Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD OF ENHANCING AND/OR INDUCING NEURONAL MIGRATION
USING ERYTHROPOIETIN
RELATED APPLICATIONS
This application claims the benefit of United States Provisional Application
Serial Number 60/399,395, filed July 31, 2002. The entire disclosure of this
priority
application is hereby incorporated by reference.
FIELD OF THE INVENTION
This invention relates to methods of enhancing and/or inducing the migration
of
multipotent neural stem cells and their progeny by exposing the stem cells and
their
progeny to erythropoietin. In a preferred embodiment, additional growth
factors are also
utilized.
REFERENCES
U.S. Patent No. 4,703,008.
U.S. Patent No. 5,128,242.
U.S. Patent No. 5,198,542.
U.S. Patent No. 5,208,320.
U.S. Patent No. 5,326,860.
U.S. Patent No. 5,441,868.
U.S. Patent No. 5,547,935.
U.S. Patent No. 5,547,993.
U.S. Patent No. 5,621,080.
U.S. Patent No. 5,623,050.
U.S. Patent No. 5,750,376.
U.S. Patent No. 5,801,147.
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U.S. Patent No. 5,955,346.
U.S. Patent No. 6,165,783.
U.S. Patent No. 6,191,106.
U.S. Patent No. 6,242,563.
U.S. Patent No. 6,294,346.
U.S. Patent No. 6,376,218.
U.S. Patent No. 6,429,186.
U.S. Patent No. 6,618,698.
International PCT Application No. WO 93/01275.
International PCT Application No. WO 94/10292.
International PCT Application No. WO 03/040310.
S.A. Bayer, "Neuron production in the hippocampus and olfactory bulb of the
adult rat brain: addition or replacement?" N.Y. Acad. Sci. 457:163-173 (1985).
S. Bernichtein et al., "S179D-human PRL, a pseudophosphorylated human PRL
analog, is an agonist and not an antagonist," Endocrinology 142(9):3950-3963
(2001).
C.G. Craig et al., "In vivo growth factor expansion of endogenous subependymal
neural precursor cell populations in adult mouse brain," J. Neurosci.
16(8):2649-58
(1996).
C.R. Freed et al., "Survival of implanted fetal dopamine cells and neurologic
improvement 12 to 46 months after transplantation for Parkinson's Disease," N.
Engl. J.
Med. 327:1549-1555 (1992).
M.S. Kaplan, "Neurogenesis in the 3-month old rat visual cortex," J. Comp.
Neurol. 195:323-338 (1981)
D. van der Kooy and S. Weiss, "Why stem cells?" Science 287:1439-41 (2000).
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M.J. Perlow et al., "Brain grafts reduce motor abnormalities produced by
destruction of nigrostriatal dopamine system," Science 204:643-647 (1979).
C.S. Potten and Loeffler, "Stem cells: attributes, cycles, spirals, pitfalls
and
uncertainties. Lessons for and from the Crypt," Development 110:1001-1020
(1990).
P. Rakic, "Limits of neurogenesis in primates," Science 227:1054-1056 (1985).
B.A. Reynolds and S. Weiss, "Generation of neurons and astrocytes from
isolated
cells of the adult mammalian central nervous system," Science 255:1707-1710
(1992).
R. Rietze et al. , "Mitotically active cells that generate neurons and
astrocytes are
present in multiple regions of the adult mouse hippocampus," J. Comp. Neurol.
424(3):397-408 (2000)
T. Shingo et al., "Erythropoietin regulates the in vitro and in vivo
production of
neuronal progenitors by mammalian forebrain neural stem cells," J. Neurosci.
21 (24) : 973 3-9743 (2001 ) .
D.D. Spencer et al."Unilateral transplantation of human fetal mesencephalic
tissue
into the caudate nucleus of patients with Parkinson's Disease," N. Engl. J.
Med.
327:1541-1548 (1992).
H. Widner et al., "Bilateral fetal mesencephalic grafting into two patients
with
Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),"
N.
Engl. J. Med. 327:1556-1563 (1992).
All of the publications, patents, and patent applications cited in this
application are
hereby incorporated by reference in their entirety to the same extent as if
the disclosure of
each individual publication, patent, or patent application was specifically
and individually
indicated to be incorporated by reference in its entirety.
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BACKGROUND OF THE INVENTION
Neurogenesis in mammals is largely complete early in the postnatal period.
While it was previously thought that cells of the adult mammalian central
nervous system
(CNS) have little or no ability to undergo mitosis and generate new neurons,
recent studies
have demonstrated that the mature nervous system does have some limited
capability to
produce new neurons. (Craig et al., 1996; Rietze et al., 2000; review in van
der Kooy and
Weiss, 2000). Several mammalian species.(e.g., rats) exhibit the limited
ability to
generate new neurons in restricted adult brain regions such as the dentate
gyros and
olfactory bulb (Kaplan, 1981; Bayer, 1985). However, the generation of new CNS
neurons in adult primates does not normally occur (Rakic, 1985). This relative
inability to
produce new neural cells in most mammals (and especially primates) may be
advantageous
for long-term memory retention; however, it is a distinct disadvantage when
the need to
replace lost neuronal cells arises due to an injury or disease.
The role of neural stem cells in the adult is to replace cells that are lost
by
natural cell death, injury or disease. Until recently, the low turnover of
cells in the
mammalian CNS together with the inability of the adult mammalian CNS to
generate new
neuronal cells in response to the loss of cells following an injury or disease
had led to the
assumption that the adult mammalian CNS does not contain multipotent neural
stem cells.
