U.S. patent application number 12/398888 was filed with the patent office on 2010-01-28 for methods and compositions for delivery of exogenous factors to nervous system sites.
This patent application is currently assigned to Regenerative Research Foundation. Invention is credited to Akhilesh Banerjee, Susan K. Goderie, Ravindra Kane, Natalia Lowry, Supriya Punyani, Jeffrey Stern, Sally Temple.
Application Number | 20100021422 12/398888 |
Document ID | / |
Family ID | 41056655 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100021422 |
Kind Code |
A1 |
Temple; Sally ; et
al. |
January 28, 2010 |
METHODS AND COMPOSITIONS FOR DELIVERY OF EXOGENOUS FACTORS TO
NERVOUS SYSTEM SITES
Abstract
The present invention relates to treatment methods and methods
for sustained delivery of one or more exogenous factors to desired
nervous system sites. In certain embodiments, the invention relates
to the use of biodegradable microspheres to deliver exogenous
factors, such as the morphogenic factor, sonic hedgehog (Shh), to
the site of spinal cord injury. In certain embodiments, the
Shh-releasing microspheres are administered together with stem
cells, which may be spinal cord neural stem cells. In certain
embodiments, the invention relates to regrowth of neural cells in
both the central and peripheral nervous systems.
Inventors: |
Temple; Sally;
(Slingerlands, NY) ; Lowry; Natalia; (Albany,
NY) ; Stern; Jeffrey; (Slingerlands, NY) ;
Goderie; Susan K.; (Ballston Spa, NY) ; Kane;
Ravindra; (Niskayuna, NY) ; Punyani; Supriya;
(New Delhi, IN) ; Banerjee; Akhilesh; (Lucknow,
IN) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
Regenerative Research
Foundation
Rensselaer
NY
Albany Medical College
Albany
NY
Rensselaer Polytechnic Institute
Troy
NY
|
Family ID: |
41056655 |
Appl. No.: |
12/398888 |
Filed: |
March 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61034068 |
Mar 5, 2008 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/93.7; 424/94.62; 514/1.1 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 9/19 20130101; A61K 38/18 20130101; C12N 5/0623 20130101; A61P
25/00 20180101; A61K 35/30 20130101; A61K 9/0019 20130101 |
Class at
Publication: |
424/85.2 ;
514/12; 424/93.7; 424/94.62 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 38/16 20060101 A61K038/16; A61K 35/12 20060101
A61K035/12; A61K 38/46 20060101 A61K038/46; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part in the course of research
sponsored by the New York State Spinal Cord Injury Research Trust
Fund through New York State Department of Health Contract #
C020922. New York State may have certain rights in this invention.
Claims
1. A method for treating a nervous system injury in a mammal
comprising administering to a mammal in need of such treatment an
effective amount for treating the nervous system injury of a
pharmaceutical formulation comprising at least one exogenous
factor, said pharmaceutical formulation providing sustained
delivery of the at least one exogenous factor for at least about 7
days.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 1, wherein the nervous system injury is
selected from the group consisting of spinal cord injury,
amyotrophic lateral sclerosis (ALS), peripheral nerve injury, and
spinal nerve injury.
4. The method of claim 1, wherein the sustained delivery
composition comprises a plurality of biodegradable
microspheres.
5. The method of claim 1, wherein the pharmaceutical formulation
further comprises an effective amount of stem cells, wherein the
amount of stem cells in combination with said at least one
exogenous factor is effective for treating a nervous system
injury.
6. The method of claim 1, wherein the at least one exogenous factor
is a growth factor selected from the group consisting of Nerve
Growth Factor (NGF), Glial Cell Line-Derived Growth Factor (GDNF),
Neurotrophin (NT) 3, NT 4/5, NT 6, Ciliary Neurotrophic Factor
(CNTF), Leukemia Inhibitory Factor (LIF), Interleukin 6 (IL6),
Interleukin 11(IL11), Cardiotrophin 1, a growth factor hormone,
hyaluronidase, chondroitinase ABC(CABC), basic fibroblast growth
factor (bFGF), insulin-related growth factor (IGF-I), brain-derived
neurotrophic factor (BDNF), epidermal growth factor (EGF), and
sonic hedgehog (Shh).
7. The method of claim 5, wherein the stem cells are neural stem
cells.
8. The method of claim 1, wherein the pharmaceutical formulation is
administered by at least one injection, wherein a first injection
is administered at the site of nervous system injury.
9. The method of claim 8, wherein a second injection is
administered at a site rostral to the site of nervous system
injury.
10. The method of claim 1, wherein the stem cells are
endothelial-expanded stem cells.
11. The method of claim 10, wherein the endothelial-expanded stem
cells are pre-treated with sonic hedgehog.
12. The method of claim 10, wherein the endothelial-expanded stem
cells are pre-treated with sonic hedgehog and retinoic acid.
13. A method for increasing neural cell growth or regenerating
neural cells in a mammal which comprises administering to a mammal
in need of such treatment an effective amount for increasing neural
cell growth or regenerating neuronal cells of a sustained release
pharmaceutical formulation comprising at least one exogenous
factor, said pharmaceutical formulation continuously delivering the
at least one exogenous factor for at least about 7 days.
14. The method of claim 13, wherein the mammal is a human.
15. The method of claim 13, wherein the neural cell is selected
from the group consisting of neurons, glial cells, and progenitor
cells.
16. The method of claim 13, wherein the sustained delivery
composition comprises a plurality of biodegradable
microspheres.
17. The method of claim 13, wherein the pharmaceutical formulation
further comprises an effective amount of stem cells, wherein said
amount in combination with said at least one exogenous factor is
effective for increasing neural cell growth or regenerating neural
cells.
18. The method of claim 13, wherein the at least one exogenous
factor is a growth factor selected from the group consisting of
Nerve Growth Factor (NGF), Glial Cell Line-Derived Growth Factor
(GDNF), Neurotrophin (NT) 3, NT 4/5, NT 6, Ciliary Neurotrophic
Factor (CNTF), Leukemia Inhibitory Factor (LIF), Interleukin 6
(IL6), Interleukin 11(IL 11), Cardiotrophin 1, a growth factor
hormone, hyaluronidase, chondroitinase ABC(CABC), basic fibroblast
growth factor (bFGF), insulin-related growth factor (IGF-1),
brain-derived neurotrophic factor (BDNF), epidermal growth factor
(EGF), and sonic hedgehog (Shh).
19. The method of claim 17, wherein the stem cells are neural stem
cells.
20. The method of claim 13, which comprises administering at least
one injection of the pharmaceutical formulation at a site at which
it is desired to increase neural cell growth or regenerate neural
cells.
21. The method of claim 20, which comprises administering a second
injection containing said pharmaceutical formulation at a site
rostral to the first site of injection.
22. The method of claim 13, wherein the stem cells are
endothelial-expanded stem cells.
23. The method of claim 22, wherein the endothelial-expanded stem
cells are pre-treated with sonic hedgehog.
24. The method of claim 22, which comprises pre-treating
endothelial-expanded stem cells with sonic hedgehog and retinoic
acid.
25. A pharmaceutical formulation which comprises an effective
amount for increasing neuronal cell growth or regenerating neuronal
cells of at least one sustained delivery composition comprising at
least one exogenous factor selected from the group consisting of
Nerve Growth Factor (NGF), Glial Cell Line-Derived Growth Factor
(GDNF), Neurotrophin (NT) 3, NT 4/5, NT 6, Ciliary Neurotrophic
Factor (CNTF), Leukemia Inhibitory Factor (LIF), Interleukin 6
(IL6), Interleukin 11(IL11), Cardiotrophin 1, a growth factor
hormone, hyaluronidase, chondroitinase ABC(CABC), basic fibroblast
growth factor (bFGF), insulin-related growth factor (IGF-1),
brain-derived neurotrophic factor (BDNF), epidermal growth factor
(EGF), and sonic hedgehog (Shh), wherein the composition provides
sustained delivery of at least one exogenous factor for at least 7
days, and a pharmaceutically acceptable excipient.
26. The pharmaceutical formulation of claim 25, further comprising
an effective amount of stem cells, wherein said amount in
combination with said at least one exogenous factor is effective
for increasing neural cell growth or regenerating neural cells.
27. The pharmaceutical formulation of claim 25, wherein the at
least one sustained delivery composition comprises a plurality of
biodegradable microspheres.
28. The pharmaceutical formulation of claim 26, wherein the stem
cell is a neural stem cell.
29. The pharmaceutical formulation of claim 26, wherein the stem
cell is an endothelial-expanded stem cell.
30. The pharmaceutical formulation of claim 25, wherein the
endothelial-expanded stem cell is pre-treated with sonic
hedgehog.
31. The pharmaceutical formulation of claim 25, wherein the
endothelial-expanded stem cell is pre-treated with sonic hedgehog
and retinoic acid.
32. The pharmaceutical formulation of claim 25, wherein the
sustained delivery composition releases from about 1 ng/ml to about
20 ng/ml Shh for at least 7 days.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119 to U.S. Provisional Application Ser. No. 61/034,068,
filed Mar. 5, 2008, which is hereby incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to treatments and methods for
sustained delivery of one or more exogenous factors to
predetermined sites in the mammalian nervous system. In certain
embodiments, the invention relates to the use of biodegradable
microspheres to deliver exogenous factors, such as the morphogenic
factor, sonic hedgehog (Shh), to the site of spinal cord injury. In
other embodiments, the Shh-releasing microspheres are administered
together with stem cells, which may be spinal cord neural stem
cells.
BACKGROUND OF THE INVENTION
[0004] Spinal cord injury (SCI) causes loss of spinal cord cells,
damage to ascending and descending axonal tracts and loss of
myelination, resulting in paralysis. After SCI, axonal regeneration
is prevented by the lack of matrix that supports growth through
production of important growth and morphogenic factors (Harel and
Strittmatter 2006; Lu and Tuszynski 2007). Successful treatment of
SCI will include approaches that aid in the regeneration of damaged
axons and/or in the replacement of oligodendrocytes to improve
myelination.
[0005] Stem cell therapy has been envisioned as a treatment that
may serve to prevent and/or reverse SCI by replacing damaged or
lost spinal cord cells, delivering factors conducive to spinal cord
repair, and providing a physical scaffold for instructing and
enabling axon regrowth. However, at present, the technology to
successfully direct stem cell differentiation into the appropriate
or desired cell type in vivo is lacking. Specifically, research
studying stem cells in the context of treating SCI has shown that
transplanted neural stem cells (NSC) do not differentiate into the
appropriate cell types for neuron regeneration, such as
oligodendrocytes and neurons. NSCs instead differentiate into
primarily astrocytes in vivo, thereby limiting functional recovery
(Enzmann et al. 2006).
[0006] One reason functional recovery from SCI is limited is
because the microenvironment of the adult spinal cord lacks the
necessary biological cues for proper differentiation of NSCs into
neurons and oligodendrocytes. The spinal cord microenvironment
instead favors astrogliogenesis, (the growth of astrocytes),
thereby adding to the astroglial scar, which is believed to be an
impermeable barrier to recovering, outgrowing axons (Silver and
Miller 2004). It has been demonstrated in vitro that exogenous
factors are needed to direct NSC differentiation toward the cell
types useful for SCI treatment, including oligodendrocytes and
neurons (Cattaneo 1990; Gage 2000).
[0007] Achieving delivery of soluble growth factors to the site of
SCI is a challenging problem. If injected once, soluble factors
flow away quickly from the injury site after injection.
Furthermore, long-term pumps, which have been used in other
applications to deliver soluble factors, are difficult to use in
SCI patients, as the human spinal cord moves significantly as a
result of respiratory variations and the pulse, thus catheters tend
to migrate. A need exists for a method that can provide sustained
delivery of important growth factors to the site of injury over a
prolonged period of time.
[0008] Sonic hedgehog (Shh) is a multifunctional factor that acts
as a morphogen early in spinal cord development, when different
cell types are established (Jessell 2000), and as a guidance factor
for the commissural axons at later developmental stages (Charron et
al. 2003). Specifically, Shh influences the glial choice by
inducing oligodendrocyte differentiation and inhibiting the
astrocyte lineage (Tekki-Kessaris et al. 2001; Sussman et al. 2002;
Agius et al. 2004); Shh treatment in vitro results in the
enhancement of neurite outgrowth from dorsal root ganglion neurons
(So et al. 2006). Direct injection of soluble Shh into the spinal
cord at the time of injury results in improved nerve-to-muscle
conductivity, although no functional recovery is observed.
Functional recovery can be measured in terms of improved motor or
sensory function, usually assessed with behavioral tests, such as
measuring the ability of mice to walk on a horizontal ladder. The
failure to improve function is likely due to rapid clearance of Shh
from the central nervous system (CNS) (Bambakidis and Miller
2004).
[0009] Spinal cord injuries are not only common, but they are at
present difficult to treat, because NSCs do not differentiate on
their own into oligodendrocytes and neurons. While some growth
factors, such as Shh, are known to drive this differentiation, it
has up to now not been known how to harness this beneficial effect
in vivo. Thus, there is a longstanding unfulfilled need for
effective spinal cord injury treatments, and also, more generally,
for treatments useful in the CNS that would provide or result in
the delivery of an effective amount of a desired exogenous factor
to the spinal cord or nervous system location to facilitate neural
cell recovery (e.g. to provide a niche for growth and repair in
this environment).
SUMMARY OF THE INVENTION
[0010] The present invention provides methods for treating spinal
cord injury in a patient in need of such treatment, by
administering to the site of spinal cord injury a sustained
delivery composition that includes one or more exogenous factors.
In other embodiments, the method is useful for treating a nervous
system injury or for increasing neural cell growth in a desired
target location.
[0011] In yet another embodiment, the invention relates to a method
for delivering one or more exogenous factors to a neural cell or
nervous system site by administering an effective amount of a
sustained delivery composition comprising one or more exogenous
factors and, optionally, an effective amount of stem cells to the
neural cell or nervous system site.
[0012] In yet another embodiment, the invention relates to a method
for treating a nervous system injury in a mammal by administering
to a mammal in need of such treatment an effective amount for
treating the nervous system injury of a pharmaceutical formulation
containing at least one exogenous factor, said pharmaceutical
formulation providing sustained delivery of the at least one
exogenous factor for at least about 7 days.
[0013] In yet another embodiment, the invention relates to a method
for increasing neural cell growth or regenerating neural cells in a
mammal by administering to a mammal in need of such treatment an
effective amount for increasing neural cell growth or regenerating
neuronal cells of a sustained release pharmaceutical formulation
containing at least one exogenous factor, said pharmaceutical
formulation continuously delivering the at least one exogenous
factor for at least about 7 days.
[0014] In certain embodiments, the mammal is a human.
[0015] In certain embodiments, the nervous system injury is
selected from the group consisting of spinal cord injury,
amyotrophic lateral sclerosis (ALS), peripheral nerve injury, and
spinal nerve injury.
[0016] In yet another embodiment, the sustained delivery
composition comprises a plurality of biodegradable
microspheres.
[0017] In yet another embodiment, the pharmaceutical formulation
further contains an effective amount of stem cells, wherein the
amount of stem cells in combination with said at least one
exogenous factor is effective for treating a nervous system
injury.
[0018] In yet another embodiment, the at least one exogenous factor
is a growth factor selected from the group consisting of Nerve
Growth Factor (NGF), Glial Cell Line-Derived Growth Factor (GDNF),
Neurotrophin (NT) 3, NT 4/5, NT 6, Ciliary Neurotrophic Factor
(CNTF), Leukemia Inhibitory Factor (LIF), Interleukin 6 (IL6),
Interleukin 11(IL11), Cardiotrophin 1, a growth factor hormone,
hyaluronidase, chondroitinase ABC(CABC), basic fibroblast growth
factor (bFGF), insulin-related growth factor (IGF-I), brain-derived
neurotrophic factor (BDNF), epidermal growth factor (EGF), and
sonic hedgehog (Shh).
[0019] In yet another embodiment, the stem cells are neural stem
cells.
[0020] In yet another embodiment, the pharmaceutical formulation is
administered by at least one injection, wherein a first injection
is administered at the site of nervous system injury.
[0021] In yet another embodiment, a second injection is
administered at a site rostral to the site of nervous system
injury.
[0022] In yet another embodiment, the stem cells are
endothelial-expanded stem cells.
[0023] In yet another embodiment, the endothelial-expanded stem
cells are pre-treated with sonic hedgehog.
[0024] In yet another embodiment, the endothelial-expanded stem
cells are pre-treated with sonic hedgehog and retinoic acid.
[0025] In yet another embodiment, the invention relates to a
pharmaceutical formulation containing an effective amount for
increasing neuronal cell growth or regenerating neuronal cells of
at least one sustained delivery composition containing at least one
exogenous factor selected from the group consisting of Nerve Growth
Factor (NGF), Glial Cell Line-Derived Growth Factor (GDNF),
Neurotrophin (NT).sub.3, NT 4/5, NT 6, Ciliary Neurotrophic Factor
(CNTF), Leukemia Inhibitory Factor (LIF), Interleukin 6 (IL6),
Interleukin 11(IL11), Cardiotrophin 1, a growth factor hormone,
hyaluronidase, chondroitinase ABC(CABC), basic fibroblast growth
factor (bFGF), insulin-related growth factor (IGF-I), brain-derived
neurotrophic factor (BDNF), epidermal growth factor (EGF), and
sonic hedgehog (Shh), wherein the composition provides sustained
delivery of at least one exogenous factor for at least 7 days, and
a pharmaceutically acceptable excipient.
[0026] In certain embodiments, the effective amount of stem cells,
wherein the amount in combination with the at least one exogenous
factor is effective for increasing neural cell growth or
regenerating neural cells.
[0027] In certain embodiments, the at least one sustained delivery
composition contains a plurality of biodegradable microspheres.
[0028] In yet another embodiment, the stem cell is a neural stem
cell. In yet another embodiment, the stem cell is an
endothelial-expanded stem cell. In yet another embodiment, the
endothelial-expanded stem cell is pre-treated with sonic hedgehog.
In yet another embodiment, the endothelial-expanded stem cell is
pre-treated with sonic hedgehog and retinoic acid.
