Title of Invention	BACTERIAL DELIVERY SYSTEM
Abstract	Helicobacter based preparations coraprising a pharmacologically active molecule of interest are disclosed, as well as methods of preparing and using said preparations. In particular, Helicobacter pylori vectors, vector plasmids and recombinant cells that include a sequence encoding a pharmacologically active molecule of interest useful in therapeutic treatments and/or vaccination against disease are provided. Delivery of the pharmacologically active molecules is provided at the mucosal surface, such as the gastric mucosa or nasal membranes, to provide effective and continuous delivery of a pharmacologically active agent. Vectors and shuttle vector constructs are also provided.

Title of Invention

BACTERIAL DELIVERY SYSTEM

Abstract

Helicobacter based preparations coraprising a pharmacologically active molecule of interest are disclosed, as well as methods of preparing and using said preparations. In particular, Helicobacter pylori vectors, vector plasmids and recombinant cells that include a sequence encoding a pharmacologically active molecule of interest useful in therapeutic treatments and/or vaccination against disease are provided. Delivery of the pharmacologically active molecules is provided at the mucosal surface, such as the gastric mucosa or nasal membranes, to provide effective and continuous delivery of a pharmacologically active agent. Vectors and shuttle vector constructs are also provided.

Full Text	BACTERIAL DELIVERY SYSTEM FIELD OF THE INVENTION The present invention relates to the delivery of biologically active agents in vivo. In particular, the present invention relates to treating, palliating or preventing diseases using a bacterial delivery system for delivering biologically active agents directly to an anatomical site in vivo. In one embodiment, the bacterial delivery system comprises Helicobacter or bacterium exhibiting Helicobacter features for delivering heterologous nucleic acid into an animal or animal cell, wherein the heterogonous nucleic acid is expressed. In a further embodiment the Helicobacter has been engineered to contain a DNA vector that encodes heterogonous nucleic acid, wherein upon infection the nucleic acid vector is expressed such that the biologically active agent is delivered to the animal body, especially at the mucosa. BACKGROUND TO THE INVENTION There is a continuing need for long-term delivery of pharmacologic and immunologic agents to individuals with both congenital and acquired diseases. In most cases, this involves repeated administration of therapeutic compounds several times per day, daily or at intervals. However, it is appreciated that any long-term therapeutic or preventive regime has problems such as compliance, side effects and drug resistance. Consequently, there is a constant need to identify treatments, which will be usually 100% effective, free from side effects and cheap. One form of delivery that has been investigated in recent times for various therapeutic and prophylactic agents has been the use of microorganisms. Genes of interest from various organisms including bacteria, viruses, parasites as well as mammals have, for example, been cloned into a variety of bacteria, viruses and mycobacteria for the purpose of directing these micro-organisms to express foreign protein or impart certain desired properties. These microorganisms have been used in vaccination programs, gene-replacement therapies and therapeutic composition delivery. Microorganisms have also been used to transform animal (host) cells in vivo. Host cell transformation can be accomplished using gene-delivery vectors comprising replication incompetent viruses (see for example U.S. Pat. No. 5,824,544), naked DNA, (see for example U.S. Pat. No. 6,261,834), liposomes containing recombinant expression cassettes (see for example U.S. Pat. No. 6,271,207). Other molecular-based therapeutic composition delivery approaches include using replication incompetent recombinant viruses designed to express heterogonous surface proteins (see, for example, U.S. Pat. No. 6,376,236). In recent times, pharmaceutical researchers have also attempted to develop methods for in vivo therapeutic composition expression using recombinant organism-based vectors, inanimate vectors and naked DNA. Examples of recombinant organism-based vectors include recombinant bacteria (see for example U.S. Pat. No. 5,547,664) and viruses such as alphaviruses (see for example U.S. Pat. No. 6,391,632), vaccinia viruses (see for example U.S. Pat. No. 6,267,965), adenoviruses (see for example U.S. Pat. No. 5,698,202) and adenovirus associated virus (AAV) (see for example U.S. Pat. No. 6,171,597). Inanimate vectors include lipidic gene delivery vector constructs such as DNA/cationic liposome complexes, DNA encapsulated in neutral or anionic liposomes, and liposome-entrapped, polycation-condensed DNA (LPDI arid LPDII) . (see Ropert, 1999, Braz J Med Bio1 Res, 32 (2):163-9). Examples of other genes that have been delivered by various bacteria include cloning the invasion genes of Shigella into the normally non-invasive E. coli rendering the E. coli invasive and therefore more suitable for use as a vaccine strain, or cloning of Plasmodium falciparum malaria genes into Salmonella typhimurium which subsequently express these malaria proteins and, following oral administration of the bacteria, induce specific cytotoxic T cell immunity and protection in mice against malaria challenge (see, for example, Hone et al., 1991, Vaccine, 9:810-816; Tacket et al., 1992, Infect. Immun., 60:536-541; Hone et al., 1992, J. Clin. Invest., 90:412-420; Chatfield et al., 1992, Vaccine, 10:8-11; Tacket et al., 1992, Vaccine, 10:443-446; and Mims et al., 1993, In: Medical Microbiology, Eds., Mosby-Year Book Europe Ltd., London; Sadoff et al., 1988, Science, 240:336-338; Aggrawal et al., 1990, J. Exp. Wed., 172:1083-1090). Attenuated or less virulent Shigella (see, for example, Noriega et al., 1994, Infect. Immun., 62:5168-5172; US Pat. Appl. No. 20020176848), Salmonella (see, for example, US Pat. No. 6,531,313; US Pat. Appl. No. 20030170211), Listeria (see, for example, Schafer et al., 1992, J. Immunol., 149:53-59; US Pat. Appl. No. 20030008839), and , other bacteria have been given orally to immunise against subsequent infection with more virulent forms of these bacteria. Likewise, attenuated bacterial and mycobacterial organisms such as Bacille Calmette-Guerin (BCG) (Lagranderie et al., 1993, Vaccine, 11:1283-1290; Flynn, 1994, Cell. Molec. Biol., 40(Suppl. l):31-36) have been administered parenterally to protect against related organisms such as M. tuberculosis. Some of the other bacterial species that have been used as vectors include yersinia enterocolitica (van Damme et al., 1992, Gastroenterol., 103:520-531) and Vibrio cholerae (Levine et al., 1994, In: Vibrio cholerae, Molecular to Global Perspective's, Wachsmuth et al.r Eds, ASM Press, Washington, D.C., pages 395-414). Despite all of the research all of the above bacterial delivery systems have technical difficulties, which need to be overcome before these can be used in vivo. Indeed, most of the bacterial systems require the bacteria themselves to produce functional molecules and are dependent on a bacterium which is sufficiently attenuated to be safe for use in humans, but still able to produce biologically active agents. However, all of the attenuated strains of bacteria used previously are not capable' of surviving lengthy periods in vivo, without causing side effects. More importantly, many of these bacterial species are not capable of delivering therapeutic or prophylactic agents to the mucosal epithelial cells of an animal. However, many of the most important therapeutic agents are taken up via the mucosa; therefore there is a need for a bacterial delivery system capable of delivering biologically active agents directly to the mucosa. U.S. Pat. No. 5,877,159 to Powell et al.r describes live bacteria that can invade mucosal cells without establishing a productive infection or causing disease to thereby introduce a eukaryotic expression cassette encoding an antigen capable of being expressed by the animal cells. While this method allows delivery of the DNA vaccine to mucosal surfaces, including easy administration, a concern for vaccine delivery in developing countries, it does not have the advantage of providing amplifiable mRNA encoding the gene of interest. Moreover, the bacterium disclosed in Powell et-al. is not capable of sustained delivery. Non-pathogenic, non-colonising, non-invasive food-grade bacterium Lactococcus lactis has been used previously in the past to deliver agents to the mucosa (see, for example, UK patent GB-2278358B) . However, while Lactococcus lactis is non-invasive it is not capable of establishing a chronic infection, which is capable of delivering continuous therapeutic agents directly into mucosal epithelial cells. Consequently, there is a continuing need for a delivery system, which is capable of establishing a non-invasive, chronic infection in close proximity to the mucosal epithelial cells of an animal such that biologically active agents can be delivered thereto. Moreover, there is a continuing need for improved delivery mechanisms for pharmacologically active molecules at the mucosal surface sufficient to elicit a useful and beneficial immunogenic response. Such would provide an effective in vivo delivery system for pharmacological active agents, as well as an effective method for immunization, i.e., antigen exposure at mucosal surface sufficient to elicit a general humoral and mucosal immune response. SUMMARY OF THE INVENTION The present invention is directed to overcoming the abovementioned challenges and is exemplified in a number of implementations and applications, some of which are summarized below. The inventors have now surprisingly found that Helicobacter and in particular Helicobacter pylori, which is capable of forming a chronic infection, can be used to produce continuous drug delivery. Moreover, Helicobacter of the present invention can be activated and inactivated at various times and because of its chronicity, the Helicobacter can be used to deliver drugs through the gastric mucosa. Helicobacter pylori is a gram-negative spiral shaped bacterium found almost exclusively in the human gastric mucosa. The acidity of the human stomach is an effective barrier to colonization by essentially all bacteria, with the exception of Helicobacter species. H. pylori has the unique ability to colonize and -persist for decades within the human gastric mucosa, despite development of a mucosal inflammatory and immune response. This characteristic renders H. pylori an interesting candidate for the delivery of selected agents though the mucosa. In accordance with some aspects, compositions, methods and systems are provided for preparing and using a Helicobacter-based construct comprising a Helicobacter sequence having a promoter region and a non-Helicobacter sequence encoding a non-Helicobacter pharmacologically active molecule. This construct in some embodiments is described as a vector or a plasmid vector, wherein the promoter sequence is capable of controlling the expression of the non-Hell cojbacter pharmacologically active molecule of interest. Accordingly, in a first aspect the present invention provides a method of delivering one or more biologically active agents to a subject comprising the step of administering to a subject an effective amount, preferably a therapeutically or prophylactically effective amount of, Helicobacter cells or bacterial cells having the features of Helicobacter, which cells express said one or more biologically active agents. In a second aspect, the present invention provides a noninvasive or non-pathogenic Helicobacter cell expressing one or more heterologous biologically active agents. Preferably, the Helicobacter is of the species H. pylori. The biologically active agent can be either homologous to the Helicobacter genus or heterologous to cells of the genus or species of the Helicobacter cells used for delivery. Heterologous agents may be derived from either eukaryotic or prokaryotic sources. In some aspects, the HeJicojbacter-based vector and vector plasmid constructs comprise biologically active agents such as an antigen, organic or inorganic molecule or substance, a pharmacological agent eg a therapeutic agent or prophylactic agent, such as a gene product or gene sequence (isolated nucleic acid). By way of example, such agents may include an immunqregulatory agent, hormone, ligand, an enzyme or an anti-sense RNA, a catalytic RNA, a protein, a peptide or any other molecule which can be present on or released from the bacterial cells so that it may be delivered to an animal or to an animal cell. In some embodiments, the biologically active agent is encoded by a nucleic acid molecule, which is preferably obtained in isolated form, and subsequently inserted into the bacterial delivery vehicle of this invention. It will be appreciated by those skilled in the art that the isolated nucleic acid molecule of the present invention may be cDNA, genomic DNA, RNA, or a hybrid molecule thereof. Preferably, the nucleic acid is cDNA. By way of example, a protein and/or peptide of interest may comprise ghrelin, amylin, insulin, motilin, (3-glucosidase, a chemical