Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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~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-
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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-
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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
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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
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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
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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
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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
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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
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~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
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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
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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
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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
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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-
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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
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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
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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.
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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
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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
CA 02220472 1997-11-26
W O 96/35793 19 PCT/CA96/00297
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-
CA 02220472 1997-11-26
W 096/3S793 20 PCT/CA96/00297
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,
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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
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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:
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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.
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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
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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
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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
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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.
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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).
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W096/35793 29 PCT/CA96100297
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.
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W096/35793 30 PCT/CA96/00297
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
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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
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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
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W 096/35793 33 PCT/CA96/00297
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-
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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.
CA 02220472 1997-11-26
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
CA 02220472 1997-11-26
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
CA 02220472 1997-11-26
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.
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W 096/35793 - 38 - PCT/CA96/00297
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
CA 02220472 1997-11-26
W 096/35793 39 PCT/CA96/00297
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-
CA 02220472 1997-11-26
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%
CA 02220472 1997-11-26
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.
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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
W O 96/35793 PCTICA96/00297
- 43 -
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
CA 02220472 1997-11-26
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:
GCGCTCACTT TATAGTTCCC CAA 23