[0029] In yet another embodiment, the sustained delivery
composition releases from about 1 ng/ml to about 20 ng/ml Shh for
at least 7 days. In certain embodiments, the sustained delivery of
one or more exogenous factors is preferably about at least 7 days,
and more preferably, about at least 2 weeks.
[0030] In certain embodiments, the exogenous factor or factors are
growth factors or morphogenic factors. In one embodiment, Shh is
the exogenous factor.
[0031] In a still further embodiment, the invention provides a
pharmaceutical composition containing a biodegradable microsphere
loaded with sonic hedgehog (Shh) or an active Shh fragment in which
the biodegradable microsphere provides sustained release of Shh for
at least about 7 days, and more preferably, at least about 2 weeks.
In certain embodiments, the pharmaceutical composition further
comprises neural stem cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGS. 1A-B show effects of Shh release. FIG. 1A demonstrates
the continuous release of Shh by microsphere compositions over time
in vitro. FIG. 1B demonstrates that microspheres co-cultured with
spinal cord NSCs are not toxic to the NSCs.
[0033] FIGS. 2A-L illustrate that direct treatment with Shh and
treatment with Shh-releasing microspheres induces increased
proliferation and neurogenesis of spinal cord NSCs. E9 spinal cord
cells were cultured for 6 days. Shh protein or supernatant from
control (PLGA only) or Shh-releasing microspheres was added to the
indicated cultures daily. Cultures were stained for nestin
(progenitor cells), .beta.-tubulin III (neurons) and DAPI
(nuclei).
[0034] FIGS. 3A-D illustrate motor recovery in mice with SCI
following treatment with Shh-containing microspheres with or
without Shh-treated spinal cord neural stem cells.
[0035] FIGS. 4A-B show that injection of Shh-containing
microspheres into mice at site of SCI results in reduced astroglial
scar formation.
[0036] FIGS. 5A-I demonstrate that transplanted NSCs graft
successfully in the spinal cord in all experimental groups.
[0037] FIGS. 6A-C show that transplantation of Shh-containing
microspheres into the site of SCI promotes corticospinal tract
fiber sprouting and growth in the caudal spinal cord.
[0038] FIGS. 7A-C show motor recovery after transplantation of
Shh/RA-BPAE-expanded NSCs into spinal cords of mice in a murine
model of SCI.
[0039] FIG. 8 shows motor recovery after transplantation of CABC
containing microspheres into the site of SCI.
[0040] FIG. 9 shows activity of CABC released from 10% loaded PLGA
microspheres.
DETAILED DESCRIPTION
[0041] Spinal cord injury (SCI) in mammals causes loss of spinal
cord cells, damage to ascending and descending axonal tracts and
loss of myelination, resulting in paralysis. After SCI, axonal
regeneration is prevented by the lack of matrix that supports
growth through production of important growth and morphogenic
factors (Harel and Strittmatter 2006; Lu and Tuszynski 2007).
Successful treatment of SCI will include approaches that aid in the
regeneration of damaged axons.
[0042] Regeneration of damaged axons encompasses several types of
neuronal response to injury; direct regrowth of severed axons
represents `true` axonal regeneration, whereas sprouting from
nearby uninjured fibers or proximal locations along severed axons
has a compensatory role. Adult nerve fibers often display haphazard
growth and are unable to efficiently reform functional circuits. To
maximize the effectiveness of repair of the damaged spinal cord, a
more faithful recapitulation of developmental pathfinding and
circuit refining mechanisms would be beneficial. At least two
approaches to recapitulating development in the injured CNS may be
employed: (1) re-establishing crucial developmental cues in the
correct pattern to guide regenerating axons, and (2) maximizing the
sprouting and plasticity of intact fibers through sensory feedback
rehabilitation techniques. See, Harrel and Strittmatter,
(2006).
[0043] For neuronal differentiation and migration, a set of
diffusible signaling molecules directs the differentiation of
ectodermal tissue into discrete regions along the early neural
tube. Molecules that inhibit bone morphogenetic protein 4 signaling
nudge ectodermal tissue down the neural pathway. Basic fibroblast
growth factors (bFGFs) and WNT proteins stimulate differentiation
into anterior neural structures, whereas retinoids stimulate
posterior neural fates. In the developing spinal cord, the floor
plate and nearby notochord secrete sonic hedgehog (Shh), which
signals the ventral cord to differentiate into motor neurons and
ventral inter neurons. Many of these morphogens (such as growth
factors) have been shown to also function as axon guidance
molecules. In addition, several morphogens persist after
development, when they might continue to regulate stem cell
division and differentiation. The role of adulthood morphogens in
the context of CNS injury is not well characterized. Id.
[0044] Whereas cell-autonomous mechanisms contribute to limiting
adult axon growth, extrinsic factors appear to have an even more
crucial role in blocking adult CNS regeneration. Classic
experiments have demonstrated the more inhibitory nature of the CNS
for axon outgrowth. Subsequent experiments have suggested that this
inhospitable milieu results primarily from the presence of CNS
myelin-specific inhibitory factors rather than a lack of positive
factors. Furthermore, the age at which most species lose the
ability to regenerate after SCI coincides with spinal cord
myelination. However, myelin is not the only extrinsic barrier to
adult CNS regeneration. CNS injury induces reactive astrocytes to
release many molecules that inhibit regeneration, including
chondroitin sulphate proteoglycans (CSPGs), carbohydrate-rich
extracellular molecules with inhibitory effects on neurite
outgrowth produced predominantly by astrocytes, and other glial
scar components. Furthermore, breakdown of the blood-brain barrier
results in the recruitment of inflammatory cells and cytokines that
have a more complicated effect on CNS regeneration. Interestingly,
as with many axon guidance molecules, several myelin-associated
inhibitors (MAIs) and CSPGs are expressed during development as
well as in the adult. For example, Nogo isoforms are expressed by
both central and peripheral neurons at developmental stages before
the onset of oligodendrocyte Nogo expression. Id.
[0045] Depending on the type of CNS injury, attempts at
regeneration might need to recapitulate all or only some of the
stages of development described above. For example, full
regeneration after stroke or neurodegenerative disease would
require the replacement of lost neurons, followed by the
regeneration and guidance of projections over the entire distance
covered by the absent tracts. By contrast, recovery from SCI could
occur through encouraging sprouting and guidance from spared
tracts, as well as maximizing plasticity of spared and regenerated
circuits. The present invention provides methods for achieving
recovery from CNS injury, for example, by stimulating neuron
sprouting, regrowth, development and/or circuit plasticity in the
adult CNS.
[0046] Stimulating neuronal cell growth/preventing neuronal cell
degeneration, can be beneficial for other conditions that affect
the central spinal cord and the spinal nerves. For example, in ALS
motor neurons die, and it has been shown that delivery of growth
factors such as VEGF can be beneficial to prevent loss of these
cells in a mouse model (Storkebaum, 2005). Motor neurons project
out to the periphery and there is evidence that damage to
peripheral nerves can involve Shh in repair processes. For example,
in sciatic nerve injury, a common condition, it has been shown
recently that shh is upregulated in schwann cells adjacent to
crush-injured sciatic nerve, and that this is followed by an
increase in brain-derived neurotrophic factor (BDNF) expression.
They found that administration of cyclopamine, a hedgehog
inhibitor, to the injured site prevented the increase in BDNF
expression and deteriorated motor neuron survival after sciatic
nerve injury. When peripheral Schwann cells were treated with
exogenous Shh, BDNF was increased suggesting that adding Shh could
help promote a beneficial repair environment (Hashimoto, 2008). In
a different study more substantial peripheral nerve injuries were
found to elicit extensive axon sprouting that was coincident with
an increase in shh mRNA, raising the possibility that an exogenous
supply of Shh can help promote peripheral nerve sprouting after
injury (Xu, Zochodne 2008). Based on the results described below,
it is expected that addition of Shh via microbeads into sciatic
nerve will augment the repair process.
[0047] Certain aspects of the present invention encompass the
therapeutic application of an Shh-containing microsphere to
increase or enhance survival and outgrowth of neurons and other
neuronal cells in the central nervous system. The ability of Shh to
regulate neuronal differentiation during development of the nervous
system and also presumably in the adult state indicates that Shh
can be reasonably expected to facilitate control of adult neurons
and other nervous system cells. This includes but is not limited to
the maintenance, functional performance, and aging of normal cells,
the repair and regeneration processes in chemically or mechanically
lesioned cells, and the prevention of degeneration and premature
death resulting from loss of differentiation in certain
pathological conditions.
[0048] In light of this understanding, the present invention
specifically contemplates applications of the subject method to the
treatment of (prevention and/or reduction of the severity of)
neurological conditions deriving from: (i) acute, (e.g., SCI),
subacute, or chronic injury to the nervous system, including
traumatic injury, chemical injury, basal injury and deficits (such
as the ischemia resulting from stroke). In acute injury, cells
including neurons, astrocytes and oligodendrocytes die and there is
an inflammatory reaction. With time, a fluid-filled cyst can appear
that forms a barrier to axon growth. With time, damaged axons can
die back and further degeneration of neurons and of the associated
glial cells, such as the myelinating cells occurs. In addition, a
scar consisting of astrocytes and chondroitin sulfact proteoglycans
can build up, both of which are inhibitory to new axon growth. In a
chronic situation then, there may be irreversible loss of spinal
cord cells, and the creation of barriers, so that repair will be
more involved and require more cells and regrowth than in the acute
injury situation. (Anderberg L, Aldskogius H, Holtz A. Spinal cord
injury--scientific challenges for the unknown future. Ups J Med.
Sci. 2007; 112(3):259-88.)
[0049] In an illustrative embodiment, the subject method is used to
treat amyotrophic lateral sclerosis (ALS), a disease of the nerve
cells in the brain and spinal cord that control voluntary muscle
movement. In ALS, neuronal cells waste away or die, and can no
longer send messages to muscles. This eventually leads to muscle
weakening, twitching, and an inability to move the arms, legs, and
body. The condition slowly gets worse. When the muscles in the
chest area stop working, it becomes hard or impossible to breathe
on one's own. ALS affects approximately 1 out of every 100,000
people. ALS patients may present with progressive spinal muscular
atrophy, progressive bulbar palsy, primary lateral sclerosis, or a
combination of these conditions. The major pathological abnormality
is characterized by a selective and progressive degeneration of the
lower motor neurons in the spinal cord and the upper motor neurons
in the cerebral cortex. The therapeutic application of
Shh-containing microspheres with or without NSCs in the spinal cord
may reverse motor neuron degeneration in ALS patients.
[0050] In yet additional embodiments, microspheres of different
types or sizes, or the same microspheres containing different
exogenous factors or different combinations of exogenous factors
are mixed, allowing for a combinatorial treatment regime, which is
another desirable treatment option for SCI treatment. For example,
microspheres carrying scar-digesting enzymes are mixed with
microspheres carrying growth factors. In an additional embodiment,
microspheres are used to create an artificial environment or niche
to instruct stem cell differentiation, such as would be useful
after implantation of spinal cord NSCs into the site of SCI.
Non-limiting examples of methods using combinational therapy are
described in detail in Lu et al. (2008) and Ashton et al.
(2007).
[0051] In certain aspects the present invention encompasses methods
for the stimulation of endogenous neural stem cells (NSC). In some
aspects the methods involve time-release mechanisms for the release
of morphogenic factors, such as, e.g., Shh, to neural sites.
Non-limiting examples of time-release mechanisms include
microspheres, which, herein, may also be referred to as `beads` or
"microbeads," alginate gels, and nanoparticles. [See, e.g., Ashton,
et al. (2007) Biomaterials, 28, 36, 5518; Drury, J. L. et al.
(2003) Biomaterials; 24:4337-4351; U.S. Pat. No. 7,226,617 to Ding
et al.; Simmons, C. A. et al. (2004) Bone; 35:562-569 As used
herein, "nanoparticles" are often defined as small particles that
are sized in the range of 1-100 nanometers (nm), but also include
sub-micron as well as larger particles encompassing the range of
1-1000 nm. See, for example: Ya-Ping Li, et al., PEGylated PLGA
nanoparticles as protein carriers: Synthesis, preparation, and
biodistribution in rats. J. of Controlled Release, 71, 2001, pages
203-211; A novel controlled release formulation for the anticancer
drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E
TPGS. J. of Controlled release, 86, 2003, 33-48; and Govender et
al. PLGA nanoparticles prepared by nanoprecipitation: drug loading
and release studies of a water soluble drug. J. of Controlled
Release 57, 1999, pages 171-185.
[0052] A time release schedule can be achieved by a series of
scheduled multiple injections. In one aspect of the present
invention, a niche that maintains NSC in an active state is
reconstructed or mimicked, for example, for a specific length of
time. Doing so facilitates the control of the number of cells
created from endogenous NSC and in some examples, the type of
progeny (i.e., cell type) that can be derived from or produced by
the activated NSC. Activated NSC is the NSC that is actively
proliferating and generating progeny.
[0053] Contemplated in the present invention are methods for the
treatment of diseases or injuries to nervous system sites including
spinal cord, peripheral nervous system, and spinal nerve injuries.
These include the following, non-limiting examples.
[0054] I) Spinal Cord Injury: [0055] Shh loaded microspheres
according to the present invention can be used to stimulate
endogenous NSC to repair spinal cord injury. As described below, a
mouse model for spinal cord injury is applicable to humans and also
to other nerve injuries, as set forth in Examples II-IV, below.
Other diseases of the spinal cord, such as ALS and spinal cord
tumors may also be treated according to methods of the present
invention. The present invention also provides methods for inducing
the maintenance, growth, or regeneration of neuronal cells, such
as, e.g., motor neurons.
[0056] II) Peripheral Nerve Injury: [0057] Peripheral nerve injury,
such as seen in sciatica and other peripheral neuropathies, can be
treated according to the methods of the present invention.
Microspheres according to the embodiments of the present invention
are injected into the area of damage to this nerve. Shh increases
after peripheral nerve injury and that this is important for the
repair process, based for example on evidence that inhibition of
hedgehog signaling is inhibitory to peripheral repair, while
addition of shh increases beneficial growth factors such as BDNF.
There are a number of different hedgehogs described to date--sonic,
desert and Indian for example, but they all converge on a similar
signaling system and are all inhibited by cyclopamine. Thus,
compositions and methods of the present invention, namely injection
of sustained release Shh microspheres or other suitable
compositions are expected to provide improvements for peripheral
nerve injury. In some embodiments, sustained release Shh
compositions may be held in place by putting them in alginate gels,
which together with the beads will eventually dissolve.
[0058] The present methods encompass in part a technique for
transplantation or administration of endothelial-expanded spinal
cord stem cells that were treated with Shh and retinoic acid to the
site of a spinal cord injury. The results show that the methods of
the present invention result in functional recovery from SCI as
exemplified by the studies in mouse models described in detail
below. It is determined that treatment according to methods of the
present invention results in motor recovery when at least about 80%
of motility is regained, following SCI. Further, it is determined
that treatment according to the methods of the present invention
results in sensory recovery when at least about 80% of sensory
ability is regained, such as determined by the tape removal assay.
Further, recovery is determined to be achieved if the increase in
motor or sensory function is statistically significant compared to
the negative control (e.g. mock-treated mice having SCI).
[0059] In certain embodiments, the present invention relates to a
method for sustained delivery of one or more exogenous factors,
such as Shh, in vivo using biodegradable microspheres engineered to
slowly release the desired factor(s) over about at least a
seven-day period. The data show that delivery of biodegradable
microspheres releasing Shh administered alone, as well as
administration of a combination of Shh-releasing microspheres and
endothelial-expanded Shh-treated spinal cord NSCs, provide
beneficial results in a murine, dorsal column model of SCI.
[0060] In additional embodiments, methods described herein for
treatment of neural cell damage, inducing neural cell regrowth, and
for inhibiting astrogliogenesis, are applied to mammals, and
preferably to humans. Mouse models of SCI closely mimic
characteristics of human SCI and provide a useful tool for
understanding human SCI, as well as related neural injuries.
[0061] After spinal cord injury, loss of spinal cord cells and
damage of sensory and motor tracts leads to paralysis. While it is
thought that the use of stem cells may present a viable treatment,
the natural microenvironment of the spinal cord does not facilitate
differentiation of NSCs into appropriate neural cell types. Certain
growth factors, such as Shh, must be present for a sustained period
of time, of about at least one week, in order for correct
differentiation to occur. The inventors have developed a method
that allows delivery of biologically active factors to the site of
the injury by incorporation of these factors into biodegradable
microspheres. This creates a minimally invasive method for
prolonged growth factor delivery.
[0062] Shh regulates various aspects of embryonic development both
in vertebrates and invertebrates (for reviews see Perrimon, N.
(1995) Cell 80, 517-520 and Johnson, R. L., and Tabin, C (1995)
Cell 81, 313-316). It is involved in anterior-posterior patterning,
formation of an apical ectodermal ridge, hindgut mesoderm, spinal
column, distal limb, rib development, and lung development, and
inducing ventral cell types in the spinal cord, hindbrain and
forebrain.
[0063] While the mechanism of action of Shh is not fully
understood, the most recent biochemical and genetic data suggest
that the receptor for Shh is the product of the tumor suppressor
gene, patched (Marigo, V., et al. (1996) Nature 384, 176-179;
Stone, D. M., et al. (1996) Nature 384, 129-134) and that other
proteins; smoothened (Stone, D. M., et al. (1996) Nature 384,
129-134; Alcedo, J., et al. (1996) Cell 86, 221-232), Cubitus
interruptus (Dominguez, M., et al. (1996) Science 272, 1621-1625;
Alexandre, C., et al. (1996) Genes & Dev. 10, 2003-2013), and
fused (Therond, P. P., et al. (1996) Proc. Natl. Acad. Sci. USA 93,
4224-4228) are involved in the Shh signaling pathway.
