Note: Descriptions are shown in the official language in which they were submitted.
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,
NRG-2 NUCLEIC ACID MOLECULES, POLYPEPTIDES,
AND DIAGNOSTIC AND THERAPEUTIC METHODS
Field of the Invention
The invention relates to neuregulins and methods for their use.
Background of the Invention
Neuregulins (NRGs) and their receptors constitute a growth factor-receptor
tyrosine kinase system for cell-cell signalling that has been implicated in
organogenesis in
nerve, muscle, epithelia, and other tissues (Lemke, Mol. Cell. Neurosci. 7:
247-262, 1996;
Burden et aL , Neuron 18: 847-855, 1997). The three known NRG genes, NRG-1,
NRG-2, and
NRG-3, map to distinct chromosomal loci (Pinkas-Kramarski et al., Proc. Natl.
Acad. ScL
USA 91: 9387-91, 1994; Carraway etal., Nature 387: 512-516, 1997; Chang et
al., Nature
387:509-512, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94: 9562-9567,
1997), and
collectively encode a diverse array of NRG proteins. The NRG protein family
includes at
least 20 (and perhaps 50 or more) secreted and membrane-bound isoforms
containing
epidermal growth factor-like (EGFL), immunoglobulin (Ig), and other
recognizable domains.
The most thoroughly studied NRG proteins to date are the gene products of
NRG-1, which include a group of approximately 15 distinct structurally-related
isoforms
(Lemke, Mol. Cell. Neurosci. 7: 247-262, 1996 and Peles and Yarden, BioEssays
15: 815-824,
1993). Isoforms of NRG-1 include Neu Differentiation Factor (NDF; Peles etal.,
Cell 69,
205-216, 1992 and Wen etal., Cell 69, 559-572, 1992), Heregulin (HRG; Holmes
et al.,
Science 256: 1205-1210, 1992), Acetylcholine Receptor Inducing Activity (ARIA;
Falls et al.,
Cell 72: 801-815, 1993), and the glial growth factors GGF1, GGF2, and GGF3
(Marchionni et
al., Nature 362: 312-8, 1993).
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The NRG-2 gene was identified by homology cloning (Chang et al.,
Nature 387:509-512, 1997; Carraway et at., Nature 387:512-516, 1997; and
Higashiyama etal., .1. Biochem. 122: 675-680, 1997) and through genomic
approaches (Busfield et al., MoL Cell: Biol. 17:4007-4014, 1997). NRG-2
isoforms include Neural-and Thymus-Derived Activator of erbB Kinases (NTAK;
Genbank Accession No. AB005060), Divergent of Neuregulin (Don-1), and
Cerebellum-Derived Growth Factor (CDGF; PCT application WO 97/09425).
Cells expressing erbB4 or erbB2/erbB4 receptors may show a particularly robust
response to NRG-2 (Pinkas-Kramarski et at., MoL Cell. Biol. 18: 6090-6101,
1998). The NRG-3 gene product (Zhang et at., Proc. Natl. Acad. Sc!. USA 94:
9562-9567, 1997) is also known to bind and activate erbB4 receptors (Hijazi et
al., Int. J. Oncol. 13:1061-1067, 1998).
The EGFL domain, present at the core of NRG isoforms, is required for
binding and activating NRG receptors, which belong to the epidermal growth
factor receptor (EGFR) family, and include EGFR (or erbB1), erbB2, erbB3, and
erbB4, also known as HER! through HER4, respectively, in humans (Meyer et
al., Development 124: 3575-3586, 1997; Orr-Urtreger et at., Proc. Natl. Acad.
ScL USA 90: 1867-71, 1993; Marchionni etal., Nature 362: 312-8, 1993; Chen et
al., J. Comp. Neurol. 349: 389-400, 1994; Corfas etal., Neuron 14: 103-115,
1995; Meyer etal., Proc. Natl. Acad. ScL USA 91:1064-1068, 1994; and Pinkas-
Kramarski etal., Oncogene 15: 2803-2815, 1997). High-affinity binding of the
NRGs may be mediated principally via either the erbB3 or erbB4 receptors.
Binding of NRG ligands leads to dimerization with other erbB subunits and
transactivation by phosphorylation on specific tyrosine residues.
NRG proteins have diverse biological properties, making them
potentially useful in the development of novel therapies for a wide range of
diseases and disorders.
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Summary of the Invention
The invention provides methods of treatment and diagnosis using
NRG-2 polypeptides, nucleic acid molecules, and antibodies. The invention also
provides novel NRG-2 polypeptides and nucleic acid molecules.
In the first aspect, the invention provides a method for increasing the
mitogenesis, survival, growth, or differentiation of a cell by administering a
NRG-2 polypeptide to the cell in an amount effective for increasing the
mitogenesis, survival, growth, or differentiation of the cell, where the cell
expresses an erbB receptor that is selective for a NRG-2 polypeptide. In
preferred embodiments of this aspect, the erbB receptor is an erbB4 homodimer,
an erb132/erbB4 heterodimer, or an erbBl/erbB3 heterodimer. In other preferred
embodiments of the first aspect, the cell is a neuronal cell, a neuronal
progenitor
cell, a neuronal-associated cell, or a muscle cell. In other preferred
embodiments
of the first aspect, the neuronal-associated cell is a Schwann cell, an
astrocyte, an
oligodendrocyte, an 0-2A progenitor cell, a glial cell, a microglial cell, an
olfactory bulb ensheathing cell, or a sensory organ cell, and the muscle cell
is a
myoblast, a satellite cell, a myocyte, a skeletal muscle cell, a smooth muscle
cell,
or a cardiac muscle cell.
In a second aspect, the invention provides a method of stimulating
mitogenesis of a glial cell by contacting the glial cell with a recombinant
NRG-2
polypeptide. In a preferred embodiment of the second aspect, the glial cell is
an
oligodendrocyte, a microglial cell, a myelinating glial cell, an olfactory
bulb
ensheathing cell, or a glial cell in an adult.
In a third aspect, the invention provides a method for inducing
myelination of a neuronal cell by a glial cell by contacting the glial cell
with a
NRG-2 polypeptide, such that the contacting is sufficient to induce
myelination of
the neuronal cell by the glial cell.
In a fourth aspect, the invention provides a method of increasing the
cardiomyocyte survival, cardiomyocyte proliferation, cardiomyocyte growth, or
cardiomyocyte differentiation, in a mammal in need thereof, by administering a
NRG-2 polypeptide to the mammal in an amount effective for increasing the
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cardiomyocyte survival, cardiomyocyte proliferation, cardiomyocyte growth, or
cardiomyocyte differentiation. In preferred embodiments of the fourth aspect,
the
mammal is a human. In other preferred embodiments of the fourth aspect, the
mammal has a pathophysiological condition that affects cardiac muscle, for
example, cardiomyopathy (e.g., a degenerative congenital disease), cardiac
trauma, heart failure, or ischemic damage, or the mammal has a
pathophysiological condition that affects smooth muscle, for example,
atherosclerosis, vascular lesion, vascular hypertension, or degenerative
congenital
vascular disease. In another preferred embodiment of the fourth aspect the
mammal is a patient with myasthenia gravis.
In a fifth aspect, the invention provides a method of affecting cellular
communication between a neuronal-associated cell and a neuronal cell in a
mammal by administering a NRG-2 polypeptide to the mammal, such that the
neuregulin interacts with the neuronal-associated cell, resulting in the
production
of at least one neurotrophic agent by the neuronal-associated cell, and the
neurotrophic agent or agents affect the mitogenesis, survival, growth,
differentiation, or neurite outgrowth of the neuronal cell. In a preferred
embodiment of the fifth aspect, the mammal is a human. In other preferred
embodiments of the first aspect, the neuronal-associated cell is a Schwann
cell, an
astrocyte, an oligodendrocyte, an 0-2A progenitor cell, a glial cell, an
olfactory
bulb ensheathing cell, a microglial cell, a sensory organ cell, or a muscle
cell
(e.g., a skeletal muscle cell, a smooth muscle cell, or a cardiac muscle
cell). In
other preferred embodiments of the fifth aspect, the cellular communication is
affected in the central nervous system or the peripheral nervous system of a
mammal. In other preferred embodiments of the fifth aspect, the administering
includes administering a purified NRG-2 polypeptide-producing cell.
In a sixth aspect, the invention provides a method for the treatment or
prophylaxis of a pathophysiological condition of the nervous system in a
mammal, by administering a therapeutically-effective amount of a recombinant
NRG-2 polypeptide to the mammal. In preferred embodiments of the sixth
aspect, the pathophysiological condition is a condition of the peripheral
nervous
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system or the central nervous system; the pathophysiological condition is
demyelination of nerve cells, damage of Schwann cells, loss of Schwann cells,
or
a neurodegenerative disorder; the pathophysiological condition is a peripheral
neuropathy (e.g., a sensory nerve fiber neuropathy, a motor fiber neuropathy,
or
both); or the pathophysiological condition is multiple sclerosis, amyotrophic
lateral sclerosis, spinal muscular atrophy, nerve injury, Alzheimer's Disease,
Parkinson's Disease, cerebellar ataxia, or spinal cord injury. In another
preferred
embodiment of the sixth aspect, the treatment or prophylaxis requires neural
regeneration or neural repair. In another preferred embodiment of the sixth
aspect, the NRG-2 polypeptide interacts with neuronal-associated cells,
resulting
in production of at least one neurotrophic agent by the neuronal-associated
cells,
and the neurotrophic agent or agents affect the mitotic activity, survival,
differentiation or neurite outgrowth of neuronal cells. In another preferred
embodiment of the sixth aspect, the administering is sufficient to induce
myelination of a neuronal cell by a glial cell (e.g., a Schwann cell or an
oligodendrocyte). In another preferred embodiment of the sixth aspect, the
administering includes administering a purified NRG-2 polypeptide-producing
cell to the mammal. The NRG-2 polypeptide-producing cell of the invention may
contain a recombinant DNA sequence, where the DNA sequence includes a
NRG-2 polypeptide-encoding sequence, and where the NRG-2 polypeptide-
encoding DNA sequence is operably-linked to a promoter.
In a seventh aspect, the invention provides a method for the treatment
of a tumor (e.g., a glial tumor) by inhibiting the proliferation of a tumor
cell by
administering an effective amount of an antibody, that inhibits binding of a
NRG-
2 polypeptide to a receptor present on the surface of the tumor cell, to a
subject in
need of such treatment. In a preferred embodiment of the seventh aspect, the
tumor cell expresses an erbB receptor that is selective for a NRG-2
polypeptide.
In an eighth aspect, the invention provides a method for the treatment
of neurofibromatosis by inhibiting glial cell mitogenesis by administering an
effective amount of an antibody, that inhibits binding of a NRG-2 polypeptide
to
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a receptor present on the surface of a glial tumor cell in an individual with
neurofibromatosis, to a subject in need of such treatment.
In a ninth aspect, the invention provides a method for inhibiting
proliferation of a cell by contacting the cell with an effective amount of an
antibody that inhibits binding of a NRG-2 polypeptide to a receptor present on
the
surface of the cell.
In a tenth aspect, the invention provides a method for stimulating
proliferation of a cell by administering a NRG-2 polypeptide to the cell.
In preferred embodiments of the ninth and tenth aspects, the cell is a
neuronal cell, a neuronal-associated cell, or a muscle cell.
The NRG-2 polypeptide of any of the above aspects or embodiments of
the invention may include, or consist of, the amino acid sequences set forth
in
SEQ ID NOs: 2 or 4, or be encoded by the nucleic acid sequences set forth in
SEQ ID NOs: 1 or 3.
In an eleventh and a twelfth aspect, the invention provides a
substantially pure NRG-2 polypeptide including, or consisting of, the amino
acid
sequences set forth in SEQ ID NOs: 2 or 4. In a thirteenth aspect, the
invention
provides a substantially pure nucleic acid molecule including a sequence
encoding a polypeptide including the amino acid sequences set forth in SEQ ID
NOs: 2 or 4. In preferred embodiments, the invention provides a vector (e.g.,
a
gene therapy vector) including the nucleic acid molecule of the thirteenth
aspect,
operably linked to a promoter; a cell containing a gene therapy vector that
contains the nucleic acid molecule of the thirteenth aspect; and a non-human
transgenic animal containing the nucleic acid molecule of the thirteenth
aspect.
In fourteenth and fifteenth aspects, the invention provides a
substantially pure nucleic acid molecule including, or consisting of, a
nucleic acid
sequence that is substantially identical to the nucleic acid sequences set
forth in
SEQ ID NOs: 1 or 3. In a sixteenth aspect, the invention provides a nucleic
acid
molecule including a sequence that is antisense to the coding strand sequence
of
the nucleic acid sequence set forth in SEQ ID NOs: 1 or 3, or a fragment
thereof.
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In a seventeenth aspect, the invention provides a non-human animal having a
knockout
mutation in one or both alleles encoding the NRG-2 polypeptide including the
amino acid
sequence set forth in SEQ ID NOs: 2 or 4. In a preferred embodiment of the
seventeenth aspect,
the invention provides a cell from the non-human animal of the seventeenth
aspect.
In an eighteenth aspect, the invention provides an antibody that specifically
binds to a
NRG-2 polypeptide that includes the amino acid sequences set forth in SEQ ID
NOs: 2 or 4. In a
preferred embodiment of the eighteenth aspect, the invention provides a method
of detecting the
presence of a NRG-2 polypeptide in a sample by contacting the sample with the
antibody of the
eighteenth aspect, and assaying for binding of the antibody to the
polypeptide. In a preferred
embodiment of the eighteenth aspect, the invention provides a kit for the
analysis of a NRG-2
polypeptide of a test subject, where the kit includes the antibody of the
eighteenth aspect.
In a nineteenth aspect, the invention provides a method of diagnosing an
increased
likelihood of developing a NRG-2-related disease or condition in a test
subject (e.g., a human) by
analyzing nucleic acid molecules of the test subject, to determine whether the
test subject contains
a mutation in NRG-2 gene that encodes a NRG-2 polypeptide including the amino
acid sequence
set forth in SEQ ID NOs: 2 or 4, where the presence of the mutation is an
indication that the test
subject has an increased likelihood of developing a NRG-2-related disease.
Various embodiments of the claimed invention provide use of a human neuregulin-
2
(NRG-2) polypeptide comprising SEQ ID NO: 4, for protecting dopaminergic
neurons under
stress from damage, wherein contact of the dopaminergic neurons with NRG-2
polypeptide
promotes survival of the dopaminergic neurons.
