EP0828839A1 - Animal gene therapy - Google Patents

Abstract

The present 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 which have been transformed with the DNA and which are suitable for implantation into a host mammal. The gene therapy of infectious diseases can be effected in situ in targeted tissue or systemically.

Description

ANIMAL GENE THERAPY

BACKGROUND OF THE INVENTION

(a) Field of the Invention The invention relates to DNA sequences, expres¬ sion 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 which have been transformed with the DNA and which are suitable for implantation into a host mammal.

(b) Description of Prior Art

At the highest level, transgenic animals are the principal way to confer transmissible resistance to diseases in animals. Only few years after the first successful gene transfer into mice the new technique was used in farm animals. Several genetic treats have been targeted for the application of transgenesis in domestic animals, but one of those important aspects is the improvement of animal health and disease resistance by gene transfer means. Transient as well as stable genetic improvement leading to disease resistance and treatment achieved by recently developed techniques in molecular biology may contribute considerably to reduce the problem of diseases.

Resistance to infections in animals elicited at various levels. Constitutional and phagocytic mecha¬ nisms (innate immunity) serve as a first line of defense. If these are ineffective the infected organ¬ ism can respond by means of specific (acquired) immu¬ nity. Thus, candidates for gene therapy applications include all genes known to modulate non-specific and specific host defense mechanisms, i.e. cytokines, major histocompatibility complex (MHC) proteins, T-cell receptors (TCR) and proteins conferring specific dis- ease resistance. Increased protection against patho¬ gens can be conferred also by other strategies such as "intracellular immunization", genetic immunization, antisense sequences as anti-pathogenic agents and dis- ruption of disease susceptibility genes.

Gene modulating Immune Responses

Cytokine orchestrate immune responses through their role as soluble mediators of cell communication. Initially identified to direct viability, prolifera¬ tion, differentiation and homing of leukocytes, they were also found to regulate the production of function or one another. In addition, cytokines interact with, and are produced by cells other than leukocytes, thus providing a means of communication between the immune system and other tissues and organs. Cytokines repre¬ sent a rapidly growing number of regulatory peptide factors including growth factors, interleukins, chemokines, colony-stimulating factors and interferons. Their functions are mediated through binding to cell surface receptors on their target cells. Cytokines have been shown to contribute directly to the develop¬ ment of pathology during infectious diseases and tumorigenesis. Different cytokines have been reported to both positively and negatively influence host defense mechanisms.

Interferon (IFNs) are a well characterized class of cytokines eliciting antiviral and antiprolif- erative activity as well as modulating cell growth, differentiation and immune responses. As well as their more characterized antiviral activity, IFNs are instru¬ mental in counteracting non-viral pathogens mostly through their effects on macrophage activation. The proteins known to be involved in the antiviral and bac- tericidal actions of interferon and their inhibitory mechanisms are numerous. The potency of IFNs to posi- tively influence host susceptibility to viral infec¬ tions was tested in transgenic mice and cell lines. Transgenic organisms overexpressing IFN-β gene con¬ structs were shown to exhibit enhanced viral resis- tance.

Recent progress in the understanding of signal transduction pathways and transcription factors acti¬ vated by IFNs and a variety of other cytokines promises to open up new therapeutic approaches as well as novel strategies of gene transfer treatments aiming at the improvement of the immune response; i.e. the transfer of cytokine encoding genes per se of distinct "cytokine-specific" signaling components. Constitutive expression of an interferon-stimulated gene factor (ISGF2) also termed interferon regulatory factor (IRF- 1) transgenes has been reported to result in IFN-inde- pendent activation of various IFN-inducible genes and enhanced resistance to viral infection.

Specific Disease Resistance Genes

Other improvement which can be brought to ani¬ mals by local gene transfer is specific disease resis¬ tance. A well examined specific disease resistance gene is the Mxl gene product of certain mouse strains. The mouse Mxl protein belongs to a family of polypeptides with GTPase activity synthesized in IFN-treated verte¬ brate cells. Some Mx proteins have been shown to block the multiplication of certain negative-stranded RNA viruses, as for example Influenza virus , VSV, rhado virus and Thogoto virus. Synthesis of mouse Mxl pro¬ tein in various cell lines and transgenic mice demon¬ strated that it is both necessary and sufficient to promote resistance to influenza A viruses in previously susceptible cells and animals. The cloning and func- tional characterization of this specific disease resis¬ tance gene enabled a gene transfer program to study whether Mxl transgenic pigs would show reduced suscep¬ tibility to influenza infections.

Natural resistance of certain inbred mouse strains to infection with antigenetically unrelated microorganisms such as Mycobacteria , Salmonellae and Leishmania is controlled by a dominant locus on chromo¬ some 1 called Beg, Lsh or Ity respectively. The locus affects the capacity of the host to restrict prolifera¬ tion of these infectious pathogens during the non-spe- cific macrophage-dependent phase of infection. A posi¬ tional cloning approach resulted in the isolation of a candidate Beg gene designated Nramp. The reduction of susceptibility to Salmonella infections by transgenesis or gene therapy ( in vivo or ex vivo) means is of great value for animal production, especially poultry. Large difference in resistance to Salmonella in chicken inbred lines have been observed. Furthermore, natural resistance or susceptibility to infection with Mycobacteria in humans and Brucella in cattle has been shown to be under genetic control similar to that observed in inbred mice and governed by Beg. Chronic infection of cattle with Brucella abortus causes the spontaneous abortion of fetal calves, threatening the economic well-being of the dairy and beef industries. Genetic resistance to certain retroviruses has been observed as a polymorphic trait in several experi¬ mental species. One of the identified loci in mice, Fv-4 , resembles the 3' half of a murine leukemia virus extending from the end of the pol gene through a com- plete env gene. Expression of Fv-4 encoding only the viral envelope protein in transgenic mice conferred resistance to infection with ecotropic retroviruses. The mechanism of Fv-4 resistance is thought to be related to the phenomenon of viral interference, i.e. competition of the synthesized envelope protein with exogenous virus for the virus receptor. Similar mecha¬ nisms are used in antiviral strategies known as "intracellular immunization".

Expression of a transgene encoding an immu- noglobulin specific for a common pathogen can provide immunity for that pathogen. As shown by many investi¬ gations, cloned genes coding for monoclonal antibodies can be expressed in large amounts in genetically manipulated mice. These mice produce antibodies against specific antigens without prior contact or immunization.

Intracellular Immunization

The concept of "intracellular immunization" essentially involves overexpression in the host of an aberrant form (dominant-negative mutant) of a viral protein that is able to interfere strongly with the replication of the wild type virus. Elegant studies in cultured cells resulting in acquired resistance to various viruses include strategies preventing virus attachment to the target cells, blocking the formation of virus-host transcription complexes, expressing domi- nant-negative viral trans-activators or interfering with the assembly of infectious viral particles. Endogenous mouse mammary tumor virus (MMTV) proviruses have been found to co-segregate genetically with loci termed self-superantigens identical to a pro¬ tein encoded in the long terminal repeat of MMTV. Genetically manipulated mice expressing high levels of this self-superantigen were shown to be protected from viral infection by deletion of a specific class of T- cells which is the target for infection.

The definition of "intracellular immunization" is also applied for antiviral strategies described in different connections such as expression of specific resistance genes, antisense RNAs or other antiviral components.

Recently, an "intracellular immunization" approach carried out in farm animals was reported. Transgenic sheep were produced and were shown express¬ ing the visna virus envelope ( env) gene. The visna virus belongs to a subfamily of ovine retroviruses that cause encephalitis, pneumonia and arthritis in sheep. The env glycoprotein is responsible for the binding of this virus to host cells. The target cell for visna virus replication in infected sheep is the macrophage. The expression of env protein on the cell surface of visna-infected cells induces immune responses to the virus. Expression of a gene construct consisting of the visna U3 enhancer region fused to the env gene in transgenic sheep had no obvious deleterious effect. Thus, the genetically manipulated sheep lines provide an evidence for the potential of a retroviral env gly¬ coprotein to prevent infection and/or to modulate dis- ease in its natural host after virus challenge.

