U.S. patent application number 14/936425 was filed with the patent office on 2017-07-13 for neurodegenerative diseases and methods of modeling.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Francesco Paolo Di Giorgio, Kevin C. Eggan.
Application Number | 20170196839 14/936425 |
Document ID | / |
Family ID | 42196899 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170196839 |
Kind Code |
A1 |
Eggan; Kevin C. ; et
al. |
July 13, 2017 |
NEURODEGENERATIVE DISEASES AND METHODS OF MODELING
Abstract
The invention relates to methods for neuroprotection, promoting
survival of motor neurons and the treatment of motor neuron
diseases by preventing cell signaling through the classic
prostaglandin D2 receptor DP1.
Inventors: |
Eggan; Kevin C.; (Boston,
MA) ; Di Giorgio; Francesco Paolo; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
42196899 |
Appl. No.: |
14/936425 |
Filed: |
November 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12570476 |
Sep 30, 2009 |
9180114 |
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14936425 |
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61200293 |
Nov 26, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4166 20130101;
A61P 25/00 20180101; A61K 31/403 20130101; Y02A 50/30 20180101;
A61K 31/7052 20130101; Y02A 50/465 20180101; A61K 31/4164 20130101;
A61K 31/40 20130101 |
International
Class: |
A61K 31/4166 20060101
A61K031/4166 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under RO1
HD046732-01A1 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1-19. (canceled)
20. A method of treating a subject with a motor neuron disease, the
method comprising administering to a subject in need thereof an
inhibitor of a prostaglandin D2 DP1 receptor.
21. The method of claim 20, wherein the inhibitor of a
prostaglandin D2 DP1 receptor is selected from the group consisting
of: a small molecule, a nucleic acid molecule, a protein and
combinations thereof.
22. The method of claim 20, wherein the inhibitor of a
prostaglandin D2 DP1 receptor is a small molecule.
23. The method of claim 20, wherein the inhibitor of the
prostaglandin D2 DP1 receptor comprises the compound of formula
(I): ##STR00015## wherein: R.sup.1 is cycloalkyl, heterocycloalkyl,
aryl, or heteroaryl, each of which can be optionally substituted;
R.sup.2 is H, halo, alkyl, alkenyl or alkynyl, each of which can be
optionally substituted; R.sup.3 is H, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which
can be optionally substituted; and X is NH, C(O) or CH.sub.2.
24. The method of claim 23, wherein the inhibitor of the
prostaglandin D2 DP1 receptor is the compound: ##STR00016##
25. The method of claim 20, wherein the inhibitor of the
prostaglandin D2 DP1 receptor is the compound: ##STR00017##
26. The method of claim 20, wherein the motor neuron disease is
selected from a group consisting of amyotrophic lateral sclerosis
(ALS), primary lateral sclerosis (PLS), progressive muscular
atrophy (PMA), pseudobulbar palsy, progressive bulbar palsy, spinal
muscular atrophy (SMA) and post-polio syndrome.
27. The method of claim 20, wherein the motor neuron disease is
amyotrophic lateral sclerosis (ALS).
28. The method of claim 20, wherein the motor neuron disease is
spinal muscular atrophy (SMA).
29. The method of claim 20, wherein the motor neuron disease is
associated with a SOD1 mutation in at least one allele.
30. The method of claim 20, wherein the inhibitor of the
prostaglandin D2 DP1 receptor counteract the toxic affects of a
glial cell carrying a SOD1 mutation in at least one allele.
31. The method of claim 20, wherein the inhibitor of the
prostaglandin D2 DP1 receptor inhibits cell death caused by glial
cells carrying a SOD1 mutation in at least one allele.
32. The method of claim 20, wherein the inhibitor of the
prostaglandin D2 DP1 receptor rescues motor neuron loss.
33. The method of claim 20, wherein the subject is a mammal.
34. The method of claim 20, wherein the subject is a human.
35. A method of treating a subject with a motor neuron disease
associated with a SOD1 mutation in at least one allele, the method
comprising administering to a subject in need thereof an inhibitor
of a prostaglandin D2 DP1 receptor and thereby treating the motor
neuron disease, wherein the inhibitor of the prostaglandin D2 DP1
receptor is the compound: ##STR00018##
36. The method of claim 35, wherein the motor neuron disease is
wherein the motor neuron disease is amyotrophic lateral
sclerosis.
37. The method of claim 35, wherein the inhibitor of the
prostaglandin D2 DP1 receptor inhibits cell death caused by glial
cells carrying a SOD1 mutation in at least one allele.
38. The method of claim 35, wherein the inhibitor of the
prostaglandin D2 DP1 receptor rescues motor neuron loss.
39. The method of claim 35, wherein the subject is a human.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U. S. C.
.sctn.119(e) of U.S. Provisional Application No. 61/200,293, filed
Nov. 26, 2008. The entire contents of which is hereby incorporated
by reference.
BACKGROUND
[0003] Amyotrophic Lateral Sclerosis ("ALS"), also known as Lou
Gehrig's disease, is a progressive neurodegenerative disease
characterized by the loss of upper and lower motor neurons,
culminating in muscle wasting and death from respiratory failure
(Boillee, S., Vande Velde, C. & Cleveland, D. W. ALS: a disease
of motor neurons and their nonneuronal neighbors. Neuron 52, 39-59,
2006). The majority of ALS cases are apparently sporadic, with 90%
of patients presenting disease symptoms without a family history of
ALS. The remaining 10% of ALS patients are diagnosed with familial
ALS (Boillee et al., 1996; Brown, R. H., Jr. Amyotrophic lateral
sclerosis. Insights from genetics. Arch Neurol 54, 1246-50, 1997;
Cole, N. & Siddique, T. Genetic disorders of motor neurons.
Semin Neurol 19, 407-18, 1999). Approximately 25% of the familial
cases of ALS are caused by dominant mutations in the gene encoding
super oxide dismutase (SOD1) (Rosen, D. R. et al. Mutations in
Cu/Zn superoxide dismutase gene are associated with familial
amyotrophic lateral sclerosis. Nature 362, 59-62, 1993).
Identification of pathogenic alleles of SOD1 has led to the
production of transgenic mouse and rat models for the study of ALS
(Gurney, M. E. et al. Motor neuron degeneration in mice that
express a human Cu, Zn superoxide dismutase mutation. Science 264,
1772-5, 1994; Nagai, M. et al. Rats expressing human cytosolic
copper-zinc superoxide dismutase transgenes with amyotrophic
lateral sclerosis: associated mutations develop motor neuron
disease. J. Neurosci. 21, 9246-54, 2001; Bruijn, L. I. et al.
ALS-linked SOD1 mutant G85R mediates damage to astrocytes and
promotes rapidly progressive disease with SOD1-containing
inclusions. Neuron 18, 327-38, 1997; Wong, P. C. et al. An adverse
property of a familial ALS-linked SOD1 mutation causes motor neuron
disease characterized by vacuolar degeneration of mitochondria.
Neuron 14, 1105-16, 1995). Overproduction of pathogenic human SOD1
protein in mice and rats leads to late onset, progressive
neurodegenerative disease (Gurney et al., 1994; Bruijn et al.,
1997; Wong et al., 1995). Studies of the SOD1 animal models have
led to the identification and study of intrinsic pathogenic
characteristics of ALS motor neurons including the formation of
protein aggregates, cytoskeletal abnormalities, proteosome
dysfunction and increased sensitivity to cell death signals
(Boillee et al., 2006; Bruijn, L. I., Miller, T. M. &
Cleveland, D. W. Unraveling the mechanisms involved in motor neuron
degeneration in ALS. Annu Rev Neurosci 27, 723-49, 2004).
[0004] Studies of chimeric mice suggest that non-cell autonomous
processes contribute to motor neuron death in ALS (Clement, A. M.
et al. Wild-type nonneuronal cells extend survival of SOD1 mutant
motor neurons in ALS mice. Science 302, 113-7, 2003). In animals
bearing both wild-type cells and cells harboring the SOD1G93A
transgene, wild-type neurons surrounded by transgenic non-neuronal
cells acquired cellular phenotypes characteristic of ALS (Clement
et al., 2003). Conversely, transgenic neurons associated with
wild-type non-neuronal cells were increasingly spared. However,
these animal studies did not identify which cells were involved in
the pathological interactions with motor neurons due to the complex
cellular milieu of both the spinal chord and the muscle.
Conditional mutagenesis experiments in which the SOD1 transgene was
specifically removed from motor neurons and microglial cells led to
an increase in animal lifespan, again suggesting the SOD1 protein
can have both cell autonomous and non-cell autonomous affects in
the disease (Boillee, S. et al. Onset and progression in inherited
ALS determined by motor neurons and microglia. Science 312,
1389-92, 2006). However, these experiments could not address the
direct effect of cellular interactions with motor neurons in the
disease because of the use of death as an endpoint.
BRIEF SUMMARY OF THE INVENTION
[0005] This invention relates to methods for neuroprotection,
promoting survival of motor neurons and the treatment of motor
neuron diseases by preventing cell signaling through the classic
prostaglandin D2 receptor DP1.
[0006] Embodiments of the present invention provide methods of
treating motor neuron disease (MND), the methods comprise
administering an inhibitor of a prostaglandin D2 DP1 receptor (also
known as AS1, ASRT1, DP, DP1, MGC49004) to a subject in need
thereof, wherein the inhibitor inhibits expression or activity the
prostaglandin D2 DP1 receptor.
[0007] The inhibitor of a prostaglandin D2 DP1 receptor is selected
from the group consisting of: a small molecule, a nucleic acid
molecule, a protein e. g. an activity-blocking antibody or a
peptidominetic, and combinations thereof. For example, a small
molecule can be a DP1 receptor specific antagonist; a nucleic acid
can be a RNA interference molecule that inhibits the expression of
the PTGDR gene; and an anti-DP1 receptor specific antibody can be
an antibody or fragment thereof that blocks the receptor-ligand
binding, the ligand being prostaglandin.
[0008] In one embodiment, the inhibitor is selected from a group
consisting of an anti-DP1 antibody, an anti-PGD2 antibody, a DP1
specific RNA interfering agent, MK-0524, BWA868C, ONO-4127Na and
resveritrol. Combinations of these inhibitors can be administered
to the subject. A combination of routes of administration is also
contemplated.
[0009] In one embodiment, the inhibitor is administered with
therapeutics typically used for the treatment of MND, e. g.
riluzole.
[0010] In one embodiment of the methods described herein further
comprising selecting a subject diagnosed with motor neuron disease.
The subject is a mammal having motor neurons, e, g. humans, dogs,
cats etc.
[0011] In some embodiments, the motor neuron disease includes but
is not limited to amyotrophic lateral sclerosis (ALS), primary
lateral sclerosis (PLS), progressive muscular atrophy (PMA),
pseudobulbar palsy, progressive bulbar palsy, spinal muscular
atrophy (SMA) and post-polio syndrome.
[0012] In one embodiment, the motor neuron disease is associated
with a SOD1 mutation in at least one allele.
BRIEF DESCRIPTION OF THE DRAWING
[0013] FIGS. 1A-1B show one embodiment of derivation of Hb9GFP;
SOD1 mouse ES cell lines. PCRs; (FIG. 1A) for human SOD1 and 112,
and (FIG. 1B) for GFP in Hb9::GFP, Hb9::GFP; SOD1 and Hb9GFP;
SOD1G93A ES cell lines.
[0014] FIGS. 2A-2F show the effect of genetic background on motor
neuron survival. Number of GFP positive cells derived from (FIG.
2A) Hb9GFP and (FIG. 2B) SOD1G93A ES cell lines present 15, 30, 45
and 60 days after dissociation of EBs plated at two different
concentrations (8.times.10.sup.5 and 4.times.10.sup.5 per well).
(FIG. 2C) Number of GFP positive motor neurons derived from Hb9GFP
and SOD1G93A 15 and 30 days after EB dissociation plated at the
concentration of 8.times.10.sup.5 (C, D) and 4.times.10.sup.5 (E,
F) per well. (FIG. 2D, FIG. 2F). Same experiments in (FIG. 2C, FIG.
2D) analyzed as percent of GFP positive motor neurons derived from
Hb9GFP and SOD1G93A cell lines present at day 15, which still
remain at 30 days.
[0015] FIG. 3 shows intracellular aggregation of SOD1 protein in
cultured motor neurons. Percentage of GFP-Positive motor neurons
with SOD1 inclusions present after 21 days in culture.
[0016] FIG. 4 shows the graph shows percentage of Hb9GFP positive
cells over time in all the conditions studied. Glial cell genotype
directly affects motor neuron survival in culture. Experiments were
made in triplicate and results were normalized to the number of
cells found at 7 days in vitro.
[0017] FIGS. 5A-5D show the percentage of differentiating EB cells
that express GFP. FACS analysis of cells dissociated from EBs after
5 days of treatment with retinoic acid and shh. (FIG. 5A) Non
transgenic cell line, (FIG. 5B) Hb9GFP, (FIG. 5C) Hb9GFP; SOD1,
(FIG. 5D) Hb9GFP; SOD1G93A. The dot plots are representative of one
experiment, but the percentages are the average of three different
experiments. Calcein blue was used to assay the viability of cells
during sorting.
[0018] FIGS. 6A-6D show quantitative and qualitative analysis of
SOD1 protein inclusions in ES cell derived motor neurons. (FIG. 6A)
Average area of SOD1 inclusions in SOD1 and SOD1G93A derived motor
neurons. (FIG. 6B) Average length of SOD1 inclusions in SOD1 and
SOD1G93A derived motor neurons. (FIG. 6C) Integrated Optical
Density of SOD1 inclusions in SOD1 and SOD1G93A derived motor
neurons. (FIG. 6D) Distribution of inclusion bodies per cell in
SOD1 and SOD1G93A derived motor neurons. Results are graphed as
mean+/-S.E.M.
[0019] FIGS. 7A-7B show characterization of primary glial
monolayers derived from SOD1 and SOD1G93A mice. (FIGS. 7A, 7B)
Summary of immuno-fluorescent analysis of glia markers GFAP, S100,
RC2, Vimentin, CD 11 b, CNPase for both wt glia (FIG. 7A) and
SOD1G93A glia (FIG. 7B) at different time points.
[0020] FIGS. 8A-8C show one embodiment of differentiation of human
ES cells into motor neurons. (FIG. 8A) Diagram outlining the
protocol used to differentiate human ES cells into motor neurons:
Undifferentiated human ES cell colonies are dissociated in
collagenase, and grown as EBs for the first 14 days in EB media,
then are induced to a rostrocaudal identity with retinoic acid (RA)
and Shh for another 14 days. Finally, EBs are matured in the
presence of GDNF for 14 more days. At this point the EBs can either
be plated whole or dissociated with papain and then plated. (FIG.
8B) Percentage of cells immuno-reactive for HB9 after treatment
with or without RA and Shh. (FIG. 8C) Percentage of cells
immuno-reactive for HB9 after 42 days of differentiation in
different HuES cell lines.
[0021] FIG. 9 shows characterization of the Hb9::GFP human ES cell
line. DNA construct used for the electroporation of human ES
cells.
[0022] FIGS. 10A-10D show the effect of glial cells over expressing
SOD1G93A on human ES cell-derived motor neurons. (FIG. 10A)
Experimental design: embryonic stem cells were differentiated into
motor neurons, and an equal number of cells were plated on two
different glial monolayers; one derived from mice over-expressing
SOD1G93A, and the other derived from non-transgenic mice (WT).
Motor neurons were counted after 10 and 20 days in co-culture.
(FIG. 10B) Number of HB9 positive cells 10 days after plating on
SOD1G93A or non-transgenic (WT) glia. (FIG. 10C) Number of HB9
positive cells 20 days after plating on SOD1G93A or non-transgenic
(WT) glia. (FIG. 10D) Number of Hb9::GFP cells 20 days after
plating on SOD1G93A glia or non-transgenic (WT) glia or glia
over-expressing the wild type form of human SOD1 (SOD1WT).
[0023] FIGS. 11A-11E show the specificity of the toxic effect of
glia overexpressing SOD1G93A on motor neurons. (FIG. 11A)
Experimental design: embryonic stem cells were differentiated into
motor neurons, and an equal number of cells were plated on two
different glial monolayers; one derived from mice over-expressing
the mutation SOD1G93A, and the other derived from non-transgenic
mice (WT). Human ES cell derived interneurons were counted after 20
days in co-culture using two different markers, CHX10 and LHX2.
(FIG. 11B) Number of LHX2 positive cells 20 days after plating on
SOD1G93A glia or non-transgenic (WT) glia. (FIG. 11C) Number of
CHX10 positive cells 20 days after plating on SOD1G93A or
non-transgenic (WT) glia. (FIG. 11D) Experimental design: embryonic
stem cells were differentiated into motor neurons and same number
of cells was plated on two different MEF monolayers; one derived
from mice over-expressing the mutation SOD1 G93A, and the other
derived from non-transgenic mice (WT). (FIG. 1E) Motor neurons were
counted after 20 days to compare the two conditions.
[0024] FIGS. 12A-12B show neuronal marker expression at different
time points during one embodiment of differentiation from human ES
cells toward the motor neuron fate. (FIG. 12A) Percent of sectioned
EBs (n=20) staining positive for PAX6, NKX6.1, ISL1/2, or HB9 at
day 0, day 14, day 28, and day 42 of differentiation. (FIG. 12B)
Percent of cells per sectioned EB (n=3) staining positive for PAX6,
NKX6.1, ISL1/2, or HB9 at day 0, day 14, day 28, and day 42 of
differentiation.
