MXPA97008615A - Genes therapy anima - Google Patents

Abstract

The present invention relates to DNA sequences, expression cassettes and DNA construction for use in therapy, specifically in gene therapy for the treatment of infectious diseases such as mastitis. Also included are pharmaceutical and veterinary compositions that contain constructs and cells that have been transformed with DNA and that are suitable for implementation in a host mammal. Gene therapy of infectious diseases can be done in situ in white or systemic tissue

Description

ANTI-GENE THERAPY BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to DNA sequences, expression cassettes and DNA constructs for use in therapy, specifically in gene therapy for the treatment of infectious diseases such as mastitis. . Also included are pharmaceutical and veterinary compositions containing the constructs and cells that have been transformed with the DNA and that are suitable for the implantation of a host mammal. (b) Description of the Prior Art At the higher level, transgenic animals are the main way to confer transmissible resistance to diseases in animals. Only a few years after the first successful gene transfer in mice, the new technique was used in farm animals. Several genetic treatises have been pointed out for the application of transgenesis in domestic animals, but one of those important aspects is the improvement of animal health and resistance to the disease by means of gene transfer. The temporary and stable genetic improvement that leads to disease resistance and the treatment achieved by techniques recently developed in molecular biology can contribute considerably to reproduce the problem of diseases. Resistance to infections in animals produced at various levels The constitutional and phagocytic mechanisms (innate immunity) serves as a first line of defense If they are not effective the infected organism can respond by means of specific (acquired) immunity. Therefore, candidates for Gene therapy applications include all known genes to modulate non-specific and specific modulated host defense mechanisms, ie, cytokines, major histocompatibility protein complex (HCM), T cell receptors (TCR) and proteins conferring resistance to the specific disease Increased protection against pathogens can also be conferred by other strategies such as intracellular immunization, genetic immunization, antisense sequences as anti-pathogenic agents and disruption of disease susceptibility genes. Immune Response of Gene Modulation. The immune responses of cytokine orchestra through its role as soluble mediators of cellular communication Identified imcially to direct viability, it was also found that the proliferation, differentiation and lodging of leukocytes, regulate the production of function or other In addition, cytokines interact with, and are produced by cells other than leukocytes, thereby providing a means of communication between the immune system and other tissues and organs Cytokines represent a rapidly growing number of regulatory peptide factors including growth factors, interleukins, chemokines, stimulation of colonies and interferons Their functions are mediated through binding to cell surface receptors in their target cells. It has been shown that cytokines contribute directly to the development of pathology during infectious diseases and tumorigenesis. Different cytokines have been reported for both host defense mechanisms of positive and negative influence. Interferon (IFNs) is a well-characterized class of cytokines producing antiviral and antiproliferative activity as well as cell growth modulation, differentiation and immune responses. As well as its most characterized antiviral activity. As well as its most characterized antiviral activity, the IFNs are instrumental in non-viral pathogens of opposite action, most of them through their effects on macrophage activation. The proteins that are known to be involved in the antiviral and bactericidal actions of interferon and its inhibitory mechanisms are numerous. The power of IFNs was tested to positively influence host susceptibility to viral infections in transgenic mice and cell lines. Transgenic organisms that overexpress IFN-β gene constructs exhibit increased viral resistance. Recent advances in the understanding of transduction pathways are signals and transcription factors activated by IFNs and a variety of other cytokines promises to open up new therapeutic approaches as well as novel strategies of gene transfer treatments helping to improve the immune response; that is, the transfer of genes that code for cytokine by themselves from different signaling components specific for cytokines. "The constitutive expression of a factor of genes stimulated by interferon (ISGF2) which are also called interfering regulatory factor transgenes (IRF- 1) has been reported to result in IFN-independent activation of several genes that can be induced by IFN and increased resistance to viral infection.Specific Disease Resistance Genes Another improvement that can be made in animals by local gene transfer is disease-specific resistance A specific disease-resistance gene that is well examined is the gene product of Mx1 from certain strains of mice The Mx1 protein from mice to a family of polypeptides with GT Pasa activity synthesized in vertebrate cells treated with IFN. Some Mx proteins have been shown to block the multiplication of C Negative RNA RNA viruses, such as Influenza virus, VSV, rhado virus and Thogoto virus. Mx1 protein synthesis of mice in several cell lines and transgenic mice demonstrated that it is both necessary and sufficient to promote resistance to influenza A virus in previously susceptible cells and animals. The cloning and functional characterization of this gene for resistance to a specific disease allowed a gene transfer program to study whether transgenic MX1 pigs could show reduced susceptibility to influenza infections. The natural resistance of certain strains of rabies mice to infection with antigenically unrelated microorganisms such as Mycobacteria, Salmonellae and Leishmania is controlled by a dominant locus on chromosome 1 called Bcg, Lsh or Ity respectively. The locus affects the ability of the host to restrict the proliferation of these infectious pathogens during the macrophage-dependent phase of non-specific infection. A positional cloning approach resulted in the isolation of a candidate Bcg gene designated Nramp. The reduction of susceptibility to Salmonella infections by transgenesis or gene therapy (in vivo or ex vivo) means that it is of great value for the production of animals, especially poultry. A large difference in resistance to Salmonella has been observed in broiler chicken lines. In addition, the natural resistance or susceptibility to infection with Mycobacteria in humans and Brucella in cattle has shown that it is under genetic control similar to that observed in mice of pups and regulated by Bcg. The chronic infection of cattle with Brucella abortus causes the spontaneous abortion of the fetus, threatening the economic benefit of the dairy and meat industries. Genetic resistance to certain retroviruses has been observed as a polymorphic trace in several experimental species. One of the sites identified in mice, Fv-4, resembles the 3 'half of a murine leukemia virus that extends from the end of the pol gene through a complete gene. The expression of Fv-4 that encodes only the viral protective protein in the resistance conferred to transgenic mice to the infection with ecotropic retroviruses. The mechanism of resistance of Fv-4 is thought to refer to the phenomenon of viral interference, that is, the competition of the coat protein synthesized with exogenous viruses for the virus receptor. Similar mechanisms are used in antiviral strategies known as intracellular immunization. "The expression of a transgene that encodes an immunoglobulin specific for a common pathogen can provide immunity for that pathogen, as shown by many investigations, the cloned genes that code for monoclonal antibodies are can express in large quantities in genetically engineered mice, these mice produce antibodies against antigens specific without prior contact or immunization. Intracellular Immunization The concept of "intracellular immunization" essentially involves the over-expression in the host of an aberrant form (dominant-negative mutant) of a viral protein that is capable of strongly interfering with the replication of the wild-type virus. Smart studies in cultured cells that result in acquired resistance to several viruses include strategies that prevent virus binding to target cells, blocking the formation of transcription complexes of the host-virus, expressing dominant-negative viral trans-activators or interfering with the set of infectious viral particles. Endogenous mouse mammary tumor virus (MTV) proviruses have been found to genetically cosegregate loci called self-priming identical to a protein encoded in the long terminal repeat of MMTV. Genetically engineered mice that express high levels of this self-superantigen were shown to be protected from viral infection by suppressing a specific class of T cells that is the target of infection. The definition of "intracellular immunization" also applies antiviral strategies described in different connections such as expression of specific resistance genes, antisense RNA other antiviral components. Recently, an "intracellular immunization" approach carried out on farm animals was reported. Transgenic sheep were produced and were shown expressing the cover gene (en). The visna virus belongs to a subfamily of sheep retrovirus that causes encephalitis, pneumonia and arthritis in sheep. The glycoprotein in is responsible for the binding of this virus to the host cells. The target cell for the replication of the virus seen in infected sheep is the macrophage. The expression of protein on the cell surface of infected cells with sight induces immune responses to the virus. The expression of a gene construct consisting of the U3 visna enhancer region fused to the gene in transgenic sheep had no obvious detrimental effect. Therefore, genetically engineered sheep lines provide evidence of the retroviral glycoprotein to prevent infection and / or to modulate the disease in its natural host after virus challenge. Nonsense RNA The use of nonsense RNA to inhibit RNA function within cells with whole organisms has provided a valuable molecular biological method. The antisense RNA functions by binding in a highly specific manner for complementary sequences, thus blocking the ability of the attached RNA to be processed and / or translated. The contradictory sequences are considered an attractive alternative for conventional drugs in the therapy of microbial infections, cancer, autoimmune diseases and other malfunctions. The experiments of gene transfer with constructions of contrasense have been carried out in mice and rabbits. Mice genetically engineered expressing antisense RNAs indicated to the retroviral packaging sequences of Molony murine leukemia virus did not develop leukemia following confrontation with infectious viruses. Transgenic rabbits were produced by expressing a construction of antisense complementary to the adenovirus h5 RNA. Mainly it was found that the cells of these rabbits are 90-98% more resistant to adenovirus infection than the cells of control animals.