Antisense RNA

The use of antisense RNA to inhibit RNA func¬ tion within cells or whole organisms has provided a valuable molecular biological method. Antisense RNA functions by binding in a highly specific manner to complementary sequences, thereby blocking the ability of the bound RNA to be processed and/or translated. Antisense sequences are considered an attractive alter- native to conventional drugs in the therapy of micro- bial infections, cancer, autoimmune diseases and other malfunctions. Gene transfer experiments with antisense constructs have been carried out in mice and rabbits. Genetically manipulated mice expressing antisense RNA targeted to the retroviral packaging sequences of Molony murine leukemia virus did not develop leukemia following challenge with infectious viruses. Trans¬ genic rabbits expressing an antisense construct comple¬ mentary to adenovirus h5 RNA were produced. Primary cells from these rabbits were found to be 90-98% more resistant to adenovirus infection than cells from con¬ trol animals.

The use of antisense RNAs as anti-parasitho- genic agents can be developed to result not only in RNA-RNA hybrids but catallytically cleave a phosphodi- ester bound in the target RNA strand. Four structural motifs (hammerhead and hairpin first identified in plant RNA pathogens, the delta motif found in human hepatitis delta virus and a less well characterized motif from Neuspora ) have thus far been described as intermediates in these self-cleavage reactions. By flanking the hammerhead motif of this ribozyme family with antisense sequences, the cleavage of specific tar¬ get RNAs has been demonstrated. A large number of sub¬ strate molecules can be processed by the catalytic RNA because the ribozyme per se is not consumed during the cleavage reaction. Bovine leukemia virus (BLV), a retrovirus , causes persistent lymphocytosis and B-lym- phocyte lymphoma in cattle and sheep. A hammerhead ribozyme flanked by antisense sequences directed against regulatory proteins of BLV was shown to inhibit BLV expression in persistently infected cells. This demonstrates the possibility of generating localized ( in vivo or ex vivo) or generalized (transgenic ani¬ mals) gene therapies that will be resistant to BLV- induced diseases.

Somatic Gene Transfer Approaches

Somatic gene transfer into farm animals will become more significant. Ex vivo and more recently in vivo gene therapy has been applied for several genetic diseases in human. Current therapies developed for — o —

more than 10 gene human disorders, such as failing genes coding normally for the adenosine deaminase, LDL receptor, glucocerebrosidase, blood clotting factor VIII, phenylalanine hydroxydase, dystrophin and others. The efficiency of the gene therapy approach has no more to be proved.

Novel methods for gene transfer into somatic cells promise to be highly efficient. These include viral vectors for delivering gene constructs and non- viral technologies, such as micro-bombarding or injec¬ tion of DNA particles or solutions into tissues or blood vessels. Although most efforts are directed pri¬ marily towards the possibility of treating human dis¬ eases, 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", i.e. application of DNA constructs encoding immunogens, has at least two powerful uses. One is to simplify the procedure and to shorten the time required to produce antibodies to particular proteins by eliminating the steps for protein purification. it would be more rapid again to introduce a gene encoding directly a neutral¬ izing or bacteriocid antibody in the organism. The second is the genetic vaccination of animals against infections by producing foreign antisense encoded by appropriate gene construct.

The somatic gene transfer approach can now be applied also both to cure and prevent an infectious diseases by releasing in the organ, or in the organism, a protein which is lethal and absolutely specific for the targeted microorganism and without any affinity or effect for the animal.

Such proteins or peptides having a high and specific antimicrobial activity are divided into two families, one including the bacteriocins and the other the lanthionines, also called lantibiotics. The appli¬ cation of biotechnology to animal treatment, particu¬ larly farm animals, is opening up new avenues of pre- vention and control that will have important implica¬ tions. The bacteriocins consist of enzymes and other bactericidal proteins. They act as catalysts and are very specific to a single chemical reaction. Bacte¬ riocins kill targeted organisms rapidly by lysing the cell wall, and they do not require that the organism undergo cell division. They are produced naturally by bacteria as a means of population control. These pro¬ teins are larger molecules than antibiotics and are expected to persist in the treated organ longer. One of these well known bacteriocins is lysostaphin, which is produced by Staphylococcus simulans biovar staphylolyticus . Unlike antibiotics, the rapid action of bacteriocins reduces the likelihood of an induced resistance in target and non-target organisms. For example, current research conducted so far seems to indicate that bacteriocins used for mastitis treatment are non-toxic to other organisms.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against several patho- genie bacteria. The ribosomal origin of lantibiotics was first shown by the isolation of the structural gene, epiA, 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 prepeptide which consists of an N-terminal leader sequence that is followed by the C-terminal propeptide from which the lantibiotic is matured and a characteristic proteolytic processing site with proline at position -2. Nisin, produced by several Lactococcus lactis strains, is a prominent mem¬ ber of the group of lanthionines.

Other bacteriocins and lanthionines are ambi- cins, defensins, cecropins, thionins, mellitins, magainins, attacines, diphterins, saponins, cacrutins, xenopins, subtilins, epidermins, pep5, lacticin 481, ancovenins, duramycins, gallidermins, cinnamycins, andropins and mastoparans.

Another new class of molecule complexes which can be secreted by the transgene, i.e. the genetic con¬ struct used for a gene therapy application is the immu- noadhesins. The therapeutic potential of antibodies has long been recognized Human antibodies should be minimally immunogenic to the patient; they should therefore be safe for chronic or repeated use. How¬ ever, it can be difficult to generate useful human antibodies for several reasons: it is ethically impos¬ sible to immunize human beings for experimental pur¬ poses, thus the available human antibodies are limited to the products of inadvertent immunization or vaccina¬ tion. Furthermore, there have been technical difficul¬ ties in the immortalization of human cell lines. Per¬ haps the most refractory technical problem is that many applications require antibodies to human antigens; since human antibodies with the desired specificity.

Several potential approaches exist to circum¬ venting these problems. One approach is to engineer the desired specificity of binding into human antibody variable(V) regions. This can be done by deriving the complementary determining regions either from mouse antibodies, or from in vi tro recombination combined with selection (e.g. combinatorial libraries and phage display technology). An alternative approach, which sometimes has advantages, is to create an antibody-like molecule by combining a binding site, derived from a human protein such as a cell-surface receptor or cell- adhesion molecule, with antibody constant domains. Such molecules are known as immunoadhesins.

Immunoadhesins can possess many of the desired chemical and biological properties of antibodies. Examples exist of immunoadhesins that can bind to Fc receptors, mediate antibody-dependent cellular cytotox- icity, and show active transport across the primate placenta. Since the immunoadhesin is constructed from a receptor sequence linked to an appropriate hinge and Fc sequence, the binding specificity of interest can be achieved using entirely human components. Another potential foreign sequence is that in the joining region. One of .well studied immunoadhesins is CD4-IgG which as been found entirely non-immunogenic in human clinical trials. A second candidate for clinical use is a rumor necrosis factor receptor immunoadhesin (THFR-IgG); this molecule is particularly interesting, since the soluble receptor itself is found naturally in the body and has been considered as a possible thera¬ peutic. While soluble receptors are valid clinical candidates, the IgG fusion form may well confer advan¬ tages such as longer half-life and improved avidity and affinity. Some receptors or immunoinducers that have been joined to the Fc part of IgG to form immunoadhes¬ ins are reported in the literature: T cell receptor, CD4, 1-selectin, CD44, CD28, B7, CTLA-4, CD22;, TNF receptor, NP receptor, IgE receptor, INF-γ receptor. These immunoadhesins should be useful in antigen recog¬ nition, reception to HIV, lymphocyte adhesion, receptor for hyluronidase, interaction B and T lymphocytes, inflammation, septic shock, homeostasis and allergy.

