deliver nucleic acids according to the methods of the present invention. Suitable delivery systems and devices are described in U.S. Patent Nos. 5,492,534, 5,562,654, 5,637,095, 5,672,167, and 5,755,691. One of skill in the art will also appreciate that the infusion rate for delivery of the nucleic acid may be varied. Suitable infusion rates may be from about 0.005 ml/min to about 1 ml/minute, preferably from about 0.01 ml/min to about 0.8 ml/min., more preferably from about 0.025 ml/min. to about 0.6 ml/min. It is particularly preferred that the infusion rate is about 0.05 ml/min.

[115] In accordance with the present invention, the nucleic acid is administered with a formulant that enhances transfection efficiency. Suitable formulants include, for example, divalent transition metals, polyionic compounds, and peptides. Suitable divalent transition metal compounds include, for example, zinc halide, zinc oxide, zinc acetate, zinc selenide, zinc telluride, and zinc sulfate. Preferred suitable divalent transition metal compounds include, for example, ZnCl₂, CuCl₂, CoCl , NiCl₂, and MgSO (Shiokawa et al, Biochem J. 326:675 (1997) and Torriglia et al, Biochimie 79:435 (1997)). Other suitable divalent transition metals are described in U.S. Patent No. 6,372,722, U.S. Patent Application No. 09/766,320, filed January 18, 2001, and International Publication WO 01/52903, filed January 19, 2001. Suitable polyanionic compounds include, for example, poly-L-glutamate. Suitable peptides include, for example, ID2 and peptides based on it such as, for example ID2-2, ID2-3, ID2-4 (Sperinde et al, J. Gene Med. 3:101 (2001)). Other suitable formulants include, for example, polyvinyl alcohol and nuclease inhibitors (Glasspool-Malone, et al. Mol. Ther. 2(2): 140 (2000)), sodium citrate, and G-actin (Shiokawa et al. (1997), supra). [116] The current invention also relates to a process for localizing a nucleic acid in a secretory gland cell of a mammal, comprising contacting a secretory gland cell with a nucleic acid transfection composition, the nucleic acid transfection composition comprising a polyionic organic acid and nucleic acid, thereby localizing the nucleic acid in the secretory gland cell of the mammal. A preferred process for localizing a nucleic acid in a secretory gland cell includes a secretory gland cell that is a salivary gland cell. [117] The nucleic acid composition of the present invention can also be localized in the secretory gland cell of the mammal by a process of delivering the nucleic acid composition to the mammal by direct administration or intraductal delivery. A preferred process for localizing the nucleic acid composition in a secretory gland is by elecfroporation administration. Preferably, the process for localizing a nucleic acid composition in a secretory gland cell includes an elecfroporation administration that comprises contacting the salivary gland with an electrode comprising at least two needles and pulsing the salivary gland.

[118] Nucleic acids administered according to the compositions and methods of the present invention may encode proteins that have local or systemic effects. Proteins encoded by nucleic acids administered according to the methods of the present invention can be used, for example, to treat or prevent any disorder amenable to treatment or prevention by expression of a therapeutic protein into the blood stream, by secretion of a therapeutic protein to the gastrointestinal tract (e.g. by secretion of the protein into the saliva), or by expression of the therapeutic protein by the transfected cell, tissue, gland, or organ. The subject can be a mammal such as, for example, a mouse, a rat, a guinea pig, a cat, a dog, a sheep, a goat, a cow, a horse, a non-human primate, or a human; or a non-mammal, such as, for example, a frog, a toad, a lizard, a snake, a turtle, a tortoise, or a salamander. [119] The present invention also provides a method for neutralizing a viras, the method comprising: administering a polyionic organic acid optionally in combination with a nucleic acid to an animal infected with a virus, thereby neutralizing the viras. The method can further comprise administering an ionizable or ionized fransition metal enhancer in combination with the polyionic organic acid and optionally the nucleic acid. Suitable ionizable or ionized transition metal enhancers include, but are not limited to, ZnCl₂, NiCl₂, CoCl₂, CuCl₂, A1C1₂, and GaCl₂. Additionally, the present invention relates to a method for neutralizing a viras comprising administering to an animal infected with a viras a polyionic organic acid, thereby neutralizing the viras. The present invention also provides a method for determining whether a polyionic organic acid directly neutralizes a viras. [120] Viruses that can be neutralized according to these methods include, but are not limited to, HIV, Epstein Barr viras, heφes simplex viras, hepatitis A, hepatitis B, hepatitis C, hepatitis E, mumps, measles, polio, and chicken pox. Preferably, the viras to be neutralized is HIV. Suitable polyionic organic acids include, but are not limited to, dyes such as Evans Blue, Congo Red, ponceau S, Congo corinth, Sirius red F3B, ponceau 6R, amido black 10B, biebrich scarlet, and aurintricarboxylic acid. Preferably, the polyionic organic acid is Congo Red and/or aurintricarboxylic acid. Suitable nucleic acids include, but are not limited to, DNA and plasmid DNA encoding a viral protein. Preferably, the nucleic acid encodes a viral envelope protein.

[121] Without being bound to any particular theory, it is believed that the present method for neutralizing a viras using a polyionic organic acid and a nucleic acid employs one or more mechanisms. These mechanisms include, but are not limited to, the polyionic organic acid: (i) promoting B cell maturation and increasing the affinity of an antibody for the virus; (ii) increasing the expression of the protein encoded by the nucleic acid; (iii) directly inhibiting the virus; and (iv) any combination of the foregoing. For example, the polyionic organic acid neutralizes a virus directly by binding to the virus or inhibiting viral reverse transcriptase, to inactivate the viras. A. Diseases and Disorders

[122] Using the compositions and methods of the present invention, the disease or disorder to be prevented or treated include autoimmune disorders, blood disorders, cardiovascular disorders, central nervous system disorders, gastrointestinal disorders, metabolic disorders, neoplastic diseases, pulmonary disorders, and bacterial and viral diseases.