chaperone, or other molecule useful in the treatment of Gauchers disease, cell wasting, human immunodeficiency disease (AIDS), appetite suppression, preparations useful in the treatment of diabetes, etc. The isolated nucleic acid is preferably incorporated into an expression vector, which can be transformed into and maintained in the Helicobacter cells. Accordingly, in a third aspect, the present invention provides a recombinant vector for delivering biologically active agents directly to an anatomical site in need thereof. The Hellcobacter cell of the present invention has preferably been adapted to secrete and/or express on its surface the biologically active agents. In a fourth aspect, the present invention provides a vector or construct for delivering in vivo an effective amount of a biologically active agent, comprising: a) a nucleotide sequence encoding a biologically active agent; b) operatively linked thereto, a control or regulatory sequence capable of controlling the expression of the nucleotide sequence of (a) such that the biologically active agent is produced in vivo when the delivery vehicle comprising the vector is delivered to a subject. The vector maybe modified chemically by means of chemical or enzymatic treatment, or in vivo by means of recombinant DNA technology to produce a modified or variant vector. Such a construct may differ from those disclosed, for example, by virtue of one or more nucleotide substitutions, deletions or insertions, but substantially retain a desired biological activity of the construct or nucleic acid molecule, or its encoded product, in accordance with this invention. In another embodiment of the present invention, the Helicobacter vector or construct is provided with a reporter gene(s) expressed from a constitutive promoter cloned into the expression vector and used as a screening tool. Non-limiting examples of reporter genes suitable for use herein include green fluorescent protein (GFP), \|Jgalactosidase, amylase, and chloramphenicol acetyl transferase (CAT). In another embodiment of the present invention the Helicobacter cell is used to deliver a heterologous gene of interest to a subject in need thereof. The gene of interest may encode a therapeutic product (a transgene product), including, but not limited to a peptide hormone (such as, but not limited to a-melanocyte-stimulating hormone (a-MSH), insulin, growth hormone, and parathyroid hormone), a cytokine including, but not limited to an interferon, interleukin (IL)-2, IL-4, IL-10, IL-12, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and erythropoietin (EPO). In still another embodiment, the present invention provides methods for treating, palliating or preventing a disease in a subject. These methods are facilitated with the use of a therapeutic or prophylactic composition. Accordingly, in a fifth aspect, the present invention provides a pharmaceutical composition comprising (a) live nonpathogenic Helicobacter cells expressing and/or secreting a biologically active agent, together with (b) a therapeutically effective carrier. In a sixth aspect the present invention provides a method of treating, preventing or palliating a disease comprising administering to a subject in need thereof an effective amount of a Helicobacter cells of the present invention that express the biologically active agent. Non-limiting examples of subjects in which the present invention may be used, include mammals such as primates, equines, bovines, porcines, ovines and rodents. Also intended are fish and birds. In one embodiment of the present invention the disease being treated, prevented or palliated is cancer, a disease or condition of the immune/hematopoietic system, a disease or condition of the reproductive system, a disease or condition of the musculoskeletal system, a disease or condition of the cardiovascular system, a disease or a condition described as mixed fetal, a disease or a condition of the excretory system, a disease or a condition of the neural/sensory system, a disease or a condition of the endocrine system, a disease or condition of the respiratory system, a disease or condition of the digestive system and a disease or condition associated with connective/epithelial tissue or disease or conditions caused by bacterial, viral or parasitic infection. The present invention provides a variety of pharmaceutically acceptable preparations formulated for delivery to a patient, such as, gastrically, orally, or intranasaly. In particular embodiments, the composition are suitable for delivery at a mucosal surface. In particular embodiments, the composition is suitable for delivery to the mucosal surface of the gut. By way of example, the mucosa may be that of the gastric, vaginal, nasal, oral, or ocular surface, or any other of the body characterized by the presence of a penetrable mucosal surface or lining. In some embodiments, the mucosal surface is the gastric mucosal surface. The various delivery forms of the compositions are readily prepared for use in the practice of the present invention given the specific types and ratios of specific Helicobacter, Helicobacter constructs and other delivery vehicles described herein, and those formulation techniques known to those in the formulary arts, such as are described in Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, which text is specifically incorporated herein by reference. lt is envisioned that the delivery system may be employed in animals, particularly primates, including humans, equines, bovines, ovines, and rodents, fish and birds. It is also anticipated that the preparations may be used on both infants and adults, as well as parentally or for administration to pregnant or lactating animals. The preparations and methods may be further described as suitable for both male and female animals. In yet another aspect, a method is provided for vaccinating an animal. In some embodiments, the method comprises administering a composition comprising a vaccine comprising cells transformed with the Helicobacter- based plasmid vector and/or plasmid vectors as described herein. In other embodiments, the method provides for the delivery of an effective amount of the pharmacologically active molecule of interest sufficient to eliminate or inhibit a disease or physiological condition in the animal, or sufficient to elicit an immune response specific for the pharmacologically active molecule of interest. By way of example, the non-Helicobacter pharmacologically active molecule of interest useful in the vaccine may comprise a mammalian protein, peptide, enzyme, hormone, or any combination of these. In particular embodiments, the pharmacologically active molecule of interest is further defined as a human pharmacologically active molecule of interest. In some embodiments the pharmacologically active molecule of interest is a human pathogen molecule/antigen, human protein antigen, such as amylin or an analog or derivative thereof, or ghrelin, or an analog or derivative thereof. In particular embodiments, the vaccine conveys immunity against the human pathogen, Ebola virus, HIV virus, Marburg virus, influenza virus, and the like. Replication competent vaccines based on attenuated recombinant vesicular stomatitis virus vectors have been described by Jones et al. (2005, Wat. Afed. 11: 786-90) that include Ebola glycoprotein and Marburg glycoprotein. Hence, it is envisioned that constructs using the HelicoJbacter-based vector systems and plasmid vector systems with these and other glycoprotein's associated with human pathogens may also be provided according to the present invention together with the disclosure provided herein. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic diagram of the plasmid construct pHPAl (2.8kb). Figure 2 is a schematic diagram of the plasmid construct pHP3 (3.4kb). Figure 3 is a schematic diagram of the plasmid vector pTMI03-8. Figure 4 shows the structure of sulfasalazine (SSN). Figure 5 is a schematic diagram showing the use of ion exchange resin (Amberlite XE-96) conjugated with a dye (Azure-A). BRIEF DESCRIPTION OF THE SEQUENCES The following nucleic acid and amino acid sequences are referenced throughout the description of the present invention: SEQ ID NO: 1 - Nucleotide sequence of plasmid pHPl (2796 nucleotides); SEQ ID NO: 2 - Nucleotide sequence of pHPl (2796 nucleotides); SEQ ID NO: 3 - Nucleotide sequence of plasmid pHP3 (3444 nucleotides)/ SEQ ID NO: 4 - Hepatitis C virus are antigen (HCV) nucleotide Sequence; SEQ ID NO: 5 - Nucleotide sequence 135 bp (45 amino acids) immunogenic coding sequence from the Hepatitis C virus (HCV) core antigen/ SEQ ID NO: 6 - Nucleotide sequence of the surface exposed loop of the HopE gene (at nt504, aa position 168) of H. pylori; SEQ ID NO: 7 - Upstream primer (29 nucleotides); SEQ ID NO: 8 - Downstream Primer (28 nucleotides); SEQ ID NO: 9 - Oligonucleotide Primer (15 nucleotides). DEFINITION OF TERMS Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter. An "antibiotic resistance gene" as defined herein includes heterologous nucleic acid sequences purposely provided to a vector and used as a selection system. The term "antibiotic resistance gene" does not include other mechanisms or genes that impart antibiotic resistance to naturally occurring micro-flora organisms. The term "attenuated" as used herein for example to describe a bacterial strain, particularly an E. coli or a Helicobacter strain such as Helicobacter pylori, is defined - 14 - as a strain that is less virulent and/or toxic (invasive) that a native, wild type bacterial strain. The term "biologically active" as used herein refers to ability to perform a biological function and with reference to a polypeptide implies that the polypeptide adopts a stable conformation ("folded form") which is the same or closely analogous to its native conformation. When folded correctly or substantially correctly, for example with formation of proper folded units a-helices, p-sheets, domains, disulphide bridges etc., a polypeptide should have the ability to perform its natural function. Generally, the unit of function in a polypeptide is a domain. Mere ability to be bound by an antibody or other receptor, either with or without elicitation of an immune response, is passive or does not constitute "biological activity". Any antigen has the ability to be bound by an antibody but is not necessarily biologically active. "Clinical grade vector" as used herein means a plasmid or other expression vector that is capable of being expressed in Helicobacter or a non-pathogenic bacterium engineered to have features of Helicobacter. The clinical grade vectors of the present invention do not use antibiotic resistance markers for selection and/or have been modified to prevent replication outside the host e.g. such as a suicide vector. An example of suicide system in H. pylori has been described by Panthel et al. 2003 (Infection 109-116). This system introduces a plasmid into H. pylori which contains the PhiXl74 lysis gene E. To eradicate the strain, incubation at 42°C for 5 hours was used. In vivo this would mean that the animal would consume a drink at 45-50°C to raise the temperature of the gastric environment above 42°C. A second example is the L-Dap selection system, commonly used to allow survival of bacterial mutants on supplemented plates (see, for example, Kirata et al, 1997 (Infection & Immunity, 65: 4158-4164). In this system the animal subject must supplement their diet with a missing substrate i.e. diamino-pimelic-acid (DAP), in order for the DapE deficient H. pylori mutant to survive. In order to eradicate the then DAP consumption is ceased. A third possible system relates to metronidazole sensitivity of H. pylori because of its rdxA gene. Excessive replication of the rdxA gene is harmful to mammalian cells and E. coli. However, duplication may be tolerated by the bacterium. Therefore a Helicobacter species of the present invention can be engineered to contain two copies of rdxA which prevent the normal mutation-dependant rdxA loss. The introduction of at least , two functional rdxA genes into the Helicobacter genome should result in a Helicobacter strain, which is permanently sensitive to metronidazole. Jeong et al. 2000 (J. Bacteriol., 182: 5082-5090) showed that the nitroreductase produced by a functional rdxA gene converts metronidazole from a prodrug to a bactericidal compound. The mode of action of the active compound is to cause DNA breaks of the Helicobacter genome. "Detectable immune response" as used herein is either an antibody (humoral) or cytotoxic (cellular) response formed in an animal in response to an antigen that can be measured using routine laboratory methods including, but not limited to enzyme-linked immunosorbant assays (ELISA), radio-immune assays (RIA), Enzyme-linked ImmunoSPOT•(ELISPOT), immunofluorescent assays (IFA), complement fixation assays (CF), Western Blot (WB) or an equivalent thereto. "Gene of interest" as used herein refers to any nucleic acid sequence encoding for a polypeptide or protein whose expression is desired. The gene of interest may or may not include the promoter or other regulatory components. The gene of interest also includes constructs capable of producing anti-sense RNA. "Gene therapy" as used herein is defined as the delivery of a gene of interest to an animal in need thereof using a recombinant