CA2220472A1 - Genetically treated animals - 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

~NI~U~L GENE THEEU~PY

RACKGRO D OF ~'HE lN V r .. '1 lON
(a) Field of the Invention The invention re:Lates to DNA sequences, expres-sion cassettes and DNA constructs for use in therapy, specifically in gene t:herapy for the treatment of in~ectious 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 malmm~l (b) DescriPtiorl 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 trans~er into mice the new technique was used in farm An;m~ls. Several genetic treats have been targeted for the application of transgenesis in domestic anima]!s, but one of those important aspects is the improvement: of ~n; ~A 1 health and disease resistance by gene transfer means. Transient as well as stable genetic improvement lea~ing to disease resistance and treatment achLeved by recently developed techniques in molecular bio]ogy 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, c:andidates for gene therapy applications include all genes known to modulate non-specific and specific host ~lefense ~ch~n;sms, i.e. cytokines, major histocompatibiL:Lity com]?lex (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 I une Respon~ec Cytokine orchestrate immune responses through their role as soluble mediators of cell communication.
Initially identified to direct viability, prolifera-tion, di~erentiation and homing o~ 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 o~ regulatory peptide factors including growth factors, interleukins, çh~kines, colony-stimulating factors and interferons.
Their ~unctions 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 inhibitorym~chAn;sms are numerous. The potency o~ IFNs to posi-W O 96/3~793 3 PCT/CA96100297 tively in~luence host susceptibility to viral in~ec-tions was tested in transgenic mice and cell lines.
Transgenic organisms overexpressing IFN-~ gene con-structs were ~;hown to exhibit enhanced viral resis-tance.
Recent progress in the understanding o~ 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 trans~er of cytokine encoding genes per se o~ distinct "cytokine-specific" signaling components. Constitutive expression of an inter~eron-stimulated gene ~actor (ISGFZ) also termed inter~eron regulatory ~actor (IRF-1) transgenes h~as been reported to result in IFN-inde-pendent activa~t:ion of various IFN-inducible genes and enhanced resistance to v:iral in~ection.
ZO Specific Disease Resistallce Genes Other :improvement which can be brought to ani-mals by local gene transfer is speci~ic disease resis-tance. A well ~examined specific disease resistance gene is the Mxl gene product o~ certain mouse strains. The mouse Mxl protein belongs to a ~amily o~ 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 ~o:r example Influenza virus , VSV, rhado virus and Thogoto virus. Synthesis o~ mouse Mxl pro-tein in various cell lines and transgenic mice demon-strated that it is both necessary and sufficient to promote resistance to in~luenza A viruses in previously susceptible cells and A~;~AlS. The cloning and func-tional characterization of this specific disease resis-tance gene en~bled a gene trans~er 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 Leishm~nia is controlled by a dominant locus on chromo-some 1 called Bcg, 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 Bcg 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 Bcg. 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 W 096/3~793 5 PCT/CA96/00297 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 provideimmunity 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 specii-ic antigens without prior contact or immunization.
Intracellular li,iunizati.on The cc)ncept of "intracellular immunization"
essentially involves overexpression in the host oi- an aberrant i-orm (dominant-negative mutant) oi- a viral protein that is able t:o interfere strongly with the replication of the wild type virus. Elegant studies in cultured cel:Ls resulting in acquired resistance to various viruse~s include strategies preventing virus att~chme~t 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 ini~ectious viral particles.
Endog~ ous mouse mAmm~ry 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-supe,rantigen 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 oi- "intracellular ; ~;zation"
is also applied for antiviral strategies described in different corlnections such as expression of specific W 096/35793 6 PCT/CA9'~C297 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 W 096/35793 7 PCTICA9~ 297 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 a~enovirus in~ection than cells ~rom con-trol An i m~ 1 S .
The use of antisense RNAs as anti-parasitho-genic agents c:an be developed to result not only in RNA-RNA hybrid,, but cat~llytically cleave a phosphodi-ester bound in the target RNA strand. Four structuralmotifs (h~mmerhead and hairpin first identi~ied in plant RNA pat;hogens, t]le delta moti~ found in human hepatitis delta virus and a less well characterized motif ~rom NeLlspora ) have thus ~ar been described as intermediates in these self-cleavage reactions. By flanking the hammerhead motif of this ribozyme family with antisense sequences, the cleavage o~ specific tar-get RNAs has been demonstrated. A large number of sub-strate molecu:Les can be processed by the catalytic RNA
because the r:ibozyme pe~ se is not consumed during the cleavage reaction. Bovine leukemia virus (BLV), a retrovirus , c:auses perc~istent lymphocytosis and B-lym-phocyte lymphcma in cattle and sheep. A h~mm~rhead ribozyme flanked by antisense sequences directed against regulatory proteins of BLV was shown to inhibit BLV expression in persistently infected cells. This demonstrates the possihility 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 ~ransfer Appro~he~
Somatic gene transfer into farm ~n;m~l S willbecome more significant Ex vivo and more recently in vivo gene therapy has been applied for several genetic diseases in human. Current therapies developed for 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 somatiG
cells promise to be highly efficient. These include viral vectors ~or 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 lS 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 An;m~l s 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 An;mAl Such proteins or peptides having a high and specific antimicrobial activity are divided into two ~amilies, one including the bacteriocins and the other the lanthionines, also called lantibiotics. The appli-cation of biotechnology to An; mA 1 treatment, particu-larly ~arm animals, is opening up new avenues o~ pre-vention and coIltrol that will have important implica-tions. The ba~_teriocins consist of enzymes and other bactericidal proteins. They act as catalysts and are very specific to a single chemical reaction. Bacte-riocins kill t:argeted organisms rapidly by lysing the cell wall, an~ 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 pe]~sist in l;he treated organ longer. One of these well }~nown 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, curre]nt research conducted so far seems to indicate that bacteriocins used for mastitis treatment are non-toxic to other organisms.