In animals, the advent of molecular biology techniques allow to create an immunoadhesin which could has two specific activities. For example, in the goal to eliminate a contamination with Staphylococcus aureus it can be possible to have an immunoadhesin composed of a lytic enzyme, like the lysostaphin, linked to the Fc part of the human IgG which has a high affinity for the protein A at the surface of the bacteria. Once the Fc is linked to the protein A on Staphylococcus aureus, the lytic part, the lysostaphin, can lyse the bacteria. The gene therapy treatments can be applied in such a way that the gene included in the constructs transferred could be coding for an immunomodulator, such as interleukins, chemokines, interferons, leu- kotriens, and certain growth factors. As explained before, the immunomodulators can makes the animal more resistant to several microorganisms.

SUMMARY OF THE INVENTION

The present invention relates to the animal gene therapy. Animal gene therapy means an approach by which a DNA construct involving an inducible or consti¬ tutive promoter linked to a gene coding for a curative or protective protein or antisense RNA or peptide which acts against infectious or potentially infectious microorganisms responsible of the diseases. Disclosed is a method for expressing a protein or antisense RNA or peptide which directly or indirectly has a therapeu¬ tic or prophylactic effects against infectious microor¬ ganisms in an animals. The invention is useful for producing a heterologous or homologous protein or antisense RNA or peptide which is tethered to a spe¬ cific tissue or organ and which can act on a microor¬ ganisms infecting the animal. The method involves inducing a liquid complex including a genetic construct into a determined 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 taken up by secretory cells of the animals.

The most costly infectious disease in animals is mastitis caused by the infection of the mammary gland. Among others, this invention relates to a method of treating mastitis. More particularly, this invention relates to the use of DNA constructs designed to be transcribed in a therapeutic protein after inser- tion into the mammary gland of both lactating or non- lactating animals.

Bovine, caprine, ovine and porcine mastitis remain some of the most costly diseases in animal agri¬ culture. Mastitis represents a significant economic loss to the diary industry, approximately 70 to 80 per¬ cent of which can be attributed to a decrease in milk production. Many infective agents have been implicated as causes of mastitis and these are dealt with sepa¬ rately as specific entities in cows, sheep, goats and pigs.

Despite significant progress in mastitis con¬ trol due to widespread adoption of post-milking teat antiseptisis, many herds continue to be plagued by this disease. A variety of different procedures have been described and used to cure mastitis caused by bacteria and yeast. These procedures include the systemic immu¬ nization of the infected animals with whole or partial protein extracts of the infective agents in order to stimulate the immune response of the treated animal to these agents. Antibodies generally produced in this way act against a membrane protein, a binding protein or a toxin secreted by the microorganisms. Hence these antibodies act as anti-adhesive, anti-toxin, neutraliz¬ ing or opsonic molecules (Nordhaug et al., 1994, J Dairy Sci . , 77:1267 & 1276). Nevertheless, the blood- milk barrier prevents all but a very small proportion of circulation IgG antibodies from reaching mammary secretion during lactation.

Other procedures have been carried out in order to stimulate the diapedesis and phagocytosis of con¬ taminating agents by leukocytes, more particularly polymorphonuclear neutrophils and macrophages. The stimulating molecules, which have been administered by intramammary injection, are cytokines, interleukin-lβ,, interleukin 2, interferon-γ, tumor necrosis factor-α.

The most widely used procedure to cure infec¬ tious diseases is administration of antibiotics. How¬ ever, this approach inflicts a lot of side effects to the animal and particularly in the case of dairy ani- mals, the milk must be discarded during the treatment period. Unfortunately, all current procedures are very short-lasting and consequently relatively inefficient. For example, none of the gram positive bacteria are entirely eliminated from the udder after treatments with antibiotics.

For these reasons a gene therapy procedure is desired that allows a gene to be integrated into a tar¬ geted tissue, such as mammary gland, and provides for the elimination, by genetic therapy, of the contaminat- ing microorganisms. In addition, gene therapy of the mastitic gland eliminates all the side effects of other procedures, enabling also an inserted gene to synthe¬ size inductively or constitutively in a permanent man¬ ner an effective amount of its therapeutic protein, peptide or RNA antisense product. Therefore, this invention allows a much more specific and effective system of infectious diseases treatment than is cur¬ rently possible.

Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of the invention as presently perceived.

The present invention provides a recombinant DNA which comprises a nucleotide sequence which encodes a protein or polypeptide which is useful in the prophy¬ laxis or treatment of mastitis, and at least one regu¬ latory control element which allows for expression of said nucleotide sequence in a mammary gland. Suitable regulatory control elements include transcription and translation regulatory sequences. Transcription and translation regulatory sequences are those DNA sequences necessary for efficient expression of the product. In general, such 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". Hence the invention further provides an expression cassette containing the above-mentioned recombinant DNA. An expression cassette will typically contain, in addition to the coding nucleotide sequence, a pro¬ moter region, a translation initiation site and a translation termination sequence.

Unique endonuclease restriction sites may also be included at the end of an expression cassette to allow the cassette to be easily inserted or removed when creating DNA constructs for use in transformations as is known in the art.

In particular the invention provides a DNA con- struct designed to express a protein or polypeptide which is useful in the prophylaxis or treatment of infectious diseases after insertion into the targetted tissues. Suitably the DNA construct comprises an inducible or constitutive promoter which is linked to a coding nucleotide sequence or gene and thereby expresses a therapeutic or protective protein which acts against infectious or potentially infectious microorganisms responsible for the diseases of animals. For example, such DNA constructs can be admin- istered to both lactating or non-lactating animals for the prophylaxis or treatment or mastitis. Hence the invention further provides a method for the prophylaxis or treatment of mastitis which comprises transforma¬ tion of mammary gland tissue with a DNA construct as described above.

The present applicants have found that expres¬ sion of proteins in mammary glands over an extended time period is possible and that a gene therapy approach to the problem of mastitis is feasible. Inte- gration of a gene which encodes a therapeutic protein or polypeptide into mammary gland tissue would allow, for example, for the elimination of infective microor¬ ganisms by genetic therapy. In addition, gene therapy of the mastitis gland eliminates all side effects of other procedures, also enabling an inserted gene to synthesize permanently and inductively or constitu- tively an effective amount of its therapeutic protein product. A gene therapy approach would be a much more specific and effective system of mastitis treatment than is currently available.

Transformation of mammary gland tissue gener¬ ally requires that the DNA be physically placed within the host gland. Current transformation procedures use a variety of techniques to introduce naked DNA into a cell and these can be used to transform a mammary gland. For example, the DNA can be injected directly into glands through the use of syringe. Alternatively, high velocity ballistics can be used to propel small DNA associated particles into the gland through an udder's skin incision. The DNA can also be introduced into a mammary gland by insertion of other entities which contain DNA. These entities include minicells, cells (e.g. fibro- blasts, adipocytes, epithelial cells, myoepithelial cells, mammary carcinoma cells, kidney cells), liposomes (e.g. natural or synthetic lipid vehicles, cationic liposomes) or other fusible lipid-surfaced bodies. The entities are transformed in vitro prior to insertion using the above-described DNA constructs. Thus the invention also provides a cell which 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 reimplantation into a mammary gland to produce the desired proteins or polypeptides.

Furthermore the invention provides a liposome which incorporates the above-described DNA construct.

Introduction of the naked or complexed DNA con¬ structs into the mammary gland can be performed by direct injection through a skin incision of the udder or through the teat canal.

Where appropriate, the DNA construct is admin¬ istered in the form of a pharmaceutically or veterinary acceptable composition in combination with a suitable carrier or diluent. Suitable carriers are liquid car¬ riers such as water, salts buffered saline or any other physiological solutions. These compositions form a further aspect of the invention.

The protein or polypeptides produced should be effective prophylaxis or treatment of mastitis. Such proteins or polypeptides include mucolytic proteins such as enzymes, antibiotics, antibodies, cytokines, tumor necrosis factors as well as proteins which can induce an immune response to infective or potentially infective agents and those which activate polymorphonu- clear neutrophils, or macrophages.

