Description

Methods and Compositions for Inducing Protective Responses to Antigenic Toxins

Technical Field

The present invention is directed to the induction of protective responses against antigenic toxins and, more particularly, to the in vivo delivery of nucleic acid sequences as a means to provide such responses.

Background of the Invention

The administration of dominant negative proteins exhibiting toxin inhibitory activity has been proposed as a method of treatment for subjects infected with toxin producing microorganism(s) .

Various dominant negative proteins exhibiting anti-toxin activity have been identified for numerous microorganism related toxins, including toxins produced by Bacillus anthracis (Singh Y, Chaudhary VK, Leppla SH. A deleted variant of Bacillus anthracis protective antigen is nontoxic and blocks anthrax toxin action in vivo. J Biol Chem. 1989 Nov 15; 264(32):19103-7., Yan M, Collier RJ. Characterization of dominant-negative forms of anthrax protective antigen. MoI Med. 2003 Jan-Feb; 9(l-2):46-51), Escherichia coli (Wai SN, et al. Characterization of dominantly negative mutant CIyA cytotoxin proteins in Escherichia coli. JBacteήol. 2003 Sep; 185(18):5491), and Helicobacter pylori (Vinion-Dubiel AD, et al. A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J Biol Chem. 1999 Dec 31; 274(53):37736-42. McClain MS, et al. A 12-amino-acid segment, present in type s2 but not type si Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation. J Bacteriol. 2001 Nov; 183(22):6499-508, Sundrud MS, et al. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion. Proc Natl Acad Sci USA. 2004 May 18; 101(20):7727-32).

The administration of sufficient quantities of a recombinant form of the dominant negative protein has been shown to have the capability to ameliorate the deleterious effects of toxin exposure in a subject organism (Singh Y, Khanna H, Chopra AP, Mehra V. A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo. /

Biol Chem. 2001 Jun 22; 276(25):22090-4, Sellman BR, Mourez M, Collier RJ. Dominant- negative mutants of a toxin subunit: an approach to therapy of anthrax. Science. 2001 Apr 27; 292(5517):695-7).

In addition it has been demonstrated that, despite the introduction of mutations in the protein, dominant negative versions of toxin proteins can retain the capacity for eliciting immune responses reactive to the native form of the toxin (Aulinger BA, Roehrl MH, Mekalanos JJ, Collier RJ, Wang JY. Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines. Infect Immun. 2005 Jun; 73(6):3408-14). In spite of the promise associated with the development of these protein-based agents capable of inhibiting the activity of antigenic toxins, there are significant limitations in the practical value of the approaches described to date. Of foremost importance is the need to provide the patient with sustained, high level exposure to the dominant negative protein at the sites of toxin distribution/localization for the duration of toxin production. For many microorganisms, the required duration of treatment can extend for several weeks or longer.

Unfortunately, with a half-life on the order of hours, these protein-based agents typically exhibit rapid degradation and clearance in vivo. Thus, frequent, serial administrations of the protein are necessary in order to achieve the desired level and duration of protection. Such an administration sequence is likely to be costly and inconvenient. In addition, the production of the large amounts of the dominant negative protein required for these serial administrations may be prohibitively expensive for the proposed indications.

Although studies that have demonstrated that administration of a dominant negative toxin inhibitor can elicit immune responses reactive to the native toxin (Aulinger BA, Roehrl MH, Mekalanos JJ, Collier RJ, Wang JY. Combining anthrax vaccine and therapy: a dominant- negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines. Infect Immun. 2005 Jun; 73(6):3408-14), the formulation as well as the route and schedule of immunization utilized to achieve immune response (intraperitoneal administration of protein formulated with aluminum hydroxide adjuvant administered 3 times at a two week interval) are not conducive to that capable of inducing direct toxin inhibition (more frequent systemic administration without an immunostimulatory adjuvant). Therefore it is unlikely that a single protein based product will be sufficient to provide direct toxin inhibition and long term induction of toxin protective immune response.

In light of the limitations of protein based delivery of dominant negative toxin inhibitors, the investigation of alternative methods for achieving the desired protection against toxin producing microorganisms is warranted.

Disclosure of the Invention

The present invention provides methods and compositions for inducing protective responses that are capable of reducing the effects in subject individuals of exposure to protein toxins.

In one aspect, the present invention provides a method for inducing a protective response to an antigenic toxin in a subject individual comprising delivering to a tissue of said individual a DNA sequence encoding a promoter region and at least one fragment of said antigenic toxin, wherein the fragment contains at least one dominant negative mutation. The DNA sequence is delivered under conditions that induce the expression of the fragment in the cells of the tissue in a manner sufficient to block at least a portion of the activity of the antigenic toxin. An alternative embodiment of the invention further includes a processing signal in the DNA sequence that promotes the desired distribution or tissue localization of the expressed toxin fragment containing the dominant negative mutation.