Lantib:iotics are peptide-derived antibiotics with high antimicrobial activity against several patho-genic bacteria The ribosomal origin of lantibioticswas first shown by the isolation of the structural gene, epiA, f~r epidermin, a lantibiotic produced by Staphylococcus epideImidis. The general structure of lantibiotic genes is t:he 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 pro:Line 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 r; n; r~ 1 ly 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 manyapplications 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 W O 96/35793 - 11 - PCT/CA~GJ~ 97 human protein such as a cell-surface receptor or cell-adhesion molecule, with antibody constant ~om~;ns.
Such molecules are known as im~Lunoadhesins.
Immunoadhesins can possess many of the desired chemical and biological properties of antibodies.
Examples exist of im~Lunoadhesins that can bind to Fc receptors, mediate antibody-dependent cellular cytotox-icity, and show active transport across the primate placenta. SiIlce the i~nunoadhesin is constructed ~rom a receptor seqllence linked to an appropriate hinge and Fc sequence, the binding specificity of interest can be achieved usinc! entirely human components. Another potential fore!ign sequence is that in the joining region.
One of ,well stu~ied imlnunoadhesins is CD4-IgG
which as been found entirely non-immunogenic in human clinical trials. A second candidate for clinical use is a rumor necrosis 3actor receptor immunoadhesin (THFR-IgG); th:Ls molecu]e 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 ]onger half-life and improved avidity and affinity. Some recepto:cs or immunoinducers that have been joined to the Fc part of IgG to ~orm immunoadhes-ins are reported in the literature: T cell receptor, CD4, l-selectin, CD44, CD28, B7, CTLA-4, CD22;, TNF
receptor, NP 3eceptor, IgE receptor, INF-r 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 A~irl~ls, the advent of molecular biology techniques allow to create an immunoadhesin which could W 096/3S793 - 12 - PCTICA9'1'~297 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 sur~ace 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 TE~E lNVL lON
The present invention relates to the An; m~ l 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 ~n; ~l s~ 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 ~n;m~l, The method involves inducing a liquid complex including a genetic construct into a determined tissue of the ~n;m~l, If desired, the infused genetic construct can be treated with a polycationic compound and/or a lipid to improve the e~iciency with which it is taken up by secretory cells of the animals.
The most costly infectious disease in ~n;mAls is mastitis caused by the in~ection o~ the mAmmA~y gland. Among others, this invention relates to a method of treating mast:itis. More particularly, this invention relates to the use o~ DNA constructs designed to be transcribed in a therapeutic protein a~ter inser-tion into the In~mmAry gLand of both lactating or non-lactating animals.
Bovine/ caprine, ovine and porcine mastitis remain some of the most costly diseases in An i m~ 1 agri-culture. Mastitis represents a signi~icant economic loss to the diary industry, approximately 70 to 80 per-cent of which can be attributed to a decrease in milk production. Mcmy infective agents have been implicated as causes o~ mastitis and these are dealt with sepa-rately as specific entities in cows, sheep, goats and pigs.
Despite signi~icant progress in mastitis con-trol due to widespread adoption of post-milking teat antiseptisis, rnany herds continue to be plagued by this disease. A variety of different procedures have been described and used to cllre mastitis caused by bacteria and yeast. These procedures include the systemic immu-nization of th~e in~ecte~l animals with whole or partial protein extracts of the infective agents in order to stimulate the immune response of the treated An;mAl to these agents. Antibodies generally produced in this way act against a membrane protein, a binding protein or a toxin secreted by t:he microorganisms. Hence these antibodies act as anti-adhesive, anti-toxin, neutraliz-ing or opsonLc molecul~es (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-l~, interleukin 2, interferon-~, tumor necrosis factor-a.
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 treatmentperiod. 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 mA Ary gland, and provides for the elimination, by genetic therapy, of the contAm;n~t-ing microorganisms. In addition, gene therapy of themastitic 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 W 096/3~793 - 15 - PCT/CA96/00297 the art upon considera1:ion of the ~ollowing detailed description o~ pre~erred embodiments exempli~ying the best mode of 1:]~e invention as presently perceived.
The present invention provides a recombinant DNA which comp:rises a nucleotide sequence which encodes a protein or polypeptide which is use~ul in the prophy-laxis or treatment of mastitis, and at least one regu-latory control element which allows ~or expression o~
said nucleotide sequence in a m~mm~ry gland.
Suitable regulatory control elements include transcription and translation regulatory sequences.
Transcription and translation regulatory sequences are those DNA sequences necessary for efficient expression o~ the product:. In general, such regulatory elements can be operab:Ly linked to any nucleotide sequence to control the expression of the sequence, the entire unit being re~erred 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 t:o the cod:ing 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 o~ 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 polypeptidewhich is usel-ul in the prophylaxis or treatment o~
infectious di.seases after insertion into the targetted tissues. Suitably the DNA construct comprises an inducible or c:onstitutive promoter which is linked to ~
coding nucle~tide 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 m~ ry gland tissue with a DNA construct as described above.
The present applicants have found that expres-sion of proteins in m~ ry 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 proteinor polypeptide into m~mm~ry gland tissue would allow, for example, for the elimination of in~ective 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 r~mm~ry 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 ~mm~ry 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.