In a preferred embodiment, the invention pro¬ vides 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: bovine, caprine, ovine, and porcine, but can concern also lower mammals or lower milk producers: rabbit, camel and bison. The invention can also be used in humans to eliminate par¬ ticularly most Staphylococci .

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates examples of DNA constructs in accordance with the present invention; and

Fig. 2 illustrates the rate of synthesis of human growth hormone in milk's sheep after injection of cationic liposome-DNA complex into the mammary gland.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one preferred embodiment of the present invention, animal gene therapy of infec¬ tious diseases consists in transfecting a targeted tis¬ sue with DNA sequences designed to produce molecules which will be relargued into the organ or the organism, this would than protect the animal against the infect- ing or potentially infecting microbial agents.

In accordance with another embodiment of the present invention, mastitis gene therapy of mammals consists of transfecting the mammary glands with DNA sequences designed to produce molecules which will be relargued into the udder, this would than protect the animal against the infecting or potentially infecting microbial agents.

The targeted tissue can also be transformed with other DNA sequences such as gene transcription and translation regulatory sequences. Transcription and translation regulatory sequences are those DNA sequences necessary for efficient expression of the gene product. In general such regulatory elements can be operably linked to any gene to control the gene's expression, the entire 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. Unique endonuclease restriction sites may also be included at the ends of an expression cassette to allow the cassette to be eas¬ ily inserted or removed when creation DNA constructs.

The expression of a gene is primarily directed by its own promoter, although other DNA regulatory ele- ments are necessary for efficient expression of a gene product. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. By convention, the transcription start site is designated +1. Sequences expending in the 5' (upstream) direction are given negative numbers and sequences extending in the 3 * (downstream) direction are given positive numbers. Promoters can be either constitutive or induc- ible. A constitutive promoter controls transcription of a gene at a constant rate during the life of a cell, whereas an inducible promoter's activity fluctuates as determined by the presence (or absence) of a specific inducer. The regulatory elements of an inducible pro- moter are usually located further upstream of the tran¬ scription start site than the TATA box. Ideally, for experimental purposes, an inducible promoter should possess each of the following properties: a low to non- existent basal level of expression in the absence of inducer, a high level of expression in the presence of inducer, and an induction scheme that does not other¬ wise alter the physiology of the cells. The basal transcription activity of all promoters can be increased by the presence of "enhancer" sequences. Although the mechanism is unclear, certain defined enhancer regulatory sequences are known, to those familiar with the art, to increase a promoter's tran¬ scription rate when the sequence is brought in proxim- ity to the promoter.

Constitutive promoters can activate the tran¬ scription of its linked gene in a tissue specific man¬ ner, such as those naturally actives in the epithelial cells of a mammary gland. For example, strong consti- tutive promoters are those controlling the expression of caseins, lactoglobulins, lactoferrin, lactalbumin, lysosymes, whey acidic proteins (WAP) coding genes in mammary glands. Preferentially, the promoters origi¬ nates from domestic animals, bovine, caprine, ovine or porcine species. Alternatively, specific mammary gland promoters can originates from smaller animals, lagomor- phes, rodents, felines or canines. Other constitutive promoters regulating expression of the cytoplasmic β- actin or ubiquitin genes can be used. Viral or retroviral promoters can be used also, like Cytomegalovirus (CMV), Simian virus 40 (SV40) or mouse mammary tumor virus (MMTV, which is additionally inducible) .

Inducible promoters include any promoter capa- ble 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 with the art and a variety exist that could conceivably be used to drive expression of the protective or curative molecule's gene.

Two preferred inducible promoters are the heat shock promoter (HST) and the glucocorticoid system. Promoters regulated by heat shock, such as the promoter normally associated with the gene encoding the 70 kDa heat shock protein, can increase expression several- fold after exposure to elevated temperatures. The heat shock promoter could be used as an environmentally inducible promoter for controlling transcription of the protective or curative molecule's gene. The glucocor- ticoid system also functions well in triggering the expression of genes including protective or curative molecule's gene. The system consists of a gene encod¬ ing glucocorticoid receptor protein (GR) which in the presence of a steroid hormone forms a complex with the hormones. This complex then binds to a short nucleo¬ tide sequence (26 bp) named the glucocorticoid response element (GRE), and this binding activates the expres¬ sion of linked genes. The glucocorticoid system can be included in the DNA transformation construct as a means to induce protective or curative molecule's expression. Once the constructs have been inserted the systemic steroid hormone or glucocorticoid will associate with the constitutively produced GR protein to bind to the GRE elements, thus stimulating expression of the pro- tective or curative molecule's genes (e.g. antibodies or enzymes) .

Presumably the targeted tissue will allow the inserted gene (naked, liposome, cell-enclosed or coated solid particle) to produce its protein product in an amount sufficient to produce the desired effect. The inserted gene's products must cure or protect the organ or the organism in which it is expressed against infec¬ tious or potentially infectious microorganisms respon¬ sible or potentially responsible of the disease. The transformation of an animal tissue requires that the DNA be physically placed within the host ani¬ mal. Current transformation procedures utilize a vari¬ ety of techniques to introduce naked DNA into a cell, that can be used to transformed a targeted tissue. In one form of transformation, the DNA is injected directly into the tissue though the use of syringe. Alternatively, high velocity ballistics can be used to propel small DNA associated particles into the tissue through a skin's incision. In other forms, the DNA can also be introduced into a targeted tissue by insertion of other entities which 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, lympho- cytes, leukocytes), liposomes (e.g. natural or syn¬ thetic lipid vehicles, cationic liposomes) or other fusible lipid-surfaced bodies.

The invention is concerned when a neutralizing, lytic or opsonic molecules are synthesized from the gene used for the infectious disease's gene therapy. Preferentially, in the case of the mastitis, the gene coding for a mucolytic protein (e.g. bacteriocins and lanthionins) can be used to eliminates the Gram posi¬ tive bacteria (mostly cocci ) . The gene products can serve as an immunomodulator and to induce an immu- nologic response, the activation of polymorphonuclear neutrophils, or macrophages for example. The product can be a cytosin or other immunomodulator. Alterna¬ tively, the genes can be used for 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-leu- cocidin, anti-protein A, anti-collagen, anti- fibronectin binding protein, anti-laminim, anti-α-toxin and anti-β-toxin antibodies; opsonic antibodies and antibodies raised against cell fusion viral protein;

3. Cytokines, interleukines, chemokines, growth factors;

4. Interferons;

5. Tumor necrosis factors; and

6. Immunoadhesins or immunotoxins.

While antibiotics are not very suitable, it can be alternatively used with inducible promoters.

Microorganisms which can be responsible of the mastitis and be eliminated by the gene therapy approach are:

In cattle Streptococcus agalactiae, Str. ube, Str. zooepidemicus, Str. dysgalactiae, Str. fae- calis and Str. pneumoniae, Straphylococcus aureus, Escherichia coli , Klebsiella spp. , Corynebacterium pyogenes , Cor. bovis, Myco- bacterium tuberculosis, Mycobacterium spp. , Bacillus cereus, Pasteurella multocida,

Pseudomonas pyocyaneus, Sphaerophorus necrophorus, Serratia marcescens, Myco- plasma spp. , Nocardia spp. , a fungus Trichosoporon spp. , yeasts Candida sp. , Cryptococcus neoformans, Saccharomyces, and

Torulopsis spp. . In sheep: Pasteurella haemolytica, Staph . Aureus, ActinoBacillus lignieresi , E. coli , Str. uberis and Str. agalactiae, and Cor. pseu- dotuberculosis. In goats: Str. agalactiae, Str. dysgalactiae, Str. pyogenes, and Staph . aureus . In pigs: Aerobacter aerogenes, E. coli , Klebsiella spp. , Pseudomonas aeruginosa, coagulase- positive Staphylococci , Str. agalactiae,

Str. dysgalactiae, and Str. uberis . In horses: Corynebacterium pseudotuberculosis, Str. zooepidemicus, and Str. egui . Other microorganisms and diseases which can be eliminated from exotic animals by the method of gene therapy are those causing: In primate: oliomylltis, Measles, Mumps, Rubella, DPT,

Tetanus. In canidae:Can. distemper, Can. adenovirus, Can. par- vovirus, Can. parainfluenza, Rabies, Lepto- spire bacterin. In felidae:Fel. panleukopenia, Fel. rhinotracheitis,

Fel. caliciviruses, Rabies. In Artiodactyla: BVD, 8-way Clos. bacterin, 5-way Lepto. bacterin, Parainfluenza 3,

Prions, Scatters.