The use of antisense RNAs as anti-parasitological agents can be developed to result not only in RNA-RNA hybrids but catalytically separate a phosphodiester bound in the target DNA strand. Four structural motifs (hammer head and hair pin first identified in the RNA patches of the plant, the delta motif found in human hepatitis delta virus and a less well-characterized motif of Neurospora) have therefore been described as intermediaries in these self-healing reactions. Looking at the hammerhead motif of this family of ribozyme contradictory consequences, it has been shown to overcome specific white RNA. A large number of substrate molecules can be processed by the catalytic RNA since the ribozyme itself is not consumed during the separation reaction. The bovine leukemia virus (VLB), a retrovirus, causes persistent lymphocytosis and B-lymphocyte lymphoma in cattle and sheep. A hammerhead ribozyme turned over by the missense sequences directed against BLV regulatory proteins showed that they inhibit the expression of BLV in persistently infected cells. This demonstrates the possibility of generating (in vivo or ex vivo) or generalized (transgenic animals) gene therapies that will be resistant to diseases induced by BLV.

Somatic Gene Transfer Approaches The transfer of somatic genes in farm animals will be more significant. Gene therapy ex vivo and more recently in vivo has been applied to several genetic diseases in humans. Current therapies developed more than 10 human disorders in genes, such as the failure of the genes to encode normally discourages adenosine, IDL receptor, glucocerebrosidase, factor VI I I of blood clot formation, phenylalanine hydroxidase, dystrophin and others. The efficiency of the gene therapy approach has not been proven.

The novel methods for gene transfer in somatic cells promises to be highly efficient. These include viral vectors for delivering gene constructs and non-viral technologies, such as micro-bombardment or injection of DNA particles or solutions in tissues or blood vessels. Although most efforts have been directed mainly towards the possibility of treating human diseases, some applications of somatic gene transfer could be of great value in veterinary medicine. It makes direct "genetic immunization" and other methods of immunomodulation possible. "Genetic immunization", that is, the application of DNA constructs that encode immunogens, has at least two strong uses. U is not to simplify the procedure and shorten the time required to produce antibodies to particular proteins by eliminating the steps of protein purification. It could be faster to re-introduce a gene that directly codes for a neutralizing or bactericidal antibody in the body. The second is the genetic vaccination in animals against infections by producing foreign counter-sense encoded by the appropriate construction of genes. The somatic gene transfer approach can now be applied either to cure or to prevent an infectious disease by releasing into the organ, or in the organism, a protein that is lethal and absolutely specific to the indicated microorganism and without any affinity or effect for the organism. animal. Said proteins or peptides having a high and specific antimicrobial activity are divided into two families, one including the baterycins and the other the lanthionines, also called antibiotics. The application of biotechnology for animal treatment, particularly of farm animals, is to open new avenues of prevention and control that will have important implications. Bacteriocins consist of enzymes and other bactericidal proteins. They act as catalysts and are very specific for a single chemical reaction. Bacteriocins rapidly kill targeted organisms using the cell wall and do not require the organism to undergo cell division. They are naturally produced by bacteria as a means of population control. These proteins are larger molecules than antibiotics and are expected to persist longer in the treated organ. One of these well-known bacteriocins is lysostaphin, which is produced by Staphyloliticus, which is a biovar of Staphyococcus simulans. Unlike lantibiotics, the rapid action of bacteriocins reduces the likelihood of induced resistance in white and non-target organisms. For example, current research conducted so far seems to indicate that bacteriocins used for mastitis treatment are not toxic to other organisms. Lantibiotics are antibiotics derived from peptides with high antimicrobial activity against several pathogenic bacteria. The ribosomal origin of lantibiotics was first shown by the isolation of the structure gene, ep / 'A, for epidermin, a lantibiotic produced by Staphylococcus epidermidis. The general structure of lantibiotic genes is the same in all lantibiotics described so far. The primary transcript of linear lantibiotics is a propeptide which consists of an N-terminal leader sequence that is followed by the C-terminal propeptide from which the lantibiotic is matured and a prolyl-characteristic proteolytic processing site in position -2. Nisin, produced by several strains of Lactococcus lactis, is a prominent member of the lanthionine group. Other bacteriocins and lanthionines are ambicines, defensins, cecropins, thionines, melitins, magainins, atacinas, dipterinas, saponinas, cacrutinas, xenopinas, subtilinas, epidermines, pep5, lacticina 481, encoveninas, duramicinas, galiderminas, cinnamycin, endropinas and mastoparanas.