vector. The gene of interest can be a transgene encoding for a therapeutic or prophylactic protein or polypeptide including, but not limited to cytokines, antiinflammatories, anti-proliferatives, antibiotics, metabolic inhibitors/activators and immunologically active antigens and fragments thereof. Furthermore, "gene therapy" as used herein also includes gene replacement technologies directed at both inherited and non-inherited disorders. The term Helicobacter includes all bacteria of the genus Helicobacter including H. pylori and H. mustelae. The term also includes bacteria that have similar biology to H. pylori in that they are capable of residing on the gastric mucosa of primates and/or capable of establishing a chronic, but isolated infection of the mucosa. The term also encompasses bacteria that have been modified so that the bacterium has H. pylori features such as the ability to reside on the gastric mucosa. A "heterologous" polypeptide is one not native to Helicobacter, i.e., not expressed by Helicobacter in nature or prior to introduction into Helicobacter, or an ancestor thereof, of encoding nucleic acid for the biologically active agent. "Host" as used herein defines the intended recipient of a therapeutic composition of the present invention. Host includes all animals. Specifically, hosts include, but are not limited to, primates (including man), bovine, equine, canine, feline, porcine, ovine, rabbits, rodents, birds and "Immunologically inert" as used herein shall mean any substance, including microorganisms such as microflora that does not provoke a significant immune response in its host. Examples of immunologically inert materials as used herein include stainless steel, biocompatible polymers such as poly-L-lactide, medical grade plastics and the microflora organisms of the present invention. A "significant immune" response is any immune response that would limit or restrict the in vivo utility of a material or organism used in accordance with the teachings of the present invention. A detectable immune response is not necessarily a "significant immune response." An "isolated nucleic acid" is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA molecule which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii> transfected cells, and (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library. "Percent identity (homology)" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, word.length=3, to obtain amino acid sequences homologous to a reference polypeptide (eg., SEQ ID NO: 2). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilised as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilising BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg., XBLAST and NBLAST) are used. These maybe found on the World Wide Web at the URL "ncbi.nim.nih.gov." The term "reporter gene" as used herein is a nucleic acid sequence incorporated into (or adjacent to) the heterologous nucleic acid encoding for the gene of interest that provides the transformed vector expressing the gene of interest an identifiable phenotype. Non-limiting examples of reporter genes include GFP, p-galactosidase, amylase, and CAT. "Screening marker" as used herein refers to an identifying characteristic (phenotype) provided to a transformed vector made in accordance with the teachings of the present invention. In one embodiment of the present invention the screening marker is a reporter gene. "Selectable marker," "selectable gene," "reporter gene" and "reporter marker" (referred to hereinafter as a "selectable marker") as used herein refer to nucleic acid sequences encoding for phenotypic traits that permit the rapid identification and isolation of a transformed bacterial vector. Generally, bacterial vectors deemed "clinical grade" and made in accordance with the teachings of the present invention are those vectors having selectable markers that do not encode for antibiotic resistance. "Transgene" as used herein refers to a gene that is inserted, using cDNA technology, into a cell in a manner that ensures its function, replication and transmission as a normal gene. "Transforming nucleic acid sequence" as used herein means a plasmid, or other expression cassette containing a nucleic acid sequence encoding a gene of interest. In some embodiments of the present invention the nucleic acid sequence can encode for one or more therapeutic agents. "Transforming nucleic acid sequence" can also be used to mean a "transgene" in accordance with' certain embodiments of the present invention. In another embodiment of the present invention the transforming nucleic acid sequence includes nucleic acid sequence encoding for a promoter and/or other regulatory elements. The term "cancer" as used herein refers to neoplastic diseases (e.g., leukemia, cancers and "hyperproliferative disorders"). The neoplasm may be located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital. In one embodiment the term "cancer" also encompasses preneoplastic conditions selected from the group consisting of hyperplasia (e.g., endometrial hyperplasia), metaplasia (e.g., connective tissue metaplasia and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia). In another embodiment, the term "cancer" also encompasses benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, and tissue hypertrophy. The term "a disease or condition of the immune/haematopoietic system" as used herein refers to a disease or condition selected from the group consisting of: anaemia, pancytopenia, leukopenia, thrombocytopenia, leukemia, Hodgkin's disease, non-Hodgkin1s lymphoma, acute lymphocytic anaemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, haemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, Celiac disease and allergies. The term "a disease or condition of the reproductive system" as used herein refers to a disease or condition selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumours, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumours, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhoea, syphilis, testicular torsion, vasitis nodosa, germ cell tumours, stromal tumours, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhoea, Gushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome. The term "a disease or condition of the musculoskeletal system" as used herein refers to a disease or condition selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumours, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis. The term "a disease or condition of the cardiovascular system" as used herein refers to a disease or condition selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurism, arteriosclerosis, peripheral vascular disease), hyponatremia/ hypematremia, hypokalemia, and hyperkalemia. The term "a disease or condition described as mixed fetal" as used herein refers to a disease or condition selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel- Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli- Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumour, neuroblastoma, and retinoblastoma. The term "a disease or condition of the excretory system" as used herein refers to a disease or condition selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy). The term "a disease or condition of the neural/sensory system" as used herein refers to a disease or condition selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumours, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders. The term "a disease or condition of the respiratory system" as used herein refers to a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy. The term "a disease or condition of the endocrine system" as used herein refers to a disease or condition selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g., male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Gushing's Syndrome), kidney cancer (e.g., hypemephroma, transitional cell cancer, and Wilm's tumour), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis. The term "a disease or condition of the digestive system" as used herein refers to a disease or condition selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumours of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumours of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, haemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha 1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice. The term "a disease or condition of the connective / epithelial" as used herein refers to a disease or condition selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anaemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderina, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch- Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa. The term "a" and "the" as used in the present descriptive is intended to include both one (the singular) and more than one (plural). A "therapeutically effective amount" of an active agent or combination of agents as described herein is understood to comprise an amount effective to elicit the desired response but insufficient to cause a toxic reaction. A desired response, for example, may constitute the formation of a sufficient and/or acceptable detectable antibody titer level in a blood sample. The dosage and duration of treatment of the preparation to be administered to a subject will be determined by the health professional attending the subject in need of treatment, and will consider the age, sex, weight, extent of existing diseased state and/or tissue damage of the subject, and specific formulation of Helicobacter and the gene of interest product being used as the treatment for the subject. The phrase, "effective level" refers to the level of the desired activity of the molecules and not necessarily limited to the number of molecules. For example, the effective level of amyliri may be decreased to stimulate ghrelin secretion by using amylin antagonists, without a necessary concomitant decrease in the amount of free amylin present in a subject. The phrase "ghrelin-associated diseases and disorders" refers to any condition that can be treated prevented or ameliorated through the modulation of ghrelin activity. These include conditions that are enhanced, exacerbated or stimulated by ghrelin, for example, growth hormone release or drive to eat. The physiological actions of ghrelin are considered to include, by way of example, the stimulation of growth hormone release, the stimulation of hormone secretion from lactotrophs and corticotropes, orexigenic and cardiovascular actions, anti-proliferative effects on thyroid and breast tumors and regulation of gastric motility and acid secretion through vagal mediation. (See WO 2005021026). Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided herein, unless specifically indicated. DETAILED DESCRIPTION OF THE INVENTION Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, arid is not intended to be limiting which will be limited only by the appended We Claim: 1. A bacterial delivery composition comprising a Helicobacter pylori cell comprising a Helicobacter vector or plasmid comprising: (a) a non-Helicobacter sequence encoding a non-Helicobacter pharmacologically active molecule of interest linked to a secretory signal peptide; and (b) a Helicobacter sequence having a promoter region capable of controlling expression of the non-Helicobacter pharmacologically active molecule; wherein the pharmacologically active molecule is expressed on the surface of the Helicobacter pylori cell or secreted therefrom; wherein the Helicobacter pylori cell chronically colonizes the mucosa of a mammal upon administration and wherein the Helicobacter pylori is not a DapE mutant strain" 2. The bacterial delivery composition as claimed in claim 1, wherein the Helicobacter pylori cell is attenuated. 3. The bacterial delivery composition as claimed in claim 1 or claim 2, wherein the Helicobacter pylori cell is non-pathogenic. 4. The bacterial delivery composition as claimed in any one of claims 1 to 3, wherein the Helicobacter pylori is strain 26695. 5. The bacterial delivery composition as claimed in any one of claims 1 to 4, wherein the promoter is an inducible promoter. 6. The bacterial delivery composition as claimed in any one of claims 1 to 5, wherein the promoter is an arabinose inducible promoter. 7. The bacterial delivery composition as claimed in any one of claims 1 to 6, wherein the promoter is promoter from the Helicobacter pylori histidine kinase HP 165. 8. The bacterial delivery composition as claimed in any one of claims 1 to 7, wherein the promoter is a FlaB sigma 54 promoter. 9. The bacterial delivery composition as claimed in any one of claims 1 to 8, wherein the promoter is a T7 promoter. 10. The bacterial delivery composition according to any one of claims 1 to 9, wherein the pharmacologically active molecule of interest comprises a protein, a peptide or a nucleic acid molecule. 11. The bacterial delivery composition according to any one of claims 1 to 10, wherein the Helicobacter based construct is a vector or a plasmid vector. 12. The bacterial delivery composition as claimed in claim 11, wherein the plasmid vector is pHP1(SEQ ID NO. 1 and 2). 13. The bacterial delivery composition as claimed in claim 11, wherein the plasmid vector is pHP3(SEQ ID N0.3). 14. The bacterial delivery composition as claimed in claim 13, wherein the plasmid vector is pTMI03-8.