The present methods and compositions include those wherein DNA sequences encoding modified forms of one or more components of a toxin are expressed and processed in the cells of a subject individual in a manner effective to modulate and preferably block the activity of the antigenic toxin. Additionally, DNA sequences encoding antigenic toxin components capable of stimulating cellular and/or humoral immune responses may be employed to provide enhanced protection against the antigenic toxin by inducing immune effectors against toxin components and, when relevant, against microorganisms producing the antigenic toxins. As an additional aspect of the invention, the DNA sequence includes appropriate means for facilitating the desired distribution of the dominant negative toxin fragment within the body of the subject individual. In addition, the invention includes methods for enhancing the toxin protective response achieved through expression of the dominant negative toxin fragment. Other aspects of the invention include compositions comprising DNA sequences encoding promoter regions, processing signals, dominant negative toxin fragments and/or fusion proteins.

Brief Description of the Drawings

Figure 1 is a graphic depiction comparing the survival results of the subjects of experimental Group 1 with the negative control Group 7; Figure 2 is a graphic depiction comparing the survival results of the subjects of experimental Group 2 with the negative control Group 7;

Figure 3 is a graphic depiction comparing the survival results of the subjects of experimental Group 3 with the negative control Group 7;

Figure 4 is a graphic depiction comparing the survival results of the subjects of experimental Group 4 with the negative control Group 7;

Figure 5 is a graphic depiction comparing the survival results of the subjects of experimental Group 5 with the negative control Group 7;

Figure 6 is a graphic depiction comparing the survival results of the subjects of experimental control Group 6 with the negative control Group 7; and Figure 7 is a graphic depiction of the antibody titers developed in the subjects of experimental Groups 1-4.

Detailed Description of the Invention

The present invention provides methods and compositions for inducing protective responses in a subject individual against the effects of protein toxins.

In one aspect, the present invention provides a method for inducing a protective response to an antigenic toxin in a subject individual comprising delivering to a tissue of said individual a DNA sequence encoding a promoter region and at least one fragment of said antigenic toxin, wherein the fragment contains at least one dominant negative mutation. The DNA sequence is delivered under conditions that induce the expression of the fragment in the cells of the tissue in a manner sufficient to block at least a portion of the activity of the antigenic toxin. An alternative embodiment of the invention further includes a processing signal in the DNA sequence that promotes the desired distribution or tissue localization of the expressed toxin fragment containing the dominant negative mutation.

The present methods and compositions include those wherein DNA sequences encoding modified forms of one or more components of a toxin are expressed and processed in the cells of a subject individual in a manner effective to modulate and preferably block the activity of the antigenic toxin. Additionally, DNA sequences encoding antigenic toxin components capable of stimulating cellular and/or humoral immune responses may be employed to provide enhanced protection against the antigenic toxin by inducing immune effectors against toxin components and, when relevant, against microorganisms producing the antigenic toxins. As an additional aspect of the invention, the DNA sequences include appropriate means for facilitating the desired distribution or tissue, localization of the dominant negative toxin fragment within the body of the subject individual. In one embodiment, the desired distribution or tissue localization is achieved using an encoded processing signal associated with a sequence encoding the dominant negative toxin fragment. The specific type of processing signal (e.g. secretion, endosomal location, membrane anchoring) is selected to facilitate the distribution or tissue localization of the expressed dominant negative toxin fragment to a target location in the tissues and/or intracellular compartments of the subject individual.

Alternatively, the desired distribution or tissue localization of the dominant negative toxin fragment may be achieved through the use of a fusion protein, wherein the dominant negative toxin fragment is fused to an appropriate targeting signal, comprising a ligand or antibody with affinity for specific tissue sites in the body of the subject individual. Methods for

the generation of suitable targeting fusion proteins are described in Nissim, A et al, Methods for targeting biologicals to specific disease sites. Trends MoI Med. 2004 Jun;10(6):269-74.

In addition, the invention includes methods and compositions for enhancing the toxin protective response achieved through expression of the dominant negative toxin fragment. These methods include means for enhancing the stability of the dominant negative toxin fragment in vivo. Degradation of the dominant negative protein to an inactive state would have significant implications for the efficacy of the procedure. Increasing the half life of the protein under physiologic conditions in the body of the subject individual offers the prospect for increasing the probability of favorable interaction between the dominant negative toxin fragment and the native toxin and/or relevant receptors. Embodiments of the invention include the use of DNA sequences encoding fusion proteins that comprise the dominant negative toxin fragment linked to a protein sequence designed to increase the stability of the protein (e.g. albumin).

The toxin protective response may also be enhanced by utilizing nucleotide sequences encoding antigenic toxin fragments. The antigenic portions of the dominant negative mutant are selected such that resulting immune responses are reactive to the native form of the toxin protein. In this manner, the toxin protective response can be enhanced through activation of cellular and humoral effectors of the immune system wherein said effectors are capable of further neutralizing the activity of the toxin. By eliciting immune response against the native toxin, embodiments of the invention utilizing these methods and compositions can improve the quality and duration of the protective response achieved.

In cases where the subject individual is infected with a toxin producing microorganism, the disclosed methods may also be combined with additional therapeutic interventions directed at the microorganism itself. These interventions can include the use of additional DNA sequences designed to elicit immune response against the microorganism, as well as the administration of appropriate pharmacological agents capable of inhibiting the microorganism.

