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WO1996035793A1 - Therapie genique animale - Google Patents

Therapie genique animale Download PDF

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Publication number
WO1996035793A1
WO1996035793A1 PCT/CA1996/000297 CA9600297W WO9635793A1 WO 1996035793 A1 WO1996035793 A1 WO 1996035793A1 CA 9600297 W CA9600297 W CA 9600297W WO 9635793 A1 WO9635793 A1 WO 9635793A1
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WIPO (PCT)
Prior art keywords
cells
dna
expression
gene
protein
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PCT/CA1996/000297
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English (en)
Inventor
Marc Gagne
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Immunova
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Publication date
Application filed by Immunova filed Critical Immunova
Priority to JP8533627A priority Critical patent/JPH11505113A/ja
Priority to NZ307139A priority patent/NZ307139A/xx
Priority to EP96913403A priority patent/EP0828839A1/fr
Priority to AU56416/96A priority patent/AU724905B2/en
Publication of WO1996035793A1 publication Critical patent/WO1996035793A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/102—Caprine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/103—Ovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated

Definitions

  • 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.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • TCR T-cell receptors
  • Interferon Interferon
  • IFNs Interferon
  • IFNs Interferon
  • 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- ⁇ gene con ⁇ structs were shown to exhibit enhanced viral resis- tance.
  • IFNs interferon-stimulated gene factor
  • IRF-1 interferon regulatory factor
  • 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.
  • 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".
  • transgene encoding an immu- noglobulin specific for a common pathogen can provide immunity for that pathogen.
  • 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.
  • MMTV mouse mammary tumor virus
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the hammerhead motif of this ribozyme family By flanking the hammerhead motif of this ribozyme family with antisense sequences, the cleavage of specific tar ⁇ get RNAs has been demonstrated.
  • Bovine leukemia virus (BLV), a retrovirus , causes persistent lymphocytosis and B-lym- phocyte lymphoma in cattle and sheep.
  • BLV bovine leukemia virus
  • 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.
  • 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.
  • 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.
  • 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 .
  • lysostaphin is produced by Staphylococcus simulans biovar staphylolyticus .
  • 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.
  • 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.
  • 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 ⁇ Illustrative. 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- ⁇ receptor.
  • 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.
  • an immunoadhesin which could has two specific activities.
  • 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.
  • 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.
  • an immunomodulator such as interleukins, chemokines, interferons, leu- kotriens, and certain growth factors.
  • the immunomodulators can makes the animal more resistant to several microorganisms.
  • 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.
  • 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.
  • the stimulating molecules which have been administered by intramammary injection, are cytokines, interleukin-l ⁇ ,, interleukin 2, interferon- ⁇ , tumor necrosis factor- ⁇ .
  • 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.
  • 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.
  • 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".
  • 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.
  • 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.
  • 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.
  • such DNA constructs can be admin- istered to both lactating or non-lactating animals for the prophylaxis or treatment or mastitis.
  • 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.
  • 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.
  • the DNA can be injected directly into glands through the use of syringe.
  • 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.
  • 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.
  • 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.
  • 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.
  • the protein or polypeptides produced should be effective prophylaxis or treatment of mastitis.
  • 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.
  • 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 .
  • Fig. 1 illustrates examples of DNA constructs in accordance with the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • strong consti- tutive promoters are those controlling the expression of caseins, lactoglobulins, lactoferrin, lactalbumin, lysosymes, whey acidic proteins (WAP) coding genes in mammary glands.
  • WAP whey acidic proteins
  • the promoters origi ⁇ nates from domestic animals, bovine, caprine, ovine or porcine species.
  • specific mammary gland promoters can originates from smaller animals, lagomor- phes, rodents, felines or canines.
  • CMV Cytomegalovirus
  • SV40 Simian virus 40
  • MMTV mouse mammary tumor virus
  • 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.
  • HST heat shock promoter
  • 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.
  • GR glucocorticoid receptor protein
  • the glucocorticoid response element 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) .
  • 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.
  • high velocity ballistics can be used to propel small DNA associated particles into the tissue through a skin's incision.
