Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTICALLY ACTIVE PROTEINS IN PLANTS
The present invention generally relates to transgenic plants expressing
therapeutically
active proteins. In particular, the present invention relates to plants
expressing
therapeutically active proteins in subcellular organelles, preferably in
vacuoles or more
preferably in plant plastids. The present invention also relates to the
therapeutic uses of
such transgenic plants.
A wide variety of diseases afflict animals, including humans, pets and
livestock. These
diseases not only cause great suffering, but they also translate into major
economic losses.
Sick workers are unable to work or have a reduced performance, and
agricultural yields can
be dramatically decreased by diseases affecting farm animals. Every human
civilization has
reacted to diseases with its own medications and although many diseases have
been and
are still combated successfully by current medicines, many other diseases are
still resisting
treatments or can be only partially treated.
Among the problems impacting the treatment of diseases are the costs of a
large number of
medications or their limited supply. Thus, in some cases, only a small number
of patients
can be efficiently treated, particularly in developing countries, and
expensive treatments are
a heavy burden on the healthcare systems throughout the world. Moreover, in
the
agricultural field, some diseases resulting in lower yields are inadequately
treated because
the costs greatly exceed the benefits gained by an efficient treatment. An
important
contribution to the high cost of some medications is the lack of economic
production
methods, particularly for protein or peptide based medications. Another
sometimes
overlooked but crucial problem of some current medicines resides in the
administration of
therapeutically effective amounts of the medicine to the desired host's organs
or body parts.
Indeed, in some cases, currently available delivery methods are not completely
satisfactory,
as for example, for the treatment of allergies or autoimmune diseases. An
additional
problem connected with medications is their transport, particularly in hot
climates, which
requires expensive refrigeration and expensive formulations, thus further
increasing costs.
The availability of a wide array of medicines without having to incur
expensive
transportation or administration costs would be of great advantage.
Plants are attractive candidates for the expression of therapeutically active
proteins
because they may be edible and have high biomass yields. Expression of
therapeutically
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active proteins in cellular compartments sequestered away from cytoplasmic
proteases is a
particularly appealing way for producing therapeutically active compounds in
plants.
Overall, there is therefore a constant and unfulfilled need for novel
medications, available in
large and inexpensive supply, which can be administered to a patient or host
through a
wide variety of methods and which are easily accessible throughout the world.
The present invention addresses the need for large and inexpensive supplies of
medications, in particular protein-based medications, for the prevention or
treatment of
diseases. Accordingly, the present invention provides transgenic plants
capable of
expressing therapeutically active proteins, in particular transgenic plants
capable of
expressing therapeutically active proteins in subcellular organelles,
preferably in vacuoles or
more preferably in plant plastids. The plants of the present invention are
particularly useful
in the context of the suppression or reduction of an undesired immune
response. Proteins
expressed in transgenic plants can be administered to a host by a wide range
of delivery
methods, preferably orally. For example, in some cases and as described infra,
plants or
plant material containing therapeutically active proteins can be ingested
orally without prior
extraction or purification, or with minor extraction or purification. Such
prevention or
treatment of diseases through the diet is convenient and reduces the cost of a
medication
considerably. Alternatively, the proteins expressed in transgenic plants can
be extracted
and administered using methods well known in the medical arts.
In particular, the present invention relates to transgenic plants expressing
therapeutically
active proteins from their plastid genome. Expression levels in plastids
according to the
present invention regularly exceed those of nuclear expressed transgenes. Such
high levels
of expression result in high concentrations of protein per gram of plant
tissue thereby
fulfilling a long-felt but heretofore unfulfilled need by allowing the use of
plants or plant
material derived from such plants in a wide range of therapeutic applications.
Furthermore,
transgene expression levels are stable over time due to the absence of gene
silencing, and
position effect variation is not an issue because of the insertion of the
transgene at a
precise, targeted position on the plastid genome by homologous recombination
Also,
proteins expressed within plant plastids remain sequestered in the organelle
and are thus
conveniently protected from degradation, in particular from degradation in the
digestive
system when the plant is ingested orally. Sequestration in the organelle also
prevents
plastid-expressed transgenes from interacting with the cytoplasmic
environment. This
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feature is also essential if the protein expressed in plants also shows
activity against some
components of the plant. Also, inducible plastid expression systems are
available (see WO
98/11235), allowing the restriction of the expression of the transgene to a
desired time-point
and thus limiting potentially detrimental effects of high transgene expression
levels over
long periods of time. Similarly, proteins expressed from the nuclear genome
which are
targeted to other subcellular compartments such as the vacuole are also
protected from
degradation or from adverse interactions with the cytoplasmic environment.
The present invention also provides therapeutic compositions comprising plant
matter
derived from such transgenic plants, novel methods for expressing
therapeutically active
proteins and novel methods for improving the condition of a wide range of
hosts including
human, livestock, pets and other animals. Methods for administering plant-
derived
therapeutic agents are also provided.
The invention therefore provides:
A plant comprising in its plastid genome at least one DNA molecule encoding at
least one
protein that is therapeutically active when administered to a host, preferably
a host in need
thereof, in a therapeutically effective amount, wherein said plant is capable
of expressing
said protein or proteins. In a preferred embodiment, the therapeutically
active protein
accumulates in the plastids of said plant. In another preferred embodiment,
the protein is
expressed in edible parts of the plants. In another preferred embodiment, the
protein is
administered orally to the host. In another preferred embodiment, the host is
a vertebrate,
preferably a mammal, more preferably human, bovine, ovine, porcine, canine or
feline. In a
further preferred embodiment, the therapeutically active protein is an
antigen, preferably an
immunologically active antigen. Accordingly, the present invention provides a
plant
comprising in its plastid genome a DNA molecule encoding an antigen,
preferably an
antigen that is immunologically active, wherein the plant is capable of
expressing the
antigen. Preferably, the antigen is expressed in the plant and, more
preferably, an immune
response to the antigen is reduced after ingestion by the host of the plant.
In another preferred embodiment, the antigen is capable of suppressing or
reducing the
immune response of the animal to the antigen, preferably by inducing tolerance
of the host
to the antigen, such as e.g. by contact or uptake by the gut mucosa. A
preferred antigen is
an allergen, preferably an airborne allergen, preferably a pollen allergen.
Examples of
preferred allergens are Der f I, Der f II, Der p I and Der p II, Can f II, Lol
p V, Sor h I, Amb a
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!, preferably Amb a l, l, Amb a II, Aln g I, Cor a I, Bet v I, Fel d 1, or
rAed a 1 and rAed a 2.
For example, an allergen expressed in a plant of the present invention is not
glycosylated.
Another preferred antigen is an autoantigen, such as collagen, preferably type
I or type III
collagen, type II collagen, myelin basic protein, myelin proteolipid protein,
interphotoreceptor
binding protein, acetylcholine receptor, an S-antigen, insulin, glutamic acid
dehydrogenase,
an islet cell-specific antigen or thyroglobulin, or a transplantation antigen,
such as an alto- or
xeno-transplantation antigen, for example a MHC protein, preferably a MHC
class II protein,
preferably an a or a b chain. In an alternate preferred embodiment, an
immunologically
active antigen is capable of inducing immunization of the animal against the
antigen. In yet
another preferred embodiment, the therapeutically active protein is a blood
protein, a
hormone, a growth factor, a cytokine, an enzyme, a receptor, a binding
protein, an immune
system protein, a translation or transcription factor, an oncoprotein or proto-
oncoprotein, a
milk protein, a muscle protein, a myeloprotein, a neuroactive peptide or a
tumor growth
suppressing protein or peptide, for example angiostatin or endostatin, both of
with inhibit
angiogenesis. In another preferred embodiment of the invention, the
therapeutically active
protein is an anti-sepsis peptide, such as BPI (bactericidal permeability-
increasing protein).
In yet another embodiment, the immune system protein is an antibody. The DNA
molecule
according to the invention is operably linked to a promoter capable of
expressing said DNA
molecule in the plastids of said plant. In a preferred embodiment the promoter
is a clpP
promoter, in another preferred embodiment a 16S r-RNA gene promoter. In a
particularly
preferred embodiment of the invention, said promoter is a transactivator-
mediated promoter,
particularly a T7 gene 10 promoter. In addition, the invention provides a
plant further
comprising a heterologous nuclear expression cassette comprising a DNA
sequence
encoding a transactivator. In a preferred embodiment the transactivator is a
T7 polymerase.
The invention further provides a plant comprising in its nuclear genome a DNA
molecule
encoding a protein that is therapeutically active when administered to a host
in need thereof
in a therapeutically effective amount, wherein said therapeutically active
protein is selected
from the group consisting of the mosquito allergens rAed a 1 and rAed a 2,
bactericidal
permeability-increasing protein (BPI) and the pollen allergens Amb a I, Amb a
L 1, Amb a ll,
Amb t V, Aln g I, Cor a I, Lol p V, Sor h and Bet v I.
The invention also provides a plant comprising in its nuclear genome a DNA
molecule
encoding a protein that is therapeutically active when administered to a host
in need thereof
in a therapeutically effective amount, wherein said therapeutically active
protein is targeted
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to a subcellular organelle of said plant. In a preferred embodiment, the
therapeutically
active protein is targeted to the vacuole. Said therapeutically active protein
which is targeted
to the vacuole preferably is selected from a group consisting of allergens
such as Der f I,
Der f II, Der p I and Der p ll, Can f ll, Lol p V, Sor h I, Amb a I,
preferably Amb a 1.1, Amb a
ll, Amb t V, Aln g I, Cor a I, Bet v l, Fel d I, or rAed a 1 and rAed a 2,
autoantigens such as
collagen, preferably type I or type III collagen, type II collagen, myelin
basic protein, myelin
proteolipid protein, interphotoreceptor binding protein, acetylcholine
receptor, an S-antigen,
insulin, glutamic acid dehydrogenase, an islet cell-specific antigen or
thyroglobulin, or a
transplantation antigen, such as an allo- or xeno-transplantation antigen, for
example a
MHC protein, preferably a MHC class II protein, preferably an a or a b chain,
or from the
group consisting of a blood protein, a hormone, a growth factor, a cytokine,
an enzyme, a
receptor, a binding protein, an immune system protein, a translation or
transcription factor,
an oncoprotein or proto-oncoprotein, a milk protein, a muscle protein, a
myeloprotein, a
neuroactive peptide or a tumor growth suppressing protein or peptide, for
example
angiostatin or endostatin, both of with inhibit angiogenesis. In another
preferred
embodiment the therapeutically active protein targeted to the vacuole is an
anti-sepsis
peptide, such as BPI (bactericidal permeability-increasing protein).
In a further preferred embodiment, the plant according to the invention is an
edible plant. In
another preferred embodiment the plant is a dicotyledonous plant, preferably
tobacco,
tomato, soybean or spinach. In an alternate embodiment, the plant is a
monocotyledonous
plant, preferably maize or rice.
In a further preferred embodiment, the expression of the protein in the plant
is regulatable,
preferably chemically regulatable. Alternatively, the expression of the
protein is constitutive,
tissue specific or developmentally regulated.
This also includes the seed for such a plant, which seed is optionally treated
(e.g., primed or
coated) and/or packaged, e.g. placed in a bag or other container with
instructions for use.
The host according to the invention is a vertebrate, particularly a mammal,
more particularly
a human, bovine, ovine, porcine, canine or feline.
The invention further provides:
A composition comprising a plant according to the invention or plant matter
derived from
said plant, wherein said composition comprises a therapeutically effective
amount of said
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protein and a composition wherein said plant is processed prior to being
administered to
said host.
The invention further provides methods wherein:
- a host in need thereof is being administered a composition according to the
invention in
an amount effective to improve the condition of said host
- said composition is administered orally to said host
said protein is an antigen, whereby an immune response of said host against
said
antigen is suppressed or reduced
- said antigen particularly is an allergen, an autoantigen or a
transplantation antigen
The invention also provides a method of treating or preventing a disease,
comprising
administering to an host in need thereof a therapeutically effective amount of
a plant
according to the invention or plant matter derived from said plant.
In a specific embodiment said disease is an allergy, an autoimmune disease or
the rejection
of a transplantation. In yet another specific embodiment said therapeutically
effective
amount is administered orally to said host.
The invention further provides:
A plant of the present invention for use as a pharmaceutical, preferably to
treat or prevent
diseases, e.g. allergies, autoimmune diseases or transplantations, for example
by induction
of tolerance, e.g. oral tolerance, in a patient in need thereof, or for the
immunization of a
host.
The invention further provides:
A plant of the present invention for use as a medical food, preferably to
treat or prevent
diseases, e.g. allergies, autoimmune diseases or transplantations, for example
by induction
of tolerance, e.g. oral tolerance, in a patient in need thereof, or for the
immunization of a
host.
The invention further provides:
A plastid transformation vector comprising a DNA molecule encoding a protein
that is
therapeutically active when administered to a host in need thereof in a
therapeutically
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effective amount, wherein said DNA molecule is operably linked to a promoter
capable of
directing the expression of said DNA molecule in a plant plastid. In a
preferred embodiment,
the protein accumulates in the plastids of said plant. In another preferred
embodiment, a
promoter in the plastid transformation vector is a clpP promoter, a 16S r-RNA
gene
promoter, a psbA promoter, a rbcL promoter or a transactivator-mediated
promoter
regulated by a nuclear transactivator (e.g., the T7 gene 10 promoter when the
transactivator
is T7).
A plastid comprising a transformation vector as described above.
A plant cell comprising a plastid as described above, wherein said plant cell
is capable of
producing said protein.
The invention furthermore provides:
A plant comprising
a heterologous nuclear expression cassette comprising a promoter, e.g., an
inducible
promoter, e.g., a wound-inducible or chemically-inducible promoter, for
example the tobacco
PR-1a promoter, or a tissue- or organ-specific promoter or a developmentally
regulated
promoter, operably linked to a DNA sequence coding for a transactivator
(preferably a
transactivator not naturally occurring in plants, preferably a RNA polymerase
or DNA
binding protein, e.g., T7 RNA polymerase), said transactivator being
optionally fused to a
plastid targeting sequence, e.g., a chloroplast targeting sequence (e.g., a
plant expressible
expression cassette); and
a heterologous plastid expression cassette comprising a transactivator-
mediated promoter
regulated by the transactivator (e.g., the T7 gene 10 promoter when the
transactivator is T7
RNA polymerase) and operably linked to a DNA molecule encoding a
therapeutically active
protein of the present invention;
also including the seed for such a plant, which seed is optionally treated
(e.g., primed or
coated) and/or packaged, e.g. placed in a bag or other container with
instructions for use.
The invention further provides:
A composition comprising a plant or plant matter as described above, wherein
said
composition comprises an amount of said protein that is therapeutically
effective when
administered to a host in need thereof. In a preferred embodiment, the plant
matter is
processed prior to being administered to the host. The processing is
preferably a type of
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processing routinely used in the food or feed industry. In another preferred
embodiment, a
composition of the present invention comprises an amount of an antigen that is
immunologically effective.
The invention further provides:
Pharmaceutical compositions comprising a plant or plant matter of the present
invention
and medical food compositions comprising a plant or plant matter of the
present invention.
The invention also further provides:
A method comprising transforming the plastid genome of a plant with a
transformation
vector as described above, preferably further comprising expressing a
therapeutically active
protein in said plant. In a preferred embodiment, the therapeutically active
protein is an
antigen, preferably an immunologically active antigen.
The invention also further provides:
A method comprising administering to a host in need thereof a composition as
described
above in an amount effective to improve the condition of the host. Preferably,
the method
comprises oral administration of the composition to the host.
The invention further provides:
A method of treating or preventing a disease, e.g. allergies, autoimmune
diseases or
rejections of transplantations, for example by induction of tolerance in a
host in need
thereof, or for the immunization of a host, comprising administering a
therapeutically
effective amount of a plant of the present invention or plant matter derived
thereof to the
host.
The present invention further provides:
The use of a plant of the present invention in the manufacture of a medication
for the
treatment or prevention of diseases, e.g. for the treatment of allergies,
autoimmune
diseases or transplantations, for example by induction of tolerance in a host
in need
thereof, or for the immunization of a host.
The present invention further provides:
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The use of a plant of the present invention in the manufacture of a medical
food for the
treatment or prevention of diseases, e.g. for the treatment of allergies,
autoimmune
diseases or transplantations, for example by induction of tolerance in a host
in need
thereof, or for the immunization of a host.
The present invention further provides:
The use of a plant of the present invention for the production of an antigen
for
determination of immunological activity.
The present invention further provides:
An antibody specific for an antigen expressed in a plant of the present
invention.
An antibody that interferes with the binding of an antibody specific for an
antigen expressed
in a plant of the present invention with the expressed antigen.
The present invention further provides:
A food product comprising an edible portion of a plant comprising a DNA
molecule
according to the invention, wherein said food product is therapeutically
active when
administered to a host in need thereof in a therapeutically effective amount.
The present invention further provides:
An agricultural product derived from a plant or plant part comprising a DNA
molecule
according to the invention, wherein said agricultural product is
therapeutically active when
administered to a host in need thereof in a therapeutically effective amount.
The present invention also further provides:
All novel products, processes, and utilities as described herein.
DEFINITIONS
In a broad sense, a "therapeutically active protein" contributes to the
condition of a host in a
positive manner when administered to the host in a therapeutically effective
amount. A
therapeutically active protein has healing, curative or palliative properties
against a disease
and may be administered to ameliorate, relieve, alleviate, reverse, or lessen
the severity of
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the disease. A "therapeutically active protein" also has prophylactic
properties and is used
to prevent the onset of a disease or to lessen the severity of such disease or
pathological
condition when it does emerge. The term "therapeutically active protein"
comprises an entire
protein or peptide, or therapeutically active fragments thereof. It also
comprises
therapeutically active analogs of the protein or peptide, or analogs of
fragments of the
protein or peptide. The term "therapeutically active protein" also refers to a
plurality of
proteins or peptides that act cooperatively or synergistically to provide a
therapeutic benefit.
An "analog" of a therapeutically active protein includes proteins that are so
structurally
related to the protein that they possess the same biological activity as the
protein.
An "immune response" is referred to as the physiological responses stemming
from the
activation of the immune system by antigens. In the present invention, the
immune
response may be suppressed through the induction of tolerance based on
exposure to the
antigen, particularly when the antigen is orally administered.
"Immunologically active" means herein capable of modulating the immune system,
for
example by stimulating an immune response or by suppressing or reducing an
immune
response or an inflammatory condition.
An "antigen" is a substance which interacts with the immune system, preferably
with
products of specific humoral or cellular immunity to stimulate an "immune
response". An
antigen is preferably a polypeptide and is a "therapeutically active protein".
An antigen
comprises the polypeptide in its entirety or a portion of the polypeptide.
Such portion of the
polypeptide is for example an epitope or an antigenic determinant of the
antigen. An
antigen may comprise one or more than one epitopes or antigenic determinants.
An antigen
also comprises "analogs" of the antigen including molecules that are so
structurally related
to the antigen that they possess the same biological activity as the antigen,
i.e. the same
immunological activity. In the context of the present invention, an antigen
encompasses e.g.
allergens, autoantigens and transplantation antigens.
An "epitope" is a portion of an antigen that determines its capacity to
combine with the
specific combining site of a corresponding antibody in a antigen-antibody
interaction.
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An "antigenic determinant" is the portion of an antigen that determine the
specificity of the
immune response stimulated by the antigen.
An "adjuvant" is a substance, preferably oily, which, when mixed and
administered with an
antigen, in particular when mixed and injected with an antigen,
nonspecifically enhances an
immune response to the antigen. A typical adjuvant is the complete Freund's
adjuvant
(CFA) or the incomplete Freund's adjuvant (Lando et al. (1981 ) J.P. immunol.
126: 1526}.
An "autoantigen" is any substance normally found within an animal that, in an
abnormal
situation such as an autoimmune disease, is no longer recognized as part of
the animal
itself by the immune system of that animal, and is therefore attacked by the
immune system
as though it were a foreign substance.
An "allergen" is an antigen that induces an allergic reaction of a host.
"Immunization" of a host against a pathogen or a toxin is one aspect of
stimulating an
immune response and refers herein to as a protection of the host against the
pathogen or
toxin by an induction of the immune system. Preferably both an immediate
immune
response and an immunological memory are induced, preferably providing for
immediate
and a long-term protection of the host. An antigen of the host of the toxin
are used for the
immunization. A vaccination is also encompassed by the term immunization.
"Administration" of therapeutically active protein to a host in need thereof
is intended as
providing the therapeutically active protein to such host in a manner which
retains the
therapeutic effectiveness of such protein for a length of time sufficient to
provide a desired
beneficial effect to such host.
"Oral administration" of a therapeutically active protein means primarily
administration by
way of the mouth, preferably by eating, but also intends to include any
administration which
provides such proteins to the host's stomach or digestive tract. In a
preferred embodiment,
oral administration results in contact of the therapeutically active protein
with the gut
mucosa.
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A "host" is an animal to whom a therapeutically active protein of the present
invention is
administered. The term "animal" covers all life forms that have an immune
system, including
humans, bovines, ovines, porcines, canines or felines.
"Food" or "food product" means herein liquid or solid food or foodstuff or
feedstuff and is a
plant, plant part or plant matter derived thereof which is ingested by humans
and other
animals. This term is intended to include raw plants or plant material which
may be fed
directly to humans and other animals or any processed plant matter together
with a
nutritional carrier which is fed to humans and other animals. Materials
obtained from a plant
are intended to include any component of a plant which is eventually ingested
by a human
or other animal.
A "medical food" comprises a composition that is eaten or drunk by a host and
has a
therapeutic effect on the host. A medical food comprises for example a plant
of the present
invention or plant matter derived thereof. Medical food may be ingested alone
or may be
administered in combination with a pharmaceutical composition well-known in
the medical
arts. A medical food also comprises the equivalent feed-stuff for non-human
animals.
"Agricultural product" as used herein refers to a plant that is eaten as a
whole such as
alfalfa sprouts, radish sprouts, wheat sprouts and the like or to an edible
portion of a plant
which is consumed by humans in either raw or cooked form. The edible portion
may be a
root, such as rutabaga, beet, carrot, and sweet potato; a tuber or storage
stem, such as
potato, Jerusalem artichoke and taro; the stem, as in asparagus and kohlrabi;
a bud, such
as Brussels sprouts; a bulb, such as onion and garlic; a petiole or leafstalk,
such as celery
and rhubarb; a leaf, such as cabbage, lettuce, parsley and spinach; an
immature flower,
such as cauliflower, broccoli and artichoke; a seed; the immature fruit, such
as eggplant,
cucumber, and sweet corn (maize); or the mature fruit, such as tomato, pepper,
apple, pear,
banana, orange, berries and the like.
A "plant" refers to any plant particularly to seed plants.
"Plant cell" refers to the structural and physiological unit of the plant,
comprising a protoplast
and a cell wall. The plant cell may be in form of an isolated single cell or a
cultured cell, or
as a part of higher organized unit such as, for example, a plant tissue, or a
plant organ.
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"Plant material" refers to leaves, stems, roots, seeds, flowers or flower
parts, fruits, pollen,
pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds,
cuttings, cell or
tissue cultures, or any other part or product of a plant.
"Plant matte" refers to any part of a plant at any stage of development,
preferably such
parts that can be administered orally. Plant matter includes edible parts of a
plant, such as
leaves, seeds, fruits, tubers, or other plant parts that can be ingested raw
or unprocessed.
Plant matter also includes isolated fractions of the plants, such as
subcellular organelles,
e.g. plastids or vacuoles. Plant matter also includes parts of a plant that
have been
subjected to various types of processing steps, in particular processing steps
commonly
used in the food or feed industry. Such steps include but are not limited to
concentration or
condensation of the solid matter of the plant to form for example a pellet,
production of a
paste, drying, or lyophilization, or by fragmentation of the plant to various
extents by cutting
or grinding, or by extraction of the liquid part of the plant to produce a
soup, a syrup or a
juice. A processing step can also include cooking the plant or plant matter.