[0025] FIG. 13 shows characterization of a Hb9::GFP human ES cell
line. Number of Hb9::GFP cells that are immunoreactive to Hb9
antibody (Hb9+Hb9::GFP).
[0026] FIG. 14A is a Venn Diagram presenting the overlap among
transcripts selectively over expressed in SOD1G93A glia and in
SOD1WT glia with respect to WT glia.
[0027] FIG. 14B is a table listing a subset of genes over expressed
in SOD1G93A glia but not in SOD1WT glia or WT glia.
[0028] FIG. 14C is a graph showing the percentage of Hb9::GFP cells
remaining on non-transgenic (WT) glia after 20 days of treatment
with GMFb, Rantes, Cxcl 7, Mcp 2, Shh or PGD2 compared to the
untreated condition (Ctrl) (n=3).
[0029] FIG. 14D is a graph showing the percentage of Hb9::GFP cells
remaining on WT glia or SOD1G93A glia after 20 days of treatment
with the inhibitor of Prostaglandin D2 receptor, a DP1 antagonist
MK 0524 (n=3).
[0030] FIG. 14E is a graph showing the percentage of Hb9::GFP cells
remaining on WT glia or SOD1G93A glia after 10 days of treatment
with the inhibitor of Prostaglandin D2 receptor, a DP2 antagonist
BAY-u3405.
[0031] FIG. 14F is a graph showing the percentage of Hb9::GFP cells
remaining on WT glia or SOD1G93A glia after 10 days of treatment
with the inhibitor of Prostaglandin D2 receptor, a DP1 antagonist
BW868C.
DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0032] Embodiments of the present invention provide methods of
treating motor neuron disease (MND), the methods comprise
administering an inhibitor of a prostaglandin D2 DP1 receptor (also
known as AS1, ASRT1, DP, DP1, MGC49004) to a subject in need
thereof, wherein the inhibitor inhibits expression or activity the
prostaglandin D2 DP1 receptor.
[0033] The inventors demonstrated that the inhibitor of
Prostaglandin D2 receptor (MK 0524) and BW868C significantly
reduced neuronal cell death for motor neurons cultured on SOD1G93A
glia.
[0034] In some embodiments, the inhibitor of a prostaglandin D2 DP1
receptor is selected from the group consisting of: a small
molecule, a nucleic acid molecule, a protein, e.g. an
activity-blocking antibody or a peptidomimetic, and combinations
thereof. For example, a small molecule can be a DP1 receptor
antagonist; a nucleic acid can be a RNA interference molecule that
inhibits the expression of the PTGDR gene; an anti-DP1 antibody can
be an antibody that blocks the receptor-ligand binding.
[0035] In one embodiment, the inhibitor is:
##STR00001##
[0036] In another embodiment, the inhibitor is:
##STR00002##
[0037] In another embodiment, the inhibitor is:
##STR00003##
[0038] In one embodiment, the inhibitor is selected from a group
consisting of an anti-DP1 antibody, an anti-PGD2 antibody, a DP1
specific RNA interfering agent, DP1 antagonist such as MK-0524,
BWA868C, ONO-4127Na and resveritrol. Combinations of these
inhibitors can be administered to the subject. A combination of
routes of administration is also contemplated.
[0039] The present invention also provides a method for treating
motor neuron disease (MND) comprising administering the compound of
formula (I):
##STR00004##
[0040] wherein
[0041] R.sup.1 is cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl, each of which can be optionally substituted;
[0042] R.sup.2 is H, halo, alkyl, alkenyl or alkynyl, each of which
can be optionally substituted;
[0043] R.sup.3 is H, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl, each of which can be
optionally substituted; and
[0044] X is NH, C(O) or CH.sub.2.
[0045] In some embodiments, X is NH.
[0046] In some embodiments, R.sup.1 is an aryl or heteroaryl. In
some embodiments, R.sup.1 is a bicyclic aryl or heteroaryl. In some
embodiments, R.sup.1 is
##STR00005##
wherein R.sup.4 is halo, C.sub.1-C.sub.6 alkyl, OR.sup.5,
NHR.sup.5, NO.sub.2, CF.sub.3 or CN; R.sup.5 is H or optionally
substituted alkyl; and m is 0-5. In some embodiments m is 0 or 1.
In some embodiments, R.sup.1 is
##STR00006##
[0047] In some embodiments, R.sup.5 is H.
[0048] In some embodiments, halo is F.
[0049] In some embodiments, R.sup.2 is an alkyl, e.g.,
C.sub.1-C.sub.10 alkyl. In some embodiments, R.sup.2 is a
substituted C.sub.1-C.sub.10 alkyl and the substituent is selected
from the group consisting of OR.sup.5, halo, .dbd.O,
CO.sub.2R.sup.5, NHR.sup.5, NO.sub.2, CN or CF.sub.3; and R.sup.5
is H or optionally substituted alkyl. In some embodiments, R.sup.2
is --(CH.sub.2).sub.pCO.sub.2H, wherein p is 0-9. In some
embodiment p is 6.
[0050] In some embodiments, R.sup.3 is C.sub.1-C.sub.6 alkyl.
[0051] In some other embodiments, R.sup.3 is a substituted alkyl.
In some embodiments, R.sup.3 is an alkyl substituted with an
optionally substituted cycloalkyl substituent.
[0052] In certain embodiments, R.sup.3 is a disubstituted alkyl. In
some embodiments, R.sup.3 is an alkyl substituted with an
optionally substituted cycloalkyl substituent and one other
substituent selected from the group consisting of OR.sup.5, halo,
.dbd.O, CO.sub.2R.sup.5, NHR.sup.5, NO.sub.2, CN or CF.sub.3; and
R.sup.5 is H or optionally substituted alkyl. In some embodiments,
R.sup.3 is an alkyl substituted by two substituents at the same
backbone carbon.
[0053] In some embodiment, R.sup.3 is:
##STR00007##
[0054] In one embodiment, compound of formula (I) is:
##STR00008##
[0055] In another embodiment, the present invention provides a
method for treating motor neuron disease (MND) comprising
administering the compound of formula (II):
##STR00009##
[0056] wherein:
[0057] R.sup.1 is H or C.sub.1-C.sub.6 alkyl;
[0058] R.sup.2 is aryl or heteroaryl, each of which can be
optionally substituted;
[0059] R.sup.3 and R.sup.4 are each independently is halo,
--CF.sub.3, --CN, --NO.sub.2, --S(.dbd.O)alkyl, --SO.sub.2alkyl,
C.sub.1-C.sub.6 alkyl; --C(O)alkyl, --CH(OH)alkyl; and
[0060] m is 1, 2 or 3.
[0061] In some embodiments, m is 1 or 2.
[0062] In some embodiments, R.sup.1 is H.
[0063] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl, e.g., a monosubstituted aryl. In some embodiments, R.sup.2 is
a substituted phenyl. In some embodiments, R.sup.2 is a substituted
aryl, wherein the substituent is selected from the group consisting
of OR.sup.5, halo, .dbd.O, CO.sub.2R.sup.5, NHR.sup.5, NO.sub.2, CN
or CF.sub.3; and R.sup.5 is H or optionally substituted alkyl. In
some embodiments, R.sup.2 is
##STR00010##
[0064] In some embodiments, the compound of formula (II) is:
##STR00011##
[0065] In some embodiments, the compound of formula (II) is:
##STR00012##
[0066] In some embodiments, R.sup.3 and R.sup.4 is halo and other
is --S(O.sub.2)CH.sub.3. In some embodiment, R.sup.3 is halo and
R.sup.4 is --S(O.sub.2)CH.sub.3. In some embodiments, R.sup.3 is F
and R.sup.4 is --S(O.sub.2)CH.sub.3. In some embodiments, R.sup.4
is F and R.sup.3 is --S(O.sub.2)CH.sub.3.
[0067] In some embodiments, both R.sup.3 and R.sup.4 are halo. In
some embodiments, R.sup.3 is F and R.sup.4 is Br.
[0068] In some embodiments, one of R.sup.3 and R.sup.4 is halo and
other is --C(.dbd.O)CH.sub.3. In some embodiments, R.sup.3 is halo
and R.sup.4 is --C(.dbd.O)CH.sub.3. In some embodiments, R.sup.3 is
F and R.sup.4 is --C(.dbd.O)CH.sub.3.
[0069] In some embodiments, one of R.sup.3 and R.sup.4 is halo and
other is --CH(OH)CH.sub.3. In some embodiments, R.sup.3 is halo and
R.sup.4 is --CH(OH)CH.sub.3.
[0070] In some embodiments, one of R.sup.3 and R.sup.4 is
--S(O.sub.2)CH.sub.3 and other is --C(.dbd.O)CH.sub.3 or
--CH(OH)CH.sub.3. In some embodiments, R.sup.3 is
--S(O.sub.2)CH.sub.3 and R.sup.4 is --C(.dbd.O)CH.sub.3. In some
embodiments, R.sup.3 is --S(O.sub.2)CH.sub.3 and R.sup.4 is
--CH(OH)CH.sub.3:
[0071] In some embodiments, the compound of formula (II) is:
##STR00013##
[0072] In some embodiments, the compound of formula (II) is:
##STR00014##
[0073] In one embodiment, the method described herein comprise
administering therapeutics typically used for the treatment of MND,
e. g. riluzole.
[0074] In one embodiment of the methods described herein further
comprising selecting a subject diagnosed with motor neuron
disease.
[0075] In some embodiments, the motor neuron disease includes but
is not limited to amyotrophic lateral sclerosis (ALS), primary
lateral sclerosis (PLS), progressive muscular atrophy (PMA),
pseudobulbar palsy, progressive bulbar palsy, spinal muscular
atrophy (SMA) and post-polio syndrome.
[0076] In one embodiment, the motor neuron disease is associated
with a SOD1 mutation in at least one allele.
[0077] In certain embodiments, the present invention provides
compositions and methods for detailed mechanistic studies of the
interactions between cells such as, without limitation, motor
neurons and other cells such as, without limitation, glia. In
certain embodiments, a motor neuron of the present invention
comprises a mutant motor neuron comprising an allele associates
with a neurodegenerative disease, such as, without limitation, ALS.
In certain embodiments, a mutant motor neuron comprises a SOD1
mutant allele associated with ALS. For example, a mutant motor
neuron may comprise a SOD1G93A allele. In certain embodiments,
compositions and methods of the present invention provide an assay
for diffusible factor(s), agent(s), etc. toxic to motor neurons. In
certain embodiments, the present invention provides a high
throughput cell based assay for small molecules that promote
survival of mutant SOD1 motor neurons. The present disclosure
validates the use of ES cells carrying disease-causing genes to
study disease mechanisms.
[0078] In certain embodiments, the present invention provides an
embryonic stem cell comprising a mutation in a gene involved in
motor neuron development and/or maintenance. In certain
embodiments, the present invention provides a motor neuron
generated by differentiating such an embryonic stem cell under
conditions wherein the embryonic stem cell adopts a motor neuron
cell fate. In certain embodiments, the present invention provides
an embryonic stem cell and/or a motor neuron comprising a mutation
in the SOD1 gene. For example, an embryonic stem cell and/or motor
neuron of the present invention may comprise a SOD1 mutation
wherein a glycine is substituted for the wild type alanine at
position 93 of the SOD1 amino acid sequence (referred to herein as
a "SOD1G93A" mutation or allele). In certain embodiments, such a
mutation in a SOD1 gene is associated with a neurodegenerative
disease.
[0079] In certain embodiments, an embryonic stem (ES) cell is
derived from a mouse bearing a transgene comprising a SOD1 allele,
such as without limitation, a SOD1G93A allele. In certain
embodiments, such a mouse bears a transgene comprising a human
SOD1G93A allele. Such a transgenic mouse is known to recapitulate
many pathologies of the human ALS disease. In certain embodiments,
an embryonic stem (ES) cell is a human ES cell bearing a transgene
comprising a SOD1 allele, such as without limitation, a SOD1G93A
allele. In certain embodiments, transgenic ES cells are
differentiated into motor neurons in large numbers (e.g., such as
by one or more methods described in Wichterle, H., Lieberam, I.,
Porter, J. A. & Jessell, T. M. Directed differentiation of
embryonic stem cells into motor neurons. Cell 110, 385-97, 2002)
and co-cultured either with ES-derived cells that arise during the
differentiation process and/or with other cells that contribute to
the survival, maintenance and/or differentiation of such transgenic
ES cells. For example, transgenic ES cells may be differentiated
into motor neurons in the presence of primary mouse and/or human
glial cells. In certain embodiments, such primary mouse and/or
human glial cells comprise a wild-type genotype. In certain
embodiments, such primary mouse and/or human glial cells comprise a
non-wild-type genotype. For example, such glial cells may comprise
a mutant SOD1 allele, e.g., a SOD1G93A allele. Such a mutant SOD1
allele may be provided as a transgene.
[0080] In certain embodiments, motor neurons of such cultures
display one or more abnormalities typical of a phenotype observed
in a particular disease. In certain embodiments, motor neurons of
such cultures display one or more abnormalities typical of a
phenotype observed in a neurodegenerative disease. For example,
such motor neurons may display abnormalities typical of those seen
in the motor neurons of ALS patients and/or ALS transgenic
animals.
[0081] In certain embodiments, the present invention provides novel
in vitro model systems to study ALS and/or other neurodegenerative
diseases, in which the factors directly influencing motor neuron
development, differentiation and/or survival can be investigated.
Certain of such systems are based on the differentiation of
embryonic stem (ES) cells derived from mice comprising a mutant
SOD1 allele. Certain of such systems are based on the
differentiation of human embryonic stem (ES) cells comprising a
mutant SOD1 allele. An exemplary mutant SOD1 allele that can be
used in accordance with methods and compositions of the present
invention is the SOD1G93A mutation, although systems of the present
invention are not limited to this mutation.
[0082] In certain embodiments, in vitro model systems of the
present invention are used to screen for a test agent that affects
the development, differentiation and/or survival of motor neurons.
In certain embodiments, such in vitro model systems are used to
screen for a test agent that affects the survival of wild type
motor neurons. In certain embodiments, such in vitro model systems
are used to screen for a test agent that affects the development,
differentiation and/or survival of motor neurons that comprise one
or more mutations. For example, such mutant motor neurons may
comprise a mutation associated with a neurodegenerative disease,
such as for example, ALS. In certain embodiments, such mutant motor
neurons comprise a mutation in the SOD1 gene, e.g. a SOD1G93A
mutation. In certain embodiments, the invention provides methods of
identifying an agent that affects the survival of a mutant motor
neuron. For example, certain of such methods comprise providing a
mutant motor neuron comprising a SOD1 mutant allele, providing a
test agent, contacting the mutant motor neuron with the test agent,
and determining the effect of the test agent on survival of the
mutant motor neuron by comparing the survival of the mutant motor
neuron to the survival of a control motor neuron lacking the SOD1
mutant allele, which control motor neuron is contacted with the
test agent for a period of time and under conditions identical to
that of the SOD1 mutant motor neuron. In certain embodiments, a
test agent is a cell, a small molecule, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an antibody, an amino acid,
and/or a virus. In certain embodiments, the test agent is an agent
that reduces the expression or activity of a gene or a product of a
gene in Table 2 (e.g., a product of a gene in Table 2 which is
involved in inflammation, an immune response, transcription,
signaling, or a metabolic pathway). In certain embodiments, the
test agent is an agent that reduces the expression or activity of a
prostaglandin D receptor.
[0083] In certain embodiments, the invention provides methods of
identifying a factor that has a non-cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a motor neuron, identifying a first cell, which
first cell negatively affects survival of the motor neuron,
identifying a second cell, which second cell does not negatively
affect survival of the motor neuron, isolating a factor from the
either the first or second cell, wherein the factor is either: i) a
factor from the first cell that contributes to the negative effect
on survival of the motor neuron; or ii) a factor from the second
cell that contributes to survival of the motor neuron. In certain
embodiments, the first cell, second cell or both is a glial cell.
In certain embodiments, the first cell, second cell or both
comprises a mutation that is associated with amyloid lateral
sclerosis, e.g. a SOD1 mutation such as without limitation a
SOD1G93A allele.
[0084] In certain embodiments, the invention provides methods of
identifying a factor that has a non-cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a motor neuron, culturing the motor neuron in
the presence of a test cell such that survival of the motor neuron
is negatively affected as compared to survival of a motor neuron
cultured in the presence of a control cell, and identifying a
factor present in the test cell, which factor contributes to the
negative effect on survival of the motor neuron. In certain
embodiments, the invention provides methods of identifying a factor
that has a non-cell autonomous effect on the survival of a motor
neuron, which methods comprise providing a motor neuron, culturing
the motor neuron in the presence of a test cell such that survival
of the motor neuron is negatively positively affected as compared
to survival of a motor neuron cultured in the presence of a control
cell, and identifying a factor that is absent in the test cell,
which factor contributes to the positive effect on survival of the
motor neuron.
[0085] In certain embodiments, the invention provides methods of
identifying a test agent that modulates the non-cell autonomous
effect of a test cell on the survival of a motor neuron. For
example, certain of such methods comprise providing a motor neuron,
culturing the motor neuron in the presence of a (i) test cell such
that survival of the motor neuron is negatively affected as
compared to survival of a motor neuron cultured in the presence of
a control cell, and (ii) a test agent, wherein a change in the
survival of the motor neuron in the presence of the test agent as
compared to the survival of the motor neuron in the absence of the
test agent indicates that the test agent modulates the non-cell
autonomous effect of a test cell. In certain embodiments, the test
agent is an agent that reduces the expression or activity of a gene
or a product of a gene in Table 2 (e.g., a product of a gene in
Table 2 which is involved in inflammation, an immune response,
transcription, signaling, or a metabolic pathway). In certain
embodiments, the test agent is an agent that reduces the expression
or activity of a prostaglandin D receptor.