Examples of infectious diseases which could be cured or prevented by the application of gene therapy are: anemia, arthritis, rhinotracheitis, bronchitis, bulbar paralysis, bursal diseases, hepatitis, cloaci- tis, coryza, enterohepatitis, hemopoietic necrosis, jaundice, keratoconjunctivitis, laryngotracheitis, myxomatosis, necrotic hepatitis, ophthalmia, pancreatic necrosis, pododernatitis, polyarthritis, pustular balanoposthitis, vulvovaginitis, serositis, sinusitis, stomatitis, synovitis, thromboembolic meningitis, and tracheobronchitis.

The present invention concerns a gene therapy approach with both curative and prophylactic activities on causing diseases infectious microorganisms. The invention concerns in particular DNA sequences, expres¬ sion vectors, DNA carriers (lyposome, solid particles) and cells allowing to make use of the process.

The invention concerns equally the cells (e.g. Mac-T, lung, kidney, muscle cells) genetically trans¬ formed in vitro with the gene of interest and reim- planted into the originating tissues to produce the curative or prophylactic proteins, peptide or antisense RNA against microorganisms responsible or potentially responsible of the diseases.

The invention concerns more particularly domes¬ tic animals: bovine, caprine, ovine, porcine, feline, canine and birds, but can concerns also more exotic animals such as rabbit, camel and bison. The present invention will be more readily un¬ derstood by referring 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 mammary glands

Mammary-gland promoters have been used in transgenic animals to limit transgene expression to the mammary gland. Gene therapy techniques to target just one organ for introduction of a foreign gene have also been demonstrated. Most efforts toward postnatal gene therapy have relied on new genetic information into tissues: target cells are removed from the body, infected with viral vectors carrying the new genetic information, and then reimplanted into the body. For some applications, direct introduction of genes into tissues in vivo, with or without the use of viral vec¬ tors, would be useful. Direct in vivo gene transfer into postnatal animals has been achieved with formula¬ tions of DNA encapsulated in liposomes, DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Nicolau et al., 1983, PNAS USA, 80:1068), calcium phosphate-coprecipitated DNA (Benvenisty et al., 1986, PNAS USA, 83:9551), and DNA coupled to a polylysine-glycoprotein carrier complex (Wu and Wu, 1988, J. Biol . Chem. , 263:14621). In vivo infectivity of cloned viral DNA sequences after direct intrahepatic injection with or without formation of calcium phos¬ phate coprecipitates has also been described (Seeger et al., 2984, PNAS USA, 81:5849). With the use of cat¬ ionic lipid vesicles (Feigner et al., 1989, PNAS USA, 84:7413), mRNA sequences containing elements that enhance stability can be efficiently translated in tis¬ sue culture cells (Malone et al., 1989, PNAS USA, 86:6077) and in Xenopus laevis embryos (Malone, 1989, Focus 11:61). It is demonstrated here that injection of pure DNA complexed to cationic liposomes directly into sheep mammary gland results in significant expres¬ sion of reporter gene within the gland. Preparation of plasmid-liposome mixture

Plasmid pCR3 (InVitrogen) was used as mammalian expression vector. After PCR amplification, the human growth hormone (hGH) cDNA was inserted into pCR3. This resulted in plasmid construct pCR3. Plasmid-Lipofect- AMINE™ (BRL) mixture was prepared as described by the manufacturer (GibcoBRL). Briefly, 50 ug of pCR3-hGH suspended in 500 μl sterile phosphate buffered saline (PBS), was mixed to 100 μl of LipofectAMINE™ also pre¬ viously diluted into 500 μl of PBS, and kept at room temperature at 1 hour.

Infusion of the plasmid-liposome complexes into sheep mammary gland

The circular pCR3-hGH plasmid-LipofectAMINE™ mixture was loaded into a glass syringe. Just after dropping, by using a 20-gauge needle, the DNA-liposome complex was infused directly through the udder's skin into the mammary parenchyma. One ml was injected into the right quarter of two ewes. The milk of the left glands was used as negative controls.

Analysis of sheep milk

Sheep were milked once daily by hand with the milk kept at -80°C until analyzed. The amount of hGH was measured by immunoassay (Immunocorp) after deter- mining that the milk did not affect the accuracy of the assay. Aliquots (100 ul) of milk samples were ana¬ lyzed.

RESULTS hGH synthesized by injecting pCR3-hGH into the mammary gland was detected all along the lactating period, meaning about 60 days, as illustrated in Fig. 2. The concentration of hGH in the sheep's milk was relatively high during the first 5 days. At that time it was of 300 to 400 ng/ml (± 43 ng/ml). hGH con¬ centrations in the milk from the left (control) gland was from 10 to 15 ng/ml for the two sheep everyday of the experiment. No important differences of concentra¬ tion of hGH in milk samples were found between each ewes.

Conclusion

These results demonstrate that expression from plasmid DNA can persist in a sheep's mammary gland for at least 60 days. The unprecedented ability of plasmid DNA to stably express a foreign gene in a mammary gland throughout the lactating period of a sheep has impor¬ tant implications for gene therapy. The stable expres¬ sion of circular plasmid DNA suggest that foreign acceleration or by viral transduction should also be stably maintained. EXAMPLE II

Human growth hormone (hGH) secretion in goats¹ milk after direct transfer of the hGH gene into the mammary gland

An alternative route of introducing genes into the mammary parenchyma is through expansion of gene therapy techniques. In this study two Gibbon ape leu- kemia virus (GaLV) pseudotype retroviral vectors were used to transfer reporter genes into a goat's mammary secretory epithelial cells in vitro and in vivo.

Cells and tissue culture MDBKs, a bovine kidney cell line and Mac-T cells, a bovine mammary epithelial cell line were used. Retroviral packaging cell lines used (φCre, PA317, and PG13/LNc8) were acquired from ATCC. Cells were main¬ tained in Dulbecco's modified Eagle's medium (DMEM) supplemented with gentamycin (54 mg/ml) and 10% fetal calf serum, 37°C with 5% CO2 /95% air.

Establishment of producer cell lines

A construct carrying the JR-gal neo- (Wang et al., 1991, Cancer Res . , 51:2642) was transfected into the ecotropic packaging cell line φCre by particle bombardment at 1 μg of DNA per mg of gold beads. Two days after bombardment, the supernatant was removed from these cells and centrifuged, and after the addi- tion of Polybrene at 4 μg/ml, the retroviral solution was used to infect both amphotropic and GaLV pseudotype packaging cell lines. A plasmid carrying the retrovirus vector, MFG-hGH was cotransfected with pSV2neo at a ratio of 50:1 via particle bombardment into PA317s and PG13/LN cδs. Packaging cells producing retrovirus containing the hGH gene were selected by

G418 resistance (400 μg/ml). Virus producing cells

The PG13/LN c8 clones that yielded the highest levels of hGH produced from the target cell lines were chosen for the infusions into a goat's mammary glands. Each clone was passed three times into 200 100 mm-plates. Cellular supernatant was collected over a 3-day period, concentrated, and resuspended in DMDM with Gentamycin.