W 096/35793 - 17 ~ PCT/CA~G~297 The DNA can also be introduced into a m~ ry gland by insertion of other entities which contain DNA.
These entities include minicells, cells (e.g. fibro-blasts, adipocytes, epithelial cells, myoepithelial cells, m~ ry carcinoma cells, kidney cells), liposomes (e.g. natural or synthetic lipid vehicles, cationic lipoc,omes) or other fusible lipid-surfaced bodies. The entities are transformed in vitro prior to insertion Usi~ the abo~Te-described DNA constructs.
Thus t:he invent:ion also provides a cell which has been transformed using a DNA construct as described above. Examp]Les of such cells include Mac-T cells.
Genetically transformed cells of this type are suitable for reimplantation into a mA ~ry gland to produce the desired proteins or polypeptides.
Furthe!rmore the invention provides a liposome which incorporates the above-described DNA construct.
IntrocLuction 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 appropriat:e, the DNA construct is admin-istered in the form of a pharmaceutically or veterinary acceptable contposition 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 e~fective prophylaxis or treatment of mastitis. Such proteins or polypeptides include mucolytic proteins such as enzymes, antibiotics, antibodies, cytokines, tumor necrosLs factors as well as proteins which can induce an immune response to infective or potentially W 096/35793 - 18 ~ PCT/CA96/00297 infective agents and those which activate polymorphonu-clear neutrophils, or macrophages.
In a pre~erred 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 AmmAls or lower milk producers: rabbit, camel and bison. The invention can also be used in humans to eliminate par-ticularly most Staphyl ococci .
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 o~
cationic liposome-DNA complex into the mA =Iry gland.

