Note: Descriptions are shown in the official language in which they were submitted.
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FIELD OF INVENTION
[0001 ] The present invention relates to the production of GM-CSF in plants.
[0002] BACKGROUND OF THE INVENTION
s [0003] At present, the majority of recombinant protein-based medicines are
produced
in mammalian cells or single cell organisms such as bacteria and yeast.
However, the
capital investment and operational costs associated with these systems are
very high.
For example, a mammalian cell-based manufacturing plant can cost upwards of
$250
million. To achieve greater cost savings, and to address a capacity deficit in
the global
1o demand for recombinant protein-based pharmaceuticals, plants are being
explored as
alternative protein productions hosts (Giddings et al., 2000 ; Staub et al.,
2000;
Daniell et al., 2001; Walmsley et al., 2003). Different plant tissues such as
leaves,
seeds and tubers have been engineered for producing useful recombinant
proteins
(Vandekerckhove et al., 1989; Sijmons et al., 1990; Pen et al., 1992; Herbers
et al.,
15 1995; Ma et al., 1995; van Rooijen et al., 1995; Arakawa et al., 1998; Y
Kusnadi et
al., 1998; Zeitlin et al., 1998; Farran et al., 2002 ; Tackaberry et al.,
1999). In a
number of studies, tobacco has been used as a host plant but has some major
drawbacks, including that tobacco is not a major food substance in a mammalian
diet.
[0004] Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine
of
2o clinical importance. The mature GM-CSF is a polypeptide of 127 amino acid
residues
(Cantrell et al., 1985; Lee et al., 1985; Wong et al., 1985) and it regulates
production
and function of white blood cells (granulocytes and monocytes), which are
important
in fighting infections (Metcalf, 1991 ). GM-CSF is now an integral part of the
clinical
management for life-threatening neutropenia, the most common toxicity of
cancer
25 chemotherapy (Dale, 2002). Other oncology applications include treatment of
febrile
neutropenic conditions and support following bone marrow transplantation
(Dale,
2002). Potential applications are also under evaluation in patients with
pneumonia,
Crohn's fistulas, diabetic foot infections and a variety of other infectious
conditions
including HIV-related opportunistic infections (Dale, 2002). The high cost of
human
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GM-CSF in prior culture systems has placed practical limits on its widespread
use
(Dale, 2002). Previously, human GM-CSF has been produced by recombinant means
in COS (along et al., 1985), yeast (Ernst et al., 1987) and Namalwa cells
(Okamoto et
al., 1990). GM-CSF has also been expressed in tobacco, but at very low levels
(James
et al., 2000 ; Sardana et al., 2002).
[0005] US Patent 5,677,474 (Rogers) teaches a method of producing foreign
polypeptides in the seeds of cereal crops, including rice. Transformation of
barley
plants with a GUS reporter gene is disclosed. No transgenic plants containing
GM-
CSF were produced.
[0006] US Patent 5,889,189 (Rodriguez et al.) teaches a method of producing
heterologous peptides in monocots including rice. Expression of a GUS reporter
gene
in transgenic rice seed is disclosed. No transgenic plants containing GM-CSF
were
produced.
[0007] James et al. (2000) used transformed tobacco cell suspensions to
produce and
t5 secrete GM-CSF, which was then isolated from the growth medium. Yields were
low
(maximum of 250 microgram/L) and a complicated process of adding stabilizing
proteins and increasing salt concentration of the growth media was necessary
to
enhance recovery of secreted GM-CSF. No transgenic cereal crops containing GM-
CSF were produced.
2o [0008] Sardana et al. (2002) disclose the production of GM-CSF in
transgenic tobacco
seed. Yields were low with seed extracts containing recombinant human GM-CSF
protein up to a level of 0.03% of total soluble protein. No transgenic cereal
crops
containing GM-CSF were produced.
SUMMARY OF THE INVENTION
25 [0009] The present invention relates to the production of GM-CSF in plants.
[0010] It is an object of the invention to provide an improved method of
producing
GM-CSF in plants.
2
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[0011 J According to an embodiment of the present invention, there is provided
a
method of producing granulocyte-macrophage colony stimulating factor (GM-CSF)
in
a cereal crop comprising growing a cereal crop that has a stably integrated
genetic
construct that includes a regulatory region functional in a cereal crop
operably
associated with GM-CSF coding sequence, or a fragment, or derivative thereof,
operably associated with a transcriptional terminator.
[0012] According to the present invention there is provided a transgenic
cereal crop
plant comprising a stably integrated genetic construct that includes a
regulatory region
functional in a cereal crop operably associated with GM-CSF coding sequence,
or a
to fragment, or derivative thereof, operably associated with a transcriptional
terminator.
[0013] According to the present invention there is provided a genetic
construct
comprising a regulatory region functional in a cereal crop operably associated
with a
GM-CSF coding sequence optimized for expression in a cereal crop operably
associated with a transcriptional terminator.
15 [0014] Cereal crops belong,to the family Poaceae, and include graminoids or
non-
graminoids. In some instances cereal crops from the Avena, Zea, Triticum,
Secale or
Hordeum will be desirable. Commonly farmed cereal crops include, but are not
limited to, rice, wheat, oats, rye, corn, sorghum, and barley. Each of the
commonly
farmed cereal crops can be classified into various cultivars. Rice (Oryza
sativa), for
2o example, includes a japonica cultivar and an indica cultivar. In a
particularly
preferred embodiment of the invention the cereal crop is Oryza sativa,
japonica cv.
Xiushui 11.
[001 S] In an aspect of the present invention regulatory regions that are
preferentially
active within certain organs or tissues at specific developmental stages are
25 contemplated. These regulatory regions may also be active in a
developmentally
regulated manner, or at a basal level in other organs or tissues within the
plant as well.
A number of regulatory regions of seed protein coding sequences have been
identified and characterized. For example, glutelin (Gt), which represents the
major
reserve endosperm protein in rice seeds, is encoded by a small multigene
family with
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subfamilies designated Gtl, Gt2, Gt3, etc. The glutelin regulatory regions
have been
shown to be preferentially active in seed/endosperm tissue.
[0016] In another aspect of the present invention the GM-CSF coding sequence
is
optimized for expression in a cereal crop. For example, the GM-CSF coding
sequence
is optimized for expression in rice, japonica cultivar. In a particularly
preferred
embodiment of the present invention the GM-CSF coding sequence is SEQ ID NO: l
.
[0017] In another aspect of the present invention the GM-CSF coding sequence
encodes an N-terminal methionine residue.
[0018] In another aspect of the present invention the GM-CSF coding sequence
is
operably linked to a signal sequence. For example, the signal sequence is the
glutelin
1 signal sequence.
[0019] In another aspect of the present invention there is provided a method
of
producing granulocyte-macrophage colony stimulating factor (GM-CSF) in a plant
comprising, transforming the plant with a genetic construct comprising a
regulatory
region functional in the plant, operably associated with a GM-CSF coding
sequence,
or a fragment or a derivative thereof, operably associated with a
transcriptional
terminator, and; expressing the GM-CSF.
[0020] In another embodiment, there is provided a method as defined above
wherein
the GM-CSF is human GM-CSF, a fragment or a derivative thereof. Preferably the
2o GM-CSF exhibits between about 60% to 100%, preferably 80% to 100%, more
preferably 95% to 100% of the activity of human GM-CSF.
[0021 ] The present invention also provides a method as defined above wherein
the
plant is a cereal plant, preferably rice. The rice may be, but is not limited
to japonica
cultivar.
[0022] The present invention also provides a method as defined above, wherein
the
genetic construct, or portion of the genetic construct is integrated into the
genome of
the plant. Alternatively, the construct may be extrachromosomal.
4
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[0023] The present invention also provides a transgenic plant comprising a
genetic
construct comprising a regulatory region functional in the plant, operably
associated
with a plant optimized GM-CSF coding sequence or a fragment or a derivative
thereof, operably associated with a transcriptional terminator.