"Expression» refers to the transcription and/or translation of an endogenous
gene or a
transgene in plants. In the case of antisense constructs, for example,
expression may refer
to the transcription of the antisense DNA only.
"Expression cassette" as used herein means a DNA sequence capable of directing
expression of a particular nucleotide sequence in an appropriate host cell,
comprising a
promoter operably linked to the nucleotide sequence of interest which is
optionally operably
finked to 3' sequences, such as 3' regulatory sequences or termination
signals. It also
typically comprises sequences required for proper translation of the
nucleotide sequence.
The coding region usually codes for a protein of interest but may also code
for a functional
RNA of interest, for example antisense RNA or a nontranslated RNA that, in the
sense or
antisense direction, inhibits expression of a particular gene, e.g., antisense
RNA. The
expression cassette comprising the nucleotide sequence of interest may be
chimeric,
meaning that the nucleotide sequence is comprised of more than one DNA
sequences of
distinct origin which are fused together by recombinant DNA techniques
resulting in a
nucleotide sequence which does not occur naturally, and which particularly
does not occur
in the plant to be transformed. The expression cassette may also be one which
is naturally
occurring but has been obtained in a recombinant form useful for heterologous
expression.
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Typically, however, the expression cassette is heterologous with respect to
the host, i.e.,
the particular DNA sequence of the expression cassette does not occur
naturally in the host
cell and must have been introduced into the host cell or an ancestor of the
host cell by a
transformation event. The expression of the nucleotide sequence in the
expression cassette
may be under the control of a constitutive promoter or of an inducible
promoter which
initiates transcription only when the host cell is exposed to some particular
external
stimulus. In the case of a multicellular organism, such as a plant, the
promoter can also be
specific to a particular tissue or organ or stage of development. A nuclear
expression
cassette is usually inserted into the nuclear genome of a plant and is capable
of directing
the expression of a particular nucleotide sequence from the nuclear genome of
said plant. A
plastid expression cassette is usually inserted in to the plastid genome of a
plant and is
capable of directing the expression of a particular nucleotide sequence from
the plastid
genome of said plant. In the case of a plastid expression cassette, for
expression of the
nucleotide sequence from a plastid genome, additional elements, i.e. ribosome
binding
sites, or 3' stem-loop structures that impede plastid RNA polyadenylation and
subsequent
degradation may be required.
"Gene" refers to a coding sequence and associated regulatory sequences wherein
the
coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense
RNA
or antisense RNA. Examples of regulatory sequences are promoter sequences, 5'
and 3'
untranslated sequences and termination sequences. Further elements that may be
present
are, for example, introns.
"Heterologous" as used herein means of different natural or of synthetic
origin. For
example, if a host cell is transformed with a nucleic acid sequence that does
not occur in
the untransformed host cell, that nucleic acid sequence is said to be
heterologous with
respect to the host cell. The transforming nucleic acid may comprise a
heterologous
promoter, heterologous coding sequence, or heterologous termination sequence.
Alternatively, the transforming nucleic acid may be completely heterologous or
may
comprise any possible combination of heterologous and endogenous nucleic acid
sequences. Similarly, heterologous refers to a nucleotide sequence derived
from and
inserted into the same natural, original cell type, but which is present in a
non-natural state,
e.g. a different copy number, or under the control of different regulatory
elements.
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"Marker Gene" a gene encoding a selectable or screenable trait.
A regulatory DNA sequence is said to be "operably linked to" or "associated
with" a DNA
sequence that codes for an RNA or a protein if the two sequences are situated
such that
the regulatory DNA sequence affects expression of the coding DNA sequence.
"Regulatory elements" refer to sequences involved in conferring the expression
of a
nucleotide sequence. Regulatory elements. comprise a promoter operably linked
to the
nucleotide sequence of interest and, optionally, 3' sequences, such as 3'
regulatory
sequences or termination signals. They also typically encompass sequences
required for
proper translation of the nucleotide sequence.
"Subcellular organelles" refers to intracellular organs of characteristic
structure and function.
Subcellular organelles are for example vacuoles, plastids, mitochondria, the
cell nucleus,
the endoplasmic reticulum or the plasma membrane.
"Homoplasmic" refers to a plant, plant tissue or plant cell wherein all of the
plastids are
genetically identical. This is the normal state in a plant when the plastids
have not been
transformed, mutated, or otherwise genetically altered. In different tissues
or stages of
development, the plastids may take different forms, e.g., chloroplasts,
proplastids,
etioplasts, amyloplasts, chromoplasts, and so forth.
A "promoter" refers to a DNA sequence that initiates transcription of an
associated DNA
sequence. The promoter region may also include elements that act as regulators
of gene
expression such as activators, enhancers, and/or repressors.
An "inducible promoter" is a promoter which initiates transcription only when
the plant is
exposed to some particular external stimulus, as distinguished from
constitutive promoters
or promoters specific to a specific tissue or organ or stage of development.
Particularly
preferred for the present invention are chemically-inducible promoters and
wound-inducible
promoters. Chemically inducible promoters include plant-derived promoters,
such as the
promoters in the systemic acquired resistance pathway, for example the PR
promoters, e.g.,
the PR-1, PR-2, PR-3, PR-4, and PR-5 promoters, especially the tobacco PR-1a
promoter
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and the Arabidopsis PR-1 promoter, which initiate transcription when the plant
is exposed to
BTH and related chemicals. See US Patent 5,614,395, incorporated herein by
reference,
and WO 98/03536, incorporated herein by reference. Chemically-inducible
promoters also
include receptor-mediated systems, e.g., those derived from other organisms,
such as
steroid-dependent gene expression, for example the glucocorticoid,
progesterone and
estrogen receptor systems, copper-dependent gene expression, such as that
based on
ACE1, tetracycline-dependent gene expression, the Lac repressor system and the
expression system utilizing the USP receptor from Drosophila mediated by
juvenile growth
hormone and its agonists, described in WO 97/13864, incorporated herein by
reference, as
well as systems utilizing combinations of receptors, e.g., as described in WO
96/27673,
incorporated herein by reference. Additional chemically-inducible promoters
include elicitor-
induced promoters, safener-induced promoters as well as the alcA/alcR gene
activation
system that is inducible by certain alcohols and ketones (WO 93/21334; Caddick
et af.
(1998) Nat Biotechnol 16:177-180, the contents of which are incorporated
herein by
reference). Wound inducible promoters include promoters for proteinase
inhibitors, e.g., the
proteinase inhibitor II promoter from potato, and other plant-derived
promoters involved in
the wound response pathway, such as promoters for polyphenyl oxidases, LAP and
TD.
See generally, C. Gatz, "Chemical Control of Gene Expression", Annu. Rev.
Plant Physiol.
Plant Mol. Biol. (1997) 48: 89-108, the contents of which are incorporated
herein by
reference.
A "transactivator" is a protein which, by itself or in combination with one or
more additional
proteins, is capable of causing transcription of a coding region under control
of a
corresponding transactivator-mediated promoter. Examples of transactivator
systems
include bacteriophage T7 gene 10 promoter, the transcriptional activation of
which is
dependent upon a specific RNA polymerase such as the phage T7 RNA polymerase.
The
transactivator is typically an RNA polymerase or DNA binding protein capable
of interacting
with a particular promoter to initiate transcription, either by activating the
promoter directly or
by inactivating a repressor gene, e.g., by suppressing expression or
accumulation of a
repressor protein. The DNA binding protein may be a chimeric protein
comprising a binding
region (e.g., the GAL4 binding region) linked to an appropriate
transcriptional activator
domain. Some transactivator systems may have multiple transactivators, for
example
promoters which require not only a polymerase but also a specific subunit
(sigma factor) for
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promoter recognition, DNA binding, or transcriptional activation. The
transactivator is
preferably heterologous with respect to the plant or to the subcellular
organelle or
component of the plant cell in which induction is effected.
A "minimal promoter" comprises promoter elements, particularly a TATA element,
that are
inactive or that have greatly reduced promoter activity in the absence of
upstream
activation. In the presence of suitable upstream activating sequences fused to
the minimal
promoter and of corresponding transcription factor, the minimal promoter
functions to permit
transcription.
"Recombinant DNA technology" refers to procedures used to join together DNA
sequences
as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY:
Cold Spring
Harbor Laboratory Press.
A "screenable marker gene" refers to a gene whose expression does not confer a
selective
advantage to a transformed cell, but whose expression makes the transformed
cell
phenotypically distinct from untransformed cells.
A "selectable marker gene" refers to a gene whose expression in a plant cell
gives the cell a
selective advantage. The selective advantage possessed by the cells
transformed with the
selectable marker gene may be due to their ability to grow in the presence of
a negative
selective agent, such as an antibiotic or a herbicide, compared to the growth
of non-
transformed cells. The selective advantage possessed by the transformed cells,
compared
to non-transformed cells, may also be due to their enhanced or novel capacity
to utilize an
added compound as a nutrient, growth factor or energy source. Selectable
marker gene
also refers to a gene or a combination of genes whose expression in a plant
cell gives the
cell both, a negative and a positive selective advantage.
"Synthetic" refers to a nucleotide sequence comprising structural characters
that are not
present in the natural sequence. For example, an artificial sequence that
resembles more
closely the G+C content and the normal codon distribution of dicot and/or
monocot genes is
said to be synthetic.
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'Transformation" refers to introduction of a nucleic acid into a cell. In
particular, the stable
integration of a DNA molecule into the genome of an organism of interest.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEG1UENCE LISTING
SEQ ID No:1 oligonucleotide
SEQ ID No:2 oligonucleotide
SEQ ID No:3 oligonucleotide
SEQ ID No:4 oligonucleotide
SEQ ID No:Soligonucleotide
SEQ ID No:6oligonucleotide
SEQ ID No:7oligonucleotide
SEQ ID No:8oligonucleotide
SEQ !D No:9oligonucleotide
SEQ ID No:lOoligonucleotide
SEQ ID No:l1oligonucleotide
SEQ ID No:l2oligonucleotide
SEQ ID No:l3oligonucleotide
SEQ ID No:l4oligonucleotide
SEQ ID No:lSoligonucleotide
SEQ ID No:l6oligonucleotide
SEQ ID No:l7oligonucleotide
SEQ ID No:l8oligonucleotide
SEQ ID No:l9oligonucleotide
SEQ ID No:20oligonucleotide
SEQ ID No:21oligonucleotide
SEQ ID No:22oligonucleotide
SEQ ID No:23oligonucleotide
SEQ ID No:24oligonucleotide
SEQ ID No:25oligonucleotide
SEQ ID No:26oligonucleotide
SEQ ID No:27oligonucleotide
T73a_U
SEQ ID No:28oligonucleotide
T73a_L
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_19_
SEQ ID No:29oligonucleotide
minpsb_U
SEQ ID No:30oligonucleotide
minpsb_L
SEQ ID No:31oligonucleotide
SEQ ID No:32oligonucleotide
SEQ ID No:33oligonucleotide
SEQ ID No:34oligonucleotide
SEQ ID No:35oligonucleotide
SEQ ID No:36oligonucleotide
SEQ ID No:37oligonucleotide
ErspU
SEQ ID No:38oligonucleotide
ErspL
SEQ ID No:39oligonucleotide
ErspovL
SEQ ID No:40oligonucleotide
AmboeU
The present invention discloses transgenic plants expressing therapeutically
active proteins,
in particular antigens. DNA molecules encoding such proteins are expressed
from the
plastid genome of the plant or are expressed in the cell nucleus and the
proteins are
targeted to the cell cytosol or to subcellular organelles, such as vacuoles.
Plants of the
present invention are able to express the proteins in a cost effective manner
and are easily
accessible. Such plants are able to express large amounts of such proteins in
a cost
effective manner. Furthermore, therapeutically active proteins expressed in
plastids are
conveniently packaged, making purification and processing especially easy.
Also, such
compartmentalization allows the therapeutic molecules to be protected during
digestion,
thereby favoring oral administration. Therapeutically active proteins
expressed in transgenic
plants according to the present invention can be administered by a wide range
of methods
to hosts, including human, pets or livestock, to prevent or treat a variety of
diseases, and
thereby improve the condition of the treated host. In particular, transgenic
plants of the
present invention or plant material derived from such plants can be ingested
orally by a host
and can be used for example to treat allergies, autoimmune diseases or to
prevent the
rejection of transplantations, preferably by induction of tolerance of the
host to antigens.
The invention also discloses compositions, such as pharmaceutical
compositions,
comprising such plants, or plant matter derived from such plants, as well as
methods to
improve the condition of a host by administration of a composition of the
present invention.
Therapeutically Active Proteins Expressed in Transgenic Plants
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Proteins of the present invention are preferably able to modulate a host's
immune
response, examples are given below. Therefore, they can be used to treat or
prevent
undesired immune responses, for example to suppress or reduce an immune
response of
the host, for example by inducing tolerance. Alternatively, they can also
stimulate the
immune system of a host and thereby contribute or result in an immunization of
the host
against a disease, for example a bacterial, parasitic or viral disease.
In a preferred embodiment, a single protein is expressed in a transgenic
plant. In this case,
if several proteins are desired for a particular treatment, a mixture of
plants each expressing
a different protein is used. In an alternate preferred embodiment, several
different proteins
are expressed in the same transgenic plant. In this case, the DNA molecules
encoding the
different proteins are included in different expression cassettes which are
transformed into
the plant or, alternatively, the DNA molecules are included in the same
expression cassette.
For expression from the plastid genome, for example, the DNA molecules
encoding the
different therapeutically active proteins can be engineered into an expression
cassette to
form a single, polycistronic messenger RNA. For simplicity and clarity
purposes, "a"
therapeutically active protein as mentioned throughout the text refers to as
"at least one"
therapeutically active protein, meaning one or more proteins. Also, a
therapeutically
effective amount of a therapeutically active protein of the present invention
may be
obtained from a single plant or from plant matter derived from a single plant,
or may be
obtained from a plurality of plants, for examples siblings of the plant.
Plants transformed in accordance with the present invention may be monocots or
dicots and
include, but are not limited to, maize, wheat, barley, rye, sweet potato,
bean, pea, chicory,
lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus,
onion, garlic,
pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon,
plum, cherry,
peach, nectarine, apricot, strawberry, grape, raspberry, blackberry,
pineapple, avocado,
papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet,
sunflower,
rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant,
cucumber,
Arabidopsis, turfgrasses, ornamentals and woody plants such as coniferous and
deciduous
trees. Once a desired gene has been transformed into a particular plant
species, it may be
propagated in that species or moved into other varieties of the same species,
particularly
including commercial varieties, using traditional breeding techniques.
Also included in the present invention are edible algae, such as unicellular
green algae (e.g.
Chlamydomonas), multicellular green algae (e.g. Ulva), unicellular red algae
(e.g.
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Porphyridium) and multicellular red algae (e.g. Porphyra), which contain
plastid genomes
substantially similar to those of higher plant that may be transformed in a
similar manner.
Expression of Therapeutically Active Proteins in Plant Plastids
The present invention particularly relates to the expression of
therapeutically active proteins
from the plastid genome of a plant. In this case, some or all of the several
thousand copies
of the circular plastid genome present in each plant cell are transformed with
a DNA
molecule encoding a therapeutically active protein of the present invention.
The enormous
transgene copy number typical of plastids permits expression levels that
usually greatly
exceed expression levels commonly obtained from nuclear-expressed genes. Such
high
levels of expression further reduce the cost of production of a
therapeutically active protein
in plants, and also permits the use of such transgenic plants in applications
requiring high
levels of the protein in plant material, as for example when the plant or
plant material is
ingested orally to treat allergies, autoimmune diseases or to prevent the
rejection of
transplantations. Plastid gene expression also has a number of additional
advantages. For
example, transgene expression levels are stable over time due to the absence
of gene
silencing and position effect variation, polycistronic operons can be
expressed in a
coordinated manner from a single promoter or regulatory sequence allowing the
production
of equimolar amounts of several proteins, uniparental plastid gene inheritance
prevents
pollen transmission of foreign DNA in most economically important crops and
hence
reduces the possibility of lateral transfer to wild or cultivated plants.
Also, plastid transgene
integration occurs via a homologous recombination process, meaning that
precise targeted
engineering and gene replacement is readily performed. Furthermore, proteins
expressed
within the plastid remain sequestered in the organelle and thus are prevented
from
interacting with the cytoplasmic environment. This feature is essential if the
protein
expressed in plants also shows activity against or interacts adversely with
some
components of the plant cytosol.
Plastid transformation technology is described extensively in U.S. Patent Nos.
5,451,513,
5,545,817, 5,545,818 and 5,576,198; in PCT application nos. WO 95/16783 and WO
97/32977; and in McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305
(1994), all of
which are incorporated herein by reference. Plastid transformation via
biolistics was
achieved initially in the unicellular green alga Chlamydomonas reinhardtii
(Boynton et al.
(1988) Science 240: 1534-1537, incorporated herein by reference) and this
approach,
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using selection for cis-acting antibiotic resistance loci
(spectinomycin/streptomycin
resistance) or complementation of non-photosynthetic mutant phenotypes, was
soon
extended to Nicotiana tabacum (Svab et al. (1990) Proc. Natl. Acad. Sci. USA.
87: 8526-
8530, incorporated herein by reference).
The basic technique for tobacco plastid transformation involves the particle
bombardment of
leaf or callus tissue or PEG-mediated uptake of plasmid DNA in protoplasts
with regions of
cloned plastid DNA flanking a selectable antibiotic resistance marker. The 0.5
to 1.5 kb
flanking regions, termed targeting sequences, facilitate homologous
recombination with the
plastid genome and thus allow the replacement or modification of specific
regions of the
156 kb tobacco plastid genome. Initially, point mutations in the chloroplast
16S rDNA and
rpsl2 genes conferring resistance to spectinomycin and/or streptomycin were
utilized as
selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga,
P. (1990)
Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992)
Plant Cell 4,
39-45, incorporated herein by reference). This resulted in stable homoplasmic
transformants at a frequency of approximately one per 100 bombardments of
target leaves.
The presence of cloning sites between these markers allowed creation of a
plastid targeting
vector for introduction of foreign genes (Staub, J.M., and Maliga, P., EMBO J.
12: 601-606
(1993), incorporated herein by reference). Substantial increases in
transformation
frequency were obtained by replacement of the recessive rRNA or r-protein
antibiotic
resistance genes with a dominant selectable marker, the bacterial aadA gene
encoding the
spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab,
Z., and
Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917, incorporated herein
by
reference). Previously, this marker had been used successfully for high-
frequency
transformation of the plastid genome of the green alga Chlamydomonas
reinhardtii
(Goldschmidt-Clermont, M. (1991 ) Nucl. Acids Res. 19, 4083-4089, incorporated
herein by
reference). Recently, plastid transformation of protoplasts from tobacco and
the moss
Physcomitrella patens has been attained using polyethylene glycol (PEG)
mediated DNA
uptake (O'Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996) Planfa
199: 193-201,
both of which are incorporated herein by reference). Both particle bombardment
and
protoplast transformation are appropriate in the context of the present
invention.
A DNA molecule encoding a therapeutically active protein of the present
invention is
inserted into a plastid expression cassette comprising a promoter capable of
expressing the
DNA molecule in plant plastids. A preferred promoter capable of expression in
a plant
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plastid is a promoter isolated from the 5' flanking region upstream of the
coding region of a
plastid gene, which may come from the same or a different species, and the
native product
of which is typically found in a majority of plastid types including those
present in non-green
tissues. Gene expression in plastids differs from nuclear gene expression and
is related to
gene expression in prokaryotes (described in Stern et al. (1997) Trends in
Plant Sciences 2:
308-315, incorporated herein by reference). Plastid promoters generally
contain the -35 and
-10 elements typical of prokaryotic promoters and some plastid promoters are
recognized
by a E. cohlike RNA polymerase mostly encoded in the plastid genome and are
called PEP
(plastid-encoded RNA polymerase) promoters while other plastid promoters are
recognized
by a nuclear-encoded RNA polymerase (NEP promoters). Both types of plastid
promoters
are suitable for the present invention. Examples of plastid promoters are
promoters of clpP
genes, such as the tobacco clpP gene promoter (WO 97/06250, incorporated
herein by
reference) and the Arabidopsis clpP gene promoter (comprised between positions
71882
and 72,371 in the Arabidopsis plastid genome, the sequence of which was made
available
by Takakazu Kaneko and Satoshi Tabata of the Kazusa DNA Research Institute at
the
URL: ftp://genome-ftp.stanford.edu/pub/arabidopsis/chloroplast/
). Another promoter that is capable of expressing a DNA molecule in plant
plastids comes
from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et
al., Microbiol.
f?ev. 58:700-754 (1994), Shinozaki et al., EMBO J. 5:2043-2049 (1986), both of
which are
incorporated herein by reference). Other examples of promoters that are
capable of
expressing a DNA molecule in plant plastids are a psbA promoter or a rbcL
promoter. A
plastid expression cassette also preferably further comprises a plastid gene
3' untranslated
sequence (3' UTR) operatively linked to a DNA molecule of the present
invention. The role
of untranslated sequences is preferably to direct the 3' processing of the
transcribed RNA
rather than termination of transcription. Preferably, the 3' UTR is a plastid
rpsl6 gene 3'
untranslated sequence or the Arabidopsis plastid psbA gene 3' untranslated
sequence. In a
further preferred embodiment, a plastid expression cassette comprises a poly-G
tract
instead of a 3' untranslated sequence. A plastid expression cassette also
preferably further
comprises a 5' untranslated sequence (5' UTR) functional in plant plastids
operatively linked
to a DNA molecule of the present invention.
A plastid expression cassette is comprised in a plastid transformation vector,
which
preferably further comprises flanking regions for integration into the plastid
genome by
homologous recombination. The plastid transformation vector may optionally
comprise at
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least one chloroplast origin of replication. The present invention also
encompasses a plant
plastid transformed with such a plastid transformation vector, wherein the DNA
molecule is
expressible in the plant plastid. The invention also encompasses a plant or
plant cell,
including the progeny thereof, comprising this plant plastid. In a preferred
embodiment, the
plant or plant cell, including the progeny thereof, is homoplasmic for
transgenic plastids.
Other promoters that are capable of expressing a DNA molecule in plant
plastids are
transactivator-regulated promoters, preferably heterologous with respect to
the plant or to
the subcellular organelle or component of the plant cell in which expression
is effected. In
these cases, the DNA molecule encoding the transactivator is inserted into an
appropriate
nuclear expression cassette which is transformed into the plant nuclear DNA.
The
transactivator is targeted to plastids using a plastid transit peptide. The
transactivator and
the transactivator-driven DNA molecule are brought together either by crossing
to a
selected plastid-transformed line a transgenic line containing a DNA molecule
encoding the
transactivator supplemented with a plastid-targeting sequence and operably
linked to a
nuclear promoter, or by directly transforming a plastid transformation vector
containing the
desired DNA molecule into a transgenic line containing a DNA molecule encoding
the
transactivator supplemented with a plastid-targeting sequence and operably
linked to a
nuclear promoter. If the nuclear promoter is an inducible promoter, in
particular a chemically
inducible promoter, expression of the DNA molecule in the plastids of plants
is activated by
foliar application of a chemical inducer. Such inducible transactivator-
mediated plastid
expression system is preferably tightly regulatable, with no detectable
expression prior to
induction and exceptionally high expression and accumulation of protein
following induction.
A preferred transactivator is for example viral RNA polymerase. Preferred
promoters of this
type are promoters recognized by a single sub-unit RNA polymerase, such as the
T7 gene
promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA
polymerase. The gene encoding the T7 polymerase is preferably transformed into
the
nuclear genome and the T7 polymerase is targeted to the plastids using a
plastid transit
peptide. Promoters suitable for nuclear expression of a gene, for example a
gene encoding
a viral RNA polymerase such as the T7 polymerase, are described infra.