Full Text

BACTERIAL DELIVERY SYSTEM
FIELD OF THE INVENTION
The present invention relates to the delivery of
biologically active agents in vivo. In particular, the
present invention relates to treating, palliating or
preventing diseases using a bacterial delivery system for
delivering biologically active agents directly to an
anatomical site in vivo. In one embodiment, the bacterial
delivery system comprises Helicobacter or bacterium
exhibiting Helicobacter features for delivering
heterologous nucleic acid into an animal or animal cell,
wherein the heterogonous nucleic acid is expressed. In a
further embodiment the Helicobacter has been engineered to
contain a DNA vector that encodes heterogonous nucleic
acid, wherein upon infection the nucleic acid vector is
expressed such that the biologically active agent is
delivered to the animal body, especially at the mucosa.
BACKGROUND TO THE INVENTION
There is a continuing need for long-term delivery of
pharmacologic and immunologic agents to individuals with
both congenital and acquired diseases. In most cases, this
involves repeated administration of therapeutic compounds
several times per day, daily or at intervals. However, it
is appreciated that any long-term therapeutic or preventive
regime has problems such as compliance, side effects and
drug resistance. Consequently, there is a constant need to
identify treatments, which will be usually 100% effective,
free from side effects and cheap.
One form of delivery that has been investigated in recent
times for various therapeutic and prophylactic agents has
been the use of microorganisms. Genes of interest from
various organisms including bacteria, viruses, parasites as
well as mammals have, for example, been cloned into a
variety of bacteria, viruses and mycobacteria for the
purpose of directing these micro-organisms to express
foreign protein or impart certain desired properties. These
microorganisms have been used in vaccination programs,
gene-replacement therapies and therapeutic composition
delivery.
Microorganisms have also been used to transform animal
(host) cells in vivo. Host cell transformation can be
accomplished using gene-delivery vectors comprising
replication incompetent viruses (see for example U.S. Pat.
No. 5,824,544), naked DNA, (see for example U.S. Pat. No.
6,261,834), liposomes containing recombinant expression
cassettes (see for example U.S. Pat. No. 6,271,207). Other
molecular-based therapeutic composition delivery approaches
include using replication incompetent recombinant viruses
designed to express heterogonous surface proteins (see, for
example, U.S. Pat. No. 6,376,236).
In recent times, pharmaceutical researchers have also
attempted to develop methods for in vivo therapeutic
composition expression using recombinant organism-based
vectors, inanimate vectors and naked DNA. Examples of
recombinant organism-based vectors include recombinant
bacteria (see for example U.S. Pat. No. 5,547,664) and
viruses such as alphaviruses (see for example U.S. Pat. No.
6,391,632), vaccinia viruses (see for example U.S. Pat. No.
6,267,965), adenoviruses (see for example U.S. Pat. No.
5,698,202) and adenovirus associated virus (AAV) (see for
example U.S. Pat. No. 6,171,597). Inanimate vectors include
lipidic gene delivery vector constructs such as
DNA/cationic liposome complexes, DNA encapsulated in
neutral or anionic liposomes, and liposome-entrapped,
polycation-condensed DNA (LPDI arid LPDII) . (see Ropert,
1999, Braz J Med Bio1 Res, 32 (2):163-9).
Examples of other genes that have been delivered by various
bacteria include cloning the invasion genes of Shigella
into the normally non-invasive E. coli rendering the E.
coli invasive and therefore more suitable for use as a
vaccine strain, or cloning of Plasmodium falciparum malaria
genes into Salmonella typhimurium which subsequently
express these malaria proteins and, following oral
administration of the bacteria, induce specific cytotoxic T
cell immunity and protection in mice against malaria
challenge (see, for example, Hone et al., 1991, Vaccine,
9:810-816; Tacket et al., 1992, Infect. Immun., 60:536-541;
Hone et al., 1992, J. Clin. Invest., 90:412-420; Chatfield
et al., 1992, Vaccine, 10:8-11; Tacket et al., 1992,
Vaccine, 10:443-446; and Mims et al., 1993, In: Medical
Microbiology, Eds., Mosby-Year Book Europe Ltd., London;
Sadoff et al., 1988, Science, 240:336-338; Aggrawal et al.,
1990, J. Exp. Wed., 172:1083-1090).
Attenuated or less virulent Shigella (see, for example,
Noriega et al., 1994, Infect. Immun., 62:5168-5172; US Pat.
Appl. No. 20020176848), Salmonella (see, for example, US
Pat. No. 6,531,313; US Pat. Appl. No. 20030170211),
Listeria (see, for example, Schafer et al., 1992, J.
Immunol., 149:53-59; US Pat. Appl. No. 20030008839), and ,
other bacteria have been given orally to immunise against
subsequent infection with more virulent forms of these
bacteria. Likewise, attenuated bacterial and mycobacterial
organisms such as Bacille Calmette-Guerin (BCG)
(Lagranderie et al., 1993, Vaccine, 11:1283-1290; Flynn,
1994, Cell. Molec. Biol., 40(Suppl. l):31-36) have been
administered parenterally to protect against related
organisms such as M. tuberculosis.
Some of the other bacterial species that have been used as
vectors include yersinia enterocolitica (van Damme et al.,
1992, Gastroenterol., 103:520-531) and Vibrio cholerae
(Levine et al., 1994, In: Vibrio cholerae, Molecular to
Global Perspective's, Wachsmuth et al.r Eds, ASM Press,
Washington, D.C., pages 395-414).
Despite all of the research all of the above bacterial
delivery systems have technical difficulties, which need to
be overcome before these can be used in vivo. Indeed, most
of the bacterial systems require the bacteria themselves to
produce functional molecules and are dependent on a
bacterium which is sufficiently attenuated to be safe for
use in humans, but still able to produce biologically
active agents. However, all of the attenuated strains of
bacteria used previously are not capable' of surviving
lengthy periods in vivo, without causing side effects. More
importantly, many of these bacterial species are not
capable of delivering therapeutic or prophylactic agents to
the mucosal epithelial cells of an animal. However, many
of the most important therapeutic agents are taken up via
the mucosa; therefore there is a need for a bacterial
delivery system capable of delivering biologically active
agents directly to the mucosa.
U.S. Pat. No. 5,877,159 to Powell et al.r describes live
bacteria that can invade mucosal cells without establishing
a productive infection or causing disease to thereby
introduce a eukaryotic expression cassette encoding an
antigen capable of being expressed by the animal cells.
While this method allows delivery of the DNA vaccine to
mucosal surfaces, including easy administration, a concern
for vaccine delivery in developing countries, it does not
have the advantage of providing amplifiable mRNA encoding
the gene of interest. Moreover, the bacterium disclosed in
Powell et-al. is not capable of sustained delivery.
Non-pathogenic, non-colonising, non-invasive food-grade
bacterium Lactococcus lactis has been used previously in
the past to deliver agents to the mucosa (see, for example,
UK patent GB-2278358B) . However, while Lactococcus lactis
is non-invasive it is not capable of establishing a chronic
infection, which is capable of delivering continuous
therapeutic agents directly into mucosal epithelial cells.
Consequently, there is a continuing need for a delivery
system, which is capable of establishing a non-invasive,
chronic infection in close proximity to the mucosal
epithelial cells of an animal such that biologically active
agents can be delivered thereto. Moreover, there is a
continuing need for improved delivery mechanisms for
pharmacologically active molecules at the mucosal surface
sufficient to elicit a useful and beneficial immunogenic
response. Such would provide an effective in vivo delivery
system for pharmacological active agents, as well as an
effective method for immunization, i.e., antigen exposure
at mucosal surface sufficient to elicit a general humoral
and mucosal immune response.
SUMMARY OF THE INVENTION
The present invention is directed to overcoming the abovementioned
challenges and is exemplified in a number of
implementations and applications, some of which are
summarized below.
The inventors have now surprisingly found that Helicobacter
and in particular Helicobacter pylori, which is capable of
forming a chronic infection, can be used to produce
continuous drug delivery. Moreover, Helicobacter of the
present invention can be activated and inactivated at
various times and because of its chronicity, the
Helicobacter can be used to deliver drugs through the
gastric mucosa.
Helicobacter pylori is a gram-negative spiral shaped
bacterium found almost exclusively in the human gastric
mucosa. The acidity of the human stomach is an effective
barrier to colonization by essentially all bacteria, with
the exception of Helicobacter species.
H. pylori has the unique ability to colonize and -persist
for decades within the human gastric mucosa, despite
development of a mucosal inflammatory and immune response.
This characteristic renders H. pylori an interesting
candidate for the delivery of selected agents though the
mucosa.
In accordance with some aspects, compositions, methods and
systems are provided for preparing and using a
Helicobacter-based construct comprising a Helicobacter
sequence having a promoter region and a non-Helicobacter
sequence encoding a non-Helicobacter pharmacologically
active molecule. This construct in some embodiments is
described as a vector or a plasmid vector, wherein the
promoter sequence is capable of controlling the expression
of the non-Hell cojbacter pharmacologically active molecule
of interest.
Accordingly, in a first aspect the present invention
provides a method of delivering one or more biologically
active agents to a subject comprising the step of
administering to a subject an effective amount, preferably
a therapeutically or prophylactically effective amount of,
Helicobacter cells or bacterial cells having the features
of Helicobacter, which cells express said one or more
biologically active agents.
In a second aspect, the present invention provides a noninvasive
or non-pathogenic Helicobacter cell expressing one
or more heterologous biologically active agents.
Preferably, the Helicobacter is of the species H. pylori.
The biologically active agent can be either homologous to
the Helicobacter genus or heterologous to cells of the
genus or species of the Helicobacter cells used for
delivery. Heterologous agents may be derived from either
eukaryotic or prokaryotic sources.
In some aspects, the HeJicojbacter-based vector and vector
plasmid constructs comprise biologically active agents such
as an antigen, organic or inorganic molecule or substance,
a pharmacological agent eg a therapeutic agent or
prophylactic agent, such as a gene product or gene sequence
(isolated nucleic acid). By way of example, such agents may
include an immunqregulatory agent, hormone, ligand, an
enzyme or an anti-sense RNA, a catalytic RNA, a protein, a
peptide or any other molecule which can be present on or
released from the bacterial cells so that it may be
delivered to an animal or to an animal cell.
In some embodiments, the biologically active agent is
encoded by a nucleic acid molecule, which is preferably
obtained in isolated form, and subsequently inserted into
the bacterial delivery vehicle of this invention. It will
be appreciated by those skilled in the art that the
isolated nucleic acid molecule of the present invention may
be cDNA, genomic DNA, RNA, or a hybrid molecule thereof.
Preferably, the nucleic acid is cDNA.
By way of example, a protein and/or peptide of interest may
comprise ghrelin, amylin, insulin, motilin, (3-glucosidase,
a chemical chaperone, or other molecule useful in the
treatment of Gauchers disease, cell wasting, human
immunodeficiency disease (AIDS), appetite suppression,
preparations useful in the treatment of diabetes, etc.
The isolated nucleic acid is preferably incorporated into
an expression vector, which can be transformed into and
maintained in the Helicobacter cells.
Accordingly, in a third aspect, the present invention
provides a recombinant vector for delivering biologically
active agents directly to an anatomical site in need
thereof. The Hellcobacter cell of the present invention has
preferably been adapted to secrete and/or express on its
surface the biologically active agents.
In a fourth aspect, the present invention provides a vector
or construct for delivering in vivo an effective amount of
a biologically active agent, comprising:
a) a nucleotide sequence encoding a
biologically active agent;
b) operatively linked thereto, a control or
regulatory sequence capable of controlling the expression
of the nucleotide sequence of (a) such that the
biologically active agent is produced in vivo when the
delivery vehicle comprising the vector is delivered to a
subject.
The vector maybe modified chemically by means of chemical
or enzymatic treatment, or in vivo by means of recombinant
DNA technology to produce a modified or variant vector.
Such a construct may differ from those disclosed, for
example, by virtue of one or more nucleotide substitutions,
deletions or insertions, but substantially retain a desired
biological activity of the construct or nucleic acid
molecule, or its encoded product, in accordance with this
invention.
In another embodiment of the present invention, the
Helicobacter vector or construct is provided with a
reporter gene(s) expressed from a constitutive promoter
cloned into the expression vector and used as a screening
tool. Non-limiting examples of reporter genes suitable for
use herein include green fluorescent protein (GFP), |Jgalactosidase,
amylase, and chloramphenicol acetyl
transferase (CAT).
In another embodiment of the present invention the
Helicobacter cell is used to deliver a heterologous gene of
interest to a subject in need thereof. The gene of interest
may encode a therapeutic product (a transgene product),
including, but not limited to a peptide hormone (such as,
but not limited to a-melanocyte-stimulating hormone
(a-MSH), insulin, growth hormone, and parathyroid hormone),
a cytokine including, but not limited to an interferon,
interleukin (IL)-2, IL-4, IL-10, IL-12, granulocyte colony
stimulating factor (G-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF) and erythropoietin (EPO).
In still another embodiment, the present invention provides
methods for treating, palliating or preventing a disease in
a subject. These methods are facilitated with the use of a
therapeutic or prophylactic composition. Accordingly, in a
fifth aspect, the present invention provides a
pharmaceutical composition comprising (a) live nonpathogenic
Helicobacter cells expressing and/or secreting a
biologically active agent, together with (b) a
therapeutically effective carrier.
In a sixth aspect the present invention provides a method
of treating, preventing or palliating a disease comprising