[0024] The present invention also provides a genetic construct comprising a
regulatory region functional in a plant, operably associated with a GM-CSF
coding
sequence optimized for expression in a plant, operably associated with a
transcriptional terminator.
[0025] The transgenic plant may be, but is not limited to a cereal plant,
preferably
1o rice. However, other types of cereal plants are also contemplated. Further,
the rice
may be, but is not limited to japonica cultivar.
[0026] The present invention also provides a plant seed comprising the genetic
construct comprising a regulatory region functional in a plant, operably
associated
with a GM-CSF coding sequence optimized for expression in a plant, operably
15 associated with a transcriptional terminator.
[0027] The present invention also provides a plant cell comprising the genetic
construct comprising a regulatory region functional in a plant, operably
associated
with a GM-CSF coding sequence optimized for expression in a plant, operably
associated with a transcriptional terminator.
20 [0028] This summary of the invention does not necessarily describe all
features of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] These and other features of the invention will become more apparent
from the
following description in which reference is made to the appended drawings
wherein:
25 [0030] FIGURE 1 shows a map of a genetic construct comprising a GM-CSF
coding
sequence operably associated with a Gtl regulatory region in accordance with
an
embodiment of the present invention. The mature human GM-CSF sequence (384 bp)
is fused in-frame with the rice glutelin signal sequence. The coding sequence
is under
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the control of a 1.8 kb glutelin Gtl promoter from rice. The NOS-TER fragment
is
260 bp.
[0031] FIGURE 2 shows PCR products and a Southern blot on DNA from transgenic
rice plants in accordance with a further embodiment of the present invention.
(Figure
2A) PCR. Lane designations: M, 100-by ladder as a marker; GM-CSF, positive
control plasmid; NT, DNA from a non-transformed rice plant; NO DNA, negative
control lacking template DNA; lanes marked as # 1 to #6 represent six
independent
transgenic rice plants. (Figure 2B): Southern blot. Lane 1 and 2: positive
control as
HindIII insert released from the construct shown in Figure 1. Lanes 3-8:
HindIII-
to cleaved genomic DNA from independent transgenic rice plants (#1- #6
respectively).
NT refers to DNA from non-transformed rice plant.
[0032] FIGURE 3 shows a Western blot analysis detecting human GM-CSF protein
in rice seed extracts in accordance with a further embodiment of the present
invention.
The blots for two independent transgenic rice plants are shown. Lane
designations: M,
prestained molecular weight marker; lanes 1 and 2, E. coli-derived commercial
GM-
CSF at two different concentrations; lanes 3 and 4, seed extract from a non-
transformed rice plant; lanes 5-7, seed extracts at different concentrations
from
transgenic rice plants. The left panel is for transgenic rice plant # 1 and
the right panel
is for the transgenic rice plant # 6.
2Q [0033] FIGURE 4 shows biological activity of seed expressed human GM-CSF in
accordance with a further embodiment of the present invention. Bioassays were
done
using TF-1 cells. The TF-1 cells grown as suspension cultures in RPMI 1640
medium
were pipetted into duplicate wells (lx 105 cells/well) of a tissue culture
plate. The
cells were incubated in the absence or presence of seed extracts from
transformed (#1
plant) and non-transformed (NT) plants, extraction buffer or E. coli. derived
GM-
CSF. Cell proliferation was determined using haemocytometry/trypan blue
exclusion.
Plot designations: (~---~): Medium + GM-CSF; (x---x): Medium + Rice Extract;
(0---
-O): Medium Alone; (0----C7): Medium + NT Extract; (O----O): Medium +
Extraction
Buffer.
6
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[0034] FIGURE 5 shows a DNA alignment between a non-optimized GM-CSF
coding sequence (GMCSF/Ori; SEQ ID N0:3) and its derivative (SEQ ID NO:I)
optimized for expression in rice (O. sativa, japonica). Sequence differences
are
indicated by "o".
[0035] FIGURE 6 shows a protein alignment of the GM-CSF derivatives encoded by
the GMCSF/Ori and GMCSF/Opti shown in Figure 5. The protein sequences of
GMCSF/Ori and GMCSF/Opti are identical. The N-terminal of the naturally
occurring form of mature human GM-CSF is indicated by an arrow. An N-terminal
methionine that is fused to the naturally occurring mature human GM-CSF is
to indicated by an asterisk.
DETAILED DESCRIPTION
[0036] The following description is of a preferred embodiment.
[0037] GM-CSF has previously been produced in tobacco cells (James et al.
2000;
Sardana et al., 2002). However, tobacco is inconvenient as an additive to a
mammalian diet. Furthermore, GM-CSF yields from transgenic tobacco were low.
The present invention provides an improved method of producing GM-CSF in
plants.
[0038] The production of heterologous proteins in edible plants can simplify
the
subsequent processing required for preparation of medicament. In some cases,
an
edible transgenic plant containing a protein of interest may be added to an
animal diet
2o without any extraction of the protein from plant tissues. Alternatively,
the
heterologous protein may be purified or semi-purified from the plant.
[0039] Cereal crops form a natural part of the mammalian diet. Cereal crops
belong to
the family Poaceae, and include graminoids or non-graminoids. In some
instances
cereal crops from Avena, Zea, Triticum, Secale or Hordeum are desirable and
contemplated by the present invention. Cereal crops of interest include, but
are not
limited to, rice, wheat, oats, rye, corn, sorghum, and barley. Rice and
certain other
cereal crops are self pollinating, and therefore provide an advantage of self
containment of heterologous coding sequences of interest.
CA 02451001 2003-11-26
_8.
[0040] The present invention provides a method of producing GM-CSF comprising
growing a cereal crop that has stably integrated a construct that includes a
GM-CSF
coding sequence.
[0041 ] An aspect of an embodiment of the present invention relates to
transforming a
plant with a genetic construct that comprises a GM-CSF, a fragment, or a
derivative
thereof in a cereal crop plant to produce a transformed cereal crop plant.
With respect
to coding sequence "fragment" means any 5', 3', or both 5' and 3' deletion.
With
respect to a protein or polypeptide, "fragment" means any N-terminal, C-
terminal, or
both N-terminal and C-terminal truncation. With respect to both coding
sequence and
1o encoded polypeptide, "derivative" means any addition, substitution, or
deletion of
nucleotide or amino acid residues, respectively. For example, a codon
optimized GM-
CSF coding sequence is a derivative of the naturally occurring GM-CSF coding
sequence. As another example, a mature GM-CSF polypeptide having an N-terminal
methionine residue is a derivative of the naturally occurring form that does
not
15 possess the N-terminal metionine. Preferably, the GM-CSF is a mammalian GM-
CSF. More preferably, the GM-CSF is human GM-CSF (hGM-CSF). Even more
preferably, the hGM-CSF is modified to optimize expression in cereal crop
tissues.
Therefore the present invention includes cereal crops, cereal crop cells or
cereal crop
seeds comprising a nucleotide sequence which encode GM-CSF, a fragment or a
2o derivative thereof.
[0042] It is preferable that the GM-CSF, fragment or derivative thereof
encoded by
the plant exhibit substantially the same activity as natural or wild-type GM-
CSF,
preferably human GM-CSF. Preferably, it exhibits at least 50% of the activity,
more
preferably at least 80% and still more preferably at least 95% of the activity
of human
25 GM-CSF. It is also contemplated that the plant produced recombinant GM-CSF
may
exhibit a higher specific activity than that of wild type human GM-CSF.
Various
assays to measure activity of GM-CSF are known in the art, and any of these
assays
may be employed to compare the activity of plant produced recombinant GM-CSF
with that of human GM-CSF.