Various embodiments of the claimed invention provide use of a human neuregulin-
2
(NRG-2) polypeptide comprising SEQ ID NO: 4, for increasing mitogenesis,
survival,
growth, or differentiation of a cell, wherein said cell is a muscle cell, and
wherein said cell
expresses an epidermal growth factor B (erbB) receptor that is selective for
the NRG-2
polypeptide.
Various embodiments of the claimed invention provide use of a human neuregulin-
2
(NRG-2) polypeptide comprising SEQ ID NO: 4, for preparation of a composition
comprising
said polypeptide and a pharmaceutically acceptable diluent, carrier or
excipient, said
composition being for increasing mitogenesis, survival, growth, or
differentiation of a cell,
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wherein said cell is a muscle cell, and wherein said cell expresses an
epidermal growth factor
B (erbB) receptor that is selective for the NRG-2 polypeptide.
Various embodiments of the claimed invention provide use of a human
recombinant
neuregulin-2 (NRG-2) polypeptide for promotion of cerebellar granule neuron
proliferation,
survival, or both, wherein said recombinant NRG-2 polypeptide comprises SEQ ID
NO: 4.
Various embodiments of the claimed invention provide use of a human
recombinant
neuregulin-2 (NRG-2) polypeptide, wherein said recombinant NRG-2 polypeptide
comprises
SEQ ID NO: 4, for preparation of a composition comprising said recombinant NRG-
2
polypeptide and a pharmaceutically acceptable diluent, carrier or excipient,
wherein said
composition is for protecting dopaminergic neurons under stress from damage,
wherein
contact of the dopaminergic neurons with said recombinant NRG-2 polypeptide
promotes
survival of the dopaminergic neurons.
Various embodiments of the claimed invention provide use of Use of a
neuregulin-2
(NRG-2) polypeptide for increasing cardiomyocyte survival, cardiomyocyte
proliferation,
cardiomyocyte growth, or cardiomyocyte differentiation in a mammal, wherein
said NRG-2
polypeptide comprises SEQ ID NO: 4.
Various embodiments of the claimed invention provide use of a neuregulin-2
(NRG-2)
polypeptide for preparation of a composition comprising said polypeptide and a
pharmaceutically acceptable diluent, carrier or excipient, said composition
being for
increasing cardiomyocyte survival, cardiomyocyte proliferation, cardiomyocyte
growth, or
cardiomyocyte differentiation in a mammal, wherein said NRG-2 polypeptide
comprises SEQ
ID NO: 4.
Various embodiments of the claimed invention provide an isolated neuregulin-2
(NRG-2) polypeptide consisting of the amino acid sequence set forth in SEQ ID
NO: 4.
Various embodiments of the claimed invention provide an isolated nucleic acid
molecule comprising a nucleic acid sequence that is at least 95% identical to
the nucleic acid
sequence set forth in SEQ ID NO: 3.
Various embodiments of the claimed invention provide an isolated nucleic acid
molecule consisting of the nucleic acid sequence set forth in SEQ ID NO: 3.
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Various embodiments of the claimed invention provide a nucleic acid molecules
that
are antisense to nucleic acid molecules as described above.
Various embodiments of the claimed invention provide vectors comprising
nucleic
acid molecules as described above, and cells transformed or transfected with
such vectors.
Various embodiments of the claimed invention provide a composition comprising
a
human neuregulin-2 (NRG-2) polypeptide and a pharmaceutically acceptable
diluent, carrier
or excipient, for use in increasing mitogenesis, survival, growth, or
differentiation of a cell,
wherein said cell is a muscle cell; wherein said NRG-2 polypeptide comprises
SEQ ID NO: 4;
and wherein said cell expresses an epidermal growth factor B (erbB) receptor
that is selective
for the NRG-2 polypeptide.
Various embodiments of the claimed invention provide a composition comprising
a
human neuregulin-2 (NRG-2) polypeptide and a pharmaceutically acceptable
diluent, carrier
or excipient, for use in promotion proliferation and/or survival of a
cerebellar granule neuron,
wherein said NRG-2 polypeptide is a recombinant NRG-2 polypeptide that
comprises SEQ ID
NO: 4.
Various embodiments of the claimed invention provide a composition comprising
a
neuregulin-2 (NRG-2) polypeptide and a pharmaceutically acceptable diluent,
carrier or
excipient, for use in increasing cardiomyocyte survival, cardiomyocyte
proliferation,
cardiomyocyte growth, or cardiomyocyte differentiation in a mammal, wherein
said NRG-2
polypeptide comprises SEQ ID NO: 4.
Various embodiments of the claimed invention provide a purified recombinant
NRG-2
polypeptide producing cell for use in protecting dopaminergic neurons under
stress from
damage, wherein the cell produces a recombinant neuregulin-2 (NRG-2)
polypeptide
comprising SEQ ID NO: 4, wherein said recombinant NRG-2 polypeptide upon
contact of the
dopaminergic neurons promotes survival of the dopaminergic neurons.
Various embodiments of the claimed invention provide a purified recombinant
NRG-2
polypeptide producing cell for use in promoting cerebellar granule neuron
proliferation and/or
survival, wherein the cell produces a recombinant neuregulin-2 (NRG-2)
polypeptide
comprising SEQ ID NO: 4, wherein said recombinant NRG-2 polypeptide promotes
cerebellar granule neuron proliferation and/or survival.
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By "neuregulin" or "NRG" is meant a polypeptide that is encoded by a NRG-1,
NRG-2,
or NRG-3 gene or nucleic acid molecule (e.g., a cDNA), and binds to and
activates an erbB
receptor or combinations thereof. Generally, a neuregulin possesses
recognizable domains, such
as an epidermal growth factor-like (EGFL) domain, that binds to and activates
an erbB receptor or
combinations thereof, and an immunoglobulin (Ig) domain. An EGFL domain bears
structural
similarity to the EGF receptor-binding domain as disclosed in Holmes et al.
(Science 256:1205-
1210, 1992), U.S. Patent No. 5,530,109, U.S. Patent No. 5,716,930, U.S. Serial
No. 08/461,097,
Hijazi etal. (Int. J. Oncol. 13:1061-1067, 1998), Chang etal. (Nature 387:509-
512, 1997),
Carraway et at. (Nature
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87:512-516, 1997), Higashiyama etal. (J. Biochem. 122: 675-680, 1997), and PCT
publication WO 97/09425.
By "neuregulin 2" or "NRG-2" is meant a polypeptide encoded by a NRG-2 gene,
or a NRG-2 nucleic acid molecule, and is described in the specification
herein, and in, for
example, Carraway etal., Nature 387: 512-516, 1997; Chang etal., Nature 387:
509-511,
1997; Higashiyama etal., I Biochem. 122: 675-680, 1997; and Busfield etal.,
Mol. Cell.
Biol. 17:4007-4014, 1997. NRG-2 isoforms include Neural-and Thymus-Derived
Activator
of ErbB Kinases (NTAK; Genbank Accession No. AB005060; Higashiyama et al., J
Biochem. 122: 675-680, 1997), Divergent of Neuregulin (don-1; PCT publication
WO
98/07736), and Cerebellum-Derived Growth Factor (CDGF; PCT publication WO
97/09425;
U.S.P.N. 5,912,326), and the NRG-2 molecules described herein. Generally, the
erbB1
receptor (EGFR) is a preferred dimerization partner for a NRG-2 polypeptide.
Preferred
receptor combinations for NRG-2 polypeptides are erbB4 homodimers, erbB2/erbB4
heterodimers, or erbBl/erbB3 heterodimers. A CDGF, don-1, or NTAK polypeptide
or
nucleic acid molecule, as set forth in the amino acid and nucleic acid
sequences disclosed in
Higashiyama et al., J. Biochem. 122: 675-680, 1997, WO 98/07736, WO 97/09425,
and
U.S.P.N. 5,912,326, may be specifically excluded from certain aspects of the
invention. For
example, one or more of a CDGF, don-1, or NTAK polypeptide or nucleic acid
molecule,
may be excluded from the methods for increasing the mitogenesis, survival,
growth, or
differentiation of a cell; for increasing the cardiomyocyte survival,
cardiomyocyte
proliferation, cardiomyocyte hypertrophy, or cardiomyocyte differentiation in
a mammal; for
affecting cellular communication between a neuronal-associated cell and a
neuronal cell; for
stimulating mitogenesis of a glial cell; for inducing myelination of a
neuronal cell by a glial
cell; for the treatment or prophylaxis of a pathophysiological condition of
the nervous system
in a mammal; for the treatment of a tumor; for the treatment of
neurofibromatosis; for
inhibiting proliferation of a cell; or for stimulating proliferation of a
cell.
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By "erbB receptor" is meant erbB1 (EGFR), erB2, erbB3, and erbB4
(also HER-1, HER-2, HER-3, and HER-4 of human) existing as monomeric or
multimeric (e.g., homodimeric or heterodimeric) cell surface receptor tyrosine
kinases that bind to and/or are activated by one or more neuregulins (Meyer et
al.,
Development 124: 3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci.
USA 90: 1867-71, 1993; Marchionni et al., Nature 362: 312-8, 1993; Chen et
at.,
.1. Comp. Neurol. 349: 389-400, 1994; Corfas et al., Neuron 14: 103-115, 1995;
Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; and
Pinkas-Kramarski et at., Oncogene 15: 2803-2815, 1997). Preferably, the erbB
receptors are erbB4 homodimers, erbB2/erbB4 heterodimers, erbBl/erbB3
heterodimers, or any receptor combination that is selective for a NRG-2
polypeptide over a NRG-1 polypeptide or a NRG-3 polypeptide.
By "selective" is meant the preferential binding of an erbB receptor or
combination thereof to a NRG-2 polypeptide over a NRG-1 or a NRG-3
polypeptide. More specifically, preferential binding is defined as an increase
in
the affinity of an erbB receptor to a NRG-2 polypeptide of at least 1.5 fold,
more
preferably at least 2 fold, relative to the affinity of an erbB receptor to a
NRG-1
or NRG-3 polypeptide.
By "neuronal cell" is meant a neuron, nerve cell; neurocyte, or
neuronal progenitor cell. A neuronal cell is the morphological and functional
unit
of the central nervous system and the peripheral nervous system, and includes
cholinergic neurons and non-cholinergic neurons.
By "neuronal-associated cell" is meant any non-neuronal cell that is
capable of affecting the function of a neuron, or whose function can be
affected
by a neuron. A neuronal-associated cell includes, but is not limited to, a
muscle
cell or a nervous system support cell, including a Schwann cell, an astrOcyte,
an
oligodendrocyte, an 0-2A progenitor cell, a glial cell (e.g., a radial glial
cell or a
Bergmann glial cell), a microglial cell, an olfactory bulb ensheathing cell,
or a
sensory organ cell (e.g., a retinal cell).
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By "muscle cell" is meant any cell that contributes to muscle tissue.
Muscle tissue is a primary tissue, consisting mainly of specialized
contractile
cells, and is generally classified as skeletal muscle, cardiac muscle, or
smooth
muscle. Myoblasts, satellite cells, myotubes, myocytes (e.g., cardiomyocytes),
and myofibril tissues are all included in the term "muscle cells," and may all
be
treated according to the methods of the invention. Muscle cell effects may be
induced within skeletal, cardiac, and smooth muscle.
By "neurotrophic agent" or "neurotrophic factor" is meant a substance
that elicits a trophic effect in one or more neuronal cells. These effects
include,
but are not limited to, survival, mitosis, and differentiation. Neurotrophic
agents
include, but are not limited to, neurotrophins, nerve growth factor, ciliary
neurotrophic factor, and brain-derived neurotrophic factor.
By "affecting" is meant the induction of a quantitative change in the
response of a target cell, as a result of an interaction with a NRG-2
polypeptide or
nucleic acid molecule.
By "cellular communication" is meant the synthesis of a substance
(e.g., a neurotrophic agent) in a first cell type (e.g., a neuronal-associated
cell)
and the interaction of that substance with a second cell type (e.g., a
neuronal cell),
such that the substance elicits a change in the first or second cell type.
Cellular
communication includes, but is not limited to, secretion of the substance from
a
cell. Cellular communication can occur reciprocally or non-reciprocally with
one
or more cell types.
By "mitogenesis" is meant any cell division that results in the
production of new cells in the patient. More specifically, mitogenesis in
vitro is
defined as an increase in mitotic index, relative to untreated cells, of 50%,
more
preferably 100%, and most preferably 300%, when the cells are exposed to
labeling agent for a time equivalent to two doubling times. The mitotic index
is
the fraction of cells in culture that have labeled nuclei when grown in the
presence of a tracer that only incorporates during S phase (e.g., BrdU), and
the
doubling time is defined as the average time required for the number of cells
in
the culture to increase by a factor of two. By "inhibiting mitogenesis" is
meant a
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decrease in the mitotic index, relative to untreated cells, of 50%, more
preferably
100%, and most preferably 300%, when the cells are exposed to labeling agent
for a time equivalent to two doubling times. Inhibiting mitogenesis also means
a
cessation of any increase in the mitotic index, relative to control cells.
An effect on mitogenesis in vivo is defmed as an increase in cell
activation as measured by the appearance of labeled cells in the tissue of a
mammal exposed to a tracer that only incorporates during S phase (e.g., BrdU).
An useful therapeutic is defined in vivo as a compound that increases cell
activation relative to a control mammal by at least 10%, more preferably by at
least 50%, and most preferably by more than 200% when the mammal is exposed
to labeling agent for a period of greater than 15 minutes and tissues are
assayed
between 10 hours and 24 hours after administration of the mitogen at the
therapeutic dose. For example, in muscle cells, satellite cell activation in
vivo
may be detected by monitoring BrdU incorporation. Alternatively, satellite
cell
activation in vivo may be detected by the appearance of the intermediate
filament
vimentin by immunological or RNA analysis methods. When vimentin is
assayed, the useful mitogen is defmed as one which causes expression of
detectable levels of vimentin in the muscle tissue when the therapeutically
useful
dosage is provided. Mitogenesis may be induced in, for example, muscle cells
of
skeletal, cardiac, and smooth muscle, and in glial cells.
By "survival" is meant any process by which a cell avoids death. The
term survival as used herein also refers to the prevention of cell loss as
evidenced
by necrosis, apoptosis, or the prevention of other mechanisms of cell loss.