Other aspects of the invention include compositions comprising DNA sequences encoding promoter regions, processing signals, dominant negative toxin fragments and/or fusion proteins.

Examples illustrative of the methods of the present invention are provided. Specifically, in vivo delivery of a DNA sequence encoding a dominant negative mutant form of a subunit of anthrax toxin is shown to inhibit the activity of the native form of the multi-component, pore- forming anthrax toxin.

Baccillus anthracis is but one example of an organism which produces a toxin that can cause severe illness in humans. Other organisms which produce multi-component toxins that assemble on the host cell surface in a manner comparable to anthrax toxin include Clostridium spirqforme, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, and Clostridium botulinum.

Furthermore, there are important cytolytic toxins, such as the α-hemolysin from Staphylococcus aureus and aerolysin from Aeromonas hydrophila, which, like anthrax toxin, assemble into heptameric ring structures at the host cell surface and form pores in the cell membrane. Other bacterial toxins such as such as cholera toxin and Shigella toxin, are assembled within the confines of the bacterium, and so interference by mutant dominant-negative subunits is rendered more difficult, since it must be based solely on competitive binding with the relevant toxin receptors expressed on host cells.

Still other bacteria, such as Listeria monocytogenes and Bordello, are capable of infecting cells and producing toxins which act intracellularly. In these more problematic cases, practice of the present invention necessitates the use of means for delivering the DNA sequence to the infected or vulnerable cell types with an appropriate processing signal enabling the dominant negative toxin fragment to be distributed to the appropriate intracellular compartment.

The methods and compositions of the invention can be applied to ameliorate the effects of a variety of protein toxins including zootoxins, phytotoxins, bacterial toxins, and mycotoxins.

In general, the methods and compositions described herein are appropriate for use in subject individuals that are at risk for exposure to the toxins or toxin producing organisms (i.e. prophylactic use) as well as those already exposed to the toxins or toxin producing organisms (i.e. therapeutic use). The subject individual is most preferably a human. However, the methods and compositions are also appropriate for use in animal species, preferably mammalian species. As used herein:

The term "toxin" is defined as a protein-based agent produced by the expression of a polynucleotide sequence in a plant, animal, or pathogenic microorganism which engenders at least one deleterious effect in the host individual. The term "toxin fragment" refers to a polypeptide comprising at least a portion of the amino acid sequence of a toxin. Specifically, a "toxin fragment" includes any contiguous portion

of the polypeptide sequence at least 7 amino acids in length up to and including the entire polypeptide sequence.

The term "antigenic toxin" refers to a toxin, that, when administered to a subject individual in the proper context, is capable of inducing an immune response. The term "mutation" refers to an alteration in the amino acid sequence characteristic of the native form of a given polypeptide. A "deletion mutation" is an alteration in the native form of a polypeptide sequence resulting from the eliminiation of an amino acid in the sequence. A ' "substitution mutation" is an alteration in the native form of a polypeptide sequence resulting from a change from one amino acid for another (e.g. lysine for asparatic acid). The term "dominant negative mutation" is a change in the amino acid sequence of a biologically active polypeptide which produces an inactive variant of the polypeptide, which, by interacting with active forms of the polypeptide and / or the cellular machinery of a subject individual, alters the interaction of the active polypeptide with the cellular machinery, thereby modulating the activity of the active polypeptide in the subject individual. The term "cellular machinery" refers to intracellular and extracellular components relevant to the cellular processes including metabolism, molecular transport, protein expression and processing, and replication.

The term "fusion protein" refers to the product of the expression of two or more nucleic acid sequences isolated from multiple genes and joined such that they retain their correct reading frames but make a single protien.

The term "processing signal" refers to a polynucleotide sequence which, when expressed in conjection with a desired polypeptide in the cells of a subject individual provides the cellular machinery with instructions regarding the distribution or tissue localization of the polypeptide.

DNA Based Production of Toxin Fragments with Dominant Negative Mutations

In accordance with the present invention, the subject individual is treated by delivering a DNA sequence to cells of one or more target tissues of the subject individual. The DNA sequence includes a promoter region and a region coding for the expression of least one fragment of the toxin of interest wherein one or more dominant negative mutations have been introduced. The objectives of introducing negative mutations in the toxin are two fold: First, the mutation(s) ensure that the mutant toxin fragment is inactive and cannot lead to pathology in the subject

individual. Second, the mutation(s) should allow the mutant toxin fragment to interact and/or compete with the native form of the toxin in a manner that modulates the activity of the toxin.

Numerous dominant negative mutations have already been identified for relavant toxins, including toxins produced by Bacillus anthracis (Singh Y, Chaudhary VK, Leppla SH. A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo. J Biol Chem. 1989 Nov 15; 264(32):19103-7., Yan M, Collier RJ. Characterization of dominant-negative forms of anthrax protective antigen. MoI Med. 2003 Jan-Feb; 9(l-2):46-51), Escherichia coli (Wai SN, et al. Characterization of dominantly negative mutant CIyA cytotoxin proteins in Escherichia coli. J Bacteriol. 2003 Sep; 185(18):5491), and Helicobacter pylori (Vinion-Dubiel AD, et al. A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J Biol Chem. 1999 Dec 31; 274(53): 37736-42. McClain MS, et al. A 12-amino-acid segment, present in type s2 but not type si Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation. J Bacteriol. 2001 Nov; 183(22):6499-508, Sundrud MS, et al Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion. Proc Natl Acad Sd USA. 2004 May 18; 101(20):7727-32).