administering to a subject in need thereof an effective
amount of a Helicobacter cells of the present invention
that express the biologically active agent.
Non-limiting examples of subjects in which the present
invention may be used, include mammals such as primates,
equines, bovines, porcines, ovines and rodents. Also
intended are fish and birds.
In one embodiment of the present invention the disease
being treated, prevented or palliated is cancer, a disease
or condition of the immune/hematopoietic system, a disease
or condition of the reproductive system, a disease or
condition of the musculoskeletal system, a disease or
condition of the cardiovascular system, a disease or a
condition described as mixed fetal, a disease or a
condition of the excretory system, a disease or a condition
of the neural/sensory system, a disease or a condition of
the endocrine system, a disease or condition of the
respiratory system, a disease or condition of the digestive
system and a disease or condition associated with
connective/epithelial tissue or disease or conditions
caused by bacterial, viral or parasitic infection.
The present invention provides a variety of
pharmaceutically acceptable preparations formulated for
delivery to a patient, such as, gastrically, orally, or
intranasaly. In particular embodiments, the composition
are suitable for delivery at a mucosal surface. In
particular embodiments, the composition is suitable for
delivery to the mucosal surface of the gut.
By way of example, the mucosa may be that of the gastric,
vaginal, nasal, oral, or ocular surface, or any other of
the body characterized by the presence of a penetrable
mucosal surface or lining. In some embodiments, the
mucosal surface is the gastric mucosal surface.
The various delivery forms of the compositions are readily
prepared for use in the practice of the present invention
given the specific types and ratios of specific
Helicobacter, Helicobacter constructs and other delivery
vehicles described herein, and those formulation techniques
known to those in the formulary arts, such as are described
in Remington's Pharmaceutical Sciences, 20th edition, Mack
Publishing Company, which text is specifically incorporated
herein by reference.
lt is envisioned that the delivery system may be employed
in animals, particularly primates, including humans,
equines, bovines, ovines, and rodents, fish and birds. It
is also anticipated that the preparations may be used on
both infants and adults, as well as parentally or for
administration to pregnant or lactating animals. The
preparations and methods may be further described as
suitable for both male and female animals.
In yet another aspect, a method is provided for vaccinating
an animal. In some embodiments, the method comprises
administering a composition comprising a vaccine comprising
cells transformed with the Helicobacter- based plasmid
vector and/or plasmid vectors as described herein. In
other embodiments, the method provides for the delivery of
an effective amount of the pharmacologically active
molecule of interest sufficient to eliminate or inhibit a
disease or physiological condition in the animal, or
sufficient to elicit an immune response specific for the
pharmacologically active molecule of interest.
By way of example, the non-Helicobacter pharmacologically
active molecule of interest useful in the vaccine may
comprise a mammalian protein, peptide, enzyme, hormone, or
any combination of these. In particular embodiments, the
pharmacologically active molecule of interest is further
defined as a human pharmacologically active molecule of
interest. In some embodiments the pharmacologically active
molecule of interest is a human pathogen molecule/antigen,
human protein antigen, such as amylin or an analog or
derivative thereof, or ghrelin, or an analog or derivative
thereof.
In particular embodiments, the vaccine conveys immunity
against the human pathogen, Ebola virus, HIV virus, Marburg
virus, influenza virus, and the like. Replication competent
vaccines based on attenuated recombinant vesicular
stomatitis virus vectors have been described by Jones et
al. (2005, Wat. Afed. 11: 786-90) that include Ebola
glycoprotein and Marburg glycoprotein. Hence, it is
envisioned that constructs using the HelicoJbacter-based
vector systems and plasmid vector systems with these and
other glycoprotein's associated with human pathogens may
also be provided according to the present invention
together with the disclosure provided herein.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic diagram of the plasmid construct
pHPAl (2.8kb).
Figure 2 is a schematic diagram of the plasmid construct
pHP3 (3.4kb).
Figure 3 is a schematic diagram of the plasmid vector
pTMI03-8.
Figure 4 shows the structure of sulfasalazine (SSN).
Figure 5 is a schematic diagram showing the use of ion
exchange resin (Amberlite XE-96) conjugated with a dye
(Azure-A).
BRIEF DESCRIPTION OF THE SEQUENCES
The following nucleic acid and amino acid sequences are
referenced throughout the description of the present
invention:
SEQ ID NO: 1 - Nucleotide sequence of plasmid pHPl (2796
nucleotides);
SEQ ID NO: 2 - Nucleotide sequence of pHPl (2796
nucleotides);
SEQ ID NO: 3 - Nucleotide sequence of plasmid pHP3 (3444
nucleotides)/
SEQ ID NO: 4 - Hepatitis C virus are antigen (HCV)
nucleotide Sequence;
SEQ ID NO: 5 - Nucleotide sequence 135 bp (45 amino acids)
immunogenic coding sequence from the Hepatitis C virus
(HCV) core antigen/
SEQ ID NO: 6 - Nucleotide sequence of the surface exposed
loop of the HopE gene (at nt504, aa position 168) of H.
pylori;
SEQ ID NO: 7 - Upstream primer (29 nucleotides);
SEQ ID NO: 8 - Downstream Primer (28 nucleotides);
SEQ ID NO: 9 - Oligonucleotide Primer (15 nucleotides).
DEFINITION OF TERMS
Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of
certain terms that will be used hereinafter.
An "antibiotic resistance gene" as defined herein includes
heterologous nucleic acid sequences purposely provided to a
vector and used as a selection system. The term "antibiotic
resistance gene" does not include other mechanisms or genes
that impart antibiotic resistance to naturally occurring
micro-flora organisms.
The term "attenuated" as used herein for example to
describe a bacterial strain, particularly an E. coli or a
Helicobacter strain such as Helicobacter pylori, is defined
- 14 -
as a strain that is less virulent and/or toxic (invasive)
that a native, wild type bacterial strain.
The term "biologically active" as used herein refers to
ability to perform a biological function and with reference
to a polypeptide implies that the polypeptide adopts a
stable conformation ("folded form") which is the same or
closely analogous to its native conformation. When folded
correctly or substantially correctly, for example with
formation of proper folded units a-helices, p-sheets,
domains, disulphide bridges etc., a polypeptide should have
the ability to perform its natural function. Generally, the
unit of function in a polypeptide is a domain.
Mere ability to be bound by an antibody or other receptor,
either with or without elicitation of an immune response,
is passive or does not constitute "biological activity".
Any antigen has the ability to be bound by an antibody but
is not necessarily biologically active.
"Clinical grade vector" as used herein means a plasmid or
other expression vector that is capable of being expressed
in Helicobacter or a non-pathogenic bacterium engineered to
have features of Helicobacter. The clinical grade vectors
of the present invention do not use antibiotic resistance
markers for selection and/or have been modified to prevent
replication outside the host e.g. such as a suicide vector.
An example of suicide system in H. pylori has been
described by Panthel et al. 2003 (Infection 109-116). This system introduces a plasmid into H. pylori
which contains the PhiXl74 lysis gene E. To eradicate the
strain, incubation at 42°C for 5 hours was used. In vivo
this would mean that the animal would consume a drink at
45-50°C to raise the temperature of the gastric environment
above 42°C.
A second example is the L-Dap selection system, commonly
used to allow survival of bacterial mutants on supplemented
plates (see, for example, Kirata et al, 1997 (Infection &
Immunity, 65: 4158-4164). In this system the animal subject
must supplement their diet with a missing substrate i.e.
diamino-pimelic-acid (DAP), in order for the DapE deficient
H. pylori mutant to survive. In order to eradicate the then
DAP consumption is ceased.
A third possible system relates to metronidazole
sensitivity of H. pylori because of its rdxA gene.
Excessive replication of the rdxA gene is harmful to
mammalian cells and E. coli. However, duplication may be
tolerated by the bacterium. Therefore a Helicobacter
species of the present invention can be engineered to
contain two copies of rdxA which prevent the normal
mutation-dependant rdxA loss. The introduction of at least ,
two functional rdxA genes into the Helicobacter genome
should result in a Helicobacter strain, which is
permanently sensitive to metronidazole. Jeong et al. 2000
(J. Bacteriol., 182: 5082-5090) showed that the
nitroreductase produced by a functional rdxA gene converts
metronidazole from a prodrug to a bactericidal compound.
The mode of action of the active compound is to cause DNA
breaks of the Helicobacter genome.
"Detectable immune response" as used herein is either an
antibody (humoral) or cytotoxic (cellular) response formed
in an animal in response to an antigen that can be measured
using routine laboratory methods including, but not limited
to enzyme-linked immunosorbant assays (ELISA), radio-immune
assays (RIA), Enzyme-linked ImmunoSPOT•(ELISPOT),
immunofluorescent assays (IFA), complement fixation assays
(CF), Western Blot (WB) or an equivalent thereto.
"Gene of interest" as used herein refers to any nucleic
acid sequence encoding for a polypeptide or protein whose
expression is desired. The gene of interest may or may not
include the promoter or other regulatory components. The
gene of interest also includes constructs capable of
producing anti-sense RNA.
"Gene therapy" as used herein is defined as the delivery of
a gene of interest to an animal in need thereof using a
recombinant vector. The gene of interest can be a transgene
encoding for a therapeutic or prophylactic protein or
polypeptide including, but not limited to cytokines, antiinflammatories,
anti-proliferatives, antibiotics, metabolic
inhibitors/activators and immunologically active antigens
and fragments thereof. Furthermore, "gene therapy" as used
herein also includes gene replacement technologies directed
at both inherited and non-inherited disorders.
The term Helicobacter includes all bacteria of the genus
Helicobacter including H. pylori and H. mustelae. The term
also includes bacteria that have similar biology to H.
pylori in that they are capable of residing on the gastric
mucosa of primates and/or capable of establishing a
chronic, but isolated infection of the mucosa. The term
also encompasses bacteria that have been modified so that
the bacterium has H. pylori features such as the ability to
reside on the gastric mucosa.
A "heterologous" polypeptide is one not native to
Helicobacter, i.e., not expressed by Helicobacter in nature
or prior to introduction into Helicobacter, or an ancestor
thereof, of encoding nucleic acid for the biologically
active agent.
"Host" as used herein defines the intended recipient of a
therapeutic composition of the present invention. Host
includes all animals. Specifically, hosts include, but are
not limited to, primates (including man), bovine, equine,
canine, feline, porcine, ovine, rabbits, rodents, birds and
"Immunologically inert" as used herein shall mean any
substance, including microorganisms such as microflora that
does not provoke a significant immune response in its host.
Examples of immunologically inert materials as used herein
include stainless steel, biocompatible polymers such as
poly-L-lactide, medical grade plastics and the microflora
organisms of the present invention. A "significant immune"
response is any immune response that would limit or
restrict the in vivo utility of a material or organism used
in accordance with the teachings of the present invention.
A detectable immune response is not necessarily a
"significant immune response."
An "isolated nucleic acid" is a nucleic acid the structure
of which is not identical to that of any naturally
occurring nucleic acid or to that of any fragment of a
naturally occurring genomic nucleic acid spanning more than
three separate genes. The term therefore covers, for
example, (a) a DNA molecule which has the sequence of part
of a naturally occurring genomic DNA molecule but is not
flanked by both of the coding sequences that flank that
part of the molecule in the genome of the organism in which
it naturally occurs; (b) a nucleic acid incorporated into a
vector or into the genomic DNA of a prokaryote or eukaryote
in a manner such that the resulting molecule is not
identical to any naturally occurring vector or genomic DNA;
(c) a separate molecule such as a cDNA, a genomic fragment,
a fragment produced by polymerase chain reaction (PCR), or
a restriction fragment; and (d) a recombinant nucleotide
sequence that is part of a hybrid gene, i.e., a gene
encoding a fusion protein. Specifically excluded from this
definition are nucleic acids present in mixtures of (i) DNA
molecules, (ii> transfected cells, and (iii) cell clones,
e.g., as these occur in a DNA library such as a cDNA or
genomic DNA library.
"Percent identity (homology)" of two amino acid sequences
or of two nucleic acids is determined using the algorithm
of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA,
87:2264-2268, 1990, modified as in Karlin and Altschul
(Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an
algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul et al. (J. Mol. Biol. 215:403-410,
1990). BLAST nucleotide searches are performed with the
NBLAST program, score=100, wordlength=12, to obtain
nucleotide sequences homologous to a nucleic acid molecule
of the invention. BLAST protein searches are performed with
the XBLAST program, score=50, word.length=3, to obtain amino
acid sequences homologous to a reference polypeptide (eg.,
SEQ ID NO: 2). To obtain gapped alignments for comparison
purposes, Gapped BLAST is utilised as described in Altschul
et al. (Nucleic Acids Res. 25:3389-3402, 1997). When
utilising BLAST and Gapped BLAST programs, the default
parameters of the respective programs (eg., XBLAST and
NBLAST) are used. These maybe found on the World Wide Web
at the URL "ncbi.nim.nih.gov."
The term "reporter gene" as used herein is a nucleic acid
sequence incorporated into (or adjacent to) the
heterologous nucleic acid encoding for the gene of interest
that provides the transformed vector expressing the gene of
interest an identifiable phenotype. Non-limiting examples
of reporter genes include GFP, p-galactosidase, amylase,
and CAT.
"Screening marker" as used herein refers to an identifying
characteristic (phenotype) provided to a transformed vector