Expression of the
DNA molecules in plastids can be constitutive or can be inducible Expression
of the DNA
molecules in the plastids can be also organ- or tissue-specific. These
different embodiment
are extensively described in WO 98/11235, incorporated herein by reference.
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Nuclear Expression of a Transactivator or of Therapeutically Active Proteins
For expression in a particular transgenic plant, a nucleotide sequence
according to the
invention may require modification and optimization. Low expression in
transgenic plants
may result from heterologous nucleotide sequences having codons which are not
preferred
in plants. It is known in the art that al! organisms have specific preferences
for codon
usage, and the codons of the nucleotide sequence described in this invention
can be
changed to conform with plant preferences, while maintaining the amino acids
encoded
thereby. Furthermore, high expression in the plant nucleus is best achieved
from coding
sequences which have at least 35% GC content, and preferably more than 45%.
Additionally, nucleotide sequences which have low GC contents may express
poorly in plant
nuclei due to the existence of ATTTA motifs which may destabilize messages,
and AATAAA
motifs which may cause inappropriate polyadenylation. Although preferred gene
sequences
may be adequately expressed in both monocotyledonous and dicotyledonous plant
species,
sequences can be modified to account for the specific codon preferences and GC
content
preferences of monocotyledons or dicotyledons as these preferences have been
shown to
differ (hurray et al. Nuci. Acids Res. 17: 477-498 (1989)). Also, the DNA
molecule is
screened for the existence of illegitimate splice sites which cause message
truncation. All
changes required to be made within the DNA molecule such as those described
above are
made using well known techniques of e.g. site directed mutagenesis, PCR, and
synthetic
gene construction using the methods described in the published patent
applications EP 0
385 962, EP 0 359 472, and WO 93/07278.
For efficient initiation of translation, sequences adjacent to the initiating
methionine may
require modification. For example, sequences known to be effective in plants
can be
included. Joshi has reported an appropriate consensus sequence for plant
nuclei (NAR 15:
6643-6653 (1987)) and Clontech suggests a further consensus translation
initiator
(1993/1994 catalog, page 210). These consensus sequences are suitable for use
with DNA
molecules of this invention. The sequences are incorporated into constructions
comprising
the DNA molecules, up to and including the ATG {whilst leaving the second
amino acid
unmodified}, or alternatively up to and including the GTC subsequent to the
ATG (with the
possibility of modifying the second amino acid of the transgene}.
Expression of a nucleotide sequence encoding a transactivator or a
therapeutically active
protein in transgenic plants is driven by a promoter shown to be functional in
plants. The
choice of promoter depends on the temporal and spatial requirements for
expression, and
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also depending on the host species. Expression of a DNA molecule of the
present invention
in plants can be constitutive or inducible, for example chemically inducible,
or the
expression of the DNA molecule can be organ- or tissue-specific. Although many
promoters
from dicotyledons have been shown to be operational in monocotyledons and vice
versa,
ideally dicotyledonous promoters are selected for expression in dicotyledons,
and
monocotyledonous promoters for expression in monocotyledons. However, there is
no
restriction to the provenance of selected promoters; it is sufficient that
they are operational
in driving the expression of the nucleotide sequence in the desired cell.
Preferred promoters
which are expressed constitutively include the CaMV 35S and 19S promoters, and
promoters from genes encoding actin or ubiquitin. A DNA molecule of this
invention can
also be expressed under the regulation of promoters which are chemically
regulated.
Preferred technology for chemical induction of gene expression is detailed in
patent
application EP 0 332 104 and US patent 5,614,395. A preferred promoter for
chemical
induction is the tobacco PR-1a promoter. A preferred category of promoters is
that which is
wound inducible. Numerous promoters have been described which are expressed at
wound
sites and also at the sites of phytopathogen infection. Ideally, such a
promoter is only
active locally at the sites of infection. Preferred promoters of this kind
include those
described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al.
Plant Molec.
Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989),
Rohrmeier & Lehle,
Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-
142 (1993),
and Warner et al. Plant J. 3: 191-201 (1993). Preferred tissue specific
expression patterns
include green tissue specific, root specific, stem specific, fruit specific
and flower specific.
Promoters suitable for expression in green tissue include many which regulate
genes
involved in photosynthesis, and many of these have been cloned from both
monocotyledons and dicotyledons. A preferred promoter is the maize PEPC
promoter from
the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-
589
(1989)). A preferred promoter for root specific expression is that described
by de Framond
(FEES 290: 103-106 (1991 ); EP 0 452 269) and a further preferred root-
specific promoter is
that from the T-1 gene (de Framond et al. FEBS 290: 103-106 (1991 ); EP 0 452
269). A
preferred stem specific promoter is that described in US patent 5,625,136 and
which drives
expression of the maize trpA gene. Preferred embodiments of the invention are
transgenic
plants expressing a DNA molecule in a root-specific fashion. Further preferred
embodiments are transgenic plants expressing a DNA molecule in a wound-
inducible or
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pathogen infection-inducible manner. Additional promoters are synthetic
promoters such as
the Gelvin Super MAS promoter (Ni et al. (1995) Plant J. 7: 661-676).
In addition to the selection of a suitable promoter, constructions for
expression of a DNA
molecule in the plant nucleus preferably comprise appropriate 3' sequences,
such as 3'
regulatory sequences or transcription terminators, to be operably linked
downstream of the
heterologous nucleotide sequence. Several such terminators are available and
known in the
art (e.g. tm1 from CaMV, E9 from rbcS). Any available terminator known to
function in
plants can be used in the context of this invention. Numerous other sequences
can be
incorporated into expression cassettes for a DNA molecule described in this
invention.
These include sequences which have been shown to enhance expression such as
intron
sequences (e.g. from Adh1 and bronzel) and viral leader sequences (e.g. from
TMV,
MCMV and AMV).
In another preferred embodiment, the nuclear expressed DNA molecule of the
present
invention is targeted to a subcellular location or locations in the plant. For
example, a
therapeutically active protein is secreted from the cell into the apoplast or
a therapeutically
active protein is targeted to a particular subcellular organelle, for example
to the vacuoles or
to the endoplasmic reticulum. This is achieved for example by the fusion of
the appropriate
targeting sequences to a DNA molecule of the present invention using
techniques well
known in the art. Thus for targeting to the apoplast or to the vacuole, the
protein preferably
comprises an appropriate signal peptide, preferably at its N-terminus, which
allows targeting
of the protein to the organelle. In a preferred embodiment, a protein of the
present invention
is targeted to the vacuoles. For targeting of the protein to the vacuoles, in
addition to an N-
terminal signal peptide, the protein preferably also further comprises a
vacuolar targeting
sequence, such as that from a tobacco chitinase gene, preferably at its C-
terminus
(Neuhaus et al. (1991 ) Proc. Natl. Acad. Sci. USA 88, 10362-10366). The
expression of a
protein encoded by a DNA molecule of the present invention can also be
targeted to the
plastids using an appropriate plastid transit peptide, preferably comprised at
the N-terminus
of the protein as described in details infra. A protein of the present
invention can also be
targeted to the mitochondrion, for example by fusion with a mitochondrial
targeting
sequence, such as a N. plumbaginifolia F1-ATPase ~i-subunit (Chaumont et al.
(1994) Plant
Molecular Biology 24: 631-641).
Vectors suitable for plant transformation are described elsewhere in this
specification. For
Agrobacterium-mediated transformation, binary vectors or vectors carrying at
least one T-
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DNA border sequence are suitable, whereas for direct gene transfer any vector
is suitable
and linear DNA containing only the construction of interest may be preferred.
In the case of
direct gene transfer, transformation with a single DNA species or co-
transformation can be
used (Schocher et al. Biotechnology 4: 1093-1096 (1986)). For both direct gene
transfer
and Agrobacterium-mediated transfer, transformation is usually (but not
necessarily)
undertaken with a selectable marker which may provide resistance to an
antibiotic (e.g.
kanamycin, hygromycin or methotrexate) or a herbicide (e.g.
Basta/phosphinothricin or an
inhibitor of protoporphyrinogen oxidase). The choice of selectable marker is
not, however,
critical to the invention. Examples of expression cassettes and transformation
vectors are
described in further detail infra.
The DNA molecules of the present invention are introduced into the plant cell
in a number
of well known ways. Those skilled in the art will appreciate that the choice
of method might
depend on the type of plant, i.e. monocot or dicot, targeted for
transformation. Suitable
methods of transforming plant cells include microinjection (Crossway et al.,
BioTechniques
4:320-334 (1986)), electroporation (Riggs and Bates, Proc. Natl. Acad. Sci.
USA
83:5602-5606 (1986)), Agrobacterium-mediated transformation (Hinchee et al.,
Biotechnology 6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO
J.
3:2717-2722 (1984)), and ballistic particle acceleration using devices
available from
Agracetus, Inc., Madison, Wisconsin and Dupont, lnc., Wilmington, Delaware
(see, for
example, U.S. Patent 4,945,050; and McCabe et al., Biotechnology 6:923-926
(1988); see
also Weissinger et al., Annual Rev. Genet. 22:421-477 (1988); Sanford et al.,
Particulate
Science and Technology 5:27-37 (1987)(onion); Christou et al., Plant Physiol.
87:671-674
(1988) (soybean); McCabe et al., Bio/Technology 6:923-926 (1988)(soybean);
Datta et al.,
Bio/Technology 8:736-740 (1990)(rice); Klein et al., Proc. Natl. Acad. Sci.
USA,
85:4305-4309 (1988)(maize); Klein et al., Bio/Technoiogy 6:559-563
(1988)(maize); Klein et
al., Plant Physiol. 91:440-444 (1988)(maize); Fromm et al., Bio/Technology
8:833-839
(1990); and Cordon-Kamm et al., Plant Cell 2:603-618 (1990)(maize); Koziel et
al.
(Biotechnology 11: 194-200 (1993))(maize); Shimamoto ef al. Nature 338: 274-
277
(1989)(rice); Christou et al. Biotechnology 9: 957-962 (1991 )(rice); European
Patent
Application EP 0 332 581 (orchardgrass and other Poaideae); Vasil et al.
(Biotechnology
11: 1553-1558 (1993)(wheat); Weeks et al. (Plant Physiol. 102: 1077-1084
(1993) (wheat);
Wan et aL (Plant Physiol. 104: 37-48 (1994)(barley)); Umbeck et al.,
(Bio/Technology 5:
263-266 (1987)(cotton).
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Administration of Therapeutically Active Proteins to a Host
An advantage of the present invention is the wide variety of ways by which a
therapeutically
active protein expressed in transgenic plants can be administered to a host
such as, for
example, orally, enterally, nasally, parenterally, particularly
intramuscularly or intravenously,
rectally, topically, ocularly, pulmonarilly or by contact application. In a
preferred
embodiment, an allergen expressed in transgenic plants is administered to a
host orally.
In a preferred embodiment, a therapeutically active protein expressed in a
transgenic plant
is extracted and purified, and used for the preparation of a pharmaceutical
composition.
Localization of the expressed therapeutically active protein in the plastids
greatly facilitates
such extraction and purification. For example, intact plastids are first
isolated by
centrifugation making the extraction and purification of the therapeutically
active protein
easier. In another preferred embodiment, proteins are isolated and purified in
accordance
with conventional conditions and techniques known in the art previously used
to isolate
such proteins, such as extraction, precipitation, chromatography, affinity
chromatography,
electrophoresis, or the like. Such compositions typically comprise an
effective amount of a
therapeutically active protein together with one or more organic or inorganic,
liquid or solid,
pharmaceutically suitable carrier materials. A therapeutically active protein
produced
according to the present invention is employed in dosage forms such as
tablets, troches,
dispersions, suspensions, solutions, capsules, creams, ointments, aerosols,
powder
packets, or liquid solutions as long as the biological activity of the protein
is not destroyed
by such dosage form. For example, the protein may be provided as a
pharmaceutical
composition by means of conventional mixing, granulating, dragee-making,
dissolving,
lyophilizing or similar processes. The dosage of the protein is dependent upon
the weight,
age, and physical and pharmacokinetical condition of the patient and is
further dependent
upon the method of delivery.
Where the protein is administered enterally, it may be introduced in solid,
semi-solid,
suspension or emulsion form and may be compounded with any pharmaceutically
acceptable carriers, including water, suspending agents, emulsifying agents.
The proteins
of the invention may also be administered by means of pumps, or in sustained-
release
form, especially, when administered as a preventative measure, so as to
prevent the
development of disease in a subject or when administered to ameliorate or
delay an already
established disease.
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Therapeutically active proteins produced according to the present invention
are particularly
well-suited for oral administration as pharmaceutical compositions.
Compositions for oral
administration include proteins provided as dry powders, food-stuffs, aqueous
or non-
aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents
are
propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable
organic esters.
Aqueous carriers include water, water-alcohol solutions, emulsions or
suspensions,
including saline and buffered medical parenteral vehicles including sodium
chloride solution,
Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's
solution
containing lactose, or fixed oils. In a preferred embodiment, such
compositions are ingested
orally alone or ingested together with food or feed or a beverage.
In another preferred embodiment, a transgenic plant expressing a
therapeutically active
protein of the present invention or plant matter derived from such plant are
administered
orally as medical food. Such edible compositions are consumed by eating if in
a solid form
or by drinking if in a liquid form. In a preferred embodiment, the transgenic
plant material is
directly ingested without a prior processing step or after minimal culinary
preparation. For
example, the therapeutically active protein is expressed in a plant parts of
which can be
eaten directly, such as a fruit or a vegetable. Preferably, the protein is
expressed in the
plastids of a plant part which can be eaten. All types of plastids in any
plant are suitable for
the present invention. For example, the protein is expressed in spinach or
lettuce
chloroplasts, in tomato chromoplasts or in potato amyloplasts. In an
alternative preferred
embodiment, the plant is processed and the plant material recovered after the
processing
step is ingested. Processing methods preferably used in the present invention
are methods
commonly used in the food or feed industry. The final products of such methods
still
comprise a substantial amount of the protein and can be conveniently eaten or
drunk. The
final product may also be mixed with other food or feed forms, such as salts,
carriers, flavor
enhancers, antibiotics and the like, and consumed in solid, semi-solid,
suspension, or
emulsion form. In another preferred embodiment, such methods comprise a
conservation
step, such as, e.g., pasteurization, cooking or addition of conservation and
preservation
agents. Any plant is used and processed in the present invention to produce
edible or
drinkable plant matter. When tobacco plants are used according to the present
invention,
for example, it may be necessary to treat the plants or plant material to
remove harmful
substances such as nicotine. In a preferred embodiment, a low-alkaloid tobacco
plant is
used for the expression of a protein of the present invention. In a preferred
embodiment,
the amount of therapeutically active protein in an edible or drinkable plant
matter according
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to the present invention is tested. Such amount is determined e.g. by Elisa or
Western blot
analysis using an antibody specific for the protein. This determination is
used to standardize
the amount of protein ingested. For example the amount of therapeutically
active protein in
a juice, e.g. in a tomato juice, is determined and regulated, for example by
mixing batches
of product having different levels of protein, so that the quantity of juice
to be drunk per
dose can be standardized. It is clear that the present invention provides
novel and efficient
ways to produce and administer plant-expressed therapeutically active agents
as medical
foods.
A therapeutically active protein produced in a plant and eaten by the host is
absorbed by
the digestive system. One advantage of the ingestion of a plant or plant
material,
particularly intact plants or plant material, or plant material which has been
only mildly
processed, is to provide encapsulation or sequestration of the protein in the
cells of the
plant. Thus, the protein may receive at least some protection from digestion
in the upper
digestive tract before reaching the gut or intestine and a higher proportion
of active protein
would be thus available for uptake.
When a plant used for expression of a therapeutically active protein of the
present invention
is tobacco, the crude, so-called "F2 soluble protein fraction" can for example
be used for
therapeutical administration. An additional fraction, F1, is produced by
precipitation of high
molecular weight protein complexes from a crude tobacco extract. In non-
transgenic plants,
this F1 fraction consists almost exclusively of ribulose bisphosphate
carboxylase as
disclosed in US patent 4,347,324. It is possible that some transgenic
therapeutically active
proteins which are produced by the methods of the present invention may
partition into the
F1 fraction as well. In this case, the F1 fraction could also be used for
therapeutical
administration.
Preferred hosts or recipients for therapeutically active proteins of the
present invention are
animals. Animals preferably include vertebrates, preferably mammals. Preferred
mammals
are humans, pets or companion animals, like e.g. cats, dogs, rodents, ferrets,
primates,
fishes or birds, or farm animals, like cows, hogs, poultry, or the like.
Immunization
Several current vaccines need expensive refrigeration and are therefore not
readily
available in developing countries. Also, efficient vaccines against some
pathogens have still
not been produced in large enough quantities or are still not sufficiently
safe for broad
distribution. In an attempt to improve the situation, a hepatitis B viral
surface antigen has
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been expressed in plants and administered through edible plants with the goal
of
vaccinating against hepatitis B (see for example US patents 5,484,719 and
5,612,487).
However, the genes encoding the vaccines were expressed from the plant nuclear
genome
and only relatively low yields of the vaccines could obtained, thus precluding
the efficient
production of an edible vaccine. Immunization against pathogenic
microorganisms has also
been attempted by expressing antigenic determinants of the pathogen in
transgenic plants
and administering the plant orally to a host (US patents 5,654,184, 5,679,880,
5,686,079).
However, in these cases too, relatively low levels of expression of the
transgene form the
plant nuclear genome have limited the commercial success of such approaches.
It is therefore a preferred embodiment of the present invention to express
antigens capable
of inducing an immunization of a host against a pathogen, for example a
bacterial, a
parasitic or a viral pathogen or to immunize a host against a toxin, in
transgenic plants, in
particular in subcellular organelles, preferably in vacuoles, more preferably
in plant plastids.
Plants of the present invention are used for the immunization of humans
against for
example poliomyelitis, measles, mumps, rubella, smallpox, yellow fever, viral
hepatitis B,
influenza, rabies, adenoviral infections, Japanese B encephalitis, varicella,
diarrhea, acute
respiratory infections, malaria, pertussis, diphteria, tetanus or neonatal
tetanus. Such
proteins are also advantageously used for the immunization of animals to
prevent diseases
such as for example equine infections, canine distemper, rabies, canine
hepatitis,
parvovirus, and feline leukemia, Newcastle, Rinderpest, hog cholera, blue
tongue and foot-
mouth, brucellosis, fowl cholera, anthrax and black leg, as well as diseases
resulting from
infections with protozoans and hefminths. Plants of the present invention are
also used for
the immunization of animals, including humans, against various toxins or
irritants, such as
snake or bee venom, mosquito saliva, poison ivy. The host becomes immunized
upon
administration of the antigen, preferably by oral ingestion of the plant or of
plant matter
derived from the plant. An adjuvant, as well-known in the immunological art,
may be added
to an composition comprising an antigen of the present invention in an amount
such as to
improve the immunological activity of the composition and thereby the
therapeutical
effectiveness of the composition. Information relating to the immune system
and to
immunological responses are also found in Hood et al. (1984) Immunology. The
Benjamin/Cummings Publishing Co, Inc. Menlo Park, CA.
Tolerization
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The field of the medical arts relating to the suppression or reduction of
undesired immune
responses also needs improvements. An animal's immune system is an intricate
network of
specialized cells and organs acting upon various specific signals. One of its
major functions
is to discriminate between "self" and "non-self". This basic property allows
for protection of
the host against invading pathogens without provoking detrimental immune
responses
against the host itself. Typically, an immune response is characterized by a
cellular
response provided by certain cells of the lymphoid system and a humoral
response
provided by antibodies, and is initiated by. the encounter of a non-self
determinant by the
immune system resulting in the destruction of the non-self. However, in some
cases, the
discrimination between self and non-self becomes deficient, resulting in an
immunological
response against some parts of the self and leading to the development of an
autoimmune
disease. In some other cases, a immunological response against specific non-
self antigens
results in undesired effects such as, for example, the rejection of a non-
autologous tissue or
organ transplant, or the development of an allergic response against an
environmental
determinant. In these cases, an effective suppression or reduction of the
immune response
is desirable. Several strategies have been attempted to reach this goal. For
example,
certain autoimmune diseases have been prevented by aerosol administration of
autoantigens (US patents 5,641,473 and 5,641,474). Other treatment attempts
have been
based on oral tolerization of the host to an antigen, a mechanism by which
ingested
proteins cause a suppression or reduction of the usual immune response to
specific foreign
substances or autoantigens (see for example Weiner (1994) Proc. Natl. Acad.
Sci. 91,
10,762-10,765; Friedman et al. (1994) in Grandstein RD (ed): Mechanisms of
Immune
Regulation, Chem. Immunol. Basel, Karger, Vol 58, 259-290). There are multiple
mechanism involved in oral tolerance, two of the primary ones being active
cellular
suppression or clonal anergy. This is thought to be associated with absorption
by mucosa of
the small intestine. Antigens are taken up in the gut and presented to
specialized mucosal
tissues, such as Peyer's patch cells, which are the point of entry to the gut-
associated
immune system. This induced immunological hypo- or unresponsiveness is related
to the
suppression of food allergy that occurs normally in most individuals and is
the primary
means of tolerization to the numerous potential antigens which enter the
digestive tract as
components of the diet. In the therapeutical area, oral administration of
insulin has provided
some relief in the treatment of type I diabetes (US patent 5,763,396) and oral
administration
of type I or type III collagen had some success in the treatment of autoimmune
arthritis (US
patent 5,733,547). Additionally, attempts to suppress the rejection of a
transplant by oral
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administration of polymorphic class II MHC allopeptides has been made (US
patent
5,593,698). In these previous cases, the antigen was purified from natural
sources.
However, for an oral tolerization to be effective, high amount of antigens
have to be
ingested. Such high amounts are difficult or expensive to obtain for many
antigens, in
particular for airborne antigens, and cannot be easily prepared in an orally
ingestible
composition. Thus, the success of many of such strategies has been hampered by
the lack
of sufficient amounts of protein available for oral ingestion. Recently, the
expression in
transgenic plants of certain transplantation antigens and autoantigens and
their enteral or
oral administration has been attempted (WO 95/08347). However, here again
relatively low
levels of nuclear transgene expression are likely to preclude a successful
outcome of these
approaches.
Therefore, in a preferred embodiment, antigens capable of suppressing or
reducing an
immune response or an inflammatory condition of a host are expressed in
plants, in
particular in plant plastids. Preferably such antigens are administered orally
to the host.
Transgenic plants expressing such antigens are particularly beneficial in the
treatment,
prevention or amelioration of diseases such as for example allergies,
autoimmune diseases
or rejection of transplantations, preferably by inducing tolerance of the host
to one or
multiple antigens. For oral ingestion, the plant expressing the antigen or
plant matter
derived from such plants is either eaten raw or eaten or drunk after a
processing step, for
example a processing step described supra. In a preferred embodiment, the
result of the
oral ingestion of the plant or plant matter is an induction of tolerance of
the host against the
antigen. The present invention for the first time provides for a plentiful and
cheap source of
antigens which can be used for oral tolerization by expressing such antigens
from the
plastid genome of a plant. In a preferred embodiment, edible plants engineered
to
overexpress specific peptide antigens as described in the present invention
are an ideal
vehicle for an oral tolerization therapy based on medical food. The use of
transgenic plants
for oral tolerance has a number of potential advantages over current
approaches
(biochemical purification or recombinant expression in cell culture systems
and protein
expression in transgenic milk). These advantages include low production cost
per dose,
minimal risk of transmitting mammalian pathogens, no or limited need for
purification,
processing or encapsulation when antigen-containing plants or plant material
are consumed
directly as food.