[0086] In certain embodiments, the invention provides methods of
identifying a factor that has a cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a mutant motor neuron comprising a first SOD1
mutation, providing a control motor neuron lacking the first SOD1
mutation, culturing the mutant motor neuron, determining the effect
of the first SOD1 mutation on survival of the mutant motor neuron
by comparing the survival of the mutant motor neuron to the
survival of the control motor neuron, which control motor neuron is
cultured for a period of time and under conditions identical to
that of the mutant motor neuron, and isolating a factor from the
either the mutant motor neuron or the control motor neuron, wherein
the factor is either: i) a factor from the mutant motor neuron that
contributes to the negative effect on survival of the motor neuron;
or ii) a factor from the control motor neuron that contributes to
survival of the motor neuron.
[0087] In certain embodiments, in vitro model systems of the
present invention are used to screen for a factor that has a
non-cell autonomous effect on the development, differentiation
and/or survival of a motor neuron, e.g. a SOD1 mutant motor neuron
such as a SOD1G93A mutant motor neuron. In certain embodiments,
such a factor comprises a factor originating from another motor
neuron. In certain embodiments, such a factor comprises a factor
originating from another cell that is not a motor neuron. In
certain embodiments, such a factor originates from a glial cell.
Such a factor may have a negative effect on the development,
differentiation and/or survival of a motor neuron. Alternatively,
such a factor may contribute to the development, differentiation
and/or survival of a motor neuron such that its absence negatively
affects survival of the motor neuron.
[0088] In vitro model systems of the present invention are useful
for the identification of factors, agents, etc. that both
positively and negatively affect motor neuron survival. In certain
embodiments, one or more factors, agents, etc. that affect motor
neuron survival are identified by using SOD1 mutant glial cells,
e.g. glial cells comprising a SOD1G93A mutation. In certain
embodiments, in vitro model systems of the present invention
utilize human motor neurons and/or other cell types derived from
human ES cells. Such in vitro model systems may be advantageously
employed for the human physiological validation of findings from
animal models, such as, without limitation, animal models that
recapitulate neurodegenerative diseases such as ALS and/or other
neurodegenerative diseases. Additionally or alternatively, such in
vitro model systems may be advantageously employed to identify
novel factors, agents, etc. that affect human motor neuron
development and/or contribute to a disease state, such as, without
limitation, a neurodegenerative disease, e.g. ALS, in the absence
of an animal model. Additionally or alternatively, such in vitro
model systems may be advantageously employed to illuminate the
target, efficacy, toxicity, mode of action, etc. of factors,
agents, etc. that affect human motor neuron development and/or
contribute to a disease state, such as, without limitation, a
neurodegenerative disease, e.g. ALS.
[0089] Certain embodiments of the present invention are discussed
in detail below. Those of ordinary skill in the art will
understand, however, that various modifications to these
embodiments are within the scope of the appended claims. It is the
claims and equivalents thereof that define the scope of the present
invention, which is not and should not be limited to or by this
description of certain embodiments.
Definitions
[0090] Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
Unless explicitly stated otherwise, or apparent from context, the
terms and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the
claims.
[0091] For simplicity, chemical moieties are defined and referred
to throughout can be univalent chemical moieties (e.g., alkyl,
aryl, etc.) or multivalent moieties under the appropriate
structural circumstances clear to those skilled in the art. For
example, an "alkyl" moiety can be referred to a monovalent radical
(e.g. CH.sub.3--CH.sub.2--), or in other instances, a bivalent
linking moiety can be "alkyl," in which case those skilled in the
art will understand the alkyl to be a divalent radical (e.g.,
--CH.sub.2--CH.sub.2--), which is equivalent to the term
"alkylene." Similarly, in circumstances in which divalent moieties
are required and are stated as being "alkoxy", "alkylamino",
"aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic",
"alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those
skilled in the art will understand that the terms "alkoxy",
"alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl", "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl" refer to the corresponding divalent moiety.
[0092] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0093] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents. Exemplary acyl groups include, but are
not limited to, (C.sub.1-C.sub.6)alkanoyl (e.g., formyl, acetyl,
propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),
(C.sub.3-C.sub.6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,
cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),
heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,
pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl,
piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g.,
benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl,
thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl,
1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,
benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl,
cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl
group may be any one of the groups described in the respective
definitions.
[0094] The term "alkyl" refers to saturated non-aromatic
hydrocarbon chains that may be a straight chain or branched chain,
containing the indicated number of carbon atoms (these include
without limitation methyl, ethyl, propyl, allyl, or propargyl),
which may be optionally inserted with N, O, S, SS, SO.sub.2, C(O),
C(O)O, OC(O), C(O)N or NC(O). For example, C.sub.1-C.sub.6
indicates that the group may have from 1 to 6 (inclusive) carbon
atoms in it.
[0095] The term "alkenyl" refers to an alkyl that comprises at
least one double bond. Exemplary alkenyl groups include, but are
not limited to, for example, ethenyl, propenyl, butenyl,
l-methyl-2-buten-1-yl and the like.
[0096] The term "alkynyl" refers to an alkyl that comprises at
least one triple bond.
[0097] The term "alkoxy" refers to an --O-alkyl radical.
[0098] The term "aminoalkyl" refers to an alkyl substituted with an
amino.
[0099] The term "mercapto" refers to an --SH radical.
[0100] The term "thioalkoxy" refers to an --S-alkyl radical.
[0101] The term "aryl" refers to monocyclic, bicyclic, or tricyclic
aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring
may be substituted by a substituent. Examplary aryl groups include,
but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl,
fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl,
and the like.
[0102] The term "arylalkyl" refers to alkyl substituted with an
aryl.
[0103] The term "cyclyl" or "cycloalkyl" refers to saturated and
partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, for example, 3 to 8 carbons, and, for example, 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Exemplary cycloalkyl groups include, but
are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,
and the like.
[0104] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Examplary heteroaryl groups include,
but are not limited to, pyridyl, furyl or furanyl, imidazolyl,
benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl,
pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the
like.
[0105] The term "heteroarylalkyl" refers to an alkyl substituted
with a heteroaryl.
[0106] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring may be
substituted by a substituent. Examplary heterocyclyl groups
include, but are not limited to piperazinyl, pyrrolidinyl,
dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
[0107] The term "haloalkyl" refers to an alkyl group having one,
two, three or more halogen atoms attached thereto. Exemplary
haloalkyl groups include, but are not limited to chloromethyl,
bromoethyl, trifluoromethyl, and the like.
[0108] The term "optionally substituted" means that the specified
group or moiety, such as an aryl group, heteroaryl group and the
like, is unsubstituted or is substituted with one or more
(typically 1-4 substituents) independently selected from the group
of substituents listed below in the definition for "substituents"
or otherwise specified.
[0109] The term "substituents" refers to a group "substituted" on
an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl,
heteroaryl, acyl, amino group at any atom of that group. Suitable
substituents include, without limitation, halo, hydroxy, oxo,
nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl,
alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl,
aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkylthio,
CF.sub.3, N-morphilino, phenylthio, alkanesulfonyl, arenesulfonyl,
alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,
alkylcarbonyl, acyloxy, cyano or ureido. In some embodiments,
substituent can itself be optionally substituted. In some cases,
two substituents, together with the carbons to which they are
attached to can form a ring.
[0110] An "antibody" that can be used according to the methods
described herein includes complete immunoglobulins, antigen binding
fragments of immunoglobulins, as well as antigen binding proteins
that comprise antigen binding domains of immunoglobulins. Antigen
binding fragments of immunoglobulins include, for example, Fab,
Fab', F(ab').sub.2, scFv and dAbs. Modified antibody formats have
been developed which retain binding specificity, but have other
characteristics that may be desirable, including for example,
bispecificity, multivalence (more than two binding sites), and
compact size (e.g., binding domains alone). Single chain antibodies
lack some or all of the constant domains of the whole antibodies
from which they are derived. Therefore, they can overcome some of
the problems associated with the use of whole antibodies. For
example, single-chain antibodies tend to be free of certain
undesired interactions between heavy-chain constant regions and
other biological molecules. Additionally, single-chain antibodies
are considerably smaller than whole antibodies and can have greater
permeability than whole antibodies, allowing single-chain
antibodies to localize and bind to target antigen-binding sites
more efficiently. Furthermore, the relatively small size of
single-chain antibodies makes them less likely to provoke an
unwanted immune response in a recipient than whole antibodies.
Multiple single chain antibodies, each single chain having one VH
and one VL domain covalently linked by a first peptide linker, can
be covalently linked by at least one or more peptide linker to form
multivalent single chain antibodies, which can be monospecific or
multispecific. Each chain of a multivalent single chain antibody
includes a variable light chain fragment and a variable heavy chain
fragment, and is linked by a peptide linker to at least one other
chain. The peptide linker is composed of at least fifteen amino
acid residues. The maximum number of linker amino acid residues is
approximately one hundred. Two single chain antibodies can be
combined to form a diabody, also known as a bivalent dimer.
Diabodies have two chains and two binding sites, and can be
monospecific or bispecific. Each chain of the diabody includes a VH
domain connected to a VL domain. The domains are connected with
linkers that are short enough to prevent pairing between domains on
the same chain, thus driving the pairing between complementary
domains on different chains to recreate the two antigen-binding
sites. Three single chain antibodies can be combined to form
triabodies, also known as trivalent trimers. Triabodies are
constructed with the amino acid terminus of a VL or VH domain
directly fused to the carboxyl terminus of a VL or VH domain, i.e.,
without any linker sequence. The triabody has three Fv heads with
the polypeptides arranged in a cyclic, head-to-tail fashion. A
possible conformation of the triabody is planar with the three
binding sites located in a plane at an angle of 120 degrees from
one another. Triabodies can be monospecific, bispecific or
trispecific. Thus, antibodies useful in the methods described
herein include, but are not limited to, naturally occurring
antibodies, bivalent fragments such as (Fab').sub.2, monovalent
fragments such as Fab, single chain antibodies, single chain Fv
(scFv), single domain antibodies, multivalent single chain
antibodies, diabodies, triabodies, and the like that bind
specifically with an antigen.
[0111] Antibodies can also be raised against a polypeptide or
portion of a polypeptide by methods known to those skilled in the
art. Antibodies are readily raised in animals such as rabbits or
mice by immunization with the gene product, or a fragment thereof.
Immunized mice are particularly useful for providing sources of B
cells for the manufacture of hybridomas, which in turn are cultured
to produce large quantities of monoclonal antibodies. Antibody
manufacture methods are described in detail, for example, in Harlow
et al., 1988 in: Antibodies, A Laboratory Manual, Cold Spring
Harbor, N.Y. While both polyclonal and monoclonal antibodies can be
used in the methods described herein, it is preferred that a
monoclonal antibody is used where conditions require increased
specificity for a particular protein.
[0112] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional nucleic
acid segments can be ligated. Another type of vector is a viral
vector, wherein additional nucleic acid segments can be ligated
into the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors", or more simply "expression vectors." In
general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, lentiviruses, adenoviruses and adeno-associated
viruses), which serve equivalent functions. In one embodiment,
lentiviruses are used to deliver one or more siRNA molecule of the
present invention to a cell.
[0113] Within an expression vector, "operably linked" is intended
to mean that the nucleotide sequence of interest is linked to the
regulatory sequence(s) in a manner which allows for expression of
the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a target cell when the
vector is introduced into the target cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). Furthermore, the RNA
interfering agents may be delivered by way of a vector comprising a
regulatory sequence to direct synthesis of the siRNAs of the
invention at specific intervals, or over a specific time period. It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the target cell, the level of expression of siRNA desired, and the
like.
[0114] The expression vectors of the invention can be introduced
into target cells to thereby produce siRNA molecules of the present
invention. In one embodiment, a DNA template, e.g., a DNA template
encoding the siRNA molecule directed against the mutant allele, may
be ligated into an expression vector under the control of RNA
polymerase III (Pol III), and delivered to a target cell. Pol III
directs the synthesis of small, noncoding transcripts which 3' ends
are defined by termination within a stretch of 4-5 thymidines.
Accordingly, DNA templates may be used to synthesize, in vivo, both
sense and antisense strands of siRNAs which effect RNAi (Sui, et
al. (2002) PNAS 99(8):5515).
[0115] "Agent", "Test agent": The terms "agent" and "test agent" as
used herein refer to a compound or other entity that is tested to
determine whether it has an effect on the differentiation,
development and/or survival of a cell. As non-limiting examples, a
test agent may comprise a cell, a small molecule, a hormone, a
vitamin, a nucleic acid molecule, an enzyme, an amino acid, and/or
a virus. Those of ordinary skill in the art will be aware of other
test agents that may be tested for their effect(s) on
differentiation, development and/or survival of a cell. In certain
embodiments, a differentiating cell is subjected to a test agent
before, during and/or after differentiation to determine its
effect(s) on differentiation, development and/or survival of a
cell. For example, an embryonic stem cell undergoing
differentiation into a cell type of interest may be subjected to a
test agent before, during and/or after differentiation. In certain
embodiments, an embryonic stem cell undergoing differentiation into
a motor neuron is subjected to a test agent before, during and/or
after differentiation. In certain embodiments, a test agent that is
identified as having one or more effects on the differentiation,
development and/or survival of a cell is used in the treatment,
prevention and/or cure of a disease of interest.
[0116] "Embryonic stem cell", "ES cell": The terms "embryonic stem
cell" and "ES cell" as used herein refer to an undifferentiated
stem cell that is derived from the inner cell mass of a blastocyst
embryo and is pluripotent, thus possessing the capability of
developing into any organ or tissue type or, at least potentially,
into a complete embryo. Embryonic stem cells appear to be capable
of proliferating indefinitely, and of differentiating into all of
the specialized cell types of a mammal, including the three
embryonic germ layers (endoderm, mesoderm, and ectoderm), and all
somatic cell lineages and the germ line. As non-limiting examples,
embryonic stem cells have been shown to be capable of being induced
to differentiate into cardiomyocytes (Paquin et al., Proc. Nat.
Acad. Sci., 99:9550-9555, 2002), hematopoietic cells (Weiss et al.,
Hematol. Oncol. Clin. N. Amer., 11(6):1185-98, 1997; also U.S. Pat.
No. 6,280,718), insulin-secreting beta cells (Assady et al.,
Diabetes, 50(8):1691-1697, 2001), and neural progenitors capable of
differentiating into astrocytes, oligodendrocytes, and mature
neurons (Reubinoff et al., Nature Biotechnology, 19:1134-1140,
2001; also U.S. Pat. No. 5,851,832). One of ordinary skill in the
art will be aware of other cell types that have been derived from
embryonic stem cells.
[0117] "SOD1": As will be clear from context, the term "SOD1" as
used herein refers to either the gene encoding superoxide dismutase
1 or the enzyme encoded by this gene. The SOD1 gene or gene product
is known by other names in the art including, but not limited to,
ALS1, Cu/Zn superoxide dismutase, indophenoloxidase A, IPOA, and
SODC_HUMAN. Those of ordinary skill in the art will be aware of
other synonymous names that refer to the SOD1 gene or gene product.
The SOD1 enzyme neutralizes supercharged oxygen molecules (called
superoxide radicals), which can damage cells if their levels are
not controlled. The human SOD1 gene maps to cytogenetic location
21q22.1. Certain mutations in SOD1 are associated with ALS in
humans including, but not limited to, Ala4Val, Gly37Arg and
Gly93Ala, and more than one hundred others. Those of ordinary skill
in the art will be aware of these and other human mutations
associated with ALS. Certain compositions and methods of the
present invention comprise or employ cells comprising a SOD1
mutation.
[0118] "SOD1 mutations" refer to mutations in the human SOD1 gene
(NC_000021.8; NT_011512.11; AC_000064.1; NW_927384.1; AC_000153.1;
NW_001838706.1 NM_000454.4; NP_000445.1 and NCBI Entrez GenelD:
6647) including but are not limited to Ala4Val, Cys6Gly, Val7Glu,
Leu8Val, Gly10Val, Gly12Arg, Val14Met, Gly16Ala, Asn19Ser,
Phe20Cys, Glu21Lys, Gln22Leu, Gly37Arg, Leu38Arg, Gly41Ser,
His43Arg, Phe45Cys, His46Arg, Val47Phe, His48Gln, Glu49Lys,
Thr54Arg, Ser59Ile, Asn65Ser, Leu67Arg, Gly72Ser, Asp76 Val,
His80Arg, Leu84Phe, Gly85Arg, Asn86Asp, Val87Ala, Ala89Val,
Asp90Ala, Gly93Ala, Ala95Thr, Asp96Asn, Val97Met, Glu100Gly,
Asp101Asn, Ile104Phe, Ser105Leu, Leu106Val, Gly108Val, Ile112Thr,
Ile113Phe, Gly114Ala, Arg115Gly, Val118Leu, Ala140Gly, Ala145Gly,
Asp124Val, Asp124Gly, Asp125His, Leu126Ser, Ser134Asn, Asn139His,
Asn139Lys, Gly141Glu, Leu144Phe, Leu144Ser, Cys146Arg, Ala145Thr,
Gly147Arg, Val148Gly, Val148Ile, Ile149Thr, Ile151Thr, and
Ile151Ser. SOD1 is also known as ALS, SOD, ALS1, IPOA, homodimer
SOD1. "SOD1 mutation" databases can be found at Dr. Andrew C. R.