While it will be understood that the development of an appropriate dominant negative toxin fragment will be required in order to practice the methods of the current invention, the process for identifying a dominant negative mutant is straightforward. One of ordinary skill in the art can readily assess whether a specific toxin is amenable to a dominant negative mutation strategy based on the toxin's mode of action in a subject individual.

Once a candidate toxin or toxin component has been identified, one can then utilize established methods for creating and screening candidate mutants for the desired dominant negative activity (see, for example, U.S. Pat. No. 5,580,723; Sambrook et ah, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, and Mourez M, et al. Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis. Proc Natl Acad Sd USA. 2003 Nov 25; 100(24):13803-8). The primary objective of in vitro screening is to identify dominant negative candidates exhibiting appropriate binding characteristics and favorable neutralizing activity. Candidates exhibiting sufficient activity in vitro are assembled into DNA vector sequences capable of being expressed in vivo. Final selection of candidate mutants is performed based on the administration of these vector sequences encoding the dominant negative toxin fragments in animal models to assess their

activity in vivo. Preferably, these studies will utilize the method and route of DNA administration intended for use in humans. The principal. criteria for selection of candidates is toxin neutralizing activity as demonstrated using accepted in vivo challenge models (see, for example, Stiles BG, Garza AR, Ulrich RG, Boles JW. Mucosal vaccination with recombinantly attenuated staphylococcal enterotoxin B and protection in a murine model. Infect Immun. 2001 Apr; 69(4):2031-6., Zhao P, Liang X, Kalbfleisch J, Koo HM, Cao B. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum Antibodies. 2003; 12(4): 129-35., and Woody MA, Krakauer T, Stiles BG. Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model. Vaccine. 1997 Feb; 15(2): 133-9.) as well as an acceptable safety profile as assessed using standard toxicological readouts (see U.S. FDA Guidance for Industry Considerations for Plasmid DNA Vaccines for Infectious Disease Indications, www.fda.gov). Secondary selection criteria include the antigenicity / immunogenicity of the encoded protein. It should be noted that other similar methods for creating and testing dominant negative variants of a toxin will be apparent to one of ordinary skill in the art and can be utilized without departing from the scope of the present invention.

Preferably, the DNA sequence is administered orally, by inhalation, or by intravascular, intramuscular, subcutaneous, or intradermal injection. Although the methods of the invention can be practiced using unformulated DNA (i.e. "naked" DNA), preferably, means for enhancing the intracellular uptake and expression of the DNA sequence are utilized. Appropriate means for enhancing DNA delivery include but are not limited to: transfection facilitating formulations, particle driven bombardment, and in vivo electroporation, as well as the use of recombinant viral vectors (e.g. adenovirus, adeno-associated virus, vaccinia virus, and pox virus). Once delivered to the cells in the target tissue(s), the DNA sequence is expressed, resulting in the production of one or more dominant negative toxin fragments.

During an episode of toxin exposure, the dominant negative toxin fragment(s) produced from the DNA sequence in the subject individual's cells will interact with the cellular machinery of the subject individual and / or the native toxin to modulate the activity of the toxin, thereby ameliorating at least a portion of the deleterious effects of toxin exposure on the subject individual.

In order to maximize the degree of protection afforded the subject individual, the methods and compositions of the present invention should be applied prior to or as soon after the

onset of toxin exposure as can be practically accomplished. The beneficial effects of the procedure are dependent on engendering sufficient interaction between the dominant negative toxin fragment(s) and the native toxin to reduce the deleterious effects of the toxin on the subject individual. Variables which determine whether a protective response can be achieved using the described methods include (but are not limited to): the magnitude and duration of dominant negative toxin fragment production, the tissues to which the fragment is distributed, the affinity of the dominant negative fragment for the native toxin as well as the molar fraction of dominant negative toxin fragment necessary to neutralize the native toxin, the sensitivity of the subject individual's species to the native toxin, and the mechanism by which the toxin exerts its deleterious effects .

In order to accomplish the intended toxin neutralization, it is necessary for the dominant negative mutant to interact with the native toxin and/or relevant host cells. This interaction is achieved by expressing the dominant negative fragment at sufficient levels either in the tissue where the toxin is present (e.g. blood, liver, lung, skin etc.) or alternatively in a remote tissue followed by distribution of the dominant negative fragment to the tissue where the toxin is present. This distribution is facilitated through the use of an appropriate protein processing signal associated with the toxin fragment nucleotide sequence. The specific processing signal sequence is selected such that the toxin fragment is distributed appropriately within the tissues of the subject in order to best counteract the effects of the toxin. Selection of an appropriate processing signal is dependent on a number of factors including, but not limited to: the tissues susceptible to toxin activity (e.g. cells of the lung, liver, or intestine, or general non-specific cytotoxicity), the distribution of the toxin within the body (e.g. local, regional, or systemic), the mechanism for toxin activity (e.g. intracellular or extracellular), and the tissues in which the DNA sequence is expressed. In order to maximize the likelihood of proper processing by the cells of the subject organism, the signal(s) included in the DNA sequence are isolated from species related to that of the subject organism, with a close relation preferable (e.g. the utilization of secretion signal from a mammalian protein to treat a human subject and more preferably a human protein to treat a human subject).