[0064] The full-length human Shh protein has been described and has
protein accession number NP.sub.--000184 (SEQ ID NO:1). The mouse
Shh protein has also been described and has protein accession
number CAA53922 (SEQ ID NO:3). Coding sequences for Shh include
accession numbers AC.sub.--000068 (human, SEQ ID NO:6) and X76290
(murine, SEQ ID NO:7). Additional mammalian Shh proteins, such as
SEQ ID NO:4, SEQ ID NO:5, and those described in U.S. Pat. Nos.
6,664,075; 6,271,363; 6,165,747. Any full-length Shh proteins, or
Shh active fragments are useful in the present invention.
Additionally, in the presently described experiments, human Shh
amino terminal active fragment was utilized, but similar
experiments using mouse Shh showed that the active fragments both
functioned in a similar manner. It is contemplated that human Shh
and active fragments of the protein will be used for pharmaceutical
formulations to be administered in humans.
[0065] Shh is produced as a 47-49 kDa-secreted (depending on
species) protein that post-translationally cleaves to give two
mature proteins: an approximately 19-kDa amino-terminal fragment
that remains cell associated and a 29-31-kDa carboxy terminal
fragment that is released from the cell. The membrane-associated
amino-terminal fragment contains the signaling portion of the
molecule. The mouse Shh (mShh) precursor carboxy terminus encodes
the autoprocessing domain which acts only in cis. The N-terminal
peptide is both necessary and sufficient for short- and long-range
Shh signaling activities, and therefore, fragments of Shh are also
biologically active and important (Porter, J. A. et al. (1995)
Nature 374:363: Lai et al. (1995) Development 121:2349-2360;
Roelink, H. et al. (1995) Cell 81:445-455; Porter, J. A. et al.
(1996) Science 274:255; Fietz, M. J. et al. (1995) Drosophila.
Curr. Biol. 6:643-650; Fan, C. M. et al. (1995) Cell 81:457-465;
Mart`, E., et al. (1995) Nature 375:322-325; Lopez-Martinez et al.
(1995) Curr. Biol 5:791-795; Ekker, S. C. et al. (1995) Development
121:2337-2347; Forbes, A. J. et al. (1996) Development 122:112;
Goetz et al. (2006) J. Biol. Chem. February 17; 281(7):4087-93).
Furthermore, Shh may be modified post-translationally while
maintaining its functional activity. For example, certain
hydrophobic modifications of Shh, such as the addition of a
long-chain fatty acid at the N-terminus and cholesterol at the
C-terminus, greatly activate Shh (Taylor et al. (2001).
Biochemistry. April 10; 40(14):4359-71). Any such modified Shh
proteins, while exhibiting functional activity are useful in the
present invention.
[0066] In certain embodiments, the Shh protein comprises SEQ ID
NO:1. The Shh protein may also comprise a fragment of SEQ ID NO:1,
wherein the fragment may be any N-terminal or other active fragment
of Shh. In other embodiments, the Shh N-terminal fragment comprises
SEQ ID NO: 2. In yet further embodiments, the Shh protein comprises
SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
[0067] In yet another embodiment, the Shh protein is encoded by any
one of the nucleic acid sequences selected from the group
consisting of SEQ ID NO: 6, SEQ ID NO:7, and SEQ ID NO:8.
[0068] In certain embodiments, the Shh protein can comprise a full
length protein, such as represented in the sequence listings, or it
can comprise a fragment of, for instance, at least 5, 10, 20, 50,
100, 150, 200, 250, or 300 amino acids in length. Preferred
hedgehog polypeptides include Shh sequences corresponding
approximately to the natural proteolytic fragments of the hedgehog
proteins, such as from about Cys-24 through about the region that
contains the proteolytic processing site, e.g., Ala-194 to Gly-203,
or from about Cys-198 through Ala-475 of the human Shh protein, or
analogous fragments thereto.
[0069] In certain embodiments, Shh protein may be a protein with an
N-terminal cysteine that is appended with at least one hydrophobic
moiety, a protein with an N-terminal amino-acid that is not a
cysteine appended with at least one hydrophobic moiety, or a
protein with at least one hydrophobic moiety substituted for the
N-terminal amino acid; wherein the protein binds to patched and has
at least 80% amino acid identity, or at least 90%-95% identity to a
hedgehog amino acid sequence comprising SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In certain embodiments,
the Shh protein may comprise any of the sequences identified by the
accession numbers NT.sub.--07956.2, AC.sub.--000050.1, or
NW.sub.--923796.1. In certain embodiments, the active agent in the
sustained release composition may include a molecule with
"Shh-like" activity (e.g., the Curis agonist: Wichterle H, Lieberam
I, Porter J A, Jessell T M. Directed differentiation of embryonic
stem cells into motor neurons. Cell. 2002 Aug. 9;
110(3):385-97.)
[0070] The interaction of Shh with one of its cognate receptors,
patched (ptc), sets in motion a cascade involving the activation
and inhibition of downstream effectors, the ultimate consequence of
which is, in some instances, a detectable change in the
transcription or translation of a gene. Shh and its cognate
receptor patched (ptc) are expressed in the epithelial and/or
mesenchymal cell components of the skin (i.e., the hair follicle).
See Parisi et al., (1998) Cell Res 8, 15-21; St. Jacques et al.,
(1998) Current Biology, 8, 1058-1068; and Dahmane et al., (1997)
Nature, 389, 876-880. The two-way interaction between epithelial
and the dermal mesenchymal cells directs the subsequent development
of hair follicles. Disrupting this interaction might lead to a
modulation of proliferation and/or differentiation events that give
rise to hair and/or epithelial tissue structures such as the
gut.
[0071] Another embodiment of the invention concerns the therapeutic
application of Shh or other morphogenic factor-containing
microspheres to specifically control the type of cell that
differentiates from NSC in vivo. For example, the amount of Shh
contained within the microsphere can be adjusted to specifically
control and induce differentiation of NSC into floor plate, motor
neurons, or oligodendrocytes. For example, in vitro, 7-16 nM Shh
induces floor plate differentiation, and 4 nM Shh induces the
differentiation of motor neurons. See, Roelink et al, 1995 Cell 81
445-455; Ericson et al, 1997; Cell. 1997 Jul. 11; 90(1):169-80.
[0072] Moreover, in the spinal cord, oligodendrocyte precursors
(OLPs) emerge from the ventral ventricular zone in a restricted
domain near the floor plate--the ventral motor neuron progenitor
(pMN) domain, composed of neural progenitors that express the olig
gene bHLH transcription factors, which generate motorneurons (MNs)
during early development of the neural tube (Lu et al., 2000, 2002;
Takebayashi et al., 2002; Zhou and Anderson, 2002). MNs and
oligodendrocytes are not produced simultaneously from these
progenitors; specification of these lineages occurs in two
successive waves, with MNs produced first OLP specification takes
place at a time long after dorsoventral neuronal patterning is
completed. OLP specification from ventral neural progenitors is
optimal at concentrations of Shh much higher than those reported to
induce MNs from neural progenitors (12-25 nM) (Danesin C, Agius E,
Escalas N, Ai X, Emerson C, Cochard P, Soula C Ventral neural
progenitors switch toward an oligodendroglial fate in response to
increased Sonic hedgehog (Shh) activity: involvement of Sulfatase 1
in modulating Shh signaling in the ventral spinal cord. J.
Neurosci. 2006 May 10; 26(19):5037-48.)
[0073] Thus, controlling cell type differentiation based on the
concentration of Shh is a useful approach for the treatment of
diseases of the nervous system in which specific cell types should
be induced to proliferate. For example, Shh creates the floor plate
and dopaminergic neurons in the midbrain arise from the floorplate.
(See, Ono et al., (2007) Development and Disease,
134:3213-3125).
[0074] Yet another aspect of the present invention concerns the
therapeutic application of an Shh-containing microsphere to enhance
survival and outgrowth of neurons and other neuronal cells in both
the central nervous system and the peripheral nervous system. In
the course of developing the methods of the present invention, a
mouse model of SCI was utilized. In this model, SCI was produced in
adult mice by performing an operation under anesthetic: the spinal
cord was cut halfway through from the back (dorsal) side. At the
same time, biodegradable microspheres were injected into the injury
site. The microspheres were engineered to release Shh over at least
a two week period, at a concentration known to be active in the
developing spinal cord. The animals were tested for behavioral
motor and sensory skills prior to sacrificing and assessing changes
in the cord. The animals receiving the Shh containing microspheres
were compared to those that received the microspheres alone
(control).
[0075] As used herein, the term "behavioral recovery" is understood
to include motor (locomotor) and sensory recovery, where motor
recovery may be measured, for example, by the horizontal ladder
test described herein, and sensory recovery may be measured, e.g.,
by the tape test discussed in Example 5, below. However, it is
understood by those of ordinary skill in the art that the term
"behavioral recovery" may also be used interchangeably with the
term "motor recovery" (i.e., when only motor, but not sensory,
recovery has been assessed).
[0076] It was discovered that mice receiving an injection of
biodegradable microspheres that release the growth factor Shh into
the site of SCI, exhibited motor recovery after injury. This
recovery could be explained by the decreased scarring at the site
of injury, and by the increased regrowth of a major motor
tract--the corticospinal tract. Shh has not previously been shown
to stimulate sprouting/increased growth of central nervous system
neuronal axon in vivo, hence this is an unexpected action of Shh
treatment in vivo.
[0077] In one set of experiments, transplantation of Shh-treated
stem cells to the injured mouse spinal cord helped improve
behavioral outcomes. In another set of experiments, it was found
that adding both microspheres releasing Shh and Shh-treated stem
cells is even more beneficial than either alone. The release by
microspheres of growth factors into the environment around the stem
cells can help create a specialized environment or `niche` to
regulate stem cell behavior. This can help drive the stem cells to
generate cell fates beneficial for spinal cord injury, such as
neurons and oligodendrocytes.
[0078] Thus, in a further embodiments, the present compositions and
methods will be useful for stimulating endogenous stem cells.
Stimulating these endogenous stem cells in the adult spinal cord
will provide a therapeutic `niche` such that damaged neural cells
are repaired or replaced, along the lines of Shh function in the
embryo.
[0079] In a further embodiment, the present compositions and
methods will provide benefits for treating individuals with injured
or damage to a spinal disc. Such spinal disc damage or injury
includes when the disk slips and the nerve roots become contused
and/or pinched and damaged. Injection of sustained release Shh
compositions or other factors into the nerve root are expected to
be beneficial.
[0080] In summary, the inventors have developed a method for
treating SCI in which microspheres releasing growth factors over a
prolonged period are beneficial to the injured site. Specifically,
Shh reduces scarring and enhances axon sprouting and outgrowth from
the corticospinal tract. Moreover, this method allows a further
beneficial effect of adding microspheres plus stem cells, which
enhances recovery from SCI and provides a specialized environment
around the injured site that is conducive to neuronal cell growth
and/or recovery. Thus, in certain embodiments, the compositions
and/or methods of the present invention provide a niche for growth
and in certain instances expansion of endogenous progenitor/stem
cells.
[0081] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, protein
expression and purification, antibody, and recombinant DNA
techniques within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Sambrook et al.
(2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al.
eds. (2005) Current Protocols in Molecular Biology. John Wiley and
Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current
Protocols in Cell Biology. John Wiley and Sons Inc.: Hoboken, N.J.;
Coligan et al. eds. (2005) Current Protocols in Immunology, John
Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005)
Current Protocols in Microbiology, John Wiley and Sons, Inc.:
Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in
Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna
et al. eds. (2005) Current Protocols in Pharmacology, John Wiley
and Sons, Inc.: Hoboken, N.J.; Nucleic Acid Hybridization, Hames
& Higgins eds. (1985); Transcription And Translation, Hames
& Higgins, eds. (1984); Animal Cell Culture Freshney, ed.
(1986); Immobilized Cells And Enzymes, IRL Press (1986); Perbal, A
Practical Guide To Molecular Cloning (1984); and Harlow and Lane.
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press: 1988).
[0082] The electronic version of the sequence listing containing
SEQ ID NOs1-8 is hereby incorporated by reference in its
entirety.
DEFINITIONS
[0083] The following definitions are provided for clarity and
illustrative purposes only, and are not intended to limit the scope
of the invention.
[0084] As used herein, the term "spinal cord injury (SCI)" in
understood to include injury to the spinal cord which causes loss
of spinal cord cells, damage to ascending and descending axonal
tracts and/or loss of myelination. SCI can result in decreased limb
function and/or paralysis. In certain cases, SCI involves acute,
subacute or chronic injury to the spinal cord. An example of acute
injury includes injury or trauma that occurs less than 12 hours
after the injury. An example of subacute injury includes injury or
trauma that occurs about 12 hours to about 30 days after injury. An
example of chronic injury to the spinal cord includes injury or
trauma that occurs over 12 months after spinal cord injury (from
the U.S. clinical trials website).
[0085] As used herein, the term "stem cell" refers to a cell that
retains the ability to renew itself through mitotic cell division
and can differentiate into a diverse range of specialized cell
types.
[0086] As used herein, the term "neural stem cell (NSC)" describes
cells that are the self-renewing, multipotent cells that generate
phenotypes of the nervous system.
[0087] The term "growth factor" can be a naturally occurring,
endogenous or exogenous protein, or recombinant protein, capable of
stimulating cellular proliferation and/or cellular
differentiation.
[0088] As used herein, the term "morphogenic factor" refers to a
substance governing the pattern of tissue development and, in
particular, the positions of the various specialized cell types
within a tissue.
[0089] As used herein, "neural" means the nervous system and
includes glial cells and neurons.
[0090] As used herein, "central nervous system" includes brain
and/or the spinal cord of a mammal. The term may also include the
eye and optic nerve in some instances.
[0091] The term "neuron" as used herein describes a nerve cell
capable of receiving and conducting electrical impulses from the
central nervous system. A nerve cell or "neuron" may typically
include a cell body, an axon, axon terminals, and dendrites.
[0092] The term "exogenous factor" describes those compounds
capable of inducing differentiation of a stem cell into a neuronal
cell. These compounds include, but are not limited to antioxidants,
trophic factors, morphogenic factors, and growth factors.
[0093] As used herein, the term "sustained delivery" includes
delivery of an exogenous factor in vivo over a period of time
following administration, preferably at least a week or several
weeks. Sustained delivery of the exogenous factor can be
demonstrated by, for example, the continued outgrowth of CNS
neurons over time. Alternatively, sustained delivery of the
exogenous factor, such as Shh, can be demonstrated by detecting the
presence of the exogenous factor in vitro over time.
[0094] Expression Construct
[0095] By "expression construct" is meant a nucleic acid sequence
comprising a target nucleic acid sequence or sequences whose
expression is desired, operatively associated with expression
control sequence elements which provide for the proper
transcription and translation of the target nucleic acid
sequence(s) within the chosen host cells. Such sequence elements
may include a promoter and a polyadenylation signal. The
"expression construct" may further comprise "vector sequences". By
"vector sequences" is meant any of several nucleic acid sequences
established in the art which have utility in the recombinant DNA
technologies of the invention to facilitate the cloning and
propagation of the expression constructs including (but not limited
to) plasmids, cosmids, phage vectors, viral vectors, and yeast
artificial chromosomes.
[0096] Expression constructs of the present invention may comprise
vector sequences that facilitate the cloning and propagation of the
expression constructs. A large number of vectors, including plasmid
and fungal vectors, have been described for replication and/or
expression in a variety of eukaryotic and prokaryotic host cells.
Standard vectors useful in the current invention are well known in
the art and include (but are not limited to) plasmids, cosmids,
phage vectors, viral vectors, and yeast artificial chromosomes. The
vector sequences may contain a replication origin for propagation
in E. coli; the SV40 origin of replication; an ampicillin,
neomycin, or puromycin resistance gene for selection in host cells;
and/or genes (e.g., dihydrofolate reductase gene) that amplify the
dominant selectable marker plus the gene of interest.
[0097] Express and Expression
[0098] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g., the resulting protein, may also be
said to be "expressed" by the cell. An expression product can be
characterized as intracellular, extracellular or secreted. The term
"intracellular" means something that is inside a cell. The term
"extracellular" means something that is outside a cell. A substance
is "secreted" by a cell if it appears in significant measure
outside the cell, from somewhere on or inside the cell.
[0099] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" means the
introduction of a "foreign" (i.e. extrinsic or extracellular) gene,
DNA or RNA sequence to a cell, so that the host cell will express
the introduced gene or sequence to produce a desired substance,
typically a protein or enzyme coded by the introduced gene or
sequence. The introduced gene or sequence may also be called a
"cloned" or "foreign" gene or sequence, may include regulatory or
control sequences, such as start, stop, promoter, signal,
secretion, or other sequences used by a cells genetic machinery.
The gene or sequence may include nonfunctional sequences or
sequences with no known function. A host cell that receives and
expresses introduced DNA or RNA has been "transformed" and is a
"transformant" or a "clone". The DNA or RNA introduced to a host
cell can come from any source, including cells of the same genus or
species as the host cell, or cells of a different genus or
species.
[0100] Expression System
[0101] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g. for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell.
[0102] Gene or Structural Gene
[0103] The term "gene", also called a "structural gene" means a DNA
sequence that codes for or corresponds to a particular sequence of
amino acids which comprise all or part of one or more proteins or
enzymes, and may or may not include regulatory DNA sequences, such
as promoter sequences, which determine for example the conditions
under which the gene is expressed. Some genes, which are not
structural genes, may be transcribed from DNA to RNA, but are not
translated into an amino acid sequence. Other genes may function as
regulators of structural genes or as regulators of DNA
transcription.
[0104] A coding sequence is "under the control of" or "operatively
associated with" expression control sequences in a cell when RNA
polymerase transcribes the coding sequence into RNA, particularly
mRNA, which is then trans-RNA spliced (if it contains introns) and
translated into the protein encoded by the coding sequence.
[0105] The term "expression control sequence" refers to a promoter
and any enhancer or suppression elements that combine to regulate
the transcription of a coding sequence. In a preferred embodiment,
the element is an origin of replication.