Martin website at the University College of London (the World Wide
Web address at "www" "period" bioinf "period" org "period" uk), the
ALS/SOD1 consortium website (the World Wide Web address at "www"
"period" also "period" org) and the human gene mutation database
(HGMD.RTM. at the Institute of Medical Genetics at Cardiff, United
Kingdom.
[0119] "Stem-cell producing condition": The term "stem-cell
producing condition" as used herein refers to a condition or set of
conditions that permits and/or drives a cell to become a stem cell.
In certain embodiments, an embryonic cell is permitted and/or
driven to become an embryonic stem cell by subjecting such an
embryonic cell to a stem-cell producing condition. For example, an
embryonic blastomere may permitted and/or driven to become an
embryonic stem cell by isolating the embryonic blastomere from the
inner cell mass of a blastocyst and culturing the embryonic
blastomere under stem-cell producing conditions, such that at least
one blastomere proliferates into a pluripotent embryonic stem cell.
In certain embodiments, a transgenic embryonic stem cell is
generated by producing a transgenic cell according to one or more
methods of the present invention, allowing the transgenic cell to
develop into a transgenic blastocyst comprising a plurality of
transgenic blastomeres, isolating one or more transgenic
blastomeres from the inner cell mass of the transgenic blastocyst,
and culturing the isolated transgenic blastomere(s) under stem-cell
producing conditions such that at least one transgenic blastomere
develops into a pluripotent transgenic embryonic stem cell.
[0120] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0121] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the invention.
[0122] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0123] In the context of the invention, the term "treating" or
"treatment", as used herein, refers to a method that is aimed at
delaying or preventing the onset of a pathology (e.g. death of
motor neurons), at reversing, alleviating, inhibiting, slowing down
or stopping the progression, aggravation or deterioration of the
symptoms of the pathology. Treating or treatment mean to relieve or
alleviate at least one symptom associated with such condition, or
to slow or reverse the progression or anticipated progression of
such condition, at bringing about ameliorations of the symptoms of
the pathology. In one embodiment, the symptom of a motor neuron
disease is alleviated by at least 20%, at least 30%, at least 40%,
or at least 50%. In one embodiment, the symptom of a motor neuron
disease is alleviated by more that 50%. In one embodiment, the
symptom of a motor neuron disease is alleviated by 80%, 90%, or
greater.
[0124] The pharmaceutical compositions of the invention are
administered in a therapeutically effective amount. As used herein,
the phrase "therapeutically effective amount" refers to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pathological processes mediated by PGD2 DP1
expression or activity (e.g. death of motor neurons) or an overt
symptom of pathological processes mediated by PGD2 DP1 expression
or activity. The specific amount that is therapeutically effective
can be readily determined by an ordinary medical practitioner, and
may vary depending on factors known in the art, such as, for
example, the patient's history and age, the stage of pathological
processes, and the administration of other agents that inhibit
pathological processes in motor neuron disease.
Embryonic Stem Cells and their Generation
[0125] Stem cells typically share two important characteristics
that distinguish them from other types of cells. First, they are
unspecialized cells that are capable of maintaining their
unspecialized state and of renewing themselves for long periods
through cell division. Second, under appropriate conditions, they
can be induced to differentiate into cells with specialized
functions. Several types of stem cells have been identified
including adult stem cells, umbilical cord stem cells, and
embryonic stem cells.
[0126] Embryonic stem cells may be characterized by any of several
criteria, which criteria will be known by those of ordinary skill
in the art. For example, embryonic stem cells are typically capable
of continuous indefinite replication in vitro. Continued
proliferation for a long period of time (e.g., 6 months, one year
or longer) of culture is a sufficient evidence for immortality, as
primary cell cultures without this property fail to continuously
divide for such a length of time (Freshney, Culture of animal
cells. New York: Wiley-Liss, 1994). In certain embodiments,
embryonic stem will continue to proliferate in vitro under
appropriate culture conditions for longer than one year, and
maintain the developmental potential to contribute all three
embryonic germ layers throughout this time. Such developmental
potential can be demonstrated by the injection of embryonic stem
cells that have been cultured for a prolonged period (over a year)
into SCID mice and then histologically examining the resulting
tumors. However, length of time in culture is not the sole criteria
that may be used to identify an embryonic stem cell, and even
though cells have grown in culture for less than 6 months, such
cells may nevertheless be embryonic stem cells.
[0127] Additionally or alternatively, embryonic stem cells may be
identified by the expression of certain markers, including but not
limited to cell surface markers. As will be understood by those of
ordinary skill in the art, embryonic stem cells from different
species will exhibit species-specific markers on their cell
surfaces. For example, Thomson (U.S. Pat. Nos. 5,843,780 and
6,200,806, each of which is incorporated herein in its entirety by
reference) discloses certain cell surface markers that may be used
to identify embryonic stem cells derived from primates.
Furthermore, Stage Specific Embryonic Antigens (SSEAs) are
monoclonal antibodies that recognize defined carbohydrate epitopes
and may also be used to identify embryonic stem cells. Embryonic
stem cells derived from different species exhibit different
patterns of SSEAs. For example, undifferentiated primate ES cells
(including human ES cells) express SSEA-3 and SSEA-4, but not
SSEA-1. Conversely, undifferentiated mouse ES cells express SSEA-1,
but not SSEA-3 or SSEA-4. Additionally or alternatively, markers
that are not exhibited on the surface of a cell may be used to
identify an embryonic stem cell. For example, the homeodomain
transcription factor Oct 4 (also termed Oct-3 or Oct3/4) is
frequently used as a marker for totipotent embryonic stem cells.
Those of ordinary skill in the art will be aware of cell surface
and other markers that are useful in identifying embryonic stem
cells, including markers diagnostic of a given species that can be
used to identify an embryonic stem cell from that species.
[0128] Additionally or alternatively, embryonic stem cells may be
identified by the capacity to develop into all of the specialized
cell types of a mammal, including the three embryonic germ layers
(endoderm, mesoderm, and ectoderm), and all somatic cell lineages
and the germ line. Additionally and/or alternatively, embryonic
stem cells may be identified by the capacity to participate in
normal development when transplanted into a preimplantation embryo
to generate a chimeric embryo.
[0129] Cultured cells that have proliferated in cell culture for a
long period of time (e.g., six or more months) without
differentiating, are pluripotent, and appear genetically normal are
typically considered to be embryonic stem cells. In certain
embodiments, an embryonic stem cell of the present invention
comprises a human embryonic stem cell. In certain embodiments, an
embryonic stem cell of the present invention comprises a non-human
embryonic stem cell. For example, a non-human embryonic stem cell
of the present invention may include, but is not limited to, a
mouse, rat, pig, sheep, goat, and/or a primate stem cell. Those of
ordinary skill in the art will be aware of other non-human stem
cells that may be used in accordance with the present
invention.
[0130] The capacity of embryonic stem cells (ES) to self renew in
culture, while retaining their pluripotent potential, provides the
opportunity to produce virtually unlimited numbers of
differentiated cell types to replenish those lost as a consequence
of disease (Evans, M. J. & Kaufman, M. H. Establishment in
culture of pluripotential cells from mouse embryos. Nature 292,
154-6, 1981; Martin, G. R. Isolation of a pluripotent cell line
from early mouse embryos cultured in medium conditioned by
teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634-8,
1981). An alternative, but equally important potential of ES cells
is to provide insights into disease mechanisms (Lerou, P. H. &
Daley, G. Q. Therapeutic potential of embryonic stem cells. Blood
Rev 19, 321-31, 2005; Ben-Nun, I. F. & Benvenisty, N. Human
embryonic stem cells as a cellular model for human disorders. Mol
Cell Endocrinol 252, 154-9, 2006). ES cells carrying the genes
responsible for a particular disease can be induced to
differentiate into the cell types affected in that disease. Studies
of the differentiated cells in culture could provide important
information regarding the molecular and cellular nature of events
leading to pathology.
[0131] In certain embodiments, this approach is used to develop an
in vitro model of Amyotrophic Lateral Sclerosis ("ALS"). As
described more fully below in the Examples section of the present
application, embryonic stem cell lines were derived from normal
mice, and from mice that over-express either the wild-type human
SOD1 transgene or the mutant SOD1G93A transgene, the latter of
which is responsible for one type of familial ALS (see Example
section). Using the methods established by Wichterle et al (2002)
the three ES cell lines were differentiated into motor neurons in
culture. The wild-type SOD1 and the mutant SOD1G93A motor neurons
produce high levels of the corresponding human SOD1 proteins, and
they both display properties that characterize bone fide motor
neurons. These motor neurons could be maintained in long-term
culture, providing the opportunity to detect differences between
the mutant SOD1G93A ES cell-derived motor neurons and those derived
from control cell lines.
[0132] In certain embodiments, embryonic stem cells are generated
by culturing cells from the inner cell mass in a culture dish that
is coated with a feeder layer comprising mouse embryonic skin cells
that have been treated so they will not divide. Such a feeder layer
gives the inner cell mass cells a sticky surface to which they can
attach and also releases nutrients into the culture medium. In
certain embodiments, cells from the inner cell mass are cultured in
a culture dish that is not coated with a feeder layer. Such
embodiments provide certain advantages including reduction of the
risk that viruses or other macromolecules in the mouse cells may be
transmitted to the cultured cells.
[0133] In certain embodiments, embryonic stem cells are generated
by subjecting cells to stem-cell producing conditions. Stem-cell
producing conditions are known to those of ordinary skill in the
art and can often vary between species. For example, leukemia
inhibitory factor (LIF) is necessary and sufficient to prevent
differentiation of mouse embryonic stem cells and to allow them to
grow in an undifferentiated state indefinitely. Conversely, for
primate embryonic stem cells, at least one group has reported that
growth on a fibroblast feeder layer is required to prevent them
from differentiating (see e.g., U.S. Pat. Nos. 5,843,780 and
6,200,806, incorporated herein by reference in their entirety). One
of ordinary skill in the art will be aware of appropriate stem-cell
producing conditions including, but not limited to, culture media
and/or culturing conditions that permit and/or drive a cell of a
given species to become a stem cell.
[0134] In certain embodiments, embryonic stem cells of the present
invention are generated by any of a variety of methods disclosed in
U.S. Provisional Patent Application No. 60/926,525, filed Apr. 26,
2007, which is incorporated herein by reference in its entirety.
For example, in certain embodiments, an embryonic stem cell is
generated by transferring nuclear-derived genetic material from a
donor cell to a recipient cell to generate a transgenic cell, after
which the transgenic cell is allowed to develop into a blastocyst
and a blastomere cell from the inner cell mass is isolated and/or
cultured (and optionally passaged for several generations) under
stem-cell producing conditions, resulting in generation of an
embryonic stem cell syngenic with the nuclear-derived genetic
material removed from the donor cell used to generate the
transgenic cell.
[0135] In certain embodiments, embryonic stem cells of the present
invention are generated such that the generated embryonic stem
cells comprise a mutation associated with a disease of interest.
For example, embryonic stem cells may be generated which contain a
mutation associated with a neurodegenerative disease. Exemplary
neurodegenerative diseases include, but are not limited to: ALS,
Parkinson's disease, and Alzheimer's disease. Those of ordinary
skill in the art will be aware of other neurodegenerative diseases
of interest, as well as mutations associated with such
diseases.
[0136] In certain embodiments, an embryonic stem cell is generated
that comprises a mutation associated with ALS. For example, an
embryonic stem cell may be generated that comprises a mutation in
the SOD1 gene, e.g., Ala4Val, Gly37Arg and/or Gly93Ala. In certain
embodiments, an embryonic stem cell is generated that comprises a
SOD1G93A allele. In certain embodiments, an embryonic stem cell is
generated that comprises a human SOD1G93A allele.
[0137] In certain embodiments, an embryonic stem cell comprises a
mutation in a gene associated with neurodegenerative disease, which
gene is present as a transgene. For example, an endogenous gene
associated with a neurodegenerative disease may be deleted or
otherwise inactivated in such an embryonic stem cell by any of a
variety of techniques known to those skilled in the art, and a
transgene comprising a mutant copy of the endogenous gene may be
introduced into the embryonic stem cell by any of a variety of
techniques known to those skilled in the art. In certain
embodiments, such a transgene is integrated into the genome of the
embryonic stem cell. In certain embodiments, such a transgene is
not integrated into the genome of the embryonic stem cell.
[0138] Once embryonic stem cell lines are established, batches of
such embryonic stem cell lines can be frozen and for future
culturing and/or experimentation.
Differentiation into Motor Neurons
[0139] In certain embodiments of the present invention, an
embryonic stem cell is subjected to conditions that result in the
embryonic stem cell differentiating into a motor neuron. For
example, embryonic stem cells may be dissociated into a single-cell
suspension, allowed to spontaneously aggregate into embryoid bodies
over a first period of time (e.g. 48 hours, although such a period
of time may be increased or decreased depending on other conditions
to which the embryonic stem cells are subjected), and then treated
with a suitable differentiation factor or factors for a second
period of time such that the embryonic stem cells differentiate
into motor neurons. By way of example, such differentiation factors
may include retinoic acid (RAc) and soluble sonic hedgehog (Shh),
which may be administered for, e.g., 5 days. Other differentiation
factor(s) and condition(s) will be known to those of ordinary skill
in the art.
[0140] In certain embodiments, a motor neuron differentiated from
an embryonic stem cell comprises a mutation in a gene associated
with neurodegenerative disease. As non-limiting examples, such a
neurodegenerative disease may include ALS, Parkinson's disease,
Alzheimer's disease or any number of other neurodegenerative
diseases known to those of skill in the art. A variety of genes are
known to be associated with neurodegenerative diseases. As one
non-limiting example, mutations in the SOD1 gene are known to be
associated with the neurodegenerative disease ALS. For example, in
humans, Gly92Ala, Ala4Val and Gly37Arg mutations are associated
with the onset and progression of ALS. Those of ordinary skill in
the art will be aware of other SOD1 mutations associated with ALS.
In certain embodiments, compositions and method of the present
invention comprise or employ human motor neurons comprising a
SOD1G93A mutation such as Gly92Ala, Ala4Val and/or Gly37Arg.
[0141] In mice, the dominant SOD1G93A mutation is associated with
ALS-like phenotype. Thus, in certain embodiments, the present
invention comprises mouse motor neurons comprising a SOD1G93A
mutation. In certain embodiments, the present invention comprises
human motor neurons comprising a SOD1G93A mutation.
[0142] A number of changes characteristic of neurodegeneration in
ALS were observed in mouse mutant SOD1G93A motor neurons between 14
and 28 days (for additional detail, see Examples section below).
First, the SOD1G93A protein changed its intracellular localization,
forming inclusions that increased in size and density. Second, the
levels of ubiquitin increased. Third, some motor neurons expressed
activated caspase-3 and displayed cytoplasmic staining with
cytochrome c antibodies. Finally, a significant difference in
survival was observed between mutant SOD1G93A motor neurons and the
controls. Thus, many of the late onset pathologies observed in both
human ALS and SOD1G93A mice are recapitulated in this in vitro
model, including the loss of motor neurons, which is ultimate cause
of symptoms in patients.
[0143] In certain embodiments, methods of the present invention
comprise using human and/or non-human SOD1 mutant motor neurons to
screen for test agents that affect motor neuron differentiation,
development and/or survival. In certain embodiments, methods of the
present invention comprise using such SOD1 mutant motor neurons to
identify a factor that has a non-cell autonomous effect on the
differentiation, development and/or survival of a motor neuron.
[0144] In certain embodiments, a motor neuron differentiated from
an embryonic stem cell comprises a mutation in a gene associated
with neurodegenerative disease, which gene is present as a
transgene. For example, an endogenous gene associated with a
neurodegenerative disease may be deleted or otherwise inactivated
in an embryonic stem cell from which such a motor neuron is derived
by any of a variety of techniques known to those skilled in the
art, and a transgene comprising a mutant copy of the endogenous
gene may be introduced into the embryonic stem cell by any of a
variety of techniques known to those skilled in the art. In certain
embodiments, such a transgene is integrated into the genome of the
differentiated motor neuron. In certain embodiments, such a
transgene is not integrated into the genome of the differentiated
motor neuron.
Conditions Affecting Motor Neuron Differentiation, Development
and/or Survival
[0145] The present invention encompasses the recognition that
proper differentiation, development and/or survival of a cell can
be influenced by its environment. For example, non-cell autonomous
processes can contribute to the differentiation, development and/or
survival of a cell. In certain embodiments, the present invention
provides novel system and compositions for studying such non-cell
autonomous processes and for identifying factors that mediate such
non-cell autonomous processes.
[0146] In certain embodiments, methods and compositions of the
present invention are used to study non-cell autonomous processes
that contribute to the proper differentiation, development and/or
survival of a motor neuron. For example, both autonomous defects in
motor neurons and toxic non-cell autonomous interactions with other
cell types in the spinal cord have been implicated in ALS pathology
(Bruijn et al., 2004; Clement et al., 2003; Boillee et al., 1995;
Beers, D. R. et al. Wild-type microglia extend survival in PU.1
knockout mice with familial amyotrophic lateral sclerosis. Proc
Natl Acad Sci USA 103, 16021-6, 2006). Methods and compositions of
the present invention are well suited to the identification and
study of factors that mediate non-cell autonomous effects of other
cell types on motor neurons, leading to ALS.