. The desired distribution of the dominant negative mutant may also be facilitated through the association of a nucleotide sequence encoding a targeting polypeptide with the nucleotide sequence encoding the dominant negative toxin fragment. The hybrid gene sequence is constructed such that expression of this hybrid sequence results in the formation of a fusion

protein retaining the desired activity of the dominant negative fragment. By fusing a targeting polypeptide with affinity for a target tissue (or tissues) to the dominant negative toxin fragment, the uptake and retention of the dominant negative polypeptide at relevant disease sites can be improved. Methods for identification of appropriate targeting polypeptides and construction of fusion proteins are straightforward and well known in the art (see for example Nissim A, et al. Methods for targeting biologicals to specific disease sites. Trends MoI Med. 2004 Jun; 10(6):269-74).

In one embodiment, the methods and compositions of the present invention can be applied to address the pathology associated with exposure to a multimeric toxin, wherein multiple polypeptides interact to form the active toxin molecule. The multimeric toxin may be comprised of two or more polypeptides of a single type (e.g. dimer, trimer, or tetramer toxin) or may be formed through the arrangement of two or more different polypeptides (multi-component or multi-gene toxins). The methods of the present invention can be used to provide a protective response to a given multimeric toxin by providing one or more mutated toxin polypeptides which interact with the native polypeptides to prevent formation of the active toxin.

For example, the neurotoxin α-latrotoxin is a multimeric toxin produced by the black widow spider, α-latrotoxin monomers associate to form dimers and tetramers, the structure of which have been defined (Orlova EV, Rahman MA, Gowen B, Volynski KE, Ashton AC, Manser C, van Heel M, Ushkaryov YA Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores. Nat Struct Biol. 2000 Jan; 7(1):48- 53). These tetramer structures are capable of forming pores in lipid bilayers. Toxin activity is based on pore formation in neurons, the pores interfere with normal calcium ion transport, thereby disrupting the function of the affected neurons resulting in adverse cardiovascular and neuromuscular symptoms. The in vivo expression of a dominant negative form of the α- latrotoxin polypeptide according to the methods disclosed in the present invention would interfere with the formation of functional pore structures and/or interaction of the tetramer with the membrane of target cells.

In addition to multimeric toxins comprised of a oligomers of a single polypeptide, there are many multimeric toxins comprised of multiple components wherein one component (subunit A) mediates the enzymatic activity of the toxin, while another component (subunit B) binds to a receptor on the membrane of the host cell, thereby allowing the subunit A to be transferred into

the host's cells. Toxin activity is dependent on appropriate interaction between the toxin subunits themselves as well as proper interaction with the host cell.

In one embodiment, the present invention may be practiced by utilizing a mutated form of the subunit A wherein the mutation(s) produce an enzymatically inactive form of the subunit which retains the ability to bind to subunit B. In another embodiment, a mutated form of subunit B is utilized. Desirable modifications to subunit B activity would include retention or enhancement of binding to host cell receptors with a reduction in binding affinity for subunit A. Alternatively, subunit B can be modified to reduce binding affinity to the host cell while retaining affinity for active subunit A. In each of these embodiments, the mutated form of the specific subunit reduces toxin activity through intereaction and/or competition with the active subunit. It should be noted that the use of multiple dominant negative subunits may be useful to further reduce toxin activity, but the mutations must be selected for compatibility or they may interact with each other, thereby reducing the protective effect.

By way of example, the bacterium Baccillus anthracis produces two exotoxins, the edema toxin (EdTx) and the lethal toxin (LeTx). The toxins are made of several components that are non-toxic individually, but act in combinations to produce disease-like symptoms and death. LeTx is a combination of protective antigen (PA) and lethal factor (LF). EdTx is a combination of protective antigen (PA) and edema factor (EF). LF and EF enzymatically modify molecular targets within the host cell cytosol, while PA transports them from the cell surface to that compartment. The three components of the toxins are secreted individually from the bacterium and interact at the surface of host cells to enable intracellular uptake of LF and EF.

The skin is the most frequent route of infection with B. Anthracis, resulting in a local or loco-regional infection usually successfully treated with antibiotics. Rarely, gastrointestinal anthrax is observed after ingestion of contaminated meat; this disease can be lethal, due to toxin- induced local hemorrhagic necrosis, as well as septicemia. Finally, the inhalation of spores can result in pulmonary anthrax, which is often fatal: inhaled spores multiply in the mediastinal lymph nodes, resulting in local hemorrhagic necrosis, fever, severe respiratory distress and shock. Although concentrated at the site of infection, the toxin components can be taken up into the blood and lymph and circulated within the organism, leading to destruction of tissues away from the infection site.