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In another preferred embodiment, an antigen of the present invention is co-
administered to
a host with a tolerizing adjuvant. Such an adjuvant is for example a toxin,
such as a cholera
toxin B subunit (as described for example in Arakawa et al. (1998) Nat
Biotechnol 16: 934-
8). Such toxin subunit molecules are known in the art to promote oral
tolerance as opposed
to mucosal immunity (Sun et al PNAS (1996) 93:7196-201 ). Another tolerizing
adjuvant
used in the present invention is an E. coli labile enterotoxin B. In a
preferred embodiment, a
nucleic acid sequence encoding the toxin is fused to a nucleic acid sequence
encoding the
antigen to produce a DNA molecule encoding a fusion protein. A plant
comprising such
DNA molecule is produced as described in the present invention and the fusion
protein is
administered to the host as described in the present invention. in another
preferred
embodiment, the toxin and the antigen are expressed in a plant as separate
polypeptides
and administered to the host as described in the present invention.
Alternatively, the toxin
and the antigen are expressed in different plants and combined prior to
administration to
the host. Alternatively, the toxin is obtained from a different organism,
preferably purified,
and then combined with the antigen prior to administration to the host.
Allergens
Allergies cause a major discomfort in a large proportion of the population and
are suspected
to also have adverse effects on animals. The range of magnitude of allergic
reactions in a
human population ranges from mild symptoms to heavy and dramatic appearances,
such as
anaphylactic shocks, and common pathological appearances include food
allergies, skin
reactions (urticaria or atopic dermatitis), allergic reactions of the upper
respiratory tract (e.g.
hayfever or allergic rhinitis) or allergic reactions of the lower respiratory
tract that are a
major cause of asthma (for a review, see O'Hehir et al. (1991 ) Annu. Rev.
Immunol. 9: 67-
95). Allergic responses are typically due to exaggerated IgE responses to
various types of
allergens (for example pollen, animal dander, insect fecal matter or dust as
well as food
allergens) and are often associated with inflammatory responses. The
pathological
manifestations of such IgE-antigen interactions are due in particular to mast-
cell
degranulation that results in the release of histamine, heparin and
leukotrienes. Present
medications are mainly unspecific and are in the form of a treatment with e.g.
antihistamines or epinephrine, an antagonist of mast-cell degranulation. More
specific
treatments have also been developed and are based on periodic injections of
suballergic
doses of the allergen, resulting in a desensitization of the host against the
allergen.
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However, such injections are cumbersome and desensitization addresses barely
or not at
all the T cell response usually associated with allergies. Injections for
desensitization are
therefore often of limited efficacy. The present invention provides novel,
specific methods
for the treatment of allergies by expressing DNA molecules encoding allergens
in plants and
administering such plants or plant matter derived from such plants to a host.
Such plants or
plant matter are preferably administered orally to the host who thereby
develops tolerance
to the allergen. Treatment of infants or young individuals whose pedigree
suggests that
they may be at risk for the onset of allergic.diseases later in life may also
be undertaken in
a prophylactic manner to prevent the development of allergy. Alternatively,
allergens
expressed in transgenic plants of the present invention are extracted and
purified according
to methods well-known in the art and used for desensitization of a host
against the allergen
by periodic injections of suballergic doses of the allergen.
Suitable allergens for use in the context of the present invention includes
food allergens,
drug allergens, venom allergens, plant allergens, fungal allergens, bacterial
allergens,
animal allergens, other allergens from naturally-occurring or synthetic
substances and
extracts thereof. Food allergens include, for example, seafood, strawberries,
fresh fruit and
vegetables, peanuts and cow's milk. Drug allergens include, for example,
penicillin and
insulin. Venom allergens include, for example, bee, wasp and mosquito venoms.
A
preferred allergen is bee venom peptide PLA-2. Plant allergens include, for
example
allergens from pollen, such as, tree pollen, grass pollen and weed pollen.
Fungal allergens
include, for example, mold spores. Animal allergens include, for example,
dander or saliva
from dogs, cats, horses, etc. Preferred allergens of the present invention are
house dust
mite allergens, such as allergens from Dermatophagoides farinae and
Dermatophagoides
pteronyssinus, preferably the Der f I, Der f 11, Der p 1 and Der p II
allergens (US patents
5,552,142, 5,770,202 and 5,798,099). Alternative preferred allergens are
derived from
ryegrass pollen, e.g from Lolium perenne and include isolated peptides of Lol
p V (US
patent 5,710,126). Further alternate preferred allergens include allergens
derived from
Johnson grass pollen, such as Sor h l, a major allergen derived from Sorghum
halepense
(US patents 5,480,972 and 5,691,167). Further preferred allergens include
ragweed pollen
allergen, such as the Amb a I and Amb a II allergens (US patent 5,698,204).
Further
preferred allergens also include the Aln g I allergen of alder, Alnus sp., the
Cor a I allergen
of hazel (Corylus sp.) and the Bet v I allergen of birch (Befula sp.),
described in US patent
5,693,495. Further allergens include cat antigens, such as the Fel d I
allergen (US patent
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5,328,991 ), and dog dander allergens, such as Can f II (US patent 5,939,283).
Further
allergen also include the rAed a 1 and rAed a 2 allergens from the mosquito
Aedes aegypty
(WO 98/04274) and antigens of the honey bee venom (Lomnitzer and Rabson (1986)
J.
Allergy Clin. Immunol. 78: 25-30) or murine urinary proteins (Gurka et al.
(1989) J. Allergy
Clin. Immunol. 83: 945-954).
Furthermore, it has been suggested that the anaphylactic reaction and the IgE-
mediated
reaction are separable and that disulfide bridges are largely responsible for
anaphylaxis.
The removal of cystein residues involved in forming disulfide bridges in
Dermatophagoides
farinae Der f ll allergen has been shown to greatly reduce the anaphylactic
reaction without
altering the allergenic epitopes (Takai et ai. (1997) Nature Biotechnology 15:
754-758). The
present invention also encompasses the expression of allergens modified in
this manner so
as to lack disulfide bridges. A DNA molecule encoding an allergen is cloned in
a plastid
transformation vector using techniques well-know in the art and transgenic
plants are
produced. Plants or plant matter of the present invention are administered to
a host alone or
in combination with other therapies against allergies.
Antigens Expressed in Transgenic Plants
It is a preferred embodiment of the present invention to express antigens in
transgenic
plants, in particular in plant plastids. Preferably, such antigens are capable
of modulating
the immune system of a host in need thereof, resulting in the desired
therapeutic effect.
Transgenic plants expressing such antigens are particularly beneficial in the
treatment,
prevention or amelioration of diseases such as for example allergies,
autoimmune diseases
or rejection of transplantations, preferably by inducing tolerance of the host
to one or
multiple antigens, or in immunization procedures. Such transgenic plants are
administered
to a host by different methods known in the art, preferably orally, preferably
by eating or
drinking a plant of the present invention or plant matter derived from such
plants.
Antigens in Autoimmune Diseases or Self-Antigens
An autoimmune disease is a malfunction of the immune system of an animal,
including a
human, in which the immune system fails to distinguish between foreign
substances within
the animal and substances which are part of the animal's normal composition.
As a failure
of immunological tolerance, T cells or B cells, or both, emerge bearing
receptors allowing
them to recognize and attack self components. This results in an autoimmune
disease in
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the host bearing such T cells or B cells. Numerous diseases have been
diagnosed to be
caused at least in part by autoantigens. For example, blood cells are the most
common type
of cells affected, leading to diseases such as thrombocytopenic purpura, in
which
antibodies against platelets are formed, or agranulocytosis, where
autoantibodies are
formed against polymorphonuclear leukocytes. Hemolysis and anemia has also
been
reported to be due in some cases to autoantibodies directed at the surface of
erythrocytes.
Antibodies to cell-surface receptors can also cause diseases by interfering
with receptor
function, such as in the case of myasthenia gravis, where antibodies are
formed against
acetylcholine receptors, thus impeding neuromuscular transmission. The
opposite effect,
the stimulation of a receptor by an anti-receptor autoantibody has also been
discovered in
Grave's disease or hyperthyroidism. Further examples of diseases where a
failure of
immunological tolerance is suspected or known include for example multiple
sclerosis,
diabetes mellitus, systemic lupus erythematosus, polychondritis, systemic
sclerodoma,
Wegener's granulamatosis, dermatomyositis, chronic active hepatitis,
psoriasis, Steven-
Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease
(including
e.g. ulcerative colitis and Crohn's disease), sarcoidosis, primary biliary
cirrhosis, uveitis
(anterior and posterior), keraconjunctivitis sicca and vernal
keraconjunctivitis, interstitial lung
fibrosis, psoriatic arthritis and glomerulonephritis (with and without
nephrotic syndrome, e.g.
including idiopathic nephrotic syndrome or minimal change nephropathy), and
arthritis (for
example rheumatoid arthritis, arthritis chronica progrediente and arthritis
deformans).
For autoimmune diseases as for allergies, present medications largely lack
specificity and
are often connected with unpleasant side effects, such as global
immunosuppression of the
host. It is therefore a preferred embodiment of the present invention to
provide for novel
treatments for autoimmune diseases by expressing self-antigens that are
targeted by
autoantibodies in plant plastids. Such plants or plant matter derived from
such plants is
administered to a host in need thereof, preferably orally, preferably by being
eaten or
drunken by the host. Tolerance of the host to the specific autoantibodies then
develops
without affecting the general immune response abilities of the host. Plants or
plant matter of
the present invention are administered to a host alone or in combination with
immunosuppressant or anti-inflammatory agents, including cyclosporins,
rapamycins, FK
506 and steroids.
Preferred self-antigens of the present invention are for example collagen,
preferably type I
or type III collagen, to treat or prevent arthritis, preferably rheumatoid
arthritis (see US
patent 5,733,547), myelin basic protein to treat or prevent multiple
sclerosis, S-antigen to
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treat or prevent autoimmune uveoretinitis, insulin to treat or prevent type I
diabetes (see US
patent 5,763;396), gfutamic acid decarboxylase or an islet cell-specific
antigen to treat or
prevent diabetes, thyroglobulin to treat or prevent autoimmune thyroiditis,
acetylcholine
receptor to treat or prevent myasthenia gravis.
A DNA molecule encoding an autoantigen is cloned in a plastid transformation
vector or into
a transformation vector for nuclear transformation using techniques well-know
in the art and
transgenic plants are produced.
Transplantation Antigens
There is a great need for grafts or transplantations for the replacement of
severely injured
tissues and organs. Grafts from an individual to himself (autografts)
generally succeed but
grafts from genetically dissimilar individuals of the same species (allogeneic
grafts) or
between individuals of different species (xenogeneic grafts) do not normally
succeed
without immunosuppressive drugs. Without continued immunosuppressive drug
therapy, the
organ will be rejected. A general method to induce tolerance to the
transplanted organ and
to improve the chances of success of an alto- or xenogeneic graft has
therefore great
potential and is addressed in the present invention.
A transplant rejection is initiated by an immune response to the cell-surface
antigens that
distinguish donor from host. Such cell-surface antigens mainly belong to the
histocompatibility antigens, in particular to the major histocompatibility
complex (MHC).
Products of the class I and II MHC genes are involved in presenting antigens.
They are
therefore particularly important in the recognition of nonself antigens by T
cells and play an
important role in transplantation rejection. Class I MHC complexes comprise a
transmembrane glycoprotein homodimer (heavy chain, HC) attached to a b2
microglobulin
moiety (Bjorkman et al. (1987) Nature 329: 506). Three class la loci (HLA-A, B
and C in
humans, H-2K, H-2D and H-2L in mice) and several class Ib loci encoding the HC
moiety
have been identified (York and Rock (1996) Annu Rev Immunol 14: 369-396).
Class II MHC
are heterodimers composed of two glycoproteins, the a and b chains (Brown et
al. (1993)
Nature 364: 33). Since the MHC loci are conserved among vertebrates, in
particular among
mammals (Klein (1986) Natural History of the Major Histocompatibility Complex.
Oxford:
Blackwell Sci.), DNA molecules encoding MHC antigens can also be isolated from
various
mammals, including pig, a preferred donor for xenotransplantations.
The importance of MHC antigens in transplantation rejection has been further
established
by showing that targeting of class II molecules by monoclonal antibodies
attenuates
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transplant rejection. Therefore, MHC antigens are good candidates for a
therapy involving
tolerization of a graft recipient against specific MHC determinant of a donor.
Recently, the
expression in transgenic plants of certain transplantation antigens and
autoantigens and
their enteral or oral administration, has been attempted (WO 95/08347).
However, here
again relatively low levels of nuclear transgene expression are likely to
preclude a
successful outcome of these approaches.
In the present invention, MHC genes, in particular a gene encoding a class I
HC moiety and
class II MHC genes encoding the a or b chain, are expressed from the plastid
genome or
the nuclear genome of a plant. Transgenic plants comprising such plastids are
administered
to a host, preferably prior to and/or after a tissue- or organ transplantation
(alto- or xeno-
transplantation), to induce tolerization of the host. Preferably, such plants
or matter derived
from such plants is administered orally to the host to induce an oral
toferization of the host.
DNA molecules encoding the different known allotypes of MHC class I HC gene
and class II
MHC genes encoding the a or b chain are introduced in transgenic plants. For
use in a
particular transplantation, allotypes of both donor and recipient are
determined and the
recipient is administered, preferably orally, plant matter comprising selected
MHC antigens
of the donor. Plants or plant matter of the present invention are administered
to a host
alone or in combination with immunosuppressant, including e.g. cyclosporins.
Other Therapeutically Active Proteins
Other therapeutically active proteins expressed in plants in the context of
the present
invention are e.g., but not limited to, blood proteins (e.g. clotting factors
VIII and IX,
complement factors and complements, hemoglobins or other blood proteins, serum
albumin, and the like), hormones (e.g. insulin, growth hormone, thyroid
hormone,
catechoiamines gonadotrophines, PMSG, trophic hormones, prolactin, oxytocin,
dopamine,
bovine somatotropin, leptins and the like), growth factors (e.g. EGF, PDGF,
NGF, IGF, and
the like), cytokines (e.g. interleukines, CSF, G-CSF, GMCSF, EPO, TNF, TGFa,
TGFb,
interferons and the like), enzymes (e.g. tissue plasrninogen activator,
streptokinase,
cholesterol biosynthetic or degradative, steroidogenic enzymes, kinases,
phosphodiesterases, methylases, de-methylases, dehydrogenases, cellulases,
proteases,
lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate
cyclases,
neuraminidases and the like), hormones or other receptors (e.g. steroid
protein, peptide,
lipid or prostaglandin, and the like), binding proteins (e.g. steroid binding
proteins, growth
hormone or growth factor binding proteins, and the like), immune system
proteins (e.g.
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antibodies, antibody fragments, chimeric antibodies, variable regions, or the
like, or MHC
genes), antigens (e.g. bacterial, parasitic, viral, allergens, antigens in
autoimmune diseases,
transplantation antigens, and the like), translation or transcription factors,
oncoproteins or
proto-oncoproteins, milk proteins (e.g. caseins, lactalbumin, whey, and the
like), muscle
proteins (e.g. myosin, tropomyosin, and the like), myeloproteins, neuroactive
peptides (e.g.
enkephalins), collagen, anti-sepsis peptides (e.g. BPI (bactericidal
permeability-increasing
protein) or tumor growth suppressing proteins or peptides, for example
angiostatin or
endostatin, both of with inhibit angiogenesis.
Expression of Bactericidal Permeability-Increasing (BPI) Protein in Plants
The present invention also relates to the expression of BPI, or fragments
thereof, in
transgenic plants, in particular in subcellular organelles, preferably in
vacuoles or more
preferably in plant plastids. BPI is a protein isolated from the granules of
mammalian
polymorphonuclear leukocytes (PMN) which are blood cells that are essential in
the defense
against invading microorganisms in mammals (US patent 5,641,874; Wilde et al.
(1994) J.
Biol. Chem. 269: 17,411-17,416). BPI is a potent bactericidal agent active
against a broad
range of gram-negative bacterial species (see for example US patents
5,753,620,
5,827,816 and 5,763,567, incorporated herein by reference in their entirety).
It exhibits a
high degree of specificity in its cytotoxic effect, i.e. 10-40 nM (0.5-2.0
micrograms},
producing greater than 90°/a killing of 107 sensitive bacteria whereas
100-fold higher
concentrations of BPI are non-toxic for other microorganisms and eukaryotic
cells. BPI
isolated from both human and rabbit PMN has been purified to homogeneity. The
molecular
weight of human BPI is approximately 58,000 Daltons (58 kDa) and that of
rabbit BPI is
approximately 50 kDa. Due to its exquisite selectivity and lack of
cytotoxicity toward cells
other than gram-negative bacteria, the BPI fragments of the present invention
are
particularly useful as specific therapeutic agents. Currently gram-negative
bacterial
infections, such as those caused by Escherichia coli, various species of
Salmonella,
Klebsiella or Pseudomonas are treated with antibiotics, such as penicillin
derivatives,
aminoglycosides and chloramphenicol. The effectiveness of antibiotics is
limited due to the
fact that gram-negative bacilli tend to display significant intrinsic
resistance to many
currently available antibiotics and to readily develop further resistance due
to the acquisition
of resistance factor plasmids. However, production of BPI in recombinant
systems has been
problematic and purification from mammalian cells is not cost-effective given
that doses in
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the gram range are preferable for therapeutic efficacy. One reason for the
high dose
required is the rapid clearance rate of BPI from the bloodstream. It is
therefore a preferred
embodiment of the present invention to express a DNA molecule encoding BPI or
a BPI
fragment from the plastid genome of a plant. Expression in plastids with its
high yields is
particularly advantageous in this case.
When employed to treat bacteremia (i.e. the presence of bacteria in the blood
stream) or
sepsis (bacterial contamination of bodily fluids) caused by gram-negative
bacteria, BPI or
BPI fragments produced according to the present invention are preferably
administered
parenterally, and most preferably intravenously in amounts broadly ranging
between about
1 microgram and 1000 micrograms and preferably between 10 and about 250
micrograms
per treatment. The duration and number of treatments may vary from individual
to
individual, depending upon the severity of the illness. A typical treatment
regime may
comprise intravenous administration of about 100 micrograms of the BPI
fragments three
times a day. To help avoid rapid inactivation, BPI or BPI fragments may be
coupled with
physiologically-acceptable carriers, such as normally occurring serum proteins
(e.g. albumin
or lysozyme). The BPI or BPI fragments of the present invention can also be
employed
topically to treat mammals suffering from skin infections caused by
susceptible gram-
negative bacteria which occur in bedridden patients suffering from decubitus
ulcers (bed
sores) or in burn patients. In a preferred embodiment, the BPI or BPI
fragments of the
present invention in amounts ranging between 1 microgram and 1000 micrograms
per dose,
may be mixed with antibiotics. In another preferred embodiment of the present
invention,
pharmaceutical formulations for treating mammals suffering from gram-negative
bacterial
infections may contain the BPI fragments of the present invention in addition
to standard
amounts (well-known in the art) of antibiotics such as Penicillin-G (available
from E. R.
Squibb and Sons, Inc., Princeton, N.J.) or cephalosporins (available from Eli
Lily & Co.,
Indianapolis, Ind.). In a particularly preferred embodiment, the BPI fragments
of the present
invention may be mixed with hydrophobic antibiotics, such as rifampicin, and
hydrophobic
penicillins such as Penicillin-V Benzathine (Lederle Labs, Pearl River, N.Y.).
The increased
permeability of gram-negative bacteria after BPI treatment is expected to
enhance the
effectiveness of such antibiotics which cannot easily enter non-permeabilized
bacteria.
The BPI or BPI fragments of the present invention are expected to be ideally-
suited for co-
treatment using any antibiotic, immune system cells or factors such as T-cells
or interleukin-
2, cytotoxic agents or the like, effective against gram-negative bacteria.
Because of the
increased sensitivity to the fragments of the present invention of the more
pathogenic,
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smooth, gram-negative bacteria, the BPI fragments of the present invention are
expected to
decrease resistance of such bacteria to such factors. Substantially
simultaneous
administration of the fragments of the present invention and the antibiotic of
choice is
preferred. In another preferred embodiment, BPi or BPI fragments are ingested
orally
according to the embodiments described supra, preferably to treat bacterial
infections of the
digestive system. The protein is administered as a pharmaceutical composition
or is
ingested as a medical food.
BPI has also been shown to be active in the treatment of conditions including
the
neutralization of the anti-coagulant activity of heparin, inhibition of
angiogenesis, tumor and
endothelial cell proliferation, and treatment of chronic inflammatory diseases
(see US patent
US5807818, incorporated herein by reference).
The invention will be further described by reference to the following detailed
examples.
These examples are provided for purposes of illustration only, and are not
intended to be
limiting unless otherwise specified.
EXAMPLES
Standard recombinant DNA and molecular cloning techniques used here are well
known in
the art and are described by J. Sambrook, E. F. Fritsch and T. Maniatis,
Molecular Cloning:
A Laborator)r manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY
(1989) and by
T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions,
Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al.,
Current
Protocols in Molecular Bioloay, pub. by Greene Publishing Assoc. and Wiley-
Interscience
(1987).
Example 1: Isolation of the Amb a I. i cDNA from short ragweed (Ambrosia
artemisiifolia) pollen by RT-PCR amplification
Total RNA from 100 mg of defatted pollen (Greer Laboratories) is isolated by
the
phenol/SDS method (Current Protocols in Molecular, Biology). First-strand cDNA
from 0.5
mg of total RNA is synthesized with the Advantage RT-for-PCR kit (Clontech)
using an oligo
(dT),e primer. The single-stranded DNA is used as a template for PCR
amplification of the
Amb a L 1 cDNA (Rafnar et al. (1991 ) J. Biol. Chem. 266: 1229-1236) with the
Pfu Turbo
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DNA Polymerase kit (Stratagene) using the following oligonucleotides: the "top
strand"
primer adds a Ncol site to the translational start site of Amb a 1.1 (5'-GCG
GCC ATG GGG
ATC AAA CAC TGT TGT TA-3', SEQ ID N0:1 ) and the "bottom strand" primer adds a
Xbal
site after the stop codon in the 3' untranslated region (5'-GCG GTC TAG ATC
ATT ATA
AGT GCT TAG T-3', SEQ ID N0:2). The 1.2 kb band is digested with Ncol and Xbal
for
subcloning and the entire cDNA is sequenced and compared to GenBank accession
number M80558. Seven by differences are observed but none of the differences
changes
the amino acid composition.
Example 2: Construction of vector for homologous recombination into the
tobacco
plastid genome
The trnV and rpsl2/7 intergenic region of the tobacco plastid genome is
modified for
insertion of chimeric genes by homologous recombination. A 1.78 kb region
(positions
139255 to 141036, Shinozaki et al., (1986) EMBO J 5: 2043-2049) is PCR
amplified from
the tobacco plastid genome and a Psti site is inserted after position 140169,
yielding 915 by
and 867 by of flanking plastid DNA 5' and 3' of the Psti insertion site. PCR
amplification
(PfuTurbo DNA Polymerase, Stratagene, La Jolla, CA) is performed with a primer
pair
inserting a Bs~EI site before position 139255 (5'-TAA CGG CCG CGC CCA ATC ATT
CCG
GAT A-3', SEQ ID N0:3) and a Pstl site after position 140169 (5'-TAA CTG CAG
AAA GAA
GGC CCG GCT CCA A-3', SEQ ID N0:4). PCR amplification is also performed with a
primer pair inserting a Pstl site before position 140170 (5'-CGC CTG CAG TCG
CAC TAT
TAC GGA TAT G-3', SEQ ID N0:5) and a BsNV l site after position 141036 (5'-CGC
CGT
ACG AAA TCC TTC CCG ATA CCT C-3', SEQ ID N0:6). The Psfl - BsiEl fragment is
inserted into the Psti - Sadl sites of pBluescript SK+ (Stratagene), yielding
pAT216 and the
Pst1-BsNVI fragment is inserted into the Psfl-Acc651 sites of pBluescript SK+,
yielding
pAT215. PAT218 contains the 1.78 kb of plastid DNA with a Psfi site for
insertion of
chimeric genes and selectable markers and is constructed by ligation of the
2.0 kb Pst1-Scal
fragment of pAT215 and the 2.7 kb Psfl-Scal band of pAT216.