[0147] Several studies have suggested that cells within the spinal
cord may have pathological, non-cell autonomous affects on motor
neurons or on the rate of disease progression (Clement et al.,
2003; Boillee et al., 2006). However, these studies were of limited
utility since they were not able to resolve the identity of cell
types that caused these affects and/or were not able to determine
whether they acted directly to affect motor neuron survival. The
present invention encompasses the discovery and recognition that
cultures of ES cell derived motor neurons contain other cell types,
including astroglia, and that these ES cell derived cells have a
non-cell autonomous affect on motor neuron survival in vitro (see
Examples section below). The effects of co-culturing motor neurons
with primary glia from SOD1G93A mice and mice expressing the
wild-type SOD1 protein were systematically examined. It was
discovered that mutant SOD1G93A glia reduced the survival of both
wild type and mutant motor neurons. However, the effect was
significantly greater on mutant SOD1G93A motor neurons. Therefore,
the presently described studies show for the first time that an ALS
genotype in glial cells directly and negatively affects the
survival of motor neurons and they confirm that there is a cell
autonomous component to motor neuron degeneration.
[0148] Consistent with the present disclosure, Nagai et al. have
shown that primary astroctye cultures expressing ALS-associated
mutant SOD1 proteins contain diffusible factor(s) that are toxic to
both primary and ES cell-derived motor neurons (Makiko Nagai, D. B.
R., Tetsuya Nagata, Alcmene Chalazonitis, Thomas M. Jessell, Hynek
Wichterle, Serge Przedborski. Astrocytes expressing ALS-associated
SOD1 mutants release factors selectively toxic to spinal motor
neurons. Nature Neurosciencc, 2007). In Nagai et al.'s study, motor
neurons were the only cell types affected by these mutant glial
cells and only SOD1G93A glial cells, not muscle cells or
fibroblasts, adversely affected motor neuron survival. Although in
Nagai et al's study, mutant primary neurons exhibited morphometric
alterations, their survival up to 14 days in culture was
indistinguishable from that of their wild-type counterparts. In the
presently described studies, differences in survival between
wild-type SOD1 and mutant SOD1G93A ES cell-derived motor neurons
were observed at 14 and 28 days in culture. The differences between
the two studies may originate in the source (embryo or ES
cell-derived) or number of the motor neurons used and the timeframe
of the investigations.
[0149] In certain embodiments, the present invention provides
methods for identifying and studying non-cell autonomous factors
produced by glial and/or other cells, which factors influence the
differentiation, development and/or survival of motor neurons. For
example, motor neurons may be cultured in the present of mutant
glial cells, and the survival of such motor neurons may be compared
to the survival of motor neurons cultured in the presence of wild
type glial cells. A difference in survival of motor neurons
indicates that a mutation present in such a glial cell is important
in mediating proper survival of motor neurons. In certain
embodiments, such a mutation in a glial cell results in an
alteration in the quantity and/or quality of a protein encoded by a
gene in which the mutation is located, which protein may be a
factor that contributes to proper survival of motor neurons. In
certain embodiments, a mutation in a glial cell results in an
alteration in the quantity and/or quality of a protein that is not
encoded by gene in which the mutation is located. For example, a
mutation may alter the quantity and/or quality of a produced
transcription factor, which transcription factor contributes to the
proper regulation and/or expression of a second protein, which
second protein may be a factor that mediates proper survival of
motor neurons. In certain embodiments, a mutation in a glial cell
results in an alteration in the quantity and/or quality of a factor
that contributes to proper survival of motor neurons, which factor
is not a protein (e.g. a small molecule, a lipid, a hormone, etc.).
Those of ordinary skill in the art will be aware of a variety of
other ways in which a mutation in a particular gene may affect a
factor that contributes to the proper survival of motor
neurons.
[0150] In certain embodiments, embryonic stem cells are induced to
differentiate into motor neurons in the presence of glial cells.
Such embodiments are useful in the study of normal motor neuron
differentiation, development and/or survival, and can be expected
to provide useful insights into possible causes, treatments and/or
cures of various neurodegenerative diseases.
[0151] In certain embodiments, embryonic stem cells are induced to
differentiate into motor neurons in the presence of mutant glial
cells, and differentiation, development and/or survival of such
motor neurons may be compared to differentiation, development
and/or survival of motor neurons cultured in the presence of wild
type glial cells. A difference in differentiation, development
and/or survival of the motor neurons indicates that a mutation
present in the glial cell contributes to proper differentiation,
development and/or survival of motor neurons. In certain
embodiments, a mutation in a glial cell results in an alteration in
the quantity and/or quality of a protein encoded by gene in which
the mutation is located, which protein is a factor that contributes
to proper differentiation, development and/or survival of motor
neurons. In certain embodiments, a mutation in a glial cell results
in an alteration in the quantity and/or quality of a protein that
is not encoded by gene in which the mutation is located. For
example, a mutation may alter the quantity and/or quality of a
produced transcription factor, which transcription contributes to
the proper regulation and/or expression of a second protein, which
second protein is a factor that mediates proper differentiation,
development and/or survival of motor neurons. In certain
embodiments, a mutation in a glial cell results in an alteration in
the quantity and/or quality of a factor that is important for
proper differentiation, development and/or survival of motor
neurons, which factor is not a protein (e.g. a small molecule, a
lipid, a hormone, etc.). Those of ordinary skill in the art will be
aware of a variety of other ways in which a mutation in a
particular gene may affect a factor that is important in the proper
survival of motor neurons.
[0152] In certain embodiments, mutant glial cells to be used in
accordance with the present invention to identify and/or study
factors that contribute to proper differentiation, development
and/or survival of motor neurons comprise a mutation in a gene
associated with a neurodegenerative disease. As but a few
non-limiting examples, such a neurodegenerative disease may include
ALS, Parkinson's disease, Alzheimer's disease or any number of
other neurodegenerative diseases known to those of skill in the
art. A variety of genes are known to be associated with
neurodegenerative diseases. As one non-limiting example, mutations
in the SOD1 gene are known to be associated with the
neurodegenerative disease ALS. Thus, in certain embodiments, mutant
glial cells to be used in accordance with the present invention to
identify and/or study factors that contribute to proper
differentiation, development and/or survival of motor neurons
comprise a mutation in the SOD1 gene. In humans, Gly92Ala, Ala4Val
and Gly37Arg mutations are associated with the onset and
progression of ALS. Thus, in certain embodiments, mutant glial
cells to be used in accordance with the present invention to
identify and/or study factors that contribute to proper
differentiation, development and/or survival of motor neurons
comprise a SOD1G93A mutation such as Gly92Ala, Ala4Val and/or
Gly37Arg. Those of ordinary skill in the art will be aware of a
variety of other SOD1 mutant alleles associated with ALS, which
mutant alleles can be advantageously used in accordance with one or
more of the embodiments described herein.
[0153] In certain embodiments, the present invention provides
methods for identifying and/or studying non-cell autonomous factors
produced by non-glial cells, which factors influence the
differentiation, development and/or survival of motor neurons.
Non-limiting examples of such non-glial cells that can influence
differentiation, development and/or survival of motor neurons
include microglial cells, oligodendrocytes, astrocytes, other
neuronal cell types in spinal cords (e.g. interneurons) and/or
other cells that are in contact with neurons (e.g. muscle cells).
Those of ordinary skill in the art will be aware of a variety of
other non-glial cells that can influence the differentiation,
development and/or survival of motor neurons.
[0154] In certain embodiments, methods of the present invention
comprise identifying a factor that has a non-cell autonomous effect
on survival of a motor neuron. In certain embodiments, such methods
comprise providing a motor neuron, identifying a first glial cell,
which first glial cell negatively affects survival of the motor
neuron, identifying a second glial cell, which second glial cell
does not negatively affect survival of the motor neuron, isolating
a factor from the either the first or second glial cell, wherein
the factor is either: i) a factor from the first glial cell that
contributes to the negative effect on survival of the motor neuron;
or ii) a factor from the second glial cell that contributes to
survival of the motor neuron.
[0155] Factors that influence differentiation, development and/or
survival can be identified by any of a variety of methods known to
those of ordinary skill in the art. In certain embodiments, such a
factor is directly identified after determining that a given wild
type or mutant cell contributes to proper differentiation,
development and/or survival of a cell of interest such as, e.g., a
motor neuron. Non-limiting examples of such methods include
fractionation, mass spectrometry, protein chip analysis (e.g., if
such a factor comprises a protein), chromatography, etc. Those of
ordinary skill in the art will be aware of and will be able to
employ suitable techniques for directly identifying such a
factor.
[0156] In certain embodiments, such a factor is identified
indirectly. For example, a factor may comprise a protein encoded by
a gene. In such embodiments, a gene that encodes such a factor may
be identified by any of a variety of techniques such as, for
example, differential display, gene chip analysis, RT-PCR, direct
sequencing, etc. Those of ordinary skill in the art will be aware
of and will be able to employ suitable techniques for identifying
such a factor indirectly.
[0157] In certain embodiments, a combination of two or more factors
may together contribute to differentiation, development and/or
survival of a cell of interest such as, for example, a motor
neuron. Methods and compositions of the present invention may be
advantageously used to identify such a combination of factors.
Disease Modeling and Drug Screening
[0158] In certain embodiments, the present invention offers great
potential for developing better models for the study of human
disease and/or better methods of treatment. In certain embodiments,
methods of the present invention employ embryonic stem cells and/or
differentiated cells that comprise alterations (e.g., deletions,
rearrangements, duplications, substitutions, etc.) in genes
associated with a particular disease. In certain embodiments, a
disease of interest is a neurodegenerative disease. Exemplary
neurodegenerative diseases that can be studied using compositions
and methods of the present invention include, but are not limited
to, ALS, Parkinson's disease, and Alzheimer's disease. Those
skilled in the art will be aware of a number of other
neurodegenerative diseases that can be studied using compositions
and methods the present invention.
[0159] In certain embodiments, a disease of interest is modeled
and/or studied by inducing an embryonic stem cell line (that has,
for example, been generated by any of the variety of methods of the
present invention to contain one or more alterations in one or more
genes associated with a disease of interest) to differentiate by
culturing such a cell line under appropriate differentiation
conditions. For example, an embryonic stem cell line may be
generated that contains one or more alterations in one or more
genes associated with a neurological degenerative disease, e.g.
ALS, or any other neurodegenerative disease of interest. Such an
embryonic cell line may be induced to differentiate into motor
neurons by subjecting it to appropriate differentiation conditions.
Those of ordinary skill in the art will be aware of appropriate
differentiation conditions. By observing differentiation,
development and/or survival of such a motor neuron and comparing it
to differentiation, development and/or survival of a motor neuron
derived from an embryonic stem cell line that does not contain the
genetic alteration(s) associated with the neurodegenerative disease
of interest, the practitioner can achieve a better understanding of
the genetic basis of disease progression and pathogenesis.
[0160] Additionally or alternatively, an embryonic stem cell line
that is generated to contain one or more alterations in one or more
genes associated with a disease of interest may be used to screen
for agents (e.g., a cell, a small molecule, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an antibody, an amino acid, a
virus, etc.) that can be used, for example, in the treatment,
prevention and/or cure of that disease. For example, such an
embryonic stem cell line may be induced to differentiate into a
cell type associated with the disease of interest by placing it
under appropriate differentiation conditions. Before, during and/or
after differentiation, such a cell may be subjected to a test agent
in order to determine whether that agent has an effect on
differentiation, development and/or survival of the cell. In
certain embodiments, an embryonic stem cell comprising a mutation
associated with a neurodegenerative disease is induced to
differentiate into a motor neuron, which motor neuron is subjected
to an agent before, during and/or after differentiation.
[0161] In certain embodiments, compositions and methods of the
present invention are useful in studying and/or modeling diseases
that to date, have not been amenable to such study and/or modeling.
For instance, in many cases, by the time a patient is diagnosed
with a particular disease, the early events of disease progression
and pathogenesis have already occurred, making it difficult or
impossible to determine and track the molecular, cellular, or other
changes that occur during the course of the disease. Using
inventive methods and compositions disclosed herein, researchers
will now be able to determine and study such molecular, cellular,
or other changes, leading to a better understanding of disease
progression and pointing the way to more effective treatments.
[0162] In certain embodiments, methods of the present invention
comprise identifying an agent that affects the survival of a SOD1
mutant motor neuron. In certain embodiments, such methods comprise
providing a SOD1 mutant motor neuron, providing a test agent,
contacting the SOD1 mutant motor neuron with the test agent, and
determining the effect of the test agent on survival of the SOD1
mutant motor neuron by comparing the survival of the SOD1 mutant
motor neuron to the survival of a control motor neuron lacking the
SOD1 mutant allele, which control motor neuron is contacted with
the test agent for a period of time and under conditions identical
to that of the SOD1 mutant motor neuron. In certain embodiments, a
SOD1 motor neuron used in such methods is derived from an embryonic
stem cell. In certain embodiments, a SOD1 motor neuron used in such
methods comprises a SOD1 mutation associated with a
neurodegenerative disease of interest, e.g. ALS.
[0163] In certain embodiments, compositions and methods of the
present invention are used to study and/or model diseases and/or to
screen for agents that can be used in the treatment, prevention
and/or cure of diseases, which compositions or methods comprise or
make use of human embryonic stem cell lines and/or differentiated
cells derived from such human embryonic stem cell lines.
[0164] In certain embodiments, compositions and methods of the
present invention are used to study and/or model diseases and/or to
screen for agents that can be used in the treatment, prevention
and/or cure of diseases, which compositions or methods comprise or
make use of non-human embryonic stem cell lines and/or
differentiated cells derived from such non-human embryonic stem
cell lines. Use of non-human stem cell lines and/or differentiated
cells derived from them is advantageous when ethical and/or
practical limitations prevent the use of human stem cell lines.
Non-limiting examples of non-human stem cell lines (and/or
differentiated cells derived from them) that may be used in
accordance with the present invention to study and/or model
diseases and/or to screen for agents that can be used in the
treatment, prevention and/or cure of diseases include mouse, rat,
and primate stem cell lines. Those of ordinary skill in the art
will be aware of a variety of other non-human stem cell lines that
will be useful, and those of ordinary skill in the art will be able
to generate such stem cell lines by employing one or more methods
of the present invention.
[0165] Compositions and methods of the present invention may be
used to study and/or model of any of a variety of diseases or
conditions. Non-limiting examples of such diseases or conditions
include childhood congenital malformations, sickle cell anemia,
neurological diseases such as amyotrophic lateral sclerosis (also
known as Lou Gehrig's disease), Parkinson's disease, Alzheimer's
disease or any of a variety of other neurological diseases, Down
syndrome (a condition that arises in patients with trisomy for
chromosome 21 resulting in dysregulated signaling through the
NFAT/calcineurin pathway), etc. One of ordinary skill in the art
will be aware of a variety of other disease conditions that may be
modeled and/or studied by generating embryonic stem cells according
to one or more methods of the present invention.
[0166] In certain embodiments, compositions and methods of the
present invention comprise or make use of human embryonic stem cell
lines and/or differentiated cells derived from a patient suffering
from and/or predicted to suffer from a disease of interest.
Patient-specific, immune-matched human embryonic stem cells have
the potential to be of great biomedical importance for studies of
disease and development. For example, certain patients may respond
better to a given therapy or drug regimen than other patients.
Additionally or alternatively, certain patients may experience
fewer and/or less severe side effects after being administered a
given therapy or drug regimen than other patients. By utilizing
embryonic stem cells that contain the genetic complement of a
patient suffering from and/or predicted to suffer from a disease of
interest, and permitting such cells to differentiate into a cell
type associated with that disease, it will be possible to better
predict which therapy or drug regimen will be most beneficial
and/or result in the least detrimental side effects.
[0167] Furthermore, patient-specific human embryonic stem cells
have the potential to be of great biomedical importance for the
discovery and/or development of patient-specific agents that can be
used to prevent and/or treat a disease of interest. By utilizing
embryonic stem cells containing the genetic complement of a patient
suffering from a disease of interest, permitting such cells to
differentiate into a cell type associated with that disease, and
subjecting such cells to one or more test agents before, during
and/or after differentiation, discovery and/or development of an
agent that will be most beneficial and/or result in the least
detrimental side effects for that particular patient will be
facilitated. Those of ordinary skill in the art will be able to
apply methods and compositions of the present invention to the
discovery and/or development of an agent specific for a patient
and/or disease of interest.
[0168] In certain embodiments, a disease of interest is modeled
and/or studied by inducing an embryonic stem cell line to
differentiate into a cell type of interest by culturing such a cell
line under appropriate differentiation conditions, wherein the
embryonic stem cell line is differentiated in the presence of one
or more different cell types that contribute to proper
differentiation, development and/or survival of the cell type of
interest. For example, an embryonic stem cell line containing one
or more alterations in one or more genes associated with a
neurological degenerative disease, e.g. ALS or any other
neurodegenerative disease of interest, may be induced to
differentiate into a motor neuron in the presence of one or more
glial cells. By observing the differentiation, development and/or
survival of such a motor neuron and comparing it to the
differentiation, development and/or survival of a motor neuron
derived from an embryonic stem cell line that does not contain the
genetic alteration(s) associated with the neurodegenerative disease
of interest, the practitioner can achieve a better understanding of
the genetic basis of disease progression and pathogenesis.
[0169] In certain embodiments, an embryonic stem cell line
containing a wild type genome is induced to differentiate into a
cell type of interest in the presence of one or more different cell
types that contribute to proper differentiation, development and/or
survival of the cell type of interest. By observing the
differentiation, development and/or survival of such a cell type of
interest and comparing it to the differentiation, development
and/or survival of a cell type of interest in the absence of such
different cell types, the practitioner can achieve a better
understanding of the genetic basis of normal differentiation,
development and/or survival a cell type of interest.