The critical identifying feature of a stem cell is its ability to exhibit self
renewal or to
generate more of itself. The simplest definition of a stem cell would be a
cell with the
capacity for self maintenance. A more stringent (but still simplistic)
definition of a stem
cell is provided by Potten and Loeffler (1990) who have defined stem cells as
"undifferentiated cells capable of a) proliferation, b) self maintenance, c)
the production of
a large number of differentiated functional progeny, d) regenerating the
tissue after injury,
and e) a flexibility in the use of these options."
CNS disorders encompass numerous afflictions such as neurodegenerative
diseases (e.g., Alzheimer's and Parkinson's), brain injury (e.g., stroke, head
injury,
cerebral palsy) and a large number of CNS dysfunctions (e.g., depression,
epilepsy, and
schizophrenia). In recent years, neurodegenerative disease has become an
important
concern due to the expanding elderly population which is at the greatest risk
for these
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disorders. These diseases, which include Alzheimer's Disease, Parkinson's
Disease,
Huntington's Disease, Multiple Sclerosis (MS), and Amyotrophic Lateral
Sclerosis, have
been linked to the degeneration of neuronal cells in particular locations of
the CNS,
leading to the inability of these cells or the brain region to carry out their
intended
function.
Degeneration in a brain region known as the basal ganglia can lead to diseases
with various cognitive and motor symptoms, depending on the exact location.
The basal
ganglia consists of many separate regions, including the striatum (which
consists of the
caudate and putamen), the globus pallidus, the substantia nigra, substantia
innominate,
ventral pallidum, nucleus basalis of Meynert, ventral tegmental area and the
subthalamic
nucleus. Many motor deficits are a result of neuronal degeneration in the
basal ganglia.
Huntington's Chorea is associated with the degeneration of neurons in the
striatum, which
leads to involuntary jerking movements in the host. Degeneration of a small
region called
the subthalamic nucleus is associated with violent flinging movements of the
extremities in
a condition called ballismus, while degeneration in the putamen and globus
pallidus is
associated with a condition of slow writhing movements or athetosis. In the
case of
Parkinson's Disease, degeneration is seen in another area of the basal
ganglia, the
substantia nigra gars compacta. This area normally sends dopaminergic
connections to the
dorsal striatum which are important in regulating movement. In the case of
Alzheimer's
Disease, there is a profound cellular degeneration of the forebrain and
cerebral cortex. In
addition, upon closer inspection, a localized degeneration in an area of the
basal ganglia,
the nucleus basalis of_Meynert, appears to be selectively degenerated. This
nucleus
normally sends cholinergic projections to the cerebral cortex which are
thought to
participate in cognitive functions including memory.
Other forms of neurological impairment can occur as a result of neural
degeneration, such as cerebral palsy, or as a result of CNS trauma, such as
stroke and
epilepsy.
In addition to neurodegenerative diseases, brain injuries often result in the
loss
of neurons, the inappropriate functioning of the affected brain region, and
subsequent
behavior abnormalities. Probably the largest area of CNS dysfunction (with
respect to the
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number of affected people) is not characterized by a loss of neural cells but
rather by an
abnormal functioning of existing neural cells. This may be due to
inappropriate firing of
neurons, or the abnormal synthesis, release, and/or processing of
neurotransmitters.
These dysfunctions may be the result of well studied and characterized
disorders such as
depression and epilepsy, or less understood disorders such as neurosis and
psychosis.
Other forms of neurological impairment can occur as a result of neural
degeneration, such as amyotrophic lateral sclerosis and cerebral palsy, or as
a result of
CNS trauma such as stroke and epilepsy.
Demyelination of central and peripheral neurons occurs in a number of
pathologies and leads to improper signal conduction within the nervous system.
Myelin is
a cellular sheath, formed by glial cells, that surrounds axons and axonal
processes that
enhances various electrochemical properties and provides trophic support to
the neuron.
Myelin is formed by Schwann cells in the peripheral nervous system and by
oligodendrocytes in~ the central nervous system. Among the various
demyelinating
diseases, MS is the most notable.
To date, treatment for CNS disorders has been primarily via the administration
of pharmaceutical compounds. Unfortunately, this type of treatment has been
fraught with
many complications including limited ability to transport drugs across the
blood-brain
barrier and drug-tolerance acquired by patients to whom these drugs are
administered
long-term. For instance, partial restoration of dopaminergic activity in
Parkinson's
patients has been achieved with levodopa, which is a dopamine precursor able
to cross the
blood-brain barrier. However, patients become tolerant to the effects of
levodopa, and
therefore, steadily increasing dosages are needed to maintain its effects. In
addition, there
are a number of side effects associated with levodopa such as increased and
uncontrollable
movement.
Recently, the concept of neurological tissue grafting has been applied to the
treatment of neurological diseases such as Parkinson's Disease. Neural grafts
may avert
the need not only for constant drug administration, but. also for complicated
drug delivery
systems which arise due to the blood-brain barrier. However, there are
limitations to this
technique as well. First, cells used for transplantation which carry cell
surface molecules
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of a differentiated cell from another host can induce an immune reaction in
the host. In
addition, the cells must be at a stage of development where they are able to
form normal
neural connections with neighboring cells. For these reasons, initial studies
on
neurotransplantation centered on the use of fetal cells. Several studies have
shown
improvements in patients with Parkinson's Disease after receiving implants of
fetal CNS
tissue. Implantation of embryonic mesencephalic tissue containing dopamine
cells into the
caudate and putamen of human patients was shown by Freed et al. ( 1992) to
offer long-
term clinical benefit to some patients with advanced Parkinson's Disease.