Other new classes of molecule complexes that can be secreted by the transgene, ie, the construct used for a gene therapy application, are the immunoadhesins. The therapeutic potential of antibodies has been recognized for a long time. The human antibodies should be minimally immunogenic for the patient; therefore, they must be safe for chronic or repeated use. However, it can be difficult to generate useful human antibodies for several reasons: it is ethically impossible to immunize humans for experimental purposes, therefore, the available human antibodies are limited to immunization products or inadvertent vaccination. In addition, there have been technical difficulties in the immortalization of human cell lines. Perhaps, the most refractory technical problem is that many applications require antibodies to human antigens, as well as human antibodies with the desired specificity. There are several potential approaches to avoid these problems. One approach is to treat the desired binding specificity in variable regions (V) of antibodies. This can be done by deriving complementarity that determines regions of either mouse antibodies, or in vitro recombination combined with selection (eg, combinatorial libraries and phage display technology). An alternative approach, which sometimes has advantages, is to create a molecule similar to antibodies by combining a binding site, derived from a human protein such as a cell surface receptor or cell adhesion molecule, with constant domains of antibodies. These molecules are known as immunoadhesins. Immunoadhesins can have many desired chemical and biological properties of antibodies. There are examples of immunoadhesins that can bind to Fe receptors and show active transport through primate placenta. Since the immunoadhesin is constructed from a receptor sequence bound to an appropriate hinge and Fe sequence, the binding specificity of interest can be achieved using fully human components. Another potential strange sequence is that in the junction region. One of the well-studied immunoadhesins is CD4-IgG that has been found completely non-immunogenic in human clinical trials. A second candidate for clinical use is an immunoadhesin of tumor necrosis factor receptor (THFR-IgG); This molecule is particularly interesting, given that the soluble receptor itself is naturally found in the body and has been considered as a possible therapeutic. While soluble receptors are valid clinical candidates, the fusion form of IgG may well confer benefits such as longer half-life and improved strength and affinity. Some receptors or immunoinductors that have been linked to the Fe part of IgG to form immunoadhesins are reported in literature: T cell receptor, CD4, 1-selectin, CD44, CD28, B7, CTLA-4, CD22; TNF receptor, NP receptor, IgE receptor, INF- ?. These immunoadhesins should be useful in antigen recognition, HIV reception, lymphocyte adhesion, hyaluronidase receptor, B and T lymphocyte interaction, inflammation, peptide shock, homeostasis and allergy. In animals, the arrival of molecular biology techniques allows the creation of an immunoadhesin that could have two specific activities. For example, in order to eliminate a contamination with Staphylococcus aureus it may be possible to have an immunoadhesin composed of a lytic enzyme, similar to lysostaphin, attached to the Fe part of human IgG that has a high affinity for protein A on the surface of the bacteria. Once the Fe has been bound to protein A in Staphylococcus aureus, the lytic part, lysostaphin, can lyse the bacteria. Gene therapy treatments can be applied in such a way that the gene included in the transferred constructs could be encoded for an immunomodulator, such as interleukins, chemokines, interferons, leukotrienes and certain growth factors. As explained above, immunomodulators can make the animal more resistant to several microorganisms. COMPENDIUM OF THE INVENTION The present invention relates to the therapy of animal genes The therapy of animal genes means an approach by which the construction of DNA involving an inducible or constitutive promoter linked to a gene encoding a protective or protective protein or RNA of contrasense or peptide that acts against infectious or potentially infectious microorganisms responsible for diseases. A method is described for expressing a protein or RNA of contrasence or peptide that directly or indirectly has therapeutic or prophylactic effects against infectious microorganisms in animals. The invention is useful for producing a heterologous or homologous protein or sense RNA or peptide that binds to a specific tissue or organ and that can act on a microorganism that infects the animal. The method involves inducing a liquid complex that includes a genetic construct in a given tissue of the animal. If desired, the infused genetic construct can be treated with a polycationic compound and / or a lipid to improve the efficiency with which it is absorbed by secretory cells of the animals. The most expensive infectious disease in animals is mastitis caused by infection of the mammary gland. Among others, this invention relates to a method for treating mastitis. More particularly, this invention relates to the use of DNA constructs designed to be transcribed into a therapeutic protein after insertion into a mammary gland of lactating or non-lactating animals. The mastitis of cattle, goats, sheep and pigs is still one of the most expensive diseases in animal agriculture. Mastitis represents a significant economic loss for the dairy industry, approximately 70 to 80 percent of which can be attributed to a decrease in milk production. Many infectious agents have been implicated as a cause of mastitis and these are treated separately as specific entities in cows, sheep, goats and pigs. Despite significant progress in mastitis control due to the widespread adoption of antisepsis of the teats after milking, many flocks remain plagued by this disease. A variety of different procedures have been described and used to cure mastitis caused by bacteria and yeast. These methods include the systemic immunization of the infected animals with whole or partial protein extracts of the infectious agents in order to stimulate the immune response of the treated animal to these agents. The antibodies generally produced in this manner act against a membrane protein, a binding protein or a toxin secreted by microorganisms. Therefore, these antibodies act as anti-adhesive, anti-toxin, neutralizing or opsonic molecules (Nordhaug et al., 1994, J Dairy Sci., 77: 1267 and 12176). However, the milk barrier prevents not all but a very small proportion of IgG antibody circulation from reaching breast secretion during lactation. Other procedures have been carried out in order to stimulate diapedesis and phagocytosis of contaminating agents by leukocytes, more particularly neutrophils and polymorphonuclear macrophages. The stimulation molecules, which have been administered by intermammary injection, are cytokines, interleukin-1β, interleukin 2, interferon-α tumor necrosis factor-a. The most widely used procedure to cure infectious diseases is the administration of antibiotics. However, this approach imposes a lot of lateral effects on the animal and particularly in the case of animals that give milk, milk must be discarded during the treatment period. Unfortunately, all current procedures are of short duration consequently they are relatively inefficient. For example, none of the gram positive batteries are completely removed from the udder after antibiotic treatments. For these reasons, a gene therapy method that allows a gene to integrate into a target tissue, such as the mammary gland and provide for the elimination by gene therapy of the contaminating microorganisms is desired. In addition, gene therapy of the mastitic gland eliminates all the alterative effects of other procedures, also allowing an inserted gene to synthesize inductively or constitutively in a permanent form an effective amount of its therapeutic protein, peptide or RNA counter-sense product. Therefore, this invention allows a much more specific and effective system of treatment of infectious diseases than is currently possible. The additional objects, aspects and advantages of the invention will be apparent to those skilled in the art upon considering the following detailed description of the preferred embodiments exemplifying the best mode of the invention as it is currently perceived. The present invention provides a recombinant DNA comprising a nucleotide sequence that encodes a protein or polypeptide that is useful in the prophylaxis or treatment of mastitis and at least one regulatory control element that allows the expression of said nucleotide sequence in a gland mammary Suitable regulatory control elements include the transcriptional and translational regulatory sequences. Transcriptional and translational regulatory sequences are those DNA sequences necessary for the efficient expression of the product. In general, said regulatory elements can be operably linked to any nucleotide sequence to control the expression of the sequence, the entire unit being referred to as the "expression cassette". Therefore, the invention also provides an expression cassette containing the recombinant DNA mentioned above. An expression cassette will typically contain in addition to the coding nucleotide sequence, a promoter region, a translation initiation site and a translation termination sequence. Unique endonuclease restriction sites can also be included at the end of an expression cassette for allowing the cassette to be easily inserted or removed when creating DNA constructs for use as transformations as is known in the art. In particular, the invention provides a DNA construct designed to express a protein or polypeptide that is useful for the prophylaxis or treatment of infectious diseases after insertion into the designated tissues. Suitably, the DNA construct comprises an inducible or constitutive promoter that is linked to a coding nucleotide sequence or gene thereby expressing a therapeutic or protective protein that acts against infectious or potentially infectious microorganisms responsible for animal diseases. For example, said DNA constructs can be administered to either lactating or non-lactating animals for the prophylaxis or treatment of mastitis. Therefore, the invention also provides a method for the prophylaxis or treatment of mastitis comprising the transformation of mammary gland tissue with a DNA construct as described above. The present applicants have found that expression of proteins in mammary glands over an extended period is possible and that a gene therapy approach towards the problem of mastitis is possible. The integration of the gene encoding a therapeutic protein or polypeptide in mammary gland tissue could allow, for example, the elimination of infectious microorganisms by genetic therapy. In addition, gene therapy of the mastitis gland eliminates all side effects of other procedures., thus allowing an inserted gene to synthesize permanently and inductively or constitutively an effective amount of its therapeutic protein product. A gene therapy approach could be a much more specific and effective mastitis treatment system than is currently available. The transformation of mammary gland tissue generally requires that the DNA be physically placed within the host gland. Current transformation procedures use a variety of techniques to introduce pure DNA into a cell and these can be used to transform a mammary gland. For example, DNA can be injected directly into glands through the use of syringes. Alternatively, high-speed ballistics can be used to propel associated small DNA particles into the gland through an incision of the udder skin. DNA can also be introduced into a mammary gland by inserting other entities which contain DNA. These entities include minicells, cells (e.g., fibroblasts, adipocytes, epithelial cells, myoepithelial cells, mammary carcinoma cells, kidney cells), liposomes (e.g., natural or synthetic lipid carriers, cationic liposomes) or other surface bodies of meltable lipids. The entities are transformed in vitro before insertion using the DNA constructs described above.

Therefore, the invention also provides a cell that has been transformed using a DNA construct as described above. Examples of such cells include Mac-T cells. Genetically transformed cells of this type are suitable for producing the desired proteins or polypeptides. In addition, the invention provides a liposome incorporating the DNA construct described above. The introduction of pure or complexed DNA constructions into the mammary gland can be done by direct injection through an incision in the skin of the udder or through the nipple canal. Where apriate, the DNA construct is administered in the form of an acceptable pharmaceutical or veterinary composition in combination with a suitable vehicle or diluent. Suitable vehicles are liquid vehicles such as water, salt solution with pH regulated with salts or any other physiological solution. These compositions form a further aspect of the invention. The protein or polypeptides produced must be the prophylaxis or effective treatment of mastitis. Said proteins or polypeptides include mucolytic proteins such as enzymes, antibiotics, antibodies, cytokines, tumor necrosis factors as well as proteins that can induce an immune response for ineffective or potentially ineffective agents and those that activate polymorphonuclear neutrophils, or macrophages.