  • 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.
  • the gene coding for a mucolytic protein e.g. bacteriocins and lanthionins
  • 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.
  • the genes can be used for in-situ synthesis of the following therapeutic polypeptides: 1. Enzymes or mucolytic proteins, such as lysostaphin and mucolysins;
  • Antibodies such as anti-hemolysins, anti-leu- cocidin, anti-protein A, anti-collagen, anti- fibronectin binding protein, anti-laminim, anti- ⁇ -toxin and anti- ⁇ -toxin antibodies; opsonic antibodies and antibodies raised against cell fusion viral protein;
  • Cytokines interleukines, chemokines, growth factors
  • 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:
  • 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 .
  • 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,
  • 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.
  • cells e.g. Mac-T, lung, kidney, muscle cells
  • 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.
  • 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.
  • 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- AMINETM (BRL) mixture was prepared as described by the manufacturer (GibcoBRL). Briefly, 50 ug of pCR3-hGH suspended in 500 ⁇ l sterile phosphate buffered saline (PBS), was mixed to 100 ⁇ l of LipofectAMINETM also pre ⁇ viously diluted into 500 ⁇ l of PBS, and kept at room temperature at 1 hour.
  • PBS sterile phosphate buffered saline
  • the circular pCR3-hGH plasmid-LipofectAMINETM 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.
  • 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.
  • hGH Human growth hormone
  • GaLV Gibbon ape leu- kemia virus
  • DMEM Dulbecco's modified Eagle's medium
  • a construct carrying the JR-gal neo- was transfected into the ecotropic packaging cell line ⁇ Cre by particle bombardment at 1 ⁇ g 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 ⁇ g/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 c ⁇ s.
  • Packaging cells producing retrovirus containing the hGH gene were selected by
  • 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.
  • Polybrene was added to concentrated PG13/LN c8 MFG-hGH viral stock at 80 ⁇ g/ml and loaded into a syr- inge.
  • 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.
  • the concentration of hGH in the medium removed from Mac-T and MDBK cells 2 days after infection with retrovirus packaged by PG13/LN c ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Lysostaphin is an endopeptidase produced by
  • Staphylococcus simulans 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).
  • 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.
  • mice 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.
  • 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 .
  • S. aureus (Newbould 305) was eliminated from glands of guinea pigs 48 hrs post-infection by 125 ⁇ g of lysostaphin in 14/16, 25 ⁇ g in 5/8, 5 ⁇ g in 5/10, 1 ⁇ g in 0/1, and 0 ⁇ g 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.
  • 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.
  • rLYS recombinant lysostaphin
  • penicillin G retained less than 10% of its bacteriostatic activity over the same incubation time.
  • 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.
  • 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.
  • 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- Pigic to the veterinarian.
  • 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.
  • 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.
  • 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.
  • hGH human growth hormone
  • CMV human cytomegalovirus immediate early gene 1 pro- moter/enhancer region
  • 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.
  • 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 was used for the challenge in transfected eukaryotic cells. Staphylococcal strains were grown in Brain Heart Infusion (BHI) medium.
  • BHI Brain Heart Infusion
  • 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.
  • 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.
  • CMV human cytomegalovirus immediate early gene 1 promoter/enhancer region
  • 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 ⁇ g 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
  • the wells containing the transfected 293 cells were infected with 10 2 or 10- ⁇ of Staphylococcus aureus Newbould. Samples of lOO ⁇ l 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.
  • CFU colony forming units
  • 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.
  • 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 10 2 bac ⁇ teria were added to the wells containing the trans ⁇ fected cells.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 1: TTAAGGTTGA AGAAAACAAT T 21
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 2: GCGCTCACTT TATAGTTCCC CAA 23

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Abstract

La présente invention concerne des séquences d'ADN, des cassettes d'expression et des produits de synthèse d'ADN à usage thérapeutique, notamment pour la thérapie génique de maladies infectieuses telles que la mammite. Elle concerne également des compositions pharmaceutiques et vétérinaires contenant ces produits de synthèse, ainsi que des cellules qui ont été transformées avec l'ADN et qui se prêtent à l'implantation chez un mammifère hôte. La thérapie génique de maladies infectieuses peut s'appliquer in situ à des tissus ciblés ou de manière systémique.
PCT/CA1996/000297 1995-05-10 1996-05-10 Therapie genique animale WO1996035793A1 (fr)

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JP8533627A JPH11505113A (ja) 1995-05-10 1996-05-10 動物遺伝子治療
NZ307139A NZ307139A (en) 1995-05-10 1996-05-10 Method of genetically treating and preventing mastitis and other diseases in animals.
EP96913403A EP0828839A1 (fr) 1995-05-10 1996-05-10 Therapie genique animale
AU56416/96A AU724905B2 (en) 1995-05-10 1996-05-10 Animal gene therapy