CA 02451001 2003-11-26
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[0043] The protein produced by the method of the present invention may
comprise
full-length mature GM-CSF or a fragment or derivative thereof. As will be
appreciated by someone of skill in the art, an entire protein may not be
required for
the biological efficacy of EGF within a mammal, but rather, it may be possible
that a
smaller fragment of the protein may be used. As will also be recognized by the
person skilled in the art, various derivatives such as altered glycosylation
derivatives,
or derivatives with additional N-terminal or C-terminal residues, or
derivatives which
alter the strength of association (Ka) or disassociation (Kd) between GM-CSF
and its
receptor, may also be employed without eliminating biological activity, and
may even
1o increase biological efficacy. An example of a GM-CSF produced by a cereal
crop
plant is full-length mature GM-CSF having about 127 amino acids. However, the
actual length of the amino acid sequence may vary depending upon the signal
sequences, added N-terminal or C-terminal amino acid residues, ER retention
sequences, or protein purification tag sequences that may be added to the GM-
CSF
15 sequence. Any of such sequences, as would be known in the art, may be
employed in
the present invention.
[0044] The protein produced by the method of the present invention may be
partially
or completely purified from the plant. In addition, the protein may be
formulated into
a form for oral use or an injectable dosage form. Furthermore, the protein
produced by
2o the method of the present invention may be used for administration to a
mammal, for
example a human, in need thereof.
[0045] The protein produced by the method of the present invention, which
comprises
GM-CSF or fragments or derivatives thereof may have a variety of uses
including, but
not limited to the production of biologically active proteins for use as oral
proteins,
25 for systemic administration, for general research purposes, or combinations
thereof.
Further, the protein produced by the method of the present invention may be
produced
in large quantities in cereal crops, isolated and optionally purified at
potentially
reduced costs compared to other conventional methods of producing proteins
such as,
but not limited to, those which employ cell culture processes.
CA 02451001 2003-11-26
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(0046) When preparing the genetic constructs and transgenic plants provided by
the
present invention several factors may be considered in order to optimize
expression of
heterologous coding sequences of interest. Increased expression of GM-CSF in
cereal
crops may be obtained by utilizing a modified or derivative nucleotide
sequence.
Examples of such sequence modifications include, but are not limited to, an
altered
G/C content to more closely approach that typically found in plants, and the
removal
of codons atypically found in plants commonly referred to as codon
optimization.
Other modifications include alteration of premature poly-A signals, mRNA
destabilizing sequences and intron-like sequences. Preferential expression of
GM-
1o CSF in specific tissues, constituitively or at specific times is also
contemplated. For
example, seeds are known to store stable proteins for long periods of time and
can
accumulate high levels of proteins. Furthermore, strategies relating to
targeting the
protein encoded by a transgene to specific compartments within the cell, for
example
but not limited to the ER, can be adopted to address the problem of low levels
of
15 foreign protein expression in genetically transformed plants. At a
subcellular level,
organelles may also be targeted as required and may include targeting the
transgene
protein to the endoplasmic reticulum (ER), vacuole, apoplast, or chloroplast.
Expression may also be increased through the use of translational fusions. For
example, the transgene-encoded protein may be fused with a signal peptide that
2o directs protein synthesis in plants into a desired cellular compartment,
for example the
ER. Optionally, the transgene fusion could comprise a second signal peptide
that
allows for retention of proteins in the ER or targeting of proteins to the
vacuole. A
non-limiting example of a signal sequence that may be used to target and
retain the
protein within the ER is the H/KDEL sequence (Schouten et al 1996, Plant
Molec.
25 Biol. 30, 781-793). Without wishing to be considered limiting in any
manner, or
bound by theory, replacing a secretory signal sequence with a plant secretory
signal
may also ensure targeting to the endoplasmic reticulum (Denecke et al 1990,
Plant
Cell 2, 51-59).
[0047] The choice of 3' and 5' untranslated regions operatively associated
with a
3o coding sequence are also factors which can affect expression levels.
Generally, but not
exclusively, transcriptional, translational, or both transcriptional and
translational
initiation regulatory regions will be found in 5' untranslated regions, while
CA 02451001 2003-11-26
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transcriptional termination signals are found in 3' untranslated regions.
Regulatory
regions and transcriptional terminators of the present invention will, at
least, be
functional in a cereal crop plant.
[0048] By "regulatory region" or "regulatory element" it is meant a portion of
nucleic
acid typically, but not always, upstream of the protein coding region of a
gene, which
may be comprised of either DNA or RNA, or both DNA and RNA. When a
regulatory region is active, and in operative association with a coding
sequence of
interest, this may result in expression of the coding sequence of interest. A
regulatory region may be spliced in vitro to be operatively associated with a
coding
sequence of interest. Alternatively, a coding sequence of interest may be
integrated
downstream of an endogenous regulatory region located within a plant genome. A
regulatory element may be capable of mediating organ specificity, or
controlling
developmental or temporal gene activation. A "regulatory region" includes
promoter
elements, core promoter elements exhibiting a basal promoter activity,
elements that
are inducible in response to an external stimulus, elements that mediate
promoter
activity such as negative regulatory elements or transcriptional enhancers.
"Regulatory region", as used herein, also includes elements that axe active
following
transcription, for example, regulatory elements that modulate gene expression
such as
translational and transcriptional enhancers, translational and transcriptional
repressors,
2o upstream activating sequences, and mRNA instability determinants. Several
of these
latter elements may be located proximal to the coding region.
[0049] In the context of this disclosure, the term "regulatory element" or
"regulatory
region" typically refers to a sequence of DNA, usually, but not always,
upstream (5')
to the coding sequence of a structural gene, which controls the expression of
the
coding region by providing the recognition for RNA polymerase and/or other
factors
required for transcription to start at a particular site. However, it is to be
understood
that other nucleotide sequences, located within introns, or 3' of the sequence
may also
contribute to the regulation of expression of a coding region of interest. An
example
of a regulatory element that provides for the recognition for RNA polymerase
or other
3o transcriptional factors to ensure initiation at a particular site is a
promoter element.
Most, but not all, eukaryotic promoter elements contain a TATA box, a
conserved
11
CA 02451001 2003-11-26
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nucleic acid sequence comprised of adenosine and thymidine nucleotide base
pairs
usually situated approximately 25 base pairs upstream of a transcriptional
start site. A
promoter element comprises a basal promoter element, responsible for the
initiation of
transcription, as well as other regulatory elements (as listed above) that
modify gene
expression.
[0050] There are several types of regulatory regions, including those that are
developmentally regulated, inducible or constitutive. A regulatory region that
is
developmentally regulated, or controls the differential expression of a gene
under its
control, is activated within certain organs or tissues of an organ at specific
times
to during the development of that organ or tissue. However, some regulatory
regions
that are developmentally regulated may preferentially be active within certain
organs
or tissues at specific developmental stages, they may also be active in a
developmentally regulated manner, or at a basal level in other organs or
tissues within
the plant as well. A number of regulatory regions of seed protein coding
sequences
15 have been identified and characterized. For example, glutelin (Gt), which
represents
the major reserve endosperm protein in rice seeds, is encoded by a small
multigene
family with subfamilies designated Gtl, Gt2, Gt3, etc. The glutelin promoters
have
been shown to be preferentially active in seed/endosperm tissue in controlling
the
expression of various reporter genes in transgenic plant systems, resulting in
2o preferential expression in seed/endosperm tissue, and further expression
that may be
developmentally regulated. By "preferential expression in seeds" is meant that
the
encoded product of a coding sequence is, on average, present in higher levels
in
mature seeds than in other portions of the mature plant.
[0051 ] An inducible regulatory region is one that is capable of directly or
indirectly
25 activating transcription of one or more DNA sequences or genes in response
to an
inducer. In the absence of an inducer the DNA sequences or genes will not be
transcribed. Typically the protein factor, that binds specifically to an
inducible
regulatory region to activate transcription, may be present in an inactive
form which is
then directly or indirectly converted to the active form by the inducer.