According to the methods of the present invention, the toxin activity can be neutralized through the administration of a DNA sequence encoding one or more dominant negative forms of the anthrax toxin subunits. Since it binds to both EF and LF and is necessary for the toxin to achieve uptake into host cells, the PA subunit is an appealing target for development of a dominant negative inhibitor.

Desirably, the DNA sequence encoding the dominant negative form of the toxin fragment can be administered directly to the tissues at the site of infection (e.g. inhaled delivery to the lungs following inhalational exposure to anthrax spores). However, practical or technical circumstances may limit delivery to surface accessible tissues such as muscle or skin. In such cases, the DNA sequence should be designed to allow a sufficient amount of the expressed toxin fragment to be distributed from the transfected cells to tissue locations of the body likely to have significant presence of the toxin (e.g. lung tissue, blood, lymph, liver, and kidney). This is preferably accomplished through the use of a processing signal appropriate for secretion of the dominant negative toxin fragment from the transfected cells. The toxin fragment can then be taken up from the extracellular space surrounding the transfected cell into the lymph and/or blood, thereby allowing systemic circulation of the protein. The desired toxin protective effect is achieved through the interaction of the dominant negative fragment of the PA gene with the native PA protein, thereby inhibiting formation of the functional heptameric ring structure As an another example, a toxin produced by the bacterium Vibrio cholerae (cholera toxin) binds to the membranes of enteric cells allowing a toxin subunit to be taken up into the cell thereby resulting in changes in water and ion transport that lead to severe diarrhea, which if untreated can rapidly lead to death due to dehydration / hypotension. Unlike anthrax toxin, the cholera toxin subunits (A and B) are assembled in the bacterium prior to secretion. Thus, the methods of the present invention would be most effectively applied by providing a mutated toxin multimer; an effective dominant negative mutant for cholera toxin would likely require competiteive binding with the appropriate toxin binding site located on the hosts' enteric cells (for humans, the oligosaccharide of GMl ganglioside). If the methods of the present invention were practiced by using an appropriate method for intramuscular delivery of a DNA sequence (reviewed in Herweijer H, Wolff JA. Progress and prospects: naked DNA gene transfer and therapy. Gene Ther. 2003 Mar; 10(6):453-8., McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscles: current status. BioDrugs. 2004; 18(3): 155-65. , and Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene Ther. 2002 Dec; ^«

9(24): 1647-52.) encoding a dominant negative form of cholera toxin, then preferably a strong secretion signal would be included in the DNA sequence, thereby allowing the dominant negative polypeptide to be taken up into systemic circulation and distributed to the intestinal tract, where it can compete with the active toxin to modulate toxin uptake into the cells of the mucosal epithelium. In contrast, if the DNA sequence was delivered orally to the cells of the intestinal lining (see reference Rothman S, Tseng H, Goldfine I. Oral gene therapy: a novel method for the manufacture and delivery of protein drugs. Diabetes Technol Ther. 2005 Jun; 7(3):549~57), then the processing signal would preferably be selected for retention of the dominant negative polypeptide in the local tissues (e.g. using a membrane binding or localization signal to faciliate interaction between the negative mutant and the binding site for the toxin).

An additional illustrative example of the present invention involves the neutralization of an intracellular toxin. The bacterium Listeria monocytogenes is capable of surviving and proliferating in phagocytes at least partially through the activity of the toxin listeriolysin O. Thus, neutralization of the listeriolysin O toxin is best achieved by targeting a nucleic acid sequence encoding the toxin to the phagocytes of the subject organism. To best achieve the desired toxin neutralization, the nuclieic acid sequence must encode an intracellular processing signal directing the dominant negative toxin fragment to the appropriate intracellular compartment (e.g. the phagosome or lysosome).

Enhancement of the Toxin Protective Response

It should be noted that the kinetics (i.e. magnitude and duration) of endogenous expression of the toxin fragment will depend on a number of factors including but not limited to the functional components utilized in the DNA sequence (e.g. promoter, enhancer), the species of the subject individual, the method of DNA sequence delivery, the DNA dose, and the target tissue. Typically, expression of the toxin fragment will persist at target levels for 1 - 60 days and most commonly from 5 - 21 days. Although effective use of a given DNA sequence encoding a dominant negative toxin is facilitated through analysis of the magnitude and duration of expression following in vivo administration, the process for characertizing the duration of gene expression is straightforward. After constructing the appropriate DNA sequence, the sequence can be administered in one or more animal models using the delivery approach and route of administration most closely modeling that intended for use in humans. The level of expression