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WO1998012353A3 (fr) * 1996-09-20 1998-05-28 Texas A & M Univ Sys Methode d'identification d'animaux resistants ou sensibles a des maladies telles que la melitococcie, la tuberculose, la maladie de johne et la salmonellose
WO1999005289A1 (fr) * 1997-07-23 1999-02-04 9071-7125 Québec Inc. Analogues de lysostaphine recombinants
WO1999005527A3 (fr) * 1997-07-22 1999-11-25 Novimmune Sa Methodes diagnostiques et therapeutiques d'une maladie auto-immune telle que le diabete sucre insulinodependant mettant en oeuvre des superantigenes retroviraux
US6875903B2 (en) 1998-06-22 2005-04-05 University Of Vermont Treatment of Staphylococcus infections
US7091332B1 (en) 1998-06-22 2006-08-15 University Of Vermont Treatment of staphylococcus infections
JP2009183304A (ja) * 1997-01-17 2009-08-20 Scripps Res Inst:The 組換え分子の発現に有用なｒｎａ結合タンパク質及び結合部位

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EP0448511A1 (fr) * 1990-03-12 1991-09-25 Ciba-Geigy Ag Combinaisons antipathogènes contenant les peptides lytiques et les enzymes hydrolytiques
WO1991018512A1 (fr) * 1990-05-25 1991-12-12 Washington State University Research Foundation, Inc. Procede d'induction de mecanismes de defense a des plantes
WO1993025567A1 (fr) * 1992-06-15 1993-12-23 Gene Pharming Europe B.V. Production de polypeptides recombines par des especes bovines et des procedes transgeniques

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EP0279582A2 (fr) * 1987-02-17 1988-08-24 Pharming B.V. Séquences d'ADN pour diriger des protéines vers les glandes mammaires afin d'être sécrétées efficacement
EP0448511A1 (fr) * 1990-03-12 1991-09-25 Ciba-Geigy Ag Combinaisons antipathogènes contenant les peptides lytiques et les enzymes hydrolytiques
WO1991018512A1 (fr) * 1990-05-25 1991-12-12 Washington State University Research Foundation, Inc. Procede d'induction de mecanismes de defense a des plantes
WO1993025567A1 (fr) * 1992-06-15 1993-12-23 Gene Pharming Europe B.V. Production de polypeptides recombines par des especes bovines et des procedes transgeniques

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WILLIAMSON ET AL: "EXPRESSION OF THE LYSOSTAPHIN GENE OF STAPHYLOCOCCUS SIMULANS IN A EUKARYOTIC SYSTEM", APPLIED AND ENVIROMENTAL MICROBIOLOGY, vol. 60, no. 3, March 1994 (1994-03-01), pages 771 - 776, XP000579799 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012353A3 (fr) * 1996-09-20 1998-05-28 Texas A & M Univ Sys Methode d'identification d'animaux resistants ou sensibles a des maladies telles que la melitococcie, la tuberculose, la maladie de johne et la salmonellose
US6114118A (en) * 1996-09-20 2000-09-05 Texas A&M University System Method of identification of animals resistant or susceptible to disease such as ruminant brucellosis, tuberculosis, paratuberculosis and salmonellosis
JP2009183304A (ja) * 1997-01-17 2009-08-20 Scripps Res Inst:The 組換え分子の発現に有用なｒｎａ結合タンパク質及び結合部位
WO1999005527A3 (fr) * 1997-07-22 1999-11-25 Novimmune Sa Methodes diagnostiques et therapeutiques d'une maladie auto-immune telle que le diabete sucre insulinodependant mettant en oeuvre des superantigenes retroviraux
US6800469B1 (en) 1997-07-22 2004-10-05 Novimmune S.A. Methods for diagnosis and therapy of autoimmune disease, such as insulin dependent diabetes mellitus, involving retroviral superantigens
WO1999005289A1 (fr) * 1997-07-23 1999-02-04 9071-7125 Québec Inc. Analogues de lysostaphine recombinants
US6875903B2 (en) 1998-06-22 2005-04-05 University Of Vermont Treatment of Staphylococcus infections
US7091332B1 (en) 1998-06-22 2006-08-15 University Of Vermont Treatment of staphylococcus infections

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