[0106] Heterologous
[0107] The term "heterologous" refers to a combination of elements
not naturally occurring. For example, heterologous DNA refers to
DNA not naturally located in the cell, or in a chromosomal site of
the cell. Preferably, the heterologous DNA includes a gene foreign
to the cell. For example, the present invention includes chimeric
DNA molecules that comprise a DNA sequence and a heterologous DNA
sequence which is not part of the DNA sequence. A heterologous
expression regulatory element is such an element that is
operatively associated with a different gene than the one it is
operatively associated with in nature. In the context of the
present invention, a gene encoding a protein of interest is
heterologous to the vector DNA in which it is inserted for cloning
or expression, and it is heterologous to a host cell containing
such a vector, in which it is expressed.
[0108] Homologous
[0109] The term "homologous" as used in the art commonly refers to
the relationship between nucleic acid molecules or proteins that
possess a "common evolutionary origin," including nucleic acid
molecules or proteins within superfamilies (e.g., the
immunoglobulin superfamily) and nucleic acid molecules or proteins
from different species (Reeck et al., (1987) Cell; 50: 667). Such
nucleic acid molecules or proteins have sequence homology, as
reflected by their sequence similarity, whether in terms of
substantial percent similarity or the presence of specific residues
or motifs at conserved positions.
[0110] Host Cell
[0111] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown or used or manipulated in
any way for the production of a substance by the cell. For example,
a host cell may be one that is manipulated to express a particular
gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can
further be used for screening or other assays that are described
infra. Host cells may be cultured in vitro or one or more cells in
a non-human animal (e.g., a transgenic animal or a transiently
transfected animal). Suitable host cells include but are not
limited to Streptomyces species and E. coli.
[0112] Treating or Treatment
[0113] "Treating" or "treatment" of a state, disorder or condition
includes:
[0114] (1) preventing or delaying the appearance of clinical or
sub-clinical symptoms of the state, disorder or condition
developing in a mammal that may be afflicted with or predisposed to
the state, disorder or condition but does not yet experience or
display clinical or subclinical symptoms of the state, disorder or
condition; or
[0115] (2) inhibiting the state, disorder or condition, i.e.,
arresting, reducing or delaying the development of the disease or a
relapse thereof (in case of maintenance treatment) or at least one
clinical or sub-clinical symptom thereof; or
[0116] (3) relieving the disease, i.e., causing regression of the
state, disorder or condition or at least one of its clinical or
sub-clinical symptoms.
[0117] The benefit to a subject to be treated is either
statistically significant or at least perceptible to the patient or
to the physician.
[0118] Patient or Subject
[0119] "Patient" or "subject" refers to mammals and includes human
and veterinary subjects.
[0120] Therapeutically Effective Amount
[0121] A "therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating a state,
disorder or condition, is sufficient to effect such treatment. The
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight,
physical condition and responsiveness of the mammal to be
treated.
[0122] About or Approximately
[0123] The term "about" or "approximately" means within an
acceptable range for the particular value as determined by one of
ordinary skill in the art, which will depend in part on how the
value is measured or determined, e.g., the limitations of the
measurement system. For example, "about" can mean a range of up to
20%, preferably up to 10%, more preferably up to 5%, and more
preferably still up to 1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
5-fold, and more preferably within 2-fold, of a value. Unless
otherwise stated, the term `about` means within an acceptable error
range for the particular value.
[0124] Dosage
[0125] The dosage of the therapeutic formulation will vary widely,
depending upon the nature of the disease, the patient's medical
history, the frequency of administration, the manner of
administration, the clearance of the agent from the host, and the
like. The initial dose may be larger, followed by smaller
maintenance doses. The dose may be administered as infrequently as
weekly or biweekly, or fractionated into smaller doses and
administered daily, semi-weekly, etc., to maintain an effective
dosage level.
[0126] Carrier
[0127] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous solution saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Alternatively, the
carrier can be a solid dosage form carrier, including hut not
limited to one or more of a hinder (for compressed pills), a
glidant, an encapsulating agent, a flavorant, and a colorant.
Suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0128] Isolated
[0129] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. Isolated nucleic acid molecules
include, for example, a PCR product, an isolated mRNA, a cDNA, or a
restriction fragment. Isolated nucleic acid molecules also include,
for example, sequences inserted into plasmids, cosmids, artificial
chromosomes, and the like. An isolated nucleic acid molecule is
preferably excised from the genome in which it may be found, and
more preferably is no longer joined to non-regulatory sequences,
non-coding sequences, or to other genes located upstream or
downstream of the nucleic acid molecule when found within the
genome. An isolated protein may be associated with other proteins
or nucleic acids, or both, with which it associates in the cell, or
with cellular membranes if it is a membrane-associated protein.
[0130] Mutant
[0131] As used herein, the terms "mutant" and "mutation" refer- to
any detectable change in genetic material (e.g., DNA) or any
process, mechanism, or result of such a change. This includes gene
mutations, in which the structure (e.g., DNA sequence) of a gene is
altered, any gene or DNA arising from any mutation process, and any
expression product (e.g., protein or enzyme) expressed by a
modified gene or DNA sequence. As used herein, the term "mutating"
refers to a process of creating a mutant or mutation.
[0132] Nucleic Acid Hybridization
[0133] The term "nucleic acid hybridization" refers to
anti-parallel hydrogen bonding between two single-stranded nucleic
acids, in which A pairs with T (or U if an RNA nucleic acid) and C
pairs with G. Nucleic acid molecules are "hybridizable" to each
other when at least one strand of one nucleic acid molecule can
form hydrogen bonds with the complementary bases of another nucleic
acid molecule under defined stringency conditions. Stringency of
hybridization is determined, e.g., by (i) the temperature at which
hybridization and/or washing is performed, and (ii) the ionic
strength and (iii) concentration of denaturants such as formamide
of the hybridization and washing solutions, as well as other
parameters. Hybridization requires that the two strands contain
substantially complementary sequences. Depending on the stringency
of hybridization, however, some degree of mismatches may be
tolerated. Under "low stringency" conditions, a greater percentage
of mismatches are tolerable (i.e., will not prevent formation of an
anti-parallel hybrid). See Molecular Biology of the Cell, Alberts
et al., 3rd ed., New York and London: Garland Publ., 1994. Ch.
7.
[0134] Typically, hybridization of two strands at high stringency
requires that the sequences exhibit a high degree of
complementarity over an extended portion of their length. Examples
of high stringency conditions include: hybridization to
filter-bound DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1 mM EDTA at
65.degree. C., followed by washing in 0.1.times.SSC/0.1% SDS at
68.degree. C. (where 1.times.SSC is 0.15 M NaCl, 0.15 M Na citrate)
or for oligonucleotide molecules washing in 6.times.SSC/0.5% sodium
pyrophosphate at about 37.degree. C. (for 14 nucleotide-long
oligos), at about 48.degree. C. (for about 17 nucleotide-long
oligos), at about 55.degree. C. (for 20 nucleotide-long oligos),
and at about 60.degree. C. (for 23 nucleotide-long oligos)).
Accordingly, the term "high stringency hybridization" refers to a
combination of solvent and temperature where two strands will pair
to form a "hybrid" helix only if their nucleotide sequences are
almost perfectly complementary (see Molecular Biology of the Cell,
Alberts et al., 3rd ed., New York and London: Garland Publ., 1994,
Ch. 7).
[0135] Conditions of intermediate or moderate stringency (such as,
for example, an aqueous solution of 2.times.SSC at 65.degree. C.;
alternatively, for example, hybridization to filter-bound DNA in
0.5 M NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing
in 0.2.times.SSC/0.1% SDS at 42.degree. C.) and low stringency
(such as, for example, an aqueous solution of 2.times.SSC at
55.degree. C.), require correspondingly less overall
complementarity for hybridization to occur between two sequences.
Specific temperature and salt conditions for any given stringency
hybridization reaction depend on the concentration of the target
DNA and length and base composition of the probe, and are normally
determined empirically in preliminary experiments, which are
routine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 2, ch.
9.50, CSH Laboratory Press, 1989; Ausubel et al. (eds.), 1989,
Current Protocols in Molecular Biology, Vol. I, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3).
[0136] As used herein, the term "standard hybridization conditions"
refers to hybridization conditions that allow hybridization of
sequences having at least 75% sequence identity. According to a
specific embodiment, hybridization conditions of higher stringency
may be used to allow hybridization of only sequences having at
least 80% sequence identity, at least 90% sequence identity, at
least 95% sequence identity, or at least 99% sequence identity.
[0137] Nucleic acid molecules that "hybridize" to any desired
nucleic acids of the present invention may be of any length. In one
embodiment, such nucleic acid molecules are at least 10, at least
15, at least 20, at least 30, at least 40, at least 50, and at
least 70 nucleotides in length. In another embodiment, nucleic acid
molecules that hybridize are of about the same length as the
particular desired nucleic acid.
[0138] Nucleic Acid Molecule
[0139] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear (e.g., restriction fragments) or circular DNA
molecules, plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0140] Orthologs
[0141] As used herein, the term "orthologs" refers to genes in
different species that apparently evolved from a common ancestral
gene by speciation. Normally, orthologs retain the same function
through the course of evolution. Identification of orthologs can
provide reliable prediction of gene function in newly sequenced
genomes. Sequence comparison algorithms that can be used to
identify orthologs include without limitation BLAST, FASTA, DNA
Strider, and the GCG pileup program. Orthologs often have high
sequence similarity. The present invention encompasses all
orthologs of the desired protein.
[0142] Operatively Associated
[0143] By "operatively associated with" is meant that a target
nucleic acid sequence and one or more expression control sequences
(e.g., promoters) are physically linked so as to permit expression
of the polypeptide encoded by the target nucleic acid sequence
within a host cell.
[0144] Percent Sequence Similarity or Percent Sequence Identity
[0145] The terms "percent (%) sequence similarity", "percent (%)
sequence identity", and the like, generally refer to the degree of
identity or correspondence between different nucleotide sequences
of nucleic acid molecules or amino acid sequences of proteins that
may or may not share a common evolutionary origin (see Reeck et
al., supra). Sequence identity can be determined using any of a
number of publicly available sequence comparison algorithms, such
as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program
Manual for the GCG Package, Version 7, Madison, Wis.), etc.
[0146] To determine the percent identity between two amino acid
sequences or two nucleic acid molecules, the sequences are aligned
for optimal comparison purposes. The percent identity between the
two sequences is a function of the number of identical positions
shared by the sequences (i.e., percent identity=number of identical
positions/total number of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are, or
are about, of the same length. The percent identity between two
sequences can be determined using techniques similar to those
described below, with or without allowing gaps. In calculating
percent sequence identity, typically exact matches are counted.
[0147] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A non-limiting
example of a mathematical algorithm utilized for the comparison of
two sequences is the algorithm of Karlin and Altschul, Proc. Natl.
Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul,
Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et
al., J. Mol. Biol. 1990; 215: 403. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to sequences of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3, to obtain amino acid sequences
homologous to protein sequences of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., Nucleic Acids Res. 1997, 25:3389.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationship between molecules. See Altschul
et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and
PSI-Blast programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See
ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS 1988; 4:
11-17. Such an algorithm is incorporated into the ALIGN program
(version 2.0), which is part of the GCG sequence alignment software
package. When utilizing the ALIGN program for comparing amino acid
sequences, a PAM120 weight residue table, a gap length penalty of
12, and a gap penalty of 4 can be used.
[0148] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the algorithm of Needleman
and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been
incorporated into the GAP program in the GCG software package
(Accelrys, Burlington, Mass.; available at accelrys.com on the
WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix,
a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of
1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the
percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package using a
NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a
length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set
of parameters (and the one that can be used if the practitioner is
uncertain about what parameters should be applied to determine if a
molecule is a sequence identity or homology limitation of the
invention) is using a Blossum 62 scoring matrix with a gap open
penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5.
[0149] In addition to the cDNA sequences encoding various desired
proteins, the present invention further provides polynucleotide
molecules comprising nucleotide sequences having certain percentage
sequence identities to any of the aforementioned sequences. Such
sequences preferably hybridize under conditions of moderate or high
stringency as described above, and may include species
orthologs.
[0150] Variant
[0151] The term "variant" may also be used to indicate a modified
or altered gene. DNA sequence, enzyme, cell, etc., i.e., any kind
of mutant.
[0152] Pharmaceutically Acceptable
[0153] When formulated in a pharmaceutical composition, a
therapeutic compound of the present invention can be admixed with a
pharmaceutically acceptable carrier or excipient. As used herein,
the phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are generally believed to be
physiologically tolerable and do not typically produce an allergic
or similar untoward reaction, such as gastric upset, dizziness and
the like, when administered to a human.
[0154] Pharmaceutically Acceptable Derivative
[0155] The term "pharmaceutically acceptable derivative" as used
herein means any pharmaceutically acceptable salt, solvate or
prodrug, e.g. ester, of a compound of the invention, which upon
administration to the recipient is capable of providing (directly
or indirectly) a compound of the invention, or an active metabolite
or residue thereof. Such derivatives are recognizable to those
skilled in the art, without undue experimentation. Nevertheless,
reference is made to the teaching of Burger's Medicinal Chemistry
and Drug Discovery, 5th Edition, Vol 1: Principles and Practice,
which is incorporated herein by reference to the extent of teaching
such derivatives. Preferred pharmaceutically acceptable derivatives
are salts, solvates, esters, carbamates, and phosphate esters.
Particularly preferred pharmaceutically acceptable derivatives are
salts, solvates, and esters. Most preferred pharmaceutically
acceptable derivatives are salts and esters.
[0156] Pharmaceutical Compositions and Administration
[0157] While it is possible to use a composition provided by the
present invention for therapy as is, it may be preferable to
administer it in a pharmaceutical formulation, e.g., in admixture
with a suitable pharmaceutical excipient, diluent, or carrier
selected with regard to the intended route of administration and
standard pharmaceutical practice. Accordingly, in one aspect, the
present invention provides a pharmaceutical composition or
formulation comprising at least one active composition, or a
pharmaceutically acceptable derivative thereof, in association with
a pharmaceutically acceptable excipient, diluent, and/or carrier.
The excipient, diluent and/or carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0158] The compositions of the invention can be formulated for
administration in any convenient way for use in human or veterinary
medicine. In one embodiment, the one or more exogenous factors,
(i.e., active ingredient) can be delivered in a vesicle, including
as a liposome (see Langer, Science, 1990; 249:1527-1533; Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365
(1989); Lopez-Berestein, ibid., pp. 317-327; see generally
ibid.).
[0159] In yet another embodiment, the exogenous factor(s) can be
delivered in a controlled release form. For example, one or more
exogenous factors, (e.g., Shh) may be administered in a polymer
matrix such as poly(lactide-co-glycolide) (PLGA), in a microsphere
or liposome implanted subcutaneously, or by another mode of
delivery (see Cao et al., 1999, Biomaterials, February;
20(4):329-39). The microspheres of the present invention may also
be composed of PLGA and anhydrous poly-vinyl alcohol (PVA). Edlund
et al. "Degradable Polymer Microspheres for Controlled Drug
Delivery", Advances in Polymer Science Vol. 157, 2002, 67) lists on
page 77 a number of different degradable polymers investigated for
controlled drug delivery applications (e.g. polyglycolide,
polylactide, etc.). Thus, suitable controlled or continuous release
formulations useful in the present invention could be made using
these other degradable polymers. PVA is one of a range of possible
substances that can be used to stabilize microspheres produced by
emulsion solvent evaporation techniques. PVA is used as a
stabilizing/emulsifying agent. Varying the concentration of PVA can
enable the size of the microspheres to be varied, which in turn can
influence the release profile (e.g. See, Zhao et al. BioMagnetic
Research and Technology 2007, 5:2 "Process and formulation
variables in the preparation of injectable and biodegradable
magnetic microspheres. PLGA microspheres of the present invention
may range in size from 10-40 .mu.m, with an average diameter of 20
.mu.m. The size can be controlled by varying the speed of the
homogenizer, etc. In certain embodiments, larger particles may be
used; varying the size of the microspheres can be guided by
clinical considerations. The degradation of microspheres is based
on the hydrolysis of the ester linkages in the PLGA polymer. In
general, biodegradable polymers have been classified into
surface-eroding and bulk-eroding (See Biomaterials, 2002,
23:4221-4231). PLGA is reported to be a bulk-eroding polymer (Id.).
As described in the Examples, exemplary pharmaceutical formulations
of the present invention have achieved release of biologically
active Shh from microspheres over a 7 day period, with an
indication that Shh continues to be released for a time beyond the
7 day test period.
[0160] Another aspect of delivery includes the suspension of
microspheres in an alginate hydrogel, which is considered
biocompatible and is compatible with stem cells. The bioactive
factor would be released from the microsphere present in the
hydrogel, therefore, its rate of release can be adjusted in the
same way as when there is no hydrogel (e.g., by changing the
composition/molecular weight of polymers used to make the
microsphere, changing protein loading in the microsphere,
microsphere size, etc.). It was shown in Ashton et al., that the
incorporation of microspheres containing alginate lyase into the
hydrogel enable controlled release of this enzyme which in turn
provides control over the rate of degradation of the hydrogel
(Ashton et al. 2007; Piantino et al., 2006, Exp Neurol.,
201(2):359-67; See also, U.S. Pat. No. 7,226,617 to Ding et al. Yet
another aspect of the invention includes the use of nanoparticles
for the delivery of exogenous factors to nervous system sites.
Another example of controlled release compositions include an
amorphous carbohydrate glass matrix, as described in detail in PCT
publication number WO 93/10758, in which a bioactive agent such as
Shh is incorporated into the carbohydrate glass matrix and
controlled release or degradation is adjusted by addition of a
hydrophobic substance.
[0161] In certain embodiments, the microspheres of the present
invention are injected at the site of spinal cord injury. For
injections into the spinal cord, a 1 .mu.l microsphere suspension
containing 0.13 mg microspheres per .mu.l media is used. In certain
embodiments, the microspheres are loaded with 0.65 .mu.g of Shh
(recombinant human Shh from R&D Systems, Minneapolis, Minn.,
corresponding to SEQ ID NO:2, the active N-terminal Shh fragment
(Cys24-Gly 197 of SEQ ID NO: 1) per .mu.l of microsphere
suspension. In a preferred embodiment, the microspheres of the
present invention release bioactive levels of at least one
exogenous factor, e.g., from about 1 ng/ml to about 20 ng/ml Shh,
more preferably from about 1 ng/ml to about 10 ng/ml Shh, and most
preferably from about 2 ng/ml to about 7 ng/ml Shh. In certain
embodiments, microsphere compositions of the present invention
release a preferred concentration of about 5 ng/ml Shh over a
course of at least about 7 days. In the present invention, any
controlled release formulation may be used in lieu of microspheres
that have a similar controlled release profile (e.g., releasing at
least one exogenous factor in the preferred range over a course of
at least about 7 days).