Increasing survival as used herein indicates a decrease in the rate of cell
death by
at least 10%, more preferably by at least 50%, and most preferably by at least
100% relative to an untreated control. The rate of survival may be measured by
counting cells capable of being stained with a dye specific for dead cells
(e.g.,
propidium iodide) in culture. The rate of survival may be measured by counting
cells stainable with a dye specific for dead cells (such as propidium iodide)
in
culture when the cells are 8 days post-differentiation (i.e., 8 days after the
medium is changed from 20% to 0.5% serum).
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By "growth" is meant the increase in size or number of a cell type
relative to a control cell. The therapeutic usefulness of growth increases the
size
or number of a cell in diseased tissue by at least 10% or more, more
preferably by
50% or more, and most preferably by more than 100% relative to the equivalent
tissue in a similarly treated control animal. Growth can be measured by, for
example, an increase in net weight, protein content, or cell diameter. Muscle
growth may occur by the increase in the fiber size and/or by increasing the
number of fibers.
By "differentiation" is meant a morphological and/or chemical change
that results in the generation of a different cell type or state of
specialization. The
differentiation of cells as used herein refers to a cellular development
program
that specifies one or more components of a cell type. The therapeutic
usefulness
of differentiation increases the quantity of any component of a cell in
diseased
tissue by at least 10% or more, more preferably by 50% or more, and most
preferably by more than 100% relative to the equivalent tissue in a similarly
treated control animal.
By "proliferation" is meant the growth or reproduction of similar cells.
By "inhibiting proliferation" is meant the decrease in the number of similar
cells
by at least 10%, more preferably by at least 20%, and most preferably by at
least
50%. By "stimulating proliferation" is meant an increase in the number of
similar
cells by at least 10%, more preferably by at least 20%, and most preferably by
at
least 50%.
By "inducing myelination" is meant the acquisition, development, or
formation of myelin sheath around a nerve fiber. The useful therapeutic for
inducing myelination confers an increase in the density of a myelin sheath by
at
least 10%, more preferably by at least 20%, and most preferably by at least
50%,
relative to a control nerve fiber. By "demyelination" is meant the loss of the
myelin sheath around a nerve fiber.
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By "interacts" is meant contact of a NRG-2 polypeptide with a receptor
or other molecule on a target cell.
By "pathophysiological condition" is meant a disturbance of function
and/or structure of a living organism, resulting from an external source, a
genetic
predisposition, a physical or chemical trauma, or a combination of the above,
including, but not limited to, any mammalian disease.
By "neuropathy" is meant any disorder affecting the nervous system.
A neuropathy may be, for example, a peripheral neuropathy, such a sensory
nerve
fiber neuropathy or motor fiber neuropathy.
By "cardiomyopathy" is meant a disease that affects the heart muscle.
Cardiomyopathy may be primary, i.e., mainly affecting cardiac muscle, or
secondary, i.e., affecting cardiac muscle secondary to a systemic disease,
infection, or metabolic disease.
By "ischemic damage" is meant damage resulting from decreased
blood circulation to cardiac muscle.
By "degenerative congenital disease" is meant a disease that exists at
birth, which may be hereditary or due to an influence occurring during
gestation,
that results in a pathological change in cells or tissues.
By "treatment" is meant the medical management of a patient with the
intent to cure, ameliorate, stabilize, or prevent a disease, pathological
condition,
or disorder. This term includes active treatment, that is, treatment directed
specifically toward the improvement or associated with the cure of a disease,
pathological condition, or disorder, and also includes causal treatment, that
is,
treatment directed toward removal of the cause of the associated disease,
pathological condition, or disorder. In addition, this term includes
palliative
treatment, that is, treatment designed for the relief of symptoms rather than
the
curing of the disease, pathological condition, or disorder; preventative
treatment,
that is, treatment directed to minimi7ing or partially or completely
inhibiting the
development of the associated disease, pathological condition, or disorder;
and
supportive treatment, that is, treatment employed to supplement another
specific
therapy directed toward the improvement of the associated disease,
pathological
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condition, or disorder. The phrase "treatment" also includes symptomatic
treatment, that is, treatment directed toward constitutional symptoms of the
associated disease, pathological condition, or disorder.
By "therapeutically-effective amount" is meant an amount of a NRG-2
polypeptide or nucleic acid molecule sufficient to produce a healing,
curative,
stabilizing, or ameliorative effect in the treatment of a disorder.
By "neurodegenerative disorder" is meant any pathophysiological
condition that is characterized by the degeneration of neuronal cells or
neuronal-
associated cells. The degeneration may include, for example, a decrease in
cell
number or size, an increase in cell apoptosis or death, or a decrease in cell
growth, survival or differentiation.
By "neural regeneration or neural repair" is meant the treatment of a
pathophysiological condition by, for example, an increase in neuronal cell or
neuronal-associated cell number or size, a decrease in neuronal cell or
neuronal-
associated cell apoptosis or death, or an increase in neuronal cell or
neuronal-
associated cell growth, survival or differentiation.
By "inhibits binding" is meant preventing or reducing the binding of a
NRG-2 polypeptide to a receptor. The binding is preferably reduced by at least
10%, more preferably by at least 50%, and most preferably by at least 100%
relative to a control sample.
By "polypeptide" or "polypeptide fragment" is meant a chain of two or
more amino acids, regardless of any post-translational modification (e.g.,
glycosylation or phosphorylation), constituting all or part of a naturally or
non-
naturally occurring polypeptide. By "post-translational modification" is meant
any change to a polypeptide or polypeptide fragment during or after synthesis.
Post-translational modifications can be produced naturally (such as during
synthesis within a cell) or generated artificially (such as by recombinant or
chemical means). A "protein" can be made up of one or more polypeptides.
The term "identity" is used herein to describe the relationship of the
sequence of a particular nucleic acid molecule or polypeptide to the sequence
of a
reference molecule of the same type. For example, if a polypeptide or nucleic
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acid molecule has the same amino acid or nucleotide residue at a given
position,
compared to a reference molecule to which it is aligned, there is said to be
"identity" at that position. The level of sequence identity of a nucleic acid
molecule or a polypeptide to a reference molecule is typically measured using
sequence analysis software with the default parameters specified therein, such
as
the introduction of gaps to achieve an optimal alignment (e.g., Sequence
Analysis
Software Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, or
PIT FUP/PREITYBOX programs). These software programs match identical or
similar sequences by assigning degrees of identity to various substitutions,
deletions, or other modifications. Conservative substitutions typically
include
substitutions within the following groups: glycine, alanine, valine,
isoleucine, and
leucine; aspartic acid, glutasnic acid, asparagine, and glutamine; serine and
threonine; lysine and arginine; and phenylalanine and tyrosine.
A nucleic acid molecule or polypeptide is said to be "substantially
identical" to a reference molecule if it exhibits, over its entire length, at
least
90%, preferably at least 95%, more preferably at least 97%, and most
preferably
99% identity to the sequence of the reference molecule. For polypeptides, the
length of comparison sequences is at least 16 amino acids, preferably at least
20 amino acids, more preferably at least 25 amino acids, and most preferably
at
least 35 amino acids. For nucleic acid molecules, the length of comparison
sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more
preferably at least 75 nucleotides, and most preferably at least 110
nucleotides.
A nucleic acid molecule or polypeptide is "analyzed" or subject to
"analysis" if a test procedure is carried out on it that allows the
determination of
its biological activity or whether it is wild type or mutated. For example,
one can
analyze the genes of an animal (e.g., a human) by amplifying genomic DNA of
the animal using the polymerase chain reaction, and then determining whether
the
amplified DNA contains a mutation, e.g., by nucleotide sequence or restriction
fragment analysis.
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By a "substantially pure polypeptide" is meant a polypeptide (or a
fragment thereof) that has been separated from proteins and organic molecules
that naturally accompany it. Typically, a polypeptide is substantially pure
when it
is at least 60%, by weight, free from the proteins and naturally-occurring
organic
molecules with which it is naturally associated. Preferably, the polypeptide
is a
NRG-2 polypeptide that is at least 75%, more preferably at least 90%, and most
preferably at least 99%, by weight, pure. A substantially pure NRG-2
polypeptide can be obtained, for example, by extraction from a natural source
(e.g., cerebellum), by expression of a recombinant nucleic acid molecule
encoding a NRG-2 polypeptide, or by chemical synthesis. Purity can be
measured by any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components
when it is separated from those proteins and organic molecules that accompany
it
in its natural state. Thus, a protein that is chemically synthesized or
produced in a
cellular system different from the cell in which it is naturally produced is
substantially free from its naturally associated components. Accordingly,
substantially pure polypeptides not only include those derived from eukaryotic
organisms, but also those synthesized in E. coli or other prokaryotes.
An antibody is said to "specifically bind" to a polypeptide if it
recognizes and binds to the polypeptide (e.g., a NRG-2 polypeptide), but does
not
substantially recognize and bind to other molecules (e.g., non-NRG-2 related
polypeptides) in a sample, e.g., a biological sample, that naturally includes
the
polypeptide.
By a "transgene" is meant a DNA molecule that is inserted by artifice
into a cell (e.g., the nuclear genome of a cell), and is incorporated into the
genome of an organism that develops from the cell. Such a transgene can be
partly or entirely heterologous (ie., foreign) to the transgenic organism, or
can be
a gene that is homologous to an endogenous gene of the organism. An organism
or animal (e.g., a mammal, such as a mouse, rat, or goat) can be said to be
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"transgenic" if it developed from a cell that had a transgene inserted into it
by
artifice.
By a "knockout mutation" is meant an artificially-induced alteration in
a nucleic acid molecule (created by recombinant DNA technology or deliberate
exposure to a mutagen) that reduces the biological activity of the polypeptide
normally encoded therefrom by at least 80% relative to the unmutated gene. The
mutation can be, without limitation, an insertion, deletion, frarneshift
mutation, or
a missense mutation. A "knockout animal" is preferably a mammal, and more
preferably a mouse, containing a knockout mutation, as defined above.
By "vector" is meant a genetically engineered plasmid or virus, derived
from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus,
herpesvirus,
or artificial chromosome, that is used to transfer a polypeptide (e.g., a NRG-
2
polypeptide) coding sequence, operably linked to a promoter, into a host cell,
such that the encoded peptide or polypeptide is expressed within the host
cell.
By "promoter" is meant a minimal sequence sufficient to direct or
control transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression controllable for cell
type
or physiological status (e.g., hypoxic versus normoxic conditions), or
inducible
by external signals or agents; such elements may be located in the 5' or 3' or
internal regions of the native gene.
By "operably linked" is meant that a nucleic acid encoding a
polypeptide (e.g., a cDNA) and one or more regulatory sequences are connected
in such a way as to permit gene expression when the appropriate molecules
(e.g.,
transcriptional activator proteins) are bound to the regulatory sequences.
By "NRG-2 polypeptide producing cell" is meant a cell (or a
descendent of a cell) into which a DNA molecule encoding a NRG-2 polypeptide
has been introduced, by means of recombinant DNA techniques or known gene
therapy techniques.
The invention provides several advantages. For example, it provides
methods and reagents that can be used in the diagnosis and treatment of
diseases
that are sensitive to the bioactivities of NRG-2 polypeptides. Other features
and
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advantages of the invention will be apparent from the detailed description of
the
invention, the drawings, and the claims.
Brief Description of the Drawings
Fig. 1 shows a schematic diagram of NRG-2 gene products and human
cDNA clones.
Fig. 2 shows a schematic diagram of the mammalian expression vector
pRc/CMV2.
Fig. 3A shows a western blot analysis of conditioned media (cm) and
cell lysates (cells) from mock- (vector) or rhNRG-2a-transfected CHO/S cells.
Fig. 3B shows a western blot analysis of conditioned media from
mock- (vec), rhNRG-2a (a), or rhNRG-213-transfected (0) CHO/S cells.
Fig. 3C shows a western blot analysis of conditioned media from CHO
cells expressing rat NRG-213.
Figs. 4A-B show western blot analyses of receptor phosphorylation on
tyrosine residues in cells responsive to treatment with NRG-2 polypeptides.
Fig. 5 shows a western blot analysis of the expression of recombinant
human NRG-2a and NRG-2(3.
Fig. 6 shows the nucleic acid sequence for human NRG-2a (SEQ ID
NO: 1).
Fig. 7 shows the amino acid sequence for human NRG-2cc (SEQ ID
NO: 2).
Fig. 8 shows the nucleic acid sequence for human NRG-211 (clone 2b7)
(SEQ ID NO: 3).
Fig. 9 shows the amino acid sequence for human NRG-213 (clone 2b7)
(SEQ ID NO: 4).
Fig. 10 shows a western blot analysis of stable transfectants used for
methotrexate selection.
Fig. 11A shows a Coomassie stained polyacrylamide gel of purified
rhNRG-213.
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Fig. 11B shows a scan of the gel of Fig. 11A.
Fig. 12 shows a gold stained polyacrylamide gel of total protein and
purified rhNRG-2p.
Fig. 13 shows a western blot analysis of receptor.phosphorylation of
tyrosine residues in cells responsive to HPLC purified rhNRG-2f3.
Fig. 14A shows a photograph of midbrain dopaminergic neurons
pretreated with rhNRG-213 and challenged with 6-0HDA.
Fig. 14B shows a photograph of untreated control midbrain
dopaminergic neurons challenged with 6-01-EDA.
Fig. 15A shows a photograph indicating BrdU incorporation in neural
cells, including neural progenitor cells, cultured with rhNRG-213.
Fig. 15B shows a bar graph indicating the effect of rhNRG-2I3 on BrdU
incorporation.
Fig. 16A shows a photograph of cerebellar granule neurons migrating
on a glial cell process.
Fig. 16B shows a bar graph indicating the effect of NRG-2 on neuronal
migration.
Fig. 17 shows a western blot analysis of the activation of p42/44
MAPk(Erk) and Akt by NRG-2 proteins.
Fig. 18 shows a line graph of 3H-Lencine uptake into neonatal rat
ventricular myocytes treated with NRG proteins.
Detailed Description of the Invention
The invention provides NRG-2 polypeptides and nucleic acid
molecules, antibodies that bind these NRG-2 polypeptides, and therapeutic and
diagnostic methods employing NRG-2 polypeptides and nucleic acid molecules.