In a preferred embodiment, the invention provides a recombinant DNA sequence which comprises a nucleotide sequence which encodes a lytic protein or antibody under the control of a mammary gland-specific promoter, or any ubiquitous or inducible non-mammary promoter. The invention is particularly applicable for the treatment of farm animals: cattle, goats, sheep, and pigs, but may also refer to lower mammals or lower milk producers: rabbits, camels or bison. The invention can also be used in humans to eliminate particularly the majority of Staphylococci. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates examples of DNA constructions according to the present invention, and Figure 2 illustrates the human growth hormone synthesis regime in sheep giving milk after the invention of liposome complex. cationic-DNA in the mammary gland. DETAILED DESCRIPTION OF THE INVENTION According to a preferred embodiment of the present invention, the gene therapy of infectious disease animals consists of the transfection of a target tissue with DNA sequences designed to produce molecules that are reinserted into the organ or organism. This could then protect the animal against infectious or potentially infectious microbial agents. According to another embodiment of the present invention, the mammalian mastitis gene therapy consists of transfecting the mammary glands with DNA sequences designed to produce molecules that will be reinserted into the udder, this could then protect the animal against the infectious microbial agents or potentially infectious. The white tissue can also be transformed with other DNA sequences such as regulatory sequences of transcription and translation of genes. The transcriptional and translational regulatory sequences are those DNA sequences necessary for the efficient expression of the gene product. In general, said regulatory elements can be operably linked to any gene to control the expression of the genes, the whole unit being referred to as the "expression cassette". An expression cassette will typically contain, in addition to the coding sequence, a promoter region, a translation initiation site and a translation termination sequence. Single endonuclease restriction sites can also be included at the ends of an expression cassette to allow the cassette to be easily inserted or removed when creating the DNA constructs. The expression of a gene is mainly driven by its own promoter, although other DNA regulatory elements are necessary. Promoter sequence elements include the consensual sequence of the gene box (TATAAT), which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most cases the TATA box is required for the start of precise transcription. For convenience, the transcription start site is designated +1. The sequences that extend in the 5 'direction (upstream) are given negative numbers and the sequences that extend in the 3' direction (downstream) are given positive numbers. The promoters can be both constitutive and inductive.

A constitutive promoter controls the transcription of a gene at a constant rate during the life of a cell, while an inducible activity of the promoter fluctuates as determined by the presence (or absence) of a specific inducer. The regulatory elements of an inducible promoter are usually located upstream as well from the transcription start site as the TATA box. Ideally, for experimental purposes, an inducible promoter must possess each of the following properties: a basal level of low to nonexistent expression in the absence of inducer, a high level of expression in the presence of an inducer and an induction scheme that it does not alter in any way the physiology of the cells. The basal transcription activity of all promoters can be increased by the presence of the "enhancer" sequences. Although the mechanism is unclear, certain defined enhancer regulatory sequences are known to those familiar in the art, in order to increase a transcription rate of the promoter when the sequence approaches the promoter. Constitutive promoters can achieve transcription of their ligated gene in a tissue specific form of a mammary gland. For example, strong constitutive promoters are those that control the expression of caseins, lactoglobulins, lactoferrin, lactalbumin, lysosomes, wheat acid proteins (PAT) that encode genes in mammary glands. Preferentially, the promoters originate from domestic animals, cattle, goats, sheep or porcine species. Alternatively, the promoters of the specific mammary gland can originate from smaller animals, lagomorphs, rodents, felines or canines. Other constitutive promoters that regulate the expression of cytoplasmic ß-actin or ubiquitin genes can be used. Viral or retroviral promoters can also be used, such as Cytomegalovirus (CMV), Simian virus 40 (SV40) or mouse mammary tumor virus (MMTV, which is additionally inducible). Inducible promoters include any promoter capable of increasing the amount of gene product produced by a given gene, in response to exposure to an inducer. Inducible promoters are known to those familiar in the art and there is a variety that could conceivably be used to boost gene expression of protective or curative molecules. Two preferred inducible promoters are the heat shock promoter (TCC) and the system. glucocorticoid Promoters regulated by heat shock, such as the promoter normally associated with the gene encoding the heat shock protein of 70 kDa, may increase expression several times after exposure to elevated temperatures. The heat shock promoter could used as an environmentally inducible promoter to control gene transcription of the protective or curative molecule The glucocorticoid system also works well to drive expression of genes that include protective or curative molecule gene The system consists of a gene encoding glucocorticoid receptor protein ( RG) that in the presence of a Spheroidal hormone forms a complex with the hormones This complex is then linked to a sequence of short nucleotides (26 bp) named the glucocorticoid response element (ERG) and this binding activates the expression of linked genes. The glucocorticoid system can be included in the construction of transformation of DNA as a means to induce protective or curative molecule expression Once the constructs have been inserted, the systemic steroid or glucocorticoid hormone will be associated with the RG protein constitutively produced to bind to the ERG elements, thereby stimulating gene expression of protective or curative molecules (v gr, antibodies or enzymes) Presumably, the indicated tissue will allow the inserted gene (pure, of liposomes, solid particle enclosed in the cell or coated) will produce its protein product in an amount sufficient to produce the desired effect. The products of the inserted gene must heal or protect the organ or organism in which it is expressed against infectious or potentially infectious microorganisms responsible for the disease. The transformation of an animal tissue requires that the DNA be physically placed inside the host animal. Current transformation procedures use a variety of techniques to introduce pure DNA into a cell, which can be used to transform a designated tissue. In a transformation form, the DNA is injected directly into the tissue through the use of a syringe. Alternatively, high-speed ballistics can be used to propel small associated DNA particles into the tissue through an incision in the skin. In other forms, DNA can also be introduced into a designated tissue by inserting other entities that contain DNA. These entities include minicells, cells (e.g., fibroblasts, adipocytes, Mac-T cells, myoepithelial cells, mammary carcinoma cells, kidney cells, liver cells, lung cells, lymphocytes, leukocytes), liposomes (v. gr., natural or synthetic lipid vehicles, cationic liposomes) or other meltable bodies with lipid surface.

The invention relates to when neutralizing, lytic or opsonic molecules are synthesized from the gene used for the therapy of infectious disease genes. Preferably, in the case of mastitis, the gene encoding a mucolytic protein (e.g., bacteriocins and lenthionines) can be used to eliminate Gram-positive bacteria (mostly cocci). The gene products can serve as an immunomodulator and to induce an immune response, the activation of polymorphonuclear neutrophils, or for example macrophages. The product can be a cytosine or another immunomodulator. Alternatively, the genes can be used for the in situ synthesis of the following therapeutic polypeptides: 1. Enzymes or mucolytic proteins, such as lysostaphin and mucolysins; 2. Antibodies, such as anti-hemolysins, anti-leukocidin, anti-protein A, anti-collagen, antifibronectin binding protein, anti-paminime, antibodies against toxin a and against beta-toxin; opsonic antibodies and antibodies that arise against viral fusion protein; 3. Cytokines, interleukins, chemokines, growth factors; 4. Interferons; 5. Tumor necrosis factors; and 6. Immunoadhesins or immunotoxins.

While antibiotics are not very suitable, they can alternatively be used with inducible promoters. The microorganisms that may be responsible for mastitis and can be eliminated by the gene therapy approach are: In cattle Streptocuccus agalactiae, Str. Ube, STr. zooepidemicus, Str, dysglactiae, S Tr. faecalis and Str. pneumoniae, Staphylococcus aureus, Escherichia coli, Klebsiella spp. , Corinebacterium pyogenes, Cor. bovis, Mycobacterium tuberculosis, Mycobacterium spp. , Bacillus cereus, Pasteurella multocida, Pseudomonas pyocianeus, Sphaerophorus necrophorus, Serratia marcescens, Mycoplasma spp. , Nocardia spp. , a fungus Trichosoporon spp. , levadu ras Candida sp. , Cryptococcus neoformans, Saccharomyces and Torulopsis spp.