Example 3: Amplification of the tobacco 16S rRNA gene promoter and rbs of the
rbcL
gene
The 16S rRNA gene promoter is PCR amplified from tobacco DNA (N. tabacum cv.
Xanthi)
and fused to a synthetic ribosome binding site (rbs) of the tobacco plastid
rbcL gene. The
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"top strand" primer inserts an EcoRl site at the 5' end of the 16S rRNA gene
promoter
before position 102568 (5'-GCC AGA ATT CGC CGT CGT TCA ATG AGA ATG-3', SEO ID
N0:7). The "bottom strand" primer amplifies up to position 102675 of the 16S
rRNA gene
promoter, removes two upstream ATG's by changing positions 102661 (A to C) and
102670
(A to C), adds the rbs of the rbcL gene (positions 57569-57584) as a 5'
extension of the
primer and inserts a BspHl site at the 3' end of the rbs (5'-GCC TTC ATG ATC
CCT CCC
TAC AAC TAT CCA GGC GCT TCA GAT TCG-3', SEQ ID N0:8). The 142 by amplification
product is gel purified and cleavage with ~EcoRl and BspHl yields a 128 by
fragment
containing the tobacco 16S rRNA gene promoter fused to the rbs of the rbcL
gene.
Example 4: isolation of the Arabidopsis 16S rRNA gene promoter region
Isolation of the Arabidopsis 16S rRNA gene promoter region is facilitated by
the likelihood
that gene order in the Arabidopsis plastid genome is conserved relative to
that of Nicotiana
tabacum, a plant for which the entire plastid genome is known. in Sinapis
alba, a closely
related species to Arabidopsis, the 16S rRNA gene and valine tRNA are oriented
as in
tobacco (GenBank accession number CHSARRN1 ). The Arabidopsis 16S rRNA gene
promoter region is isolated by PCR amplification (PfuTurbo DNA Polymerase,
Stratagene,
La Jolla, CA) using total A. thaliana (cv "Landsberg erecta") as template and
the following
primers that are conserved in both Nicotiana and Sinapis albs: "top strand"
primer (5'-CAG
TTC GAG CCT GAT TAT CC-3', SEO ID N0:9) and the "bottom strand" primer (5'-GTT
CTT
ACG CGT TAC TCA CC-3', SEQ ID N0:10). The predicted 379 by amplification
product
comprising the Arabidopsis 16S rRNA gene promoter region corresponding to
nucleotides
102508 to 102872 of the tobacco plastid genome (Shinozaki et a1. (1986) EMBO J
5: 2043-
2049) is blunt end ligated into the EcoRV site of pGEMSZf(-) (Promega) and
sequence
analysis and comparisons to the tobacco 16S rRNA gene promoter is performed.
Example 5: Amplification of the tobacco plastid rpsl6 gene 3' untranslated RNA
sequence (3' UTR)
The tobacco plastid rpsl6 3' UTR is PCR amplified from tobacco DNA (N. tabacum
cv.
Xanthi) using the following oligonucleotide pair: a Spel site is added
immediately after the
stop codon of the plastid rpsl6 gene encoding ribosomal protein S16 with the
"top strand"
primer (5'-CGC GAC TAG TTC AAC CGA AAT TCA AT-3', SEQ ID N0:11 ) and a Psfl
site is
added at the 3' end of the rpsl6 3' UTR with the "bottom strand" primer (5'-
CGC TCT GCA
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GTT CAA TGG AAG CAA TG-3', SEQ ID N0:12). The amplification product is gel
purified
and digested with Spel and Pstl, yielding a 163 by fragment containing the
tobacco rpsl6 3'
UTR (positions 4941 to 5093 of the tobacco plastid genome, Shinozaki et al.
(1986) EMBO
J 5: 2043-2049) flanked 5' by a Spel site and 3' with a Pstl site.
Example 6: Construction of a 16S rRNA gene promoter :: aadA gene :: rpsl6 3'
UTR
cassette for plastid transformation selection
The coding sequence of the aadA gene, a bacterial gene encoding the enzyme
aminoglycoside 3" adenyltransferase that confers resistance to spectinomycin
and
streptomycin, is isolated from pRL277 (Black et al. (1993) Molecular
Microbiology 9:77-84
and Prentki et al. (1991 ) Gene 103: 17-23). The 5' major portion of the aadA
coding
sequence is isolated as a 724 by BspHl-BssHll fragment from pRL277 (the
starting codon is
at the BspHl site) and the 3' remainder of the aadA gene is modified by adding
a Spel site
20 by after the stop codon by PCR amplification using pRL277 as template and
the
following oligonucleotide pair: the "top strand" primer (5'-ACC GTA AGG CTT
GAT GAA-3',
SE(~ ID N0:13) and the "bottom strand" primer which added a Spel site (5'-CCC
ACT AGT
TTG AAC GAA TTG TTA GAC-3', SEQ ID N0:14). The 658 by amplification product is
gel
purified, digested with BssHlf, Spel and the 89 by fragment is ligated to the
5' portion of the
aadA gene carried on a 724 by BspHl-BssHll fragment, the 16S rRNA gene
promoter and
rbs of rbcL carried on a 128 by EcoRl-BspHl PCR amplified fragment and EcoRl-
Spel
digested pLITMUS28 vector (New England Biolabs), yielding pAT223. A three-way
ligation
is performed on an EcoRl- Spel 0.94 kb fragment of pAT223 containing the 16S
rRNA gene
promoter-rbs driven aadA gene, a 163 by Spel, Pstl digested PCR fragment
containing the
rpsl6 3' UTR and pUCl9 (New England Biolabs) cut with EcoRl, Psfl to obtain
pAT229
containing the 16S rRNA gene promoter driving the aadA gene with the rpsl6 3'
UTR.
Example 7: Amplification of the bacteriophage T7 gene 10 promoter
The bacteriophage T7 gene 10 promoter is PCR amplified from pET-3d
(Stratagene) using
the following oligonucleotide pair: the "top strand" primer inserts an EcoRl
site at the 5' end
of the T7 promoter (5'-CCC GAA TTC ATC CCG CGA AAT TAA TA-3', SEQ ID N0:15)
and
the "bottom strand" primer inserts a Ncol site at the 3' end (5'-CGG CCA TGG
GTA TAT
CTC CTT CTT AAA GTT AAA-3', SEQ ID N0:16). The amplification product is gel
purified
and cleavage with EcoRl, Ncol produces a 96 by fragment containing the T7
promoter.
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Example 8: Amplification of the bacteriophage T7 gene 10 terminator
The bacteriophage T7 gene 10 terminator is PCR amplified from pET-3d
(Stratagene) using
the following oligonucleotide pair: the "top strand" primer inserts a Hindlll
site at the 5' end
of the terminator (5'-GCG AAG CTT GCT GAG CAA TAA CTA GCA TAA-3', SEQ ID
N0:17)
and the "bottom strand" primer inserts a Pstl site at the 3' end of the
terminator (5'-GCG
CTG CAG TCC GGA TAT AGT TCC TCC T-3', SEQ ID N0:18). The amplification product
is gel purified and cleavage with Hindlll Pstl produces an 86 by fragment
containing the T7
terminator.
Example 9: Amplification of the Arabidopsis fhaliana plastid psbA 3'
untranslated
RNA sequence (UTR)
The A. thaliana plastid psbA 3' UTR is PCR amplified from A. thaliana DNA
(ecotype
Landsberg erecta) using the following oligonucleotide pair: the "top strand"
primer adds a
Spel site to the 5' end of the 3' UTR and eliminates a Xbal site in the native
sequence by
mutating a G to an A (underlined) (5'-GCG ACT AGT TAG TGT TAG TCT AAA TCT AGT
T-
3', SEQ ID N0:19) and the "bottom strand" primer adds a HindlIl site to the 3'
end of the
UTR (5'-CCG CAA GCT TCT AAT AAA AAA TAT ATA GTA-3', SEQ ID N0:20). The
amplified region extends from position 1350 to 1552 of GenBank accession
number
X79898. The 218 by PCR product is gel purified, digested with Spel and Hindlll
and ligated
with the Hindlll-Psfl cut PCR fragment carrying the T7 terminator into the
Spel-Psi1 sites of
pBluescript SK- (Stratagene), yielding pPH171. Sequence analysis of the psbA
3' UTR
region of pPH171 compared to GenBank accession number X79898 reveals deletion
of
adenine nucleotides at positions 1440 and 1452.
Example 10: Construction of a vector using a polyguanosine tract as a
substitute for
a3'UTR
A polyguanosine tract has been shown to substitute functionally for the
plastid atp8 gene 3'
UTR in vivo (Drager et al. (1996) RNA 2:652-663). A poly G tract containing 18
consecutive
guanosine residues flanked by Spel, Hindlll sticky ends on the 5' and 3' ends
respectively is
assembled by annealing the following two kinased oligonucleotides: (5'-CTA GTG
GGG
GGG GGG GGG GGG GGA-3', SEGO lD N0:21 ) and (5'-AGC TTC CCC CCC CCC CCC
CCC CCA-3', SEQ ID N0:22). The polyG~etract containing Spel, Hindlll sticky
ends is
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ligated with the Hindlll, Psfl digested PCR fragment containing the T7
terminator into the
Spel, Psfl sites of pBluescript SK+ (Stratagene), yielding pAT222.
Exampie 11: Preparation of a chimeric gene containing the ragweed pollen
allergen
Amb a Li coding sequence fused to a bacteriophage T7 gene 10 promoter and
terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco piastid
transformation vector
A bacteriophage T7 gene 10 promoter :: Amb a 1.1 :: A. thaliana psbA 3' UTR ::
T7
terminator cassette is constructed by a four-way ligation of the 96 by EcoRl,
Ncol PCR
fragment containing the T7 promoter, the 1.2 kb Ncol, Xbal PCR fragment
containing the
Amb a 1.1 cDNA and the 295 by Xbal, Pstl fragment of pPHl7i containing the A.
thaliana
psbA 3' UTR and T7 terminator into the EcoRl, Psd sites of pGEM-3Z
(Stratagene), yielding
plasmid pAT230. The T7 promoter driven Amb a 1.1 gene cassette is ligated to
the aadA
selectable marker cassette by cloning the 1.1 kb Hindlll, EcoRl fragment of
pAT229
containing the 16S rRNA gene promoter-rbs :: aadA :: rpsl6 3' UTR cassette and
the 1.6 kb
EcoRl, Ps8 pAT230 fragment carrying the T7 promoter :: Amb a 1.1 :: psbA 3'
UTR :: T7
terminator cassette into the Hindlll, Psfl sites of pBluescript SK+
(Stratagene), producing
plasmid pAT234. Plastid transformation vector pAT238 is constructed by
ligating the 2.7 kb
Pstl band from pAT234 containing the Amb a 1.1 and selectable marker cassettes
into the
Psfl site of pAT218 and screening for an insert orientation where the Amb a
1.1 gene is
transcribed in the same direction as the rpsl2/70RF.
Example 12: Preparation of a chimeric gene containing the ragweed pollen
allergen
Amb a 1.1 coding sequence fused to a bacteriophage T7 gene 10 promoter and
terminator and a pofyguanosine tract in a tobacco plastid transformation
vector
The plastid transformation vector pAT239 containing the T7 gene 10 promoter ::
Amb a I. i
:: polyguanosine tract :: T7 terminator and the 16S rRNA gene promoter-rbs ::
aadA :: rpsl6
3' UTR cassette is constructed by a four-way ligation of the 96 by EcoRl, Ncol
PCR
fragment containing the T7 promoter, the 1.2 kb Ncol, Xbal PCR fragment
containing the
Amb a 1.1 cDNA and the 30 by Xbal, Hindlll fragment of pAT222 carrying the
poly G,8 tract
into the 5.9 kb EcoRl, Hindlll vector fragment of pAT238 containing the
selectable marker,
T7 terminator and flanking homologous plastid regions and screening for an
insert
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orientation where the Amb a 1.1 gene is transcribed in the same direction as
the rpsl2/7
ORF.
Example 13: Preparation of a chimeric gene containing the Dermatophagoides
allergen Der f I coding sequence fused to a bacteriophage T7 gene 10 promoter
and
terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the Derf I gene is described in US patent 5,770,202.
This
sequence is used to design PCR primers for amplification of the Der f I coding
sequence
from Dermatophagoides farinae cDNA. The cDNA is obtained by reverse
transcription (as
for Amb a 1.1) from total RNA extracted from a D. farinae dust mite
preparation (Greer
Laboratories). A bacteriophage T7 gene 10 promoter :: Der f I :: A. thaliana
psbA 3' UTR ::
T7 terminator cassette is constructed as described for pAT230. The T7 promoter
driven Der
f I gene cassette is ligated to the aadA selectable marker cassette and
inserted into the
Hindlll, Psfl sites of pBluescript SK+ (Stratagene). A plastid transformation
vector is then
constructed as described in example 11.
Example 14: Preparation of a chimeric gene containing the Dermatophagoides
allergen Der f II coding sequence fused to a bacteriophage T7 gene 10 promoter
and
terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the Der f Il gene is described in US patent 5,770,202.
This
sequence is used to design PCR primers for amplification of the Der f lI
coding sequence
from Dermatophagoides farinae cDNA. The cDNA is obtained by reverse
transcription (as
for Amb a 1.1) from total RNA extracted from a D. farinae dust mite
preparation (Greer
Laboratories). A bacteriophage T7 gene 10 promoter :: Der f ll :: A. thaliana
psbA 3' UTR ::
T7 terminator cassette is constructed as described for pAT230. The T7 promoter
driven Der
f II gene cassette is ligated to the aadA selectable marker cassette and
inserted into the
Hindlll, Psfl sites of pBluescript SK+ (Stratagene). A plastid transformation
vector is then
constructed as described in example 11.
Example 15: Preparation of a chimeric gene containing the Dermatophagoides
allergen Der p I coding sequence fused to a bacteriophage T7 gene 10 promoter
and
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terminator and the Arabidopsls plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the Der p I gene is described in US patent 5,770,202.
This
sequence is used to design PCR primers for amplification of the Der p I coding
sequence
from Dermatophagoides pteronyssinus cDNA. The cDNA is obtained by reverse
transcription (as for Amb a 1.1) from total RNA extracted from a D.
pteronyssinus dust mite
preparation (Greer Laboratories). A bacteriophage T7 gene 10 promoter :: Der p
I :: A.
thaliana psbA 3' UTR :: T7 terminator cassette is constructed as described for
pAT230. The
T7 promoter driven Der p ! gene cassette is ligated to the aadA selectable
marker cassette
and inserted into the Hindlll, Pstl sites of pBluescript SK+ (Stratagene). A
plastid
transformation vector is then constructed as described in example 11.
Example 16: Preparation of a chimeric gene containing the Dermatophagoides
allergen Der p II coding sequence fused to a bacteriophage T7 gene 10 promoter
and
terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the Der p II gene is described in US patent 5,770,202.
This
sequence is used to design PCR primers for amplification of the Der p II
coding sequence
from Dermatophagoides pteronyssinus cDNA. The cDNA is obtained by reverse
transcription (as for Amb a 1.1) from total RNA extracted from a D,
pteronyssinus dust mite
preparation (Greer Laboratories). A bacteriophage T7 gene 10 promoter :: Der p
II :: A.
thaliana psbA 3' UTR :: T7 terminator cassette is constructed as described for
pAT230. The
T7 promoter driven Der p II gene cassette is ligated to the aadA selectable
marker cassette
and inserted into the Hindlll, Psti sites of pBluescript SK+ (Stratagene). A
plastid
transformation vector is then constructed as described in example 11.
Example 17: Preparation of a chimeric gene containing the Johnson grass pollen
allergen Sor h I coding sequence fused to a bacteriophage T7 gene 10 promoter
and
terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the Sor h I gene is described in US patent 5,480,972. A
bacteriophage T7 gene 10 promoter :: Sor h 1:: A, thaliana psbA 3' UTR :: T7
terminator
cassette is constructed. The T7 promoter driven Sor h 1 gene cassette is
ligated to the
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aadA selectable marker cassette and inserted into the Hindlll, Psfl sites of
pBluescript SK+
(Stratagene). A plastid transformation vector is then constructed as described
in example
11.
Example 18: Preparation of a chimeric gene containing the birch pollen
allergen Bet V
I coding sequence fused to a bacteriophage T7 gene 10 promoter and terminator
and
the Arabidopsis plastid psbA 3' UTR in a tobacco plastid transformation vector
The coding sequence of the Bet V I gene is described in Breiteneder et al.
(1989) EMBO J.
8: 1935-1938. A bacteriophage T7 gene 10 promoter :: Bet V I :: A, thaliana
psbA 3' UTR ::
T7 terminator cassette is constructed. The T7 promoter driven Bet V I gene
cassette is
ligated to the aadA selectable marker cassette and inserted into the Hindlll,
Pstl sites of
pBluescript SK+ (Stratagene). A plastid transformation vector is then
constructed as
described in example 11.
Example 19: Preparation of a chimeric gene containing the mosquito salivary
allergen
rAed a 1 coding sequence fused to a bacteriophage T7 gene 10 promoter and
terminator and the Arabfdopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the rAed a 1 gene is described in WO 98/04274. A
bacteriophage
T7 gene 10 promoter :: rAed a 1:: A. thaliana psbA 3' UTR :: T7 terminator
cassette is
constructed. The T7 promoter driven rAed a 7 gene cassette is ligated to the
aadA
selectable marker cassette and inserted into the Hindfll, Psti sites of
pBluescript SK+
(Stratagene). A plastid transformation vector is then constructed as described
in example
11.
Example 20: Preparation of a chimeric gene containing the glutamic acid
decarboxylase (GAD) coding sequence fused to a bacteriophage T7 gene 10
promoter
and terminator and the Arabidopsis plastid psbA 3' UTR in a tobacco plastid
transformation vector
The coding sequence of the human GAD gene is obtained from Genbank (accession
number L16888). A bacteriophage T7 gene 10 promoter :: GAD:: A, thaliana psbA
3' UTR ::
T7 terminator cassette is constructed. The T7 promoter driven GAD gene
cassette is
ligated to the aadA selectable marker cassette and inserted into the Hindlll,
Psfl sites of
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pBluescript SK+ (Stratagene). A plastid transformation vector is then
constructed as
described in example 11.
Example 21: Preparation of a chimeric gene containing the thyroglobulin coding
sequence fused to a bacteriophage T7 gene 10 promoter and terminator and the
Arabidopsis plastid psbA 3' UTR in a tobacco plastid transformation vector
The coding sequence of the thyroglobulin gene is obtained from Genbank
(accession
number U93033). A bacteriophage T7 gene 10 promoter :: thyroglobulin:: A.
thaliana psbA
3' UTR :: T7 terminator cassette is constructed. The T7 promoter driven
thyroglobulin gene
cassette is ligated to the aadA selectable marker cassette and inserted into
the Hindlll, Pstl
sites of pBluescript SK+ (Stratagene). A plastid transformation vector is then
constructed
as described in example 11.
Example 22: Biolistic Transformation of the Tobacco Plastid Genome
Seeds of Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in
a 1" circular
array on T agar medium and bombarded 12-14 days after sowing with 1 l.rt~n
tungsten
particles (M10, Biorad, Hercules, CA) coated with plasmid DNA essentially as
described in
Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917. Bombarded seedlings are
incubated on
T medium for two days after which leaves are excised and placed abaxial side
up in bright
light (350-500 Nmol photons/m2/s) on plates of RMOP medium (Svab, Z.,
Hajdukiewicz, P.
and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 Ng/ml spectinomycin
dihydrochloride (Sigma, St. Louis, MO). Resistant shoots appearing underneath
the
bleached leaves three to eight weeks after bombardment are subcloned onto the
same
selective medium, allowed to form callus, and secondary shoots isolated and
subcloned.
Complete segregation of transformed plastid genome copies (homoplasmicity) in
independent subclones is assessed by standard techniques of Southern blotting
(Sambrook
et al., (1989) Molecular Clonin : A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold
Spring Harbor). BamHl/EcoRl-digested total cellular DNA (Mettler, I. J. (1987)
Plant Mol
Biol Reporier5, 346-349) is separated on 1% Tris-borate (TBE) agarose gels,
transferred
to nylon membranes (Amersham) and probed with 32P-labeled random primed DNA
sequences corresponding to a 0.7 kb BamHIIHindlll DNA fragment from pC8
containing a
portion of the rps7/12 plastid targeting sequence (see patent application WO
98/11235).
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Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA
medium
(McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the
greenhouse.
Example 23: Construction of a plastid-targeted bacteriophage T7 RNA polymerise
gene fused to the tobacco PR-1 a promoter
A synthetic oligonucleotide linker comprising an Ncol restriction site and ATG
start codon
followed by the first seven plastid transit peptide codons from the rbcS gene
(encoding the
small subunit of ribulose bisphosphate carboxylase) and endogenous Pstl
restriction site
(top strand: 5'-CAT GGC TTC CTC AGT TCT TTC CTC TGC A-3', SEQ ID N0:23; bottom
strand: 5'-GAG GAA AGA ACT GAG GAA GC-3', SEQ ID N0:24), a 2.8 kb PstllSacl
DNA
fragment of pCGN4205 (McBride, K. E. et al. (1994) PNAS 91, 7301-7305)
containing the
bacteriophage T7 RNA polymerise gene (T7 Pol) fused in frame to the 3' portion
of the
rbcS gene transit peptide coding sequence, a 0.9 kb NcollKpnl DNA fragment of
pCIB296
containing the tobacco PR-1 a promoter with an introduced Ncol restriction
site at the start
codon (Uknes et al. (1993) Plant Cell 5, 159-169) and 4.9 kb SfillKpnl and 6.6
kb SacllSfil
fragments of binary Agrobacterium transformation vector pSGCGC1 (a derivative
of
pGPTV-Hyg containing the pofylinkerfrom pGEM4 (Promega, Madison WI) cloned
into the
SacllHindlll sites) are ligated to construct pPH110.
Example 24: Introduction of the chimeric PR-1 a / T7 Pol gene into the tobacco
nuclear
genome by Agrobacterium-mediated leaf disc transformation
Hygromycin resistant NT-pPH110 tobacco plants are regenerated as described
from shoots
obtained following cocultivation of leaf disks of N. tabacum 'Xanthi' and
"NahG" (Friedrich et
al. (1995) Plant Mol Bio129:959-68) with GV3101 Agrobacterium carrying the
pPH110
binary vector. For each transgenic line duplicate leaf punches of
approximately 2-3 cm2 are
incubated for 2 days in 3 ml of BTH (5.6 mg/10 ml) or sterile distilled water
under ca. 300
NmoUm2/s irradiance. Leaf material is harvested, flash frozen and ground in
liquid nitrogen.
Total RNA is extracted (Verwoerd et al. (1989) NAR 17, 2362) and Northern blot
analysis is
carried out as described (Ward et al. (1991 ) The Plant Cell 3, 1085-1094}
using a
radiolabelled T7 RNA polymerise gene probe. Plants of nineteen NT-110X (Xanthi
genetic
background) and seven NT-110N (NahG genetic background) Ti lines showing a
range of
T7 Pol expression are transferred to the greenhouse and self pollinated.
Progeny
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segregating 3:1 for the linked hygromycin resistance marker are selfed and
homozygous T2
lines selected.
Example 25: Induction of ragweed allergen expression in plastids of transgenic
plants
Homozygous NT-11 OX and NT-11 ON plants containing the PR-1 a-T7 RNA Pol
construct are
used to pollinate homoplasmic plastid transformant lines carrying the
maternally inherited
pAT238 and pAT239 constructs. The Nt_ pAT238 x NT-110X or NT_110N, and
Nt_pAT239
x NT-110X or NT_110N F1 progeny (which were heterozygous for the PR-1/T7
polymerase
nuclear expression cassette and homoplasmic for the T7I Amb a I.1_plastid
expression
cassette) are germinated on soil. Upon reaching a height of 20-40 cm, the
plants are
sprayed with the inducer compound BTH to elicit T7 Pol-regulated expression of
the Amb a
1.1 gene that is localized to the plastids. Plant material is harvested just
prior to induction
and at 8 hours and 1, 2, 3, 7, and 14 or 28 days following induction and flash
frozen in
liquid nitrogen. Similar procedures are applied for transgenic plants
comprising the Der f I,
Sor h I, Bet V I and rAed a 1 allergen genes, and the GAD and thyroglobulin
genes.