made in accordance with the teachings of the present
invention. In one embodiment of the present invention the
screening marker is a reporter gene.
"Selectable marker," "selectable gene," "reporter gene" and
"reporter marker" (referred to hereinafter as a "selectable
marker") as used herein refer to nucleic acid sequences
encoding for phenotypic traits that permit the rapid
identification and isolation of a transformed bacterial
vector. Generally, bacterial vectors deemed "clinical
grade" and made in accordance with the teachings of the
present invention are those vectors having selectable
markers that do not encode for antibiotic resistance.
"Transgene" as used herein refers to a gene that is
inserted, using cDNA technology, into a cell in a manner
that ensures its function, replication and transmission as
a normal gene.
"Transforming nucleic acid sequence" as used herein means a
plasmid, or other expression cassette containing a nucleic
acid sequence encoding a gene of interest. In some
embodiments of the present invention the nucleic acid
sequence can encode for one or more therapeutic agents.
"Transforming nucleic acid sequence" can also be used to
mean a "transgene" in accordance with' certain embodiments
of the present invention. In another embodiment of the
present invention the transforming nucleic acid sequence
includes nucleic acid sequence encoding for a promoter
and/or other regulatory elements.
The term "cancer" as used herein refers to neoplastic
diseases (e.g., leukemia, cancers and "hyperproliferative
disorders"). The neoplasm may be located in a tissue
selected from the group consisting of: colon, abdomen,
bone, breast, digestive system, liver, pancreas, prostate,
peritoneum, lung, blood (e.g., leukemia), endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), uterus, eye, head and neck, nervous (central and
peripheral), lymphatic system, pelvic, skin, soft tissue,
spleen, thoracic, and urogenital.
In one embodiment the term "cancer" also encompasses preneoplastic
conditions selected from the group consisting of
hyperplasia (e.g., endometrial hyperplasia), metaplasia
(e.g., connective tissue metaplasia and/or dysplasia (e.g.,
cervical dysplasia, and bronchopulmonary dysplasia).
In another embodiment, the term "cancer" also encompasses
benign dysproliferative disorder selected from the group
consisting of: benign tumors, fibrocystic conditions, and
tissue hypertrophy.
The term "a disease or condition of the
immune/haematopoietic system" as used herein refers to a
disease or condition selected from the group consisting of:
anaemia, pancytopenia, leukopenia, thrombocytopenia,
leukemia, Hodgkin's disease, non-Hodgkin1s lymphoma, acute
lymphocytic anaemia (ALL), plasmacytomas, multiple myeloma,
Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune
disease, rheumatoid arthritis, granulomatous disease,
immune deficiency, inflammatory bowel disease, sepsis,
neutropenia, neutrophilia, psoriasis, immune reactions to
transplanted organs and tissues, systemic lupus
erythematosis, haemophilia, hypercoagulation, diabetes
mellitus, endocarditis, meningitis, Lyme Disease, Celiac
disease and allergies.
The term "a disease or condition of the reproductive
system" as used herein refers to a disease or condition
selected from the group consisting of: cryptorchism,
prostatitis, inguinal hernia, varicocele, leydig cell
tumours, verrucous carcinoma, prostatitis, malacoplakia,
Peyronie's disease, penile carcinoma, squamous cell
hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's
syndrome, mucopurulent cervicitis, Sertoli-leydig tumours,
ovarian cancer, uterine cancer, pelvic inflammatory
disease, testicular cancer, prostate cancer, Klinefelter's
syndrome, Young's syndrome, premature ejaculation, diabetes
mellitus, cystic fibrosis, Kartagener's syndrome,
testicular atrophy, testicular feminization, anorchia,
ectopic testis, epididymitis, orchitis, gonorrhoea,
syphilis, testicular torsion, vasitis nodosa, germ cell
tumours, stromal tumours, dysmenorrhea, retroverted uterus,
endometriosis, fibroids, adenomyosis, anovulatory bleeding,
amenorrhoea, Gushing's syndrome, hydatidiform moles,
Asherman's syndrome, premature menopause, precocious
puberty, uterine polyps, dysfunctional uterine bleeding,
cervicitis, chronic cervicitis, mucopurulent cervicitis,
cervical dysplasia, cervical polyps, Nabothian cysts,
cervical erosion, cervical incompetence, cervical
neoplasms, pseudohermaphroditism, and premenstrual
syndrome.
The term "a disease or condition of the musculoskeletal
system" as used herein refers to a disease or condition
selected from the group consisting of: bone cancers (e.g.,
osteochondromas, benign chondromas, chondroblastoma,
chondromyxoid fibromas, osteoid osteomas, giant cell
tumours, multiple myeloma, osteosarcomas), Paget's Disease,
rheumatoid arthritis, systemic lupus erythematosus,
osteomyelitis, Lyme Disease, gout, bursitis, tendonitis,
osteoporosis, osteoarthritis, muscular dystrophy,
mitochondrial myopathy, cachexia, and multiple sclerosis.
The term "a disease or condition of the cardiovascular
system" as used herein refers to a disease or condition
selected from the group consisting of: myxomas, fibromas,
rhabdomyomas, cardiovascular abnormalities (e.g.,
congenital heart defects, cerebral arteriovenous
malformations, septal defects), heart disease (e.g., heart
failure, congestive heart disease, arrhythmia, tachycardia,
fibrillation, pericardial Disease, endocarditis), cardiac
arrest, heart valve disease (e.g., stenosis, regurgitation,
prolapse), vascular disease (e.g., hypertension, coronary
artery disease, angina, aneurism, arteriosclerosis,
peripheral vascular disease), hyponatremia/ hypematremia,
hypokalemia, and hyperkalemia.
The term "a disease or condition described as mixed fetal"
as used herein refers to a disease or condition selected
from the group consisting of: spina bifida,
hydranencephaly, neurofibromatosis, fetal alcohol syndrome,
diabetes mellitus, PKU, Down's syndrome, Patau syndrome,
Edwards syndrome, Turner syndrome, Apert syndrome,
Carpenter syndrome, Conradi syndrome, Crouzon syndrome,
cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld
syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-
Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome,
Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-
Opitz syndrome, thromocytopenia-absent radius (TAR)
syndrome, Treacher Collins syndrome, Williams syndrome,
Hirschsprung's disease, Meckel's diverticulum, polycystic
kidney disease, Turner's syndrome, and gonadal dysgenesis,
Klippel-Feil syndrome, Ostogenesis imperfecta, muscular
dystrophy, Tay-Sachs disease, Wilm's tumour,
neuroblastoma, and retinoblastoma.
The term "a disease or condition of the excretory system"
as used herein refers to a disease or condition selected
from the group consisting of: bladder cancer, prostate
cancer, benign prostatic hyperplasia, bladder disorders
(e.g., urinary incontinence, urinary retention, urinary
obstruction, urinary tract Infections, interstitial
cystitis, prostatitis, neurogenic bladder, hematuria),
renal disorders (e.g., hydronephrosis, proteinuria, renal
failure, pyelonephritis, urolithiasis, reflux nephropathy,
and unilateral obstructive uropathy).
The term "a disease or condition of the neural/sensory
system" as used herein refers to a disease or condition
selected from the group consisting of: brain cancer (e.g.,
brain stem glioma, brain tumours, central nervous system
(Primary) lymphoma, central nervous system lymphoma,
cerebellar astrocytoma, and cerebral astrocytoma,
neurodegenerative disorders (e.g., Alzheimer's Disease,
Creutzfeldt-Jakob Disease, Parkinson's Disease, and
Idiopathic Presenile Dementia), encephalomyelitis, cerebral
malaria, meningitis, metabolic brain diseases (e.g.,
phenylketonuria and pyruvate carboxylase deficiency),
cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia
Complex, schizophrenia, attention deficit disorder,
hyperactive attention deficit disorder, autism, and
obsessive compulsive disorders.
The term "a disease or condition of the respiratory system"
as used herein refers to a disease or disorder selected
from the group consisting of: cancers of the respiratory
system such as larynx cancer, pharynx cancer, trachea
cancer, epiglottis cancer, lung cancer, squamous cell
carcinomas, small cell (oat cell) carcinomas, large cell
carcinomas, and adenocarcinomas. Allergic reactions, cystic
fibrosis, sarcoidosis, histiocytosis X, infiltrative lung
diseases (e.g., pulmonary fibrosis and lymphoid
interstitial pneumonia), obstructive airway diseases (e.g.,
asthma, emphysema, chronic or acute bronchitis),
occupational lung diseases (e.g., silicosis and
asbestosis), pneumonia, and pleurisy.
The term "a disease or condition of the endocrine system"
as used herein refers to a disease or condition selected
from the group consisting of: cancers of endocrine tissues
and organs (e.g., cancers of the hypothalamus, pituitary
gland, thyroid gland, parathyroid glands, pancreas, adrenal
glands, ovaries, and testes), diabetes (e.g., diabetes
insipidus, type I and type II diabetes mellitus), obesity,
disorders related to pituitary glands (e.g.,
hyperpituitarism, hypopituitarism, and pituitary dwarfism),
hypothyroidism, hyperthyroidism, goiter, reproductive
disorders (e.g., male and female infertility), disorders
related to adrenal glands (e.g., Addison's Disease,
corticosteroid deficiency, and Gushing's Syndrome), kidney
cancer (e.g., hypemephroma, transitional cell cancer, and
Wilm's tumour), diabetic nephropathy, interstitial
nephritis, polycystic kidney disease, glomerulonephritis
(e.g., IgM mesangial proliferative glomerulonephritis and
glomerulonephritis caused by autoimmune disorders; such as
Goodpasture's syndrome), and nephrocalcinosis.
The term "a disease or condition of the digestive system"
as used herein refers to a disease or condition selected
from the group consisting of: ulcerative colitis,
appendicitis, Crohn's disease, hepatitis, hepatic
encephalopathy, portal hypertension, cholelithiasis, cancer
of the digestive system (e.g., biliary tract cancer,
stomach cancer, colon cancer, gastric cancer, pancreatic
cancer, cancer of the bile duct, tumours of the colon
(e.g., polyps or cancers), and cirrhosis), pancreatitis,
ulcerative disease, pyloric stenosis, gastroenteritis,
gastritis, gastric atropy, benign tumours of the duodenum,
distension, irritable bowel syndrome, malabsorption,
congenital disorders of the small intestine, bacterial and
parasitic infection, megacolon, Hirschsprung's disease,
aganglionic megacolon, acquired megacolon, colitis,
anorectal disorders (e.g., anal fistulas, haemorrhoids),
congenital disorders of the liver (e.g., Wilson's disease,
hemochromatosis, cystic fibrosis, biliary atresia, and
alpha 1-antitrypsin deficiency), portal hypertension,
cholelithiasis, and jaundice.
The term "a disease or condition of the connective /
epithelial" as used herein refers to a disease or condition
selected from the group consisting of: connective tissue
metaplasia, mixed connective tissue disease, focal
epithelial hyperplasia, epithelial metaplasia,
mucoepithelial dysplasia, graft v. host disease,
polymyositis, cystic hyperplasia, cerebral dysplasia,
tissue hypertrophy, Alzheimer's disease,
lymphoproliferative disorder, Waldenstron's
macroglobulinemia, Crohn's disease, pernicious anaemia,
idiopathic Addison's disease, glomerulonephritis, bullous
pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic
fibrosis, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, osteoporosis, osteocarthritis, periodontal
disease, wound healing, relapsing polychondritis,
vasculitis, polyarteritis nodosa, Wegener's granulomatosis,
cellulitis, rheumatoid arthritis, psoriatic arthritis,
discoid lupus erythematosus, systemic lupus erythematosus,
scleroderina, CREST syndrome, Sjogren's syndrome,
polymyositis, dermatomyositis, mixed connective tissue
disease, relapsing polychondritis, vasculitis, Henoch-
Schonlein syndrome, erythema nodosum, polyarteritis nodosa,
temporal (giant cell) arteritis, Takayasu's arteritis,
Wegener's granulomatosis, Reiter's syndrome, Behcet's
syndrome, ankylosing spondylitis, cellulitis, keloids,
Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma
elasticum, osteogenese imperfecta, chondrodysplasias,
epidermolysis bullosa, Alport syndrome, and cutis laxa.
The term "a" and "the" as used in the present descriptive
is intended to include both one (the singular) and more
than one (plural).
A "therapeutically effective amount" of an active agent or
combination of agents as described herein is understood to
comprise an amount effective to elicit the desired response
but insufficient to cause a toxic reaction. A desired
response, for example, may constitute the formation of a
sufficient and/or acceptable detectable antibody titer
level in a blood sample. The dosage and duration of
treatment of the preparation to be administered to a
subject will be determined by the health professional
attending the subject in need of treatment, and will
consider the age, sex, weight, extent of existing diseased
state and/or tissue damage of the subject, and specific
formulation of Helicobacter and the gene of interest
product being used as the treatment for the subject.
The phrase, "effective level" refers to the level of the
desired activity of the molecules and not necessarily
limited to the number of molecules. For example, the
effective level of amyliri may be decreased to stimulate
ghrelin secretion by using amylin antagonists, without a
necessary concomitant decrease in the amount of free amylin
present in a subject.
The phrase "ghrelin-associated diseases and disorders"
refers to any condition that can be treated prevented or
ameliorated through the modulation of ghrelin activity.
These include conditions that are enhanced, exacerbated or
stimulated by ghrelin, for example, growth hormone release
or drive to eat. The physiological actions of ghrelin are
considered to include, by way of example, the stimulation
of growth hormone release, the stimulation of hormone
secretion from lactotrophs and corticotropes, orexigenic
and cardiovascular actions, anti-proliferative effects on
thyroid and breast tumors and regulation of gastric
motility and acid secretion through vagal mediation. (See
WO 2005021026).
Throughout the specification, unless the context requires
otherwise, the word "comprise" or variations such as
"comprises" or "comprising", will be understood to imply
the inclusion of a stated integer or group of integers but
not the exclusion of any other integer or group of
integers.
Where the definition of terms departs from the commonly
used meaning of the term, applicant intends to utilize the
definitions provided herein, unless specifically indicated.
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, it is to
be understood that this invention is not limited to
particularly exemplified methods and may, of course, vary.
It is also to be understood that the terminology used
herein is for the purpose of describing particular
embodiments of the invention only, arid is not intended to
be limiting which will be limited only by the appended