In sheep: Pasteurella haemolytica, Staph. aureus, ActinoBacillus lingnieresi, E. Coli, Str. uberis and Str. agalactiae, and Cor. p se udot ube reulosis. In goats: Str. Agalactiae, Str. Agalactiae, Str. Dysgalactiae, Str.

Pyogenes and Staph. A ureus. In pigs: Aerobacter aerogenes, E. coli, Klebsiella spp. , Pseudomonas aeruginosa, Staphylococci coagulase positive, Str. Agalactiae, Str. Dysgalactiae and Str. Uberis. In horses: Corynebacterium pseudotuberculosis, Str. Zooepidemicus and Str. Equi.

Other microorganisms and diseases that can be eliminated from exotic animals by the gene therapy method are those that cause: In primates: Poliomyelitis, Measles, Mumps, Rubella, DPT, Tetanus. In dogs: Canine distemper, canine adenovirus, canine parvovirus, Canine parainfluenza, Rabies, Bactrerina Leptospira. In Felines: feline panleukopenia, feline rhinotracheitis, feline calicivirus, rabies. In Artiodactílas: BVD, Bacterina de Clos. Of 8 forms, Bacterina de Lepto. Of 5 forms, Parainfluenza 3, Prions, Dissipations. Examples of infectious diseases that could be cured or avoided by the application of gene therapy are: anemia, arthritis, rhinotracheitis, bronchitis, bulbar paralysis, bursal diseases, hepatitis, cloacitis, coryza, enterohepatitis, hemopoietic necrosis, jaundice, keratoconjunctivitis, laryngotracheitis, myxomatosis, necrotic hepatitis, ophthalmia, pancreatic necrosis, pododermatitis, polyarthritis, pustular balanoposthitis, vulvovaginitis, serositis, sinusitis, stomatitis, synovitis, thromboembolic meningitis and tracheobronchitis. The present invention relates to a gene therapy approach with both curative and prophylactic activities on microorganisms that cause infectious diseases. The invention relates in particular to AD sequences, expression vectors, DNA vehicles (liposome, solid particles) and cells that make use of the process.