[0162] In yet another embodiment, one or more additional exogenous
factors either alone, or in combination with Shh can include, but
is not limited to, any one or more of the following components
loaded within a nanosphere/microsphere or other controlled release
formulation: enzymes, proteins, and antibodies. For example,
neurotrophic molecules, such as Nerve Growth Factor (NGF),
Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin (NT) 3, 4/5,
and 6, Ciliary Neurotrophic Factor (CNTF), Glial Cell Line-Derived
Growth Factor (GDNF), Leukemia Inhibitory Factor (LIF), Interleukin
6 (IL6), interleukin 11(IL11), and Cardiotrophin 1, and growth
factor hormones, epidermal growth factor (EGF) such as
interferon-.alpha. (IFNa), Interferon (IFN) and Tumor Necrosis
Factor (TNF), e.g. TNF-.alpha.(, can be incorporated into the
nanospheres/microspheres or other controlled release formulation of
the present invention. Further, proteoglycans, such as decorin, or
antibodies that block the inhibitory activity of certain
proteoglycans (such as NG2 proteoglycan) can further be
incorporated into the nanospheres/microspheres or other controlled
release formulations of the present invention. Carrier proteins,
such as Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin
(KLH), Ovalbumin (OVA), Fetal Bovine Serum (FBS), Thyroglobulin
(THY), and Human Serum Albumin (HSA), can optionally be loaded into
the controlled release formulation of the present invention. Any
combination of these nanosphere/microsphere or other controlled
release formulation loaded factors, including Shh with any of these
additional factors, can also be combined with stem cells in the
present invention.
[0163] In certain embodiments of the present invention, a first
exogenous-factor-containing microsphere or nanoparticle suspension,
or other controlled release formulation, containing at least one
exogenous factor, may be used in combination with a second
controlled release formulation (e.g., a second microsphere
suspension), containing at least one exogenous factor that is
different than that contained in the first controlled release
formulation. Such an approach may be termed "combinational therapy"
and is useful for the treatment of diseases and conditions as
described herein. For example, combinational therapy may be used
for the treatment of spinal cord injury (SCI). SCI involves glial
scar formation, loss of cells and slow dying of remaining cells.
Using a combination approach, a first controlled release
formulation (e.g., a first set of a microspheres) may be loaded
with scar-chewing enzymes, such as, e.g., chondroitinase ABC(CABC)
or hyaluronidase (Struve et al, Glia, 2005) and a second controlled
release formulation may be loaded with, e.g., Shh to promote
growth, or with GDNF to reduce cell death. In other embodiments,
these and/or other factors may be combined in a single controlled
release formulation or each in separate formulations (e.g., a
separate suspension of microspheres or nanoparticles for each
factor to be administered to a nervous system site).
[0164] These sustained or controlled release formulations or
mixtures of combinations of controlled release formulations of the
present invention are delivered by injection at the site of injury
or site where increased neural cell growth is desired. Optionally,
a second injection is delivered rostral to the first injection site
(1 mm for the mouse models, for humans the distance may be adjusted
according). In certain embodiments, the formulation(s) may also be
administered systemically, e.g., intravenously. In yet other
embodiments, the controlled release formulations each containing at
least one exogenous factor as described above are also loaded with
NSC or endothelial-expanded NSC and, optionally, retinoic acid.
[0165] In certain embodiments of the present invention, retinoic
acid (RA) is included in a controlled release composition, such as
microspheres. RA is known to induce differentiation of P19 cells
into neurons, astrocytes and oligodendrocytes, cell types which are
normally derived from the neuroectoderm. P19 cells are an embryonal
carcinoma cell line having many characteristics of embryonic stem
cells, including an ability to differentiate into different cell
types (e.g., skeletal muscle, cardiac muscle and neurons). Newman,
K. D, and M. W. McBurney (2004) Biomaterials 25:5763-5771,
describes including RA and P19 cells in PLGA microspheres.
[0166] Other examples of exogenous factors include basic fibroblast
growth factor (bFGF), hyaluronidase, and insulin-related growth
factor (IGF-I). Other growth factors and combinations of growth
factors and/or scar-digesting enzymes that are useful for promoting
growth and differentiation of NSC into nervous system cells and/or
for decreasing scar formation, are contemplated by the present
invention. For examples of combinational therapy, see e.g., Schwab
J M, et al. (2006) Prog. Neurobiol.; 78(2):91-116; Pearse D D and
Bunge M B. (2006) J. Neurotrauma; 23(3-4):438-52; Azanchi R, et al.
(2004) J. Neurotrauma; 21(6):775-88; Kim B G, et al. (2008) J.
Comp. Neurol.; 508(3):473-86; and Bretzner F, et al. (2008) Eur J.
Neurosci. (9): 1795-807.
[0167] In one embodiment, the PLGA microspheres are prepared
containing bioactive morphogenic or growth factor (e.g.,
recombinant human Shh from R&D Systems, Minneapolis, Minn.,
corresponding to SEQ ID NO:2, the active N-terminal Shh fragment
(Cys24-Gly 197 of SEQ ID NO:1). In another embodiment, the
microspheres may contain Mg hydroxide to neutralize acids generated
during PLGA degradation (See, Zhu et al. Nature Biotech, January
2000, Vol. 18(1):52-57) and/or as a cryoprotectant to improve
protein stability. (See, Jaganathan et al. International J. of
Pharmaceutics, 2005, 294: 23-32).
[0168] Preferably, the controlled release formulations of the
present invention release bioactive levels of at least one
exogenous factor, e.g., Shh, for at least about 7 days. In some
embodiments, the controlled release formulations release bioactive
amounts of at least one exogenous factor for at least about 2
weeks. The amount of exogenous factor released and the duration of
release may be determined by an in vitro assay, as described in
Example 1, infra. The extent and duration of release from
microspheres may be controlled by varying parameters such as
polymer molecular weight, composition, microsphere size, and
protein loading (See, Freiberg et al. "Polymer Microspheres for
controlled drug release," International J. of Pharmaceutics, 2004
282:1-18; Berkland et al. "PLG Microsphere Size Controls Drug
Release Rate Through Several Competing Factors" Pharm. Res. 2003,
20:1055-1062; and Ashton et al. describing the effects of varying
protein loading in the microsphere).
[0169] The effective amounts of compounds of the present invention
include doses that partially or completely achieve the desired
therapeutic, prophylactic, and/or biological effect. The actual
amount effective for a particular application depends on the
condition being treated and the route of administration. The
effective amount for use in humans can be determined from animal
models. For example, a dose for humans can be formulated to achieve
circulating and/or local concentrations that have been found to be
effective in animals.
[0170] Kits
[0171] In one embodiment, the invention relates to a kit comprising
an effective amount of a sustained delivery composition comprising
one or more exogenous factors useful for increasing neuronal cell
growth or treating spinal cord or nervous system injuries, and
optionally stem cells packaged in a manner suitable for
administration to a patient. In certain embodiments, the kits also
include instructions teaching one or more of the methods described
herein.
[0172] The abbreviations in the specification correspond to units
of measure, techniques, properties or compounds as follows: "min"
means minutes, "h" means hour(s), ".mu.L" means microliter(s), "mL"
means milliliter(s), "mM" means millimolar, "M" means molar,
".mu.l" means micriliter(s); "mmole" means millimole(s), "kb" means
kilobase, "bp" means base pair(s), and "IU" means International
Units. "Polymerase chain reaction" is abbreviated PCR; "Reverse
transcriptase polymerase chain reaction" is abbreviated RT-PCR;
"Estrogen receptor" is abbreviated ER; "DNA binding domain" is
abbreviated DBD; "Untranslated region" is abbreviated UTR; "Sodium
dodecyl sulfate" is abbreviated SDS; and "High Pressure Liquid
Chromatography" is abbreviated HPLC.
EXAMPLES
Materials and Methods
[0173] The following describes the materials and methods employed
in Examples 1-6.
[0174] Animals
[0175] The care and use of animals reported in this study was
approved and overseen by Taconic Farms, which is licensed by the US
Department of Agriculture and the New York State Department of
Health, Division of Laboratories and Research and accredited by the
American Association for the Accreditation of Laboratory Animal
Care. Mice are purchased from Taconic Farms and are either Swiss
Webster or C57-BL6.
[0176] Cell Culture
[0177] Mouse embryonic day 8-9 (E8-9) spinal cords (Swiss Webster,
Taconic Farms, or GFP transgenic mice from Jackson labs on a C57BL6
background) were dissected and enzymatically dissociated using
papain (Worthington) as described previously (Qian et al. 1997).
Briefly, spinal cord tissue was incubated in 5-7 units/ml activated
papain solution plus 32 .mu.g/ml DNase in DMEM with rocking for 20
minutes at room temperature. The tissue was rinsed 3 times with
DMEM (Gibco) and triturated with a fire-polished glass Pasteur
pipette to generate a single-cell suspension. Cells were plated at
clonal density (2000-4000 cells/well) onto poly-L-lysine coated
6-well plates and cultured in basal serum-free medium consisting of
DMEM with L-glutamine, sodium pyruvate, B-27, N-2 (Stein Cell
Inc.), 1 mM N-acetylcysteine (Sigma) and 10 ng/ml bFGF (Gibco), and
10 ng/ml LIF (Sigma).
[0178] For co-culture expansion for transplantation experiments,
bovine pulmonary artery endothelial (BPAE) cells (VEC Technologies
INC., ATCC, #CCL-209) were used at passage 10-20. Three days before
co-culturing with spinal cord cells, endothelial cells were plated
into 60 mm transwell membrane inserts (Costar) at 2000
cells/transwell, in DMEM with 10% FBS. Four hours before use, the
transwells were rinsed and transferred to serum-free medium
containing 10 ng/ml FGF2. The transwells containing feeder cells
were placed above freshly plated, low density cultures of spinal
cord cells, and the co-cultures were fed every two days with
serum-free medium., cells were co-cultured in 6-well plates with
BPAE cells. One .mu.M Shh N terminal peptide (R&D systems, SEQ
ID NO:2) and 1 .mu.M retinoic acid (RA) (Sigma) were added to the
cultures.
[0179] Preparation of Cells for Transplantation
[0180] On the day of surgery, the cells were removed from the wells
with Accutase, centrifuged, washed with HBSS, centrifuged,
dissociated into single cells, resuspended in culture media, and
counted. The concentration of viable cells (usually greater than
90%), as determined by Trypan blue exclusion, was adjusted to 105
cells/.mu.l. The cells were maintained on ice until use.
[0181] Surgical Procedures and Experimental Groups
[0182] Adult (10-12 week (wk) old) female C57BL/6 mice were deeply
anesthetized by inhalation of isoflurane vapor (3%). The surgery
was performed as described previously (Li et al. 2005). A complete
laminectomy was performed and the dorsal aspect of the spinal cord
was exposed at T8 and T9 levels. A dorsal over-hemisection was
performed at T8 using a pair of microscissors and a scalpel blade
to completely sever the dorsal and dorsolateral corticospinal
tracts. The depth of the lesion (1.0 mm) was assured by passing a
marked needle across the dorsal part of the spinal cord. The lesion
was bilaterally symmetric and extended to a depth about two thirds
of the dorso-ventral axis of the spinal cord. 0.5 .mu.l of cells or
0.5 .mu.l of microspheres, or a mix of cells and microspheres were
slowly injected into the site of the injury and into a second site
1 mm rostral to the injury site. After surgery, animals were
maintained on heating pads, closely observed until fully awake and
then returned to their home cages. Mice were allowed to recover for
4 weeks.
[0183] Experimental groups were as follows: 1) Control microspheres
(n=6); 2) Control microspheres+endothelial expanded untreated cells
(n=8); 3) Shh microspheres+endothelial expanded untreated cells
(n=7); 4) Shh microspheres+endothelial expanded Shh treated cells
(n=5); 5) Shh microspheres (n=6). In the figures, "microspheres" is
abbreviated ".mu.s."
[0184] Behavioral Testing
Horizontal Ladder Walking Test
[0185] This test assesses the ability to accurately place the hind
paws while walking on a horizontal ladder by analyzing the
frequency of failure to accurately grasp the rungs. The ladder
apparatus (adapted from (Metz and Whishaw 2002)) is 23 inches long,
and consists of side walls made of clear Plexiglas.RTM. and metal
rungs that could be inserted with minimal spacing of 3/8 inch. The
ladder was elevated 30 cm above the ground with a refuge (home
cage) al the end. The mice were videotaped and scored at a later
date by an experimenter blind to the treatment groups. The number
of foot-slips per run was recorded (as shown in FIG. 2A).
Open Field Locomotion
Basso Mouse Scale Test
[0186] This test assesses body coordination, trunk stability, and
locomotion (Basso et al. 2006). Mice are placed on a smooth surface
table and their walk is videotaped. Movies are scored by an
experimenter blind to the treatment groups (as shown in FIG.
2B).
Rearing Test
[0187] This test assesses hind limb strength. Mice are placed in a
glass cylinder and allowed to explore their environment. Rearing is
graded as: O-- mouse supports its weight on full plantar surface,
heel does not leave the ground: 1--mouse supports its weight on
toes, heel rise above the ground; 2--mouse is capable of supporting
its weight on tiptoes. The mice were videotaped and scored at a
later date by an experimenter blind to the treatment groups (as
shown in FIG. 2C).
Swim Test
[0188] This test assesses hind limb movement, forelimb dependency,
hindlimb alteration, trunk instability and body angle as described
in Smith R R, et al. (2006) J Neurotrauma. (11):1654-70. Mice were
placed in a Plexiglas.RTM. chamber that is 60 inches long, 7 inches
wide, and 12 inches deep. An adjustable Plexiglas.RTM. ramp, placed
at one end of the chamber and covered with a 5 mm-thick, soft
neoprene pad, allows animals to exit the pool. The pool is filled
to a depth of 8 inches with warm tap water (27-30.degree. C.) for
each swimming session and is thoroughly cleaned daily. Hind limb
movement, forelimb dependency, hindlimb alteration, trunk
instability and body angle were each graded as described in Smith R
R, Burke D A, Baldini A D, Shum-Siu A, Baltzley R, Bunger M,
Magnuson D S. The Louisville Swim Scale: a novel assessment of
hindlimb function following spinal cord injury in adult rats. J.
Neurotrauma. 2006 November; 23(11):1654-70. Asterisks indicate
p<0.01. The mice were videotaped and scored at a later date by
an experimenter blind to the treatment groups (as shown in FIG.
2D).
Tape removal test
[0189] The tape removal test was used to assess both sensory
impairment and paw function. Mice were scruffed and held by one
experimenter while another placed strips of tape (1/8
inch.times.0.5 inch; Fisherbrand) over the ventral surface of the
hindpaw. The mouse was placed on a smooth surface, and the timer
was started. The time taken for the mouse to notice the tape (touch
the tape with the snout) was noted. The animals were given a
maximum of 3 minutes to sense the tape. The mice were videotaped
and scored at a later date by an experimenter blind to the
treatment groups (as shown in FIG. 6C).
[0190] Immunocytochemistry
[0191] Cell cultures were fixed with 4% paraformaldehyde in PBS
(140 mM NaCl, 2.6 mM KCl, 8 mM Na.sub.2HPO.sub.4, 1.4 mM
KH.sub.2PO.sub.4 pH 7.4) for 30 minutes and rinsed three times with
PBS buffer at pH 7.4. Cells were incubated with 10% normal goat
serum (NGS) in PBS with 0.1% Triton X-100 (PBST) for 30 min. No
Triton X-100 was used when cells were immunolabeled with O4, an
antibody against a surface membrane protein. Primary antibodies
(Table 1) were added and incubated overnight at 4.degree. C.,
followed by three rinses with PBS, and then a one hour incubation
with the appropriate secondary antibodies. Alexa-conjugated
secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgM
and IgG, Invitrogen) were used at 1:500 dilution.
TABLE-US-00001 TABLE 1 Primary antibodies Species, Antibody
Expression isotype Dilution Source .beta.-tubulin III Neurons Mouse
IgG2b 1:600 Sigma GFAP Astrocytes Rabbit IgG 1:1000 Dako Ki67
Dividing cells Rabbit IgG 1:1000 Vector labs Nestin Embryonic Mouse
IgG1 1:4 DSHB progenitors NeuN Neurons Mouse IgG1 1:100 Chemicon O4
Oligodendrocytes Mouse IgM Neat DSHB RIP Oligodendrocytes Mouse
IgG1 1:50 DSHB
[0192] Cryostat Sections
[0193] Mice were deeply anesthetized and perfused transcardially
with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) solution,
pH 7.4. Spinal cord segments containing the injury sites were
dissected, rinsed in 0.1 M PB solution, and placed into 0.1 M PB
solution containing 30% sucrose for 24 h at 4.degree. C. The spinal
cord tissue was then frozen in embedding media (O.C.T. compound,
Tissue-Tek) and serially sectioned on a cryostat (20 .mu.m
sections). Tissue sections were washed in PBS and incubated for 10
min with 10% normal goat serum (NGS) in PBS with 0.3% Triton X-100.
The sections were then incubated with the appropriate primary
antibody (Table 1) overnight in 10% NGS-PBST at 4.degree. C. The
following day, sections were washed three times (5 min each) in PBS
and incubated with appropriate secondary antibodies for 1 hour.
[0194] Preparation of Bioactive Biodegradable Microspheres
[0195] Generally, the microspheres were prepared from
poly(lactide-co-glycolide) PLGA (Boehringer Ingleheim) and Shh
aseptically using the double emulsion method at room temperature.