DE'rATrr~n DESCRIPTION OF THE lWV~ lON
In accordance with one pre~erred 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 An;mAl against the infect-ing or potentially infecting microbial agents.
In accordance with another embodiment of thepresent invention, mastitis gene therapy of A1 S
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 trans~ormed 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 c~ssette" An expression cassette will typically contain, in addition to the coding sequence, a promoter region, a translation initiation site and a translation t~!lmination sequence. Unique endonuclease restriction sit;es may a]so be included at the ends o~
an expression cassette to allow the cassette to be eas-ily inserted or removed when creation DNA constructs.
The expression of a ger.e is primarily directed by its own pro~loter, although other DNA regulatory ele-ments are necessary for efficient expression of a geneproduct. 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) clLirection are given positive numbers.
Promoters can be either constitutive or induc-ible. A constit:utive promoter controls transcription of a gene at a c:onstant rate during the li~e o~ a cell, whereas an in~3Lucible promoter's activity fluctuates as determined by the presence (or absence) of a specific inducer. The regulator~r elements o~ 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 ofinducer, 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 mAmm~ry gland. For example, strong consti-tutive promoters are those controlling the expressionof caseins, lactoglobulins, lacto~errin, 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 An;mAl S, lagomor-phes, rodents, felines or canines. Other constitutive promoters regulating expression of the cytoplasmic ~-actin or ubiquitin genes can be used.
Viral or retroviral promoters can be used also, like Cytomegalovirus (CMV), Simian virus 40 (SV40) or mouse m~~Ary tumor virus (MMTV, which is additionally inducible).
Inducible promoters include any promoter capa-ble of increasing the amount of gene product produced, W 096/35793 - 21 - PCT/CA~G~297 by a given gene in response to exposure to an inducer.
Inducible promoters are known to those ~amiliar 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 regu:Lated by heat shock such as the promoter normally assotiated with the gene encoding the 70 kDa heat shock protein call 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 funrtions well in triggering theexpression of genes in~_luding 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 l~26 bp) na~med the glucocorticoid response element (GRE) and this binding activates the expres-sion of linkecl genes. The glucocorticoid system can be included in the DNA transformation construct as a means to induce pro1:ective or curative molecule's expression.
Once the con:-tructs have been inserted the systemic steroid hormone or glucocorticoid will associate with the constitutively prodwced GR protein to bind to the GRE elements thus stimulating expression of the pro-tective or curative mo]ecule's genes (e.g. antibodiesor 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 o~ the disease.
The trans~ormation o~ 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 ~orms, 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. ~ibroblasts, adipocytes, Mac-T cells, myoepithelial cells, r~mm~ry 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 nnucolysins;

2. Antibo~ies, such as anti-hemolysins, anti-leu-cocidin, anti-protein A, anti-collagen, anti-~ibronectin binding protein, anti-laminim, anti-a-toxin and anti-~-toxin antibodies;
opsonic antibodies and antibodies raised against cell ~us:ion viral protein;

3. Cytokines, interleukines, chemokines, growth factors;

4. Inter~erons;

5. Tumor necrosis ft~ctors; and 6. Immunoadhesins or immunotoxins.
While antibiotics are not very suitable, it can be alternative:Ly used with inducible promoters.
Microorganisms which can be responsible of the mastitis and b~ 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., Cr~ptococcus neoformans, Saccharomyces, and To r~ul opsis spp In sheep: Pac;t eurella haemolytica, Staph. Aureus, Act:inoBacillus lignieresi, E. coli, Str.
- uberis and Str. agalactiae, and Cor. pseu-dot:uberculosis.

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: Corynebacte~ium pseudotuberculosis, Str.
zooepidemicus, and Str. equi.
Other microorganisms and diseases which can be eliminated from exotic animals by the method of gene therapy are those causing:
In primate:Poliomylltis, 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 Z0 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, ophth~l m; a, 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 o~ the process.
The invention concerns equally the cells (e.g.
Mac-T, lung, kidney, muscle cells) genetically trans-~ormed in vitro with tlle gene o~ interest and reim-planted into t:he 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 An; mA l s: ~ovine, 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 scop~e.
EXAMPLE I
Long-term plersistenc~. of plasmid DNA and foreign exp~r,ession in sheep mammary glands MAmmAr-y-gland promoters have been used in transgenic animals to limit transgene expression to the mAmmA~y gland. Gene therapy techniques to target just one organ for introduction of a foreign gene have also been demonstrat:ed. Most: efforts toward postnatal gene therapy have relied on new genetic information into tissues: target cells are removed from the body, in~ected with viral vectors carrying the new genetic information, alld then reimplanted into the body. For some applicati~ns, direct introduction of genes into tissues in vi~, with or without the use of viral vec-tors, would be useful. Direct in vivo gene transfer 3S into postnata] An;m~ls has been achieved with formula-tions of DNA encapsulatled 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., lg86, 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 (Felgner 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 m~mm~ry gland results in significant expres-sion of reporter gene within the gland.
Preparation of plasmid-l;po~ome mixture Plasmid pCR3 (InVitrogen) was used as ~ ian 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 themanufacturer (GibcoBRL). Briefly, 50 ug of pCR3-hGH
suspended in 500 ~1 sterile phosphate buffered saline (PBS), was mixed to 100 ~1 of LipofectAMINE~ also pre-viously diluted into 500 ~1 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 W 096/357~3 - 27 - PCT/CA96100297 complex was infused directly through the udder s skin into the mAmm;lry parenchyma. One ml was injected into the right quarter of two ewes. The milk of the left glands was use~ as negative controls.
- Analysi~ of s~l~ep 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) a~ter ~eter-mining that the milk dicl not affect the accuracy of theassay. Aliquots (100 ul) of milk samples were ana-lyzed.
~UTTS
hGH synthesized by injecting pCR3-hGH into the mAm~Ary glancl was detected all along the lactating period mean:ing 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 oE 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.