However, the
3o protein factor may also be absent. The inducer can be a chemical agent such
as a
protein, metabolite, growth regulator, herbicide or phenolic compound or a
l2
CA 02451001 2003-11-26
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physiological stress imposed directly by heat, cold, salt, or toxic elements
or indirectly
through the action of a pathogen or disease agent such as a virus. A plant
cell
containing an inducible regulatory region may be exposed to an inducer by
externally
applying the inducer to the cell or plant such as by spraying, watering,
heating or
similar methods. Inducible regulatory elements may be derived from either
plant or
non-plant genes (e.g. Gatz, C. and Lenk, LR.P.,1998, Trends Plant Sci. 3, 352-
358;
which is incorporated by reference). Examples, of potential inducible
promoters
include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997,
Ann. Rev.
Plant Physiot. Plant Mol. Biol. 48, 89-108; which is incorporated by
reference),
to steroid inducible promoter (Aoyama, T, and Chua, N.H.,1997, Plant J. 2, 397-
404;
which is incorporated by reference) and ethanol-inducible promoter (Salter,
M.G., et
al, 1998, Plant Journal 16, 127-132; Caddick, M.X., et a1,1998, Nature
Biotech. 16,
177-180, which are incorporated by reference) cytokinin inducible IB6 and CKI1
genes (Brandstatter, I. and Kieber, J.J.,1998, Plant Cell 10, 1009-1019;
Kakimoto, T.,
1996, Science 274, 982-985; which are incorporated by reference) and the auxin
inducible element, DRS (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971;
which is
incorporated by reference).
[0052] The coding sequence of the invention may be operatively associated with
a
suitable 3' untranslated region that is functional in plants. A 3'
untranslated region
2o refers to a DNA segment that contains a polyadenylation signal and any
other
regulatory signals capable of effecting mRNA processing or gene expression.
The
polyadenylation signal is usually characterized by effecting the addition of
polyadenylic acid tracks to the 3' end of the mRNA precursor. Polyadenylation
signals are commonly recognized by the presence of homology to the canonical
form
5'-AATAAA-3' although variations are not uncommon.
[0053] Examples of suitable 3' untranslated regions are the 3' transcribed non-
translated regions containing a polyadenylation signal of Agrobacterium tumor
inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and
plant genes
such as the soybean storage protein genes and the small subunit of the
ribulose-1, 5-
3o bisphosphate carboxylase (ssRUBISCO) gene.
13
CA 02451001 2003-11-26
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[0054] Genetic constructs of the present invention can also include further
enhancers,
either translation or transcription enhancers, as may be required. These
enhancer
regions are well known to persons skilled in the art, and can include the ATG
(methionine) initiation codon and adjacent sequences. The initiation codon
must be in
phase with the reading frame of the coding sequence to ensure translation of
the entire
sequence. The translation control signals and initiation codons can be from a
variety of origins, both natural and synthetic. Translational initiation
regions may
be provided from the source of the transcriptional initiation region, or from
the 5'
region of the structural coding sequence, or may be derived from a source
to independent of the transcriptional initiation region or structural coding
sequence.
Translational initiation regions can be specifically selected and modified so
as to
increase translation of the mRNA.
[0055] In addition to enhancing translation of an mRNA, an N-terminal
methionine
residue may increase protein stability/yield. Tobias et al. (Science 254, 1374-
1377
(1991)) reported protein half lives of only two minutes when the following
amino
acids were present at the amino terminus: Arg, Lys, Phe, Trp, and Tyr. In a
review of
this phenomenon, termed the 'N-end rule', by Varshavsky (Proc. Natl. Acad. Sci
USA, 93: 12142-49 (1996)), Glycine, Valine, and Methionine were identified as
potential stabilizing residues that are common to all known N-end rules.
However,
2o such a result is not obtained for all proteins and thus secondary factors
may also affect
protein stability. Other derivatives of GM-CSF could confer added stability,
improve
yield, or provide a metabolic competitive advantage as compared to a wild-type
plant
or other recombinant plant transformed and expressing a gene of interest which
is not
GM-CSF. Further, other derivatives of GM-CSF may exhibit an altered,
preferably
increased strength of association between GM-CSF and its receptor. In still
another
embodiment contemplated herein, other derivatives of GM-CSF may promote
upregulation or downregulation of the GM-CSF receptor or may enhance or
inhibit
receptor internalization when used or administered to a subject, such as, but
not
limited to a human.
[0056] The present invention provides a modified GM-CSF coding sequence that
is
codon optimized for expression in plants, preferably cereal crops. An example
of a
14
CA 02451001 2003-11-26
-15-
codon optimized GM-CSF sequence is shown in SEQ ID NO:1. By "codon
optimized" is meant the selection of appropriate DNA nucleotides for use
within a
structural gene or fragment thereof that approaches codon usage within a
plant.
Therefore, an optimized gene or nucleic acid sequence refers to a gene in
which the
nucleotide sequence of a native or naturally occurring gene has been modified
in order
to utilize statistically-preferred or statistically-favored codons within a
plant. The
nucleotide sequence typically is examined at the DNA level and the coding
region
optimized for expression in plants determined using any suitable procedure,
for
example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
In this
to method, the standard deviation of codon usage, a measure of codon usage
bias, may
be calculated by first finding the squared proportional deviation of usage of
each
codon of the native GM-CSF gene relative to that of highly expressed plant
genes,
followed by a calculation of the average squared deviation. The formula used
is:
N
15 SDCU = ~ [(Xn-Yn)/Yn ]2/N
n=1
[0057] Where Xn refers to the frequency of usage of codon n in highly
expressed
plant genes, where Yn to the frequency of usage of codon n in the gene of
interest and
N refers to the total number of codons in the gene of interest. A table of
codon usage
2o from highly expressed genes of dicotyledonous plants is compiled using the
data of
Murray et al. (1989, Nuc Acids Res. 17:477-498).
[0058] Another example of a method of codon optimization is based on the
direct use,
without performing any extra statistical calculations, of codon optimization
tables
such as those provided on-line at the Codon Usage Database through the NIAS
25 (National Institute of Agrobiological Sciences) DNA bank in Japan
(http://www.kazusa.or.jp/codon/). The Codon Usage Database contains codon
usage
tables for a number of different species, with each codon usage table having
been
statistically determined based on the data present in Genbank. For example,
the
following table (located at http://www.kazusa.or.jp/codon/cgi-
3o bin/showcodon.cgi?species=Oryza+sativa+(japonica+cultivar-group)+[gbpln])
may
CA 02451001 2003-11-26
-16-
be used for codon optimization of transgenes that are to be expressed in
japonica
cultivar rice plants:
Oryza saliva (japonica cultivar-group) [gbpln]: 32630 CDS's (12783238 codons)
fields: [triplet] [frequency: per thousand] ([number])
UUU 13.6(173985)UCU 12.5(159540)UAU 10.3(131821)UGU6.5( 82520)
UUC 21.9(279329)UCC 15.6(199591)UAC 14.7(188349)UGC12.1(154274)
UUA 6.4( 82284)UCA 11.8(150624)UAA 0.6( 8057)UGA1.1( 14199)
UUG 15.0(192153)UCG 12.0(153755)UAG 0.8( 10388)UGG14.3(183072)
CUU 14.9(190177)CCU 13.8(175845)CAU 11.6(148589)CGU8.0(101835)
CUC 24.2(309923)CCC 12.3(156817)CAC 13.9(i78202)CGC16.3(208778)
CUA 8.0(102568)CCA 14.4(184035)CAA 14.3(183412)CGA7.6( 96761)
CUG 20.1(256688)CCG 17.7(226399)CAG 20.6(263543)CGG14.1(180051)
AUU 14.5(184754)ACU 11,0(140200)AAU 15.1(192829)AGU8.8(112594)
AUC 19.2(245629)ACC 15.0(191716)AAC 18.2(233034)AGC15.4(197340)
AUA 8.9(113169)ACA 11.7(148967)AAA 16.7(213264)AGA10.9(138985)
AUG 23.4(298881)ACG 11.6(148202)AAG 31.9(408318)AGG15.8(202111)
GUU 15.5(197654)GCU 19.6(250883)GAU 25.5(326196)GGU19.9(189844)
GUC 19.7(251434)GCC 30.1(385250)GAC 27.9(356336)GGC28.5(364371)
GUA 7.1( 90381)GCA 17.6(224608)GAA 22.6(289123)GGA16.4(210234)
GUG 23.8(304169)GCG 26.0(332493)GAG 38.6(493349)GGG17.2(219956)
Coding GC 55.04% 1st letter GC 58.27% 2nd letter GC 46.04% 3rd letter GC
60.81%
[0059] By using the above table to determine the most preferred or most
favored
codon(s) for each amino acid in a rice (japonica cultivar) plant, a naturally-
occurring
3o nucleotide sequence encoding a protein of interest can be codon optimized
for
expression in rice (japonica cultivar) by replacing codons that may have a low
statistical incidence in the rice (japonica cultivar) genome with
corresponding
codons, in regard to an amino acid, that are statistically more favored.