Similar success
was shown by Spencer et al. (1992). Widner et al. (1992) have shown long-term
functional improvements in patients with N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine
(MPTP)-induced Parkinsonism that received bilateral implantation of fetal
mesencephalic
tissue. Perlow et al. (1979) describe the transplantation of fetal
dopaminergic neurons into
adult rats with chemically induced nigrostriatal lesions. These grafts showed
good
survival, axonal outgrowth and significantly reduced the motor abnormalities
in the host
animals. A further discussion of tissue transplantation techniques and
drawbacks can be
found in U.S. Patent No. 6,294,346 B1.
While the studies noted above are encouraging, the use of large quantities of
aborted fetal tissue for the treatment of disease raises ethical
considerations and political
obstacles. There are other considerations as well. Fetal CNS tissue is
composed of more
than one cell type, and thus is not a well-defined source of tissue. In
addition, there are
serious doubts as to whether an adequate and constant supply of fetal tissue
would be
available for transplantation. For example, in the treatment of MPTP-induced
Parkinsonism (Widner, 1992) tissue from 6 to 8 fresh fetuses were .required
for
implantation into the brain of a single patient. There is also the added
problem of the
potential for contamination during fetal tissue preparation. Moreover, the
tissue may
already be infected with a bacteria or virus, thus requiring expensive
diagnostic testing for
each fetus used. However, even diagnostic testing might not uncover all
infected tissue.
For example, the successful diagnosis of HIV-free tissue is not guaranteed
because
antibodies to the virus are generally not present until several weeks after
infection.
While currently available transplantation approaches represent a significant
improvement over other available treatments for neurological disorders, they
suffer from
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significant drawbacks. The inability in the prior art of the transplant to
fully integrate into
the host tissue, and the lack of availability of neuronal cells in unlimited
amounts from a
reliable source for grafting are, perhaps, the greatest limitations of
neurotransplantation. A
well-defined, reproducible source of neural cells is currently available. It
has been
discovered that multipotent neural stem cells, capable of producing progeny
that
differentiate into neurons and glia, exist in adult mammalian neural tissue.
(Reynolds and
Weiss, 1992). Methods have been provided for the proliferation of these stem
cells to
provide large numbers of neural cells that can differentiate into neurons and
glia (See U.S.
Pat. No. 5,750,376, and International Application No. WO 93/01275). Various
factors
can be added to neural cell cultures to influence the make-up of the
differentiated progeny
of multipotent neural .stem cell progeny, as disclosed in published PCT
application WO
94110292. Additional methods for directing the differentiation of stem cell
progeny were
disclosed in U.S. Pat. No. 6,165,783 utilizing erythropoietin and various
growth factors.
Thus, the repair of damaged neural tissue may potentially be replaced in a
relatively non-invasive fashion, by inducing neural cells to proliferate and
differentiate
into neurons, astrocytes, and oligodendrocytes in vivo, averting the need for
transplantation. However, simply inducing neural cells to proliferate and
differentiate is
not always sufficient to treat a neurodegenerative disease or brain injury if
the new
neurons are not able to reach the lesioned or damaged area. During
development, neurons
in many regions of the brain are directed to their appropriate destinations by
migrating
along radial glia. For example, developing neurons migrate outward from the
ventricular
zone to the cortical plate. As many neural stem cells in the adult nervous
system are in
the localized areas, which may be remote from the affected areas, it is
particularly
desirable to be able to elicit migration of these cells to other affected
areas of the brain to
replace lost neurons, e.g., the basal ganglia in Parkinson's Disease.
SCARY OF THE INVENTION
Accordingly, a major object of the present invention is to provide both in
vivo
and in vitro techniques of enhancing or inducing migration of multipotent
neural stem cells
or multipotent neural stem cell progeny.
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The current invention provides a method of enhancing or inducing the migration
of multipotent neural stem cell and/or multipotent neural stem cell progeny in
a subject
comprising administering erythropoietin to a subject in an amount effective to
enhance
neural stem cell migration. In a preferred embodiment, at least one other
growth factor
besides erythropoietin is administered. In a particularly preferred
embodiment, the other
growth factor is epidermal growth factor. In another embodiment, the other
growth factor
is prolactin.
The erythropoietin and growth factors can be administered in a different
order.
In one embodiment, the erythropoietin is administered concurrently with at
least one other
growth factor. In an alternative embodiment, the erythropoietin is
administered
sequentially with at least one other growth factor. In a preferred embodiment,
at least one
other growth factor is administered prior to the administration of
erythropoietin. In an
alternative embodiment, the at least one other growth factor is administered
after the
erythropoietin.
In one embodiment, the subject is suffering from a neurodegenerative disease
or
brain injury. In various embodiments, the subject is suffering from
Alzheimer's Disease,
Multiple Sclerosis, Huntington's Disease, Amyotrophic Lateral Sclerosis,
Parkinson's
Disease, surgery, stroke, a physical accident, depression, epilepsy, neurosis,
or psychosis.