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Bioassays
NRG-2 ligands and erbB receptors are expressed in the nervous
system, in neural precursors and neurons of brain, spinal cord and retina;
skeletal
and cardiac muscle; lung; thymus, kidney; adrenal glands; skin; breast
epithelia;
and other organs during embryonic development and in adult tissues. Major
sites
of NRG-2 expression include the cerebellum (Purkinje and granule cells),
olfactory bulb, dentate gyms, pyramidal cells of the occipital cortex, lung,
and
thymus. The expression patterns of NRG-2 receptors in specific cells and
tissues
are used to identify cellular targets of NRG-2 actions, and to identify
bioactivities
that are relevant to specific NRG-2-related diseases, such as demyelinating
disorders of the peripheral and central nervous systems; neuropathies;
neurodegenerative disorders; cardiomyopathies; loss of hearing, balance or
vision; pain; neurotrauma; cancer; sensorineural hearing loss or sensorineural
balance loss from viral infection, aging, or antibiotics (e.g.
aminoglycosides);
retinopathy (e.g., hypertensive, diabetic, occlusive, macular degeneration,
retinitis
pigmentosa, optic neuropathy, injury); Guillaime Barre disease; stroke; or
brain
or spinal cord injury. Many of the NRG-2-responsive cell types in embryonic,
neonatal, and adult tissues express the receptor combinations of erbB2/erbB3,
erbB2/erbB4, or erbB4 alone. For example, peripheral nervous system (PNS) and
central nervous system (CNS) glial cell types express erbB2; Schwann cells
also
express erbB3. In the CNS, erbB4 and erbB3 have been observed on various
glial cell types, including astrocytes, oligodendrocyte progenitors, radial
glia in
the developing cortex, and Bergmann glia in the cerebellum. The erbB2/erbB4
combination is found in ventricular cardiomyocytes.
Therapeutic and diagnostic utilities for NRG-2 polypeptides are
identified by, for example, conducting bioassays in vitro. Culture systems
that
reflect NRG-2 expression patterns, along with the distribution of particular
receptors, such as erbB2/erbB4 or erbB4 alone, which are examples of erbB
receptor combinations that may show a preference for NRG-2 over NRG-1, are
selected. For example, NRG-2 bioactivities are evaluated using CNS glia, such
as oligodendrocytes and olfactory bulb ensheathing cells, mid-brain
dopaminergic
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neurons, cerebellar granule neurons, and cardiomyocytes. These cell
populations
express NRG receptors, and respond to treatment with one or more isoforms of
NRG-1 in a variety of quantitative bioassays. The activities of NRG-2 (e.g.
rhNRG-2a, rhNRG-213) and NRG-1 (e.g., rhGGF2) isoforms are compared, using
sister cultures, in various dose-response assays, including but not limited
to,
stimulation of proliferation, survival, differentiation, migration, and
morphological changes. The relative potencies of the NRG-2 and NRG-1
=
isoforms are determined on the basis of, for example, protein concentration.
Diagnostic Methods Employing NRG-2 Nucleic Acid Molecules, Polypeptidest
and Antibodies
NRG-2 nucleic acid molecules, polypeptides, and antibodies are used
in methods to diagnose or monitor a variety of diseases and conditions,
including
those involving mutations in, or inappropriate expression of, NRG-2 genes.
NRG-2 expression has been documented in a variety of tissues, as discussed
above. Thus, detection of abnormalities in NRG-2 genes or their expression are
used in methods to diagnose, or to monitor treatment or development of
diseases
of these tissues.
The diagnostic methods of the invention are used, for example, with
patients that have a cardiovascular or a neurological disease, in an effort to
determine its etiology, and thus, to facilitate selection of an appropriate
course of
treatment. The diagnostic methods are also used with patients that have not
yet
developed a cardiovascular or neurological disease, but who may be at risk of
developing such a disease, or with patients that are at an early stage of
developing
such a disease. Many cardiovascular and neurological diseases occur during
development, and thus, the diagnostic methods of the invention are also
carried
out on a fetus or embryo during development. Also, the diagnostic methods of
the invention are used in prenatal genetic screening, for example, to identify
parents who may be carriers of a recessive NRG-2 mutation.
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NRG-2 abnormalities that are detected using the diagnostic methods of
the invention include those characterized by, for example, (i) abnormal NRG-2
polypeptides, (ii) NRG-2 genes containing mutations that result in the
production
of such polypeptides, and (iii) NRG:=2 mutations that result in production of
abnormal amounts of NRG-2.
Levels of NRG-2 expression in a patient sample are determined by
using any of a number of standard techniques that are well known in the art.
For
example, NRG-2 expression in a biological sample (e.g., a blood or tissue
sample,
or amniotic fluid) from a patient is monitored by standard northern blot
analysis
or by quantitative PCR (see, e.g., Ausubel et al., Current Protocols in
Molecular
Biology, John Wiley & Sons, New York, NY, 1998; PCR Technology: Principles
and Applications for DNA Amplification, H.A. Ehrlich, Ed., Stockton Press, NY;
Yap et al. Nucl. Acids. Res. 19:4294, 1991).
Therapeutic Methods Employing NRG-2 Nucleic Acid Molecules, Polypeptides,
and Antibodies
The invention includes methods of treating or preventing NRG-2-
related diseases. Therapies are designed to circumvent or overcome a NRG-2
gene defect, or inadequate or excessive NRG-2 gene expression, and thus
modulate and possibly alleviate conditions involving defects in NRG-2 genes or
proteins. In considering various therapies, it is understood that such
therapies are,
preferably, targeted to the affected or potentially affected organs, for
example, the
heart or the nervous system. Reagents that are used to modulate NRG-2
biological activity can include, without limitation, full length NRG-2
polypeptides; NRG-2 cDNA, mRNA, or antisense RNA; NRG-2 antibodies; and
any compound that modulates NRG-2 biological activity, expression, or
stability.
Treatment or prevention of diseases resulting from a mutated NRG-2
gene is accomplished, for example, by replacing a mutant NRG-2 gene with a
normal NRG-2 gene, administering a normal NRG-2 gene, modulating the
function of a mutant NRG-2 protein, delivering normal NRG-2 protein to the
appropriate cells, or altering the levels of normal or mutant NRG-2 protein.
It is
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also possible to correct a NRG-2 defect to modify the physiological pathway
(e.g., a signal transduction pathway) in which the NRG-2 protein participates.
Gene transfer is achieved using viral vectors, as well as non-viral
means involving transfection in vitro by means of any standard technique,
including but not limited to, calcium phosphate, DEAE dextran,
electroporation,
protoplast fusion, and liposomes. Transplantation of normal genes into the
affected tissues of a patient can also be accomplished by transferring a
normal
NRG-2 gene into a cultivatable cell type ex vivo, after which the cell (or its
descendants) is injected into a targeted tissue. Another strategy for
inhibiting
NRG-2 function using gene therapy involves intracellular expression of an anti-
NRG-2 antibody or a portion of an NRG-2 antibody. For example, the gene (or
gene fragment) encoding a monoclonal antibody that specifically binds to NRG-2
and inhibits its biological activity is placed under the transcriptional
control of a
tissue-specific gene regulatory sequence. Another therapeutic approach
involves
administration of recombinant NRG-2 polypeptide, either directly to the site
of a
potential or actual disease-affected tissue (for example, by injection) or
systemically (for example, by any conventional recombinant protein
administration technique). The dosage of systemically delivered NRG-2 depends
on a number of factors, including the size and health of the individual
patient but,
generally, between about 0.006 mg/kg to about 0.6 mg/kg, inclusive, is
administered per day to an adult in any pharmaceutically acceptable
formulation.
Dosages of NRG-2 delivered by local delivery may differ from systemic
delivery,
and can be determined using standard techniques known to those of ordinary
skill
in the art.
In a patient diagnosed as having a NRG-2 mutation or NRG-2-related
disease, or as susceptible to NRG-2 mutations, aberrant NRG-2 expression (even
if those mutations or expression patterns do not yet result in alterations in
NRG-2
expression or biological activity), or to a NRG-2-related disease, any of the
above-described therapies are administered before the occurrence of the
disease
phenotype. Also, compounds shown to modulate NRG-2 expression or NRG-2
biological activity are administered to patients diagnosed with potential or
actual
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diseases by any standard dosage and route of administration. Alternatively,
gene
therapy using an antisense NRG-2 mRNA expression construct is undertaken to
reverse or prevent the gene defect prior to the development of the full course
of
the disease.
The therapeutic methods of the invention are, in some cases, targeted to
prenatal treatment. For example, a fetus found to have a NRG-2 mutation is
administered a gene therapy vector including a normal NRG-2 gene or normal
NRG-2 protein. Such treatment may be required only for a short period of time,
or may, in some form, be required throughout such a patient's lifetime. Any
continued need for treatment, however, is determined using, for example, the
diagnostic methods described above. Also as discussed above, NRG-2
abnormalities may be associated with diseases in adults, and thus, adults are
subject to the therapeutic methods of the invention as well.
Identification of Molecules that Modulate NRG-2 Biological Activity or Whose
Biological Activity is Modulated by NRG-2
Isolation of NRG-2 cDNAs (as described herein) also facilitates the
identification of molecules that increase or decrease NRG-2 biological
activity.
Similarly, molecules whose activity is modulated by NRG-2 biological activity
can be identified. According to one approach, candidate molecules are added at
varying concentrations to the culture medium of cells expressing NRG-2 mRNA.
NRG-2 biological activity is then measured using standard techniques. The
measurement of biological activity can include, without limitation, the
measurement of NRG-2 protein and nucleic acid molecule levels, and NRG-2
phosphorylation.
If desired, the effect of candidate modulators on expression can also be
measured at the level of NRG-2 protein production using the same general
approach and standard immunological detection techniques, such as western
blotting or immunoprecipitation with a NRG-2-specific antibody (see below).
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A test compound that is screened in the methods described above can
be a chemical, be it naturally-occurring or artificially-derived. Such
compounds
can include, for example, polypeptides, synthesized organic molecules,
naturally
occurring organic molecules, nucleic acid molecules, and components thereof.
Candidate NRG-2 modulators include peptide as well as non-peptide molecules
(e.g., peptide or non-peptide molecules found, e.g., in a cell extract,
mammalian
serum, or growth medium in which mammalian cells have been cultured).
Administration of NRG-2 Polypentides, NRG-2 Nucleic Acid Molecules, and
Modulators of NRG-2 Synthesis or Function
A NRG-2 protein, nucleic acid molecule, modulator, neutralizing
NRG-2 antibody, or NRG-2-inhibiting compound (e.g., antisense NRG-2 or a
NRG-2 dominant negative mutant) is administered within a pharmaceutically-
acceptable diluent, carrier, or excipient, in unit dosage form to patients or
experimental animals. Also, conventional pharmaceutical practice is employed
to
provide suitable formulations or compositions in which to administer such
molecules or compounds to patients suffering from a NRG-2-related disease,
such
as demyelinating disorders of the peripheral and central nervous systems;
neuropathies; neurodegenerative disorders; cardiomyopathies; loss of hearing,
balance or vision; pain; neurotrauma; cancer; sensorineural hearing loss or
sensorineural balance loss from viral infection, aging, or antibiotics (e.g.
aminoglycosides); retinopathy (e.g., hypertensive, diabetic, occlusive,
macular
degeneration, retinitis pigmentosa, optic neuropathy, injury); Guillaime Barre
disease; stroke; or brain or spinal cord injury. Administration can begin
before or
after the patient is symptomatic.
Any appropriate route of administration can be employed, for example,
administration can be parenteral, intravenous, intra-arterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal,
inhalation to
deep lung, aerosol, by suppositories, oral, or topical (e.g. by applying an
adhesive
patch carrying a formulation capable of crossing the dermis and entering the
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bloodstream). Preferably, the administration is local to the afflicted tissue,
such
as cardiac, lung, or nerve tissue. Therapeutic formulations can be in the form
of
liquid solutions or suspensions; for oral administration, formulations can be
in the
form of tablets or capsules; and for intranasal formulations, in the form of
powders, nasal drops, or aerosols. Any of the above formulations may be a
sustained-release formulation.
Methods that are well known in the art for making formulations are
found, for example, in Remington's Pharmaceutical Sciences, (18th edition),
ed.
A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Formulations for
parenteral administration can, for example, contain excipients; sterile water;
or
saline; polyalkylene glycols, such as polyethylene glycol; oils of vegetable
origin;
or hydrogenated napthalenes. Sustained-release, biocompatible, biodegradable
lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-
polyoxypropylene copolymers can be used to control the release of the
compounds. Other potentially useful parenteral delivery systems for NRG-2
modulatory compounds include ethylene-vinyl acetate copolymer particles,
osmotic pumps, implantable infusion systems, and liposomes. Formulations for
inhalation can contain excipients, for example, lactose, or can be aqueous
solutions containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate,
and deoxycholate, or can be oily solutions for administration in the form of
nasal
drops, or as a gel.
Synthesis of NRG-2 Proteins, Polypeptides, and Polvpeptide Fragments
Those skilled in the art of molecular biology will understand that a
wide variety of expression systems can be used to produce the recombinant NRG-
2 proteins. The precise host cell used is not critical to the invention. The
NRG-2
proteins can be produced in a prokaryotic host (e.g., E. coli) or in a
eukaryotic
host (e.g., S. cerevisiae, insect cells such as Sf) cells, or mammalian cells
such as
COS, NlEH 3T3, CHO, or HeLa cells). These cells are commercially available
from, for example, the American Type Culture Collection, Rockville, MD (see
also Ausubel et al., Current Protocols in Molecular Biology, John Wiley &
Sons,
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New York, NY, 1998). The method of transformation and the choice of
expression vehicle (e.g., expression vector) will depend on the host system
selected. Transformation and transfection methods are described, e.g., in
Ausubel
et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York,
NY, 1998, and expression vehicles can be chosen from those provided, e.g. in
Pouwels et at., Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987).
The characteristics of NRG-2 nucleic acid molecules are analyzed by
introducing such genes into various cell types or using in vitro extracellular
systems. The function of NRG-2 proteins produced in such cells or systems are
then examined under different physiological conditions. Also, cell lines can
be
produced that over-express the NRG-2 gene product, allowing purification of
NRG-2 for biochemical characterization, large-scale production, antibody
production, and patient therapy.