Example 2fi: Determination of antigen expression and content of transgenic
plants
Total RNA is extracted (Verwoerd et al. (1989) NAR 17, 2362) and Northern blot
analysis is
carried out as described (Ward et al. (1991 ) The Plant Cell 3, 1085-1094)
using
radiolabelled probes specific for the Amb a 1.1 gene. In order to determine
the amount of
Amb a L 1 present in the tissues of transgenic plants, chemiluminescent
(Amersham)
Western blot analysis is performed according to the manufacturer's
instructions and Harlow
and Lane (1988) Antibodies: A laboratory manual, Cold Spring Harbor
Laboratory, Cold
Spring Harbor using antisera raised against the Amb a 1.1 proteins and
purified Amb a l.1
protein standards. Similar procedures are applied for the Der f l, Sor h I and
Bet V I, rAed a
1 allergens, and for GAD and thyroglobulin.
Example 27: Induction of oral tolerance in rats
Female Lewis or Wistar Furth rats weighing 150 to 220 g (6-8 weeks of age) are
used. Rats
are immunized in both hind footpads with 50 ~Cg guinea pig antigen emulsified
in complete
Freund's adjuvant {CFA). In some experiments, 50 pg ovalbumin (OVA) (Sigma) is
added to
the emulsified antigen and injected similarly. For induction of oral
tolerance, rats are fed
transgenic plants of the present invention five times at three-day intervals.
Nine days after
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immunization, the rats are sacrificed and their popliteal lymph nodes are
removed. A single
cell suspension is prepared by pressing the lymph nodes through a stainless
steel mesh. A
total of 105 lymph node cells (LNC) are cultured with the indicated number of
either
irradiated (2000 Rads) or intact LNC derived from fed rats in quadruplicate in
round-
bottomed 96-well plate (Costar). Antigens (50 pg/ml) are added to the culture
in a volume of
20 NI. The cultures are incubated for 80 hours and are pulsed with 1 NCi [3 H]
TdR/well for
the last 16 hours of culture. The cultures are then harvested on an automatic
cell harvester
and read on a standard liquid scintillation counter. The percentage of primed
LNC (PLNC)
proliferation is then calculated. Proliferation Media RPMI (Gibco) is used in
all the
experiments. The medium is filtered sterile after adding 2x10-5 M 2-
mercaptoethanol, 1%
sodium pyruvate, 1 % penicillin and streptomycin, 1 % non-essential amino
acids, and 1
autologous serum.
Example 28: Measurement of antibodies
A. Serum Levels of Antibodies
A solid-phase enzyme-linked immunoabsorbent assay (ELISA) is used for
determination of
antibody titers against the antigen. Microtiter plates are incubated with 0.1
ml per well of 10
Ng antigen/ml in doubled distilled water. Plates are incubated for 18 hrs at
25° C. After 3
washes with PBS/Tween-20 (Bio-Rad), pH 7.5, plates are incubated with 3%
BSA/PBS for 2
hrs at 37° C, washed twice, and 100 NI of diluted serum is added in
quadruplicate. The
plates are incubated for 2 hrs at 37° C. After three rinses with
PBS/Tween-20, plates are
incubated with 100 NI/well of peroxidase-conjugated goat anti-rat IgG antibody
(Tago, USA)
diluted 1:1000 in 1 % BSA/PBS for 1 hr at 25° C. Color reaction is
obtained by exposure to
D-phenylenediamine (0.4 mg/ml phosphate) citrate buffer, pH 5.0) containing
30% H2O2.
The reaction is stopped by adding 0.4N H2 S04 and OD 492 nm is read on an
EL1SA
reader.
B. In Vitro Measurement of Antibody Production:
Popliteal and splenic LNC are obtained from fed, naive and challenged rats and
seeded at
a concentration of 107 cells per ml petri dish either alone or irradiated.
(2000 Rads) together
with other PLNC as indicated. The cultures are maintained in proliferation
media, with or
without antigen (20 Ng/ml), for 3 days in an incubator and then harvested. The
diluted
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supernatants are used to examine the in vitro production and secretion of IgG
antibody and
are measured for antibody production using an ELISA test as described
previously.
Example 29: Induction of oral tolerance in mice
Female Balb/c mice aged 6 to 8 weeks are used. For induction of oral
tolerance, mice are
fed extracts from tobacco plants expressing recombinant ragweed from the T71
Amb a 1.1
plastid expression cassette according to Example 22. More specifically, mice
are fed
Fraction 1 or Fraction 2 of tobacco plants. transformed with the T7/ Amb a l.1-
plastid
expression cassette or, as a control, Fraction 1 or Fraction 2 from
untransformed, control
tobacco plants. Naive animals are divided into three recombinant ragweed-fed
groups (0.1,
1, and 10 mg recombinant ragweed/day) and one untransformed tobacco-fed group
(as
control). Each group consists of 8 animals. Oral administration of antigens to
the animals
starts after an initial 10 day period of acclimatization to the vivarium
facility. Animals are
given by gavage the corresponding tobacco fraction adjusted to a final volume
of 0.2-0.5 ml
with H20 or phosphate-buffered saline once daily for 5-8 sequential days.
Induction of oral tolerization is assessed four days after the last oral
feeding of tobacco-
expressed allergen. All four groups of mice are sensitized three times by
intraperitoneal
injection of recombinant ragweed (10 p.g/animal} at two-week intervals. In
addition to
recombinant ragweed, each mouse is also sensitized to ovalbumin (10 pg/animal;
Sigma
Chemical, St. Louis, MO, USA) to demonstrate that the mice are capable of
mounting an
immune response to other antigens. Antigens are dissolved in 0.9% saline and
mixed with
aluminum hydroxide (2:1; Alu Gel S Senra, Feinbiochemica GmbH, Heidelberg,
Germany)
to give the desired concentration in a final volume of 200 p.l/mouse.
Serum samples for determination of antigen-specific IgE, IgGI, and IgG2a
levels are
collected four times: once prior to start of oral administration of antigen
and at appropriate
time points for each of the three immunizations. Sera are aliquoted and stored
at -20 oC
until assayed.
All serum samples are subjected to capture, solid-phase enzyme-linked
immunosorbent
assay (ELISA) for determination of recombinant ragweed and ovalbumin-specific
serum IgE,
IgGI, and IgG2a levels. Ninety-six well microtiter plates (Immunoplates
MaxisorpTM
Surface, Nunc, Roskilde, Denmark) are incubated with 0.1 ml per well of
corresponding
antigen (1-10 p.g/ml) in 0.015 M sodium carbonate bicarbonate buffer (pH 9)
for 2 h at 37
oC followed by overnight at 4 oC. All washings are performed with 0.8 % salt
solution
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containing 0.05% Tween-solution (pH 7.4; washing buffer; Sigma Chemical).
Unless noted,
serum samples, revealing antibodies, and reagents are diluted in washing
buffer containing
1 % heat-inactivated fetal calf serum, all incubations are performed in a
humid atmosphere,
and the reaction volume is 100 pl/well. After coating, the plates are washed
three times in
washing buffer. Serum samples are applied in three-fold serial dilutions to
the antigen-
coated wells and incubated at 37 oC for 2 h. After washing three times in
washing buffer,
corresponding biotinylated revealing antibodies (So. Biotechnology Assoc.,
Birmingham,
AL, USA; Binding Site, Birmingham, UK) are applied to the plates at 37 oC for
1 h. After
washing, horseradish peroxidase-labeled Avidin D is applied to detect
immunoreactivity
(1:3,000, 60 min, 37 oC; Vector Lab., Burlingame, CA, USA). After rewashing,
plates are
additionally washed three times using citrate buffer (substrate buffer, pH 5).
Enzyme activity
is detected with o-phenylenediamine (Sigma Chemical) in substrate buffer (0.5
mg/ml) and
hydrogen peroxide (1 p.l/mi) and stopped after 1-15 min with 4 N H2S04 (50
pl/well; Merck
KgaA, Darmstadt, Germany). Optical densities are determined
spectrophotometrically at
492 nm with an ELISA reader. Final results are expressed in relative ELISA
units (REU)
compared to a standard serum pool (arbitrarily assigned 100 REU) or in
absolute protein
concentrations (p,g/ml).
Similar procedures to test for oral tolerance are applied for transgenic
plants comprising the
Der f 1, Sor h I, Bet v I and rAed a 1 allergen genes, and the GAD and
thyroglobulin genes
or any other antigen of the invention.
Induction of oral tolerance to house dust mite extract is described in Sato et
al., Immunology
95: 193-199, 1998. House dust mite experiments in humans are described by
Passalacqua
et al., Lancet, 351: 629-632, 1998.
Example 30: Chemically-regulated expression of the human BPI gene in plant
plastids
I Construction of a plastid transformation vector containing the BPI gene
under control of a
promoter element responsive to the bacteriophag_e T7 RNA polymerase
The coding sequence of the human BPI gene {as described in US patent
5,198,541) is
cloned as a 182 by NcollEcoRl restriction fragment (containing the 5' portion
of the cDNA
and incorporating the start codon into the Ncol site) and a 1288 by EcoRllXbal
restriction
fragment (containing the 3' portion of the cDNA sequence and the termination
codon).
These fragments are gel-purified and ligated in a three-way reaction to a 7693
by NcollXbal
fragment from plasmid pT7 GUS. The resulting plasmid (pT7_BPI) is verified by
DNA
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sequencing and restriction analysis to contain the BPI gene downstream of the
T7 gene 10
promoter from plasmid pET3a (Novagen) with a chimeric 5' untranslated leader
containing
the sequence 5'-GGG AGA CCA CAA CGG TTT CCC TCT AG-3' (SEQ ID N0:25} derived
from the T7 gene 10 leader, the linker sequence 5'-CGAGG-3' derived from the
Stul linker
of plasmid pCB, and the sequence 5'-GGG AGT CCC TGA TGA TTA AAT AAA CCA AGA
TTT TAC CAT GG-3' (SEQ ID N0:26) derived from the tobacco plastid psbA gene 5'
leader
modified to include an Ncol restriction site at the start codon _(underlined).
pT7_GUS is
constructed by ligating a gel-purified 386 by EcoRllNcol fragment of plasmid
pPH142 that
contains the chimeric T7 promoter/psbA 5' leader to a dephosphorylated and gel
purified
9241 by NcollEcoRl fragment of GUS plastid transformation vector pC8
(international
Patent Application WO 98/11235). Plasmid pPH142 is constructed by digesting
plasmid
pPH136 with BsaBl (2 hr at 60° C), gel purifying and redigesting the
linearized plasmid DNA
with BsmFl (1 hr at 37° C), treating with alkaline phosphatase (1 hr at
37° C followed by
phenol extraction) and ligating the resulting gel-purified 3398 by fragment to
a synthetic
double-stranded oligonucleotide made by annealing the oligonucleotides T73a_U
(5'-CGA
TCC CGC GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC C-3',
SEQ ID N0:27) and T73a_L (5'-TAG AGG GAA ACC GTT GTG GTC TCC CTA TAG TGA
GTC GTA TTA ATT TCG CGG GAT CG-3', SEQ ID N0:28) subsequent to phosphorylation
with T4 polynucleotide kinase (1 hr at 37° C). Plasmid pPH136 DNA is
amplified in an E.
coli strain lacking dam methylase activity (DM-1, Stratagene, La Jolla,
California) in order to
permit digestion with BsaBl. pPH136 is in turn constructed by ligating a 3428
by gel-
purified SfullNcol fragment from plasmid pPH120 to a synthetic double-stranded
oligonucleotide -made by annealing the oligonucfeotides minpsb U (5'-GGG AGT
CCC TGA
TGA TTA AAT AAA CCA AGA TTT TAC-3', SEQ ID N0:29) and minpsb_L (5'-CAT GGT
AAA ATC TTG GTT TAT TTA ATC ATC AGG GAC TCC C-3', SEQ ID N0:30). This
oligonucleotide comprises a 38 nt portion of the tobacco psbA gene 5'
untranslated leader
sequence implicated in translational up-regulation of the psbA gene product in
an in-vitro
plastid translation system (Hirose and Sugiura, EMBO J. 15: 1687-1695, 1996).
pPH120
comprises a modified portion of the psbA promoter-driven aadA gene and
divergent T7-lac
promoter of plasmid pC8 (derived from Novagen plasmid pET2ld and a chimeric
ribosome
binding site from the tobacco plastid rbcL gene, into which a fragment of
yeast DNA is
inserted in order to block the low-level bidirectional transcriptional
activity of the psbA
promoter characteristic of vectors derived from plasmid pPRV111A (Allison et
al., EMBO J.
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1996 Jun 3;15(11 ):2802-9). PPH120 is constructed by (igating a 256 by
EcoRllHincll
fragment of the Saccharomyces cerevisiae LEU2 gene, a 2645 by EcoRllDralll
fragment of
pPH119, and a 569 by Dralll/Klenow-filled EcoRl fragment of pPH119. PPH119 is
in turn
constructed by digesting plasmid pPH118 with Stul and religating in order to
remove the
duplicated Stul linker present in the pC8-derived chimeric T7 promoter of
pPH118. PPH118
contains a 235 by SpellNcol fragment of pC8 that is isolated and gel purified
and then
ligated into cloning vector pGEMSZf(+)(Promega, Madison WI) that is digested
with Ncol
and Spel.
II' Biolistic transformation of the tobacco plastid qenome with plasmid pT7
BPI
Transformation of the plastid genome is carried out as described in Example
22. Resistant
shoots appearing underneath the bleached leaves three to eight weeks after
bombardment
are subcloned onto the same selective medium, allowed to form callus, and
secondary
shoots isolated and subcloned. Complete segregation of transformed plastid
genome
copies (homoplasmicity) in independent subclones is assessed by standard
techniques of
Southern blotting (Sambrook et al., (1989) Molecular Cloning: A Laboratory
Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor). BamHllNcol-digested total
cellular DNA
(Mettler, I. J. (1987) Plant Mol Biol Reporter5, 346-349) is separated on 1 %
Tris-borate
(TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with
32P-
labeled random primed DNA sequences corresponding to a 0.7 kb BamHllHindlll
DNA
fragment from pC8 containing a portion of the rps7/12 plastid targeting
sequence.
Homoplasmic shoots containing the expected 1.25 kb fragment and lacking the
wild-type
3.3 kb fragment are rooted aseptically on spectinomycin-containing MS/IBA
medium
(McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the
greenhouse.
Ill: Chemical induction of BPI expression in tobacco plants transformed with
plasmid
pT7 BPI and carryinc,~a bacteriophage T7 RNA polymerase gene with a plastid-
targeting
sequence in the nuclear qenome under control of the BTH-responsive PR-1 a
promoter.
Homoplasmic Nt pT7 BPI plants of line C35BP1-5B-4 are pollinated by tobacco
line
Nt_110X6b-5 and the seed tested for maternal inheritance of the spectinomycin
resistance
marker carried by the transforming plasmid pT7_BPI. Additional seeds of these
F1 progeny
are germinated in soil in 6 inch clay pots. Eight weeks after sowing, plants
are sprayed with
a foliar application of 1.0 mM BTH until run-off occurs. Tissue is harvested
for analysis of
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BPI accumulation at intervals of 0, 1, 2, 3, 7, 14, 21, 28 and 35 days
following BTH
application. Control plants of the same line sprayed with water or an inert
ingredient, as
well as C35BP1-58-4 plants carrying only the plastid BPI gene and not the
chemically-
inducible transactivator are germinated at the same time and assayed in the
same manner
using the same time schedule. Accumulation of BPI is assayed by standard
techniques of
Northern hybridization (mRNA) and Western blotting (protein). Additionally,
activity assays
are performed using leaf extracts in order to assess the ability of the plant-
expressed BPI to
inhibit the exponential growth in liquid medium of gram-negative bacteria
(e.g. E. colr).
Example 31: Construction of Expression Cassettes for Nuclear Expression of a
Transgene in Plants
Gene sequences intended for expression in transgenic plants are firstly
assembled in
expression cassettes behind a suitable promoter and preferably upstream of a
suitable
transcription terminator. All requirement for constructions of plant
expression cassettes
apply to DNA molecules of the present invention, in particular DNA molecules
encoding a
transactivator or a therapeutically active protein and are carried out using
techniques well-
known in the art.
Promoter Selection
The selection of promoter used in expression cassettes determines the spatial
and temporal
expression pattern of the transgene in the transgenic plant. Selected
promoters express
transgenes in specific cell types (such as leaf epidermal cells, mesophyll
cells, root cortex
cells) or in specific tissues or organs (roots, leaves or flowers, for
example) and this
selection reflects the desired location of biosynthesis of the transgene.
Alternatively, the
selected promoter may drive expression of the transgener under a light-induced
or other
temporally regulated promoter. A further alternative is that the selected
promoter be
chemically regulated. This provides the possibility of inducing the expression
of the
transgene only when desired and caused by treatment with a chemical inducer.
3' Regulator)r Sequences
A variety of 3' regulatory sequences are available for use in expression
cassettes. In the
plant nucleus, these sequences are for example responsible for the termination
of
transcription beyond the transgene and its correct polyadenylation.
Appropriate
transcriptional terminators and those which are known to function in plants
and include the
CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, and
the pea
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rhcS E9 terminator. These can be used in both monocotyledonous and
dicotyledonous
plants.
Sequences for the Enhancement or Regulation of Exoression
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the
transgene to
increase its expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly
in
monocotyledonous cells. For example, th.e introns of the maize Adh 1 gene have
been
found to significantly enhance the expression of the wild-type gene under its
cognate
promoter when introduced into maize cells. Intron 1 was found to be
particularly effective in
enhancing expression of fusion constructs made with the chloramphenicol
acetyltransferase
gene (Callis et al., Genes Develop 1: 1183-1200 (1987)). In the same
experimental system,
the intron from the maize bronzel gene had a similar effect in enhancing
expression (Callis
et al., supra). Intron sequences have been routinely incorporated into plant
transformation
vectors, typically within the non-translated leader. A number of non-
translated leader
sequences derived from viruses are also known to enhance expression, and these
are
particularly effective in dicotyledonous cells. Specifically, leader sequences
from Tobacco
Mosaic Virus (TMV, the "S2-sequence"}, Maize Chlorotic Mottle Virus (MCMV),
and Alfalfa
Mosaic Virus (AMV) have been shown to be effective in enhancing expression
(e.g. Gallie
et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec.
Biol. 15; 65-79
(1990)).
Example 32: Targeting of Gene Products within the Cell
Various mechanisms for targeting gene products are known to exist in plants
and the
sequences controlling the functioning of these mechanisms have been
characterized in
some detail. For example, the targeting of gene products to plastids, for
example
chloroplasts, is controlled by a signal sequence found at the aminoterminal
end of various
proteins and which is cleaved during chloroplast import yielding the mature
protein (e.g.
Comai et al. J. Biol. Chem. 263: 15104-15109 (1988}}. These signal sequences
can be
fused to heterologous gene products to effect the import of heterologous
products into the
chloroplast (van den Broeck et aI. Nature 313: 358-363 (1985)). DNA encoding
for
appropriate signal sequences can be isolated from the 5' end of the cDNAs
encoding the
RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein
and many
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other proteins which are known to be chforoplast localized. The signal
sequence selected
should include the known cleavage site and the fusion constructed should take
into account
any amino acids after the cleavage site which are required for cleavage. In
some cases this
requirement may be fulfilled by the addition of a small number of amino acids
between the
cleavage site and the transgene ATG or alternatively replacement of some amino
acids
within the transgene sequence. Fusions constructed for chloroplast import can
be tested for
efficacy of chloroplast uptake by in vitro translation of in vitro transcribed
constructions
followed by in vitro chloroplast uptake using techniques described by
(Bartlett et al. In:
Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp
1081-1091
(1982); Wasmann ef al. Mol. Gen. Genet. 205: 446-453 (1986)). Accordingly, any
of these
signal sequences are fused to a nucleotide sequence encoding a transactivator
to target
the transactivator into the chloroplast. The above described mechanisms for
cellular
targeting are utilized not only in conjunction with their cognate promoters,
but also in
conjunction with heterologous promoters so as to effect a specific cell
targeting goal under
the transcriptional regulation of a promoter which has an expression pattern
different to that
of the promoter from which the targeting signal derives.
Other gene products are localized to other organelles such as the
mitochondrion and the
peroxisome (e.g. Unger etaL Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs
encoding
these products can also be manipulated to effect the targeting of heterologous
gene
products to these organelles. Examples of such sequences are the nuclear-
encoded
ATPases and specific aspartate amino transferase isoforms for mitochondria.
Targeting to
cellular protein bodies has been described by Rogers ef al. (Proc. Natl. Acad.
Sci. USA 82:
6512-6516 (1985)). In addition sequences have been characterized which cause
the
targeting of gene products to other cell compartments. Aminoterminal sequences
are
responsible for targeting to the ER, the apoplast, and extracellular secretion
from aleurone
cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally,
aminoterminal sequences
in conjunction with carboxyterminal sequences are responsible for vacuolar
targeting of
gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
Example 33: Examples of Expression Cassette Construction
The present invention encompasses the expression of a DNA molecule of the
present
invention under the regulation of any promoter which is expressible in plants,
regardless of
the origin of the promoter. Therefore the DNA molecule of the present
invention is inserted
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into any of the expression cassettes using techniques well-known in the art.
These
expression cassettes can then be easily transferred to the plant
transformation vectors
described below. Furthermore, the invention also encompasses the use of any
plant-
expressible promoter in conjunction with any further sequences required or
selected for the
expression of the transgene. Such sequences include, but are not restricted
to,
transcriptional terminators, extraneous sequences to enhance expression (such
as introns
[e.g. Adh intron 1 ], viral sequences [e. g. TMV-SZ]), and sequences intended
for the
targeting of the gene product to specific organelles and cell compartments.
Constitutive Expression: the CaMV 35S Promoter
Construction of the plasmid pCGN1761 is described in the published patent
application EP
0 392 225. pCGN1761 contains the "double" 35S promoter and the fm!
transcriptional
terminator with a unique EcoRl site between the promoter and the terminator
and has a
pUC-type backbone. A derivative of pCGN1761 is constructed which has a
modified
polylinker which includes Notl and Xhol sites in addition to the existing
EcoRl site. This
derivative is designated pCGN176i ENX. pCGN1761 ENX is useful for the cloning
of cDNA
sequences or gene sequences (including microbial ORF sequences) within its
polylinker for
the purposes of their expression under the control of the 35S promoter in
transgenic plants.
The entire 35S promoter-gene sequence-tml terminator cassette of such a
construction can
be excised by Hindlll, Sphl, Sall, and Xbal sites 5' to the promoter and Xbal,
BamHl and
Bgll sites 3' to the terminator for transfer to transformation vectors.
Furthermore, the double
35S promoter fragment can be removed by 5' excision with HindIll, Sphl, Sall,
Xbal, or Pstt,
and 3' excision with any of the polylinker restriction sites (EcoRl, Notl or
Xhon for
replacement with another promoter.