In a particularly preferred embodiment, the subject is suffering from a
stroke.
In an embodiment of the invention, the multipotent neural stem cells and/or
progeny migrate towards a lesioned or damaged area of the brain of the
subject. In a
particularly preferred embodiment, the multipotent neural stem cells and/or
progeny
migrate to the basal ganglia. In one embodiment, the subject is a mammal. In a
preferred
embodiment, the subject is a human. In a particularly preferred embodiment,
the mammal
is an adult. In another embodiment, the multipotent neural stem cells and/or
progenitor
cells which are derived from the multipotent neural stem cells are
transplanted into the
subject. In a preferred embodiment, the multipotent neural stem cells and/or
progenitor
cells are incubated with erythropoietin and at least one growth factor before
being
transplanted into the subject.
Another aspect of the invention provides a method of enhancing or inducing the
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migration of multipotent neural stem cells and/or multipotent neural stem cell
progeny
comprising exogenously adding to the multipotent neural stem cells and/or
multipotent
neural stem cell progeny an amount of erythropoietin effective to cause the
multipotent
neural stem cells and/or multipotent neural stem cell progeny to migrate. In a
preferred
embodiment, at least one other growth factor is added. In a particularly
preferred
embodiment, the other growth factor is epidermal growth factor. In another
embodiment,
the at least one other growth factor is prolactin.
In another embodiment, the erythropoietin is added concurrently with the at
least
one other growth factor. In an alternative embodiment, the erythropoietin is
added
sequentially with the at least one other growth factor. In a particularly
preferred
embodiment, the other growth factor is added prior to the addition of
erythropoietin. In
another embodiment, the other growth factor is added after the addition of
erythropoietin.
Another aspect of the invention provides a method for enhancing or inducing
migration of multipotent neural stem cells and/or multipotent neural stem cell
progeny,
comprising exposing said multipotent neural stem cells and/or multipotent stem
cell
progeny to hypoxic conditions to induce expression of erythropoietin in order
to enhance
or induce migration. In a preferred embodiment, at least one other growth
factor is
exogenously added. In a particularly preferred embodiment, the other growth
factor is
epidermal growth factor. In another embodiment, the other growth factor is
prolactin. In
one embodiment, the other growth factor is added to said multipotent neural
stem cells
and/or multipotent neural stem cell progeny concurrently with hypoxic
conditions. In an
alternative embodiment, the other growth factor is added to the multipotent
neural stem
cells and/or multipotent neural stem cell progeny sequentially with hypoxic
conditions. In
a particularly preferred embodiment, the other growth factor is added to the
multipotent
neural stem cells and/or multipotent neural stem cell progeny prior to
exposure to hypoxic
conditions. In another embodiment, the other growth factor is added after
exposure to
hypoxic conditions.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Distribution of total BrdU+ cells between the subventricular zone
(SVZ) and the striatum (Str) in mice lesioned with ibotenic acid and treated
with
epidermal growth factor (EGF) and Erythropoietin (Epo). When administered to
animals
treated with EGF, Epo enhanced the number of neural progenitors in the
stratium. (* p <
0.05).
Figure Z. Number of NeuN+/BrdU+ cells (mature neurons) in the striatum of
mice lesioned with ibotenic acid and treated with EGF and Epo. EGF enhanced
the
number of NeuN+/BrdU+ cells in the striatum. When administered to animals
treated
with EGF, Epo further enhanced this effect.
Figure 3. 3A: Number of Dcx+/BrdU+ cells (immature neurons or neuronal
precursors) in the subventricular zone (SVZ) in mice lesioned with ibotenic
acid and
treated with EGF and Epo. 3B: Number of Dcx+/BrdU+ cells (immature neurons or
neuronal precursors) in the striatum of mice lesioned with ibotenic acid and
treated with
EGF and Epo. When administered to animals treated with EGF, Epo enhanced
migration
of neuronal precursors into the damaged striatum. (* p < 0.05).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of enhancing or inducing migration
of
multipotent neural stem cells or multipotent neural stem cell progeny by
utilizing
erythropoietin in conjunction with at least one other growth factor.
Prior to describing the invention in further detail, the terms used in this
application
are defined as follows unless otherwise indicated.
As used herein, the term "multipotent neural stem cell" or "neural stem cell"
refers
to an undifferentiated cell which is capable of self maintenance. Thus, in
essence, a stem
cell is capable of dividing without limit. "Progenitor cells" are non-stem
cell progeny of a
multipotent neural stem cell. A distinguishing feature of a progenitor cell is
that, unlike a
stem cell, it has limited proliferative ability and thus does not exhibit self
maintenance. It
is committed to a particular path of differentiation and will, under
appropriate conditions,
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eventually differentiate. A neuronal progenitor cell is capable of~,a limited
number of cell
divisions before giving rise to differentiated neurons. A glial progenitor
cell likewise is
capable of a limited number of cell divisions before giving rise to astrocytes
or
oligodendrocytes. A neural stem cell is multipotent because its progeny
include both
neuronal and glial progenitor cells and thus is capable of giving rise to
neurons,
astrocytes, and oligodendrocytes. Multipotent neural stem cell progeny include
neuronal
precursor cells, glial precursor cells, neurons, and glial cells.