[123] Autoimmune disorders that can be treated according to the methods of the present invention, include, for example, arthritis, diabetes, systemic lupus eryfhematosus, or Grave's disease. Other diseases and conditions include, ionizing radiation, an autoimmune disorder, Sjδgren's syndrome, graft-versus-host disease, systemic lupus erythematosis, rheumatoid arthritis, HIV-l infection, ageing, treatment with medications/drugs, autonomic dysfunction, conditions affecting the CNS, psychogenic disorder, trauma, or decrease in mastication. [124] Using the compositions and methods of the present invention, the disease or disorder to be prevented or treated include autoimmune disorders, blood disorders, cardiovascular disorders, central nervous system disorders, gastrointestinal disorders, metabolic disorders, neoplastic diseases, pulmonary disorders, and bacterial and viral diseases.

[125] Blood disorders that can be treated according to the methods of the present invention, include, for example, anemia sickle cell anemia, a globin disorder, or a clotting disorder such as hemophilia. Cardiovascular disorders that can be treated or prevented according to the methods of the present invention include, for example, high blood pressure, high cholesterol, and angina. Central nervous system disorders that can be treated according to the methods of the present invention, include, for example, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and Lou Gehrig's disease. Gastrointestinal disorders that can be treated according to the methods of the present invention include esophageal reflux, lactose deficiency, defective vitamin B12 absoφtion, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, and inflammatory bowel syndrome. Metabolic disorders that can be treated according to the methods of the present invention, include, for example, enzyme deficiencies, obesity, lysosomal storage disease, Hurler's disease, Scheie's disease, Hunter's disease, Sanfilippo diseases, Morqio diseases, Maroteaux-Lamy disease, Sly disease, or dwarfism. Neoplastic diseases that can be treated or prevented according to the methods of the present invention, include, for example, colon cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, breast cancer, skin cancer, leukemia, lymphoma, myeloma, or conditions arising out of cancer radiation treatment such as xerostomia. Pulmonary disorders that can be treated according to the methods of the present invention include, for example, cystic fibrosis, emphysema, or asthma. Bacterial diseases that can be treated or prevented according to the methods of the present invention, include, for example diphtheria, Lyme disease, meningitis, food poisoning, or pneumonia. Viral diseases that can be treated or prevented according to the methods of the present invention, include, for example, H1N, Epstein Barr viras, heφes simplex viras, hepatitis A, hepatitis B, hepatitis C, and hepatitis E, mumps, measles, polio, or chicken pox.

[126] Other conditions and symptoms include xerostomia, or dry mouth, which is a symptom associated with a decrease in salivary flow and/or alterations in salivary composition (see, U.S. Patent Application No. 60/458,793, filed March 26, 2003, incoφorated herein by reference). Xerostomia is manifested in complaints of oral dryness, burning of the tissues, difficulty eating and swallowing, irritation of the tongue and painful ulcerations as well as significantly progressive caries and periodontal disease. The burning tongue (glossodynia) associated with Xerostomia may become quite severe with chronic dryness, resulting in atrophy and painful Assuring and desquamation of the mucosa, often interfering with nutritional intake. Another condition to be freated is xerophthahnia, which is excessive drying of the conjunctiva and cornea; may be due to local disease or vitamin A deficiency.

B. Electroporation

[127] In one aspect, elecfroporation is used to enhance the efficiency of gene transfer after administration of a nucleic acid to a cell, tissue, gland or organ. In a particularly preferred embodiment, the nucleic acid is administered to a salivary gland before electroporation. Electroporation methods and techniques suitable for the present invention are described, for example, in PCT/US03/12628, filed April 21, 2003, and incoφorated herein by reference (see, also U.S. Patent Application Nos. 60/428,590, filed November 22, 2002, 60/407,375, filed August 30, 2002, and 60/453,999, filed March 11, 2003, all of which are incoφorated herein by reference). The use of electroporation in gene delivery is described in Somiari and Glasspool-Malone, Mol Ther. 2(3):178 (2000). Electroporation involves contacting cells, tissues, glands, or organs with an electrode comprising at least two needles and pulsing an electric signal through the cells, tissues, glands, or organs via the electrode. In a particularly preferred embodiment, a salivary gland is contacted with the electrode. The cells, tissues, glands, or organs may be contacted with more than two electrodes according to the methods of the present invention. If the cells, tissues, glands, or organs are contacted with more than one electrode, the contact may be simultaneous or sequential. The cells, tissues, glands, or organs may be contacted with the electrodes in multiple positions in accordance with the methods of the present invention. For example, the electrodes may be positioned vertically, longitudinally, or horizontally to come in contact with the salivary gland. The electrodes may also be positioned at angles to each other to come into contact with the cells, tissues, glands, or organs. Suitable angles include, for example, 45 degrees, 60 degrees, 75 degrees, 90 degrees, 120 degrees, 160 degrees, or 180 degrees. Preferably, the electrodes are positioned to ensure that the entire salivary gland is pulsed. One of skill in the art would understand that the position of the electrodes may be adjusted as needed to create an electric field that will extend throughout the entire salivary gland upon pulsing.

[128] The cells, tissues, glands, or organs may be contacted with electrodes that include, for example, needles, laparoscopic needles, probes, needles with paddles, needles with rotating paddles, and needles with flat plates or calipers. Methods of elecfroporation are described in U.S. Patent Nos. 6,233,482, 6,135,990, 5,993,434, and 5,704,908). Electrodes may comprise individual needles, laparoscopic needles, probes, needles with paddles, and flat plates or may comprise an anay of multiple needles, laparoscopic needles, probes, needles with paddles, and flat plates. One of skill in the art will appreciate that the space between 2 needles on the same electrode may be varied. The space between two needles may be, for example, about 0.1, 0.25, 0.4, 0.5, 0.6, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 cm. Electrodes and electrode arrays are described in WO 98/47562. Other configurations of the electrodes and electrode arrays for example, angle or shape of needle array, may be used to meet particular size and access needs according to the present invention.