However, one
or more less-favored codons may be selected to delete existing .restriction
sites, to
create new ones at potentially useful junctions (5' and 3' ends to add signal
peptide or
termination cassettes, internal sites that might be used to cut and splice
segments
together to produce a correct full-length sequence), or to eliminate
nucleotide
sequences that may negatively effect mRNA stability or expression.
[0060] The naturally-occurring or native GM-CSF encoding nucleotide sequence
may
4.o already, in advance of any modification, contain a number of codons that
correspond
to a statistically-favored codon in a particular plant species. Therefore,
codon
optimization of the native GM-CSF nucleotide sequence, may comprise
determining
which codons, within the native human GM-CSF nucleotide sequence, are not
16
CA 02451001 2003-11-26
-17-
statistically-favored with regards to a particular plant, and modifying these
codons in
accordance with a codon usage table of the particular plant to produce a codon
optimized derivative. The modified or derivative nucleotide sequence encoding
GM-
CSF may be comprised, 100 percent, of plant preferred codon sequences, while
encoding a polypeptide with the same amino acid sequence as that produced by
the
native GM-CSF coding sequence. Alternatively, the modified nucleotide sequence
encoding GM-CSF may only be partially comprised of plant preferred codon
sequences with remaining codons retaining nucleotide sequences derived from
the
native GM-CSF coding sequence. A modified nucleotide sequence may be fully or
1o partially optimized for plant codon usage provided that the protein encoded
by the
modified nucleotide sequence is produced at a level higher than the protein
encoded
by the corresponding naturally occurring or native gene. For example, the
modified
GM-CSF comprises from about 60% to about 100% codons optimized for plant
expression. As another example, the modified GM-CSF comprises from 90% to
15 100% of codons optimized for plant expression.
[0061 ] A modified nucleotide sequence that is optimized for codon usage in a
plant
may possess a GC content that is similar to the GC content of nucleotide
sequences
that occur naturally and are expressed in that plant. However, the nucleotide
sequence
of a modified gene, that has only been partially optimized for codon usage in
a plant,
2o may be further modified so as to approach the GC content of nucleic acid
sequences
that occur naturally and are expressed in that plant. For example, a modified
GM-
CSF coding sequence, that is only partially optimized for codon usage in rice,
may be
further modified so as to approach the GC content of rice nucleotide
sequences, while
encoding a polypeptide with the same amino acid sequence as that produced by
the
25 native GM-CSF coding sequence. Furthermore, a native or naturally occurring
gene
could be optimized with respect to GC content without considering codon
optimization. The modified nucleotide sequence of the present invention may be
additionally optimised to create or eliminate restriction sites, or to
eliminate
potentially deleterious processing sites, such as potential polyadenylation
sites or
3o intron recognition sites, or mRNA destabilising sequences.
1~
CA 02451001 2003-11-26
- 1g-
[0062] The present invention encompasses sequences that are similar or
substantially
identical to a coding sequence or modified coding sequence of GM-CSF. By
"substantially identical" is meant any nucleotide sequence with similarity to
the
genetic sequence of GM-CSF, or a fxagment or a derivative thereof. The term
"substantially identical" can also be used to describe similarity of
polypeptide
sequences. For example, nucleotide sequences or polypeptide sequences that are
greater than about 70%, preferably greater than about 80%, more preferably
greater
than about 70% identical to the GM-CSF coding sequence or the encoded
polypeptide, respectively, and still retain GM-CSF activity are contemplated.
To
1o determine whether a nucleic acid exhibits similarity with the sequences
presented
herein, oligonucleotide alignment algorithms may be used, for example, but not
limited to a BLAST (GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using
default parameters: Program: blastn; Database: nr; Expect 10; filter: default;
Alignment: pairwise; Query genetic Codes: Standard(1)), BLASTZ (EMBL URL:
15 http://www.embl-heidelberg.de/Services/ index.html using default
parameters: Matrix
BLOSUM62; Filter: default, echofilter: on, Expect:l0, cutoff: default; Strand:
both;
Descriptions: S0, Alignments: 50), or FASTA, search, using default parameters.
Polypeptide alignment algorithms are also available, for example, without
limitation,
BLAST 2 Sequences (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html, using default
20 parameters Program: blastp; Matrix: BLOSUM62; Open gap (11) and extension
gap
(1) penalties; gap x dropof~ 50; Expect 10; Word size: 3; filter: default).
[0063] An alternative indication that two nucleic acid sequences are
substantially ,
identical is that the two sequences hybridize to each other under moderately
stringent,
or preferably stringent, conditions. Hybridization to filter-bound sequences
under
25 moderately stringent conditions may, for example, be performed in 0.5 M
NaHP04,
7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2
x
SSC/0.1% SDS at 42°C for at least 1 hour (see Ausubel, et al. (eds),
1989, Current
Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Ine., and
John
Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to
filter-
3o bound sequences under stringent conditions may, for example, be performed
in 0.5 M
NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1 % SDS
at 68°
C for at least 1 hour (see Ausubel, et al. (eds), 1989, supra). Hybridization
conditions
18
CA 02451001 2003-11-26
19-
may be modified in accordance with known methods depending on the sequence of
interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and
Molecular
Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview
of
principles of hybridization and the strategy of nucleic acid probe assays",
Elsevier,
New York). Generally, but not wishing to be limiting, stringent conditions are
selected to be about 5°C lower than the thermal melting point for the
specific
sequence at a defined ionic strength and pH.
[0064] The present invention provides transgenic plants containing a genetic
construct comprising a GM-CSF coding sequence. Methods of regenerating whole
to plants from plant cells are known in the art, and the method of obtaining
transformed and regenerated plants is not critical to this invention. In
general,
transformed plant cells are cultured in an appropriate medium, which may
contain
selective agents such as antibiotics, where selectable markers are used to
facilitate
identification of transformed plant cells. Once callus forms, shoot formation
can be
encouraged by employing the appropriate plant hormones in accordance with
known
methods and the shoots transferred to rooting medium for regeneration of
plants.
The plants may then be used to establish repetitive generations, either from
seeds or
using vegetative propagation techniques.
[0065] The constructs of the present invention can be introduced into plant
cells
2o using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation,
micro-injection, electroporation, biolistics etc as would be known to those of
skill in
the art. For reviews of such techniques see for example Weissbach and
Weissbach,
Methods for Plant Molecular Biology, Academy Press, New York VIII, pp.
421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988);
and
Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism,
2d
Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly,
Langmans Ltd. London, pp. 561-579 (1997).