A "neurosphere" is a group of cells derived from a single neural stem cell as
the
result of clonal expansion. Primary neurospheres may be generated by plating
as primary
cultures brain tissue which contains neural stem cells. The method for
culturing neural
stem cells to form neurospheres has been described in, e.g., U.S. Patent No.
5,750,376.
Secondary neurospheres may be generated by dissociating primary neurospheres
and
allowing the individual dissociated cells to form neurospheres again.
By "growth factor" is meant a substance that affects the growth of a cell or
an
organism, including proliferation, differentiation, and increases in cell
size. A growth
factor is a polypeptide which shares substantial sequence identity with a
native mammalian
growth factor and possesses a biological activity of the native mammalian
growth factor.
In a preferred embodiment, the native mammalian growth factor is a native
human growth
factor. Having a biological activity of a native mammalian growth factor means
having at
least one activity of a native mammalian growth factor, such as binding to the
same
receptor as a particular native mammalian growth factor binds and/or eliciting
proliferation and/or differentiation and/or changes in cell size. Preferably,
the growth
factor binds to the same receptor as a particular native mammalian growth
factor. This
includes functional variants of the native mammalian growth factor.
A polypeptide which shares substantial sequence identity with a native
mammalian growth factor is at least about 30 % identical to the native
mammalian growth
factor at the amino acid level. The growth factor is preferably at least about
40 % , more
preferably at least about 60 % , and most preferably about 60 % identical to
the native
mammalian growth factor at the amino acid level. Thus, the term growth factor
encompasses analogs which are deletional, insertional, or substitutional
mutants of a native
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mammalian growth factor. Furthermore, the term growth factor encompasses the
growth
factors from other species and naturally occurring and synthetic variants
thereof.
Erythropoietin (Epo) is a growth factor. Other exemplary growth factors that
may be used in conjunction with Epo in embodiments of the present invention
include,
inter alia, platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), insulin-
like growth factor-1 and -2 (IGF-1, IGF-2), transforming growth factors a and
~i (TGF-a,
TGF-Vii), acidic and basic fibroblast growth factors (a-FGF/FGF-2, b-FGF/FGF-
2),
interleukins 1, 2, 6, and 8 (IL-1, IL-2, IL-6, IL-8), nerve growth factor
(NGF), brain-
derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), interleukin-3,
hematopoietic
colony stimulating factors (CSFs), amphiregulin, interferon-~y (INF-y),
thyrotropin
releasing hormone (TRH), pituitary adenylate cyclase activating polypeptide
(PACAP),
and prolactin. In a preferred embodiment, Epo is used in conjunction with EGF.
In
another embodiment, Epo is used in conjunction with prolaction.
It should be noted that variants or analogs of these agents, which share a
substantial identity with a native mammalian growth factor listed above and
are capable of
binging the receptor for a native mammalian growth factor, can be used in the
present
application. For example, there are two forms of mammalian PACAP, PACAP38 and
PACAP27. Any variant or analog that is capable of binding to a receptor for a
native
mammalian PACAP and shares a substantial sequence identity with either PACAP38
or
PACAP27 is suitable for use in the present invention. Particularly useful are
the analogs
and variants disclosed in, e.g., U.S. Patent Nos. 5,128,242; 5,198,542;
5,208,320;
5,326,860; 5,801,147; and 6,242,563.
Similarly, EGF variants or analogs, which share a substantial identity with a
native mammalian EGF and are capable of binding to a receptor for the native
mammalian
EGF, can be used in the present application. These EGF variants and analogs
include, but
are not limited to, the recombinant modified EGF having a deletion of the two
C-terminal
amino acids and a neutral amino acid substitution at position 51, such as
asparagine,
glutamine, serine, or alanine (particularly EGFS1N or EGFS1Q, having
asparagine (N) or
glutamine (Q) at position 51, respectively; WO 03/040310); the EGF mutein (EGF-
X16)
in which the His residue at position 16 is replaced with a neutral or acidic
amino acid
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(U.S. Patent No. 6,191,106); the 52-amino acid deletion mutant of EGF which
lacks the
amino terminal residue of the native EGF (EGF-D); the EGF deletion mutant in
which the
amino terminal residue as well as the two C-terminal residues (Arg-Leu) are
deleted
(EGF-B); the EGF-D in which the Met residue at position 21 is oxidized (EGF-
C); the
EGF-B in which the Met residue at position 21 is oxidized (EGF-A); heparin-
binding
EGF-like growth factor (HB-EGF); betacellulin; amphiregulin; neuregulin; or a
fusion
protein comprising any of the above. Other useful EGF analogs or variants are
described
in WO 03/040310, and U.S. Patent Nos. 6,191,106 and 5,547,935.
Specifically included as prolactins are the naturally occurring prolactin
variants,
prolactin-related protein, placental lactogens, S179D-human prolactin
(Bernichtein et al.,
2001), prolactins from various mammalian species, including, but not limited
to, human,
other primates, rat mouse, sheep, pig, and cattle, and the prolactin mutants
described in
U.S. Patent Nos. 6,429,186 and 5,955,346.
"Erythropoietin" refers to a polypeptide that shares substantial sequence
similarity with native mammalian erythropoietin and possesses a biological
activity of the
native mammalian erythropoietin, including recombinant erythropoietin or
epoietin.