[129] Factors to consider in determining suitable electroporation conditions include: electric field strength, pulse duration, pulse number, and pulse frequency. One of skill in the art will understand that appropriate values for each of these factors, i.e., values that enhance transfection efficiency, can be determined by standard means known in the art, i.e., without undue experimentation. (See, e.g., Canatella and Prausnitz, Gene Therapy 8:1464 (2001)). The electrode may emit an electric field strength from about 1 to about 1000 V/cm, from about 25 to about 750 V/cm, from about 50 to about 500 V/cm, form about 60 to about 300 V/CM or from about 75 to about 250 V/cm. The pulse length may be from about 1 to about 60 ms, from about 2 to about 50 ms, from about 4 to about 40 ms, from about 5 to about 30 ms, or from about 7 to about 25 ms. For example, a suitable electric field strength is typically from about 100 V/cm to about 200 V/cm and a suitable electrical pulse length is typically from about 10 ms to about 20 ms. A suitable number of pulses is typically from about 1 to about 30 pulses, from about 2 to about 20 pulses, from about 4 to about 15 pulses, from about 5 to about 12 pulses, preferably from about 5 pulses to about 6 pulses. [130] Suitable signal generators for electroporation are commercially available and include, for example, an Electro Cell Manipulator Model ECM 600 (Genetronics, Inc., San Diego, CA), an Electro Cell Manipulator Model ECM 830 (BTX, San Diego, CA), an

ElectroSquarePorator T820 (Genetronics, Inc., San Diego, CA), a PA-2000 (Cyto Pulse Sciences, Inc., Columbia, MD) or a PA-4000, (Cyto Pulse Sciences, Inc., Columbia, MD). These signal generators and methods of using them are described in U.S. Patent Nos.: 6,314,316, 6,241,701, 6,233,482, 6,135,990, 5,993,434, and 5,704,908. [131] The electrodes may be activatable in a predetermined sequence, which may include sequential or simultaneous activation of any or all of the electrodes. Suitable devices can be used, for example, with alternating current, direct current, pulsed alternating current, pulsed direct current, high- and low- voltage alternating current with variable frequency and amplitude, variable direct current waveforms, variable alternating current signals biased with variable direct current waveforms, variable alternating current signals biased with constant direct current, and square wave pulse signals. Selective control of the application of electrical signals between the individual electrodes can be accomplished for example, manually, mechanically, or electrically.

V. Examples

Example 1. Preparation of plasmid DNA-containing solutions.

[132] Stock solutions containing Evans blue, aurintricarboxylic acid, Congo red, suramin, calcium chloride, and zinc chloride of varying concentration were prepared in 0.9% sterile saline. For plasmid DNA solutions containing either Evans blue or Congo red, the required amounts of Evans Blue dye (or Congo red) and plasmid DNA were sequentially added to a polystyrene culture tube containing 0.9% sterile saline and thoroughly vortexed.

Aurintricarboxylic acid (or suramin)/zinc chloride/plasmid DNA solutions, the required amounts of aurintricarboxylic acid (or suramin), zinc chloride, and plasmid DNA are sequentially added to a polystyrene culture tube and thoroughly vortexed. Aurintricarboxylic acid/plasmid DNA solutions were prepared by sequentially adding the required amounts of aurintricarboxylic acid and plasmid DNA to a polystyrene culture tube. All other DNA solutions were prepared in a similar manner. Example 2. Intraductal instillation of a DNA construct to the rat submandibular glands.

[133] Adult, male, Sprague-Dawley rats (weighing 260-300g) received an intramuscular injection of a mixture of ketamine (30mg/kg b.wt.), xylazine (6.0mg/kg b.wt.) and acepromazine (1.0 mg kg b.wt.). Both the right and left salivary gland ducts were then cannulated with a fine polyurethane tubing (i.d. 0.005") and cemented in place with a small drop of Krazyglue®. Afropine was then administered subcutaneously (0.5 mg/kg b.wt.) and, after 10 minutes, the DNA-containing solution was injected in a retrograde manner to each submandibular gland. The rate of plasmid DNA delivery was held constant at 0.05 mL/min by using a syringe pump. The polyurethane tubing was kept in place for 10 additional minutes after plasmid DNA adminisfration.

Example 3. Collection of animal tissues and plasma samples.

[134] At 48 hours post DNA administration, the rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg b.wt.), blood was collected in heparinized tubes, and both submandibular glands were excised. Salivary gland tissues were homogenized in cold luciferase lysis buffer (1.5 ml buffer per 0.1 g tissue). All tissue homogenates and blood samples were then assayed for reporter gene expression.

Example 4. Determination of relative luciferase expression and alkaline phosphatase (SEAP) protein levels.

[135] Relative luciferase activity was determined using a L max plate luminometer (Analytical Luminescence Laboratories) by measuring light emissions over a 10 second period from 100 μl of lysis homogenate. Activity is expressed as relative light units, which is a function of luciferase concentration. Relative SEAP activity from plasma and tissue homogenate was measured via a chemiluminescent method using a plate luminometer (L max, Molecular Devices). All reagents for this assay provided by Roche Molecular Biochemicals. [136] The data presented in Figure 2 suggests that DNA solutions containing Evans Blue promote salivary gland transfection efficiency. The submandibular glands of adult, male, Sprague Dawley rats (250-300g) were each treated with 100 microliters of solution containing: (A) Evans Blue and zinc chloride; (B) zinc chloride and DNA; (C) zinc chloride, Evans Blue, and DNA; (D) Evans Blue and DNA. Each data point represents the average luciferase expression obtained from eight submandibular glands (four animals), SEM. [137] The results of figure 3 show that increasing the concentration of Evans Blue (0-6 mg/ml) enhance salivary gland transfection efficiency. 100 microliters of the DNA solutions were administered to the right and left submandibular glands of adult, male, Sprague-Dawley rats. Each data point represents the average SEAP protein content obtained from 4 animals, SEM.

[138] Figure 4 demonstrates that treatment of the salivary glands with Evans Blue leads to higher secreted hGH concentrations in plasma. This research also shows that the addition of calcium chloride to an Evans Blue/DNA solution improves the levels of observed human growth hormone protein in plasma. The submandibular glands of adult, male, Sprague Dawley rats (250-3 OOg) were each treated with 100 microliters of solution containing: (A) Evans Blue and calcium chloride; (B) calcium chloride and DNA; (C) Evans Blue and DNA; (D) Evans Blue, calcium chloride and DNA. Each data point represents the average human growth hormone protein content obtained from 4 animals, SEM.