Use of NRG-2 Antibodies
Antibodies to NRG-2 proteins (for example, those described herein) are
used to detect NRG-2 proteins or to inhibit the biological activities of NRG-2
proteins. For example, a nucleic acid molecule encoding an antibody or portion
of an antibody can be expressed within a cell to inhibit NRG-2 function. In
addition, the antibodies can be coupled to compounds, such as radionuclides
and
liposomes for diagnostic or therapeutic uses. Antibodies that inhibit the
activity
of a NRG-2 polypeptide can also be useful in preventing or slowing the
development of a disease caused by inappropriate expression of a wild type or
mutant NRG-2 gene.
Construction of Transgenic Animals and Knockout Animals
Characterization of NRG-2 genes provides information that allows
NRG-2 knockout animal models to be developed by homologous recombination.
Preferably, a NRG-2 knockout animal is a mammal, most preferably a mouse.
Similarly, animal models of NRG-2 overproduction can be generated by
integrating one or more NRG-2 sequences into the genome of an animal,
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according to standard transgenic techniques. Moreover, the effect of NRG-2
gene
mutations (e.g., dominant gene mutations) can be studied using transgenic mice
carrying mutated NRG-2 transgenes or by introducing such mutations into the
endogenous NRG-2 gene, using standard homologous recombination techniques.
A replacement-type targeting vector, which can be used to create a
knockout model, can be constructed using an isogenic genomic clone, for
example, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla, CA).
The
targeting vector can be introduced into a suitably-derived line of embryonic
stem
(ES) cells by electroporation to generate ES cell lines that carry a
profoundly
truncated form of a 1VRG-2 gene. To generate chimeric founder mice, the
targeted cell lines are injected into a mouse blastula-stage embryo.
Heterozygous
offspring can be interbred to homozygosity. NRG-2 knockout mice provide a
tool for studying the role of NRG-2 in embryonic development and in disease.
Moreover, such mice provide the means, in vivo, for testing therapeutic
compounds for amelioration of diseases or conditions involving a NRG-2-
dependent or NRG-2-affected pathway.
The following Examples will assist those skilled in the art to better
understand the invention and its principles and advantages. It is intended
that
these Examples be illustrative of the invention and not limit the scope
thereof.
EXAMPLE 1
Cloning of Human NRG-2 cDNA
A full-length cDNA encoding NRG-2a was identified from
cerebellum. Multiple probes to various regions of NRG-2 coding sequences were
designed based on rodent and human sequence data for cloning, mapping and
sequence analysis. Prior to screening libraries, the specificity of the probes
was
confirmed by analyzing human cerebellar RNA using a 3' RACE (rapid
amplification of cDNA ends) approach. Approximately 400,000 cDNAs from
two human cerebellum yt10 cDNA libraries (Clontech Laboratories, Palo Alto,
=
CA; Catalog No. HL1128a) were screened with an oligonucleotide probe: 5' GCA
TCA ACC AGC TCT CCT GC 3' (SEQ ID NO: 5) from the EGFL domain of
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NRG-2. Twenty five hybridization signals were detected; twenty of the phage
clones corresponding to these signals were cloned and further analyzed by
hybridization studies, physical mapping, and DNA sequencing. The results of
these analyses were consistent with the existence of multiple structural
variants
(isoforms) among the human NRG-2 clones that were identified. Preliminary
structural information on the clones was obtained by filter hybridization to
phage
plaques and restriction endonuclease analyses of the cDNA inserts. PCR
studies,
using internal primers, in pairs or in combination with flanking sequences,
were
used to obtain physical mapping data (see Table 1).
The primers used were as follows:
Primer 1471:5' - GCA TCA ACC AGC TCT CCT GC -3' (SEQ ID NO: 6)
Primer 1494:5' - TGC GAA CTG CTG ACA CCT GT -3' (SEQ ED NO: 7)
Primer 1527:5' - CCA CCT ITT GAG CAA GT1' CAG - 3' (SEQ ID NO: 8)
Primer 1528:5' - GAG GTG OCT TAT GAG TTC TTC -3' (SEQ ID NO: 9)
Primer 1531:5' - GGC CAC CAC ACA GAC GAT G - 3' (SEQ ID NO: 10)
First, the insert sizes, which ranged from 0.8 kb to 3.3 kb (average size
was roughly 1.7 kb), were analyzed. NRG-2 transcripts contain an EGFL domain
and cytoplasmic sequences that exhibit much of the structural diversity of
these
polypeptides, and this specific internal region was focused on next to map the
clones by PCR analysis. This analysis yielded four groups of products, and
multiple clones were identified in each group. Therefore, the four groups (A-
D)
are likely to represent the extent of structural diversity in this region
among the
NRG-2 gene products in human cerebellum. Four clones (group A) gave no
product in this experiment. This result was consistent with the data from
hybridization experiments, which had shown that these clones lacked the
sequence of the downstream primer (in the cytoplasmic domain). In the third
experiment, the orientation of the clones was determined and the distance from
the EGFL domain to the ends of the clones was estimated by using primers in
the
EGFL domain in combination with primers from flanking sequences in the phage
arms. These studies, therefore, enabled the segregation of the NRG-2 cDNAs
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into groups, and facilitated identification of potential full-length cDNAs
encoding
secretable isoforms of human NRG-2.
Table 1: Mapping human NRG-2 cerebellar cDNA clones
Group:clones Internal Largest 5' end to EGFL to
3'end4
productl clone:size2 EGFL3
A: 1,4,13,15 none 13: 3300 1400 2300
B: 3,6,7,9,11, 170 11: 1050 600 450
18,20
C: 10,12,14,16 260 14: 1500 850 650
D: 5,8,17,19 650 8: 1600 800 800
PCR analyses of cDNA clones: products were sized on 6% polyacrylamide gels;
the table shows sizes in
base pairs.
1. Upstream primer 1471 from EGFL domain; downstream primer 1531 from
cytoplasmic domain.
2. Primers 1527, 1528 from flanking sequences in 2gt10.
3. Upstream primer 1527 from flanking sequences in Agt10; downstream primer
1494 from EGFL domain.
4. Upstream primer 1471 from EGFL domain; downstream primer 1528 from flanking
sequences in gt10.
EXAMPLE 2
Human NRG-2 DNA Sequence Analysis
To obtain a more complete picture of the different structures, DNA
sequencing was undertaken on representative clones from each group using a
cycle sequencing protocol and the same primers used for the PCR analysis
described above. Comparison of the sequence contigs surrounding the EGFL
domain (from groups B-D) to each other and to rat and human NRG-2 sequences
led to several conclusions. First, the group B clones matched the NRG-20 cDNA
structure. These sequences connected the EGFL domain to the transmembrane
and cytoplasmic domains, and thus encoded membrane-attached NRG-2 protein.
Second, all of the group C structures contained both the a and the sequences,
and matched the structure of the NRG-2a cDNA. Therefore, group C clones
should encode a secreted NRG-2 protein. Clone 14 appeared to be the best
candidate for a full-length version of this structure. In group D, both a and
0
sequences were present, but they were not adjacent. A 450 bp sequence
intervening between these two known coding sequences was found, and
immediately adjacent to the regions identified as a and 0 sequences were
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canonical splice junction donor (GT) and acceptor (AG) sequences. Thus, this
structure probably represents a partially spliced transcript of the NRG-2
gene.
Given this information, it appeared that secretable forms of NRG-2
were most likely be found in the clones of groups A and C. Clone 14 served as
a
suitable representative of group C. Two group A clones were advanced in
parallel; clone 13 was selected because of the relatively large insert size
and clone
was pursued because of the presence of sequence 5' of the EGFL domain that
was detected in hybridization experiments. When sequences of clones 13, 14 and
15 were completed it became apparent that none of them alone encoded a full
10 length human NRG-2a. However, given the substantial overlap in
the structure
of these clones, it was clear that portions of each could be spliced together
to
generate one full length clone encoding NRG-2a. Fig. 1 shows a schematic
representation of the structure of these sequences; coding segments of the NRG-
2
gene are shown in the shaded boxes, and the coding sequences (solid lines)
that
15 are present in described NRG-2a and NRG-213 isoforms are drawn
above;
isoforms of NRG-2 contain GGF2-like, immunoglobulin-like (Ig), EGF-like
(EGFL), a, (3, transmembrane (M), and cytoplasmic (cyto) domains; stop codons
are indicated by (*); and putative intron sequences are represented by dashed
lines. A unique BsrGI site (B) present in the a coding segment was used to
construct a full-length human NRG-2a cDNA by connecting 5' sequences of
clone 15 to the 3' sequences of clone 14, and the sequence of the final
construct
was determined. The major open reading frame of the NRG-2a cDNA (Fig. 6,
SEQ ID NO: 1) encodes a 331 amino acid protein (Fig. 7, SEQ ID NO: 2).
EXAMPLE 3
Cloning and Construction of Human NRG-213 cDNA
The human NRG-213 cDNA is constructed partly from the human
NRG-2a cDNA (the vector and the 5' 869 bp of sequence encoding the N-
terminus of human NRG-2, which is present in both the a and the JE isoforms)
and
partly from a phage clone (e.g., phage clone 11 was shown in mapping studies
to
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=
contain the 0 sequence--see Table 1, Example 1, and Example 2) containing a
partial human cDNA encoding human NRG-20 (two 3' fragments: one contains
the (3 sequence and the other a stop codon).
The human NRG-2a cDNA (Example 2) can be digested with enzymes
Not I and Xba I (New England Biolabs, Beverly, MA) to generate a 5500 bp
vector and a 1555 bp insert containing the cDNA. Both fragments are recovered
from a gel of 1% agarose in TAE buffer using the QIAEX II Agarose Gel
Extraction kit and protocol (Qiagen, Inc., Valencia, CA). The insert fragment
(1555 bp) is further digested using Drd I (New England Biolabs, Beverly, MA)
to
generate a 5' 869 bp fragment and a 3' fragment of approximately 700 bp. The
869 bp Not I-Drd I fragment is recovered from a gel of 2% agarose in TAE
buffer
using the QIAEX II Agarose Gel Extraction kit and protocol (Qiagen, Inc.,
Valencia, CA). This 869 bp fragment contains the initiator methionine and
encodes the N-terminal portion of human NRG-20. It is ligated into the 5550 bp
vector along with two additional fragments, which are derived from cDNAs that
have sequences of human NRG-20 as described below.
The major difference between the human NRG-2a and human NRG-20
sequences reside between the single Drd I and Bsr DI sites. The a isoform
contains a 77 bp coding segment that is spliced into the 0 isoform sequence.
To
obtain the sequences encoding human NRG-2(3, a 113 bp Drd I-BsrDI fragment,
which is 77 bp shorter than the corresponding sequence of human NRG-2a, is
generated from phage clone 11 as follows. Primers (1551: 5' GTG-AGC-ACC-
ACC-CTG-TCA-TC-3', SEQ NO: 11;1546: 5'- GAG-CTA-GTC-TAG-AGT-
GGC-TTA-TGA-GTA-1TT-CTT-C-3', SEQ ID NO: 12) flanking the Drd I and
BsrDI sites are used to amplify the phage clone 11 DNA template following
methods recommended by the supplier of Taq Polymerase (Perkin Elmer/Roche,
Branchburg, NJ). The PCR product is precipitated with ethanol, then digested
sequentially using Drd I and BsrDI to produce a 113 bp fragment. Similarly,
the
3' fragment also is derived by PCR amplication of the phage clone 11 template
using primers 1550 and 1546. Primer 1550 (5' -CAG-CAG-TTC-GCA-ATG-
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GTC-AAC-TTC-TCC-TAA-GCA-CC-3', SEQ ID NO: 13) is positioned to cross
over the BsrD1 site and contains an insertion of a single T that will mutate
the
target sequence 5' -CAG-CAG-TTC-GCA-ATG-GTC-AAC-TTC-TCC-AAG-
CAC-C-3', SEQ ID NO: 14, to 5' -CAG-CAG-TTC-GCA-ATG-GTC-AAC-TTC-
5 TCC-TAA-GCA-CC-3', SEQ ID NO: 15, and thus, convert a lysine codon to a
TAA stop codon. Primer 1546 is targeted to the phage right arm cloning site
and
contains an Xba I site. Digestion of the product using BsrDI and Xba I
generates
a 425 bp fragment that becomes the 3' end of the human NRG-20 cDNA. Both
the 113 bp and 425 bp fragments are recovered from 2% agarose gels.
10 The recovery of
fragments is quantified by electrophoresis relative to
double stranded DNA markers of known length and quantity (e.g., phage lambda
Hind III digest; New England Biolabs, Beverly, MA; pGEM markers, Promega,
Madison, WI), and then each purified fragment is converted into molar
equivalents. The purified vector (100 ng) and the three fragments are ligated
15 together (T4 DNA ligase; New England Biolabs, Beverly, MA) at equimolar
ratios according to instructions provided by the supplier. The ligations are
used
to transform competent bacterial cells, such as E. coli XL1 Blue (Stratagene,
La
Jolla, CA) according to instructions provided by the supplier. Colonies
containing the vector are selected on the basis of resistance to 50 ug/ml
20 ampicillin, and the structure of the human NRG-20 cDNA is analyzed by
PCR
amplification and DNA sequencing of plasmid DNA. The major open reading
frame of the human NRG-213 cDNA (Fig. 8, SEQ ID NO: 3) encodes a protein of
298 amino acids (Fig. 9, SEQ ID NO: 4).
25 EXAMPLE 4
Expression of Human NRG-2
A vector for transient and stable expression of human NRG-2 in
mammalian cells was constructed..The pRc/CMV2 vector (Invitrogen V750-20;
see Fig. 2) was used to express human NRG-2. This 5.5 kb vector utilizes a
30 CMV promoter and a bovine growth hormone polyadenylation site to drive
high
level constitutive expression in both transient and stable transfections.
Neomycin
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selection (G418) can be used to select for stable transformants. The human
NRG2-a cDNA sequence (SEQ ID NO: 1) was cloned directionally into the
polylinker using the Hind ifi and Xba I sites. The cDNA insert of the final
construct was sequenced on both strands. The human NRG-2 expression vectors
were then expressed in CHO cells to provide a reliable source of recombinant
human protein.
Both human NRG-2 a and 13 cDNAs were transiently transfected into
CHO/S cells (Life Technologies, Inc., Rockville, MI)). Heterologous expression
of transfected genes was performed to ensure the proper functioning of the
CHO/S cell system. Mock transfections were performed in parallel.