Shh microspheres (10-40 .mu.m in diameter) containing 0.5% Shh
(Cat. No. 1314-SH/CF, recombinant human Shh from R&D Systems,
Minneapolis, Minn., corresponding to SEQ ID NO:2, the active
N-terminal Shh fragment, which corresponds to Cys24-Gly 197 of SEQ
ID NO:1), as well as control PLGA microspheres without any
incorporated Shh protein were prepared. An aqueous solution of
3.125 .mu.g of Shh was suspended in 625 .mu.g of PLGA dissolved in
methylene chloride. This solution was sonicated for 3 s at 20%
amplitude in an ice bath using a Vibra-Cell.TM. high-intensity
ultrasonic liquid processor (Sonics & Materials, Inc.) to form
a first water/oil emulsion. This emulsion was then dispersed and
stabilized in 20 ml of 0.5% (w/v) aqueous polyvinylalcohol (PVA),
and mixed at high speed (8000 rpm) with a Silverson L4RT high shear
laboratory mixer, 3/4 inch tip, for 20 sec to produce the second
water/oil/water emulsion. This emulsion was stirred for 1 hour at
room temperature allowing the microspheres to form by evaporation
of methylene chloride. The microspheres were then isolated by
centrifugation (1500 rpm, 3 min) and subsequently washed four times
with distilled deionized water to remove adsorbed PVA. To remove
water, the microspheres were collected by centrifugation, frozen in
liquid nitrogen, and lyophilized. The dried microspheres were
stored in a sealed glass vial and placed in a dessicator at
-20.degree. C. The morphology and size of the microspheres were
characterized by scanning electron microscopy. Double-emulsion
techniques used in the present invention are a variation of those
described in (Fu et al. 2003).
[0196] Statistics
[0197] Behavioral scores for all animals in each group were
averaged, the standard deviation and the standard error of the mean
were calculated, and all statistical analyses were performed using
Microsoft Excel software. BDA fibers were counted from at least
five sections for each treatment. The numbers of fibers for each
experiment were averaged, the standard deviation and the standard
error of the mean were calculated, and all statistical analyses
were performed using Microsoft Excel software.
EXAMPLES
[0198] The following examples are included to demonstrate certain
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Shh-Containing Microspheres Release Active Shh Protein In Vitro and
are not Toxic to Neural Stem Cells
[0199] To establish a source of continuous release of Shh,
biodegradable microspheres (10-40 .mu.m in diameter) of
poly(lactide-co-glycolide) (PLGA) that incorporated 0.5% of Shh as
described above (Cat. No. 1314-SH/CF, recombinant human Shh from
R&D Systems, Minneapolis, Minn., corresponding to SEQ ID NO:2,
the active N-terminal Shh fragment, which corresponds to Cys24-Gly
197 of SEQ ID NO:1) were generated. Microsphere preparation is
described above and adapted from the methods of (Fu et al. 2003).
To determine the release kinetics 0.1 mg of Shh-containing
microspheres or microspheres with PLGA alone (blank sample) were
resuspended in 1 ml PBS. 100 .mu.l aliquots were taken out each day
and analyzed for the Shh release by ELISA, as shown in FIG. 1A.
FIGS. 1A-B show biodegradable PLGA-based microspheres release
active Shh in a course of at least 7 days. These microspheres are
not toxic for neural stem cells In FIG. 1A, 0.5% Shh microspheres
were resuspended in PBS. 100 .mu.l aliquots were analyzed daily for
Shh release by ELISA. No Shh was released in the PLGA alone group,
and thus, these zero data points do not appear on the graph in FIG.
1A. The amount of Shh measured in each aliquot from the
Shh-containing microspheres group is graphed in FIG. 1A (data
points are represented by circles). This preparation of
microspheres was found to continuously release about 5 ng/ml Shh
per day in a course of at least 7 days.
[0200] Although the present examples have been conducted with this
microsphere composition, it is contemplated that any biologically
acceptable sustained release composition that can continuously
release the desired amount of one or more exogenous factors, (e.g.,
at least about 5 ng/ml Shh per day in a course of at least 7 days)
would be useful in the present methods. Suitable compositions
include for example liposomes, alginate hydrogels, sustained
release nanoparticle compositions, and amorphous carbohydrate glass
matrix compositions.
[0201] To test whether microsphere-released Shh is bioactive, Shh
released from the microspheres in culture with spinal cord stem
cells was tested. Spinal cord tissue was dissected from embryonic
day 9 (E9) mice, dissociated to single cells and plated at clonal
density in Terasaki plates (Nunc 60 well plates purchased from
Krackeler Scientific Inc.). Shh-releasing microspheres and control
microspheres (PLGA only) were resuspended in BPAE-conditioned
medium. Freshly isolated E9 spinal cord NSCs were treated daily
with Shh protein or supernatant from control or Shh-releasing
microspheres for 5 days. On day 6 cultures were fixed and stained,
as shown on FIG. 1B. It was found that spinal cord stein cells
cultured with supernatant from suspended control microspheres
displayed similar rates of growth/apoptosis as cells cultured in
conditioned medium alone (control cells); therefore PLGA
microspheres are not toxic for spinal cord stem cells. To further
prove this point spinal cord stern cells were cultured with PLGA
microspheres added directly to the cells, and similarly a lack of
toxicity from the microspheres was observed, as shown in FIG. 1B.
In FIG. 1B, White bar=20 .mu.m.
[0202] Spinal cord stem cells cultured with Shh protein and spinal
cord stem cells cultured with supernatant from Shh-containing
microspheres demonstrated increased proliferation and neurogenesis
compared to untreated cells or cells treated with the supernatant
from control microspheres, as shown in FIGS. 2A-L. In FIGS. 2A-L,
the cultured NSCs were immuno-stained for nestin (which identifies
progenitor cells FIGS. 2A-D), .beta.-tubulin III (which stains
neurons, FIGS. 2E-H), and DAPI (which stains the cell nucleus,
FIGS. 2I-L). Positive staining is indicated by bright spots in each
cell culture panel.
Example 2
Transplantation of Shh-Releasing Microspheres or a Combination of
Shh-Treated Spinal Cord Neural Stem Cells with Shh-Releasing
Microspheres Produced/Resulted in Motor Recovery
[0203] Shh-releasing microspheres and a combination of
Shh-releasing microspheres and endothelial-expanded Shh treated
cells were examined as treatments for SCI. To generate
endothelial-expanded spinal cord stem cells, E9 mouse spinal cord
stem cells were co-cultured with BPAE cells in serum-free medium
with or without the addition of 1 .mu.M Shh and 1 .mu.M retinoic
acid for 6 days, then removed and injected into adult (10-12 weeks
old) mice recipients that had a dorsal over-hemisection spinal cord
injury.
[0204] Adult mice were anaesthetized and the dorsal surface of the
cord was exposed at T8-9, and the cord was cut down to a depth of 1
mm, representing halfway through the cord from the dorsal surface.
This procedure severs the descending corticospinal and ascending
sensory spinal axons located in the dorsal columns of the spinal
cord (Li et al. 2005; Vallieres et al. 2006). Immediately after the
injury was created, microspheres with or without
endothelial-expanded E9 mouse spinal cord stem cells were
transplanted into the site of the injury. Experimental groups were
as follows: 1) Control (PLGA only) microspheres (n=6); 2) Control
microspheres+endothelial-expanded untreated E9 mouse spinal cord
stem cells (n=8); 3) Shh microspheres+endothelial expanded
untreated E9 mouse spinal cord stem cells (n=7); 4) Shh
microspheres+endothelial expanded Shh treated E9 mouse spinal cord
stem cells (n=5); 5) Shh microspheres (n=6).
[0205] The mice were allowed to recover, all of them were
ambulatory after the injury (given that the ventral cord was
intact) but had detectable hind paw deficits. After 4 weeks, the
motor behaviors of the mice were assessed. Four behavioral tests
were used: the skilled horizontal ladder walking test (FIG. 3A),
which can detect hind paw deficits (Metz and Whishaw 2002), open
field locomotion (Basso Mouse Scale) (FIG. 3B) (Basso et al. 2006),
a rearing test to determine hind leg strength (FIG. 3C), and a swim
test, which can be used to determine hind limb movement, forelimb
dependency, hindlimb alteration, trunk instability and body angle
(FIG. 3D). For horizontal ladder test the number of footslips was
scored. All the behavioral tests showed a statistically significant
motor improvement (*=p.ltoreq.0.01) between mice that had been
treated with Shh-releasing microspheres or a combination of
Shh-treated endothelial-expanded spinal cord stem cells and
Shh-releasing microspheres versus mice that received control
microspheres, as shown in FIG. 3A-D (data shown as
mean.+-.SEM).
[0206] Mice that received control microspheres had hindpaw deficits
comparable to control mice that had a spinal cord injury without a
cell transplant (mock injury control). These data (FIGS. 3A-D)
point to the benefit, i.e., functional recovery after treating SCI
model mice with Shh-releasing microspheres and a combination of
Shh-releasing microspheres and spinal cord stem cells that were
treated with Shh during their expansion phase ex vivo.
Example 3
Injection of Shh-Releasing Microspheres into SCI Resulted in a
Reduced Astroglial Scar Formation Compared to Injection of Control
Microspheres into SCI
[0207] After behavioral testing, the mice were sacrificed (four
weeks after SCI) and the spinal cords were examined by staining
longitudinal sections of the SCI site with GFAP, which is an
astrocyte marker (as shown in FIG. 4A). In FIGS. 4A-B, the spinal
cord cells appear as bright regions, since they are isolated from
GFP transgenic mice and constitutively express green fluorescent
protein (GFP). Transplantation of Shh-releasing microspheres
resulted in a decrease of the astrocytic scar at the site of
injury, as shown in FIG. 4B, demonstrated by reduced staining of
GFAP in FIG. 4B (few bright-staining cells on sections treated with
Shh-releasing microspheres) compared to FIG. 4A (control
microspheres lacking Shh and containing more brighter staining
regions). These data illustrate that injection of Shh-releasing
microspheres into SCI site resulted in reduced astroglial scar
formation compared to injection of control microspheres.
[0208] Transplanted Neural Stem Cells Grafted Well in all
Experimental Groups. (FIGS. 5A-I)
[0209] The fate of the transplanted cells in the spinal cords of
mice was also analyzed by staining longitudinal sections of the SCI
site with the astrocyte marker GFAP (middle row, FIGS. 5B, E, and
H) and the oligodendrocyte marker RIP (lower row, FIGS. 5C, F, and
I). The transplanted cells were visualized by GFP fluorescence. The
transplanted cells were found to have survived, and the graft size
was approximately the same in all experimental groups. Untreated
expanded spinal cord NSCs transplanted alone are known to
differentiate mostly into astrocytes. Thus, the addition of control
microspheres to untreated expanded spinal cord NSCs should not
alter this differentiation pathway. Consistent with this theory,
untreated endothelial-expanded spinal cord NSCs co-transplanted
with control microspheres differentiated mostly into astrocytes,
and not into oligodendrocytes (FIG. 5, A-C, left column). However,
when untreated NSCs were instead co-transplanted with Shh-releasing
microspheres, the cells differentiated largely into
oligodendrocytes (FIG. 5D-F, center column). Furthermore, when
Shh-treated NSCs were co-transplanted with Shh-releasing
microspheres, the cells also differentiated into oligodendrocytes
(FIG. 5G-I, right column). Thus, the Shh released by the
microspheres (and not the microspheres themselves) diverted or
drove spinal cord NSC differentiation from the astrocyte lineage
(undesirable) to the desired oligodendrocyte lineage. Positive
staining is indicated by brighter spots or regions of the cell
sections.
[0210] This effect is believed to be the result of Shh acting in a
time and concentration-dependent manner on spinal cord progenitor
cells: early in development high concentration of Shh promote NSC
differentiate into floor plate, slightly lower amounts promote
differentiation into neurons, in development Shh promotes NSC
differentiation into oligodendrocytes. In additional experiments,
the cell fate of the implanted Shh-treated cells treated with
Shh-releasing microspheres is determined by staining the cells with
the neuronal marker NeuN, six weeks after transplantation.
Example 4
Transplantation of Shh-Containing Microspheres into SCI Site
Induces CST Fiber Sprouting and Growth in the Caudal Spinal
Cord
[0211] To address the mechanism for the motor recovery in animals
that have received Shh-releasing microspheres and a combination of
Shh-releasing microspheres and Shh-treated endothelial-expanded
cells the corticospinal tract (CST) was analyzed by labeling the
fibers of the CST with biotinylated dextran amine (BDA) injections.
In mice that received control microspheres, no BDA-labeled CST
fibers extended beyond the injury site (FIG. 6A. arrow indicates
injury site). However, in mice that received Shh-releasing
microspheres, regenerating axons from the transected CST bypassed
the transection site and projected into the caudal spinal cord.
Sprouting, branched fibers of the CST could be observed in white
and gray matter in the caudal spinal cord, as shown in FIG. 6B (see
arrowheads). In FIG. 6B, inset shows a magnified image of branched
fibers. Axonal sprouting rostral to the lesion site was also
enhanced in the animals that received Shh-releasing microspheres
and especially in the animals that received a combination of
Shh-releasing microspheres and Shh-treated endothelial-expanded
cells. The number of BDA-positive fibers (sprouting and growing
fibers) was counted 3 mm caudal to the site of SCI. The average
number of fibers.+-.SEM per section is shown (FIG. 6C). Results
from both the Shh microspheres and Shh-treated NSCs+Shh
microspheres groups were significantly different than the control
microsphere group (p<0.05 for Shh microspheres and p<0.01 for
Shh-treated NSCs+Shh microspheres).
Example 5
Transplantation of Shh/RA-BPAE-Expanded NSC into SCI Site Results
in Enhanced Oligodendrocyte Differentiation and Functional
Recovery
[0212] To address the role of Shh/RA-endothelial (BPAE)-expanded
NSC in the recovery from SCI, these cells were transplanted into
the spinal cord following surgical induction of SCI and compared to
mice that were transplanted with control cells (BPAE-expanded cells
grown without Shh/RA). FIG. 7A is a photograph of a mouse in the
horizontal ladder test, the results of which are shown in FIG. 7B.
The number of footslips per run was scored for mice that received
Shh-endothelial (BPAE) expanded spinal cord cells (n=3) and saline
injection controls (n=5). Mice that received BPAE-expanded cells
grown without Shh (n=6) behaved as saline injection controls. FIG.
7C shows the results of the tape removal test. The time taken for
mice that had been transplanted with Shh/RA-BPAE-expanded NSC at
the site of SCI to sense the tape placed on the ventral surface of
the hindpaw and to attempt to remove the tape was measured. Both
assays show that mice receiving Shh/RA-BPAE grown cells had
improved sensory and motor scores compared to saline injection
controls. The asterisk indicates that p<0.01.
Example 6
Combinational Therapy
[0213] To address the role of CABC in the recovery from SCI, 10%
CABC formulation encapsulated in microspheres was transplanted into
the mouse spinal cord injury model. Four weeks after the
transplantation, animals had shown some motor recovery. However,
this recovery had not reached statistically significant levels.
These results are shown in FIGS. 8 and 9. FIG. 8 shows
transplantation of microspheres releasing chondroitinase ABC into
the mouse SCI model resulted in moderate behavioral improvement.
The results in FIG. 8 were scored as the number of footslips per
run for mice that received chondroitinase ABC. The difference
between the control and treated mice did not reach statistical
significance, but still indicate a moderate behavioral
improvement.
[0214] In FIG. 9 the activity of chondroitinase ABC was analyzed
over the course of 10 days from 10% loaded microspheres. The
chondroitinase ABC 10% loaded microspheres were resuspended in PBC.
100 .mu.l aliquots were analyzed daily for CABC activity. The
results in FIG. 9 illustrate the sustained and continuous release
of CABC from the loaded microspheres over the 10 day test
period.
[0215] The data from FIG. 8 utilizing sustained release CABC
microspheres is indicative that these microspheres can be combined
with the Shh microspheres and one or more additional growth factors
such as IGF-I or bFGF in order to further improve the SCI niche and
provide therapeutic factors enabling growth and/or recovery of
damaged neural cells in patients in need of such treatment, as
indicated for the mouse SCI models as described herein.
SUMMARY
[0216] The present invention demonstrates that transplantation of
endothelial-expanded NSCs, which are treated with Shh and retinoic
acid, into the dorsal hemisection in a murine model of SCI results
in locomotor and sensory recovery (Lowry et al. 2007). Furthermore,
a continuous source of Shh at the injury site was provided by
incorporating Shh into biodegradable microspheres that were capable
of releasing Shh in a sustained manner over the course of at least
7 days. It was demonstrated that Shh provided via biodegradable
microspheres is a potent therapeutic agent for treating SCI. Mice
that received single injection of Shh-releasing microspheres
exhibited motor recovery. This motor recovery is explained, in
part, by the decrease in astrocytic scar formation in animals that
receive Shh-releasing microspheres, and also by the increased
outgrowth of corticospinal tract in these animals. Treatment with a
combination of Shh-releasing microspheres and Shh-treated
endothelial-expanded cells facilitates the further enhancement of
motor recovery. Motor recovery is an aspect of behavioral recovery
and can be assessed by behavioral tests. In the present examples,
motor recovery refers to locomotion and hind limb movements.
Additionally, there was no evidence of tumor formation in any of
the treated animals.
[0217] Treatment of patients suffering from such degenerative
conditions can include the application of Shh, or other exogenous
factors which mimic its effects, in order to control, for example,
differentiation and apoptotic events which give rise to loss of
neurons (e.g. to enhance survival of existing neurons) as well as
promote differentiation and repopulation by progenitor cells in the
area affected. In preferred embodiments, a source of Shh is a
biodegradable microsphere compound releasing Shh. Optionally, this
Shh-releasing microsphere compound is administered together with
stem cells or endothelial-expanded spinal cord NSCs, which are
optionally pre-treated with Shh and/or retinoic acid, to or
proximate the area of degeneration.
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[0262] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0263] While the compositions and methods of this invention have
been described in terms of specific embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the compositions and methods and in the steps or in the
sequence of steps of the method described herein without departing
from the concept and scope of the invention. More specifically, it
will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the scope of the
invention as defined by the appended claims.