[139] Figure 5 demonstrates the results from screening DNA solutions containing different concentrations of either Evans Blue or Congo Red (0-6 mg/ml) for the ability to enhance systemic protein delivery after submandibular gland transfection. The results indicate that DNA solutions containing Congo Red enhance salivary gland transfection efficiency more when compared to DNA solutions that contain Evans Blue, at the same concentration. 100 microliters of the DNA solutions were administered to the right and left submandibular glands of adult, male, Sprague-Dawley rats. Each data point represents the average SEAP protein content obtained from 4 animals, SEM.

Example 5. Biodistribution data.

[140] Accumulation of plasmid DNA in the submandibular gland is shown in Figure 6 for DNA solutions containing H₂O, Evans Blue and Congo Red. The results indicate that solutions containing Evans Blue and Congo Red result in accumulation of plasmid DNA between 10³ - 10⁴ more than that without. Male adult Sprague Dawley rats were treated with 175 μg of plasmid DNA by retroductal infusion to the salivary glands (n=4 treated with DNA + H₂O, n=4 treated with DNA + Evans Blue[6mg/ml], n=4 treated with DNA + Congo Red [6mg/ml] , n=l naive). An aqueous solution containing 175 μg of the plasmid pSEAP was co-formulated and delivered. Tissues were harvested after 24 hours and total DNA extracted and purified. The plasmid was quantified by Q-PCR and concentrations are presented as copies of plasmid DNA per 100 ng genomic DNA.

Example 6. DNase inhibition.

[141] Experimental Design. Two different polyionic organic acids were mixed with different concentrations of DNase types I and II. The compounds were all tested at 0.67 mg/ml. The two positive controls, EDTA and cationic lipids, demonstrate that the enzyme is inhibited by removing divalent metal cofactors or by complexing the DNA. The positive confrols demonstrate that the DNA is readily degraded.

[142] Experimental Procedure. DNA (1 μg) was mixed with dye (final concentration 0.67 mg/mL) or lipid (20:1 charge ratio) on ice, then added to DNase I (Figure 7) or II (Figure 8) and incubated for 5 min at 37°C. After 5 min. the samples were immediately placed on ice, loading dye was added. The DNA integrity was determined by analyzing the samples using gel electrophoresis. Aliquots of the reaction mixture were loaded onto a 0.7% agarose gel in TRIS/acetic acid/EDTA (TAE) buffer that was treated with ethidium bromide. The agarose gels were electrophoresed at 120 V and visualized under ultraviolet light. [143] Figure 7 illustrates the effect of Polyionic Organic Acids on DNase Type I activity. DNA (1 μg) was mixed with Congo Red and Evans Blue (final concentration 0.67 mg/ml) or lipid (20: 1 charge ratio) and incubated with DNase Type I for 5 min at 37°C. The DNA integrity was then determined by analyzing the samples using gel electrophoresis. The DNA appears in three distinct forms (from top to bottom): nicked circular (A), linear (B), and digested (C). [144] Figure 8 illustrates the effect of Polyionic Organic Acids on DNase Type II activity. DNA (1 μg) was mixed with Congo Red and Evans Blue (final concentration 0.67 mg/ml) or lipid (20: 1 charge ratio) and incubated with DNase Type II for 5 min at 37°C. The DNA integrity was then determined by analyzing the samples using gel electrophoresis. The DNA appears in three distinct forms (from top to bottom): nicked circular (A), linear (B), and digested (C). Example 7. Determination of alkaline phosphatase (SEAP) protein levels after transfection with ATA and zinc.

[145] The plasmid pBATSEAP contains the secreted alkaline phosphatase (SEAP) gene operably linked to human cytomegaloviras major immediate early enhancer/promoter, which is positioned upstream of the first infron of human β-globin. The plasmid was produced by bacterial fermentation and purified with an anion exchange resin (Qiagen, Santa Clarita, CA) to yield an endotoxin-reduced, supercoiled plasmid containing less than 100 E.U. mg DNA as measured by clot LAL assay (Charles River Endosafe). Stock DNA solutions were prepared using sterile water. [146] Tissue supernatants, prepared in luciferase lysis buffer, were analyzed for SEAP concentration using the chemiluminescent SEAP Reporter Gene Assay available from Roche Diagnostics (Indianapolis, IN). Briefly, the samples were diluted 1:50 with Dilution Buffer then incubated in a water bath for 30 min at 65°C. The samples were then centrifuged for 1 min at 13,000 φm and 50 μl of the resulting supernatant was transferred to a microtiter plate. Fifty μl of the provided Inactivation Buffer was added followed by a 5-minute incubation at room temperature. The Substrate Reagent (50 μl) was added prior to a 10-minute incubation at room temperature. Light emissions from the samples were measured using an L-max plate luminometer from Molecular Devices (Sunnyvale, CA). The relative light units were converted to mass of SEAP protein based on as standard curve run simultaneously with the samples. Plasma concentrations of SEAP were measured using the same assay except that the samples were diluted 1 :7 with the Dilution Buffer. The lower sensitivity limit for determining SEAP protein concentrations in rat plasma is 50 pg/ml. [147] As shown in Figure 9, the presented data shows that the use of DNA solutions containing both ATA and zinc enhance transgene expression after salivary gland transfection and lead to higher levels of SEAP in plasma. Plasmid DNA (SEAP, 175 μg/200 μl) solutions containing zinc, ATA (4 mg/ml), or zinc/ ATA (4 mg/ml) were administered retroductally to the right and left submandibular glands of adult, male Sprague-Dawley rats. Plasma samples were collected 48 hours after plasmid DNA administration and assayed for SEAP concentration. Each data point represents the average value obtained from four animals (+/- SEM). *P<0.05.

[148] Figure 10 demonstrates that SEAP concentrations in both plasma and submandibular gland tissues increased with DNA dose, with 350 μg of plasmid DNA being approximately 10-fold more effective than 87.5 μg of plasmid DNA. Zinc (3.6 mMVATA (4 mg/ml) solutions (100 μl) containing 87.5 μg, 175 μg, or 350 μg of plasmid DNA (SEAP) were administered to the right and left submandibular glands of adult, male Sprague-Dawley rats. Plasma (white bars) and submandibular gland tissue (dark grey bars) samples were collected 48 hours after plasmid DNA administration and assayed for SEAP concentration. Each data point represents the average value obtained from four animals (+/- SEM). *P<0.05.