Induction of cell division and lactation of goats

Two 2-year-old (goats 1 and 2) and two 1-year- old (goats 3 and 4) virgin Saanen-crossbred goats were treated with exogenous steroids i.m. over a 14-day interval to induce mammogenesis and subsequent lacta- tion.

«

Infusion of viral stocks into a goat's mammary glands

Polybrene was added to concentrated PG13/LN c8 MFG-hGH viral stock at 80 μg/ml and loaded into a syr- inge. By using a 22-gauge stub adapter, the retrovi¬ ruses were infused up the right mammary teat 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, ranging from 8 to 20 ml, and was determined by the integral capacity of the gland. The left gland served as the intraanimal control and was infused with DMEM containing gentamycin. Retrovi¬ ral stock used for the infusions was then assayed on several cell lines.

Analysis of goat's milk

Goats were milked twice daily by hand with the morning milk kept at -80°C until analyzed. The amount of hGH was measured by immunoassay after determining that the milk did not affect the accuracy of the assay. Aliquots (5 μl) of milk samples diluted 1:10 in double distilled water were also analyzed by SDS/PAGE on 14% gels stained, with Coomassie blue. The protein concen¬ tration of the milk samples was determined by using BCA (Pierce et al., 1977, Anl . Biochem. , 81:478).

RESULTS

Vector production of packaging cell lines

The concentration of hGH in the medium removed from Mac-T and MDBK cells 2 days after infection with retrovirus packaged by PG13/LN cδ clone 6 was 192 and 3.8 ng/ml, respectively. Twenty-eight days after infection, hGH levels from these cells were 119.3 and 4.5 ng/ml, indicating that the provirus LTR was still functioning 4 weeks after infection.

Infusion of viral stocks into the mammary glands of goats

Viral stock infused on day 13 for goats 1 and 2 was found to contain hGH at 224 ng/ml, indication that the PG13/LN c8 packaging cell were also producing hGH.

Analysis of goat milk

Lactation commenced on day 14 of the hormonal regimen, 24 hr after the last viral infusion. Milk appeared normal throughout the lactations. The volume of milk obtained from each udder half was approximately 150 ml on the first day of lactation for goats 1 and 2 but only 10 ml for goat 3, and 35 ml for goat 4. Milk volume produced by each gland for all four goats increased daily. The levels of hGH were determined by immunoassay with unique hGH secretion patterns for each animal. In goat 1, concentration of hGH dropped stead¬ ily until day 9 of lactation when it leveled at 3-5 ng/ml, whereas goats had a more precipitous decrease in measured hGH from day 1 to day 2 of lactation, though the animal's production of hGH stabilized at 2-3 ng/ml around day 10. Milking was stopped on day 15 of lacta¬ tion for goats 1 and 2. Levels of hGH in the milk of goat 3 dropped dramatically from day 1 to 2 of lacta- tion and then increased from day 8 to day 9 where it remained at 23 ng/ml until day 16 when it began to fall again. Goat 4, in which prostaglandin E2 was infused at the end of the remaining 19-day lactation after a decline on the first 2 days. In addition, goat 4 was still secreting hGH at 5 ng/ml after 28 days. hGH con¬ centrations in the milk from the left (control) gland ranged from 0.0 to 0.6 ng/ml for the four goats at all evaluated times. These numbers are at the detection level of the assay and correlate with ones measured in two other lactating goats that had no exposure to retrovirus. The total production of hGH in the four animals ranged from 0.3 to 2 ug/day.

If the hGH gene had been stably incorporated into the stem-cell population, it would have been expected that the goats would also secrete hGH in a second lactation after the gland had undergone involu¬ tion. A second lactation was induced in two of the goats, and though goat 1 did not produce hGH, goat 2 began secreting detectable amounts of hGH starting on day 5 from the right (infused) gland and during the subsequent 10 days hGH concentrations varied from 0.4 to 2.3 ng/ml. Milk from the left control gland during this lactation always had no detectable levels of hGH.

SDS/PAGE of goat's milk sampled throughout the period of collection showed no consistent differences in the protein profiles from the retroviral-infused right glands, the control left glands, and a goat not exposed to the retrovirus. Protein concentrations measured by BCA of the milk with hGH were not statisti- cally different from the control milk, thus production of hGH by the mammary secretory epithelial cells did not appear to affect the normal cellular protein machinery. There was an indication that the milk's proteins in the treated gland were not secreted at maximal concentration on day 1 of lactation.

Conclusion

Applying gene therapy technology and replica¬ tion-defective retroviral vectors to directly introduce a foreign gene into a ruminant mammary gland has dra¬ matically reduced the time of production of pharmaceu¬ ticals in milk, from years to weeks. Although the lev¬ els of expression found are low, the methods might find application in the evaluation of different gene con- structs as a prelude to production of transgenic ani¬ mals or in the production of low levels of important proteins for evaluation purposes.

EXAMPLE III

Effect of lysostaphin on Staphylococcus aureus infec¬ tions on the mouse's mammary gland

Lysostaphin is an endopeptidase produced by

Staphylococcus simulans . It hydrolyzes the pentaglycine links 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 suc¬ cessfully in Escherichia coli and Bacillus species (Heath et al., 1987, FEMS Microbiology Letters, 44:129; Heinrich et al., 1987, Molecular and General Genetics, 209:563; Recsei et al., 1987, PNAS USA, 84:1127). The use of lysostaphin to promote lysis of Staphylococcus aureus in a variety of experimental situations is well known but the progress made in cloning and expressing the gene in other hosts raises the possibilities of producing large quantities of the enzyme relatively inexpensively. This may permit its use in vivo in new approaches to the control of staphylococcal mastitis, an economically important disease of lactating rumi- nants (Bramley et al., 1990, Res . Vet . Sci . , 49:120). This experiment shows the use of a mastitis model in the lactating mouse and clearly demonstrates potent antibacterial activity of lysostaphin against S. aureus in vivo. Lysostaphin (Sigma Chem. ) was dissolved in skimmed milk (Oxoid) to provide a range of concentra¬ tions between 0.1 and 100 μg/ml. Controls without lysostaphin were included. One ml volumes of the con¬ trols and lysostaphin dilutions were inoculated with 10* colony forming units (cfu) of S. aureus M60. This strain produces both and β toxins and was isolated from a case of bovine mastitis. Lysostaphin concentra¬ tions exceeding 2 to 3 ug/ml in milk produced a 2 to 3 log o reduction in viable S. aureus, whereas 10 ug/ml in milk reduced S. aureus from a mean of 7.95 log ιo/ml in the control to 2.0 logio/ml- Consequently a dose of 10 ug of lysostaphin was selected for use in vivo. Anaesthetized mice, of strain MF1, were inoculated in the upper pair of abdominal mammary glands (designated R4 and L4). Eight lactating mice were inoculated with 10 cfu of S. aureus in 0.1 ml saline in both R4 and L4. This was followed one hour later by the infusion of 10 ug lysostaphin in 0.1 ml saline into R4 and 0.1 ml saline into L4. After a further 30 minutes the mice were killed and the mammary glands were aseptically removed and homogenized in saline containing 0.1 mg/ml trypsin (Sigma Chem.) to destroy active lysostaphin. Ten fold dilutions were placed on 7 per cent calf blood agar (Oxoid Blood Agar Base Number 2), incubated at 37°C overnight and viable counts determined. In a fur- ther experiment using 20 mice a prophylactic use of lysostaphin was simulated by infusing 10 ug of lysostaphin intramammarily, followed either immediately or after one hour by 10- cfu of S. aureus . Control glands were infused with saline instead of lysostaphin. After 24 hours the mice were killed and dissected. Gross pathological changes were noted an viable S. aureus counts determined as described above.