EaU~MPLE II

~uman growth hormone (hGH) secretion in goat~' milk after direct transfer of the hGH gene into the mammary gland An alternative route of introducing genes into the mAmm~ry parenchyma is through expansion of gene therapy techniques. In this study two Gibbon ape leu-kemia virus (GaLV) pseudotype retroviral vectors wereused to transfer reporter genes into a goat~s mArm~ry secretory epithelial cells in vi tro and in Vl VO .
Cells and tissue culture MDBKs, a bovine kidney cell line and Mac-T
cells, a bovine m~mmAry epithelial cell line were used.
Retroviral packaging cell lines used (~Cre, 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% C02 /95% air.
E8t~hl;~hment of pro~ce~ cell lines A construct carrying the JR-gal neo- (Wang et al., 1991, Cancer ~es., 51:2642) 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 solutionwas 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 c8s. Packaging cells producing retrovirus cont~;n;ng the hGH gene were selected by G418 resistance (400 ~g/ml).

Virus pro~c~ n~ cells The PGL3/LN c8 clones that yielded the highest levels o~ hGH produced from the target cell lines were chosen ~or the in~usions into a goat's mAmm~ry glands.
Each clone WclS passed three times into 200 lO0 mm-plates. Cellular supernatant was collected over a 3-day period, concentrated, and resuspended in DMDM
with Gentamycin,.
IndLuction of Ce!ll divisi~n andL lactation of goats Two 2-year-old (goats l and 2) and two l-year-old (goats 3 and 4) virc3in Saanen-crossbred goats were treated with exogenous steroids i.m over a 14-day interval to induce mammogenesis and subsequent lacta-tion.
In~usion of vi al stocks into a goat'S mammary glands Polybrene was added to concentrated PGl3/LN c8MFG-hGH viral stock at 80 ~g/ml and loaded into a syr-inge. By using a 22-gauge stub adapter, the retrovi-ruses were infused up the right mA~m~y teat on days 3, 5, 7, 9, ll, and 13 of the hormonal regimen for goats l, 2, and 4 ancl goat 3 received infusions on days 3, 5, 7, 9, lO, ancL 13. The amount of viral solution was different for each animal, ranging from 8 to 20 ml, and was determinecL by the integral capacity of the gland.
The left gland served as the intraanimal control and was infused wLth DMEM contA;n;ng gentamycin. Retrovi-ral stock used for the infusions was then assayed on several cell li,nes.
Analy8is 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 clid not affect the accuracy of the assay.

Aliquots (5 ~1) 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).

ur TS
Vector production Of r~k~ging cell line~
The concentration of hGH in the medium removed from Mac-T and MDBK cells 2 days after infection with retrovirus packaged by PG13/LN c8 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 ma ary 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 ~n; m~ 1 . 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 ~rom day 8 to day 9 where itremained at 2:3 ng/ml unt:il day 16 when it began to fall again. Goat 4, in whic~h prostaglandin E2 was infused at the end o~ the remaining l9-day lactation a~ter a decline on the first 2 days. In addition, goat 4 was still secreting hGH at 'i 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 time!s Thes~ numbers are at the detection level of the a.ssay and correlate with ones measured in two other lac:tating goats that had no exposure to retrovirus. 'rhe 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 secreti.ng 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/Pl~GE of goat's milk sampled throughout the period of coLlection showed no consistent differences in the prote:in profiles from the retroviral-infused right glands, the control left glands, and a goat not exposed to the retrovirus. Protein concentrations measured by BC:A of the milk with hGH were not statisti-cally different from the control milk, thus production of hGH by the ~Amm~ry 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 m~m~ry gland has dra-matically reduced the time of production of pharmaceu-ticals in milk, ~rom 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 lysostArhin on Staphyloao~ c aureus infec-tions on the mouse'~ 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 lars~ quantities o~ the enzyme relatively inexpensively. This may permit its use in vivo in new approaches to the control of staphylococcal mastitis, an economicallv importallt 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 oi lysostaphin against S. aureus ill VlVO.
10Lysostaphin (Sigma Chem.) was dissolved in skimmed milk (Oxoid) to provide a range of concentra-tions between 0.1 and 100 ~g/ml. Controls without lysostaphin were includea. One ml volumes of the con-trols and lysostaphin dilutions were inoculated with 108 colony forming units (cfu) of S. aureus M60. This strain produce~ both ~ and ~ 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 10 reductic,n in viable S. aureus, whereas 10 ug/ml in milk reduce,d S. aureus from a mean of 7.95 log lo/ml in the control to 2.0 loglo/ml. Consequently a dose of 10 ug of lysostaphin was selected for use in vivo.
Anaesthetized mice, of strain MFl, were inoculated in the upper pair of ab~om;n~l mammary glands (designated R4 and L4). E,ight lactating mice were inoculated with 108 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 lysost:aphin 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 lysost~h;n.
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-W 096/357g3 _ 34 _ PCT/CA96/00297 ther experiment using 20 mice a prophylactic use o~
lysostaphin was simulated by in~using 10 ug of lysostaphin intrAmAmm~rily, followed either immediately or after one hour by 103 cfu of S. aureus. Control glands were in~used 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 oflysostaphin was administered either immediately or one hour be~ore S. aureus inoculation, recoveries after 24 hours averaged around 102 viable S. aureus per mAmmAry gland compared with approximately lOg per mA~ry gland ~or 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.