[149] The data presented in Figure 11 show a dose-dependent effect of ATA on salivary gland transfection, as the highest average concentrations of SEAP (190 ng/ml of plasma) were obtained at the highest ATA concentration (12 mg ATA/ml). Plasmid DNA (SEAP, 300 μg/50 μl) solutions containing 3.6 mM zinc chloride and ATA, at concentrations of 0, 4, 8, or 12 mg/ml, were administered retroductally to the right and left submandibular glands of adult, male Sprague-Dawley rats. Plasma samples were collected 48 hours after plasmid DNA administration and assayed for SEAP concentration. Each data point represents the average value obtained from four animals (+/- SEM). *P<0.05.

Example 8. Biodistribution data for zinc/ ATA transfections.

[150] Quantitative PCR (Q-PCR) was performed to determine the relative concentrations of plasmid DNA in salivary gland tissues following retroductal administration of DNA solutions containing ATA and zinc. The results in Figure 12 indicate an accumulation of tissue plasmid DNA and that the concentration of plasmid DNA in glands treated with zinc/ATA/DNA was approximately 500 times greater than that in glands treated with plasmid DNA in saline. Plasmid DNA (SEAP, 300 μg/50 μl) solutions containing zinc, ATA (8 mg/ml), or zinc/ ATA (8 mg/ml) were administered retroductally to the right and left submandibular glands of adult, male Sprague-Dawley rats. Submandibular gland tissues were collected 48 hours after adminisfration and the tissue associated DNA was extracted and purified. The amount of plasmid DNA was quantified by Q-PCR and concentrations are presented as copies of plasmid DNA per 100 ng of genomic DNA. Each data point represents the average value obtained from 6 samples (+/- SEM). *P<0.05. The background value for untreated salivary gland tissue is 53+/-16 copies of plasmid DNA per 100 ng of genomic DNA. Example 9. Effect of zinc/ ATA solutions on DNase inhibition.

[151] A series of DNA degradation studies were performed to investigate the effect of zinc/ ATA on nuclease activity. Plasmid DNA solutions with or without added zinc, ATA, or zinc/ATA were incubated with either DNase I, DNase II, or rat submandibular gland lysates at 37°C. Aliquots of the reaction mixture were loaded onto a 0.7% agarose-TBE gel and the DNA integrity was analyzed by agarose gel electrophoresis.

[152] As shown in Figure 13, zinc and zinc/ATA were more effective in protecting DNA from DNase I than ATA only, while ATA and zinc/ ATA were more effective in protecting DNA from DNase II than zinc only. Further, ATA and zinc/ ATA protected DNA from • endogenous DNase activity in submandibular gland lysates. These results demonstrate that a mixture of zinc and ATA is a potent general inhibitor of DNases. [153] For these experiments, DNase I and DNase II solutions were prepared in the appropriate buffers at concentrations of 0.4 milliunits/μl and 2 milliunits/μl, respectively. The tissue supernatants, prepared in PBS, were further diluted by a factor of 1 : 10 with PBS. Plasmid DNA containing the renilla luciferase gene was combined with either 3.6 mM ZnCl , a mixture containing 0.15 mM ZnCl₂ and 0.67 mg/ml ATA, or 0.67 mg/ml ATA to give a final plasmid DNA concentration of 0.1 μg/μl. At 0°C, 10 μl aliquots of the plasmid DNA solutions +/- adjuvants were mixed with 20 μl of the appropriate DNase solution or diluted salivary gland lysate. The resultant mixtures were incubated for 5 minutes at 37°C prior to the addition of sample loading buffer and electrophoresis on a 0.7%> (w/v) agarose gel. The DNA stracture is indicated as supercoiled (S), nicked (N), linear (L) in Figure 13.

Example 10. Effect of zinc/ ATA solutions on salivary gland histology after transfection. [154] Figure 14a shows that inflammatory infiltrates were observed both in the salivary gland connective tissue and capsule, and edematous changes were observed in the parenchyma after treatment with zinc/ATA/DNA. This observed inflammation was alleviated by treating the animals with dexamethasone (1.5 mg/kg b.wt.) immediately prior to administration of the zinc/ATA/DNA solution, and resulted in no detectable inflammation at 2 (Figure 14b) or 8 days (Figure 14c) following DNA administration. The acini remained intact and no stractural or edematous changes were observed. Figure 14d shows the moφhology of an untreated salivary gland. These results indicate that zinc/ATA/DNA formulations can be administered to the SG without detectible adverse effects. Arrows indicate presence of vacuoles. * indicates a region of inflammation. [155] Plasmid DNA (SEAP, 262.5 μg) solutions containing 3.6 mM zinc and 6 mg/ml ATA, with or without dexamethasone pre-treatment, were administered refroductally to the right and left submandibular glands of adult, male Sprague-Dawley rats. Two or 8 days after plasmid DNA administration, the left and right glands were removed, bisected, paraffin embedded, H&E stained, visualized on a Zeiss Axiovert 100 microscope, and photographed with a Kodak DC290 digital camera.

Example 11. Comparison of SEAP plasma concentrations after congo red- and suramin/zinc-mediated transfection in the rat submandibular gland. [156] As shown in Figure 15, fransfection of rat submandibular glands with solutions containing suramin (40 mg/mL), zinc (5 mM), and plasmid DNA (encoding for SEAP protein) result in higher observed systemic SEAP concentrations than when the salivary glands are treated with solutions containing Congo red (6 mg/ml) and the same plasmid DNA. Plasmid DNA (SEAP, 175 μg/50 μL) solutions containing either congo red (6 mg/mL) or suramin (40 mg/mL) and zinc (5 mM), were administered retroductally to the right and left submandibular glands of adult, male nude rats. Plasma samples were collected 48 hours after plasmid DNA administration and assayed for SEAP concentration. Each data point represents the average value obtained from four animals (+/- SEM).

Example 12. SEAP protein in rat salivary gland tissues observed after suramin/zinc- mediated transfection.