can be assessed by serial measurement of standard assays for the polypeptide product (e.g. ELISA) and/or mKNA expression (e.g. real time RT-PCR) to quantiate the level of expression of the dominant negative toxin fragment. This data can be utilized in conjunction with data generated from in vivo toxin challenge models in order to provide an assessment of the level and duration of protection provided by endogenous expression of the dominant negative toxin fragments). If this duration is insufficient to provide the desired toxin protective response, the quality and duration of the protective response induced in the subject individual can be improved through selection / modification of the DNA sequence encoding the dominant negative toxin fragment. In one embodiment the DNA sequence may be modified to enhance the stability of the dominant negative polypeptide under the physiologic conditions characteristic of the subject organism. Enhancing the half-life of the dominant negative toxin fragment will result in the presence of the protein at higher levels and for longer duration than achievable with the unmodified version of the protein, thereby increasing the potential for providing protective response. By way of example, the DNA sequence may be modified such that its expression results in the production of a fusion protein comprising the DNA sequence and at least a portion of a sequence encoding serum albumin. Preferably, the fusion protein is constructed using the serum albumin DNA sequence isolated from the subject organism in order to reduce complications associated with immunogenicity. Fusion proteins comprising recombinant polypeptides fused to serum albumin are straightforward to construct and have exhibited increased serum half-lives when administered by injection (see, for example, Melder RJ, Osborn BL, Riccobene T, Kanakaraj P, Wei P, Chen G, Stolow D, Halpern WG, Migone TS, Wang Q, Grzegorzewski KJ, Gallant G. Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice. Cancer Immunol Immunother. 2005 Jun, 54(6):535-47. Epub 2004 Dec 8; Sung C, Nardelli B, LaFleur DW, Blatter E,

Corcoran M, Olsen HS, Birse CE, Pickeral OK, Zhang J, Shah D, Moody G, Gentz S, Beebe L, Moore PA. An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates. J Interferon Cytokine Res. 2003 Jan, 23(l):25-36.; Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther. 2002 Nov, 303(2):540-8).

In another embodiment, the toxin protective response may be enhanced through the inclusion of toxin fragments that are antigenic. By delivering DNA sequences capable of expressing one or more antigenic toxin fragments in a manner conducive to the induction of immune response, the immune effectors of the subject individual can provide a qualitative and quantitative enhancement in the toxin protective response.

Experimental Results

In the experimental disclosure which follows, all weights are given in grams (g), milligrams (mg), micrograms (μg), nanograms (ng), or picograms (pg), all amounts are given in moles (mol), millimoles (mmol), micromoles (μmol), nanomoles (nmol), picomoles (pmol), or femtomoles (fmol), all concentrations are given as percent by volume (%), proportion by volume (v:v), molar (M), millimolar (rnM), micromolar (μM), nanomolar (nM), picomolar (pM), femtomolar (fM), or normal (N), all volumes are given in liters (L), milliliters (mL), microliters (μL), or cubic centimeters (cc), and linear measurements are given in millimeters (mm), micrometers (μm), or nanometers (nm), unless otherwise indicated. The. following examples demonstrate the practice of the present invention for the in vivo delivery of nucleotide sequences to provide a toxin protective response.

The following examples demonstrate the practice of the present invention in providing a protective response against anthrax toxin. The example is comprised of seven groups of eight Swiss- Webster mice. Groups 1-5 are administered plasmid DNA encoding the PA83 DNA sequence wherein the sequence has been codon optimized for mRNA expression and stability in mammalian cells and a dominant negative mutation has been introduced. The plasmid is administered to Groups 1, 2, and 5 by electroporation-mediated intramuscular DNA injection. The plasmid is administered to Groups 3 and 4 by high volume (~ 2 cc) intravenous injection designed transfect the liver. Group 6 is a control that is administered plasmid DNA encoding the same PA83 DNA sequence without the dominant negative mutation by electroporation mediated intramuscular injection. Group 7 is a negative control in which no DNA is administered.

In order to assess the relative potency of two different PA83 mutants, Groups 1, 3, and 5 are administered the PA83 DNA sequence in which the codon for Aspartic Acid 425 is substituted for by a Lysine codon; the gene product is denoted PA83 (D425K). Groups 2 and 4 are administered the PA83 DNA sequence in which the codon for Aspartic Acid 425 is deleted;

the gene product is denoted PA83 (D425del). The results obtained with each of these groups are compared to those observed with non-mutant PA83 (group 6).

In addition, the results obtained with intramuscular delivery of the PA83 mutants (Groups 1 and 2) are compared to those observed following intravenous delivery and uptake in the liver (Groups 3 and 4) to determine the most effective route of administration. Finally, the results from Groups 1 and 5 allow comparison of response observed with a processing signal (TPA secretion) associated with the PA83 gene to that achieved with the native form of the gene.

Procedures / Methods

Anthrax toxin is made of components that act in combination to produce disease-like symptoms (Little SF, Ivins BE, Microbes Infect 1:131-139, 1999). The combination of anthrax protective antigen (PA) and lethal factor (LF) is called lethal toxin. IV administration of lethal toxin in rodents results in the death of the animals. This experimental model has been used to evaluate the efficacy of vaccination methods as well as therapies based on administration of therapeutic agents (e.g. anti-PA monoclonal antibodies) (Gu ML, Leppla SH, Klinman DM. Vaccine 17:340-4, 1999; Price BM, Liner AL, Park S, Leppla SH, Mateczun A, Galloway DR. Infect Immun 69:4509-15, 2001; Sellman BR, Mourez M ₅ Collier RJ. Science 292:695-697, 2001 ; Tan Y, Hackett NR, Boyer JL, Crystal RG. Hum Gene Ther 14: 1673-82, 2003, Zhao P, Liang X, Kalbfleisch J, Koo HM, Cao B. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum Antibodies. 2003, 12(4): 129-35). This model is utilized to assess toxin protective response following administration of DNA sequences encoding dominant negative mutant forms of the PA83 gene.