We Claim:
1. A bacterial delivery composition comprising a Helicobacter pylori cell comprising a
Helicobacter vector or plasmid comprising:
(a) a non-Helicobacter sequence encoding a non-Helicobacter pharmacologically active molecule of interest linked to a secretory signal peptide; and
(b) a Helicobacter sequence having a promoter region capable of controlling expression of the non-Helicobacter pharmacologically active molecule;
wherein the pharmacologically active molecule is expressed on the surface of the Helicobacter pylori cell or secreted therefrom;
wherein the Helicobacter pylori cell chronically colonizes the mucosa of a mammal upon administration and wherein the Helicobacter pylori is not a DapE mutant strain"
2. The bacterial delivery composition as claimed in claim 1, wherein the Helicobacter pylori cell is attenuated.
3. The bacterial delivery composition as claimed in claim 1 or claim 2, wherein the Helicobacter pylori cell is non-pathogenic.
4. The bacterial delivery composition as claimed in any one of claims 1 to 3, wherein the Helicobacter pylori is strain 26695.
5. The bacterial delivery composition as claimed in any one of claims 1 to 4, wherein the promoter is an inducible promoter.
6. The bacterial delivery composition as claimed in any one of claims 1 to 5, wherein the promoter is an arabinose inducible promoter.
7. The bacterial delivery composition as claimed in any one of claims 1 to 6, wherein the promoter is promoter from the Helicobacter pylori histidine kinase HP 165.
8. The bacterial delivery composition as claimed in any one of claims 1 to 7, wherein the promoter is a FlaB sigma 54 promoter.
9. The bacterial delivery composition as claimed in any one of claims 1 to 8, wherein the promoter is a T7 promoter.
10. The bacterial delivery composition according to any one of claims 1 to 9, wherein the pharmacologically active molecule of interest comprises a protein, a peptide or a nucleic acid molecule.