The invention relates in the same way to cells (e.g., Mac-T, lung, kidney, muscle cells) genetically transformed in vitro with the gene of interest and reimplanted in the tissues of origin to produce the curative or prophylactic proteins, peptides or RNA of contradiction against microorganisms responsible or potentially responsible for the diseases. The invention relates more particularly to domestic animals: cattle, goats, sheep, pigs, felines, dogs and birds, but may also refer to more exotic animals such as rabbits, camels and bison. The present invention will be more readily understood by reference to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE I Long-term persistence of plasmid DNA and foreign expression in sheep's mammary glands The mammary gland promoters have been used in transgenic animals to limit the expression of transgenes to the mammary gland. Gene therapy techniques have also been shown to single out an organ for the introduction of a foreign gene. Most efforts towards postnatal gene therapy have relied on new genetic information in tissues: the target cells are removed from the body infected with viral vectors that carry the new genetic information and then reimplanted in the body. For some applications, direct introduction of genes into tissues in vivo, with or without the use of viral vectors, may be useful. Direct gene transfer in vivo in postnatal animals has been achieved with DNA formulations encapsulated in liposomes, DNA trapped in proteoliposomes containing viral protection receptor proteins (Nicolau et al., 1983, PNAS USA, 83: 9551) and DNA coupled with a polylysine-glycoprotein vehicle complex (Wu and Wu, 1988, J. Biol. Chem., 263: 14621). In vivo ineffectiveness of cloned viral DNA sequences after direct intrahepatic injection with or without formation of calcium phosphate coprecipitates has also been described (Seegar et al., 2984, PNAS USA, 81: 5849). With the use of cationic lipid vesicles (Felgner et al., 1989, PNAS USA, 86: 6077) and in Xenopus laevis embryos (Malone, 1989, focus 11:61). It is demonstrated herein that injection of pure DNA complexed with cationic liposomes directly into the sheep's mammary gland results in significant expression of the reporter gene within the gland. Preparation of Plasmid-Liposome Mixture Plasmid pCR3 (InVitrogen) was used as a mammalian expression vector. After PCR amplification, human growth hormone (hGH) cDNA was inserted into pCR3. This resulted in the construction of plasmid pCR3. The Plasmid-Lipofect-AMINE ™ (BRI) mixture was prepared as described by the manufacturer (GibcoBRL). In summary, 50 ug of pCR3-hGH suspended in 500 μl of LipofectAMINE ™ also previously diluted in 500 μl of PBS and kept at room temperature for 1 hour. Infusion of the plasmid-liposome complexes in sheep's mammary gland The mixture of pCR3-hGH circular-LípofectAMINE ™ plasmids was loaded into a glass syringe. Just after falling, using a 20-gauge needle, the DNA-liposome complex infused directly through the udder skin into the mammary parenchyma. A mi was injected into the right room of two female sheep. Milk from the left glands was used as negative controls. Analysis of sheep's milk The sheep were milked by hand once a day, maintaining the milk at -80 ° C until it was analyzed. The amount of hGH was measured by immunoanalysis (Immunocorp) after determining that the milk did not affect the accuracy of the analysis. The aliquots (100 ul) of milk samples were analyzed. RESULTS hGH synthesized was detected by injecting pCR3-hGH into the mammary gland throughout the lactation period, meaning approximately 60 days, as illustrated in Figure 2. The concentration of hGH in the milk of the sheep was relatively high during the first 5 days. At that time it was 300 to 400 ng / ml (+ 43 ng / ml). The concentrations of hGH in the milk of the left gland (control) were 10 to 15 ng / ml daily for the two sheep during the experiment. No significant differences in hGH concentration were found in milk samples between each female sheep. Conclusion These results demonstrate that the expression of plasmid DNA can persist in a mammary gland of sheep for at least 60 days. The unprecedented ability of plasmid DNA to stably express a foreign gene in a mammary gland through the period of lactation of a sheep has important implications for gene therapy. The stable expression of curricular plasmid DNA suggests that extraneous acceleration or viral transduction should also be maintained stably. EXAMPLE II Human growth hormone (hGH) in goat's milk after direct transfer of the hGH gene into the mammary gland An alternative route to introduce genes into the breast parenchyma is through the expansion of gene therapy techniques. In this studio, two retroviral vectors of Gibbon monkey leukemia virus (GalV) pseudotypes were used to transfer reporter genes into mammary secretory epithelial cells of goats in vitro and in vivo. Cells and tissue culture MDBKs, a bovine kidney cell line and Mac-T cells were used. Retroviral packaging cell lines used (fCre, PA317, and PG13 / LNc8) were purchased from ATCC. The cells were maintained in Dulbecco's modified Eagle medium (MEMD) supplemented with gentamicin (54 mg / ml) and 10% fetal calf serum, 37 ° C with 5% CO2 / 95% air. Establishment of producing cell lines A construct carrying JR-gal neo- (Wang et al., 1991, Cancer Re., 51: 2642) was transfected in fCre by particle bombardment to 1 μg of DNA per mg of gold beads. Two days after the bombardment, the supernatant was removed from these cells and centrifuged and then the addition of polybrene at 4 μg / ml, the retroviral solution was used to infect both amphotropic and GaLV pseudotype cell lines. A plasmid carrying the retrovirus vector, MFG-hGH was cotransfected with pSV2neo at a ratio of 50: 1 via bombardment of particles in PA317s and PG13 / LN cds. The packaging cells that produce retroviruses containing the hGHG gene were selected by resistance of G418 (400 μg / ml). Cells that produce viruses The clones PG13 / LN c8 that produced the highest levels of hGH produced from the target cell lines were chosen for infusions in a goat mammary gland. Each clone was passed three times in 200 100 mm plates. The cell supernatant was collected over a period of 3 days, concentrated and resuspended in DMDM with Gentamicin. Induction of cell division and lactation of goats Two goats (goats 1 and 2) of 2 years of age and two goats (3 and 4) of 1 year old virgins crossed with Saanen were treated with exogenous im steroids during a 14 day interval to induce mammogenesis and subsequent lactation Infusion of viral strains in mammary glands of a goat Pohbreno was added to the viral strain PG13 / LN c8 MFG-hGH PG13 / LN concentrated to 80 μg / ml and loaded into a syringe Using a guard adapter of caliber 22, the retroviruses were poured into the right mammary nipple on days 3, 5, 7, 9, 11, and 13 of the hormonal regimen for goats 1, 2, and 4 and goat 3 received infusions on days 3 , 5, 7, 9, 10 and 13 The amount of viral solution was different for each animal, varying from 8 to 20 ml and was determined by the integral capacity of the gland The left gland served as the intra-animal control and was poured in DMEM containing gentamicin The retroviral strain used for The infusions were then analyzed in several cell lines Analysis of goat's milk The goats were milked twice daily by hand keeping the milk in the morning at -80 ° C until analyzed The amount of hGH was measured by immunoassay after determine that the milk did not affect the accuracy of the analysis Aliquots (5 μl) of diluted milk samples were also analyzed 1 10 in double distilled water by SDS / PAGE in 14% genes stained with Coomassie blue The protein concentration of the samples of milk were determined used BCA (Pierce et al., 1977, Anl Biochem 81 478) RESULTS Production of vector packaging cell lines The concentration of hGH in the medium removed from Mac-T and MDBK cells 2 days after infection with retrovirus packed by clone 6 PG13 / LN c8 was 192 and 3.8 ng / ml, respectively. Twenty-eight days after infection, the hGH levels of these cells were 119.3 and 4.5 ng / ml, indicating that the LTR provirus is still functioning 4 weeks after infection. Analysis of goat milk Lactation started on day 14 of the hormonal regimen, 24 hr after the last viral infusion. The milk seemed normal through the lactations. The volume of milk obtained from each half udder was approximately 150 ml on the first day of lactation for goats 1 and 2 but only 10 ml for goat 3, and 35 mi for the open 4. The volume of milk produced by each gland for the four goats increased daily. The hGH levels were determined by immunoassay with unique hGH secretion patterns for each animal. In goat 1, hGH concentration decreased steadily until day 9 of lactation when it reached the level of 3-5 ng / ml, while goats had a more precipitous decrease in hGH measured from day 1 to day 2 of lactation , although hGH animal production stabilized at 2-3 ng / ml around day 10. Milking was stopped at day 15 of lactation for goats 1 and 2. HGH levels in goat's milk 3 it decreased dramatically from day 1 to 2 of lactation and then increased from day 8 to day 9 where it remained at 23 ng / ml until day 16 when it started falling again. Goat 4, in which prostaglandin D2 was infused to the remaining 19 days of lactation after a decrease in the first 2 days. In addition, goat 4 still secreted hGH at 5 ng / ml after 28 days. The concentrations of hGH in the milk of the left gland (control) varied from 0.0 to 0.6 ng / ml for the four goats in all the evaluations. These numbers are at the detection level of the analysis and correlate with those measured in the other two lactating goats that had no exposure to the retrovirus. The total production of hGH in the four animals ranged from 0.3 to 2 ug / day. If the hGH gene has been stably incorporated into the support cell population, it could have been expected that the goats could also secrete hGH in a second lactation after the gland was involuted. A second lactation was induced in two of the goats and although goat 1 did not produce hGH, goat 2 began to secrete detectable amounts of hGH starting on day 5 of the right gland (subjected to infusion) and during the subsequent 10 days concentrations ranged from 0.4 to 2.3 ng / ml. The milk from the control left gland during this lactation always had undetectable levels of hGH. SDS / PAGE of goat milk sampled through the collection period showed inconsistent differences in the protein profiles of the right glands with retroviral infusion, left control glands and a goat not exposed to the retrovirus. The protein concentrations measured by BCA of the milk with hGH were not statistically different from the control milk, thus the production of hGH by the mammary secretory epithelial cells did not seem to affect the normal cellular protein machinery. There was an indication that the milk proteins in the treated gland were not secreted at the maximum concentration on day 1 of lactation. Conclusion The application of gene therapy technology and defective replication retroviral vectors to directly introduce a foreign gene into a ruminant mammary gland has dramatically reduced the time of pharmaceutical production in milk, from years to weeks. Although the levels of expression found are low, the methods must find application in the evaluation of different gene constructions as a prelude to the production of transgenic animals or in the production of low levels of important proteins for evaluation purposes. EXAMPLE III The effect of lysostaphin on Staphylococcus aureus infections in the mammary gland of mice. Lysostaphin is an endopeptidase produced by Staphylococcus simulans. It hydrolyzes the pentaglycine bonds of the peptidoglycan of members of the genus Staphylococcus and consequently has little activity against other prokaryotes and none against eukaryotes. The lysostaphin gene has been cloned and expressed successfully in Escherichia coli and Bacillus species (Heath et al., 1987, FEMS Microbiology Letters, 44: 1127). The use of lysostaphin to promote the lysis of Staphylococcus aureus in a variety of experimental situations is well known but the progress made in the cloning and expression of the gene in other hosts raises the possibilities for producing large quantities of the relatively inexpensive enzyme. This may allow its use in vivo in new approaches for the control of staphylococcal mastitis, an economically important disease of lactating ruminants (Bramley et al., 1990, Res. Vet. Sci., 49: 120). This experiment shows the use of the mastitis model in the lactation mouse and clearly demonstrates the potent antibacterial activity of lysostaphin against S aureus in vivo. Lysostaphin (Sigma Chem.) Was dissolved in skim milk (Oxoid) to provide a scale of concentrations between 0.1 and 100 μg / ml. Controls without lysostaphin were included. Volumes of one ml of the controls and dilutions of lysostaphin were inoculated with colony forming units of 108 (cfu) of S. Aureus M60. This strain produces toxins as well as ß and was isolated from a case of cattle mastitis. The concentrations of lysostaphin exceeding 2 to 3 ug / ml in milk produced a reduction of 2 to 3 log 10 in viable S. aureus, while 10 ug / ml in milk reduced S. aureus, from a mean of 7.95 log10m? in the control to 2.0 log10 / m? Consequently, a dose of 10 ug of lysostaphin was selected for use in vivo. The anesthetized mice of the MF1 strain were inoculated into the upper pair of abdominal mammary glands (designated R4 and L4). Eight mice without lactation were inoculated with 108 cfu of S. aureus in 0.1 ml of saline in R4 and 0.1 ml of saline in L4. After an additional 30 minutes, the mice were killed and the mammary glands were removed aseptically and homogenized in saline containing 0.1 mg / ml trypsin (Sigma Chem.) To destroy active lysostaphin. One tenth dilutions were placed in 7 percent calf blood agar (Oxoid Blood Agar Base Number 2), incubated at 37 ° C overnight, and viable counts were determined. In a further experiment using 20 mice, a prophylactic use of lysostaphin was simulated by applying infusion of 10 ug of lysostaphin intramammary, followed either immediately or after one hour by 103 cfu of S. aureus. The control glands were infused with saline instead of lysostaphin. After 24 hours the mice were killed and excised. Thick pathological