Transfections were done in 100 mm dishes (in triplicate) by the
Lipofectaminen4
2000 method according to protocols supplied by the manufacturer (Life
Technologies, Inc., Rockville, MD). Cell lysates and conditioned media samples
were collected 3 or 4 days post-transfection. To prepare lysates, cell
monolayers
were washed with PBS, scraped from the dishes, and lysed by three freeze-thaw
cycles in 150 ill 0.25 M Tris HC1 pH 8. Cell debris was pelleted and the
supernatant recovered. Conditioned media samples were collected, then either
analyzed directly or concentrated and buffer-exchanged with 10 mM Tris HC1,
pH 7.4 using Centricon-10 units (Ambion). Secretion of biologically active
recombinant human NRG-2 gene product was demonstrated by stimulation of
Schwann cell proliferation following transient transfection of CHO/S cells and
detection of NRG-2 bioactivity in the conditioned media as compared to the
cell
lysate (Marchionni et al., Nature 362: 312-8, 1993).
The recombinant human NRG-2 (rhNRG-2) proteins were efficiently
expressed in the conditioned media of the transfected cells (Figs. 3A and 3B),
indicating these proteins can be successfully secreted from mammalian cells.
In
Fig. 3A, the conditioned media, but not the cell lysate from cells transfected
with
rhNRG-2a (and not from mock-transfected cells) expressed a specific
immunoreactive band running at ca. 56 kD. In Fig. 3B, both the a and (I
isoforms were secreted into the conditioned media of transfected CHO/S cells
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(note the smaller rhNRG-20 protein (298 amino acids) ran faster (ca. 47 kD)
than
the rhNRG-2a protein (331 amino acids), as expected).
After confirming the bioactivity of the expression construct in transient
transfections, stable CHO/S cell lines were generated to express rhNRG-20. The
pRc/CMV2 vector contains a Neomycin resistance gene, so stably-transformed
cells can be selected in media that contain an effective concentration of
0418.
Following transfection of recipient CHO/S cells, well-isolated colonies that
survived 11 days in the selective media were picked using cloning rings. Cell
lines showing the highest level expression of rhNRG-213 and G418 resistance
were continued for further evaluation. Three useful properties of cell lines
are
sustained viability, adaptation to serum-free (or low serum) media, and
expression level of recombinant protein. Thus, several of the lines were
expanded in parallel and tested for adaptation to serum-free growth conditions
and expression of rhNRG-20 by western blot. Western blot analysis showed that
Dulbecco's modified essential medium supplement with 2% fetal calf serum
provided for optimum expression of rhNRG-213 in these experiments. Bioactivity
was assayed on expressed material from the leading candidate lines. Two
isolates
were cloned by limiting dilution, and a single isolated cell line was used for
further studies.
In addition to generating stable CHO/S cell lines expressing NRG-2
proteins, a strategy relying on the co-amplification of integrated copies of
rhNRG-213 expression constructs and a transfected dihydrofolate reductase
(dhfr)
gene was developed. Mammalian expression vectors were constructed in
pcDNA3.1 (Invitrogen) and pMACSKk.II (Miltenyi Biotec Ltd.) under the control
of the CMV and SV40 promoters, respectively. These vectors were co-
transfected along a dhfr expression vector into CHO-dhfr cells and thirty
colonies
resistant to G418 were selected, grown up and expression levels were analyzed
by
western blot (Fig. 10) and RT/PCR. Fig. 10 Lane 1 shows rhNRG-213 control
sample, Lane 2 shows CHO SD (US) rhNRG2D 48hr supernatant, Lanes 3-5
show 24-72hrs Clone T3B2, Lanes 6-8 show 24-72hrs Clone T3B1, Lane 9 shows
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molecular weight markers (Invitrogen cat#LC5925), Lanes 10-12 show 24-72hrs
Clone T3A6, and Lanes 13-15 show 24-72hrs Clone T3A5. The T3B2, T3B1,
T3A5 clones were selected for dilution cloning and for
amplification/selection.
Gene co-amplification is induced by step-wise increase in methotrexate
concentrations and clones are monitored for increases in yield of secreted
rhNRG2f3.
EXAMPLE 5
Generation and Testing of Antisera to Detect Expressed NRG-2 Protein
A polyclonal antiserum that specifically detects expressed NRG-2
protein was generated as follows. Peptides were designed from the deduced
human and rat NRG-2 sequences to generate rabbit polyclonal antisera to be
used
to monitor NRG-2 levels in expression and purification samples.
The peptides used were as follows:
K71983M: APLERNQRYIFFLEPTEQPL'VFK (SEQ ID NO: 16)
K71984M: NSRLQFNKVKVEDAGEY (SEQ ID NO: 17)
K71985K: NGGVCYYIEGINQLS (SEQ ID NO: 18)
One of these peptides (K71984M), derived from the Ig domain
sequence, which is identical in the deduced rat and human NRG-2 sequences,
produced useful sera for western blotting recombinant rat NRG-2[3 in
conditioned
media from transfected CHO cells (see Fig. 3) and did not cross-react with
rhGGF2. 'These NRG-2 antisera were purified against the immobilized peptide.
Fig. 3C shows a western blot analysis of conditioned media from CHO cells
expressing rat NRG-21-1. The lanes contain either 20 gl of 15-fold
concentrated
conditioned medium from CHO cells expressing riNRG-213 (left) or 10 ng
rhGGF2 (right). Anti-NRG-2 serum was used at 11.1g/m1 and specifically
detected rrNRG-213, at 46 kD, but not rhGGF2, which runs at 80 kD.
Furthermore, analysis of culture media from transiently transfected
monolayers of CHO/S cells, using the expression plasmids rhNRG-2a and
rhNRG-2P and a rabbit polyclonal antibody raised against peptide K71984M,
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indicated that both rhNRG-2a and rhNRG-20 were expressed, and migrated at
approximately 55 kD and 47 kD, respectively (Fig. 5).
EXAMPLE 6
Bioassay for Assessment of Biological Activity of Expressed rhNRG-2a and
rliNRG-2(3
A bioassay for detection of biologically-active rhNRG-2a was
developed. Neuregulin signalling occurs through erbB receptor tyrosine kinases
belonging to the EGF receptor family. NRG ligand binding and receptor
activation can be detected by western blotting of treated cell lysates using
antisera
directed against phosphorylated tyrosine residues. This assay is used to study
the
interactions of NRG-2 proteins and erbB receptors in a variety of cell types
including, but not limited to, Schwann cells, oligodendrocyte progenitors,
skeletal
myotubes, cardiomyocytes, and human tumor cell lines from breast and prostate
adenocarcinomas. Biologically-active NRG-2 (e.g., conditioned medium from
CHO cells expressing recombinant rat NRG-213) can be detected using this assay
on the human breast adenocarcinoma cell line MCF-7. The results of an
experiment testing rat NRG-213 (rrNRG-213) and rhGGF2 on MCF-7 cells by a
receptor phosphotyrosine western blot is shown in Fig. 4A. MCF-7 cells were
cultured in 24 well plates (2 x 105 cell/well) and treated for 15 minutes in
DMEM-0.1% FCS containing 10 ng/ml rhGGF2, or various dilutions of medium
conditioned by CHO-cells expressing rhNRG-213. Following the treatment, the
media were removed and the cells were washed once, then the cultures were
lysed, and samples were analyzed by western blotting (Canoll et at, Neuron 17,
229-243, 1996). Phosphorylated ErbB receptors are detected with the RC2OB
phosphotyrosine antibody (Transduction Laboratories, Lexington, KY). A
positive control sample for this analysis is a lysate of A431 cells treated
with EGF
(left-most lane; Fig. 4A). When neither rhGGF2 nor rrNRG-2f3 were added to the
growth media, there were no detectable proteins phosphorylated on tyrosine.
However, addition of various concentrations of NRG-20 showed a dose
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dependent increase in phosphorylation at 185 kd. This band matched the
expected position of
the ErbB2 and ErbB3 receptors, which were also phosphorylated in response to
treatment
with rhGGF2 (10 ng/ml). Therefore, this bioassay provides a reliable method to
verify the
bioactivity of expressed and purified rhNRG-2a and rhNRG-213. This assay, when
applied to
purified recombinant protein, enables quantification of the bioactivity of NRG-
2 in dose
response curves that provide comparable data to DNA synthesis assays. Receptor
tyrosine
kinase bioassays on MCF-7 cells treated with conditioned media from CHO/S cell
transient
transfections or with purified recombinant NRG proteins are also shown in Fig.
4B.
EXAMPLE 7
Purification of Milligram Quantities of rhNRG-2a or 13
Conditioned medium harvested from a producer cell line (expressing rhNRG-2a)
is adjusted to pH 6.0 with acetic acid and loaded directly onto an S-sepharose
column
equilibrated with sodium acetate (pH 6.0). Bound material is eluted with 1M
NaCl in acetate
buffer, equilibrated in ammonium sulfate buffer and passed over a hydrophobic
interaction
column (Butyl Sepharose FFTM) in the same buffer. Bound material is eluted
with low salt
(800 mM ammonium sulfate) buffer and the rhNRG-2a peak collected. Collected
material is
buffer exchanged and concentrated to 1 mg/ml in formulation buffer (100 mM
arginine, 100
mM Sodium Sulphate, 20 mM NaAc, 1% mannitol pH 6-7) using an Amicon spiral
cartridge.
An optional, final purification step is a Sephacryl 200 HRTM column and eluted
rhNRG-2a
peak is formulated in formulation buffer. An alternative approach is to follow
the purification
scheme relying on heparin affinity, Cu-chelate and C4-reversed phase
chromatographies
(Higashiyama et al., J. Biochem. 122: 675-680, 1997).
Proteins fractions from chromatographic columns are monitored by western
blotting (e.g., see Fig. 3A-C) to identify the peak of secreted rhNRG-2a or II
Peak fractions
and final preparations are analyzed by receptor phosphorylation on MCF-7 cells
(see Fig. 4A-
B). Purity is assessed by gel electrophoresis (coommassie blue staining) and
by analytical
HPLC (Vydac C8TM column run in a gradient of acetonitrile in 0.1%
trifluoracetic acid).
Protein concentrations are determined by the bicinchoninic acid (BCA) assay
(Pierce) with
bovine serum albumin used as a standard.
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Another general plan for purification involves capture by conventional
chromatography on cation exchange followed by resolution from contaminating
proteins
through one or more steps, for example, by utilizing carboxymethyl Sepharose
chromatography followed by reverse phase HPLC.
Briefly, carboxymethyl sepharose (fast flow) columns of varying sizes were
equilibrated with 200mM NaCl 10mM Tris pH 7.4, then conditioned media samples
were
loaded, and the column was washed with approximately 3 volumes of 200mM NaC1,
10mM
Tris pH 7.4 (until the absorbance had reached baseline). Bound protein was
eluted with
500mM NaCl, 10 Mm Tris at pH 7.4. This elution was followed by a high salt
wash (1M
NaCl, 10mM Tris pH 7.4) for 3 column volumes. Fractions were collected and
analyzed by
western blot and gold or coommassie blue stained protein gels (4-20%
acrylamide Tris-
glycine-SDS). Depending on the column scale and the quantity of protein loaded
captured,
rhNRG-213 represented from 10-70% of the protein eluted in 0.5 M NaC1 from the
column.
No detectable rhNRG-2r3 was detected in the flowthrough, nor in the 0.2 M NaCl
or 1M NaCl
fractions, provided that the column was not overloaded. Significant
improvements in
recovery were obtained (>90%) by including protease inhibitors and running the
column in
the cold. The scale of capture chromatography was increased, beginning with 10
ml columns,
through 40 ml, 100 ml, and 200 ml columns. The overall results were consistent
both in terms
of recovery and purification, indicating that the scale of this step can be
adjusted to suit the
volume of starting material available.
The purification method was further developed with reverse phase HPLC using a
C4 column (Vydac 214 TP 1010, lcmX 25cm column) operated on a Biocad Perfusion
Chromatography Workstation. A series of pilot runs were performed on pooled
fractions
from several carboxymethyl sepharose columns
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that contained rhNRG-20 in 10 mM TrisHC1 pH 7.4,0.5 M NaCl. The column
was operated at a flow rate of 1 ml/min and was equilibrated in 0.2% TFA.
After
injecting the sample, a 10 min. column wash in 0.2% TFA was followed with a
30 min. linear ramp up to 90% acetonitrile, 0.2% TFA and a final 10 mm. wash
step in 90% acetonitrile, 0.2% TFA was used to complete the method. Fractions
were analyzed by western blot. Only the fractions that contained very pure
rhNRG-213 were included in the final pool. As assessed by coommassie blue
staining of the gel shown in Fig. 11, the preparation of rhNRG-2D was
approximately 92% pure. However, approximately 60-70% of the
immunoreactivity detected across the HPLC chromatograph was not included in
the rhNRG-213 pool. Therefore, although 90% purification has been achieved in
2
steps, a third step may be performed to enable more complete recovery of
rhNRG-213. This third step may include heparin sepharose and/or several
modifications of the reverse phase HPLC step (e.g., variations in solvents).
Gold staining provides another sensitive method for detecting
contaminating proteins in protein preparations, and this stain readily detects
nanogram quantities of protein. To visualize the purification process and to
further analyze the purity of rhNRG-213 samples from different stages of
purification were compared (Fig. 12). Samples were run on 4% - 20% SDS
PAGE (Novex, cat# EC 6025) gels in reduced conditions. The gels were
transferred onto PVDF membrane, and were stained for total protein with Gold
Stain (Amersham, cat # RPN490). To prevent overloading in the lane, the
starting material (serum-free conditioned media) was loaded at 1% of the
relative
amount of the purification samples. The central observation from this analysis
was that very significant purification had been achieved in two steps.
The tyrosine phosphorylation assay performed on the MCF-7 cell line
(human mammary adenocarcinoma) was used to measure the bioactivity of
purified NRG-2 samples. Briefly, cultures were challenged with test samples
(dilutions of purification samples in medium containing 0.1% FCS) for 15 min
at
37 C, then the media was aspirated, and 50 1 2X sample buffer containing DTI'
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and 1 mM sodium orthovanadate was added. Samples were then prepared for
electrophoresis and Western blotting. The control sample, lysate from A431
cells
treated with epidermal growth factor, was provided by the vendor (Transduction
Laboratories, Lexington, KY). In addition to monitoring rhNRG-20 production,
the activity of HPLC purified samples of rhNRG-2b diluted in vehicles
compatible with purification procedures such as 50% acetonitrile (AN) or PBS
was examined (Fig. 13). At the concentrations used in this experiment, AN did
not dramatically interfere with NRG signaling.