Having a biological activity of native mammalian erythropoietin means having
at least one
activity of a native mammalian erythropoietin, such as binding to the same
receptor as the
native mammalian erythropoietin binds and/or eliciting proliferation and/or
differentiation,
and/or changes in cell size. Preferably, the polypeptide binds to a native
mammalian Epo
receptor. This includes functional variants of the native mammalian
erythropoietin. The
native human erythropoietin is a glycoprotein of 165 or 166 amino acids (C-
terminal
arginine is removed in post-translational modification) and an approximate
molecular
weight of 30-40 kDa.
Erythropoietin can be generated or synthesized using genetic engineering
techniques such as those found in U.S. Patent Nos. 4,703,008; 5,441,868;
5,547,993,
5,621,080, 6,618,698, and 6,376,218. A polypeptide which shares "substantial
sequence
similarity" with the native mammalian erythropoietin is at least about 30 %
identical with
native mammalian erythropoietin at the amino acid level. The erythropoietin is
preferably
about 40 % , more preferably about 60 % , yet more preferably at least about
70 % , and most
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preferably, at least about 80 % identical with the native mammalian
erythropoietin at the
amino acid level. Thus, the term erythropoietin encompasses erythropoietin
analogs
which are deletional, insertional, or substitutional mutants of the native
mammalian
erythropoietin. Furthermore, the term erythropoietin encompasses
erythropoietins from
other species and the naturally occurring and synthetic variants thereof.
"Percent identity" or " % identity" refers to the percentage of amino acid
sequence in a protein or polypeptide which are also found in a second sequence
when the
two sequences are aligned. Percent identity can be determined by any methods
or
algorithms established in the art, such as LALIGN or BLAST.
A polypeptide possesses the "biological activity" of a growth factor,
including
erythropoeitin, if it is capable of exerting any of the biological activities
of the native
mammalian growth factor or being recognized by a polyclonal or monoclonal
antibody
raised against the native mammalian growth factor. Preferably, the polypeptide
is capable
of specifically binding to the receptor for the native growth factor in a
receptor binding
assay.
"Hypoxic conditions" or "hypoxia" refers to a decrease in normal or optimal
oxygen conditions for a cell or an organism. Normal or optimal oxygen
concentration is
135 mm Hg or 95 % air/5 % CO2. Standard hypoxic conditions comprise an oxygen
concentration of about 30-40 mm Hg.
"Migration" refers to the movement of a cell from one location to another.
Thus, a substance that "enhances" migration increases the speed, distance, or
number of
cells moving from one location to another over the speed, distance, or number
of cells
moving in the absence of the substance. For instance, the Example below
demonstrated
that the distance traveled by multipotent neural stem cells and/or multipotent
neural stem
cell progeny is much greater with Epo and EGF compared to either of these
alone. A
substance that "induces" migration elicits migration when it would not
otherwise occur in
the absence of the substance. The present invention can be used to enhance or
induce
migration of neurons to damaged areas of the CNS.
A "neurodegenerative disease or condition" is a disease or a medical condition
associated with neuron loss or dysfunction. Examples of neurodegenerative
diseases or
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conditions include neurodegenerative diseases, brain injuries or CNS
dysfunctions.
Neurodegenerative diseases include, e.g., Alzheimer's Disease, Multiple
Sclerosis,
Huntington's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease.
Brain
injuries include, e.g., injuries to the nervous system due to surgery, stroke,
and physical
accidents. CNS dysfunctions include, e.g., depression, epilepsy, neurosis, and
psychosis.
"Treating or ameliorating" means the reduction or complete removal of the
symptoms of a disease or medical condition.
An "effective amount" is an amount of a therapeutic agent sufficient to
achieve
the intended purpose. For example, an effective amount of a growth factor or
erythropoietin to enhance the migration of neural stem cells is an amount
sufficient, in
vivo or in vitro, to result in an enhancement in migration of neural stem
cells over the
speed, distance, or number in the absence of the growth factor or
erythropoietin. An
effective amount of a growth factor or erythropoietin to treat or ameliorate a
neurodegenerative disease or condition is an amount of the growth factor or
erythropoietin
sufficient to reduce or remove the symptoms of the neurodegenerative disease
or
condition. The effective amount of a given therapeutic agent will vary with
factors such
as the nature of the agent, the route of administration, the size and species
of the animal or
subject to receive the therapeutic agent, and the purpose of administration.
The effective
amount in each individual case may be determined empirically by a skilled
artisan
according to established methods in the art.
Detailed Description
In the present invention, a method of enhancing or inducing the migration of
neural stem cells and/or their progeny was discovered. As discussed in more
detail in the
Example below, Epo was able to enhance the speed, number, and distance of
migration of
neural stem cells and/or their progeny. Preferably, at least one other growth
factor is also
used. For example, when animals with striatal lesions were treated with Epo
and EGF,
greater numbers of newly generated cells were discovered in the striatum.
Additionally,
more of the newly generated cells adopted a neuronal phenotype in the damaged
striatum.
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Various embodiments of the present invention are possible. In addition to EGF,
other growth factors such as those described above can be used with Epo to
enhance or
induce migration of neural stem cells and/or their progeny. For instance,
prolactin is
another preferred embodiment of the present invention. When other growth
factors are
administered or added in conjunction with Epo, the order of administration or
addition can
be varied. Epo and the other growth factor can be administered or added
sequentially or
simultaneously. When added sequentially, Epo can be administered or added
before or
after the other growth factor.