[0066] To aid in identification of transformed plant cells, the constructs of
this
invention may be further manipulated to include plant selectable markers.
Useful
3o selectable markers include enzymes which provide for resistance to an
antibiotic
19
CA 02451001 2003-11-26
-20-
such as gentamycin, hygromycin, kanamycin; and the like. Similarly, enzymes
providing for production of a compound identifiable by colour change such as
GUS
(*-glucuronidase), or luminescence, such as luciferase are useful.
[0067] Assembly of the genetic constructs of the present invention is
performed using
standard technology know in the art. The coding sequence of interest may be
assembled enzymatically with appropriate regulatory regions and terminators,
within a
DNA vector, for example using PCR, or synthesised from chemically synthesized
oligonucleotide duplex segments. The genetic construct, for example a DNA
vector
comprising the coding sequence of interest, is then transformed to plant
genomes
1o using methods known in the art. Alternatively, a functional genetic
construct may be
assembled in planta, for example a coding sequence operably associated with a
translational initiation region may be integrated into a plant chromosome so
as to
become operably associated with an endogenous plant regulatory region. Proper
integration of the coding sequence may be determined by any method known in
the
15 art, for example Southern analysis or PCR. Expression of the coding
sequence may
be determined using methods known within the art, for example Northern
analysis,
Western analysis or ELISA.
[0068] It is contemplated that a transgenic plant comprising a heterologous
protein of
interest may be administered to any animal, including humans, in a variety of
ways
2o depending upon the need and the situation. For example, if the protein is
orally
administered, the plant tissue may be harvested and directly feed to the
animal, or the
harvested tissue may be dried prior to feeding, or the animal may be permitted
to
graze on the plant with no prior harvest taking place. It is also considered
within the
scope of this invention for the harvested plant tissues to be provided as a
food
25 supplement within animal feed. If the plant tissue is being feed to an
animal with little
or not further processing it is preferred that the plant tissue being
administered is
edible. Furthermore, the protein obtained from the transgenic plant may be
extracted
prior to its use as a food supplement, in either a crude, partially purified,
or purified
form. In this latter case, the protein rnay be produced in either edible or
non-edible
3o plants. If transgenic rice plants expressing GM-CSF are being used, then
CA 02451001 2003-11-26
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administration using whole plant tissue could be as a feed or feed additive to
humans
or other animals.
[0069] Transgenic cereal crops expressing GM-CSF, for example in
seed/endosperm
can provide several advantages with respect to preparation and administration
of
pharmaceutical proteins. Rice seed endosperm-derived flour is an example of a
food-
grade platform that may be an optimal pipeline for producing pharmaceutical-
grade
proteins. Furthermore, production in seeds eliminates the need for immediate
access
to downstream processing facilities.
[0070] Alternatively, the protein produced by the method of the present
invention
may be partially or completely processed and purified from the plant and
reformulated
into a desired dosage form. The dosage form may comprise, but is not limited
to an
oral dosage form wherein the protein is dissolved, suspended or the like in a
suitable
excipient such as but not limited to water. In addition, the protein may be
formulated
into a dosage form that could be applied topically or could be administered by
inhaler,
or by injection either subcutaneously, into organs, or into circulation. An
injectable
dosage form may include other carriers that may function to enhance the
activity of
the protein. Any suitable carrier known in the art may be used. Also, the
protein
produced by the method of the present invention may be formulated for use in
the
production of a medicament. Again, the production of proteins in seed may be
advantageous, even when further purification is contemplated. Production of
pharmaceutical proteins in seed/endosperm offers one of the most appealing
choices
as seeds naturally store stable proteins for long periods of time and there
are well-
established seed fractionation procedures for major crops (Vandekerckhove et
al.,
1989; Saalbach et al., 2001; Stoger et al., 2000; Jaeger et al., 2002).
Furthermore, the
major proportion of seed proteins belong to a limited set of protein classes,
which may
simplify the purification procedure (Jaeger et al., 2002).
[0071] The present invention will be further illustrated in the following
examples.
[0072] Examples
21
CA 02451001 2003-11-26
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[0073] Example 1: Production of Biologically Active human GM-CSF in Seeds of
Transgenic Rice Plants
[0074] Engineering the gene construct for the human GM-CSF coding sequence
under the control of rice Gtl promoter. A 1.8 kb Gtl glutelin promoter from
rice
(Zheng et al., 1993) was used to control the expression of human GM-CSF mature
coding sequence. To make the construct, standard DNA cloning and DNA
amplifications techniques were followed (Sambrook et al., 1989). A plasmid
containing the Gtl promoter (Zheng et al., 1993) with associated 72 basepair
Gtl
signal sequence was digested with NaeI enzyme that cleaved the plasmid right
after
to the Gtl signal sequence. After complete digestion, the digested plasmid DNA
was
dephosphorylated using alkaline phosphatase. The human GM-CSF coding DNA
(without its human signal sequence) was amplified from the BBG 12 plasmid
using the
polymerase chain reaction (PCR) and phosphorylated with T4 kinase. A ligation
reaction was then set up that involved above prepared plasmid with Gtl
promoter and
associated glutelin signal sequence as well as the GM-CSF DNA fragment. After
transformation of bacterial cells with an aliquot from this ligation mixture,
a
transformed colony was identified with plasmid containing Gtl promoter as well
as
glutelin signal sequence which was in-frame with the GM-CSF sequence. This
plasmid was then cleaved at BamHI and HincII sites that were present on the 3'
side
of the stop codon of the GM-CSF sequence in order to incorporate a nopaline
synthase
terminator (NOS-TER) DNA fragment with a 5'BamHI site and a 3'blunt site. In
this
constructed plasmid, an EcoRI site was present on the 5' end of the Gtl
promoter and
HindIII site was present on the 3' end of NOS terminator sequence. This
particular
plasmid was further modified to add a HindIII site on the 5' end of the Gtl
promoter
by employing the use of an adaptor with a HindIII site. The HindIII fragment
(Figure
1) encompassing the complete construct was then cloned into the binary vector
pCAMBIA 1301 (CAMBIA, Australia). This DNA vector was then transferred into
the competent LBA4404 strain of Agrobacterium.
(0075] Transgenic rice plants and integration of human GM-CSF DNA in rice
3o genome. The Agrobacterium cells containing the pCAMBIA/GM-CSF construct
were
used to transform vigorously growing rice calli. Transformed culture handling,
callus
22
CA 02451001 2003-11-26
- 23 -
induction from rice seeds (Oryza sativa cv. Xiushui 11 ), callus
transformation with
appropriate Agrobacterium cells, callus selection, maintenance and plant
regeneration
were essentially according to earlier methods (Cheng et al., 1998; Cheng et
al., 1997).
When plantlets reached about eight inches in height, and had a well-developed
root
system, they were transferred to pots of soil. Plants were grown to maturity
in a
controlled chamber at 28°C with a relative humidity of 50-60%.
[0076] A total of six independent transgenic plants were regenerated from
calli
selected on hygromycin and chosen for further investigations. To ascertain the
transgenic nature of the regenerated rice plants, DNA was extracted from leaf
tissue.
to First, to detect the presence of insert in the DNA samples from selected
rice plants,
PCR reactions were performed using primers specific to human GM-CSF sequence
coding sequence. A band of expected size was observed for all the six plants
(Figure
2A). The size of this band was identical to the one obtained for the positive
control.
No band was observed for the non-transgenic rice DNA sample. Similarly, for
the
15 negative control reaction without added DNA, no specific amplification was
observed.
For PCR, roughly 20-30 ng of rice genomic DNA was used as template for each
sample. Primers were specific to the S' and 3' termini of mature GM-CSF
sequence.