changes were observed in viable S. aureus counts as described above. RESULTS Infusion with 10 mg of lysostaphin in mammary glands was previously inoculated with reduced bacterial recoveries of S. aureus, compared with controls by more than 99 percent in 30 minutes. This reduction was statistically significant (t = 2.56; P < 0.02). When 10 ug of lysostaphin was administered either immediately in an hour before the inoculation of S. aureus, recoveries after 24 hours averaged about 102 viable S. aureus per mammary gland compared to about 109 per mammary gland for the treated controls They are saline solution. In the latter case, the control glands showed normal severe pathological changes of acute staphylococcal mastitis in the mouse. The control glands were darker and redder, had a fragile texture and some areas of liquefaction and hemolysis. The histological sections revealed a severe inflammation, infiltration of nematophils and macrophages with areas of coagulative necrosis. Large numbers of Staphylococci were visible. In contrast, the glands treated with lysostaphin remained pale and elastic with only slight redness around the base of the nipple. The histological examination showed little or no cellular infiltration, a well preserved and functioning alveolar structure and few cocci. Conclusion These experiments clearly demonstrate the anti-staphylococal activity of lysostaphin in vivo. Both therapeutic and prophylactic potentials were demonstrated. The cloning of the lysostaphin gene can make it readily available for therapeutic use at a competitive price and its relatively high specificity makes it attractive for use in animals that produce food. In addition, advances in transgenic technology allow the direction of expression of transgenes to the mammary gland of ruminants (Simons, et al., 1987, Nature, 328: 530). In general, this has been applied to the production of pharmacologically active substances for use in human medicine. However, the incorporation and expression of the lysostaphin gene in the mammary gland of lactation could potentially increase the resistance of the animal to staphylococcal mastitis. EXAMPLE IV Efficacy of lysostaphin for treatment of intramammary infection by Staphylococcus aureus The cloned-derived lysostaphin was evaluated as its bactericidal effect in intramammary infections by S. aureus (Newbould 305) was eliminated from glands of guinea pigs of India 48 hours after infection by 125 μg in 0/1, and 0 μg in 0/3. The glands infected with S. aureus 48 hours after the confrontation in untreated rabbits of india, however, the control glands 3/25 of the guinea pig of the treated treat were clarified in response to the treatment of the adjacent gland. . Somatic cells / ml in guinea pigs from India changed from 104 preinfected glands to cell counts greater than 3 x 106 after inoculation with S. aureus. The treatment with lysostaphin occasioned a neutrophilic change in the treated gland to levels exceeding 108 accompanied by an increase in the adjacent untreated gland but fell sharply to the pretreatment level. The greatest response in control glands was observed in animals receiving 125 ug which corresponded to the 2/25 free passage of S. aureus in control glands. The response of leukocytes to intramammary treatment in the cow is similar to the guinea pig model of the Indian described above. Somatic cell levels were increased tenfold in glands infected with S. aureus in the treatment after milking. The cell levels returned to the pretreatment levels or lower in the subsequent milking. An elevation in leukocytes alone can not count to give free passage to the infection. EXAMPLE V Use of a Recombinant Bacterial Enzyme (Lisostaphin) as a Therapeutic for Mastitis A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a product of Staphylococcus simulans, enzymatically degrades the cell wall of Sthaphylococus aureus and is bactericidal. Thirty Holstein-Friesian dairy cows in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all rooms. Infections were established and monitored for ommatic cell counts and colony forming units of Staphylococcus aureus 3 weeks before subsequent treatment. Infected animals were injected through the nipple canal with a single dose of recombinant lysostaphin (rLYS) (dose 1 to 500 mg) or after three milkings p.m. successive with 100 mg of rLYS in 60 ml of sterile phosphate regulated slaine solution. The animals were considered cured if the milk remained free of Staphylococcus aureus after the last treatment. RESULTS The kinetic analysis of immunologically active rLYS showed that a minimum bactericidal concentration was maintained in the milk for up to 72 hours at 37 ° C. In contrast, penicillin G retained less than 10% of its bacteriostatic activity during the same incubation time. Titration of dose and kinetics of rLYS in the bovine mammary gland In order to determine the optimal effective dose to produce long-term cures, a titration was carried out in which a single dose of rLYS was administered at concentrations of 0, 1, 10, 100, or 500 mg. The untreated quarters and the 1 mg treatment did not leave all quarters of S. aureus free. Doses of 10, 100 and 500 mg temporarily released S. aureus milk for at least one milking. In reoccurring quarters, the time in which the milk remained without S. aureus was approximately proportional to the dose administered. Fourteen days after the treatment, two quarters were cured with the dose of 100 mg and one with the dose of 500 mg. Since rLYS maintains a minimum bactericidal concentration (MBC) for approximately 24 hr and experimental infections undergo a cycle of 2 to 4 days, it was determined that multiple infusions of 100 mg rLYS are optimal during three consecutive milkings to maintain an effective dose minimum for 3 to 5 days and to produce cures Conclusion Staphylococcus aureus is one of the main etiological agents of bovine mastitis and a major cause of economic loss for the dairy industry Effective mastitis therapy for lactating dairy cow remains a necessity primary not filled Given that current therapy is only moderately effective and is expensive given that milk is discarded and infected animals are chosen, the treatment of choice adopted only during the dry period has been herd management practice. directs most infections in a lactating animal, which are chronic and subclinical nature A recombinant protein such as rLYS with bactericidal activity against S aureus could be an extremely useful therapy for veterinarians if rLYS were as effective as antibiotics, natural proteolysis and inactivation in rLYS milk, as well as inactivation During ingestion by the consumed, it could potentially minimize any issue associated with residues in the milk. The live dose titration suggested that the minimum effective therapeutic dose was 100 mg rLYS. However, therapeutically, it could be convenient to administer multiple infusions of rLYS to maintain a minimum bactericidal activity within the milk of treated glands during one to three successive milkings. The in vivo bactericidal activity of rLYS was most effectively demonstrated by the fact that 95% of the quarters left the milk of S. aureus detestable for a minimum of one milking after the last intramammary infusion. EXAMPLE VI Expression of plasmid DNA injected into a jet in the mammary gland of sheep A DNA delivery system based on jet injection has been evaluated as a means to temporarily transfect the lactating mammary gland in vivo and as a technique for DNA vaccination . The model expression plasmid contained the human growth hormone (hGH) gene driven by the promoter / enhancer region of the initial immediate human cytomegalovirus (CMV) gene 1. The expression of the pure plasmid DNA injector in the lactating mammary glands of the sheep was sufficient to be detected by Northern graph analysis when tissue was obtained 48 hours after transfection in vivo. In conclusion, the ability to temporarily transfect breast tissue in lactation in vivo circumvents the difficulties encountered with in vivo culture techniques and provides a method to examine mammary regulatory elements and test fusion gene constructs designed for the production of transgenic animal bioreactors. EXAMPLE VII Elimination of Staphylococcus aureus in a eukaryotic system expressing lysostaphin The lysostaphin gene was introduced into 293 cells (human fetal kidney cells) maintained in vitro. The recombinant bacteriocin, lysostaphin, was secreted into the culture medium and found to kill the contaminating S. aureus during the challenge. The lysostaphin gene was obtained by PCR amplification of DNA extracted from the staphylococcal biovar (NRRL B-2628) of Staphylococcus simulans, and the Newbould strain (ATCC) of Staphylococcus aureus was used for the comparison in transfected eukaryotic cells. The staphylococcal strains developed in the middle of Heart Brain Infusion (BHI). Purification of the lysostaphin gene The staphylococcal biovariety of Staphylococcus simulans was grown overnight in a shaking incubator at 37 ° C. The medium was centrifuged and the pellet resuspended in 5 ml of 50 nM EDTA-50 mM Tris-HCl (pH 7.8) containing 50 mg of lysostaphin (Sigma) mi "1 and the suspension was incubated at 37 ° C for 2 hours. The purified bacterial DNA was amplified directly by the PCR method to isolate the lysostaphin gene The group of oligonucleotide primers used were the following: 5-TTAAGGTTGAAGAAAACAATT-3 '(SEQ ID NO 1) and 5-GCGCTCACTTTATAGTTCCCCAA-3 (SEQ ID NO. NO: 2) Amplification was carried out using a Thermal DNA cyclizer and 2.5 units of taq DNA polymerase (Perkin Elmer Cetus), and a program of 30 cycles with an annealing step at 60 ° C for 30 seconds, elongation at 72 ° C for 90 seconds and denaturation at 93 ° C for 10 seconds The PCR product was composed of the entire lysostaphin sequence, including the coding gene with the amino-terminal pre-regimes and the other recombinant DNA procedures, including digestion of e restriction ndonuclease, ligation, washing with phenol-chloroform mixture, ethanol precipitation, transformation and cloning of the constructs in E. coli strain DH5a, were carried out by normal methods. All enzymes were from Boehringen Mannheim. The lysostaphin was linked to a eukaryotic expression vector including the promoter / enhancer region of the initial immediate human cytomegalovirus (CMV) gene 1 and the human interleukin-2 signal peptide. Cell culture and transfection of DNA 293 cells, a human fetal kidney cell line transformed by a mutant of simian virus 40 origin, were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 10% fetal calf serum ( vol / vol) (Gibco BTL) and glutamine (1.4 mM). The cells were seeded in wells of 30 mm to 500 000 well cells and developed in 2 ml of medium for 24 h at 37 ° C (in air atmosphere containing 5% CO2) to give 50 to 60% introduced into the cells by the calcium phosphate method with the following modifications. The precipitate containing 7.5 μg of DNA was added to 2 ml of culture medium. After 24 h, the medium was replaced with 2 ml of medium per well and the samples of the medium were collected every 24 h after the transfection to evaluate the production of the lysostaphin by the Western graph analysis and ELISA. Analysis for active biological lysostaphin Wells containing the 293 transfected cells were infected with 102 or 103 of Staphylococcus aureus Newbould. Samples of 100 μl of the infected medium were diffused on sheep blood agar. After incubation for 24 h at 37 ° C, the number of units forming the colony (CFU) was evaluated to account for the effect of inhibition of the recombinant lysostaphin on the growth of the bacterium. RESULTS Production of recombinant lysostaphin by transfected eukaryotic cells. The modified lysostaphin gene was transfected into tissue culture cells to demonstrate expression, processing and activity of the enzyme in infectious bacteria. After analysis of the culture medium, a band of approximately 25 kDa was generated; this band was similar in size to mature lysostaphin. The same result was observed in other experiments in which the expression of recombinant lysostaphin was carried out in eukaryotic cells. ELISA analyzes have revealed that recombinant lysostaphin was produced in concentrations of 100 to 250 ng / ml / 24 h depending on the clone. Activity of lysostaphin secreted by mammalian cells The activity of recombinant lysostaphin secreted by transfected mammalian cells has been observed for its efficiency in reducing or in some replicates to inhibit the growth of Staphylococcus aureus in the culture medium. Samples of media taken from non-transfected cells have not shown any inhibitory effect on the development of the bacteria present in the wells. The agar plates were completely confluent after incubation overnight. In contrast, when an initial amount of 103 bacteria was cultured in the presence of transfected eukaryotic cells, very few CFUs were counted in the plates. Less than 100 CFU were observed in our analyzes when 103 bacteria were used, whereas the presence of CFU in genes was not observed when 102 bacteria were added to the wells containing the transfected cells. While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the following invention, in general, the principles of the invention and including such deviations from the present disclosure as they enter the known or customary practice within the matter to which the invention pertains and as it may be applied to the essential aspects set forth herein and as follows in the scope of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: IMMUNOVA (B) STREET: 2750 Rue Einstein, Bureau 110 (C) CITY: Sainte-Foy (D) STATE: Quebec (E) COUNTRY: Canada (F) POSTAL CODE (ZP): G1P 4R1 (G) TELEPHONE: (418) 654-2240 (H) TELEFAX: (418) 654-2125 (ii) TITLE OF THE INVENTION: THERAPY OF GENES ANIMALS (iii) NUMBER OF SEQUENCES: 2 (iv) READABLE COMPUTER FORM: (A) TYPE OF MEDIUM: soft disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentlan Reléase # 1.0, Version # 1.30 (EPO) (v) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: GB 9509461.1 (B) DATE OF SUBMISSION: MAY 10, 1995 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) SEQUENCE DESCRIPTION: SE ID NO: 1: TTAAGGTTGA AGAAACAAT T 21 (3) INFORMATION FOR SEC ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) THREAD: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCGCTCACTT TATAGTTCCC CAÁ 23