[0264] It is further to be understood that all values are
approximate, and are provided for description.
[0265] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
Sequence CWU 1
1
81462PRTHomo sapiens 1Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val
Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Gly Pro
Gly Arg Gly Phe Gly Lys 20 25 30Arg Arg His Pro Lys Lys Leu Thr Pro
Leu Ala Tyr Lys Gln Phe Ile 35 40 45Pro Asn Val Ala Glu Lys Thr Leu
Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60Lys Ile Ser Arg Asn Ser Glu
Arg Phe Lys Glu Leu Thr Pro Asn Tyr65 70 75 80Asn Pro Asp Ile Ile
Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95Leu Met Thr Gln
Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser 100 105 110Val Met
Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp 115 120
125Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg
130 135 140Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr
Gly Met145 150 155 160Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp
Trp Val Tyr Tyr Glu 165 170 175Ser Lys Ala His Ile His Cys Ser Val
Lys Ala Glu Asn Ser Val Ala 180 185 190Ala Lys Ser Gly Gly Cys Phe
Pro Gly Ser Ala Thr Val His Leu Glu 195 200 205Gln Gly Gly Thr Lys
Leu Val Lys Asp Leu Ser Pro Gly Asp Arg Val 210 215 220Leu Ala Ala
Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr225 230 235
240Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val Ile Glu
245 250 255Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His
Leu Leu 260 265 270Phe Val Ala Pro His Asn Asp Ser Ala Thr Gly Glu
Pro Glu Ala Ser 275 280 285Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala
Leu Gly Pro Arg Ala Leu 290 295 300Phe Ala Ser Arg Val Arg Pro Gly
Gln Arg Val Tyr Val Val Ala Glu305 310 315 320Arg Asp Gly Asp Arg
Arg Leu Leu Pro Ala Ala Val His Ser Val Thr 325 330 335Leu Ser Glu
Glu Ala Ala Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly 340 345 350Thr
Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val Ile Glu 355 360
365Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu Ala His
370 375 380Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg Gly
Gly Asp385 390 395 400Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gly
Arg Val Ala Leu Thr 405 410 415Ala Pro Gly Ala Ala Asp Ala Pro Gly
Ala Gly Ala Thr Ala Gly Ile 420 425 430His Trp Tyr Ser Gln Leu Leu
Tyr Gln Ile Gly Thr Trp Leu Leu Asp 435 440 445Ser Glu Ala Leu His
Pro Leu Gly Met Ala Val Lys Ser Ser 450 455 4602174PRTHomo sapiens
2Cys Gly Pro Gly Arg Gly Phe Gly Lys Arg Arg His Pro Lys Lys Leu1 5
10 15Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro Asn Val Ala Glu Lys
Thr 20 25 30Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys Ile Ser Arg Asn
Ser Glu 35 40 45Arg Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pro Asp Ile
Ile Phe Lys 50 55 60Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu Met Thr
Gln Arg Cys Lys65 70 75 80Asp Lys Leu Asn Ala Leu Ala Ile Ser Val
Met Asn Gln Trp Pro Gly 85 90 95Val Lys Leu Arg Val Thr Glu Gly Trp
Asp Glu Asp Gly His His Ser 100 105 110Glu Glu Ser Leu His Tyr Glu
Gly Arg Ala Val Asp Ile Thr Thr Ser 115 120 125Asp Arg Asp Arg Ser
Lys Tyr Gly Met Leu Ala Arg Leu Ala Val Glu 130 135 140Ala Gly Phe
Asp Trp Val Tyr Tyr Glu Ser Lys Ala His Ile His Cys145 150 155
160Ser Val Lys Ala Glu Asn Ser Val Ala Ala Lys Ser Gly Gly 165
1703437PRTMus musculus 3Met Leu Leu Leu Leu Ala Arg Cys Phe Leu Val
Ile Leu Ala Ser Ser1 5 10 15Leu Leu Val Cys Pro Gly Leu Ala Cys Gly
Pro Gly Arg Gly Phe Gly 20 25 30Lys Arg Arg His Pro Lys Lys Leu Thr
Pro Leu Ala Tyr Lys Gln Phe 35 40 45Ile Pro Asn Val Ala Glu Lys Thr
Leu Gly Ala Ser Gly Arg Tyr Glu 50 55 60Gly Lys Ile Thr Arg Asn Ser
Glu Arg Phe Lys Glu Leu Thr Pro Asn65 70 75 80Tyr Asn Pro Asp Ile
Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp 85 90 95Arg Leu Met Thr
Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile 100 105 110Ser Val
Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly 115 120
125Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly
130 135 140Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys
Tyr Gly145 150 155 160Met Leu Ala Arg Leu Ala Val Glu Ala Gly Phe
Asp Trp Val Tyr Tyr 165 170 175Glu Ser Lys Ala His Ile His Cys Ser
Val Lys Ala Glu Asn Ser Val 180 185 190Ala Ala Lys Ser Gly Gly Cys
Phe Pro Gly Ser Ala Thr Val His Leu 195 200 205Glu Gln Gly Gly Thr
Lys Leu Val Lys Asp Leu Arg Pro Gly Asp Arg 210 215 220Val Leu Ala
Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu225 230 235
240Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val Phe Tyr Val Ile
245 250 255Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala
His Leu 260 265 270Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro Thr
Pro Gly Pro Ser 275 280 285Ala Leu Phe Ala Ser Arg Val Arg Pro Gly
Gln Arg Val Tyr Val Val 290 295 300Ala Glu Arg Gly Gly Asp Arg Arg
Leu Leu Pro Ala Ala Val His Ser305 310 315 320Val Thr Leu Arg Glu
Glu Glu Ala Gly Ala Tyr Ala Pro Leu Thr Ala 325 330 335His Gly Thr
Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val 340 345 350Ile
Glu Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu 355 360
365Ala His Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Gly Gly
370 375 380Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Ala Thr Glu Ala
Arg Gly385 390 395 400Ala Glu Pro Thr Ala Gly Ile His Trp Tyr Ser
Gln Leu Leu Tyr His 405 410 415Ile Gly Thr Trp Leu Leu Asp Ser Glu
Thr Met His Pro Leu Gly Met 420 425 430Ala Val Lys Ser Ser
4354411PRTHomo sapiens 4Met Ser Pro Ala Arg Leu Arg Pro Arg Leu His
Phe Cys Leu Val Leu1 5 10 15Leu Leu Leu Leu Val Val Pro Ala Ala Trp
Gly Cys Gly Pro Gly Arg 20 25 30Val Val Gly Ser Arg Arg Arg Pro Pro
Arg Lys Leu Val Pro Leu Ala 35 40 45Tyr Lys Gln Phe Ser Pro Asn Val
Pro Glu Lys Thr Leu Gly Ala Ser 50 55 60Gly Arg Tyr Glu Gly Lys Ile
Ala Arg Ser Ser Glu Arg Phe Lys Glu65 70 75 80Leu Thr Pro Asn Tyr
Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn 85 90 95Thr Gly Ala Asp
Arg Leu Met Thr Gln Arg Cys Lys Asp Arg Leu Asn 100 105 110Ser Leu
Ala Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg 115 120
125Val Thr Glu Gly Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu
130 135 140His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg
Asp Arg145 150 155 160Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val
Glu Ala Gly Phe Asp 165 170 175Trp Val Tyr Tyr Glu Ser Lys Ala His
Val His Cys Ser Val Lys Ser 180 185 190Glu His Ser Ala Ala Ala Lys
Thr Gly Gly Cys Phe Pro Ala Gly Ala 195 200 205Gln Val Arg Leu Glu
Ser Gly Ala Arg Val Ala Leu Ser Ala Val Arg 210 215 220Pro Gly Asp
Arg Val Leu Ala Met Gly Glu Asp Gly Ser Pro Thr Phe225 230 235
240Ser Asp Val Leu Ile Leu Leu Asp Arg Glu Pro His Arg Leu Arg Ala
245 250 255Phe Gln Val Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu Ala
Leu Thr 260 265 270Pro Ala His Leu Leu Phe Thr Ala Asp Asn His Thr
Glu Pro Ala Ala 275 280 285Arg Phe Arg Ala Thr Phe Ala Ser His Val
Gln Pro Gly Gln Tyr Val 290 295 300Leu Val Ala Gly Ala Pro Gly Leu
Gln Pro Ala Arg Val Ala Ala Val305 310 315 320Ser Thr His Val Ala
Leu Gly Ala Tyr Ala Pro Leu Thr Lys His Gly 325 330 335Thr Leu Val
Val Glu Asp Val Val Ala Ser Cys Phe Ala Ala Val Ala 340 345 350Asp
His His Leu Ala Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe His 355 360
365Ser Leu Ala Trp Gly Ser Trp Thr Pro Gly Glu Gly Val His Trp Tyr
370 375 380Pro Gln Leu Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu
Gly Ser385 390 395 400Phe His Pro Leu Gly Met Ser Gly Ala Gly Ser
405 4105437PRTMus musculus 5Met Leu Leu Leu Leu Ala Arg Cys Phe Leu
Val Ile Leu Ala Ser Ser1 5 10 15Leu Leu Val Cys Pro Gly Leu Ala Cys
Gly Pro Gly Arg Gly Phe Gly 20 25 30Lys Arg Arg His Pro Lys Lys Leu
Thr Pro Leu Ala Tyr Lys Gln Phe 35 40 45Ile Pro Asn Val Ala Glu Lys
Thr Leu Gly Ala Ser Gly Arg Tyr Glu 50 55 60Gly Lys Ile Thr Arg Asn
Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn65 70 75 80Tyr Asn Pro Asp
Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp 85 90 95Arg Leu Met
Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile 100 105 110Ser
Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly 115 120
125Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly
130 135 140Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys
Tyr Gly145 150 155 160Met Leu Ala Arg Leu Ala Val Glu Ala Gly Phe
Asp Trp Val Tyr Tyr 165 170 175Glu Ser Lys Ala His Ile His Cys Ser
Val Lys Ala Glu Asn Ser Val 180 185 190Ala Ala Lys Ser Gly Gly Cys
Phe Pro Gly Ser Ala Thr Val His Leu 195 200 205Glu Gln Gly Gly Thr
Lys Leu Val Lys Asp Leu Arg Pro Gly Asp Arg 210 215 220Val Leu Ala
Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu225 230 235
240Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val Phe Tyr Val Ile
245 250 255Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala
His Leu 260 265 270Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro Thr
Pro Gly Pro Ser 275 280 285Ala Leu Phe Ala Ser Arg Val Arg Pro Gly
Gln Arg Val Tyr Val Val 290 295 300Ala Glu Arg Gly Gly Asp Arg Arg
Leu Leu Pro Ala Ala Val His Ser305 310 315 320Val Thr Leu Arg Glu
Glu Glu Ala Gly Ala Tyr Ala Pro Leu Thr Ala 325 330 335His Gly Thr
Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val 340 345 350Ile
Glu Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu 355 360
365Ala His Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Gly Gly
370 375 380Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Ala Thr Glu Ala
Arg Gly385 390 395 400Ala Glu Pro Thr Ala Gly Ile His Trp Tyr Ser
Gln Leu Leu Tyr His 405 410 415Ile Gly Thr Trp Leu Leu Asp Ser Glu
Thr Met His Pro Leu Gly Met 420 425 430Ala Val Lys Ser Ser
43569455DNAHomo sapiens 6gcgaggcagc cagcgaggga gagagcgagc
gggcgagccg gagcgaggaa gggaaagcgc 60aagagagagc gcacacgcac acacccgccg
cgcgcactcg cgcacggacc cgcacgggga 120cagctcggaa gtcatcagtt
ccatgggcga gatgctgctg ctggcgagat gtctgctgct 180agtcctcgtc
tcctcgctgc tggtatgctc gggactggcg tgcggaccgg gcagggggtt
240cgggaagagg aggcacccca aaaagctgac ccctttagcc tacaagcagt
ttatccccaa 300tgtggccgag aagaccctag gcgccagcgg aaggtatgaa
gggaagatct ccagaaactc 360cgagcgattt aaggaactca cccccaatta
caaccccgac atcatattta aggatgaaga 420aaacaccgga gcggacaggc
tgatgactca ggtaggaacc cagcgccggg gcgtggaatg 480tgtggctttc
cagggggtta cgagaagccg aacacttcca gacttaactc tgtttgctct
540tcgggcagat aggaaggtga tttcacccgc tccttcccca cccacctgcc
cgccccccat 600ctcttcctct tcctggagga gaatggaggt caagggtcca
gctggagaag tttagggtgt 660ggtgggggtg aggacggtaa cagacgtggt
tcattatggc cctgatttga tgagtcttgc 720tacaatggcc ttccccatcc
tacctctgcc ctggcttgta acttggggag accttcactt 780tgggggcgtc
ggccctttcc aagtcaggag tggaaatgga aggagaggct gggaatcccc
840ctcccacaaa catgaagtgg tctcctggca ctgtacgaac gaacgaacgt
agccttgggc 900attggagctc agagccccca cgtttcccgt tgcctctgtg
gttttctttc ccaccactac 960ccccaccctg cacctcccca ccaaagaatt
ctcaactgga aaagccagga ggcggttctg 1020acaaaaggca ggggctccag
gggagactcc gcccgtccct gggtggctgg ctgtatcgca 1080gagctggctt
tgcgattgcg tgtccgcaat tgtgcccatc agagtgtgaa tgtattgata
1140tttctttaag gatgctcttt cgttcttcca agcccgaggt accttagggg
agggacttag 1200aacttattgg cattgcatca ctttagtttt caacctgctt
gcataagaat taagagcgaa 1260taaatattag tgtgggggga ggggaagcta
agcaaaatat gaattcctct ctctctcccc 1320acctcctttg agatttctga
gctgccaatc tcccagccaa ttctagactt tctgaaactc 1380catgcacgta
taactgaagc cagaaatggg tttccttgca aatataggtc aacatccttt
1440ttattgccct attaaaatat tcaagtccta cctttagggc taggtgcgta
cagcggctga 1500tggagtggcg ctggtggggc gcaagtgcag ggggagggta
ctgacggcag agagagagga 1560gctacctccg tgccgccctg cttcccgacc
cgattcccag gcttgcttga ggccgagaaa 1620ggcgaggggc aggcaaggta
gcctgctcca gctgtcggaa gggagaggaa tgggaaatgg 1680tcctgatttc
cttgctctcc ctcatctgct cccgaccacc ttaaatctgg accgcgagtg
1740tggacgcgcg cgccagtgcc agacagcagc gcgatccaca attaactctg
cacgggccat 1800ggggtgcccg gtgcgtgcag ctggctggag ggagttctcc
ggctagcccg aggcgcccat 1860cctctcgtca ccctcactcc ccgcggagga
ggggccttgc cagggtccct cggaacccga 1920gaggagggag gcactgcgga
gagagcggcg ggggcgtgga tacccgaggt cccagagcca 1980gagtgggtca
gcttctgccc tgctctgcgg gaggccaata ccgcagaagg ggtcctgggc
2040tcgcacacct tcccagggct tgggccttgc agccctgctg caaagctgca
agcgcacaga 2100gccgcgcagc gaggcagacg cctgcagccc cacttactcc
cgggttatcg atcccccgcg 2160gctagggttt cagtgcgcga ggggctgggc
tggagccgcc gggctctgct gctccacgcg 2220cgggagcgca ggcaccgcag
agctaacagc agcgccgggc tcgctgtagt gtccccggcg 2280gcgggggcgc
ggagatgggg gcgcccgcgc aggggccggg gcgcatcgcg ggctccgccg
2340gcctgccctg ggacgcgccc ccatccccag tccgccgcct gcctggcctc
taggcctccg 2400cgtcccagcc ggagccccca gcccgggggc ctccccccga
cccccgtccg ccctgccggg 2460ggacgcaggg cccagcggcc ccgcgcccgg
ccactctcgc cgccgcgcgc acaggcagca 2520tttgaacttc tgaccttctg
tcaacttccc tcagacgaac gaaacgaaaa caaatacttt 2580tttccttggg
cagtggctat tcccgttccc aacacaaaag gagggggaag gacggcccaa