EXAMPLE 8
NRG-2 Activities on Oligodendrocyte Progenitors
Evaluation of rhNRG-2a and rhNRG-213 effects on proliferation and
survival of cultured oligodendrocyte progenitors is performed, using rhGGF2
for
comparison. Oligodendrocyte progenitors are generated from 2 day old rat
according to the method of McCarthy and DeVellis (J. Cell Biol. 85: 890-902,
1980), and the cells are cultured in N2 defined media containing 0.5% PBS
(DM+) for one to three days to enrich for cells in the oligodendrocyte
lineage.
Purity of the cultures is established by immunofluorescence analysis using a
series of antibodies directed against GFAP, a marker for astrocytes; 0X42
monoclonal, a marker for microglia (Harlan Bioproducts for Science); anti-A2B5
monoclonal (Boehringer Mannheim), a marker for 0-2A progenitors; 04 and 01,
which recognizes early and mature oligodendrocytes respectively (Sommer et
al.,
Dev. Biol 83: 311-327, 1980); RPTP-13 (gift of J. Schlessinger, NYU Med. Ctr)
and nestin antibodies (Developmental Studies Hybridoma Bank) which
preferentially recognize early cells in the oligodendrocyte lineage (Canoll et
al.,
Neuron 17, 229-243, 1996; Gallo et al., J. Neurosci.15: 394-406, 1995).
To determine the percentage of cells synthesizing DNA in response to
rhNRG-2a, rhNRG-20, or rhGGF2 cultures are treated for 16 h and for the final
4
h in the presence of 10 euM bromodeoxyuridine (BrdU; Sigma). BrdU-labelled
cells are detected using fluoroscein-conjugated anti-BrdU immunodetection kit
(Boehringer Mannheim). The labeling index, corresponding to the ratio of
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BrdU+ cells to total cells, is determined from photomicrographs of individual
fields of BrdU labeled and Hoechst stained nuclei. To determine the labeling
index at specific stages of differentiation, BrdU staining is combined with
analysis of 04, 01 and GFAP immunofluorescence.
To assess the effect NRG-2 on cell survival, cells growing in B104
conditioned medium are changed to DM+ media for three days. They are then
switched to either N2 media or DMEM with or without rhGGF2, rhNRG-2a, or
rhNRG-213 for 12 or 24 hours and stained with the Live/Dead staining kit
(Molecular Probes, Inc) for 15 min following the manufacturer's instructions.
Morphologic criteria to quantify cell death, i.e. monitoring pyknotic cells
under
phase microscopy and the MIT assay (Sigma), are used in separate experiments.
EXAMPLE 9
NRG-2 Activities on Olfactory Bulb Ensheathing Cells
The rat olfactory bulb is an exceptional CNS tissue. Unlike other areas
of the brain, growing axons are able to enter the olfactory bulb and extend
within
this CNS environment throughout adult life. The glial cells of the olfactory
system, known as olfactory bulb ensheathing cells (OBECs), may have an
important role in CNS neural regeneration (Li et al, J. Neurosci. 18:
10514-10524, 1998). OBECs are unusual glial cells possessing properties of
both
astrocytes and Schwann cells, and may be useful cells to aid in spinal cord
regeneration. OBECs express functional NRG receptors erbB2 and erbB4
(Pollock et al. Eur. J. Neurosci. 11: 769-780, 1999). Furthermore, high levels
of
NRG-2 polypeptides are expressed in the olfactory bulb. Accordingly, these
OBECs are ideal candidates for comparing the bioactivities of NRG-1 to NRG-2
gene products.
OBECs are purified from postnatal day 7 rats by fluorescence activated
cell sorting using the 04 antibody (Barnett, In: Culture of Animal Cells, I.R.
Freshney, 3rd Edition. pp337-341. Wiley -Liss, New York, New York, 1993;
Barnett et al., Dev Biol. 155: 337-350, 1993). After sorting, cell suspensions
are
plated onto coverslips and incubated in DMEM-BS containing 10% astrocyte
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conditioned medium (ACM) overnight at 37 C (to promote cell survival) before
treatment with either growth factors or ACM. Mitogenic activity is assayed by
incorporation of BrdU into dividing cells, and cell survival and apoptosis
assays
are done as described (Pollock et al. Eur. Neurosci. 11: 769-780, 1999).
EXAMPLE 10
NRG-2 Activities on Mid-brain Dopaminergic Neurons.
The NRG receptor erbB4 is expressed in midbraim dopaminergic
neurons of the rat, mouse, and monkey. Delivery of recombinant human NRG
proteins to the striatum is useful in the treatment of Parkinson's disease.
Studies
using the exemplary proteins, rhNRG-2a, rhNRG-213, and rhGGF2 are
undertaken to further investigate the response of the dopaminergic
nigrastriatal
system to NRGs. The two NRG proteins are compared for survival-promoting
activity (i.e. protection from cell death induced by agents that induce
oxidative
stress) on dopaminergic neurons (for example, from both fetal rodents and
human
neuroblastoma cells lines, e.g., SKNNC) in vitro. Cells pre-treated with
varying
concentrations of rhNRG-2a, rhNRG-21-3, or rhGGF2 are challenged with a
twenty four hour treatment of 11.1M metadione or 100 mM
diethyldithiocarbamate to induce oxidative stress, and cell death is
quantified by
standard methods. An in vivo model of dopamine release and electrochemical
and behavioral assessments of dopaminergic function in rats can also be used.
NRG proteins were tested for survival promoting activity on rat
dopaminergic neurons in vitro. Specifically, it was determined if NRG proteins
were neuroprotective for dopaminergic neurons that were challenged in culture
with 6-hydroxydopamine (6-01-IDA). Cells pre-treated with rhNRG-213 or
rhGGF2 and untreated control cultures were exposed to 50 AM 6-0HDA for 24 h,
then cultures were stained for tyrosine hydroxylase (TH) and examined by light
microscopy (Fig. 14). Fig. 14 shows primary mesencephalic cultures
irmnunostained for tyrosine hydroxylase (TH) on day in vitro (DIV) 7. The top
panel shows a culture treated with 100 nanograms rhNRG-213 daily starting at
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DIV 0 and ending at DIV 3. On DIV 4, the culture was treated with 50 ptM 6-
OHDA. The bottom panel shows a culture that received no pretreatment, but was
treated with 50 i.tM 6-0HDA on DIV 4. Both cultures were analyzed for TH
immunoreactivity on DIV 7. Calibration bar equals 50 microns and applies to
both top and bottom panels. Similar staining patterns were observed throughout
each of the cultures. The density, number, and length of neurites of TH-
positive
neurons were reduced by 6-0HDA treatment in the culture receiving no
pretreatment. In contrast, the culture pretreated with rhNRG-2f1 shows normal
morphological development, which is comparable to the results observed with
rhGGF2. This result has been replicated in several culture experiments. The
results indicate that rhNRG-2P has beneficial effects in vivo, which can be
tested,
for example, in an animal model of Parkinson's disease.
EXAMPLE 11
Neuronal Development and Migration in the Cerebellum
Isoforms of NRG-1, NRG-2, and the erbB4 receptor are expressed at
high levels in the cerebellum (Chen et at., J Comp Neurol 349: 389-400, 1994;
Chang et at., Nature 387: 509-512, 1997; Lai et at., Neuron 6: 691-704, 1991).
RhNRG-2a, rhNRG-20, and rhOGF2 can be evaluated in cell culture assays of
migration and neurogenesis in the cerebellum. RhNRG-2a, rhNRG-2P, and
rhGGF2 are compared with respect to their effect on the rate of migration of
cerebellar granule neurons on a glial cell substrate. Imprint cultures of
postnatal
day 5 rat cerebellum containing intact Bergmann glia with migrating neurons
attached to them are made as described (Anton et al., J. Neurosci. 16: 2283-
2293,
1996). Neuronal migration is monitored using a Zeiss axiovert 135 microscope
equipped with a Zeiss W63 objective, with images recorded onto an optical
disk.
Changes in the rate and pattern of neuronal migration, neuron-glial
interactions,
and morphology are monitored in response to rhNRG-2a, rhNRG-20, and
rhGGF2.
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Effects on cerebellar granule neurogenesis are studied in dissociated
cultures of postnatal rat cerebellar granular neurons. Dividing neural
precursors
are purified from postnatal day 5 cerebellum by Percoll density gradient
centrifugation and placed into dissociated cell culture. Cultures are then
treated
with 10 iuM BrdUrd (to label dividing cells) and with varying concentrations
of
rhNRG-2a, rhNRG-20, and rhGGF2. After two to seven additional days in
culture, differentiation into neuronal and glial cell lineages is assayed by
immunostaining using cell-type specific markers, such as GFAP (glial) and TUJ1
(neuronal). For each culture condition, the total number of cells, the
BrdU-labelled cells, and the cells identified with each marker are enumerated.
Cells that entered a particular cell lineage since exposure to these growth
factors
are identified as those labelled with BrdU plus one of the markers. The
percentage of BrdU-labelled cells stained with each marker thus provides a
measure of the effects of each growth factor on the genesis and survival of
neurons and glia. Analysis of total number of cells at various time points and
the
number of apoptotic cells under different conditions are used to evaluate any
potential effect of rhNRG-2a, rhNRG-2I3, and rhGGF2 on selective survival of
neural precursors or their neuronal or glial derivatives.
Isoforms of NRG-1, NRG-2 and the erbB4 receptor are expressed at
high levels in the cerebellum, thus making in vitro studies on neural cells of
the
cerebellum an important component of these studies. Both rhNRG-20 and
rhGGF2 were evaluated in cell culture assays of migration and neurogenesis in
the cerebellum. Imprint cultures of postnatal day 5 rat cerebellum containing
intact Bergmann glia with migrating neurons attached to them were made and
analyzed. Neuronal migration was monitored using a Zeiss axiovert 135
microscope equipped with a Zeiss W63 objective, with images recorded onto an
optical disk. Changes in the rate and pattern of neuronal migration, neuron-
glial
interactions, and morphology were monitored in response to rhnrg-213 and
rhGGF2 (Fig. 16A-B). The rate of neuronal migration of cerebellar granule
neurons was measured before and after exposure to rhNRG213 (10Ong/m1),
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rhGGF2 (50ng/m1), or unsupplemented control media. Fig. 16A shows neurons
migrating on a glial cell process monitored prior to (panels to the left of
black
arrow) and after (panels to the right of black arrow) addition of growth
factors
(shown here is rhGGF2). Time elapsed between each panel is one hour. Fig. 16B
shows that exposure to rhGGF2 promoted the rate of migration of neurons by
45 2.1%. In contrast, neither control medium nor rhNRG-213 altered the rate of
migration. The asterisk indicates significance, P<0.05. Data shown are
mean SEM (n>16 for each group). Therefore, in contrast to the observed
increase in the rate of neuronal migration promoted by rhGGF2, rhNRG-2P had
no apparent effect. However, when cerebellar neural progenitors were studied
in
dissociated culture, rhNRG-213 promoted external granule (EGL) neuron
proliferation and/or survival (Fig. 15A-B). External granule layer (EGL) cells
were dissociated and cultured in neurobasal (NB)/N2 medium or in NB/N2
medium supplemented with 100 ng/ml rhNRG-2P for 5 days. 10 M BrdU was
added to all cultures from the beginning. The cells then were fixed and probed
with a polyclonal neuron- specific antibody (Tuj-1; Babco) and with anti-BrdU
monoclonal antibodies. Cells that were labeled with Tuj-1 alone (i.e.,
neurons;
asterisk[A]), BrdU+Tuj-1 (i.e., neurons generated from dividing neuroblasts in
culture; arrow [A]), and BrdU alone (i.e., non- neural cells; arrowhead[A])
were
counted. Compared to the control, more neurons (arrow, [A]) were found to have
incorporated BrdU (orange) in their nuclei when cultured in medium containing
rhNRG-20. BrdU immunoreactivity was detected with anti- mouse conjugated to
Cy3 (Red). Tuj-1 immunoreactivity was detected with anti-rabbit conjugated to
FITC. Numbers of Tuj-1 and BrdU positive cells were counted. Cell counts from
the rhNRG-20 group was normalized to that from control group to obtain fold
basal change in the number of BrdU positive neurons. This result suggests that
rhNRG-213 promotes EGL cell proliferation or the selective survival of newly
generated cerebellar granule neurons. These data therefore exemplify a
bioactivity on neural cells that is more generally applicable to neuronal
populations that express erbB4.
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EXAMPLE 12
NRG-2 Activities on Ventricular Cardiomyocytes.
To examine the role of NRG ligands and their receptors in developing
and postnatal myocardium, the ability of NRG-2 proteins to promote
proliferation,
survival and growth of isolated neonatal and adult rat cardiac myocytes was
studied.
All three of the known receptors for neuregulins, erbB2, erbB3, and erbB4, are
expressed in the developing heart at E14, after which erbB3 expression rapidly
declines while erbB2 and erbB4 expression persists in ventricular myocytes
into
adulthood. The in vitro activities of rhNRG-2a and rhNRG-2P on
cardiomyocytes are evaluated as compared to rhGGF2. Specifically, the two
growth factors are compared for effects on cardiomyocyte survival,
hypertrophy,
and contractile protein expression as described below. Neonatal rat
ventricular
myocyte (NRVM) primary cultures are prepared as described previously
(Springhorn et al., J. Biol. Chem. 267: 14360-14365, 1992). To selectively
enrich
for myocytes, dissociated cells are centrifuged twice at 500 rpm for 5 min,
preplated twice for 75 min, and finally plated at low density (0.7 - 1 x 104
cells/cm2) in DME medium supplemented with 7% FBS. Cytosine arabinoside
(AraC; 10M; Sigma) is added during the first 24-48 h to prevent proliferation
of
non-myocytes. Unless otherwise stated, all experiments are performed 36-48 h
after changing to a serum-free medium, DME plus ITS (Sigma). Using this
method, primary cultures with >95% myocytes are routinely obtained, as
assessed
by microscopic observation of spontaneous contraction and by
immunofluorescence staining with a monoclonal anti-cardiac myosin heavy chain
antibody (anti-MHC; Biogenesis, Sandown, NH).