The DNA polymerase from New England Biolabs was used. The samples were
subjected to one cycle of 95C for S minutes, 58C for 30 seconds and 72C for 90
zo seconds followed by 30 cycles of 95C for 60 seconds, S8C for 30 seconds and
72C for
90 seconds. In the final cycle, the extension time at 72C was extended to 6
minutes.
Aliquots of PCR reactions were separated on 0.8% agarose gel stained with
ethidium
bromide.
[0077] Next, to verify the integration of the intact construct into the rice
genome,
25 purified rice genomic DNA from six PCR positive plants and a non-
transformed
control rice plant as well as positive control DNA were subjected to Southern
analysis
Rice genomic DNA was isolated and purified according to published protocol.
For
Southern blot, about 10 microgram of rice DNA was digested with HindIII. The
digested DNA was separated on 0.8% agarose gel, denatured and transferred onto
a
3o nylon membrane. The membrane was probed with 32P-labelled fragment
containing
the GM-CSF sequence. The labeling was performed using a Ready to Go kit.
23
CA 02451001 2003-11-26
-24-
(Pharmacia Biotech). Hybridizations were done at 42C in 50% formamide. The
nylon
membrane was washed at room temperature with 2 X SSC, 0.1% SDS for 10 minutes.
This was followed by two washings with 1 X SSC, 0.1% SDS at 65C for 15
minutes,
and a final wash at 65C with 0.4 X SSC, 0.1% SDS for 15 minutes. The expected
fragment of 2.566 kb was observed for plant # 1, 2, 4, 5 and 6 as well as for
the
positive control (Figure 2B). An additional band was also present for plant #
1. For
plant # 3, the observed bands were not of expected size. No bands were
observed for
the non-transformed (NT) rice plant.
[0078] Human GM-CSF-specific ELISA and Western blot analysis. To detect
human GM-CSF protein in transgenic rice, extracts from seeds were made and
assayed using a human GM-CSF-specific immunoassay. For ELISA, rice seeds (100
mg) were ground to powder and 100 microliter of extraction buffer (SO mM Tris
pH
7.5, 50 mM NaCI, 1 mM EDTA, 1 mM PMSF, I% 2-mercaptoethanol, 0.1% Triton
is X-100, 1% ascorbic acid and 1% polyvinylpyrrolidone) was added. The
extracts were
clarified by brief centrifugation (14000 g) at 4C. These clear extracts were
used for
quantifying GM-CSF using a Quantikine~ kit (R&D Systems) as described
previously (Sardana et al., 2002). This kit provides for a human GM-CSF
immunoassay based on a microplate pre-coated with a monoclonal antibody
specific
2o for human GM-CSF. All samples including standards were assayed in
duplicate.
Diluted aliquots of commercial GM-CSF and of seed extracts were dispensed into
the
wells of the microplate and incubated for two hours at room temperature. The
unbound materials were washed away and GM-CSF conjugate was then added
followed by another incubation at room temperature and transfer of substrate
solution.
25 The microplate reader set at 450 nm was used for determining the optical
densities.
For each assay, standard curves were generated utilizing purified E.coli-
derived
human GM-CSF, and the test sample values were derived from these. Protein
content
in samples was determined (Bradford, 1976). ELISA data (Table I) showed that
human GM-CSF accumulated to 1.2% and 1.3% of total soluble protein in rice
seeds
3o for plants # 1 and # 6, respectively, two of the three transgenic plants
that were tested.
24
CA 02451001 2003-11-26
-25-
Table 1.
Plant GM-CSF Total Protein % GM-CSF of Total
ID (microgram/mL) (mg/mL) Soluble Protein
# 1 28 2.2 1.3
#5 5.6 2.3 0.24
#6 28 2.4 1.2
[0079] For further characterization, experiments involving Western blots were
performed. The soluble protein extracts from seeds of rice plants # 1 and 6
and a
control plant were subjected to denaturing polyacrylamide (15% SDS) gel
electrophoresis. The proteins were transferred onto PVDF membranes. The
blocking
solution consisted of 1% BSA in Tris base saline (10 mM Tris pH 7.4, 150 mM
NaCI). The membranes were probed with a 1:1000 dilution of a polyclonal rabbit
antibody to GM-CSF (R&D Systems) followed by 1:7500 diluted alkaline
to phosphatase conjugated goat anti-rabbit IgG. Protein bands were visualized
using the
NBT/BCIP substrates (Fisher Scientific, Ottawa). A distinct band of
approximatelyl8
kDa was observed in lanes containing seed extracts from transgenic rice plants
for
both the blots (Figure 3). The 18 kDa band from transgenic rice seed extract
migrated
to the same position on the gel as the corresponding E. coli-derived human GM-
CSF.
No bands were detected for the non-transformed control plants. In addition to
the 18
kDa band, other bands that ranged in size from 19-44 kDA were also detected in
the
lanes containing the transgenic rice seed extracts.
[0080] Biological activity of the rice seed-expressed recombinant human GM-
2o CSF. The biological activity of rice seed-derived human GM-CSF was tested
using a
human cell line, TF-1 (Kitamura et al., 1989) that grows only in the presence
of
medium supplemented with GM-CSF or other growth factors. TF-1 cells (Kitamura
et
al., 1989) were obtained from ATCC. These cells were grown as suspension
cultures
CA 02451001 2003-11-26
-26-
as described earlier (Sardana et al., 2002): Briefly, RPMI 1640 medium withl
ng/mL
E. coli-derived GM-CSF (R&D Systems) and fetal bovine serum (10%) was used. 1
X
PBS was used for washing the cells twice. Cells were resuspended in RPMI 1640
medium containing 10% fetal bovine serum at 2 X l OS/mL. Then 1 X 105 cells
were
dispensed to the wells of a 24-well tissue culture plate. Aliquots of 0.5 ml
RPMI
medium with 10% fetal bovine serum containing one of the following samples at
a
time were added to each of the wells: 1 ng/mL commercial GM-CSF (E. coli-
derived),
transgenic rice seed extract containing 1 ng of GM-CSF, seed extract from a
non-
transformed (NT) plant at equivalent protein concentration, seed protein
extraction
1o buffer (without mercaptoethanol). The dispensed 0.5 ml aliquots were from a
stock
solution that contained different seed extracts or commercial GM-CSF. All
experiments were performed in quadruplicate and repeated at least twice under
sterile
conditions. The cell growth was monitored and live cells were counted using
haemocytometry/trypan blue exclusion.
15 [0081] In summary, the TF-1 cells were grown in the presence or absence of
commercially available E. coli-derived recombinant human GM-CSF or aliquots of
rice seed extracts from transgenic and non-transformed control plants. Equal
final
concentrations of GM-CSF (whether positive control or seed-derived) were used.
Viable TF-1 cells were quantified using vital staining (trypan blue
exclusion).
20 [0082] The results of these in vitro assays for GM-CSF biological activity
are
presented in Figure 4. The assay medium alone (not supplemented with GM-CSF),
the
seed extract from non-transformed rice plants and the extraction buffer (EB)
added to
assay medium did not support proliferation of TF-1 cells over a period of 48
hours.
[0083] In contrast, when the seed extract from transgenic rice plant #1 was
added to
25 the medium, proliferation of TF-1 cells was observed after 48, 72 and 96
hours of
incubation. The amount of proliferation was similar to that seen in the
positive control
(E. coli-derived human GM-CSF). As the data show, this rice seed extract
resulted in
about 6-fold increase in the number of TF-1 cells over the numbers obtained
with
medium alone. Similar results were observed with the seed extract of plant # 6
(data
3o not shown).