[157] Figure 16 shows that perfusion of salivary glands with solutions containing suramin (10 mg/mL), zinc (1 mM), and plasmid DNA (encoding for the exogenous protein SEAP) results in detectable SEAP protein concentrations in salivary gland tissues for at least 3 weeks post treatment. Plasmid DNA (SEAP, 50 μg/50 μL) solutions containing suramin (10 mg/mL) and zinc (1 mM), were administered retroductally to the right and left submandibular glands of adult, male Sprague-Dawley rats. The submandibular glands were harvested at 48 hours, 7 days, 14 days, or 21 days after plasmid DNA administration, homogenized in lysis buffer, and assayed for SEAP concentration. Each data point represents the average value obtained from eight submandibular glands (n=8).

Example 13. Method for determining direct viral neutralization by a POD.

[158] This example illustrates an assay method for determining whether a polyionic organic acid directly neutralizes a virus. Preferably, the polyionic organic acid is Congo Red and/or aurintricarboxylic acid. In certain cases (e.g., aurintricarboxylic acid), a transition metal enhancer, such as zinc chloride, is added. The virus to be neutralized is preferably HIN. [159] The polyionic organic acid is administered to an animal. If the polyionic organic acid is administered along with a nucleic acid encoding a viral, fungal, or bacterial protein, the animal will generate antibodies against the protein encoded by the nucleic acid and hence become vaccinated against the protein. Next, plasma is taken from the treated animal and tested for its ability to neutralize primary and lab strains of the virus. The IC₅₀ (the concentration required for 50% inhibition of neutralization) was measured by mixing a known quantity of viras with different dilutions of plasma before adding to target cells. In a pilot study, plasma from rats vaccinated with an HIV envelope protein and either Congo Red or aurintricarboxylic acid/zinc chloride was able to generate neutralizing titers to primary and lab strains of HIV, as compared to plasma from control rats.

[160] Antibodies from the plasma are then removed, and the antibody-depleted plasma is tested for its ability to neutralize the virus by mixing plasma with viras before adding the mixture to the target cells. Further, the polyionic organic acid is mixed at different concentrations with viras, and the IC₅₀ is determined. If removal of antibodies from the plasma of animals treated with the polyionic organic acid does not change the ability of the plasma to effectively neutralize the virus, and the IC₅₀ measured for the polyionic organic acid is quite low (e.g., the polyionic organic acid is effective in a small amount), then these data suggest that the polyionic organic acid can directly neutralize a viras in an in vivo context. This demonstrates that the nucleic acid was not necessary for the observed neutralizing effect.

[161] Figure 17 presents data demonstrating direct neutralization of a viras by Congo Red. The efficacy of viral neutralization by Congo Red is measured as a decrease in the percentage of target cells infected as compared to controls without Congo Red (samples 1 and 7). The highest concentration of Congo Red tested is highly effective at inhibiting the infection of target cells by a viras, while successive 10-fold dilutions of Congo Red display reduced viral neutralization (samples 2-5). Plasma taken from animals treated with Congo Red (or Congo Red plus nucleic acid) also exhibit potent viral neutralization when compared to plasma from untreated animals (samples 6-7). Removal of antibodies from the plasma of animals treated with Congo Red does not change the ability of the plasma to effectively neutralize the viras (sample 8). As a negative control, target cells are not infected without virus (sample 9). These data indicate that Congo Red directly neutralizes the viras in an in vivo context. Example 14. Effect of POD/nucleic acid vaccination on HTV neutralization.

[162] 88 μg DNA encoding HIV envelope protein gpl20 in 200 μl distilled, deionized H₂O was retroductally delivered (50 μl/min.) using a standard syringe pump to the submandibular salivary glands of Sprague Dawley rats on weeks 0 and 3. The DNA was delivered in a formulation comprising: DOHBD:DOPE lipid (3:1)/Zn (0.125 mM), Congo Red (6 mg/ml), Evans Blue (6 mg/ml), or Congo Red (6 mg/ml )/DOHBD:DOPE lipid (3:1)/Zn (0.125 mM). Anti-gpl20 IgG titer was measured by ELISA over 17 weeks. As shown in Figure 18, all formulations were able to generate significant antibody responses to gpl20 protein, with the Zn/lipid formulation generating the lowest IgG titer compared to the other formulations. On week 9, plasma samples were collected and HIV neutralization assays were performed using HIV strains Bal and MN. Table 2 depicts data demonstrating neutralization of Bal and MN. Plasma samples from lof 2 animals treated with Congo Red and plasma samples from 1 of 2 animals treated with Congo Red/Zn/lipid were able to neutralize HIN strains Bal and MΝ. Further, a plasma sample from an animal treated with ATA/Zn was able to neutralize HIN strains Bal and MΝ. However, no neutralization was observed in untreated animals (0 of 4) or animals vaccinated intramuscularly (0 of 2).

Table 2. HIV Neutralization Assay Summary

[163] All publications and patent applications cited in this specification are herein incoφorated by reference as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

A nucleic acid transfection composition, said nucleic acid transfection composition comprising: a polyionic organic acid; and a nucleic acid.

2. The composition of claim 1, wherein said polyionic organic acid is a polyprotic polyaromatic organic compound.

3. The composition of claim 1, wherein said polyionic organic acid is a dye.

4. The composition of claim 1, wherein said polyionic organic acid is not a dye.

5. The composition of claim 1, wherein said polyionic organic acid is suramin.

6. The composition of claim 3, wherein said dye absorbs radiation of the electromagnetic spectram in the region selected from the group consisting of ultraviolet region, visible region, infrared region and combinations thereof.

7. The composition of claim 3, wherein said dye is a member selected from the group consisting of an acid dye, a direct dye, a reactive dye and a disperse dye.

8. The composition of claim 7, wherein said dye is a member selected from the group consisting of direct red, direct blue, direct yellow and direct green.

9. The composition of claim 3, wherein said dye is a member selected from the group consisting of Light Blue, Congo Red and Evans Blue.

10. The composition of claim 1, wherein said nucleic acid is selected from the group consisting of DNA, RNA, a DNA RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, viral vector DNA, and a plasmid DNA.