W 096/3~793 35 PCT/CA9~'~C297 Conclu8ion ~ hese experiments clearly demonstrate the anti-staphylococca] activity o~ lysostaphin in vivo. Both a therapeutic arld prophylactic potential were demon-strated. The cloning of the lysostaphin gene may makeit readily available for therapeutic use at a competi-tive price ancL 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 o~ the lysostaphin gene in the lactating mAmm~ry gland c:ould potentially increase the resistance of the animal t:o staphylococcal mastitis.

E~L~MP~E IV
Lysost~h;n ~efficacy for treatment of StaphylG~ us au~eus intramammary infection Cloned-derived lysostaphin was evaluated as to its bactericiclal effect on S. aureus intramammary infections. ',. 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, alld 0 ~g in 0/3. Glands infected with S.
aureus at 48 h,ours post-challenge in untreated guinea pigs persistecl, however, 3/25 control glands of treated guinea pigs cleared in response to treatment of the adjacent glancL

W 096t3S793 - 36 - PCT/CA96100297 Somatic cell/ml in guinea pig shifted from 104 pre-infected glands to cell counts greater than 3 x 106 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 intr~rAmm~ry 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 o$ a recombinant bacterial enzyme (Lysost ~rh ~ n ) as a mastitis the ~euLic 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 ~taphylococcus aureus colony-forming units 3 weeks prior to subsequent treatment. Infected ~n;~l S were injected through the teat canal with a single dose of recombinant lysostaphin (rLYS) (dose 1 WO 96/35793 _ 37 _ PCT/CA96/00297 to 500 mg) or after three successi~e p.m. milking with lO0 mg o~ rLYS in 60 m] of sterile phosphate-bu~fered saline. ~;r~,~l S were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings aft:er the last treatment.

RESULTS
Kinetic analysis o~ immunologically active rLYS
demonstrated t:hat a minimum bactericidal concentration was maintainedL in the milk for up to 72 hours at 37~C.
In contrast, penicillin ~ 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 orcler to de1;ermine the optimal effective dose to elicit long-terim cures, a titration was per-formed in which a single dose of rLYS at concentrations of 0, l, lO, lO0, or 500 mg was A~m;n;stered.
Untreated quarters and the l-mg treatment failed to clear all quarters of S. aureus. The lO- lO0- 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 lOO mg dose and one with the 500 mg dose.
Because rLYS maintaills a minimal bactericidal concentration (MBC) for approximately 24 h and the experimental infections undergo a 2- to 4- days cycling, multiple infusions of lO0 mg of rLYS over three consecutive milkin~3 were determined to be optimal to maintain a minimal eEfective dose for 3 to 5 days and to elicit cures.