11. The bacterial delivery composition according to any one of claims 1 to 10, wherein the
Helicobacter based construct is a vector or a plasmid vector.
12. The bacterial delivery composition as claimed in claim 11, wherein the plasmid vector is
pHP1(SEQ ID NO. 1 and 2).
13. The bacterial delivery composition as claimed in claim 11, wherein the plasmid vector is
pHP3(SEQ ID N0.3).
14. The bacterial delivery composition as claimed in claim 13, wherein the plasmid vector is pTMI03-8.

Documents:

1562-DELNP-2007-Abstract.pdf

1562-DELNP-2007-Claims-(10-02-2012).pdf

1562-delnp-2007-Claims-(23-05-2011).pdf

1562-DELNP-2007-Claims.pdf

1562-DELNP-2007-Correspondence Others-(10-02-2012).pdf

1562-delnp-2007-Correspondence Others-(23-05-2011).pdf

1562-delnp-2007-correspondence-others 1.pdf

1562-DELNP-2007-Correspondence-Others-(31-12-2010).pdf

1562-DELNP-2007-Correspondence-Others.pdf

1562-delnp-2007-description (complete).pdf

1562-delnp-2007-Drawings-(23-05-2011).pdf

1562-delnp-2007-drawings.pdf

1562-delnp-2007-form-1.pdf

1562-delnp-2007-form-18.pdf

1562-delnp-2007-form-2.pdf

1562-DELNP-2007-Form-3-(10-02-2012).pdf

1562-DELNP-2007-Form-3-(31-12-2010).pdf

1562-delnp-2007-form-3.pdf

1562-delnp-2007-form-5.pdf

1562-DELNP-2007-GPA-(10-02-2012).pdf

1562-delnp-2007-gpa.pdf

1562-delnp-2007-pct-304.pdf

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Patent Number

251466

Indian Patent Application Number

1562/DELNP/2007

PG Journal Number

12/2012

Publication Date

23-Mar-2012

Grant Date

19-Mar-2012

Date of Filing

27-Feb-2007

Name of Patentee

MARSHALL BARRY J.

Applicant Address

105 HAY STREET,SUBIACO,WA 6008 AUSTRALIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MARSHALL BARRY J.	105 HAY STREET,SUBIACO,WA 6008 AUSTRALIA.

PCT International Classification Number

A61K 48/00

PCT International Application Number

PCT/AU2005/001211

PCT International Filing date

2005-08-12

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/602,859	2004-08-20	U.S.A.
2	2004904564	2004-08-13	U.S.A.