[0170] In certain embodiments, a cell type of interest is induced
to differentiate in the presence of one or more different cell
types that contribute to proper differentiation, development and/or
survival of the cell type of interest, wherein the one or more
different cell types comprise one or more mutations that affect
differentiation, development and/or survival of the cell type of
interest. By observing the differentiation, development and/or
survival of the cell type of interest in such an environment and
comparing it to the differentiation, development and/or survival of
a cell type of interest differentiated in the presence of wild type
different cell types, the practitioner can achieve a better
understanding of non-cell autonomous factors and processes that
contribute to proper differentiation, development and/or survival
of the cell type of interest.
[0171] In certain embodiments, the present invention provides
systems and methods for identifying agents (e.g., a cell, a small
molecule, a hormone, a vitamin, a nucleic acid molecule, an enzyme,
an antibody, an amino acid, a virus, etc.) that can be used, for
example, in the treatment, prevention and/or cure of a disease of
interest, wherein the differentiation, development and/or survival
of a cell type of interest implicated in the onset and/or
progression of the disease of interest is influenced by one or more
different cell types. For example, such an embryonic stem cell line
may be induced to differentiate into a cell type associated with
the disease of interest by placing it under appropriate
differentiation conditions in the presence of one or more different
cell types that contribute to proper differentiation, development
and/or survival of the cell type of interest. Before, during and/or
after differentiation, such a cell may be subjected to a test agent
in order to determine whether that agent has an effect on
differentiation, development and/or survival of the cell.
[0172] In certain embodiments, an embryonic stem cell comprising a
mutation associated with a neurodegenerative disease is induced to
differentiate into a motor neuron in the presence of glial cells,
which motor neuron is subjected to an agent before, during and/or
after differentiation. In certain embodiments, such glial cells are
wild type. In certain embodiments, such glial cells comprise one or
more mutations that alter the proper differentiation, development
and/or survival of the motor neuron. For example, such glial cells
may comprise a mutation that induces the motor neuron to display a
phenotype characteristic of a disease of interest, such as for
example, a neurodegenerative disease including, without limitation,
ALS. In certain embodiments, such glial cells comprise a mutation
in the SOD1 gene, for example a SOD1G93A mutation. Thus, in certain
embodiments, the present invention provides systems and methods for
identifying agent(s) that prevent, ameliorate, or reverse the
adverse effects of such SOD1G93A mutant glial cells on the proper
differentiation, development and/or survival of a motor neuron,
including both wild type and mutant motor neurons. In certain
embodiments, such identified agents are used to prevent, treat
and/or cure ALS.
[0173] As described in the Examples herein, it has been discovered
that certain genes are overexpressed in glia having a mutation in
the SOD1 gene. Moreover, it has been discovered that agents that
target such genes or expression products of such genes can promote
survival of motor neurons (e.g., motor neurons produced and/or
cultured according to a method described herein). Accordingly, in
some embodiments, the present invention provides methods of
identifying a test agent that modulates survival of a motor neuron,
wherein the test agent targets a gene or product of a gene
overexpressed in SOD1 mutant glia. In some embodiments, a test
agent targets (e.g., inhibits expression or activity of) a gene or
product of a gene in Table 2 (e.g., a gene or product of a gene
selected from serine (or cysteine) peptidase inhibitor, clade A,
member 1b (Serpina1b); protein tyrosine phosphatase, non-receptor
type 7 (Ptpn7); poly (ADP-ribose) polymerase family, member 12
(Zc3hdc1); prostaglandin D2 receptor (Ptgdr); glia maturation
factor, beta (Gmfb); ATP-binding cassette, sub-family A (ABC 1),
member 5 (Abca5); developing brain homeobox 2 (Dbx2); RAB6B, member
RAS oncogene family (Rab6b); cut-like 1 (Cutl1); adenosine
deaminase (Ada); receptor coactivator 6 interacting protein
(Ncoa6ip); interferon-induced protein 35 (ili35); RAB, member of
RAS oncogene family-like 2A (Rabl2a); STEAP family member 4 (A
1481214); cytoglobin (cygb); Duffy blood group, chemokine receptor
(Dfy); chondrolectin (Chod1); neurexin 1 (NRXN1); defensin beta 11
(Defb11); RUN and SH3 domain containing 2 (RUSC2); matrilin-4
(matn4); X-linked lymphocyte-regulated 3A (Xlr3a); C-C motif
chemokine 8 (ccl8); T-cell immunoglobulin and mucin domain
containing 4 (Timd4); odd-skipped related 2 (osr2); RIKEN cDNA
9130213B05 gene (9130213B05Rik); reversion-inducing-cysteine-rich
protein with kazal motifs (Reck); olfactory receptor 116 (Olfr116);
protogenin homolog (Prtg; A230098A12Rik); sonic hedgehog (Shh);
formyl peptide receptor 1 (Fpr1); pro-platelet basic protein
(chemokine (C-X-C motif) ligand 7; CXCL7); DnaJ (Hsp40) homolog,
subfamily B, member 3 (DNAJB3); defensin beta 10 (Defb10);
apolipoprotein A-II (Apoa2); collagen, type I, alpha 2 (Colla2);
islet cell autoantigen 1-like (Ical1; 1700030B17Rik); ATPase, class
II, type 9A (Atp9a); chemokine (C-C motif) ligand 5 (CCL5); solute
carrier family 39 (zinc transporter), member 14 (Sc139A14); serum
amyloid A 3 (Saa3); RIKEN cDNA 3632451006 gene (3632451006Rik);
attractin like 1 (Atrnl1); Alstrom syndrome 1 (Alms 1); NK2
homeobox 2 (Nkx2-2); kallikrein related-peptidase 8 (Klk8; Prss19);
histone cluster 1, H4k (Hist1h4k); EPH receptor B2 (Ephb2);
synaptotagmin XII (Syt12); forkhead box Q1 (Foxq1); splicing
factor, arginine/serine-rich 16 (Sfrs16); LanC lantibiotic
synthetase component C-like 1 (Lanc1); and MARCKS-like 1 (Mlp)). In
some embodiments, a test inhibits expression or activity of a gene
or gene product in Table 2 which is involved in inflammation. In
some embodiments, a test agent inhibits expression or activity of a
prostaglandin D receptor.
[0174] In certain embodiments, a test agent that inhibits
expression or activity of a gene or product of a gene in Table 2
includes a small molecule, an antibody, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an amino acid, and/or a
virus.
[0175] In certain embodiments, a test agent is a nucleic acid
molecule, e.g., a nucleic acid molecule that mediates RNA
interference. RNA interference refers to sequence-specific
inhibition of gene expression and/or reduction in target RNA levels
mediated by an at least partly double-stranded RNA, which RNA
comprises a portion that is substantially complementary to a target
RNA. Typically, at least part of the substantially complementary
portion is within the double stranded region of the RNA. In some
embodiments, RNAi can occur via selective intracellular degradation
of RNA. In some embodiments, RNAi can occur by translational
repression. In some embodiments, RNAi agents mediate inhibition of
gene expression by causing degradation of target transcripts. In
some embodiments, RNAi agents mediate inhibition of gene expression
by inhibiting translation of target transcripts. Generally, an RNAi
agent includes a portion that is substantially complementary to a
target RNA. In some embodiments, RNAi agents are at least partly
double-stranded. In some embodiments, RNAi agents are
single-stranded. In some embodiments, exemplary RNAi agents can
include small interfering RNA (siRNA), short hairpin RNA (shRNA),
and/or microRNA (miRNA). In some embodiments, an agent that
mediates RNAi includes a blunt-ended (i.e., without overhangs)
dsRNA that can act as a Dicer substrate. For example, such an RNAi
agent may comprise a blunt-ended dsRNA which is >25 base pairs
length. RNAi mechanisms and the structure of various RNA molecules
known to mediate RNAi, e.g. siRNA, shRNA, miRNA and their
precursors, are described, e.g., in Dykxhhorn et al., 2003, Nat.
Rev. Mol. Cell. Biol., 4:457; Hannon and Rossi, 2004, Nature,
431:3761; and Meister and Tuschl, 2004, Nature, 431:343; all of
which are incorporated herein by reference.
[0176] In some embodiments, a nucleic acid that mediates RNAi
includes a 19 nucleotide double-stranded portion, comprising a
guide strand and an antisense strand. Each strand has a 2 nt 3'
overhang. Typically the guide strand of the siRNA is perfectly
complementary to its target gene and mRNA transcript over at least
17-19 contiguous nucleotides and typically the two strands of the
siRNA are perfectly complementary to each other over the duplex
portion. However, as will be appreciated by one of ordinary skill
in the art, perfect complementarity is not required. Instead, one
or more mismatches in the duplex formed by the guide strand and the
target mRNA is often tolerated, particularly at certain positions,
without reducing the silencing activity below useful levels. For
example, there may be 1, 2, 3, or even more mismatches between the
target mRNA and the guide strand (disregarding the overhangs).
[0177] Molecules having the appropriate structure and degree of
complementarity to a target gene will exhibit a range of different
silencing efficiencies. A variety of additional design criteria
have been developed to assist in the selection of effective siRNA
sequences. Numerous software programs that can be used to choose
siRNA sequences that are predicted to be particularly effective to
silence a target gene of choice are available (see, e.g., Yuan et
al., 2004, Nuc. Acid. Res., 32:W130; and Santoyo et al., 2005,
Bioinformatics, 21:1376; both of which are incorporated herein by
reference). Modifications that increase stability and resistance to
nuclease breakdown are well known to one skill in the art and are
contemplated.
[0178] As will be appreciated by one of ordinary skill in the art,
RNAi may be effectively mediated by RNA molecules having a variety
of structures that differ in one or more respects from that
described above. For example, the length of the duplex can be
varied (e.g., from about 17-29 nucleotides); the overhangs need not
be present and, if present, their length and the identity of the
nucleotides in the overhangs can vary (though most commonly
symmetric dTdT overhangs are employed in synthetic siRNAs).
[0179] In certain embodiments, the present invention provides
methods of modulating survival of a motor neuron by contacting a
motor neuron (in vitro or in vivo) with an agent that reduces the
expression or activity of a prostaglandin D receptor. Agents that
reduce expression or activity (e.g., antagonize) prostaglandin D
receptors include MK 0524
((3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocy-
clopenta[b]indol-3-yl]-acetic acid; Sturino et al., J. Med. Chem.
50(4):794-806, 2007) and analogs thereof, and compounds disclosed
in Mitsumori et al., Curr. Pharm. Des., 10(28):3533-8, 2004;
Beaulieu et al., Bioorg Med Chem Lett. 18(8):2696-700, 2008; Torisu
et al., Eur J Med Chem. 40(5):505-19, 2005; U.S. Pat. Pub. Nos.
20010051624, 20030055077, 20040180934, 20070244131, 20070265278,
20070265291, 20080194600, and U.S. Pat. No. 7,153,852.
Human Stem Cells and Cells Derived from them
[0180] Although mouse genetics has provided a sophisticated
understanding of the cellular and molecular mechanisms that
contribute to familial ALS, it cannot inform us as to the actual
relevance of its findings to human patients. In fact, due to the
fundamental differences between human and mouse physiology, many
observations made in mouse disease models have not translated well
to human experimental systems or to the clinic. For example,
diabetes has been "cured" many times over in the NOD mouse model of
disease. However, few of the observations and experimental
therapies developed in this mouse model have proven relevant to the
human disease (Shoda, L. K. et al. A comprehensive review of
interventions in the NOD mouse and implications for translation.
Immunity. 23, 115-26, 2005). Similarly, mutations in the RB gene
that lead to Retinoblastoma in human patients cause an independent
range of tumors in mice carrying the same genetic lesion (Goodrich,
D. W. Lee, W. H. Molecular characterization of the retinoblastoma
susceptibility gene. Biochim Biophys Acta. 1155, 43-61, 1993;
Williams B. O. et al. Extensive contribution of Rb-deficient cells
to adult chimeric mice with limited histopathological consequences.
EMBO J., 13, 4251-9, 1994). As a result many therapeutics developed
in animal, or based on drug-targets discovered in animal models,
fail in clinical trials (Shoda et al., 2005; Gawarylewski, A. The
trouble with animal models. The Scientist. 21 (7), 45-51, 2007;
Rubin, L. L. Stem cells and drug discovery: the beginning of a new
era? Cell. 132, 549-52, 2008). The cost of these failures is
substantial (Gawarylewski et al., 2007; Rubin, 2008).
[0181] Considerable time, effort and expense would be saved if
fundamental observations made in animal models could be routinely
validated in the relevant human cell types. A potential solution is
to use human embryonic stem cells as a renewable source of these
cells for the study of disease and for drug target validation. The
discoveries described herein have demonstrated the usefulness of
motor neurons derived from human ES cells in validating findings
from mouse models of ALS (see e.g., Examples 7 and 8 below). It has
been found that glia cells over-expressing the SOD1G93A mutation
negatively affect the viability of human ES cell derived motor
neurons in a time dependent manner. Such non-cell autonomous effect
of glia is specific for motor neurons, as it does not seem
interfere with the survival of human interneurons.
[0182] In certain embodiments, in vitro model systems of the
present invention utilize human motor neurons derived from human ES
cells. In certain embodiments, a human motor neuron differentiated
from a human embryonic stem cell comprises a mutation in a gene
associated with neurodegenerative disease. As non-limiting
examples, such a neurodegenerative disease may include ALS,
Parkinson's disease, Alzheimer's disease or any number of other
neurodegenerative diseases known to those of skill in the art. A
variety of genes are known to be associated with neurodegenerative
diseases. As one non-limiting example, mutations in the SOD1 gene
are known to be associated with the neurodegenerative disease ALS.
For example, in humans, Gly92Ala, Ala4Val and Gly37Arg mutations
are associated with the onset and progression of ALS. Those of
ordinary skill in the art will be aware of other SOD mutations
associated with ALS. In certain embodiments, compositions and
method of the present invention comprise or employ human motor
neurons comprising a SOD1G93A mutation such as Gly92Ala, Ala4Val
and/or Gly37Arg.
[0183] In certain embodiments, in vitro model systems of the
present invention comprising human motor neurons may be
advantageously employed for the human physiological validation of
findings from animal models, such as, without limitation, animal
models (e.g., mouse) that recapitulate in whole or in part
neurodegenerative diseases such as ALS and/or other
neurodegenerative diseases.
[0184] In certain embodiments, in vitro model systems of the
present invention comprising human motor neurons may be
advantageously employed to identify novel factors, agents, etc.
that affect motor neuron development and/or contribute to a disease
state, such as, without limitation, a neurodegenerative disease,
e.g. ALS, in the absence of an animal model. In certain
embodiments, in vitro model systems of the present invention
comprising human motor neurons may be advantageously employed to
illuminate the target, efficacy, toxicity, mode of action, etc. of
factors, agents, etc. that affect motor neuron development and/or
contribute to a disease state, such as, without limitation, a
neurodegenerative disease, e.g. ALS.
[0185] In certain embodiments, methods of the present invention
employ human mutant motor neurons that comprise a mutation in a
gene associated with a neurodegenerative disease, for example ALS.
One non-liming example of a gene associated with the
neurodegenerative disease ALS is SOD1. A variety of SOD1 mutant
alleles are known to be associated with ALS, including without
limitation, SOD1G93A. In certain embodiments, methods of the
present invention utilize human motor neurons comprising a SOD1
mutant allele (e.g., SOD1G93A) to screen for test agents that
affect motor neuron differentiation, development and/or survival.
In certain embodiments, methods of the present invention comprise
using such SOD1 mutant motor neurons to identify a factor that has
a non-cell autonomous effect on the differentiation, development
and/or survival of a motor neuron.
[0186] In certain embodiments, methods of the present invention
employ human mutant cells that are not motor neurons, which mutant
cells comprise a mutation in a gene associated with a
neurodegenerative disease, for example ALS. As but one non-limiting
example, certain methods the present invention employ glial cells
comprising a mutation in a gene associated with a neurodegenerative
disease such as ALS. In certain embodiments, methods the present
invention employ glial cells comprising a mutation in a SOD1 gene,
such as, without limitation, SOD1G93A.
[0187] Those skilled in the art will be aware of other gene
mutations associated with neurodegenerative diseases, and will be
able to use methods and compositions described herein to validate
results of animal models, to identify novel factors, agents, etc.
that affect motor neuron development and/or contribute to a disease
state, and/or to illuminate the target, efficacy, toxicity, mode of
action, etc. of factors, agents, etc. that affect motor neuron
development and/or contribute to a disease state.
Non-Human Applications
[0188] Embryonic stem cells of the present invention and/or cells
derived from them can be advantageously used in the study and/or
modeling of human diseases, although one of ordinary skill in the
art will understand that the present disclosure is not limited to
human applications. Thus, for example, non-human embryonic stem
cell lines may be used in the study and/or modeling of diseases
associated with pets (e.g., cats, dogs, rodents, etc.) as well as
commercially important domestic animals (e.g., cows, sheep, pigs,
etc.). Additionally or alternatively, non-human embryonic stem cell
lines may be used to screen for agents that can be used in the
prevention and/or treatment of diseases associated with pets and/or
commercially important domestic animals.