Expression under a Chemically Requlatable Promoter
(1) PR1-a Promoter
This section describes the replacement of the double 35S promoter in pCGN1761
ENX with
any promoter of choice; by way of example the chemically regulated f'R-1 a
promoter is
described. The promoter of choice is preferably excised from its source by
restriction
enzymes, but can alternatively be PCR-amplified using.primers which carry
appropriate
terminal restriction sites. Should PCR-amplification be undertaken, then the
promoter
should be resequenced to check for amplification errors after the cloning of
the amplified
promoter in the target vector. The chemically regulatable tobacco PR-1 a
promoter is
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cleaved from plasmid pCIB1004 (see EP 0 332 104) and transferred to plasmid
pCGN1761 ENX. pCIB1004 is cleaved with Ncol and the resultant 3' overhang of
the
linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The
fragment
is then cleaved with Hindlll and the resultant PR-1 a promoter containing
fragment is gel
purified and cloned into pCGN1761 ENX from which the double 35S promoter has
been
removed. This is done by cleavage with Xhol and blunting with T4 pofymerase,
followed by
cleavage with Hindlll and isolation of the larger vector-terminator containing
fragment into
which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX
derivative with the PR-1 a promoter and the tml terminator and an intervening
polylinker with
unique EcoRl and Notl sites.
A nucleotide sequence encoding a DNA molecule of the present invention is
inserted into
this vector, and the fusion product (i.e. promoter-gene-terminator) is
subsequently
transferred to any selected transformation vector, including those described
in this
application, thus providing for chemically inducible expression of the
transactivator.
,(2) Ethanol-Inducible Promoter
A promoter inducible by certain alcohols or ketones, such as ethanol, is also
used to confer
inducible expression of a transgene of the present invention. Such a promoter
is for
example the alcA gene promoter from Aspergillus nidulans (Caddick et al.
(1998) Nat
Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcohol
dehydrogenase I,
whose expression is regulated by the AIcR transcription factors in presence of
the chemical
inducer. For the purposes of the present invention, the CAT gene sequences in
plasmid
paIcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S
promoter
(Caddick et al. (1998) Nat Biotechnol 16:177-180) are replaced by a nucleotide
sequence
encoding a transgene to form an expression cassette having the nucleotide
sequence
encoding a transgene under the control of the alcA gene promoter. This is
carried out using
methods well known in the art. In a preferred embodiment, the minimal 35S
promoter is
replaced by any convenient minimal promoter, such as e.g. the maize Bronze-1
minimal
promoter (Roth et al. (1991 ) The Plant Cell 3: 317-325). Termination signals
in paIcA:CAT
can also be replaced by other termination signals well known in the art. The
resulting
construct is transformed into a desired plant species. The alcR gene is also
comprised in
the plant comprising the nucleotide sequence encoding a DNA molecule of the
present
invention fused to the alcA gene promoter and the expression of the alcR gene
is controlled
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by any promoter suitable for expression in plants known in the art or
described here. Thus,
tissue- or organ-specificity of the alcR gene product is achieved leading to
inducible tissue-
or organ-specificity of the transgene. Any termination signals known in the
art are also
suitable for the alcR expression cassette.
(31 Glucocorticoid-Inducible Promoter
Induction of expression of a DNA molecule of the present invention using
systems based on
steroid hormones is also contemplated. For example, a glucocorticoid-mediated
induction
system is used (Aoyama and Chua (1997) The Plant Journal 11: 605-612) and gene
expression is induced by application of a glucocorticoid, for example a
synthetic
glucocorticoid, preferably dexamethasone, preferably at a concentration
ranging from 0.1
mM to 1 mM, rnore preferably from 10 mM to 100 mM. For the purposes of the
present
invention, the luciferase gene sequences are replaced by a nucleotide sequence
encoding
a transgene to form an expression cassette having the nucleotide sequence
encoding a
transgene under the control of six copies of the GAL4 upstream activating
sequences fused
to the 35S minimal promoter. This is carried out using methods well known in
the art. In a
preferred embodiment, the minimal 35S promoter is replaced by any convenient
minimal
promoter, such as, e.g., the maize Bronze-1 minimal promoter (Both et al.
(1991 ) The Plant
Cell 3: 317-325). Termination signals can also be replaced by other
termination signals well
known in the art. The resulting construct is transformed into a desired plant
species. The
traps-acting factor comprises the GAL4 DNA-binding domain (Keegan et al.
(1986) Science
231: 699-704) fused to the transactivating domain of the herpes viral protein
VP16
(Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to the hormone-
binding domain
of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080).
The expression
of the fusion protein is controlled by any promoter suitable for expression in
plants known in
the art or described here. This expression cassette is also comprised in the
plant comprising
the nucleotide sequence encoding a transgene fused to the 6xGAL4/minimal
promoter.
Thus, tissue- or organ-specificity of the fusion protein is achieved leading
to inducible
tissue- or organ-specificity of the transgene. Any termination signals known
in the art are
also suitable for the expression cassette comprising the fusion protein.
Constitutive Expression: the Actin Promoter
Several isoforms of actin are known to be expressed in most cell types and
consequently
the actin promoter is a good choice for a constitutive promoter. In
particular, the promoter
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from the rice Act1 gene has been cloned and characterized (McElroy et al.
Plant Cell 2:
163-171 (1990)). A 1.3 kb fragment of the promoter is found to contain all the
regulatory
elements required for expression in rice protopfasts. Furthermore, numerous
expression
vectors based on the Act1 promoter have been constructed specifically for use
in
monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991 )). These
incorporate
the Act1-intron 1, Adh1 5' flanking sequence and Adh1-intron 1 (from the maize
alcohol
dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing
highest expression are fusions of 35S and the Act1 intron or the Act? 5'
flanking sequence
and the Acti intron. Optimization of sequences around the initiating ATG (of
the GUS
reporter gene) also enhanced expression. The promoter expression cassettes
described by
McElroy et aL (Mol. Gen. Genet. 231: 150-160 (1991 )) can be easily modified
for the
expression of the nucleotide sequence of the present invention and are
particularly suitable
for use in monocotyledonous hosts. For example, promoter containing fragments
can be
removed from the McElroy constructions and used to replace the double 35S
promoter in
pCGN1761 ENX, which is then available for the insertion or specific gene
sequences. The
fusion genes thus constructed can then be transferred to appropriate
transformation
vectors. In a separate report the rice Act1 promoter with its first intron has
also been found
to direct high expression in cultured barley cells (Chibbar et at. Plant Cell
Rep. 12: 506-509
{1993)).
A nucleotide sequence encoding a DNA molecule of the present invention is
inserted
downstream of such a promoter, and the fusion products (i.e. promoter-gene-
terminator) are
subsequently transferred to any selected transformation vector, including
those described in
this application.
Constitutive Expression: the Ubiquitin Promoter
Ubiquitin is another gene product known to accumulate in many cell types and
its promoter
has been cloned from several species for use in transgenic plants (e.g.
sunflower - Binet et
al. Plant Science 79: 87-94 (1991 ), maize - Christensen et al. Plant Molec.
Biol. 12: 619-632
(1989)). The maize ubiquitin promoter has been developed in transgenic monocot
systems
and its sequence and vectors constructed for monocot transformation are
disclosed in the
patent publication EP 0 342 926. Further, Taylor et al. (Plant Cell Rep. 12:
491-495 (1993))
describe a vector (pAHC25) which comprises the maize ubiquitin promoter and
first intron
and its high activity in cell suspensions of numerous monocotyledons when
introduced via
microprojectile bombardment. The ubiquitin promoter is clearly suitable for
the expression of
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the nucleotide sequence in transgenic plants, especially monocotyledons.
Suitable vectors
are derivatives of pAHC25 or any of the transformation vectors described in
this application,
modified by the introduction of the appropriate ubiquitin promoter and/or
intron sequences.
The Arabidopsis Ubiquitin 3 (UBQ3) gene promoter (Norris et al. (1993) Plant
Mol Biol
21:895-906) is also a promoter suitable for expression of a transgene of the
present
invention in plants.
A nucleotide sequence encoding a transgene is therefore inserted into any of
these vectors,
and the fusion products (i.e. promoter-gene-terminator) are used for
transformation of
plants, resulting in constitutive expression of the transgene.
Root Specific Expression
A preferred pattern of expression for a transgene of the present invention is
root
expression. A suitable root promoter is that described by de Framond (FEBS
290: 103-106
(1991 )) and also in the published patent application EP 0 452 269. This
promoter is
transferred to a suitable vector such as pCGN1761 ENX and a nucleotide
sequence
encoding a transgene is inserted into such vector. The entire promoter-gene-
terminator
cassette is subsequently transferred to a transformation vector of interest.
Wound Inducible Promoters
Wound-inducible promoters are particularly suitable for the expression of a
transgene.
Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol.
22: 573-
588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle,
Plant Molec.
Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993),
Warner et al.
Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant
invention.
Logemann et al. (supra) describe the 5' upstream sequences of the
dicotyledonous potato
wun 1 gene. Xu et al. (supra) show that a wound inducible promoter from the
dicotyledon
potato (pint) is active in the monocotyledon rice. Further, Rohrmeier & Lehle
(supra)
describe the cloning of the maize INip1 cDNA which is wound induced and which
can be
used to isolated the cognate promoter using standard techniques. Similarly,
Firek et al.
(supra) and Warner et al. (supra) have described a wound induced gene from the
monocotyledon Asparagus officinalis which is expressed at local wound and
pathogen
invasion sites. Using cloning techniques well known in. the art, these
promoters can be
transferred to suitable vectors, fused to a nucleotide sequence encoding a
transgene, and
used to express these genes at the sites of insect pest infection.
Pith Preferred Expression
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Patent application WO 93/07278 describes the isolation of the maize trpA gene
which is
preferentially expressed in pith cells. Using standard molecular biological
techniques, this
promoter or parts thereof, can be transferred to a vector such as pCGN1761
where it can
replace the 35S promoter and be used to drive the expression of a transgene of
the present
invention in a pith-preferred manner. In fact fragments containing the pith-
preferred
promoter or parts thereof can be transferred to any vector and modified for
utility in
transgenic plants. Pith preferred expression of a nucleotide sequence encoding
a transgene
is achieved by inserting the nucleotide sequence encoding a transgene in such
vector.
Pollen-Specific Expression
Patent Application WO 93/07278 further describes the isolation of the maize
calcium-
dependent protein kinase (CDPK) gene which is expressed in pollen cells. The
gene
sequence and promoter extend up to 1400 by from the start of transcription.
Using
standard molecular biological techniques, this promoter or parts thereof, can
be transferred
to a vector such as pCGN1761 where it can replace the 35S promoter and be used
to drive
the expression of a transgene of the present invention in a pollen-specific
manner. In fact
fragments containing the pollen-specific promoter or parts thereof can be
transferred to any
vector and modified for utility in transgenic plants.
Leaf-Specific Expression
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by
Hudspeth
& Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular
biological
techniques the promoter for this gene is used to drive the expression of a
transgene of the
present invention in a leaf-specific manner in transgenic plants.
Expression with Chloro~last Tarcleting
Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) have described the
successful
use of a chloroplast transit peptide for import of a heterologous transgene.
This peptide
used is the transit peptide from the rbcS gene from Nicoflana plumbaglnlfolia
(Poulsen et al.
Mol. Gen. Genet. 205: 193-200 (1986)). Using the restriction enzymes Dral and
Sphl, or
Tsp5091 and Sphl the DNA sequence encoding this transit peptide can be excised
from
plasmid prbcS-8B (Poulsen et al. supra) and manipulated for use with any of
the
constructions described above. The Dral-Sphl fragment extends from -58
relative to the
initiating rbcS ATG to, and including, the first amino acid (also a
methionine) of the mature
peptide immediately after the import cleavage site, whereas the Tsp5091-Sphl
fragment
extends from -8 relative to the initiating rbcS ATG to, and including, the
first amino acid of
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the mature peptide. Thus, these fragment can be appropriately inserted into
the polylinker
of any chosen expression cassette generating a transcriptional fusion to the
untranslated
leader of the chosen promoter (e.g. 35S, PR-1a, actin, ubiquitin etc.), whilst
enabling the
insertion of a nucleotide sequence encoding a transgene in correct fusion
downstream of
the transit peptide. Constructions of this kind are routine in the art. For
example, whereas
the Dral end is already blunt, the 5' Tsp5091 site may be rendered blunt by T4
polymerase
treatment, or may alternatively be ligated to a linker or adaptor sequence to
facilitate its
fusion to the chosen promoter. The 3' Sphl site may be maintained as such, or
may
alternatively be ligated to adaptor of linker sequences to facilitate its
insertion into the
chosen vector in such a way as to make available appropriate restriction sites
for the
subsequent insertion the nucleotide sequence encoding a transactivator.
Ideally the ATG of
the Sphl site is maintained and comprises the first ATG of the DNA molecule.
Chen &
Jagendorf (supra) provide consensus sequences for ideal cleavage for
chloroplast import,
and in each case a methionine is preferred at the first position of the mature
protein. At
subsequent positions there is more variation and the amino acid may not be so
critical. In
any case, fusion constructions can be assessed for efficiency of import in
vitro using the
methods described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in
Chloroplast
Molecular Biology, Elsevier. pp 1081-1091 (1982}} and Wasmann et al. (Mol.
Gen. Genet.
205: 446-453 (1986)). Typically the best approach may be to generate fusions
using the
DNA molecule with no modifications at the aminoterminus, and only to
incorporate
modifications when it is apparent that such fusions are not chloroplast
imported at high
efficiency, in which case modifications may be made in accordance with the
established
literature (Chen & Jagendorf, supra; Wasman et al., supra; Ko & Ko, J. Biol.
Chem. 267:
13910-13916 (1992)}.
A preferred vector is constructed by transferring the Dral-Sphl transit
peptide encoding
fragment from prbcS-8B to the cloning vector pCGN1761 ENX/Sph-. This plasmid
is cleaved
with EcoRl and the termini rendered blunt by treatment with T4 DNA polymerase.
Plasmid
prbcS-8B is cleaved with Sphl and ligated to annealed molecular adaptors. The
resultant
product is 5'-terminally phosphorylated by treatment with T4 kinase.
Subsequent cleavage
with Dral releases the transit peptide encoding fragment which is ligated into
the blunt-end
ex-EcoRl sites of the modified vector described above. Clones oriented with
the 5' end of
the insert adjacent to the 3' end of the 35S promoter are identified by
sequencing. These
clones carry a DNA fusion of the 35S leader sequence to the rbcS-8A promoter-
transit
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peptide sequence extending from -58 relative to the rbcS ATG to the ATG of the
mature
protein, and including at that position a unique Sphl site, and a newly
created EcoRl site, as
well as the existing Notl and Xhol sites of pCGN1761 ENX. This new vector is
designated
pCGN1761/CT. DNA sequences are transferred to pCGN1761/CT in frame by
amplification
using PCR techniques and incorporation of an Sphl, Nsphl, or Nlalll site at
the amplified
ATG, which following restriction enzyme cleavage with the appropriate enzyme
is ligated
into Sphl-cleaved pCGN1761/CT. To facilitate construction, it may be required
to change
the second amino acid of cloned gene, however, in almost all cases the use of
PCR
together with standard site directed mutagenesis will enable the construction
of any desired
sequence around the cleavage site and first methionine of the mature protein.
A further preferred vector is constructed by replacing the double 35S promoter
of
pCGN1761 ENX with the BamHl-Sphl fragment of prbcS-8A which contains the full-
length
light regulated rbcS-8A promoter from -1038 (relative to the transcriptional
start site) up to
the first methionine of the mature protein. The modified pCGN1761 with the
destroyed Sphl
site is cleaved with Psfl and EcoRl and treated with T4 DNA polymerase to
render termini
blunt. prbcS-8A is cleaved Sphl and ligated to the annealed molecular adaptor
of the
sequence described above. The resultant product is 5'-terminally
phosphorylated by
treatment with T4 kinase. Subsequent cleavage with BamHl releases the promoter-
transit
peptide containing fragment which is treated with T4 DNA polymerase to render
the BamHl
terminus blunt. The promoter-transit peptide fragment thus generated is cloned
into the
prepared pCGN1761 ENX vector, generating a construction comprising the rbcS-8A
promoter and transit peptide with an Sphl site located at the cleavage site
for insertion of
heterologous genes. Further, downstream of the Sphl site there are EcoRl (re-
created),
Notl, and Xhol cloning sites. This construction is designated pCGN1761
rbcS/CT.
Similar manipulations can be undertaken to utilize other GS2 chloroplast
transit peptide
encoding sequences from other sources (monocotyledonous and dicotyledonous)
and from
other genes.
Modification of pCGNi 761 ENX by Optimization of the Translational Initiation
Site
For any of the constructions described in this section, modifications around
the cloning sites
can be made by the introduction of sequences which may enhance translation.
This is
particularly useful when genes derived from microorganisms are to be
introduced into plant
expression cassettes as these genes may not contain sequences adjacent to
their initiating
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methionine which may be suitable for the initiation of translation in plants.
In cases where
genes derived from microorganisms are to be cloned into plant expression
cassettes at their
ATG it may be useful to modify the site of their insertion to optimize their
expression.
Modification of pCGN1761 ENX is described by way of example to incorporate one
of
several optimized sequences for plant expression (e.g. Joshi, supra).
pCGN1761 ENX is cleaved with Sphl, treated with T4 DNA polymerase and
religated, thus
destroying the Sphl site located 5' to the double 35S promoter. This generates
vector
pCGN1761 ENX/Sph-. pCGN1761 ENX/Sph- is cleaved with EcoRl, and ligated to an
annealed molecular adaptor. This generates the vector pCGNSENX which
incorporates the
quasi-optimized plant translational initiation sequence TAAA-C adjacent to the
ATG which is
itself part of an Sphl site which is suitable for cloning heterologous genes
at their initiating
methionine. Downstream of the Sphl site, the EcoRl, Nofl, and Xhol sites are
retained.
An alternative vector is constructed which utilizes an Ncol site at the
initiating ATG. This
vector, designated pCGN1761 NENX is made by inserting an annealed molecular
adaptor at
the pCGN1761 ENX EcoRl site. Thus, the vector includes the quasi-optimized
sequence
TAAACC adjacent to the initiating ATG which is within the Ncol site.
Downstream sites are
EcoRl, Notl, and Xhol. Prior to this manipulation, however, the twa Ncol sites
in the
pCGN1761 ENX vector {at upstream positions of the 5' 35S promoter unit) are
destroyed
using similar techniques to those described above for Sphl or alternatively
using "inside-
outside" PCR (Innes et aL PCR Protocols: A guide to methods and applications.
Academic
Press, New York (1990)). This manipulation can be assayed for any possible
detrimental
effect on expression by insertion of any plant cDNA or reporter gene sequence
into the
cloning site followed by routine expression analysis in plants.
Accordingly, a nucleotide sequence encoding a transgene is inserted into
pCGN1761 ENX
for constitutive expression under the control of the CaMV 35S promoter with an
optimized
Translation Initiation Site.
Example 34: Construction of Plant Transformation Vectors
Numerous transformation vectors are available for plant transformation, and a
nucleotide
sequence encoding a DNA molecule of the present invention is inserted into any
of the
expression cassettes described above, such that they are capable of expressing
the
transgene in desirable cells, under appropriate conditions. A resulting
expression cassette
is then incorporated into any appropriate transformation vector described
below.
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The selection of vector for use will depend upon the preferred transformation
technique and
the target species for transformation. For certain target species, different
antibiotic or
herbicide selection markers may be preferred. Selection markers used routinely
in
transformation include the nptll gene which confers resistance to kanamycin
and related
antibiotics (Vieira and Messing, Gene 19: 259-268 (1982); Bevan et al., Nature
304:184-187
(1983)), the hph gene which confers resistance to the antibiotic hygromycin
(Blochlinger &
Diggelmann, Mol Cell Biol 4: 2929-2931), the dhfrgene, which confers
resistance to
methatrexate (Bourouis and Jarry., EMBO J. 2 7 : 1099-1104 (1983)), and the
bacterial
aadA gene (Goldschmidt-Clermont, 1991, Nucl. Acids Res. 19: 4083-4089),
encoding
aminoglycoside 3'-adenylyltransferase and conferring resistance to
streptomycin or
spectinomycin. Other markers to be used include the bar gene which confers
resistance to
the herbicide phosphinothricin (White et aL, Nucl Acids Res 18: 1062 (1990),
Spencer et al.
Theor Appl Genet 79: 625-631 (1990)), a mutant EPSP synthase gene encoding
glyphosate resistance (Hinchee et al., 1988, Bio/Technology 6: 915-922), a
mutant
acetolactate synthase (ALS) gene which confers imidazolione or sulfonylurea
resistance
(Lee et al., 1988, EMBO J. 7: 1241-1248), a mutant psbA gene conferring
resistance to
atrazine (Smeda et al., 1993, Plant Physiol. 103: 911-917), or a mutant
protoporphyrinogen
oxidase gene as described in EP 0 769 059.
Selection markers resulting in positive selection, such as a phosphomannose
isomerase
gene, as described in patent application WO 93/05163, are also used.
Identification of transformed cells may also be accomplished through
expression of
screenable marker genes such as genes coding for chloramphenicol acetyl
transferase
(CAT), j3-glucuronidase (GUS), luciferase, and green fluorescent protein (GFP)
or any other
protein that confers a phenotypicalfy distinct trait to the transformed cell.
(1 ) Construction of Vectors Suitable for Agrobacterium Transformation
Many vectors are available for transformation using Agrobacterium tumefaclens.
These
typically carry at least one T-DNA border sequence and include vectors such as
pBIN19
(Bevan, Nucl. Acids Res. (1984)). Below the construction of two typical
vectors is
described.
Construction of pCIB200 andpCIB2001
The binary vectors pCIB200 and pCIB2001 are used for the construction of
recombinant
vectors for use with Agrobacterium and is constructed in the following manner.
pTJS75kan
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is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J Bacteriol.
164: 446-455
(1985)) allowing excision of the tetracycline-resistance gene, followed by
insertion of an
Accl fragment from pUC4K carrying an NPTII (Vieira and Messing, Gene 19: 259-
268
(1982); Bevan et al., Nature 304: 184-187 {1983); McBride et al., Plant
Molecular Biology
14: 266-276 (1990)). Xhol linkers are ligated to the EcoRV fragment of pCIB7
which
contains the left and right T-DNA borders, a plant selectable noslnptll
chimeric gene and
the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the Xhol-
digested
fragment is cloned into Sall-digested pTJS75kan to create pCIB200 {see also EP
0 332
104). pCIB200 contains the following unique polylinker restriction sites:
EcoRl, Sstl, Kpnl,
Bglll, Xbal, and Sall. pC182001 is a derivative of pCIB200 which created by
the insertion
into the polylinker of additional restriction sites. Unique restriction sites
in the polylinker of
pCIB200i are EcoRl, Ssfl, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll, Avrll, Apal,
Hpal, and Stul.
pCIB2001, in addition to containing these unique restriction sites also has
plant and
bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-
mediated
transformation, the RK2-derived frfA function for mobilization between E. coli
and other
hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker
is suitable
for the cloning of plant expression cassettes containing their own regulatory
signals.
Any one of the plant expression dassettes described above is inserted into
pCIB2001,
preferably using the polylinker.
Construction of pCIBlO and Hygromycin Selection Derivatives thereof
The binary vector pCIBlO contains a gene encoding kanamycin resistance for
selection in
plants, T-DNA right and left border sequences and incorporates sequences from
the wide
host-range plasmid pRK252 allowing it to replicate in both E. coli and
Agrobacterium. Its
construction is described by Rothstein ef aL (Gene 53: 153-161 (1987)).
Various
derivatives of pCIBlO have been constructed which incorporate the gene for
hygromycin B
phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These
derivatives
enable selection of transgenic plant cells on hygromycin only (pCIB743), or
hygromycin and
kanamycin (pCIB715, pCIB717). This vectors is used transform an expression
cassette of
the present invention.
(2) Construction of Vectors Suitable for non-Agrobacferium Transformation.
Transformation without the use of Agrobacferium tumefaciens circumvents the
requirement
for T-DNA sequences in the chosen transformation vector and consequently
vectors lacking
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these sequences can be utilized in addition to vectors such as the ones
described above
which contain T-DNA sequences. Transformation techniques which do not rely on
Agrobacierium include transformation via particle bombardment, protoplast
uptake (e.g.