RESULTS

Infusion with lOmg lysostaphin into mammary glands previously inoculated with S. aureus reduced bacterial recoveries, compared to the controls, by more than 99 per cent in 30 min. This reduction was statis- tically significant (t=2.56; P<0.02). When 10 ug of lysostaphin was administered either immediately or one hour before S. aureus inoculation, recoveries after 24 hours averaged around 10² viable S. aureus per mammary gland compared with approximately 10^ per mammary gland for the saline treated controls. In the latter case, the control glands showed severe pathological changes typical of acute staphylococcal mastitis in the mouse. The control glands were darker and reddened, had a brittle texture and some areas of liquefaction and haemolysis. Histological sections revealed a severe inflammation, infiltration of neutrophils and macro- phages with areas of coagulative necrosis. Large num¬ bers of Staphylococci were visible. In contrast, the lysostaphin treated glands remained pale and elastic with only slight reddening around the base of the teat. 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- staphylococcal activity of lysostaphin in vivo. Both a therapeutic and prophylactic potential were demon- strated. The cloning of the lysostaphin gene may make it readily available for therapeutic use at a competi¬ tive price and its relatively high specificity makes it attractive for use in food-producing animals. Further¬ more, advances in transgenic technology allow the direction of the expression of transgenes to the mam¬ mary 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 lactating mammary gland could potentially increase the resistance of the animal to staphylococcal mastitis.

EXAMPLE IV

Lysostaphin efficacy for treatment of Staphylococcus aureus intramammary infection

Cloned-derived lysostaphin was evaluated as to its bactericidal effect on S. aureus intramammary infections. S. aureus (Newbould 305) was eliminated from glands of guinea pigs 48 hrs post-infection by 125 μg of lysostaphin in 14/16, 25 μg in 5/8, 5 μg in 5/10, 1 μg in 0/1, and 0 μg in 0/3. Glands infected with S. aureus at 48 hours post-challenge in untreated guinea pigs persisted, however, 3/25 control glands of treated guinea pigs cleared in response to treatment of the adjacent gland. Somatic cell/ml in guinea pig shifted from 10^ pre-infected glands to cell counts greater than 3 x 10^ following S. aureus inoculation. Treatment with lysostaphin caused a neutrophilic shift in the treated gland to levels exceeding 108 accompanied by an increase in the adjacent non-treated gland but dropped sharply to pre-treatment level. The greatest response in control glands was observed in animals receiving 125 ug which corresponded to 2/25 clearance of S. aureus in control glands.

The leukocyte response to intramammary treat¬ ment in the cow is similar to the guinea pig model described above. Somatic cell levels increased ten¬ fold in S. aureus infected glands at the milking fol- lowing treatment. Cell levels returned to pre-treat¬ ment levels or lower in subsequent milking. A rise in leukocytes alone could not account for clearance of the infection.

EXAMPLE V

Use of a recombinant bacterial enzyme (Lysostaphin) as a mastitis therapeutic

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 Straphylococcus aureus and is bactericidal. Thirty Holstein-Friesian dairy cattle in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all quarters.

Infections were established and monitored for somatic cell counts and Staphylococcus aureus colony-forming units 3 weeks prior to subsequent treatment. Infected animals were injected through the teat canal with a single dose of recombinant lysostaphin (rLYS) (dose 1 to 500 mg) or after three successive p.m. milking with 100 mg of rLYS in 60 ml of sterile phosphate-buffered saline. Animals were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings after the last treatment.

RESULTS

Kinetic analysis of immunologically active rLYS demonstrated 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 over the same incubation time.

Dose titration and kinetics of rLYS in the bovine mam- mary gland

In order to determine the optimal effective dose to elicit long-term cures, a titration was per¬ formed in which a single dose of rLYS at concentrations of 0, 1, 10, 100, or 500 mg was administered. Untreated quarters and the 1-mg treatment failed to clear all quarters of S. aureus . The 10- 100- and 500- mg does transiently cleared the milk of S. aureus for at least one milking. In relapsed quarters, the length of time of the milk remained clear of S. aureus was approximately proportional to the dose administered. Fourteen days after treatment, two quarters were cured with the 100 mg dose and one with the 500 mg dose. Because rLYS maintains a minimal bactericidal concentration (MBC) for approximately 24 h and the experimental infections undergo a 2- to 4- days cycling, multiple infusions of 100 mg of rLYS over three consecutive milking were determined to be optimal to maintain a minimal effective dose for 3 to 5 days and to elicit cures. Conclusion

Staphylococcus aureus is one of the primary etiologic agents of bovine mastitis and a major cause of economic loss to the dairy industry. An effective mastitis therapy for the lactating dairy cow remains a major unfilled need. Because current therapy is only moderately efficacious and is costly because of milk discard and culling infected animals, treatment only during the dry period has been the adopted herd manage- ment practice of choice. Neither approach addresses the majority of the infections in a lactating animal, which are chronic and subclinical in nature. A recom¬ binant protein such as rLYS with bactericidal activity against S. aureus could be an extremely useful thera- peutic to the veterinarian. If rLYS was as efficacious as antibiotics, natural proteolysis and inactivation in the milk of rLYS, as well as inactivation during inges- tion by the consumer, would potentially minimize any concerns associated with residues in milk. The in vivo does titration suggested that the minimal effective therapeutic dose was 100 mg of rLYS. However, therapeutically, it would be desirable to administer multiple infusions of rLYS to maintain a minimal bactericidal activity within the milk of treated glands for one to three successive milkings. The in vivo bactericidal activity of rLYS was most effectively demonstrated by the fact that 95% of the quarters cleared the milk of detectable S. aureus for a minimum of one milking after the last intramammary infusion.

EXAMPLE VI

Expression of jet-injected plasmid DNA in the ovine mammary gland A jet-injection based DNA delivery system has been evaluated as a means to transiently transfect the — 3y —

lactating mammary gland in vivo and as a technique for DNA vaccination. The model expression plasmid con¬ tained the human growth hormone (hGH) gene driven by the human cytomegalovirus immediate early gene 1 pro- moter/enhancer region (CMV). Expression from the naked plasmid DNA jet-injector into lactating mammary glands of sheep was sufficient to be detected by Northern blot analysis when tissue was obtained 48 hours after in vivo transfection. In conclusion, the ability to tran- siently transfect lactating mammary tissue in vivo cir¬ cumvents the difficulties encountered with in vivo cul¬ ture techniques and provides a method for examining mammary regulatory elements and testing of fusion gene constructs designed for the production of transgenic animal bioreactors.

EXAMPLE VII

Elimination of Staphylococcus aureus in an eukaryotic system expressing the lysostaphin

The lysostaphin gene was introduced into 293 cells (human fetal kidney cells) maintained in vitro.

The recombinant bacteriocin, the lysostaphin, was secreted in the medium culture and was found to kill contaminant S. aureus during the challenge.

The lysostaphin gene was obtained by PCR ampli¬ fication from extracted DNA of Staphylococcus simulans biovar staphylolyticus (NRRL B-2628), and

Staphylococcus aureus strain Newbould (ATCC) was used for the challenge in transfected eukaryotic cells. Staphylococcal strains were grown in Brain Heart Infusion (BHI) medium.

Purification of the lysostaphin gene Staphylococcus simulans biovar staphylolyticus was cultured overnight in a stirring incubator at 37°C. The media was centrifuged, and the pellet was resus- pended in 5 ml of 50 mM EDTA-50mM Tris-HCL (pH 7.8) containing 50 mg of lysostaphin (Sigma) ml^-1 and the suspension was incubated at 37°C for 2 hours. Purified bacterial DNA was directly amplified by PCR method to isolated the lysostaphin gene. The set of oligonucleo- tide primers used were as followed: 5'-TTAAGGTTGAAGAAAACAATT-3' (SEQ ID N0:1) and 5'-GCGCTCACTTTATAGTTCCCCAA-3 ' (SEQ ID NO:2) . The amplification was performed by using a Thermal DNA cycler and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus), and a 30 cycles program with an annealing step at 60°C for 30 sec, elongation at 72°C for 90 sec. and denaturation at 93°C for 10 sec. The PCR product was composed by the entire lysostaphin sequence, including the coding gene with the aminoterminal pre- and pro- regions. All other recombinant DNA procedures, including restriction endonuclease digestion, ligation, washing with phenol- chloroform mixture, ethanol precipitation, transforma- tion and cloning of the constructs in E. coli strain DH5a, were carried out by standard methods. All enzymes were from Boehringer Mannheim.