Motor Neuron Disease
[0189] The motor neuron diseases (MND) are a group of neurological
disorders that selectively affect motor neurons, the nerve cells
that control voluntary muscle activity including speaking, walking,
breathing, swallowing and general movement of the body. Skeletal
muscles are innervated by a group of neurons (lower motor neurons)
located in the ventral horns of the spinal cord which project out
the ventral roots to the muscle cells. These nerve cells are
themselves innervated by the corticospinal tract or upper motor
neurons that project from the motor cortex of the brain. On
macroscopic pathology, there is a degeneration of the ventral horns
of the spinal cord, as well as atrophy of the ventral roots. In the
brain, atrophy may be present in the frontal and temporal lobes. On
microscopic examination, neurons may show spongiosis, the presence
of astrocytes, and a number of inclusions including characteristic
"skein-like" inclusions, bunina bodies, and vacuolisation. Motor
neuron diseases are varied and destructive in their effect. They
commonly have distinctive differences in their origin and
causation, but a similar result in their outcome for the patient:
severe muscle weakness. Amyotrophic lateral sclerosis (ALS),
primary lateral sclerosis (PLS), progressive muscular atrophy
(PMA), pseudobulbar palsy, progressive bulbar palsy, spinal
muscular atrophy (SMA) and post-polio syndrome are all examples of
MND. The major site of motor neuron degeneration classifies the
disorders. Common MNDs include amyotrophic lateral sclerosis, which
affects both upper and lower motor neurons. Progressive bulbar
palsy affects the lower motor neurons of the brain stem, causing
slurred speech and difficulty chewing and swallowing. Individuals
with these disorders almost always have abnormal signs in the arms
and legs. Primary lateral sclerosis is a disease of the upper motor
neurons, while progressive muscular atrophy affects only lower
motor neurons in the spinal cord. Means for diagnosing MND are well
known to those skilled in the art. Non limiting examples of
symptoms are described below.
[0190] Amyotrophic lateral sclerosis (ALS), also called Lou
Gehrig's disease or classical motor neuron disease, is a
progressive, ultimately fatal disorder that eventually disrupts
signals to all voluntary muscles. In the United States, doctors use
the terms motor neuron disease and ALS interchangeably. Both upper
and lower motor neurons are affected. Approximately 75 percent of
people with classic ALS will also develop weakness and wasting of
the bulbar muscles (muscles that control speech, swallowing, and
chewing). Symptoms are usually noticed first in the arms and hands,
legs, or swallowing muscles. Muscle weakness and atrophy occur
disproportionately on both sides of the body. Affected individuals
lose strength and the ability to move their arms, legs, and body.
Other symptoms include spasticity, exaggerated reflexes, muscle
cramps, fasciculations, and increased problems with swallowing and
forming words. Speech can become slurred or nasal. When muscles of
the diaphragm and chest wall fail to function properly, individuals
lose the ability to breathe without mechanical support. Although
the disease does not usually impair a person's mind or personality,
several recent studies suggest that some people with ALS may have
alterations in cognitive functions such as problems with
decision-making and memory. ALS most commonly strikes people
between 40 and 60 years of age, but younger and older people also
can develop the disease. Men are affected more often than women.
Most cases of ALS occur sporadically, and family members of those
individuals are not considered to be at increased risk for
developing the disease. However, there is a familial form of ALS in
adults, which often results from mutation of the superoxide
dismutase gene, or SOD1, located on chromosome 21. In addition, a
rare juvenile-onset form of ALS is genetic. Most individuals with
ALS die from respiratory failure, usually within 3 to 5 years from
the onset of symptoms. However, about 10 percent of affected
individuals survive for 10 or more years.
[0191] Progressive bulbar palsy, also called progressive bulbar
atrophy, involves the bulb-shaped brain stem--the region that
controls lower motor neurons needed for swallowing, speaking,
chewing, and other functions. Symptoms include pharyngeal muscle
weakness (involved with swallowing), weak jaw and facial muscles,
progressive loss of speech, and tongue muscle atrophy. Limb
weakness with both lower and upper motor neuron signs is almost
always evident but less prominent. Affected persons have outbursts
of laughing or crying (called emotional lability). Individuals
eventually become unable to eat or speak and are at increased risk
of choking and aspiration pneumonia, which is caused by the passage
of liquids and food through the vocal folds and into the lower
airways and lungs. Stroke and myasthenia gravis each have certain
symptoms that are similar to those of progressive bulbar palsy and
must be ruled out prior to diagnosing this disorder. In about 25
percent of ALS cases early symptoms begin with bulbar involvement.
Some 75 percent of individuals with classic ALS eventually show
some bulbar involvement. Many clinicians believe that progressive
bulbar palsy by itself, without evidence of abnormalities in the
arms or legs, is extremely rare.
[0192] Pseudobulbar palsy, which shares many symptoms of
progressive bulbar palsy, is characterized by upper motor neuron
degeneration and progressive loss of the ability to speak, chew,
and swallow. Progressive weakness in facial muscles leads to an
expressionless face. Individuals may develop a gravelly voice and
an increased gag reflex. The tongue may become immobile and unable
to protrude from the mouth. Individuals may also experience
emotional lability.
[0193] Primary lateral sclerosis (PLS) affects only upper motor
neurons and is nearly twice as common in men as in women. Onset
generally occurs after age 50. The cause of PLS is unknown. It
occurs when specific nerve cells in the cerebral cortex (the thin
layer of cells covering the brain which is responsible for most
higher level mental functions) that control voluntary movement
gradually degenerate, causing the muscles under their control to
weaken. The syndrome-which scientists believe is only rarely
hereditary-progresses gradually over years or decades, leading to
stiffness and clumsiness of the affected muscles. The disorder
usually affects the legs first, followed by the body trunk, arms
and hands, and, finally, the bulbar muscles. Symptoms may include
difficulty with balance, weakness and stiffness in the legs,
clumsiness, spasticity in the legs which produces slowness and
stiffness of movement, dragging of the feet (leading to an
inability to walk), and facial involvement resulting in dysarthria
(poorly articulated speech). Major differences between ALS and PLS
(considered a variant of ALS) are the motor neurons involved and
the rate of disease progression. PLS may be mistaken for spastic
paraplegia, a hereditary disorder of the upper motor neurons that
causes spasticity in the legs and usually starts in adolescence.
Most neurologists follow the affected individual's clinical course
for at least 3 years before making a diagnosis of PLS. The disorder
is not fatal but may affect quality of life. PLS often develops
into ALS.
[0194] Progressive muscular atrophy (PMA) is marked by slow but
progressive degeneration of only the lower motor neurons. It
largely affects men, with onset earlier than in other MNDs.
Weakness is typically seen first in the hands and then spreads into
the lower body, where it can be severe. Other symptoms may include
muscle wasting, clumsy hand movements, fasciculations, and muscle
cramps. The trunk muscles and respiration may become affected.
Exposure to cold can worsen symptoms. The disease develops into ALS
in many instances.
[0195] Spinal muscular atrophy (SMA) is a hereditary disease
affecting the lower motor neurons. Weakness and wasting of the
skeletal muscles is caused by progressive degeneration of the
anterior horn cells of the spinal cord. This weakness is often more
severe in the legs than in the arms. SMA has various forms, with
different ages of onset, patterns of inheritance, and severity and
progression of symptoms. Some of the more common SMAs are described
below.
[0196] SMA type I, also called Werdnig-Hoffmann disease, is evident
by the time a child is 6 months old. Symptoms may include hypotonia
(severely reduced muscle tone), diminished limb movements, lack of
tendon reflexes, fasciculations, tremors, swallowing and feeding
difficulties, and impaired breathing. Some children also develop
scoliosis (curvature of the spine) or other skeletal abnormalities.
Affected children never sit or stand and the vast majority usually
die of respiratory failure before the age of 2.
[0197] Symptoms of SMA type II usually begin after the child is 6
months of age. Features may include inability to stand or walk,
respiratory problems, hypotonia, decreased or absent tendon
reflexes, and fasciculations. These children may learn to sit but
do not stand. Life expectancy varies, and some individuals live
into adolescence or later.
[0198] Symptoms of SMA type III (Kugelberg-Welander disease) appear
between 2 and 17 years of age and include abnormal gait; difficulty
running, climbing steps, or rising from a chair; and a fine tremor
of the fingers. The lower extremities are most often affected.
Complications include scoliosis and joint contractures--chronic
shortening of muscles or tendons around joints, caused by abnormal
muscle tone and weakness, which prevents the joints from moving
freely.
[0199] Symptoms of Fazio-Londe disease appear between 1 and 12
years of age and may include facial weakness, dysphagia (difficulty
swallowing), stridor (a high-pitched respiratory sound often
associated with acute blockage of the larynx), difficulty speaking
(dysarthria), and paralysis of the eye muscles. Most individuals
with SMA type III die from breathing complications.
[0200] Kennedy disease, also known as progressive spinobulbar
muscular atrophy, is an X-linked recessive disease. Daughters of
individuals with Kennedy disease are carriers and have a 50 percent
chance of having a son affected with the disease. Onset occurs
between 15 and 60 years of age. Symptoms include weakness of the
facial and tongue muscles, hand tremor, muscle cramps, dysphagia,
dysarthria, and excessive development of male breasts and mammary
glands. Weakness usually begins in the pelvis before spreading to
the limbs. Some individuals develop noninsulin-dependent diabetes
mellitus.
[0201] The course of the disorder varies but is generally slowly
progressive. Individuals tend to remain ambulatory until late in
the disease. The life expectancy for individuals with Kennedy
disease is usually normal.
[0202] Congenital SMA with arthrogryposis (persistent contracture
of joints with fixed abnormal posture of the limb) is a rare
disorder. Manifestations include severe contractures, scoliosis,
chest deformity, respiratory problems, unusually small jaws, and
drooping of the upper eyelids.
[0203] Post-polio syndrome (PPS) is a condition that can strike
polio survivors decades after their recovery from poliomyelitis.
PPS is believed to occur when injury, illness (such as degenerative
joint disease), weight gain, or the aging process damages or kills
spinal cord motor neurons that remained functional after the
initial polio attack. Many scientists believe PPS is latent
weakness among muscles previously affected by poliomyelitis and not
a new MND. Symptoms include fatigue, slowly progressive muscle
weakness, muscle atrophy, fasciculations, cold intolerance, and
muscle and joint pain. These symptoms appear most often among
muscle groups affected by the initial disease. Other symptoms
include skeletal deformities such as scoliosis and difficulty
breathing, swallowing, or sleeping. Symptoms are more frequent
among older people and those individuals most severely affected by
the earlier disease. Some individuals experience only minor
symptoms, while others develop SMA and, rarely, what appears to be,
but is not, a form of ALS. PPS is not usually life threatening.
Doctors estimate the incidence of PPS at about 25 to 50 percent of
survivors of paralytic poliomyelitis.
Prostaglandin D2 Receptors
[0204] There are two identified PGD2 receptors: PGD2 receptor 1
(often known as the classic PGD2 receptor, is also called AS1, DP1,
DP, ASRT1, MGC49004, PTGDR) and G protein-coupled receptor 44 or
"chemoattractant receptor-homologous molecule expressed on TH2
cells" (also known as DP2, CD294, GPR44, and CRTH2). Both are G
protein-coupled 7-transmembrane receptors but there are distinct
differences between these two receptors with respect to their gene
encoding the receptor, cell expression and signaling responses to
ligands.
[0205] The human gene for the prostaglandin D2 receptor DP1
(NM_000953.2; NP_000944.1; NCBI Entrez GeneID: 5729) is located on
chromosome 14 and it encodes a G-protein-coupled protein
7-transmembrane receptor that has been shown to function as a
prostanoid DP receptor. The activity of this receptor is mainly
mediated by G-stimulatory proteins that stimulate adenylate cyclase
with an elevation of intracellular cAMP and Ca.sup.2+ but without
an observed increase in IP3. On the other hand, the human gene for
the prostaglandin D2 receptor CRTH2 (NM_004778.2; NP_004769.2; NCBI
Entrez GeneID: 11251) is located on chromosome 11 and it encodes a
G-protein-coupled protein 7-transmembrane receptor. The activity of
this receptor is mainly mediated by G-inhibitory proteins.
[0206] In normal subjects, DP2 is selectively expressed by T helper
2 cells but not T helper 1 cells among circulating CD4+
lymphocytes. DP2 does not mediate Nicotinic acid (NA)-induced
vasodilation; the DP2-specific agonist DK-PGD2
(13,14-dihydro-15-keto-PGD2) did not induce cutaneous vasodilation,
and DP2.sup.-/- mice have a normal vasodilatory response to NA. By
contrast, BW245C, a DP1-selective agonist, induced vasodilation in
mice, and MK-0524, a DP1-selective antagonist, blocked both PGD2-
and NA-induced vasodilation.
[0207] In response to the ligand prostaglandin D2, DP1 induces
Ca.sup.2+ influx and cAMP generation through Gas-type G protein,
which leads to vasodilation, relavation of smooth muscles, and
inhibition of dendritic cell migration. In contrast, CRTH2 is
coupled with Gai-type G protein and induces cell migration in
eosinophil, basophils, and TH2 lines.
[0208] Several literatures have indicated that activation and
signaling through the classic DP1 receptor provides neuroprotection
for motor neurons in organotypic models of ALS (Liejun Wu, et al,
2007, Neurasci. Letts. 421:253-258); provides neuroprotection
against glutamate toxicity in cultured hippocampal neurons and
organotypic slices while activation of CRTH2 promoted neuron loss
(Liang X., et al., 2005, J. Neurochem. 92:477-486); and provides
neuroprotection against ischemia-reperfusion injury in primary
cultures of corticostriatal neurons (Sofiyan Saleem, et al., 2007,
Eur. J. Neurosci. 26:73-78).
Inhibition of the Expression and/Activity of Prostaglandin D2 DP1
Receptor
[0209] In some embodiments, the methods of neuroprotecting motor
neurons, promoting survival of motor neurons and/or treating of
motor neuron diseases comprise preventing, blocking, stopping,
and/or reducing the activation and signaling from the classic
prostaglandin D2 receptor, DP1 by way of an inhibitor.
[0210] Embodiments of the invention also provide methods for
increasing motor neuron survival in a subject with motor neuron
disease comprising administering to a subject an inhibitor of
expression and/or activity of the prostaglandin D2 DP1
receptor.
[0211] In some embodiments, the inhibitor is selected from the
group consisting of: a small molecule, a nucleic acid molecule, a
protein, e.g. an activity-blocking antibody, and combinations
thereof.
[0212] In one embodiment, the inhibitor functions by inhibiting,
preventing, blocking, stopping, and/or reducing the expression of
the DP1 and the inhibitor is selected from a small molecule and a
nucleic acid. Such an inhibitor of DP1 expression would reduce the
mRNA or protein level of DP1 by at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 100%, including all the percentages between
20% to 100%. The changes in mRNA or protein level can be assessed
by any method known to one skilled in the art, e. g. quantitative
RT-PCR to determine the mRNA synthesized and Western Blot analysis
for determining the protein amount. Antibodies against the human
DP1 are commercially available, e. g. at ABNOVA Catalog #:
H00005729-B01P. In a preferred embodiment, the inhibitor is a
nucleic acid comprising a DP1 (human PTGDR) specific RNA
interference agent or a vector encoding a human PTGDR specific RNA
interference agent. In a some embodiment, the RNA interference
agent comprises one or more of the nucleotide sequences of the
following sequences:
TABLE-US-00001 (SEQ ID NO: 7) ACAGGACCUCUGAAGAAGCtt; (SEQ ID NO: 8)
AUAUGACCAGGUCAGGCAGtt; (SEQ ID NO: 9) GGGUGUCAGUAGGAAUCAAtt; (SEQ
ID NO: 10) CCAGUGUGUGACUCACUGUtt; (SEQ ID NO: 11)
AGCCCACCCAGGACUUAGCtt; (SEQ ID NO: 12) ACGCAGCUGCAACUGAAGCtt.
[0213] Alternatively, specific RNA interference agent for the human
PTGDR or a vector encoding a human PTGDR specific RNA interference
agent such as siRNA and shRNA reagents against the human PTGDR are
commercially obtainable from INVITROGEN Inc., STEALTH SELECT
RNAi.TM. siRNA (Catalog#1299003 for the set of three oligos); or
single oligos (cat. Log # HSS108762; HSS108763; HSS108764); Applied
Biosystems SILENCER.RTM. siRNAs: NM_000953; ABNOVA.
[0214] In one embodiment, the inhibitor functions by inhibiting,
preventing, blocking, stopping, and/or reducing DP1 signaling
activity. The inhibitor is an antagonist of the DP1 receptor and is
selected from the group consisting of an activity-blocking antibody
against DP1, an antibody against the ligand prostaglandin D2 (i. e.
an anti-PGD2 antibody), an antigen-binding fragment thereof of any
of the described antibodies or a small molecule antagonist of the
DP1 receptor.
[0215] In one embodiment, antibodies that specifically bind DP1 or
PGD2 can be used for the inhibition of the DP1 signaling in vivo.
Antibodies to DP1 are commercially available (e. g. ABNOVA, Catalog
#: H00005729-B01P) and can be raised by one of skill in the art
using well known methods, e. g. as disclosed in PCT publication WO
97/40072 or U.S. Application. No. 2002/0182702, which are herein
incorporated by reference. The processes of immunization to elicit
antibody production in a mammal, the generation of hybridomas to
produce monoclonal antibodies, and the purification of antibodies
may be performed by described in "Current Protocols in Immunology"
(CPI) (John Wiley and Sons, Inc.) and Antibodies: A Laboratory
Manual (Ed Harlow and David Lane editors, Cold Spring Harbor
Laboratory Press 1988) which are both incorporated by reference
herein in their entireties; Brown, "Clinical Use of Monoclonal
Antibodies," in. The DP1 inhibitory activity of a given antibody,
or, for that matter, any DP1 inhibitor, can be assessed using
methods known in the art or described herein--to avoid doubt, an
antibody that inhibits DP1 activity will cause a decrease in cAMP
production and Ca.sup.2+ influx in the presence of an
activity-blocking antibody compared to in the absence of such an
antibody. Biochemical assays for determining DP1 (PTGDR) activity
are available from INVITROGEN.TM. Inc., MILLIPORE.RTM., R&D
Systems and BIOMOL.RTM. to name a few. A decrease in cAMP
production, Ca.sup.2+ influx and/or DP1 signaling activity is at
least 10% lower, at least 20% lower, at least 30% lower, at least
40% lower, at least 50% lower, at least 60% lower, at least 70%
lower, at least 80% lower, at least 90% lower, at least 1-fold
lower, at least 2-fold lower, at least 5-fold lower, at least 10
fold lower, at least 100 fold lower, at least 1000-fold lower or
more in the presence of the anti-DP1 antibody, anti-PGD2 antibody,
and/or inhibitor of DP1 activity, including the percentages in
between herein disclosed, compared to control which is in the
absence of any DP1 antibody, PGD2 antibody, and/or DP1 activity
inhibitor.