26
CA 02451001 2003-11-26
-27-
[0084] Example 1 describes the production of a biologically active human
recombinant protein, GM-CSF, in the seeds of transgenic rice plants. The human
GM-
CSF was put under control of the 1.8 kb Gtl promoter from rice. A total of six
independent transgenic rice plants were produced using Agrobacterium-mediated
transformation procedures. Southern blot analysis suggested that five of these
plants
including plants #1 and #6 had no rearrangements in the GM-CSF construct,
indicating that the construct is present in an intact form. The mature seeds
from two of
these plants were found to contain high levels of GM-CSF (approximately 1.3%
of
total soluble protein). This is more than 4-fold higher than the
reported~expression
level in the seeds of tobacco (Sardana et al., 2002). Furthermore, even higher
levels of
GM-CSF in rice seeds may be achieved by employing a larger version of Gtl
promoter that has been shown to boost the production of phaseolin up to 4% in
rice
endosperm (Zheng et al., 1995).
[0085] The apparent molecular mass of unglycosylated GM-CSF is 15-18 kDa. Our
t5 Western blot analysis indicated that both E.coli-derived GM-CSF
(unglycosylated
form) and rice seed-derived GM-CSF migrated near the 18 kDa size marker. This
suggests that the major 18 kDa form of seed-derived GM-CSF is likely
unglycosylated. Other high molecular weight bands present at 19-44 kDa in both
rice
seeds extract may represent the glycosylated forms of GM-CSF. Furthermore, the
2o presence of l8kDa GM-CSF suggests that the rice glutelin signal peptide was
cleaved
from the human GM-CSF protein. The signal sequences of other seed storage
proteins
have been shown to be correctly processed in transgenic plants (Jaeger et al.,
2002).
[0086] The implication about the presence of unglycosylated and glycosylated
forms
of GM-CSF in rice seed extracts is in agreement with similar findings reported
on the
25 expression of GM-CSF in yeast and mammalian cells. For example, human GM-
CSF
produced in yeast ranged in size up to 50 kDa (Ernst et al., 1987); and
Namalwa cells
producing GM-CSF showed protein ranging from 16 to 35 kDa (Ok~moto et al.,
1990) as determined by Western blot analysis. There are two potential N-
glycosylation sites at Asn27 and Asn37 in the human GM-CSF protein (Cantrell
et al.,
30 1985; Lee et al., 1985; Wong et al., 1985). Most likely the smallest size
molecules
(16-18 kDa) have neither site glycosylated, the intermediate site has one site
27
CA 02451001 2003-11-26
-28-
glycosylated and the largest size has both sites glycosylated (Okamoto et al.,
1990).
Various factors such as high-volume production conditions, cellular
environment,
protein structure and molecular interactions can affect the efficiency and
state of
glycosylation. As an example, the human and mouse GM-CSF produced in yeast are
differentially glycosylated (Ernst et al., 1987). About 50% of the mouse GM-
CSF is
unglycosylated in yeast (Ernst et al., 1987). A seed storage protein is
synthesized as a
mixture of partially and fully-glycosylated protein in yeast (Vitale et al.,
1993).
[0087] Regardless of glycosylation status of the rice seed-produced GM-CSF,
the
results of assays for biological activity of seed-produced GM-CSF indicated
that the
1 o human protein is functional. This suggests that the protein produced in
seed
endosperm is maintained in an active conformation for interaction with the GM-
CSF
receptor. It is known that TF-1 cells (Kitamura et al., 1989) have specific
receptors
that bind to GM-CSF for proliferation.
[0088] Glycosylation status of rice-seed derived GM-CSF will be characterized,
15 although glycosylation is not essential for biological activity of GM-CSF,
either in
vivo or in vitro (Burgess et al., 1987; Kaushansky et al., 1987; Moonen et
al., 1987;
Quesniaux et al., 1998). The core glycans are identical in mammalian and plant
protein secretory systems, but plants have a different linkage with fucose
(alpha 1-3
linked) and have xylose residues.
20 [0089] Biologically active recombinant human GM-CSF, a protein
pharmaceutical
with many applications in medicine and research, has been preferentially
produced in
the seeds of transgenic rice plants at high levels. As rice is a self
pollinated crop, it
offers a particular attraction in terms of containment of the transgenes, in
addition to
providing advantages associated with producing protein-based medicines in
seeds.
25 (0090] Example 2: Codon Optimization of GM-CSF
[0091] In modifying the GM-CSF coding sequence to optimize expression in
plants
several factors were considered:
~ Identify preferred codons for Oryza sativa (japonica cultivar);
28
CA 02451001 2003-11-26
-29-
~ Increase G/C content;
~ Match tRNA population of Oryza sativa (japonica cultivar); and
~ Minimize secondary structure interactions.
[0092] An example of a codon optimized sequence is shown in Figure S (bottom
strand). The codon optimized sequence is aligned with a non-optimized GM-CSF.
The
G/C content of the optimized sequence is 66% compared to 40% G/C content for
the
non-optimized sequence. Both sequences encode a fusion polypeptide (see Figure
6)
comprising, in the direction of N-terminal to C-terminal:
~ a methionine residue;
~ a hexahistidine tag;
~ a 3 amino acid spacer;
~ a Factor X cleavage site;
~ a methionine residue; and
~ the mature human GM-CSF sequence.
[0093] The fusion protein is designed such that cleavage at the Factor X site
yields a
mature human GM-CSF protein with an N-terminal methionine (indicated by an
asterisk in Figure 6). The N-terminal methionine can be important for
increasing
stability and yield. Also the N-terminal methionine may confer an altered
strength of
association between GM-CSF and its receptor, or it may alter the receptor
number
2o and/or internalization kinetics of the receptor.
[0094] A genetic construct comprising the optimized sequence was prepared in
pGEM47. More specifically, the construct comprises, in the 5' to 3' direction:
~ a Glutelin 1 (Gtl) regulatory region;
a Glutelin 1 signal sequence;
29
CA 02451001 2003-11-26
-30-
the codon optimized sequence containing a sequence encoding the hexahistidine
tag, spacer, and Factor X cleavage site; and
an NOS terminator.
[0095] A SacI restriction fragment of pGEM47/His/GMCSF encompassing the
complete genetic construct with optimized GM-CSF under control of the Gtl
regulatory region was then subcloned into a binary vector pCAMBIA1301 to
produce
pCAMBIA/His/GMCSFopti.
[0096] A pCAMBIA vector comprising the non-optimized coding sequence of the
hexahistidine/GM-CSF fusion is also being produced and is being designated as
1o pCAMBIA/His/GMCSFori.
[0097] pCAMBIA vectors identical to pCAMBIA/His/GMCSFopti and
pCAMBIA/His/GMCSFori except that the mature GM-CSF coding sequence does not
encode an N-terminal methionine are also being produced.
[0098] All four of the pCAMBIA vectors are being used to transform vigorously
growing rice calli (Oiyza sativa, japonica cv. Xiushui 11) according to
methods
described in Example 1.
[0099] Protein production and biological activity of GM-CSF (with or without N-
terminal methionine) is being determined using methods described in Example 1.
[00100] All citations are hereby incorporated by reference.
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[00144] The present invention has been described with regard to one or more
embodiments. However, it will be apparent to persons skilled in the art that a
number
of variations and modifications can be made without departing from the scope
of the
invention as defined in the claims.
36
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SEQUENCE LISTING
<110> Altosaar, Illimar
Sardana, Ravinder
<120> Production of GM-CSF in Plants
<130> 08-898901CA
<140> Not Yet Known
<141> 2003-11-26
<150> Canada 2,410,702
<151> 2002-11-26
<160> 4
<170> PatentIn version 3.1
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40
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atg gcg cca gcg cgc agc ccg agc ccg tcc acc cag ccg tgg gag cac 99
Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His
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gtg aac gcg atc cag gag gcc cgc agg ctc ctc aac ctc tcc cgc gac 147
Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp
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acc gcc gcc gag atg aac gag acc gtg gag gtg atc tcc gag atg ttc 195
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37
CA 02451001 2003-11-26
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38
CA 02451001 2003-11-26
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39
CA 02451001 2003-11-26
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