The lysostaphin was linked to an eukaryotic expression vector including the human cytomegalovirus immediate early gene 1 promoter/enhancer region (CMV) and the human interleukin-2 signal peptide.

Cell culture and DNA transfection

293 cells, a human foetal kidney cell line transformed by an origin-defective mutant of simian virus 40, were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 10% (vol/vol) fetal calf serum (Gibco BRL) and glutamine (1.4 mM) . The cells were seeded into 30-mm wells at 500 000 cells par well and grown in 2 ml of medium for 24h at 37°C (in air atmosphere containing 5% CO2) to yield 50 to 60% introduced into the cells by the calcium phosphate method with the following modifications. The precipi¬ tate containing 7.5 μg of DNA was added to 2 ml of cul¬ ture medium. After 24 h, the medium was replaced with 2 ml of medium per well, and samples of the medium were harvested at each 24 h following transfection to evalu¬ ate the production of the lysostaphin by Western blot analysis and ELISA

Assay for biological active lysostaphin

The wells containing the transfected 293 cells were infected with 10² or 10-^ of Staphylococcus aureus Newbould. Samples of lOOμl of the infected medium were spreaded on sheep blood agar. After incubation for 24 h at 37°C, the number of colony forming units (CFU) was evaluated to assess the inhibition effect of the recom¬ binant lysostaphin on the growth of the bacteria.

RESULTS

Production of recombinant lysostaphin by transfected eukaryotic cells

The modified lysostaphin gene was transfected into tissue culture cells to demonstrate the expres- sion, processing and activity of the enzyme on infect¬ ing 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 has been carried out in eukaryotic cells. The ELISA assays have revealed that the recombinant lysostaphin was produced in concentrations of 100 to 250 ng/ml/24h depending of the clone. Activity of the lysostaphin secreted by mammalian cells

The activity of the recombinant lysostaphin secreted by transfected mammalian cells has been observed by its efficiency to reduce or in some repli- cates to inhibit the growth of infecting Staphylococcus aureus in the culture media. Samples of media taken from non-transfected cells have shown none inhibitory effect on the development of the bacteria present in the wells. The plates of agar were completely conflu- ent after overnight incubation. In contrast, when an initial amount of 10^ bacteria was cultured in presence of transfected eukaryotic cells, very few CFU were counted on the plates. Less than 100 CFU were observed in our assays when 10^ bacteria were used, while we did not observed the presence of CFU on gels when 10² bac¬ teria were added to the wells containing the trans¬ fected cells.

While the invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

SEQUENCE LISTING

(1) GENERJΛL INFORMATION:

(i) APPLICANT:

(A) NAME: IMMUNOVA

(B) STREET: 2750 rue Einstein, Bureau 110

(C) CITY: Sainte-Foy

(D) STATE: Quebec

(E) COUNTRY: Canada

(F) POSTVL CODE (ZIP) : G1P 4R1

(G) TELEPHONE: (418) 654-2240 (H) TELEFAX: (418) 654-2125

(A) NAME: GAGNE, Marc

(B) STREET: 913 rue Pellan

(C) CITY: St-Jean-Chrysostome

(D) STATE: Quebec

(E) COUNTRY: Canada

(F) POSTAL CODE (ZIP) : G6Z 2S8

(ii) TITLE OF INVENTION: ANIMAL GENE THERAPY (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER REMABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(Vi) PRIOR APPLICATION DATA:

(A) /APPLICATION NUMBER: GB 9509461. 1

(B) FILING DATE: 10-MAY-1995

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTAAGGTTGA AGAAAACAAT T 21

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid (C) STRΛNDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCGCTCACTT TATAGTTCCC CAA 23

Claims

WE CLAIM:

1. 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 a DNA sequence encoding for a therapeutic protein, peptide or antisense RNA operably linked to a promoter capable of directing the in vivo expression of said DNA sequence of a therapeutically effective amount of said protein, peptide or antisense RNA; and b) introducing into the animal the DNA expression system of step a) for expression of said therapeutic protein, peptide or antisense RNA.

2. The method of claim 1, wherein said treatment and/or prevention of the disease is effected in si tu and said DNA expression system is introduced in targeted tissue.

3. The method of claim 1, wherein said DNA expres¬ sion system is transgenic recombinant animal cells.

4. The method of claim 3, wherein said cells are selected from the group consisting of epithelial mam¬ mary gland cells, blood cells, lymphocytes, leukocytes, T-lymphocytes, B-lymphocytes, erythrocytes, muscle cells, hepatic cells, kidney cells, lung cells, secretory cells and non-secretory cells.

5. The method of claim 3, wherein said DNA expres¬ sion system is selected from the group consisting of a lipidic liposome, a cationic liposome, an anionic liposome.

6. The method of claims 1, 2, 3, 4 or 5, wherein said DNA sequence and said promoter are inserted into an expression vector.

7. The method of claim 6, wherein said vector is a viral vector or a retroviral vector.

8. The method of claims 1, 2, 3, 4, 5, 6 or 7, wherein said infectious diseases are caused by bacte¬ ria, virus, retrovirus, parasite, fungi, mold, yeast, prions or scrapies.

9. The method of claim 8, wherein said therapeutic protein, peptide or antisense RNA are selected from the group consisting of bacteriocins, lanthionins, lactoferrin, lysosyme.

10. The method of claim 9, wherein said bacte¬ riocins and/or lanthionins are ambicins, defensins, cecropins, thionins, mellitins, magainins, attacines, diphterins, saponins, cacrutins, xenopins, subtilins, epidermins, pep5, lacticin 481, ancovenins, duramycins, gallidermins or cinnamycins.

11. The method of claim 8, wherein said therapeutic protein, peptide or antisense RNA is selected from the group consisting of immunoglobulins, lactoglobulins, α -lactalbumin, bile-salt-stimulated lipase or ribosyme, cytokines, chemokines, growth factors, immunomodulators and major histocompatibility complex (MHC) proteins.

12. The method of claim 3, which further comprises a 5' and 3' expression regulation DNA sequence and a secretory DNA sequence functional in said animal cells and operably linked to the recombinant DNA encoding said therapeutic protein, peptide or antisense RNA.

13. A non-human transgenic animal for the produc¬ tion of a recombinant protein, peptide or antisense RNA systemically or in targeted tissue, which comprises at least an expression regulation DNA sequence and a secretory DNA sequence encoding a secretory signal sequence operatively linked to a DNA sequence encoding said protein, peptide or antisense RNA for systemic expression or for expression in targeted tissue cells of said animal.

14. The non-human transgenic animal of claim 13, wherein said expression regulation DNA sequence is selected from the group consisting of a constitutive promoter, an inducible promoter, a cytomegalo virus promoter.

15. The non-human transgenic animal of claim 14 wherein said promoter is selected from the group of DNA sequences encoding lactoferrin, serum albumin, αSl- casein, αS2-casein, β-casein, K-casein, α-lactalbumin, whey acidic protein, β-lactoglobulin, cytokines, chemokines and growth factors.

16. The non-human transgenic animal of claim 13, wherein said secretory signal sequence is selected from the group consisting of DNA sequences encoding lactoferrin, serum albumin, αSl-casein, αS2-casein, β- casein, κ-casein, α-lactalbumin, β-lactoglobulin, cytokines, chemokines or growth factors.

17. The non-human transgenic animal of claim 13, wherein the expression regulation and secretory signal sequences are from human, bovine, caprine, ovine, feline, canine, lagomorphes, birds and fishes.

18. The non-human transgenic animal of claim 13, wherein the promoter is tissue-specific for expression in targeted tissue.