The plasmid is administered intramuscularly by electroporation in the animals of groups 1, 2, 5, and 6. Briefly, the DNA injection and electric field application are administered (using the TriGrid™ integrated application system (Ichor Medical Systems, San Diego, California) consisting of a 4 electrode TriGrid™ array with 2.5 mm intraelectrode spacing interfaced with a Becton Dickinson 0.3 cc syringe. The integrated applicator is inserted into the tibialis muscle of the animal followed by the injection of DNA. Immediately thereafter electrical fields of duration and intensity sufficient to increase the intracellular uptake of DNA are propagated at the site of DNA injection.

Plasmid is administered intravenously in the animals of Groups 3 and 4. Briefly, a 2.0cc injection volume is administered over the course of 10-15 seconds into the tail vein of the animal, resulting in high level transfection of the liver. A detailed description of the technique is provided in (Feng DM, He CX, Miao CY, Lu B, Wu WJ, Ding YF, Xue JL. Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo. Clin Exp Pharmacol Physiol. 2004 Dec, 31(12): 850-5). Two days following plasmid administration, all mice receive intravenous injection of premixed 60μg of PA and 25μg of LF in PBS in a volume of lOOμL ( PA and LF from List Biological Laboratories, Campbell, CA). Based on a previous report (Ezzell JW, Ivins BE, Leppla SH. Infect Immun 45:761-767, 1984), this dose of toxin corresponds approximately to 5 time the LD ₅₀ . Following toxin administration, the mice are monitored for 30 days for general health status, including grooming, mobility, and respiratory movements. Animals displaying serious signs of morbidity are humanely euthanized.

Twenty-two days after toxin injection, blood is collected by retro-orbital bleed from the surviving animals. Serum is recovered by centrifugation. Anti-PA antibody titers are measured in each individual animal by ELISA. Briefly, serial dilutions of serum samples are added to 96 well plates coated with 100ng/well recombinant PA (obtained from List Biological Laboratories, Campbell, CA). Biotinylated anti-mouse IgG (KPL, Inc., Gaithersburg, MD), streptavidin- horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, CA) and SureBlue TMB microwell peroxidase substrate (KPL) are used for detection. OD reading at 450nm is performed using a Model 550 microplate reader (BioRad, Hercules, CA). Antibody titer is calculated as the reciprocal of the sample dilution yielding an OD ₄₅₀ of 0.600.

Results

The survival results from groups of mice receiving plasmid (filled symbols) and the group receiving no plasmid (empty symbols) are depicted in Figures 1 - 6. Analysis of the data depicted in Figures 1 - 6 indicates that a significant toxin protective response is observed in Groups 1, 2, 3, and 4 (p value < 0.05 by Wilcoxon's Rank Sum test). In contrast, the survival results for Groups 5 and 6 are not significantly different from those observed for Group 7 (no treatment).

Induction of anti-PA immune response is assessed in the surviving animals from Groups 1, 2, 3, and 4. Figure 7 depicts the anti-PA antibody titers observed approximately 3 weeks following administration of the DNA sequence. As the Figure illustrates, Groups 1 and 2 exhibit the highest antibody titers among the tested groups. Antibody responses of lower magnitude were observed in Groups 3 and 4.

Significant toxin protective response is observed for all groups (Groups 1 - 4) administered a DNA sequence encoding a dominant negative form of the PA83 gene associated with a secretion signal (TPA). The most pronounced toxin protective response is observed in Groups 1 and 3 which comprise administration of the DNA sequence encoding PA83 (D425K) with a processing signal (TPA Secretion) directing secretion of the protein from the intracellular compartment. There is no statistically significant difference in survival data observed between the two routes of DNA administration. However, there is a significant improvement in the anti- PA serum antibody level observed in Groups 1 and 2, suggesting that the intramuscular route of administration may be preferable to intravenous for achieving high level immune response.

Overall, Group 2 exhibits a lower toxin protective response than Group 1, suggesting that the PA83 (D425K) mutant toxin fragments have superior activity compared to the PA83 (D425del) mutant fragments when administered intramuscularly. However, there is no difference in activity detectable between the (D425K) and (D425del) mutants following intravenous delivery of the DNA sequence.

The decreased level of toxin protection observed in comparing Groups 1 - 4 with Group 5 indicates the enhancement in toxin protective effect associated with the presence of appropriate dominant negative mutations in the amino acid sequence of the encoded toxin fragment. Comparison of the results from Groups 1 and 6 illustrates the importance of providing a processing signal to enable appropriate distribution of the encoded fragment within the body of the subject individual.