Isolation and preparation of adult rat ventricular myocyte (ARVM)
primary cultures is carried out using techniques previously described (Berger
et
al., Am. J. Physiol. 266: H341-H349, 1994). Rod-shaped cardiac myocytes are
plated in culture medium on laminin- (10 (g/m1) precoated dishes for 60 min,
followed by one change of medium to remove loosely attached cells. The
contamination of ARVM primary cultures by non-myocytes is determined by
counting with a haemocytometer and is typically less than 5%. All ARVM
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primary cultures are maintained in a defined medium termed "ACCITT"
(Ellingsen et al., Am. J. PhysioL 265: H747-H754, 1993) composed of DME,
supplemented with 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM
tatuine, 0.1 (M insulin, and 10 nM triiodothyronine with 100 IU/ml penicillin
and
100 (g/m1 streptomycin. In experimental protocols designed to examine myocyte
survival and/or apoptosis, insulin is omitted from the defined medium, which
is
therefore termed "ACCTT".
Measurements of rates of protein synthesis ([311]1eucine uptake) are
used to monitor growth factor effects on cardiomyocyte hypertrophy. For these
experiments, 10 (M cytosine arabinoside is added to the culture medium. Cells
are grown in serum-free medium for 36 to 48 h and then stimulated with
different
doses of rhNRG-2a, rhNRG-213, or rhGGF2. After 40 h, [31111eucine (5 (Ci/ml)
is added for 8 hours, and cells washed with PBS and harvested with 10% TCA.
TCA-precipitable radioactivity is determined by scintillation counting.
Immunocytochemistry is used to examine changes in myocyte
phenotype with rhNRG-2a, rhNRG-20, or rhGGF2. For example, following
treatment with growth factors, cells are fixed in 4% (w/v) paraformaldehyde
for
30 min at room temperature, rinsed with PBS, permeabilized with 0.1% Triton
X-100 for 15 min, and then incubated with 1% FBS for another 15 rain, followed
by incubation with anti-myosin heavy chain (1:300) and visualized with
TRTTC-conjugated (NRVM) or FITC-conjugated (ARVM) second antibody.
ARVM are examined using a MRC 600 confocal microscope with a Kr/Ar laser.
The in vitro activities of rhNRG-2a and rhNRG-20 on cardiomyocytes as
compared to rhGGF2 was evaluated. Studies on cellular hypertrophy (as
monitored
by measuring protein synthesis) and activation of signalling pathways
including
p42/44 MAPK and Akt were performed (Fig. 17 and Fig. 18). Neonatal rat
ventricular myocytes isolated from 1 day old neontal rat ventricle were plated
in
24 well tissue culture plates with ¨80,000 cells/ well for 24 Ins in 10%FCS,
then
serum starved overnight. Cells were treated with recombinant neuregulins in
the
presence of 311-leucine for 241us. Cellular protein was precipitated with
5%TCA
and lysed with 0.4N NaOH. 31-1-leucine incorporation was measured with a
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scintillation counter, and presented as the average of 4 wells treated
identically
divided by the average counts in untreated cells. Neonatal rat ventricular
myocytes were plated in plOOs ¨ 2-3 million cells/plate for 24 hrs in 10%FCS,
then serum starved 24 hrs. Cells were treated with recombinant neuregulins for
10
min, then lysed with buffer containing protease and phosphatase inhibitors
(New
England Biolabs). Samples representing 70 g protein were run on 10%gel
(BioRad), and transferred to PVDF membrane for detection of phophorylated Erk
or Akt using New England Biolabs phospho-specific antibodies. Both rhGGF2
and rhNRG-20 increased protein synthesis by approximately 40% at all
concentrations examined. However, rhNRG-2a had no effect on protein
synthesis over the concentration tested. The blot shown (Fig. 17) is
representative of 2 separate experiments.
These results indicate that NR2 signalling may act to promote the
proliferation, survival, and growth of cardiac myocytes, both during and
following
myocardial trabeculation. Moreover, the persistence of NRG receptors in the
post-
natal and adult heart suggests a continuing role for neuregulins in the
myocardial
adaption to physiologic stress or injury.
EXAMPLE 13
Cell Survival Assay And Detection of Apoptosis
Cell viability is determined by the 3-(4,5-dimethylthiazol-2-y1)-2,5-
diphenyl tetrazolium bromide (MIT, Sigma) cell respiration assay. Primary
cultures of NRVM after 2 days in serum-free medium are stimulated with
different concentrations of rhNRG-2a, rhNRG-20, or rhGGF2 for either 4 or 6
days. ARVM are maintained in ACCTr medium or ACCT T medium plus
different concentrations of rhNRG-2a, rhNRG-2P, or rhGGF2 for 6 days. MTT is
then incubated with the cells for 3 h at 37 C. Living cells transform the
tetrazolium ring into dark blue formazan crystals that can be quantified by
reading the optical density at 570 nm after cell lysis with dimethylsulfoxide.
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Apoptosis is detected in neonatal and adult myocytes using the terminal
deoxynucleotidyltransferase (TdT)-mediated dUTP nick end-labeling (TUNEL)
assay. 3'-end
labelling of DNA with fluorescein-conjugated dUTP is done using an in situ
cell death
detection kit (Boehringer Mannheim) following the manufacturer's instructions.
Cells are
counterstained with an anti-MHC antibody as described above, and the nuclei
are also stained
with Hoescht 33258 (10 (M, Sigma) for 5 min. More than 500 myocytes are
counted in each
coverslip and the percentage of TUNEL-positive myocytes is calculated.
Other Embodiments
While the invention has been described in connection with specific embodiments
thereof, it will be understood that it is capable of further modifications and
this application is
intended to cover any variations, uses, or adaptations of the invention
following, in general,
the principles of the invention and including such departures from the present
disclosure come
within known or customary practice within the art to which the invention
pertains and may be
applied to the essential features hereinbefore set forth, and follows in the
scope of the
appended claims.
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SEQUENCE LISTING
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<120> NRG-2 NUCLEIC ACID MOLECULES,
POLYPEPTIDES, AND DIAGNOSTIC AND THERAPEUTIC METHODS
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Tyr Lys Ala Pro Val Val Val Glu Gly Lys Val Gin Gly Leu Val Pro
35 40 45
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Ala Gly Gly Ser Ser Ser Asn Ser Thr Arg Glu Pro Pro Ala Ser Gly
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Arg Val Ala Leu Val Lys Val Leu Asp Lys Trp Pro Leu Arg Ser Gly
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Gly Leu Gln Arg Glu Gln Val Ile Ser Val Gly Ser Cys Val Pro Leu
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Glu Arg Asn Gln Arg Tyr Ile Phe Phe Leu Glu Pro Thr Glu Gln Pro
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Leu Lys Lys Glu Val Gly Lys Ile Leu Cys Thr Asp Cys Ala Thr Arg
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Pro Lys Leu Lys Lys Met Lys Ser Gln Thr Gly Gln Val Gly Glu Lys
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Ile Lys Tyr Gly Asn Gly Arg Lys Asn Ser Arg Leu Gln Phe Asn Lys
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Val Lys Val Glu Asp Ala Gly Glu Tyr Val Cys Glu Ala Glu Asn Ile
210 215 220
Leu Gly Lys Asp Thr Val Arg Gly Arg Leu Tyr Val Asn Ser Val Ser
225 230 235 240
Thr Thr Leu Ser Ser Trp Ser Gly His Ala Arg Lys Cys Asn Glu Thr
245 250 255
Ala Lys Ser Tyr Cys Val Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly
260 265 270
Ile Asn Gln Leu Ser Cys Lys Cys Pro Asn Gly Phe Phe Gly Gln Arg
275 280 285
Cys Leu Glu Lys Leu Pro Leu Arg Leu Tyr Met Pro Asp Pro Lys Gln
290 295 300
Ser Val Leu Trp Asp Thr Pro Gly Thr Gly Val Ser Ser Ser Gln Trp
305 310 315 320
Ser Thr Ser Pro Ser Thr Leu Asp Leu Asn
325 330
<210> 3
<211> 897
<212> DNA
<213> Homo sapiens
<400> 3
atgaggcgcg acccggcccc cggcttctcc atgctgctct tcggtgtgtc gctcgcctgc 60
tactcgccca gcctcaagtc agtgcaggac caggcgtaca aggcacccgt ggtggtggag 120
ggcaaggtac aggggctggt cccagccggc ggctccagct ccaacagcac ccgagagccg 180
cccgcctcgg gtcgggtggc gttggtaaag gtgctggaca agtggccgct ccggagcggg 240
gggctgcagc gcgagcaggt gatcagcgtg ggctcctgtg tgccgctcga aaggaaccag 300
cgctacatct ttttcctgga gcccacggaa cagcccttag tctttaagac ggcctttgcc 360
cccctcgata ccaacggcaa aaatctcaag aaagaggtgg gcaagatcct gtgcactgac 420
tgcgccaccc ggcccaagtt gaagaagatg aagagccaga cgggacaggt gggtgagaag 480
caatcgctga agtgtgaggc agcagccggt aatccccagc cttcctaccg ttggttcaag 540
gatggcaagg agctcaaccg cagccgagac attcgcatca aatatggcaa cggcagaaag 600
aactcacgac tacagttcaa caaggtgaag gtggaggacg ctggggagta tgtctgcgag 660
52
CA 02887616 2015-04-08
gccgagaaca tcctggggaa ggacaccgtc cggggccggc tttacgtcaa cagcgtgagc 720
accaccctgt catcctggtc ggggcacgcc cggaagtgca acgagacagc caagtcctat 780
tgcgtcaatg gaggcgtctg ctactacatc gagggcatca accagctctc ctgcaagtgt 840
cctgtgggat acaccgggga caggtgtcag cagttcgcaa tggtcaactt ctcctaa 897
<210> 4
<211> 298
<212> PRT
<213> Homo sapiens
<400> 4
Met Arg Arg Asp Pro Ala Pro Gly Phe Ser Met Leu Leu Phe Gly Val
1 5 10 15
Ser Leu Ala Cys Tyr Ser Pro Ser Leu Lys Ser Val Gin Asp Gin Ala
20 25 30
Tyr Lys Ala Pro Val Val Val Glu Gly Lys Val Gin Gly Leu Val Pro
35 40 45
Ala Gly Gly Ser Ser Ser Asn Ser Thr Arg Glu Pro Pro Ala Ser Gly
50 55 60
Arg Val Ala Leu Val Lys Val Leu Asp Lys Trp Pro Leu Arg Ser Gly
65 70 75 80
Gly Leu Gin Arg Glu Gin Val Ile Ser Val Gly Ser Cys Val Pro Leu
85 90 95
Glu Arg Asn Gin Arg Tyr Ile Phe Phe Leu Glu Pro Thr Glu Gin Pro
100 105 110
Leu Val Phe Lys Thr Ala Phe Ala Pro Leu Asp Thr Asn Gly Lys Asn
115 120 125
Leu Lys Lys Glu Val Gly Lys Ile Leu Cys Thr Asp Cys Ala Thr Arg
130 135 140
Pro Lys Leu Lys Lys Met Lys Ser Gin Thr Gly Gin Val Gly Glu Lys
145 150 155 160
Gin Ser Leu Lys Cys Glu Ala Ala Ala Gly Asn Pro Gin Pro Ser Tyr
165 170 175
Arg Trp Phe Lys Asp Gly Lys Glu Leu Asn Arg Ser Arg Asp Ile Arg
180 185 190
Ile Lys Tyr Gly Asn Gly Arg Lys Asn Ser Arg Leu Gin Phe Asn Lys
195 200 205
Val Lys Val Glu Asp Ala Gly Glu Tyr Val Cys Glu Ala Glu Asn Ile
210 215 220
Leu Gly Lys Asp Thr Val Arg Gly Arg Leu Tyr Val Asn Ser Val Ser
225 230 235 240
Thr Thr Leu Ser Ser Trp Ser Gly His Ala Arg Lys Cys Asn Glu Thr
245 250 255
Ala Lys Ser Tyr Cys Val Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly
260 265 270
Ile Asn Gin Leu Ser Cys Lys Cys Pro Val Gly Tyr Thr Gly Asp Arg
275 280 285
Cys Gin Gin Phe Ala Met Val Asn Phe Ser
290 295
<210> 5
<211> 20
<212> DNA
<213> Homo sapiens
53
CA 02887616 2015-04-08
<400> 5
gcatcaacca gctctcctgc 20
<210> 6
<211> 20
<212> DNA
<213> Homo sapiens
<400> 6
gcatcaacca gctctcctgc 20
<210> 7
<211> 20
<212> DNA
<213> Homo sapiens
<400> 7
tgcgaactgc tgacacctgt 20
<210> 8
<211> 21
<212> DNA
<213> Homo sapiens
<400> 8
ccaccttttg agcaagttca g 21
<210> 9
<211> 21
<212> DNA
<213> Homo sapiens
<400> 9
gaggtggctt atgagttctt c 21
<210> 10
<211> 19
<212> DNA
<213> Homo sapiens
<400> 10
ggccaccaca cagacgatg 19
<210> 11
<211> 20
<212> DNA
<213> Homo sapiens
<400> 11
gtgagcacca ccctgtcatc 20
<210> 12
<211> 34
<212> DNA
<213> Homo sapiens
54
CA 02887616 2015-04-08
<400> 12
gagctagtct agagtggctt atgagtattt cttc 34
<210> 13
<211> 35
<212> DNA
<213> Homo sapiens
<400> 13
cagcagttcg caatggtcaa cttctcctaa gcacc 35
<210> 14
<211> 34
<212> DNA
<213> Homo sapiens
<400> 14
cagcagttcg caatggtcaa cttctccaag cacc 34
<210> 15
<211> 35
<212> DNA
<213> Homo sapiens
<400> 15
cagcagttcg caatggtcaa cttctcctaa gcacc 35
<210> 16
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> derived from Rattus norvegicus and Homo sapiens
<400> 16
Ala Pro Leu Glu Arg Asn Gln Arg Tyr Ile Phe Phe Leu Glu Pro Thr
1 5 10 15
Glu Gln Pro Leu Val Phe Lys
<210> 17
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> derived from Rattus norvegicus and Homo sapiens
<400> 17
Asn Ser Arg Leu Gln Phe Asn Lys Val Lys Val Glu Asp Ala Gly Glu
1 5 10 15
Tyr
CA 02887616 2015-04-08
<210> 18
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> derived from Rattus norvegicus and Homo sapiens
<400> 18
Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly Ile Asn Gin Leu Ser
1 5 10 15
56