Conclu~ion Staphylococcus aureus is one of the primary etiologic agents of bovine mastitis and a major cause o~ economic loss to the dairy industry An e~ective 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 in~ected animals, treatment only during the dry period has been the adopted herd manage-ment practice o~ choice. Neither approach addressesthe 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 use~ul thera-peutic to the veterinarian. If rLYS was as ef~icacious 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 ~or 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
Expres8ion 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 lactating m~m~ ry 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 cyto]~egalovirus immediate early gene l pro-moter/enhancer region (CMV) Expression from the nakedplasmid DNA je1_-injector into lactating r~mm~ry glands of sheep was sufficient to be detected by Northern blot analysis when tissue WclS obtained 48 hours a~ter in vivo transfection. In conclusion, the ability to tran-siently transfect lactating ~mm~ry tissue in vivo cir-cumvents the difficulties encountered with in vivo cul-ture techniques and provides a method for eXAmining m~mm~ry regulat:ory elements and testing of fusion gene constructs de~igned ~or the production of transgenic animal bioreactors.
EXAMPLE VII
Elimination cf. Staphylo~o~c aureus in an eukaryotic syst:em expressing the lysost~h; n The lyc:ostaphin gene was introduced into 293 cells (human E~etal kidn~y cells) maintained in vi tro.
The recombinaIlt bacteriocin, the lysostaphin, was secreted in the medium culture and was found to kill cont~m;nA~t S. aureus during the challenge.
The lysostaphin gene was obtained by PCR ampli-fication from extracted DNA of Staphyloeoeeus simulans biovar stap.hylolytiells (NRRL B-2628), and Staphyloeoeeuc: aureus s~rain Newbould (ATCC) was used for the challenge in transfected eukaryotic cells.
Staphylococcal strains were grown in Brain Heart Infusion (BHI) medium.
Purification o~. the lysost~hi n gene Staphy.70coecus simulans biovar staphylolytieus was cultured overnight in a stirring incubator at 37~C.
The media was centrifuged, and the pellet was resus-W 096/35793 40 PCTICA~ 297 pended in 5 ml of 50 mM EDTA-50mM Tris-HCL (pH 7.8) containing 50 mg of lysostaphin (Sigma) ml~l 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 N0:2). 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 se~uence, 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 strainDH5a, 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 cont~;n;ng 5% CO2) to yield 50 to 60%

W 096~5793 - 41 - PCT/CA96/00297 introduced into the cells by the calcium phosphate method with the following modifications. The precipi-tate containing 7.5 ~g o~ DNA was added to 2 ml o~ cul-ture medium. A~ter 24 h, the medium was replaced with 2 ml of medium per well, and samples of the medium were harvested at each 24 h i-ollowing transfection to evalu-ate the produ~c:tion o~ t:he lysostaphin by Western blot analysis and ELISA
A8say for bio:LlDgical act:ive lyso8tArh; n The we:Lls containing the trans~ected 293 cells were infected with 102 or 103 of Staphylococcus aureus Newbould. Sarnples of lOO~l of the infected medium were spreaded on s~heep blood agar. A~ter 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 lysost~?hin on the growth o~ the bacteria.
RESULTS
Production of recombina~t ly~ost~rh; n by transfected eukaryotic cel~Ls The moclified lysost~rh;n 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 lysost~rh;n has been carried out in eukaryotic cells. The ELISA assays have revealed that 1:he recombinant lysostaphin was produced in concentrations of lO0 to 250 ng/ml/24h depending of ~ the clone.

W 096/35793 - 42 - PCT/CA~G~0297 Activity of t~e lysos~rh;n secreted by mammalian cells The activity o~ the recombinant lysostaphin secreted by transfected m~mm~l ian cells has been observed by its efficiency to reduce or in some repli- r 5 cates to inhibit the growth o~ in~ecting Staphyl ococcus aureus in the culture media. Samples o~ media taken from non-transfected cells have shown none inhibitory e~ect on the development of the bacteria present in the wells. The plates of agar were completely con~lu-10 ent after overnight incubation. In contrast, when an initial amount of 103 bacteria was cultured in presence of trans~ected eukaryotic cells, very few CFU were counted on the plates. Less than 100 CFU were observed in our assays when 103 bacteria were used, while we did 15 not observed the presence of CFU on gels when 102 bac-teria were added to the wells cont~ining the trans-fected cells.
While the invention has been described in con-nection with specific embodiments thereof, it will be 20 understood that it is capable o~ ~urther 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 25 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 ~ollows in the scope of the appended claims.