PEG and electroporation), microinjection or pollen transformation (US Patent
5,629,183).
The choice of vector depends largely on the preferred selection for the
species being
transformed. Below, the construction of some typical vectors is described.
Construction of ~CIB3064
pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques
in
combination with selection by the herbicide Basta (or phosphinothricin). The
plasmid
pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli
GUS gene
and the CaMV 35S transcriptional terminator and is described in the PCT
published
application WO 93/07278. The 35S promoter of this vector contains two ATG
sequences 5'
of the start site. These sites are mutated using standard PCR techniques in
such a way as
to remove the ATGs and generate the restriction sites Sspl and Pvull. The new
restriction
sites are 96 and 37 by away from the unique Sall site and 101 and 42 by away
from the
actual start site. The resultant derivative of pCIB246 is designated pCIB3025.
The GUS
gene is then excised from pCIB3025 by digestion with Sall and Sacl, the
termini rendered
blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is
obtained from the
John Innes Centre, Norwich and the a 400 by Smal fragment containing the bar
gene from
Strepfomyces viridochromogenes is excised and inserted into the Hpal site of
pCIB3060
(Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064 which
comprises the bar gene under the control of the CaMV 35S promoter and
terminator for
herbicide selection, a gene for ampicillin resistance (for selection in E.
colt) and a polylinker
with the unique sites Sphl, Pstl, HindIll, and BamHl. This vector is suitable
for the cloning
of plant expression cassettes containing their own regulatory signals to
direct expression of
a transgene of the present invention.
Construction of pSOGl9 and pSOG35
pSOG35 is a transformation vector which utilizes the E. coli gene
dihydrofolate reductase
(DHFR) as a selectable marker conferring resistance to methotrexate. PCR is
used to
amplify the 35S promoter 0800 bp), intron 6 from the maize Adh1 gene (--550
bp) and 18
by of the GUS untransiated leader sequence from pSOGlO. A 250 by fragment
encoding
the E. coli dihydrofolate reductase type II gene is also amplified by PCR and
these two PCR
fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which
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comprised the pUCl9 vector backbone and the nopaline synthase terminator.
Assembly of
these fragments generated pSOGl9 which contains the 35S promoter in fusion
with the
intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase
terminator.
Replacement of the GUS leader in pSOGl9 with the leader sequence from Maize
Chlorotic
Mottle Virus (MCMV) generated the vector pSOG35. pSOGl9 and pSOG35 carry the
pUC
gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRl sites
available for the
cloning of foreign sequences, in particular a transgene of the present
invention.
Plastid Transformation Vectors
For constitutive expression of the nucleotide sequence of the present
invention in plant
plastids under control of the clpP gene promoter elements, plastid
transformation vector
pPH143 (example 36 of WO 97/32011 ) is used. The nucleotide sequence is
inserted into
pPH143 thereby replacing the PROTOX coding sequence. This vector is then used
for
plastid transformation and selection of transformants for spectinomycin
resistance.
Alternatively, the nucleotide sequence is inserted in pPH143 so that it
replaces the aadA
gene under control of the psbA gene promoter. In this case, transformants are
selected for
resistance to PROTOX inhibitors. In an alternative embodiment, promoters from
the plastid
16S ribosomal RNA genes of tobacco or Arabidopsis are used to express the
PROTOX
coding sequence.
Example 35: Plastid Transformation of Maize
Type I embryogenic callus cultures (Green et al. (1983) in A. Fazelahmad, K.
Downey, J.
Schultz, R.W. Voellmy, eds. Advances in Gene Technology: Molecular Genetics of
Plants
and Animals. Miami Winter Symposium Series, Vol. 20. Academic Press, N.Y.) are
initiated
from immature maize embryos, 1.5 - 2.5 mm in length, from greenhouse grown
material.
Embryos are aseptically excised from surface-sterilized ears approximately 14
days after
pollination. Embryos are either placed on D callus initiation media with 2%
sucrose and 5
mg/L chloramben (Duncan et af. (1985) Planta 165: 322-332) or onto KM callus
initiation
media with 3% sucrose and 0.75 mg/L 2,4-d (Kao and Michayluk (1975} Planta
126, 105-
110). Embryos and embryogenic cultures are subsequently cultured in the dark.
Embryogenic responses are removed from the explants after -14 days.
Embryogenic
responses from D callus initiation media are placed onto D callus maintenance
media with
2% sucrose and 0.5 mg/L 2,4-d while those of while those from KM callus
initiation media
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are placed onto KM callus maintenance media with 2% sucrose and 5 mg/L
Dicamba. After
3 to 8 weeks of weekly selective subculture to fresh maintenance media, high
quality
compact embryogenic cultures are established. Actively growing embryogenic
callus pieces
are selected as target tissue for gene delivery. The callus pieces are plated
onto target
plates containing maintenance medium with 12% sucrose approximately 4 hours
prior to
gene delivery. The callus pieces are arranged in circles, with radii of 8 and
10 mm from the
center of the target plate. Plasmid DNA is precipitated onto gold
microcarriers as described
in the DuPont Biolistics manual. Two to three p.g of each plasmid is used in
each 6 shot
microcarrier preparation. Genes are delivered to the target tissue cells using
the PDS-
1000He Biolistics device. The settings on the Biolistics device are as
follows: 8 mm
between the rupture disc and the macrocarrier, 10 mm between the macrocarrier
and the
stopping screen and 7 cm between the stopping screen and the target. Each
target plate is
shot twice using 650 psi rupture discs. A 200 X 200 stainless steel mesh
(McMaster-Carr,
New Brunswick, NJ) is placed between the stopping screen and the target
tissue.
Five days later, the bombed callus pieces are transferred to maintenance
medium with 2%
sucrose and 0.5 mg/L 2,4-d, but without amino acids, and containing 750 or
1000 nM
Formula XVII. The callus pieces are placed for 1 hour on the light shelf 4-5
hours after
transfer or on the next day, and stored in the dark at 27°C for 5-6
weeks. Following the 5-6
week primary selection stage, yellow to white tissue is transferred to fresh
plates containing
the same medium supplemented with 500 or 750 nM Formula XVII. 4-5 hours after
transfer
or on the next day, the tissues are placed for 1 hour on the light shelf and
stored in the dark
at 27°C for 3-4 weeks. Following the 3-4 week secondary selection
stage, the tissues are
transferred to plates containing the same medium supplemented with 500 nM
Formula XVII.
Healthy growing tissue is placed for 1 hour on the light shelf and stored in
the dark at 27°C.
It is subcultured every two weeks until the colonies are large enough for
regeneration.
At that point, colonies are transferred to a modified MS medium (Murashige and
Skoog
(1962) Physiol. Plant 15: 473-497) containing 3% sucrose (MS3S) with no
selection agent
and placed in the light. Either 0.25 mg/L ancymidol and 0.5 mg/L kinetin are
added to this
medium to induce embryo germination or 2 mg/L benzyl adenine is added.
Regenerating
colonies are transferred to MS3S media without ancymidol and kinetin, or
benzyl adenine,
respectively, after 2 weeks. Regenerating shoots with or without roots are
transferred to
boxes containing MS3S medium and small plants with roots are eventually
recovered and
transferred to soil in the greenhouse.
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Example 36: Preparation of a chimeric gene containing the ragweed pollen
allergen
Amb a 1.1 coding sequence fused to the constitutive Arab(dopsis UB(~3 promoter
for
nuclear expression in plants
The ragweed pollen allergen Amb a 1.1 coding sequence is fused to the
Arabidopsis UBQ3
promoter (Norris et al. (1993) Plant Mol. Biol. 21: 895-906) by inserting into
the Ncol, Xbal
sites of pPH121 the 5' half of Amb a 1.1 as a 0.83 kb Ncol, SaA fragment from
pAT230 and
the 3' end of Amb a 1.1 as a 0.39 kb SaA, Xbal PCR generated fragment designed
to
remove the dam methylated Xbal site at the 3' end of the Amb a 1.1 gene. PCR
amplification is performed using pAT230 as a template and the following primer
pair: the
"top strand" primer (5'-GTC GCT TTC AAC ACG TTC AC-3', SEQ ID N0:31 ) and the
"bottom strand" primer which removes the A before the Xbal site (5'-GCG CTC
TAG ACA
TTA TAA GTG CTT AGT-3', SEQ ID N0:32). The 452 by amplification product is gel
purified, digested with Sall and Xbal and ligated as above, producing pAT240.
The Hindlll-
Xbal of pAT240 containing the UBQ3 promoter driving the Amb a L 1 gene is
ligated into a
11.3 kb Hindlll, Xbal digested pPH108 fragment, producing a binary vector
carrying a
constitutively expressed Amb a 1.1 gene.
Example 37: Preparation of a chimeric gene containing the putative mature
ragweed
allergen Amb a I. i coding sequence fused to the constitutive Arabidopsis UBA3
promoter for nuclear expression in plants
The Amb a 1.1 has a putative signal peptide cleavage site, with the alanine at
position 26
predicted to be residue +1 (Rafnar et al. (1991 ) J. Biol. Chem. 266: 1229-
1236). The Amb
a I. i coding sequence is modified to remove the signal peptide by PCR
amplification using
pAT230 as a template and the following oligonucleotide pair: the "top strand"
primer places
an ATG before the first codon of the mature Amb a l.1 protein, the first 20 by
of the mature
protein and a Ncol site at the newly created ATG (5'-GCA CCA TGG CCG AAG ATC
TCC
AGG AAA T-3', SEQ ID N0:33) and the "bottom strand" primer (5'-CTA CCA GCC CAT
CAA CAG ACT TAC-3', SEQ ID N0:34). A 594 by amplification product is produced
comprised of the 5' end of the Amb a 1.1 gene without the signal sequence. The
fragment
is gel purified, digested with Ncol and Clal and the 0.35 kb fragment is
ligated with the 3'
end of the Amb a 1.1 gene as a 0.81 kb Clal, Xbal fragment into the Ncol, Xbal
4.4 kb
fragment of pAT240, creating a Amb a 1. l gene without the signal peptide
fused to the
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Arabidopsis UBQ3 promoter, which is cloned along with a terminator sequence
into a binary
vector.
Example 38: Preparation of a chimeric gene containing the Dermatophagoides
farinae
major allergen Der f I coding sequence fused to the constitutive Arabidopsis
UBQ3
promoter for nuclear expression in plants
The Dermatophagoides allergen Der f I coding sequence cloned as described in
example
13 is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector
as
described in example 34.
Example 39: Preparation of a chimeric gene containing the Dermatophagoides
farinae
major allergen Der f II coding sequence fused to the constitutive Arabidopsis
UBG13
promoter for nuclear expression in plants
The Dermatophagoides allergen Der f II coding sequence cloned as described in
example
13 is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector
as
described in example 34.
Example 40: Preparation of a chimeric gene containing the Dermatophagoides
pteronyssinus major allergen Der p I coding sequence fused to the constitutive
Arabidopsis UBG13 promoter for nuclear expression in plants
The Dermatophagoides allergen Der p I coding sequence cloned as described in
example
13 is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector
as
described in example 34.
Example 41: Preparation of a chimeric gene containing the Dermatophagoides
pteronyssinus major allergen Der p II coding sequence fused to the
constitutive
Arabidopsis UBQ3 promoter for nuclear expression in plants
The Dermatophagoides allergen Der p II coding sequence cloned as described in
example
13 is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector
as
described in example 34.
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Example 42: Preparation of a chimeric gene containing the Johnson grass
allergen
Sor h I coding sequence fused to the constitutive Arabidopsis UBQ3 promoter
for
nuclear expression in plants
The Johnson grass allergen Sor h I coding sequence is fused to the Arabidopsis
UB03
promoter and inserted into a binary vector as described in example 34.
Example 43: Preparation of a chimeric gene containing the birch pollen
allergen Bet V
I coding sequence fused to the constitutive Arabidopsis UBQ3 promoter for
nuclear
expression in plants
The birch pollen allergen Bet V I coding sequence is fused to the Arabidopsis
UBQ3
promoter and inserted into a binary vector as described in example 34.
Example 44: Preparation of a chimeric gene containing the mosquito salivary
allergen
rAed a 1 coding sequence fused to the constitutive Arabidopsis UBG73 promoter
for
nuclear expression in plants
The rAed a T coding sequence is fused to the Arabidopsis UBQ3 promoter and
inserted into
a binary vector as described in example 34.
Example 45: Preparation of chimeric genes for vacuolar expression containing
the
ragweed pollen allergen Amb a L 1 coding sequence fused to the constitutive
Arabidopsis UBA3 promoter
Plasmid pAT240 containing the ragweed pollen allergen gene Amb a 1.1 is used
as a
template for PCR with a "top strand" primer extending from position 775 to 799
in the Amb a
L 1 gene relative to the endogenous ATG (5'-GCA ACG GTC GCT TTC AAC ACG TTC A-
3',
SEQ ID N0:35) and a "bottom strand" primer whose sequence is homologous to the
last 21
by of the Amb a 1. l and includes 21 by of a vacuolar targeting sequence
derived from a
tobacco chitinase gene (Shinshi et al., (1990) Plant Mol. Biol. 14, 357-368,
Neuhaus et al.
(1991 ) Proc. Natl. Acad. Sci. USA 88, 10362-10366), the last colon of the
same tobacco
chitinase gene and a Xbal restriction site {5'-CGC TCT AGA TTA CAT AGT ATC GAC
TAA
AAG TCC GCA AGG TGC TCC GGG TTG GCA-3', SEQ ID N0:36). PCR amplification
generates a 447 by product which is gel purified and digested with Sall and
Xbal. The 383
by Sall, Xbal fragment containing the 3' end of Amb a 1.1 fused to a tobacco
chitinase
vacuolar targeting sequence is ligated to the 5' end of Amb a L 1 as a 0.83 kb
Ncol, Sall
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fragment from pAT240 into the 4.4 kb Ncol, Xbal fragment of pAT240 containing
the UB03
promoter and vector. The Arabidopsis UBQ3 promoter :: Amb a 1.1 coding
sequence fused
to a tobacco chitinase vacuolar targeting sequence cassette is cloned with a
terminator
sequence into a binary vector.
Plasmid pSCH10 (Shinshi et al., (1990) Plant Mol. Biol. 14, 357-368, Neuhaus
et al. (1991 )
Proc. Natl. Acad. Sci. USA 88, 10362-10366) is used as a template for PCR
amplification
with a "top strand" primer whose sequence is homologous to the first 22 by of
the tobacco
chitinase 23 amino acid N-terminal signal peptide and a BspHJ restriction site
at the ATG
(ErspU 5'-CGG TCA TGA GGC TTT GTA AAT TCA CAG-3', SEQ !D N0:37) and a "bottom
strand" primer whose sequence is homologous to the last 17 by of the tobacco
chitinase N-
terminal signal peptide fused to the first 14 by of the putative mature 5' end
of the Amb a L 1
gene (ErspL 5'-TGG AGA TCT TCG GCT GCC GAG GCA GAA AGC A-3', SEQ ID N0:38).
The 88 by amplification product is gel purified, cleaved with BspHl and Bglll
and the 76 by
fragment containing the tobacco chitinase 23 amino acid N-terminal signal
peptide fused to
the 5' end of the Amb a 1.7 gene without the native signal peptide is ligated
to the 3' end of
the Amb a I. i gene containing the tobacco chitinase vacuolar targeting
sequence as a Bglll,
Xbal fragment into the 4.4 kb NcoJ, Xbal fragment of pAT240 containing the
UBQ3
promoter and vector. The Arabidopsis UBQ3 promoter :: Amb a 1. f gene with the
native
signal peptide removed, a N-terminal tobacco chitinase signal peptide and a C-
terminal
tobacco chitinase vacuolar targeting sequence cassette is cloned with a
terminator
sequence into a binary vector.
Plasmid pSCHlO is used as a template for PCR amplification with a "top strand"
primer
whose sequence is homologous to the first 22 by of the tobacco chitinase 23
amino acid N-
terminal signal peptide and a BspHl restriction site at the ATG (ErspU) and a
"bottom
strand" primer whose sequence is homologous to the last 17 by of the tobacco
chitinase N-
terminal signal peptide fused to a 5' extension of the first 13 by of Amb a L
1 (ErspovL 5'-
AGT GTT TGA TCC CTG CCG AGG CAG AAA GCA-3', SEQ ID N0:39). Plasmid pAT240
is used as a template for PCR amplification with a "top strand" primer whose
sequence is
homologous to the first 25 by after the start codon of Amb a 1.1 and which
anneals to 13 by
of primer ErspovL (AmboeU 5'-GGG ATC AAA CAC TGT TGT TAC ATC T-3', SEQ ID
N0:40) and a "bottom strand" primer extending from positions 135 to 158 in the
Amb a L 1
gene relative to the endogenous ATG. The two PCR amplification products are
gel purified
and the PCR overlap extension technique is performed to fuse the two products
together as
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_81 _
follows: 2 ml of each purified amplification product is combined in one PCR
reaction tube
and 10 cycles are done in the absence of primers in order to anneal and extend
the fusion
product, then 5 ml of the reaction is used as a template for PCR amplification
using the
"outside" primers ErspU and AmboeL. The final amplification product consists
of the
tobacco chitinase N-terminal 23 amino acid signal peptide with a BspHl site at
the initiating
codon fused in frame to the Amb a 1.1 gene from the first amino acid after the
ATG to 155
by downstream. The product is gel purified, digested with BspHl and Bglll and
ligated to
the 3' end of the Amb a 1.1 gene containing the tobacco chitinase vacuolar
targeting
sequence as a Bglll, Xbal fragment into the 4.4 kb Ncol, Xbal fragment of
pAT240
containing the UBQ3 promoter and vector. The Arabidopsis UBQ3 promoter :: Amb
a 1.1
gene , a N-terminal tobacco chitinase signal peptide and a C-terminal tobacco
chitinase
vacuolar targeting sequence cassette is cloned with a terminator sequence into
a binary
vector.
Example 46: Preparation of chimeric genes for vacuolar expression containing
the
ragweed pollen allergen Amb a 1.1 coding sequence fused to the tobacco PR-1a
promoter
The PR-1 a promoter from PR-1 aDXhoNco (Uknes et al. (1993), The Plant Cell 5,
159-169)
is excised as a 903 by Xhol, Ncol fragment and ligated to a Ncol, Xbal
fragment containing
the Amb a 1.1 gene, a N-terminal tobacco chitinase signal peptide and a C-
terminal tobacco
chitinase vacuolar targeting sequence into the Xhol, Xbal sites of pLITMUS28
(New
England Biolabs). The PR-1 a promoter :: Amb a l.1 gene, a N-terminal tobacco
chitinase
signal peptide and a C-terminal tobacco chitinase vacuolar targeting sequence
cassette is
cloned with a terminator sequence into a binary vector. The Xhol, Ncol PR-1a
promoter
fragment is also ligated to a Ncol, Xbal fragment containing the Amb a I. i
gene with the
native signal peptide removed, a N-terminal tobacco chitinase signal peptide
and a C-
terminal tobacco chitinase vacuolar targeting sequence cassette into the Xhol,
Xbal sites of
pLITMUS28. The PR-1 a promoter :: Amb a l.1 gene with the native signal
peptide removed,
a N-terminal tobacco chitinase signal peptide and a C-terminal tobacco
chitinase vacuolar
targeting sequence cassette is cloned with a terminator sequence into a binary
vector.
Upon reaching a height of 20-40 cm, transgenic plants comprising the above
described
chimeric gene are sprayed with the inducer compound BTH to elicit expression
of the Amb
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a I. i gene. Plant material is harvested just prior to induction and at 8
hours and 1, 2, 3, 7,
and 14 or 28 days following induction and flash frozen in liquid nitrogen.
The above disclosed embodiments are illustrative. This disclosure of the
invention will place
one skilled in the art in possession of many variations of the invention. All
such obvious and
foreseeable variations are intended to be encompassed by the appended claims.
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-1 -
SEQUENCE LISTING
<110> Novartis AG
<120> Oral tolerance
<130> S-30674A/S-30675 CGC 2034/2035
<140>
<141>
<150> US 09/168231
<151> 1998-10-07
<150> US 09/167362
<151> 1998-10-07
<160> 40
<170> PatentIn Ver. 2.2
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 1
gcggccatgg ggatcaaaca ctgttgtta 29
<210> 2
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 2
gcggtctaga tcattataag tgcttagt 28
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 3
taacggccgc gcccaatcat tccggata 28
CA 02344269 2001-03-08
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-2-
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 4
taactgcaga aagaaggccc ggctccaa 28
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 5
cgcctgcagt cgcactatta cggatatg 28
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 6
cgccgtacga aatccttccc gatacctc 28
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 7
gccagaattc gccgtcgttc aatgagaatg 30
<210> 8
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
CA 02344269 2001-03-08
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-3-
<400> 8
gccttcatga tccctcccta caactatcca ggcgcttcag attcg 45
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 9
cagttcgagc ctgattatcc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 10
gttcttacgc gttactcacc 20
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 11
cgcgactagt tcaaccgaaa ttcaat 26
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 12
cgctctgcag ttcaatggaa gcaatg 26
<210> 13
<211> 18
<212> DNA
CA 02344269 2001-03-08
WO 00/20612 PCT/EP99107414
-4-
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 13
accgtaaggc ttgatgaa
18
<210> 14
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 14
cccactagtt tgaacgaatt gttagac 27
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 15
cccgaattca tcccgcgaaa ttaata 26
<210> 16
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 16
cggccatggg tatatctcct tcttaaagtt aaa 33
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 17
gcgaagcttg ctgagcaata actagcataa 30
CA 02344269 2001-03-08
PCT/EP99/07414
WO 00!20612
-5-
<210> 18
<211> 28
<212> DNA .
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 18
gcgctgcagt ccggatatag ttcctcct 28
<210> 19
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 19
gcgactagtt agtgttagtc taaatctagt t 31
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 20
ccgcaagctt ctaataaaaa atatatagta 30
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 21
ctagtggggg gggggggggg ggga 24
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
CA 02344269 2001-03-08
WO 00/20612 PCT/EP99/07414
-6-
<223> Description of Artificial Sequence:
oligonucleotide
<400> 22
agcttccccc cccccccccc ccca 24
<210> 23
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 23
catggcttcc tcagttcttt cctctgca 28
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 24
gaggaaagaa ctgaggaagc 20
<210> 25
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 25
gggagaccac aacggtttcc ctctag 26
<210> 26
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 26
gggagtccct gatgattaaa taaaccaaga ttttaccatg g 41
<210> 27
CA 02344269 2001-03-08
WO 00/20612 PCT/EP99/07414
_7_
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 27
cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc cc 52
<210> 28
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 28
tagagggaaa ccgttgtggt ctccctatag tgagtcgtat taatttcgcg ggatcg 56
<210> 29
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 29
gggagtccct gatgattaaa taaaccaaga ttttac 36
<210> 30
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 30
catggtaaaa tcttggttta tttaatcatc agggactccc
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
CA 02344269 2001-03-08
WO 00/20612 PC'T/EP99/07414
_g_
<400> 31
gtcgctttca acacgttcac 20
<210> 32
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 32
gcgctctaga cattataagt gcttagt 27
<210> 33
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 33
gcaccatggc cgaagatctc caggaaat 2g
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 34
ctaccagccc atcaacagac ttac 24
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 35
gcaacggtcg ctttcaacac gttca 25
<210> 36
<211> 54
<212> DNA
<213> Artificial Sequence
CA 02344269 2001-03-08
WO 00/20612 PCT/EP99/07414
_g_
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 36
cgctctagat tacatagtat cgactaaaag tccgcaaggt gctccgggtt ggca 54
<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 37
cggtcatgag gctttgtaaa ttcacag 27
<210> 38
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 38
tggagatctt cggctgccga ggcagaaagc a 31
<210> 39
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 39
agtgtttgat ccctgccgag gcagaaagca 30
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide
<400> 40
gggatcaaac actgttgtta catct 25