11. The composition of claim 10, wherein said nucleic acid encodes a peptide or protein.

12. The composition of claim 11, wherein said peptide or protein is an immunogenic peptide or protein.

13. The composition of claim 12, wherein said immunogenic peptide or protein is an antigen selected from the group consisting of a cancer antigen, a bacterial antigen, a viral antigen, a fungal antigen, a parasitic antigen, and an antigen overexpressed on neoplastic cells.

14. The composition of claim 11, wherein said peptide or protein is selected from the group consisting of gamma glutamyl transpeptidase, manganese superoxide dismutase, metallothionein, glutathione peroxidase (GPx)-4, and catalase.

15. The composition of claim 11 , wherein said peptide or protein is selected from the group consisting of IFN-alpha, IL- 10, sTNFR, TGF-/3, IL-4, VIP, anti-TNF antibody, ILl-RA, other antibodies to proinflammatory cytokines, soluble gp39, soluble CD40, aquaporin-1, aquaporin-5, and zsig63.

16. The composition of claim 1, wherein said composition is an aqueous solution.

17. The composition of claim 1, further comprising an ionizable or ionized transition metal enhancer.

18. The composition of claim 17, wherein said composition comprises ATA and ZnCl₂.

19. The composition of claim 17, wherein said composition comprises Suramin and ZnCl₂.

20. The composition of claim 17, wherein said ionizable or ionized transition metal enhancer is a complex, adduct, cluster or salt of an element selected from the group consisting of a d-block element, a first row f-block element, aluminum, and gallium.

21. The composition of claim 20, wherein the ionizable or ionized transition metal enhancer is a complex, adduct, cluster or salt of an element selected from the group consisting of zinc, nickel, cobalt, copper, aluminum, and gallium.

22. The composition of claim 21 , wherein the ionizable or ionized transition metal enhancer is selected from the group consisting of zinc sulfate, zinc acetate, nickel sulfate, nickel acetate, cobalt sulfate, cobalt acetate, copper sulfate, and copper acetate.

23. The composition of claim 22, wherein the ionizable or ionized transition metal enhancer is zinc acetate or zinc sulfate.

24. The composition of claim 17, wherein the ionizable or ionized transition metal enhancer is selected from the group consisting of zinc halide, nickel halide, cobalt halide, copper halide, aluminum halide, and gallium halide.

25. The composition of claim 24, wherein the ionizable or ionized transition metal enhancer is selected from the group consisting of ZnCl₂, NiCl₂, CoCl₂, CuCl₂, A1C1₂, and GaCl₂.

26. The composition of claim 1, wherein said composition further comprises a secretory gland cell.

27. The composition of claim 1 , wherein said composition further comprises a member selected from the group consisting of a cationic lipid, a cationic polymer, and a cationic peptide.

28. The composition of claim 1, wherein said composition is a nuclease inhibitor.

29. The composition of claim 28, wherein said nucleic acid of the composition has an increased half-life compared to nucleic acid alone.

30. The composition of claim 1, further comprising a nuclease inhibitor.

31. The composition of claim 30, wherein said nuclease inhibitor is a DNase inhibitor.

32. The composition of claim 31, wherein said DNase inhibitor is selected from the group consisting of a DNase I inhibitor, a DNase II inhibitor, a divalent cation chelator, a polymer, a DNA binding agent, a DNA nicking inhibitor, a detergent, a chaofropic salt, and an amino acid.

33. The composition of claim 32, wherein said DNase inhibitor is a DNase I inhibitor.

34. The composition of claim 32, wherein said DNase inhibitor is a DNase II inhibitor.

35. The composition of claim 30, wherein said composition further comprises an ionizable or ionized transition metal enhancer.

36. A method for administering a nucleic acid to a cell, said method comprising: contacting said cell with a nucleic acid transfection composition, said nucleic acid transfection composition comprising a polyionic organic acid and nucleic acid, thereby administering nucleic acid to said cell.

37. The method of claim 36, wherein said cell is a secretory gland cell.

38. The method of claim 37, wherein said secretory gland cell is selected from the group consisting of a salivary gland cell, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, and a liver cell.

39. The method of claim 38, wherein the secretory gland cell is a salivary gland cell.

40. The method of claim 38, wherein said nucleic acid is selected from the group consisting of DNA, RNA, a DNA/RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, viral vector DNA, and a plasmid DNA.

41. The method of claim 40, wherein said nucleic acid encodes a peptide or protein.

42. The method of claim 41 , wherein said peptide or protein is secreted or released from said secretory gland cell.

43. The method of claim 36, wherein said composition is delivered to said cell by retroductal delivery or direct administration.

44. The method of claim 36, wherein said composition is delivered to said cell by electroporation adminisfration.

45. The method of claim 36, wherein said composition is delivered to said cell by ultrasound administration.

46. The method of claim 36, wherein said composition is delivered to said cell by ionophoresis administration.

47. The method of claim 36, wherein the administering is by cannulation.

48. The method of claim 36, wherein the administering is by injection.

49. The method of claim 36, wherein said composition further comprises an ionizable or ionized transition metal enhancer.

50. The method of claim 49, wherein the nucleic acid transfection composition acts as a nuclease inhibitor.

51. The method of claim 49, wherein the nucleic acid transfection composition acts by perturbing the cell membrane.

52. The method of claim 51, wherein said fransfection occurs in vivo.

53. The method of claim 51, wherein said transfection occurs in vitro.

54. A method for increasing transfection efficiency in a cell, said method comprising: contacting a cell with a nucleic acid transfection composition, said nucleic acid transfection composition comprising a polyionic organic acid and nucleic acid, thereby increasing transfection efficiency.

55. The method of claim 54, wherein said cell is a secretory gland cell.

56. The method of claim 55, wherein said secretory gland cell is selected from the group consisting of a salivary gland cell, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, and a liver cell.

57. The method of claim 56, wherein the secretory gland cell is a salivary gland cell.

58. The method of claim 54, wherein said nucleic acid is selected from the group consisting of DNA, RNA, a DNA/RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, viral vector DNA, and a plasmid DNA.

59. The method of claim 58, wherein said nucleic acid encodes a peptide or protein.

60. The method of claim 59, wherein said peptide or protein is secreted or released from said secretory gland cell.

61. The method of claim 59, wherein said peptide or protein remains in said secretory gland cell.

62. The method of claim 59, wherein said peptide or protein is secreted locally from said secretory gland cell.

63. The method of claim 59, wherein said peptide or protein is secreted systemically from said secretory gland cell.

64. The method of claim 54, wherein said composition is delivered to said cell by intraductal delivery or direct administration.