The multipotent stem cells and/or their progeny can be induced to migrate or
the
migration to various areas of the brain can be enhanced. Although the Example
below
shows the migration of cells from the SVZ to the striatum, other embodiments
are also
contemplated. For example, the migration of multipotent neural stem cells or
their
progeny can be enhanced towards other areas of the basal ganglia or any other
damaged
area of the brain.
The present method can be practiced ih vivo or in vitro. For in vivo
administration, compositions containing Epo andlor other growth factors can be
delivered
via any route known in the art, such as orally, or parenterally, e.g.,
intravascularly,
intramuscularly, transdermally, subcutaneously, or intraperitoneally. In a
preferred
embodiment, the composition is administered parenterally. Alternatively, the
composition
is delivered directly to the CNS. Direct administration into the CNS can be
accomplished
via delivery into a ventricle, such as the lateral ventricle.
According to embodiments of the invention, Epo and other growth factors may be
administered in vivo to treat subjects suffering from neurodegenerative
diseases, brain
injuries, or CNS dysfunctions. Alzheimer's Disease, Huntington's Disease, and
Parkinson's Disease, inter alia, may be treated according to various
embodiments of the
invention. Alternatively, the subject may be suffering from a stroke. Because
of the
prevalence of neurodegenerative disease in adults, the preferred subject is an
adult human.
However, it is contemplated that younger subjects may also suffer from
neurodegenerative
disease, or more commonly, traumatic brain injury, and thus will benefit from
the present
invention. Additionally, while humans are particularly preferred subjects,
other species,
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such as those kept as pets, may also be treated according to an embodiment of
the
invention. Subjects may be treated with Epo and/or other growth factors, or
neural stem
cells may be exogenously treated and then transplanted into the subject. A
combination of
these approaches is also possible.
The present invention can be used ih vitro. Multipotent neural stem cells can
be
obtained from embryonic, juvenile, or adult mammalian neural tissue (e.g.,
mouse and
other rodents, and humans and other primates) or from other sources as
described in U.S.
Patent No. 6,294,346 B1. Multipotent neural stem cells can be induced to
proliferate in
vitro or in vivo using the methods disclosed in published PCT application WO
93/01275
and U.S. Pat. Nos. 5,750,376 and 6,294,346 Bl. Briefly, the administration of
one or
more growth factors can be used to induce the proliferation and
differentiation of
multipotent neural stem cells. Preferred proliferation-inducing growth factors
include
epidermal growth factor (EGF), amphiregulin, acidic fibroblast growth factor
(aFGF or
FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth
factor
alpha (TGF-cx), and combinations thereof. For the proliferation of multipotent
neural stem
cells in vitro, neural tissue is dissociated and the primary cell cultures are
cultured in a
suitable culture medium, such as the serum-free defined medium described U.S.
Pat. No.
6,165,783. A suitable proliferation-inducing growth factor, such as EGF (20
ng/ml) is
added to the culture medium to induce multipotent neural stem cell
proliferation. In
addition to proliferation-inducing growth factors, other growth factors may be
added to
the culture medium that influence proliferation and differentiation of the
cells, including
nerve growth factor (NGF), platelet-derived growth factor (PDGF), thyrotropin
releasing
hormone (TRH), transforming growth factor betas (TGF-his), insulin-like growth
factor
(IGF-1) and the like.
In the absence of substrates that promote cell adhesion (e.g. ionically
charged
surfaces such as poly-L-lysine and poly-L-ornithine coated and the like),
multipotent
neural stem cell proliferation can be detected by the formation of clusters of
undifferentiated neural cells termed "neurospheres," which after several days
in culture,
lift off the floor of the culture dish and float in suspension. Each
neurosphere results from
the proliferation of a single multipotent neural stem cell and is comprised of
daughter
multipotent neural stem cells and neural progenitor cells. The neurospheres
can be
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dissociated to form a suspension of undifferentiated neural cells and
transferred to fresh
growth-factor containing medium. This re-initiates proliferation of the stem
cells and the
formation of new neurospheres. In this manner, an unlimited number of
undifferentiated
neural stem cell progeny can be produced by the continuous culturing and
passaging of the
cells in suitable culture conditions.
Various procedures are disclosed in WO 94/10292 and U.S. Pat. Nos. 5,750,376
and 6,294, 346 B 1 which can be used to induce the proliferated neural stem
cell progeny to
differentiate into neurons, astrocytes and oligodendrocytes. Various methods
of assessing
differentiation of a particular cell type, e.g., using immunochemistry, are
described in
U.S. Pat. No. 6,294,346 B1.
The ability to manipulate the fate of the differentiative pathway of the
multipotent
neural stem cell progeny to produce more neuronal progenitor cells and neurons
is
beneficial. Cell cultures with an enriched neuronal-progenitor cell and/or
neuron
population can be used for transplantation to treat various neurological
injuries, diseases
or disorders. The neuronal progenitor cells or neurons or a combination
thereof can be
harvested and transplanted into a patient needing neuronal augmentation.
Neuronal
progenitor cells are particularly suitable for transplantation because they
are still
undifferentiated and, unlike differentiated neurons, there are no branched
processes which
can be damaged during transplantation procedures. Once transplanted, the
neuronal
progenitor cells can migrate to a damaged area of the brain and differentiate
in situ into
new, functioning neurons. Suitable transplantation methods are known in the
art and are
disclosed in U.S. Pat. Nos. 5,750,376 and 6,294,346 B1.