CA 02220472 l997-ll-26 SEQUENCE LISTING

(1) GENER~L INFOF~TION:
(i) APPLICANT:
(A) NA~E,: IMMUNOVA
(B) STPE'ET: 2750 rue Einstein, Bureau 110 (C) CII'Y: Sainte-Foy (D) STP.I'E: Quebec (E) C~UN1'KY: Canada (F) POSIAL CODE (ZIP): GlP 4Rl (G) TELE,PHONE: (418) 654-2240 (H) TELEiFAX: (418) 654-2125 (A) NAM~: GAGNE, Marc (B) STREET: 913 rue Pellan (C) CITY: St-Jean-Chrysostome (D) STA.TE: Quebec (E) COUN'LKY: Canada (F) POSTAL CODE (ZIP): G6Z 2S8 (ii) TITLE OF INVENTION: iWIMAL GENE THER~PY
(iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPER~TING 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) FILI:NG DATE: 10 MAY-1995 (2) INFORMATION F3R SEQ ID NO~ 1:
(i) S~u~ CHARACTERIS'.CICS:
(A) LENG'rH: 21 base pairs (B) TYP:E: nucleic acid (C) sTR~Nn~nN~s: single (D) TOPO:LOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETII~AL: NO
(xi) SE~u~N~ :DESCRIPTION~ SEQ ID NO: 1:
TTAAGGTTGA AGA~9A~9AT T 21 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN~;'rH: 23 base pairs (B) TYPE: nucleic acid W 096/35793 _ 44 _ PCT/CA96/00297 (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Claims (15)

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 5' expression regulation DNA sequence and a secretory DNA sequence encoding a secretory signal sequence operatively linked to a DNA sequence encoding for a therapeutic protein, peptide or antisense RNA selected from the group consisting of bacteriocins, lanthionins, lactoferrin and lysosyme, wherein said expression regulation DNA sequence and said secretory DNA sequence are 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 in targeted tissue of the animal the DNA expression system of step a) for in situ expression of said therapeutic protein, peptide or antisense RNA.

2. The method of claim 1, wherein said DNA expression system is transgenic recombinant animal cells.

3. The method of claim 2, wherein said cells are selected from the group consisting of epithelial mammary gland cells, blood cells, lymphocyted, leukocytes, T-lymphocytes, B-lymphocytes, erythrocytes, muscle cells, hepatic cells, kidney cells, lung cells, secretory cells and non-secretory cells.

4. The method of claim 3, wherein said DNA expression system is selected from the group consisting of a mary glar d ce~ s, blood ~ells, lymphocytes, leuko-~ytes, T-lympilocytes, B-lymph~cytes, erythracy~es, muscle cells, hepatlc cells, kidney cells, lur.Lg cells, secr~tory cells and non-s~cretory cells.

4 'nhe m~.hod o~ claim 3, wherein said D~ expres-sion ~y3tem is selected ~om the group consistillg o~ a lipidic liposome, a cationic liposome, an anionic liposome.

5. The method of claim 3, wherein said vector is a viral vector or a retroviral vector.

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

7. The method of claim 6, wherein said bacteriocins 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.

8. The method of claim 6, wherein said therapeutic protein, peptide or antisense RNA is selected from the group consisting of immunoglobulins, lactoglobulins, .alpha.-lactalbumin, bile-salt-stimulated lipase or ribosyme, cytokines, chemokines, growth factors and immunomodulators.

9. The method of claim 2, which further comprises a 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.

10. A non-human genetically treated animal for the production of a recombinant protein, peptide or antisense RNA systemically or in targeted tissue, which comprises a DNA expression system introduced in targeted tissue of the animal and which comprises at least a 5' expression regulation DNA sequence and a secretory DNA sequence encoding a secretory signal sequence operatively linked to a DNA sequence encoding for a therapeutic protein, peptide or antisense RNA
selected from the group consisting of bacteriocins, lanthionins, lactoferrin and lysosyme.

11. The non-human genetically treated of claim 10, wherein said expression regulation DNA sequence is selected from the group consisting of a constitutive promoter, an inductible promoyer, a cytomegalo virus promoter.

12. The non-human genetically treated animal of claim 11, wherein said promoter is selected from the group of DNA sequence encoding lactoferrin, serum albumin, .alpha.S1-casein, .alpha.S2-casein, .beta.-casein, ~-casein,.alpha.
-lactalbumin, whey acidic protein, .beta.-lactoglobulin, cytokines, chemokines and growth factors.

13. The non-huma genetically treated animal of claim 10, wherein said secretory signal sequence is selected frcm the group consisting of DNA sequences encoding lactoferrin, serum albumin, .alpha.S1-casein, .alpha.
S2-casein, .beta.-casein, ~-casein, .alpha.-lactalbumin, .beta.
-lactoglobulin, cytokines, chemokines or growth factors.

14. The non-human genetically treated animal of claim 10, wherein the expression regulatian and secretory signal sequences are from human, bovine, caprine, ovine, feline, canine, lagomorphes, birds and fishes.

15. The non-human genetically treated animal of claim 11, wherein the promoter is tissue-specific for expression in targeted tissue.