65. The method of claim 54, wherein said composition is delivered to said cell by electroporation administration.

66. The method of claim 54, wherein said composition is delivered to said cell by ultrasound adminisfration.

67. The method of claim 54, wherein the administering is by cannulation.

68. The method of claim 54, wherein the administering is by injection.

69. The method of claim 54, wherein said composition is delivered to said cell by ionophoresis administration.

70. The method of claim 54, wherein said composition further comprises an ionizable or ionized fransition metal enhancer.

71. The method of claim 54, wherein said nucleic acid is localized in said cell.

72. The method of claim 54, wherein said nucleic acid is operably linked to an expression confrol sequence.

73. The method of claim 72, wherein said expression control sequence is tissue specific.

74. The method of claim 73, wherein said tissue is intestinal epithelium.

75. The method of claim 73, wherein said tissue is liver.

76. The method of claim 54, wherein said nucleic acid encodes a therapeutic protein.

77. The method of claim 76, wherein said therapeutic protein is not expressed in an intestinal cell.

78. The method of claim 76, wherein said therapeutic protein is expressed in an intestinal cell.

79. The method of claim 76, wherein said therapeutic protein is expressed in an intestinal epithelial cell.

80. A method for stabilizing a nucleic acid, said method comprising: contacting said nucleic acid with a composition comprising a polyionic organic acid, thereby stabilizing said nucleic acid.

81. The method of claim 80, wherein said composition further comprises an ionizable or ionized fransition metal enhancer.

82. The method of claim 80, wherein said composition further comprises a nuclease inhibitor.

83. The method of claim 80, wherein said nucleic acid is in a cell.

84. The method of claim 83, wherein said nucleic acid is in a secretory gland cell.

85. The method of claim 84, wherein said secretory gland cell is selected from the group consisting of a salivary gland cell, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, and a liver cell.

86. The method of claim 85, wherein the secretory gland cell is a salivary gland cell.

87. The method of claim 80, wherein said nucleic acid is selected from the group consisting of DNA, RNA, a DNA RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, viral vector DNA, and a plasmid DNA.

88. The method of claim 87, wherein said nucleic acid is DNA.

89. The method of claim 87, wherein said nucleic acid is a plasmid DNA.

90. The method of claim 87, wherein said nucleic acid encodes a peptide or protein.

91. The method of claim 90, wherein said peptide or protein is secreted or released from said secretory gland cell.

92. The method of claim 80, wherein said stabilization occurs in vivo.

93. The method of claim 80, wherein said stabilization occurs in vitro.

94. The method of claim 80, wherein said composition acts as a nuclease inhibitor.

95. The method of claim 94, wherein the nucleic acid transfection composition increases the half-life of said nucleic acid.

96. The method of claim 94, wherein said composition increases the transfection efficiency for said nucleic acid.

97. The method of claim 94, wherein said composition increases the half- life of said nucleic acid in a cell.

98. The method of claim 80, wherein said composition is delivered to a cell by retroductal delivery or direct administration.

99. The method of claim 80, wherein said composition is delivered to a cell by electroporation administration.

100. The method of claim 80, wherein said composition is delivered to a cell by ultrasound adminisfration.

101. The method of claim 80, wherein said composition is delivered to a cell by ionophoresis administration.

102. The method of claim 80, wherein the contacting is by cannulation.

103. The method of claim 80, wherein the contacting is by injection.

104. A method for increasing the shelf life of a nucleic acid, said method comprising: contacting said nucleic acid with a composition comprising a polyionic organic acid, thereby increasing the shelf life of said nucleic acid.

105. The method of claim 104, wherein said composition further comprises an ionizable or ionized transition metal enhancer.

106. The method of claim 104, wherein said composition further comprises a nuclease inhibitor.

107. The method of claim 104, wherein said nucleic acid is selected from the group consisting of DNA, RNA, a DNA/RNA hybrid, an antisense oligonucleotide, a chimeric DNA-RNA polymer, a ribozyme, viral vector DNA, and a plasmid DNA.

108. The method of claim 107, wherein said nucleic acid is DNA.

109. The method of claim 107, wherein said nucleic acid is a plasmid DNA.

110. The method of claim 107, wherein said nucleic acid encodes a peptide or protein.

111. The method of claim 104, wherein said composition acts as a nuclease inhibitor.

112. The method of claim 104, wherein said composition stabilizes said nucleic acid.

113. The method of claim 104, wherein said increase in shelf life of a nucleic acid occurs in vitro.

114. A method for neutralizing a viras, said method comprising: administering a polyionic organic acid to an animal infected with said viras, thereby neutralizing the viras.

115. The method of claim 114, further comprising administering a nucleic acid in combination with said polyionic organic acid.

116. The method of claim 114, further comprising administering an ionizable or ionized transition metal enhancer in combination with said polyionic organic acid.

117. The method of claim 116, further comprising administering a nucleic acid.

118. The method claim 116, wherein said ionizable or ionized fransition metal enhancer is zinc chloride.

119. The method of claim 114, wherein said polyionic organic acid is a dye.

120. The method of claim 119, wherein said dye is selected from the group consisting of Congo Red, suramin, and aurintricarboxylic acid.

121. The method of claim 120, wherein said dye is Congo Red.

122. The method of claim 114, wherein said virus is selected from the group consisting of HIV, Epstein Barr viras, heφes simplex virus, hepatitis A, hepatitis B, hepatitis C, hepatitis E, mumps, measles, polio, and chicken pox.

123. The method of claim 122, wherein said viras is HIV.

124. The method of claims 115 or 117, wherein said nucleic acid encodes a viral envelope protein.

125. The method of claims 115 or 117, wherein said polyionic organic acid neutralizes said viras by increasing the affinity of an antibody for said viras.

126. The method of claims 115 or 117, wherein said polyionic organic acid neutralizes a viras by increasing the expression of the protein encoded by said nucleic acid.

127. The method of claim 114, wherein said polyionic organic acid neutralizes a viras by directly inhibiting said viras.