Alternatively, a patient's endogenous multipotent neural stem cells could be
induced to proliferate, migrate, and differentiate in situ by administering to
the patient a
composition comprising one or more growth factors, which induces the patient's
neural
stem cells to proliferate, and Epo, which instructs the proliferating neural
stem cells to
produce neuronal progenitor cells which eventually differentiate into neurons
and enhances
and/or induces migration to other brain regions. Suitable methods for
administering a
composition to a patient which induces the in situ proliferation of the
patient's stem cells
are disclosed in U.S. Pat. Nos. 5,750,376 and 6,294,346 B1.
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EXAMPLES
An in vivo mouse model of neurodegenerative disease and the use of Epo and EGF
to induce neuronal migration.
EGF has been shown to induce proliferation of neural stem cells in the
subventricular zone (SVZ). Previously, it was demonstrated that after a
unilateral striatal
lesion, newly-generated cells from both hemispheres migrated towards the
damaged area
in response to EGF. Epo is able to direct neural stem cells to differentiate
into neuronal
precursors. (Shingo et al., 2001). A mouse model of neurodegenerative disease
was used
to determine the effects of EGF and Epo on neural stem cell migration.
Following an
injury to elicit neurodegeneration, mice were infused with epidermal growth
factor (EGF)
and erythropoietin to induce proliferation, differentiation, and migration of
endogenous
neural precursor cells.
Adult male CD-1 mice were given an injection of ibotenic acid (4.0 ,ug in 1.6
~l
total volume) into the medial striatum. Within one week, many of the striatal
neurons
within the lesion area had degenerated. At this stage, a miniosmotic pump
filled with
EGF (33 pg/ml) was inserted beneath the skin above the shoulders. A small hole
was
drilled through the skull and a cannula was secured to the skull with dental
cement. The
pump was connected via tubing to the cannula, which delivers EGF into the
lateral
ventricle of the brain for a period of seven (7) days.
At the end of the seven day period, mice were injected once every two (2)
hours
over a ten-hour period with bromodeoxyuridine (BrdU)(Sigma Chemical Co.), a
marker
for cell division. On the same day, a small incision was made directly above
the pump on
the back, the tubing was cut, and the EGF pump was replaced with another pump
containing erythropoietin (1000 IU/ml). After seven days of Epo delivery, the
cannula
was removed from the skull and the wound was closed. The mice were sacrificed
immediately following Epo delivery. A series of control mice were infused with
the
delivery vehicle only, mouse serum albumin.
The mice were sacrificed via transcardial perfusion under anesthesia whereby
the
brain is fixed with 4% paraformaldehyde. The brains were removed and subjected
to a
series of postfixation and cryoprotection steps before being frozen. The
brains were cut
into 12 ,um sections and immunostained with markers for migrating immature
neurons
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(Doublecortin (Dcx) (Chemicon)) or mature neurons (NeuN (Chemicon)), and for
proliferating cells (BrdU). Once brains were sectioned and stained, total BrdU
and
NeuN/BrdU and Dcx/BrdU cells were counted on every tenth section through the
entire
forebrain. The data presented below were the results of three independent
experiments.
Infusion of EGF followed by Epo results in a greater number of newly generated
cells (BrdU+) in the striatum compared to EGF alone. Figure 1 shows the
distribution
of total BrdU+ cells between the subventricular zone (SVZ) and the striatum
(Str). These
data indicate that Epo enhances the numbers of neural progenitors in the
striatum.
Newly generated cells in the striatum adopted a neuronal phenotype in the
damaged
striatum. Some of the newly generated cells differentiated into mature neurons
(NeuN .-+/BrdU+) regardless of the infusion conditions. As can be seen in
Figure 2, all of
the NeuN + /BrdU + mature neurons are found in the striatum, indicating that
they may
have migrated from the SVZ and differentiated in the striatum.
EGF followed by Epo infusion directs the migration of neuronal progenitors
from
the SVZ into the damaged striatum. In vehicle-only-infused mice, neuronal
progenitors
(Dcx+) remain in the SVZ after two weeks of treatment. The same results are
seen in
Vehicle-Epo-infused mice. In EGF-infused mice, neuronal progenitors moved
laterally
into the striatum. In EGF-Epo-infused mice most of the neuronal progenitors
have
migrated into the striatum. Newly generated BrdU+ cells outside the SVZ
exhibited
extending processes, indicating migration laterally into the striatum (data
not shown).
Figures 3A and 3B show the distribution of Dcx+/BrdU+ cells between the SVZ
and the
striatum, respectively. The distribution of cells between the SVZ and the
striatum
indicates, surprisingly, that Epo enhanced the migration of neuronal
precursors into the
damaged striatum.
Thus, Epo, when infused in combination with EGF, resulted in increased numbers
of newly generated BrdU+ cells in the ibotenate-lesioned striatum, compared to
those
generated using EGF alone. These results show that Epo enhances both the
number and
migration rate of precursors from the lateral ventricle towards the lesioned
striatum. The
Epo-stimulated cells infiltrate the entire striatum indicating they have
migrated from their
origin in the SVZ. Epo promotes increased migration and
survival/differentiation of
newly generated neuronal precursors and thus will be useful in therapeutic
strategies
aimed at enhancing functional recovery from CNS injury or disease.
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The above example is merely illustrative of the present invention and is
considered
to be in no way limiting. The skilled artisan will appreciate numerous
variations of the
present invention.