2640gtgggggttg ggtgaggaga gccaggccgg gattatcagg cagaccccac
aaaggtcccc 2700taaaaccgag gggggtaggg gctggcagtc tgtgaggtat
ccccggttga tccctcccct 2760accttccttc tcccgattcc aggagttaag
ggtgggggag gaagggatgg ggaaggcgga 2820ggctcgggtg ctgagggcag
gggcggggtg caggaggcgg caggggagcc ccaggccggc 2880gggaggtttg
gggagcctgc tcggccgccc tcattttaaa taaccaccta ggctctgccc
2940caggtgcgtg accctcttct tctgtctccc tccctgtctc tgggtctcta
atgtgactgc 3000cgcccaagtc cctcaaccat ggcgagatcg tccccagtgg
aactttcgga gcagttccgg 3060aacgcaggag ctgccggtta atattaaccc
gggagaggaa agcgcagaca gacacgctct 3120ccccgcgcgg gcctaggtgc
caggcgaggg tgctggcggc cagggggctc ctaaggggca 3180ggaggccaga
gggccggatc tgaagcctgg agtggggtcc cgagccgcta cactaaatag
3240atttaatgtg cgctctgggg ccgccaggaa agacgctcag gtatggggtt
ggggaggggc
3300tgttccacca agcgtggggg aaaggacagt ggagagaggt gcgtttaggg
gctggggctg 3360tcttgaagct gggacgcccc cgcccccgcg ctgggggaag
cccaccggct gctggcggtg 3420acactcgccg gcgcggctcg cagatcaggg
aggtaggcgg gagctcaggc gtggggaaca 3480acttggcctc cgccgacaca
aagcccggcc ccggcggccc tgctgggctt cacggtggct 3540gcacagagtc
gggcttgatt cgcggcacac gacccaatga attaataacc ggcctgggct
3600tcccggcttt gcctgcgaca atcccgccca gcgcgggcgg aggagaggcc
gccagccgag 3660gccgcgcgga gcccgggccg gaggagggcg caaggggcgg
gggcgccaac tccagcaacc 3720ctcggcctcc gcccctcact cgcgcagcca
cctcccgtcg cggcccggct ggacccgggt 3780ctccctgccc ggggtcctcc
atgcctgccc aagtggcgca gctcacagag ctgggggcca 3840ggtcatcctc
accctgccgc cctctccctg gctgccctcc tgggaagctg tttaaagctt
3900cttcggcaca gccccagggg agggagctgc ggtggggtgg ggggcttgca
tgggggtccc 3960tgtgcgtgtt ggtggtgtgc gcctgcgcgc aacgggcctc
acatcatagc tctacactga 4020ccctggttta ctgattgatt ttcatgtaaa
acgcgttcaa tcctcaagat gacctcactc 4080aaactctgcc cttccgactt
ttttttttaa ctgctggcag gcccacaaac atgcaggcac 4140tgacctgtta
ccagggcggc ccccagccct accccacccc cagttgttgc atgttgaact
4200ctacaaccat attactgggt tttattgctg ccagatacac aggacttttc
ctgttgcgca 4260atttgtcacg tccccttaaa gcgccgcagc agtggggcca
gcgtcctcgc cccaccctct 4320cgaaagagtc cccccaaccc acgctacagt
tagggccctg gatagaagct gtccctccat 4380ggcgacaacc agactccaag
cagagcatcc ttccagactg gaggaggtta gaggtcagcc 4440ccgccctctg
cagaagtcac cttgaaattg cccctcggcc tccacttggc gcagcttctt
4500gggggatgcc accatcgtca tctgtgccag ttccccctct ttaaatcccg
tgtcccacca 4560gcagcagcag ggtaaacatc caggaagcaa gtcagtgccc
ccacaaacac acacagtgga 4620ttcaactgct ttctgtcgca tccttatctg
agggtgaccc cagaattcca ggggaacccc 4680cacaatctga atcccaggta
accccgtctg catctgccta gtcagtgttt cctgcctcct 4740cccaggcaac
ttcctgggaa actccccagg cggaggactc cgagacctca ggccttcctg
4800tctcccctcc ccctcctctc aaacccctcc ccctccctcc acctcttcag
tttgctcttc 4860aaacttgctg gacgccattc tatgctgggg ccaagaacac
aagagcggag gaagggaaca 4920ggttaaagaa aacaagaaac acaatcagac
cacagaaaag ccaggcagaa aagggttcga 4980cgggcaaaaa gaatgtggct
gtccagataa agaatgtctg tcccggcccc ggcctgtgct 5040gcaagtggca
actcacctag ccgcctgcca cccaggctcc cgcccaccgc gcagccccgc
5100cagcggcttc tcgcctcccc tctgcctcgg atagggttag ggcctgaggt
aaataaatgc 5160aaggccttca attctccaag cagtgcgcag tgcatttttc
tttatttctg ggaacttgcg 5220cccaggtctc tgtcaggcct gctgtgaggg
attctacgcg gggagaaggt ggaggctgcg 5280caggtggaga aaggggcccc
agaagggggg ctagaagtgg agggcaacgt gggggcgggg 5340cgggtatccc
agagggtgcc cctggagggt cctgtagttg atgtcttaaa catgcaggtc
5400acttgtttca gagaaacttt atttgcttct taggcctcgc taggagcatc
ggctgtttca 5460ggacctggag aaaggccccc agctctaccc tgagaggacg
tgctcctcca cgctcctccg 5520caaatgctgt ccctcttccc cagcccaggg
cccggctctt cggtgtgtct gggccattcc 5580aacccccgtc tccccacctc
tccgcatggc cctcgcgcct tgagactggg cagggcaggc 5640tgatggaggg
gccgggaggg gtggcgattg cccaggctaa cgtgtccgtc ggtgggggtc
5700cccttgtctt cgcagaggtg taaggacaag ttgaacgctt tggccatctc
ggtgatgaac 5760cagtggccag gagtgaaact gcgggtgacc gagggctggg
acgaagatgg ccaccactca 5820gaggagtctc tgcactacga gggccgcgca
gtggacatca ccacgtctga ccgcgaccgc 5880agcaagtacg gcatgctggc
ccgcctggcg gtggaggccg gcttcgactg ggtgtactac 5940gagtccaagg
cacatatcca ctgctcggtg aaagcaggta agctggccct ggccccccgg
6000atccgaccca aggaaggcca ttggcgcacc tcggcttgat tcaagagaaa
aagaaacctg 6060gggggaggct gagggccagg agcaggggcg ctgggcgatg
actgcgtttc cgcggtggaa 6120cctgccctgt gaggtgccgg cccctcgaaa
tcacccctac ctttgaggcc acagagccca 6180aggttctcca tgccccgaga
tggggtcctg tggcttcctg cccgcttctg gagcccccac 6240tgcagggggt
gggaaagcgt gactggggga ggggcgctag gcccttccag gcgagggaag
6300acagccctgc gcggttagcc aggtctgggc gagctccttc ctctcgttta
gggcttaaga 6360accaaccgcc cccacccgct atcccaagcg caggggtgtc
tatcctgccc cggagcccgc 6420gtcctggctc ctccccgccg ggcgcccgtg
gatcctaagc tgcctttggg gagaggcctg 6480gtgggcggca gtaaacccag
gggcaaccac ctccagcatc tggaggcggc gcgcccggag 6540cctgcgttcc
tactgggagc cgggccggga cgccctgggc ggcgggcagg ccccgaaacg
6600ccggcccgag tcggcgcgag gctgtcttct ctgggcctgc aacgccacac
gctgttgccg 6660gcgaggaaca gccgtggagg aggcgccatc gcgcgcacgc
aaacctccgg cccgaggctg 6720tgtgcacagc gctcttctcc gcccgcataa
attggcacgt ttagcaaagc cgttcacggt 6780gaatttcggg gaaactctgc
cttcctcaac ccccttccag gtttccctac ttgtctccta 6840aattccatgt
taatggcact atgttagtag gaaaacactg ttaaggtgtc aaggcacact
6900tgtaggtaaa ggctagagtg gcttctcgtc cccacagaaa gcaaaggcgt
ggagcggggg 6960cggcaggggc gggtgtgcgg cccggagagc tcccggctgc
aggcaggcag gaggcggcgc 7020ccccacctcg cgggctcggc ggcggcccct
gggcccaggg cgccccctgc gcaaaacctc 7080ctccccggct ccctgcccgc
ggggtccccc tagcgggggt ctccggaggc ctcctcccaa 7140gtgagcagcg
ctaatccatc ccccggatcg cgccgggaga gcggagccgc ggcgcgggag
7200ccgctcattg gcattctgag cacacgggcg ggggcgcggg gcgcagcgtg
tcaagccggg 7260ccgtgcgact cgacgactcg ggctcgccag cgcccggggt
cgcattccgg ggggctacgg 7320agggcctcca acggccagcc ccgcacttca
tgccagagaa accgatgaga agattaaaag 7380ccccctgtaa ttccagcagg
aagattcttt ctggcaatct ctatttgcaa aaagcatgat 7440cccggagatt
ggaatgcaaa gaagacggcc ctccccgccc tcctccccgg ccccctgcgc
7500tccgccccaa cttcaattat tgtcctgggg acagtgagcc tcagagagcg
acagagggct 7560cgagaaagcg ggtagtcaag gggccttgag acccggcgct
tccagcgctc cgaacaggcc 7620ccgccattta aaattcaaat acacatcttg
agtgcttgga agagaggcct ggctgtgcaa 7680atagtgcttg tgaattgcac
acggggtggg ggggggttgc acctgagcaa atagggaggg 7740ggaggcccgc
gagctgggga gagagtgagc tgagaacagg gaggggagaa aatggaagtg
7800tccccttcca agagtgtctc ctgtttatcc cagaaatcac aatgacaatg
ctgggccctt 7860tattggattt taattagaaa atccacacaa gcctcggatt
ttcacacctc ggccaatctc 7920tggaatgttt gtccagttgc tacaactact
gcagctattt ttcactcccc gcccccgccc 7980ctccgcaggc ccacgccgag
gcgcggcagg gtgctgcggg caggcgggca ggcgggcagg 8040cgggccaggg
gtttccgccg cgcagcccgg gtgctgagtg cgcgagcagg cgccgcgccc
8100cgcgccgggg cgggagggaa ggagggtgcg cccggcgccc gcgggagctc
aaggaggctt 8160cctgaggaat ccaagtgcag agcaaacacc ctctggatgg
attcgcggcg aggccgggtg 8220tgtgcggagc tgggggtggg gttggaggaa
ggcggaagga aagagtgtca ccggcctctg 8280caggaaacgc cagccaacct
ctgtgaccgc cagcccagac ttagagagtc gttaaggaat 8340gtgtcggaat
cctgtccctg gggcagtggg gttgggggag ggaggtgtgt gcgggacccg
8400tctggaatca atcgccccgc cccgcgcctt gcgcacccct ggcctaggag
cgcgggcacc 8460aagcgtgcgc cctcctcccc gagacgcgcc tccctctcgg
aactcaatgc cctgtcctct 8520cttctttccc ttctcctcac ccgcagagaa
ctcggtggcg gccaaatcgg gaggctgctt 8580cccgggctcg gccacggtgc
acctggagca gggcggcacc aagctggtga aggacctgag 8640ccccggggac
cgcgtgctgg cggcggacga ccagggccgg ctgctctaca gcgacttcct
8700cactttcctg gaccgcgacg acggcgccaa gaaggtcttc tacgtgatcg
agacgcggga 8760gccgcgcgag cgcctgctgc tcaccgccgc gcacctgctc
tttgtggcgc cgcacaacga 8820ctcggccacc ggggagcccg aggcgtcctc
gggctcgggg ccgccttccg ggggcgcact 8880ggggcctcgg gcgctgttcg
ccagccgcgt gcgcccgggc cagcgcgtgt acgtggtggc 8940cgagcgtgac
ggggaccgcc ggctcctgcc cgccgctgtg cacagcgtga ccctaagcga
9000ggaggccgcg ggcgcctacg cgccgctcac ggcccagggc accattctca
tcaaccgggt 9060gctggcctcg tgctacgcgg tcatcgagga gcacagctgg
gcgcaccggg ccttcgcgcc 9120cttccgcctg gcgcacgcgc tcctggctgc
actggcgccc gcgcgcacgg accgcggcgg 9180ggacagcggc ggcggggacc
gcgggggcgg cggcggcaga gtagccctaa ccgctccagg 9240tgctgccgac
gctccgggtg cgggggccac cgcgggcatc cactggtact cgcagctgct
9300ctaccaaata ggcacctggc tcctggacag cgaggccctg cacccgctgg
gcatggcggt 9360caagtccagc tgaagccggg gggccggggg aggggcagcg
ggagggggcg ccagctgaag 9420ccggggggcc gggggagggg cagcgggagg gggcg
945571314DNAMus musculus 7atgctgctgc tgctggccag atgttttctg
gtgatccttg cttcctcgct gctggtgtgc 60cccgggctgg cctgtgggcc cggcaggggg
tttggaaaga ggcggcaccc caaaaagctg 120acccctttag cctacaagca
gtttattccc aacgtagccg agaagaccct aggggccagc 180ggcagatatg
aagggaagat cacaagaaac tccgaacgat ttaaggaact cacccccaat
240tacaaccccg acatcatatt taaggatgag gaaaacacgg gagcagaccg
gctgatgact 300cagaggtgca aagacaagtt aaatgccttg gccatctctg
tgatgaacca gtggcctgga 360gtgaagctgc gagtgaccga gggctgggat
gaggacggcc atcattcaga ggagtctcta 420cactatgagg gtcgagcagt
ggacatcacc acgtccgacc gggaccgcag caagtacggc 480atgctggctc
gcctggctgt ggaagcaggt ttcgactggg tctactatga atccaaagct
540cacatccact gttctgtgaa agcagagaac tccgtggcgg ccaaatccgg
cggctgtttc 600ccgggatccg ccaccgtgca cctggagcag ggcggcacca
agctggtgaa ggacttacgt 660cccggagacc gcgtgctggc ggctgacgac
cagggccggc tgctgtacag cgacttcctc 720accttcctgg accgcgacga
aggcgccaag aaggtcttct acgtgatcga gacgctggag 780ccgcgcgagc
gcctgctgct caccgccgcg cacctgctct tcgtggcgcc gcacaacgac
840tcggggccca cgcccgggcc aagcgcgctc tttgccagcc gcgtgcgccc
cgggcagcgc 900gtgtacgtgg tggctgaacg cggcggggac cgccggctgc
tgcccgccgc ggtgcacagc 960gtgacgctgc gagaggagga ggcgggcgcg
tacgcgccgc tcacggcgca cggcaccatt 1020ctcatcaacc gggtgctcgc
ctcgtgctac gctgtcatcg aggagcacag ctgggcacac 1080cgggccttcg
cgcctttccg cctggcgcac gcgctgctgg ccgcgctggc acccgcccgc
1140acggacggcg ggggcggggg cagcatccct gcagcgcaat ctgcaacgga
agcgaggggc 1200gcggagccga ctgcgggcat ccactggtac tcgcagctgc
tctaccacat tggcacctgg 1260ctgttggaca gcgagaccat gcatcccttg
ggaatggcgg tcaagtccag ctga 131482727DNAMus musculus 8acaagctctc
cagccttgct accatttaaa atcaggctct ttttgtcttt taattgctgt 60ctcgagaccc
aactccgatg tgttccgtta ccagcgaccg gcagcctgcc atcgcagccc
120cagtctgggt ggggatcgga gacaagtccc ctgcagcagc ggcaggcaag
gttatatagg 180aagagaaaga gccaggcagc gccagaggga acgaacgagc
cgagcgagga agggagagcc 240gagcgcaagg aggagcgcac acgcacacac
ccgcgcgtac ccgctcgcgc acagacagcg 300cggggacagc tcacaagtcc
tcaggttccg cggacgagat gctgctgctg ctggccagat 360gttttctggt
gatccttgct tcctcgctgc tggtgtgccc cgggctggcc tgtgggcccg
420gcagggggtt tggaaagagg cggcacccca aaaagctgac ccctttagcc
tacaagcagt 480ttattcccaa cgtagccgag aagaccctag gggccagcgg
cagatatgaa gggaagatca 540caagaaactc cgaacgattt aaggaactca
cccccaatta caaccccgac atcatattta 600aggatgagga aaacacggga
gcagaccggc tgatgactca gaggtgcaaa gacaagttaa 660atgccttggc
catctctgtg atgaaccagt ggcctggagt gaagctgcga gtgaccgagg
720gctgggatga ggacggccat cattcagagg agtctctaca ctatgagggt
cgagcagtgg 780acatcaccac gtccgaccgg gaccgcagca agtacggcat
gctggctcgc ctggctgtgg 840aagcaggttt cgactgggtc tactatgaat
ccaaagctca catccactgt tctgtgaaag 900cagagaactc cgtggcggcc
aaatccggcg gctgtttccc gggatccgcc accgtgcacc 960tggagcaggg
cggcaccaag ctggtgaagg acttacgtcc cggagaccgc gtgctggcgg
1020ctgacgacca gggccggctg ctgtacagcg acttcctcac cttcctggac
cgcgacgaag 1080gcgccaagaa ggtcttctac gtgatcgaga cgctggagcc
gcgcgagcgc ctgctgctca 1140ccgccgcgca cctgctcttc gtggcgccgc
acaacgactc ggggcccacg cccgggccaa 1200gcgcgctctt tgccagccgc
gtgcgccccg ggcagcgcgt gtacgtggtg gctgaacgcg 1260gcggggaccg
ccggctgctg cccgccgcgg tgcacagcgt gacgctgcga gaggaggagg
1320cgggcgcgta cgcgccgctc acggcgcacg gcaccattct catcaaccgg
gtgctcgcct 1380cgtgctacgc tgtcatcgag gagcacagct gggcacaccg
ggccttcgcg cctttccgcc 1440tggcgcacgc gctgctggcc gcgctggcac
ccgcccgcac ggacggcggg ggcgggggca 1500gcatccctgc agcgcaatct
gcaacggaag cgaggggcgc ggagccgact gcgggcatcc 1560actggtactc
gcagctgctc taccacattg gcacctggct gttggacagc gagaccatgc
1620atcccttggg aatggcggtc aagtccagct gaagcccgac gggaccgggc
aaggggcggg 1680cggggcgggg agcgactgcg aaataaggaa ctgatgggaa
agcgcacgga aggagacttt 1740taattataag aataattcat aataataata
ataatgataa taataataat aataagtagg 1800gcagtccaaa gtagactata
aggaagcaaa aaccccgggg agttctgttg ttatgtttag 1860tttatatatt
tttttgaaat ttttcgttat tgtcttatat gggttgtttt tctcctctcc
1920tggctattta tttgtttcgt atgaatagat gttttaaaaa tatgaacgga
ccttcaagag 1980ccttaactag tttgtgtctt ggataattta ttattgtgtg
aactgtactc acagtgaggg 2040aaagattatt ttgtgaggcc aagcaacctg
ctgaaagtct atttttctac atgtcccttg 2100tcctgcgttt cagaaggcaa
acctccgcat tcctctcctg ctatgctcct gctttcccgc 2160aagtgtaaac
taaaacctgc tccatggggg tccacaaatt atatttttat acacagaatt
2220gtaaattaga tttttgagag atcaatacct aactgaatga catttcattt
tttgaaagtg 2280taaaatatga aaatatatta ttttaattta actattttcc
aatgtaatag ccgtcttctg 2340tactgccttc ttggtttgta tttgctttgt
aaccgccact ttgtcatgtt cttggaaacc 2400aagactgtta acgcacacat
atacactttt ttttttgaca gactggaaga actctgttat 2460ttttaacttc
aaagaattta ttagaaaata atatttttta aaagtgcacc tagcagcgag
2520cccacgagga tggagcctgt agtttgtaca gagaaaaaca aggatgtttt
tgcattaata 2580aactgagaag taactgctgt aaatttacta aaatgtattt
ttgaatattt tgtaatagtt 2640ttatagaaat aaagcgtgcc acacacaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaa
2727
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