Claims (15)

1. CLAIMS 1.
2. A method of treatment and / or prevention of an infectious disease in an animal, which comprises the steps of a) producing a recombinant DNA expression system comprising at least one DNA sequence of 5'y expression regulation a secretory DNA sequence encoding a secretory signal sequence operably linked to a DNA sequence encoding a therapeutic protein, peptide or antisense RNA selected from the group consisting of bactepokines, lentionmas, lactoferpan and lysosim, wherein said DNA sequence of expression regulation and said secretory DNA sequence are capable of directing the expression in vivo of said DNA sequence of a therapeutically effective amount of said protein, peptide or RNA of contradictory, and b) introducing into the soft tissue of the animal the expression system of DNA from step a) for the in situ expression of said therapeutic protein, peptide or RNA of contradictory 2 The method of claim 1, wherein said DNA expression system is transgenic recombinant animal cells.
3. The method of claim 2, wherein said cells are selected from the group consisting of epithelial mammary gland cells, blood cells, lymphocytes, leukocytes. , T lymphocytes, B lymphocytes, erythrocytes, muscle cells, liver cells, kidney cells, lung cells, secretory cells and non-secretory cells.
4. 4. The method of claim 3, wherein said DNA expression system is selected from the group consisting of a lipid liposome, a cationic liposome, an anionic liposome.
5. The method of claim 3, wherein said vector is a viral vector or a retroviral vector.
6. The method of claims 1, 2, 3, 4 or 5, wherein said infectious diseases are caused by bacteria, viruses, retroviruses, parasites, fungi, mold, yeast, prions or escarpies.
7. The method of claim 6, wherein said bacteriocins and / or lanthionines are ambicines, densins, cecropins, thionines, melitins, magainins, atacins, dipterins, saponins, cacrutins, xenopines, subtilins, epidermins, pep5, laticin 481, ancovenins , duramycins, galidermines or cinnamycins.
8. The method of claim 6, wherein said therapeutic protein, peptide or counter-sense RNA is selected from the group consisting of immunoglobulins, lactoglobulins, a-lactalbumin, lipase or ribosine stimulated by bile salts, cytokines, chemokines, growth factors of immunomodulators.
9. The method of claim 2, further comprising a 3 'expression regulation DNA sequence and a functional secretory DNA sequence in said animal cells and operably linked to the recombinant DNA encoding said therapeutic protein, peptide or RNA from contradictory.
10. 10. A non-human animal genetically treated for the production of a recombinant protein, peptide or RNA in systemic or target tissue, comprising an expression system of DNA introduced into animal white tissue comprising at least one DNA sequence of 5'-expression regulation and a secretory DNA sequence encoding a secretory signal sequence operably linked to a DNA sequence encoding a therapeutic protein, peptide or RNA of the opposite sense selected from the group consisting of bacteriocins, lanthionines, lactoferrin and lysosim .
11. 11. The genetically-treated non-human animal of claim 10, wherein said expression regulation DNA sequence is selected from the group consisting of a constitutive promoter, an inducible promoter, a cytomegalovirus promoter.
12. 12. The genetically treated non-human animal of claim 11, wherein said promoter is selected from the group of DNA sequences encoding lactoferrin, serum albumin, aS2-casein, β-casein, α-casein, α-lactalbumin, acid protein of serum, β-lactoglobulin, cytokines, chemokines and growth factors.
13. The non-human genetically treated animal of claim 10, wherein said sequence of secretory signals is selected from the group consisting of DNA sequences encoding lactoferrin, serum albumin, aS2-casein, β-casein, β-casein, a-lactalbumin, β-lactoglobulin, cytokines, chemokines or growth factors.
14. 14. The genetically treated non-human animal of claim 10, wherein the expression regulation and secretory signal sequences are from human, bovine, caprine, ovine, feline, canine, lagomorph, poultry and fish.
15. 15. The genetically-treated non-human animal of claim 11, wherein the promoter is tissue-specific for expression in white tissue.