[0216] Antibody inhibitors of DP1 or PGD2 can include polyclonal
and monoclonal antibodies and antigen-binding derivatives or
fragments thereof. Well known antigen binding fragments include,
for example, single domain antibodies (dAbs; which consist
essentially of single VL or VH antibody domains), Fv fragment,
including single chain Fv fragment (scFv), Fab fragment, and
F(ab')2 fragment. Methods for the construction of such antibody
molecules are well known in the art.
[0217] In one embodiment, the inhibitor of DP1 activity interferes
with DP1 interaction with its ligand PGD2, e. g. an anti-PGD2
antibody which serves to sequester the PGD2 away from the receptor
and thus prevent any signaling from the receptor. Anti-PGD2
antibodies are commercially available, e. g. at BIOMOL.RTM. Cat.
#905-047. An anti-DP1 antibody also serves to interfere with DP1
receptor interaction with its ligand PGD2, especially when the
antibody binds to the extracellular PGD2-binding region of the
receptor. The extracellular regions are found on amino acids 1-19,
85-103, 169-193 and 288-307 of the 360 amino acid residue of the
human DP1 protein.
[0218] In one embodiment, the inhibitor of DP1 activity is a small
molecule antagonist of PGD2 DP1 receptor function. Examples include
but are not limited to: MK-0524, BWA868C, ONO-4127Na, resveritrol,
the 2,6-substituted-4-mono substituted amino-pyrimidine compounds
described in WO/2007/121280 and the 2-phenyl-indole compounds
described in US 2009176804 and WO 2008014186. Resveritrol is a
natural compound found in grapes, mulberries, peanuts, and other
plants or food products, especially red wine that may protect
against cancer and cardiovascular disease by acting as an
antioxidant, anti-mutagen, and anti-inflammatory. Other antagonists
are described in US 2005/0215609, WO 2005/079793, and WO
2001/078697, all the patent applications are hereby incorporated by
reference in their entirety.
Administration and Formulations
[0219] In one embodiment, the methods described herein comprise
administering a pharmaceutical composition comprising of an
inhibitor of a prostaglandin D2 DP1 receptor, including anti-DP1
antibody, anti-PGD2 antibody, DP1 specific RNA interfering agents,
MK-0524, BWA868C, ONO-4127Na, resveritrol and other DP1
antagonists, and a pharmaceutically acceptable carrier or diluent.
In one embodiment, the pharmaceutical composition is administered
by injection, infusion, instillation, or ingestion. The
pharmaceutical compositions of the invention are administered in a
therapeutically effective amount.
[0220] As used herein, the term "pharmaceutically acceptable", and
grammatical variations thereof, as they refer to compositions,
carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or
upon a mammal without the production of undesirable physiological
effects such as nausea, dizziness, gastric upset and the like. Each
carrier must also be "acceptable" in the sense of being compatible
with the other ingredients of the formulation. A pharmaceutically
acceptable carrier will not promote the raising of an immune
response to an agent with which it is admixed, unless so desired.
The preparation of a pharmacological composition that contains
active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
The pharmaceutical formulation contains a compound of the invention
in combination with one or more pharmaceutically acceptable
ingredients. The carrier can be in the form of a solid, semi-solid
or liquid diluent, cream or a capsule. Typically such compositions
are prepared as injectable either as liquid solutions or
suspensions, however, solid forms suitable for solution, or
suspensions, in liquid prior to use can also be prepared. The
preparation can also be emulsified or presented as a liposome
composition. The active ingredient can be mixed with excipients
which are pharmaceutically acceptable and compatible with the
active ingredient and in amounts suitable for use in the
therapeutic methods described herein. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol or the like and
combinations thereof. In addition, if desired, the composition can
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like which enhance
the effectiveness of the active ingredient. The therapeutic
composition of the present invention can include pharmaceutically
acceptable salts of the components therein. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the polypeptide) that are formed with
inorganic acids such as, for example, hydrochloric or phosphoric
acids, or such organic acids as acetic, tartaric, mandelic and the
like. Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine and the like. Physiologically tolerable
carriers are well known in the art. Exemplary liquid carriers are
sterile aqueous solutions that contain no materials in addition to
the active ingredients and water, or contain a buffer such as
sodium phosphate at physiological pH value, physiological saline or
both, such as phosphate-buffered saline. Still further, aqueous
carriers can contain more than one buffer salt, as well as salts
such as sodium and potassium chlorides, dextrose, polyethylene
glycol and other solutes. Liquid compositions can also contain
liquid phases in addition to and to the exclusion of water.
Exemplary of such additional liquid phases are glycerin, vegetable
oils such as cottonseed oil, and water-oil emulsions. The amount of
an active agent used in the invention that will be effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. The phrase "pharmaceutically
acceptable carrier or diluent" means a pharmaceutically acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting the subject agents from one organ, or
portion of the body, to another organ, or portion of the body.
[0221] As used herein, "administered" refers to the placement of an
inhibitor of a prostaglandin D2 DP1 receptor, into a subject by a
method or route which results in at least partial localization of
the inhibitor at a desired site. An inhibitor of a prostaglandin D2
DP1 receptor, including anti-DP1 antibody, anti-PCD2 antibody, DP1
specific RNA interfering agents, MK-0524, BWA868C, ONO-4127Na,
resveritrol and other small molecule DP1 antagonists, can be
administered by any appropriate route which results in effective
treatment in the subject, i.e. administration results in delivery
to a desired location in the subject where at least a portion of
the composition delivered, i.e. at least one inhibitor of DP1, is
active in the desired site for a period of time. The period of time
the inhibitor is active depends on the half life in vivo after
administration to a subject, and can be as short as a few hours, e.
g. twenty-four hours, to a few days, to as long as several years.
Modes of administration include injection, infusion, instillation,
or ingestion. "Injection" includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion.
[0222] Inhibitor(s) of a prostaglandin D2 DP1 receptor, including
anti-DP1 antibody, anti-PCD2 antibody, DP1 specific RNA interfering
agents, MK-0524, BWA868C, ONO-4127Na, resveritrol and other DP1
antagonists, can be employed, either alone or in combination with
one or more other therapeutic agents, e. g. administered as a
"cocktail" formulation with other therapeutics typically prescribed
for MND, e. g. riluzole, massage, aromatherapy and reflexology. The
administration can be a coordinated administration for
simultaneous, sequential or separate use, of one or more inhibitors
of the invention together with one or more other active
therapeutics.
[0223] Inhibitor(s) of a prostaglandin D2 DP1 receptor, including
anti-DP1 antibody, anti-PCD2 antibody, DP1 specific RNA interfering
agents, MK-0524, BWA868C, ONO-4127Na, resveritrol and other DP1
antagonists can be administered either as the sole active
therapeutic or in a coordinated regime with one or more other
therapeutics can be administered by a variety of routes, such as
orally or by injection, e. g., intramuscular, intraperitoneal,
subcutaneous or intravenous injection, or topically such as
transdermally, vaginally and the like. Small molecule inhibitors
such as the pyrrolidine inhibitor compounds can be suitably
administered to a subject in the protonated and water-soluble form,
e.g., as a pharmaceutically acceptable salt of an organic or
inorganic acid, e.g., hydrochloride, sulfate, hemi-sulfate,
phosphate, nitrate, acetate, oxalate, citrate, maleate, mesylate,
etc. If the compound has an acidic group, e.g., a carboxy group,
base addition salts may be prepared. Lists of additional suitable
salts may be found, e.g., in Part 5 of Remington's Pharmaceutical
Sciences, 20th Edition, 2000, Marck Publishing Company, Easton,
Pa.
[0224] Inhibitor(s) of a prostaglandin D2 DP1 receptor, including
anti-DP1 antibody, anti-PCD2 antibody, DP1 specific RNA interfering
agents, MK-0524, BWA868C, ONO-4127Na, resveritrol and other DP1
antagonists can be employed, either alone or in combination with
one or more other therapeutic agents as discussed above, as a
pharmaceutical composition in a mixture with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for oral, parenteral, enteral or
topical application which do not deleteriously react with the
active compounds and are not deleterious to the recipient thereof.
Suitable pharmaceutically acceptable carriers include but are not
limited to water, salt solutions, alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, petroethral fatty acid
esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The
pharmaceutical preparations can be sterilized and if desired mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, colorings, flavorings and/or aromatic
substances and the like which do not deleteriously react with the
active compounds.
[0225] For oral administration, pharmaceutical compositions
containing Inhibitor(s) of a prostaglandin D2 DP1 receptor,
including anti-DP1 antibody, anti-PCD2 antibody, DP1 specific RNA
interfering agents, MK-0524, BWA868C, ONO-4127Na, resveritrol and
other DP1 antagonists, can be formulated as e.g., tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, syrups, elixers and the
like. Typically suitable are tablets, dragees or capsules having
talc and/or carbohydrate carrier binder or the like, the carrier
preferably being lactose and/or corn starch and/or potato starch. A
syrup, elixir or the like can be used wherein a sweetened vehicle
is employed. Sustained release compositions can be formulated
including those wherein the active component is protected with
differentially degradable coatings, e. g., by microencapsulation,
multiple coatings, etc.
[0226] For parenteral application, e.g., sub-cutaneous,
intraperitoneal or intramuscular, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Ampules are convenient unit dosages. It will be appreciated that
the actual preferred amounts of active compounds used in a given
therapy will vary according to the specific compound being
utilized, the particular compositions formulated, the mode of
application, the particular site of administration, etc. Optimal
administration rates for a given protocol of administration can be
readily ascertained by those skilled in the art using conventional
dosage determination tests conducted with regard to the foregoing
guidelines. See also Remington's Pharmaceutical Sciences, supra. In
general, a suitable effective dose of one or more 1, 2-substituted
5-pyrrolidinone compounds of the invention, particularly when using
the more potent compound (s) of the invention, will be in the range
of from 0.01 to 100 milligrams per kilogram of bodyweight of
recipient per day, preferably in the range of from 0. 01 to 20
milligrams per kilogram bodyweight of recipient per day, more
preferably in the range of 0.05 to 4 milligrams per kilogram
bodyweight of recipient per day. The desired dose is suitably
administered once daily, or several subdoses, e.g., 2 to 4
sub-doses, are administered at appropriate intervals through the
day, or other appropriate schedule. Such sub-doses may be
administered as unit dosage forms, e.g., containing from 0.05 to 10
milligrams of compound (s) of the invention, per unit dosage.
[0227] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
compositions encompassed by the invention can be made using
conventional methodologies or on the basis of in vivo testing using
an appropriate animal model, as known in the art, or as described
herein.
[0228] The nucleic acid inhibitor can comprise a delivery vehicle,
including liposomes, for administration to a subject, carriers and
diluents and their salts, and/or can be present in pharmaceutically
acceptable formulations. Methods for the delivery of nucleic acid
molecules are described in Akhtar et al., Trends in Cell Bio.
2:139, 1992; Delivery Strategies for Antisense Oligonucleotide
Therapeutics, ed. Akhtar, 1995; Maurer et al., Mol. Membr. Biol.,
16:129, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165,
1999; and Lee et al., ACS Symp. Ser. 752:184, 2000, all of which
are incorporated herein by reference. Beigelman et al., U.S. Pat.
No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe
the general methods for delivery of nucleic acid molecules. Nucleic
acid molecules can be administered to cells by a variety of methods
known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by ionophoresis, or by
incorporation into other vehicles, such as hydrogels, cyclodextrins
(see for example Gonzalez et al., Bioconjugate Chem. 10:1068,
1999), biodegradable nanocapsules, and bioadhesive microspheres, or
by proteinaceous vectors (O'Hare and Normand, International PCT
Publication No. WO 00/53722).
[0229] In the present methods, the RNA interference agent can be
administered to the subject either as naked RNA interference agent,
in conjunction with a delivery reagent, or as a recombinant plasmid
or viral vector which expresses the RNA interference agent.
Preferably, the RNA interference agent is administered as naked RNA
interference agent.
[0230] The RNA interference agent of the invention can be
administered to the subject by any means suitable for delivering
the RNA interference agent to the cells of the tissue at or near
the area of with motor neurons. For example, the RNA interference
agent can be administered by gene gun, electroporation, or by other
suitable parenteral administration routes.
[0231] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0232] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%.
[0233] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. It is further to be understood that
all base sizes or amino acid sizes, and all molecular weight or
molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." The abbreviation, "e.g." is derived from the
Latin exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0234] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0235] This invention is further illustrated by the following
example which should not be construed as limiting. The contents of
all references cited throughout this application, as well as the
figures and table are incorporated herein by reference.
EXAMPLES
Example 1: Derivation of ES Cell Lines from the SOD1G93A ALS Mouse
Model
[0236] Embryonic stem cell lines were derived by crossing
hemizygous mice carrying either the pathogenic (mutant) SOD1G93A
transgene or the non-pathogenic (wild-type) SOD1 transgene (Gurney
et al., 1994) with hemizygous mice carrying a transgenic reporter
gene in which green fluorescent protein (GFP) expression is
controlled by promoter elements from the Hb9 gene (Hb9::GFP)
(Wichterle et al., 2002). The Hb9 gene encodes a homeodomain
transcription factor that is expressed in postmitotic motor neurons
(Arber, S. et al. Requirement for the homeobox gene Hb9 in the
consolidation of motor neuron identity. Neuron 23, 659-74, 1999;
Thaler, J. et al. Active suppression of interneuron programs within
developing motor neurons revealed by analysis of homeodomain factor
HB9. Neuron 23, 675-87, 1999). This Hb9::GFP transgene provides a
marker for the differentiation of ES cells into motor neurons
(Wichterle et al., 2002). Blastocyst stage embryos were retrieved
from the progeny of these crosses and used to derive ES cell lines
that were genotyped using the polymerase chain reaction (PCR) (FIG.
1A,B). These analyses identified ES cell lines that carried only
the Hb9::GFP transgene (Hb9GFP), lines that carried both Hb9::GFP
and the wild-type SOD1 transgene (SOD1), and a line that carried
both Hb9::GFP and the mutant SOD1G93A transgene (SOD1G93A).
[0237] To determine whether these ES cells recapitulate the proper
expression pattern of the Hb9::GFP reporter transgene, we assessed
GFP fluorescence in the undifferentiated ES cells and in chimeras
created by injecting these cells into non-transgenic blastocysts.
Each of the undifferentiated cell lines lacked obvious GFP
expression (data not shown). However, in E10.5 chimeras created
with these cells, highly specific GFP expression was observed in
the developing eye, hindbrain and spinal chord where Hb9 is known
to be expressed (data not shown) (Wichterle et al., 2002; Thaler et
al., 1999). Immunostaining with antibodies that preferentially
recognize the human SOD1 protein confirmed the PCR genotyping of
the cell lines and showed that both the SOD1 and SOD1G93A
transgenes are expressed in the undifferentiated ES cells (data not
shown).
Example 2: Production and Characterization of Motor Neurons by In
Vitro Differentiation of SOD1G93A ES Cells
[0238] To determine whether pathogenic properties associated with
ALS can be recapitulated in vitro, we generated motor neurons by
differentiating the transgenic ES cell lines as previously
described (Wichterle et al., 2002). Briefly, ES cells were
dissociated into a single cell suspension, allowed to spontaneously
aggregate into embryoid bodies (EBs) over 48 hours and then treated
with retinoic acid (RA) and soluble Sonic Hedgehog (Shh)
protein.sup.1 (Wichterle et al., 2002) for 5 days.
[0239] We found that the SOD1G93A genotype does not interfere with
the initial specification or differentiation of motor neurons, as
no significant qualitative or quantitative differences were
observed in the differentiation of the three cell lines. GFP
expression in EBs derived from the different cell lines, including
SOD1G93A, first appeared 5 days after treatment with Shh and RA
(data not shown). Two days later, when EBs were dissociated with
papain and plated, GFP positive cells with an obvious neuronal
morphology could be observed (data not shown). We used fluorescence
activated cell sorting (FACS) to determine the percentage of
differentiating ES cells that expressed GFP and found no
statistically significant differences between the three cell lines
(Hb9GFP 33%+/-4%, SOD1 33%+/-9%, SOD1G93A 26%+/-6%) (see FIG.
5).
[0240] To confirm that the EB cells expressing GFP differentiated
into bone fide motor neurons, we dissociated the EBs and performed
immunostaining with antibodies that recognize proteins known to be
expressed in motor neurons (data not shown). As was previously
observed with normal ES cell lines (Wichterle et al., 2002), we
found that GFP positive cells derived from the SOD1G93A cell line
expressed a neuronal form of tubulin (data not shown), the
transcription factors Hb9 and Isl1/2 (data not shown) and the
enzymatic machinery required to generate acetylcholine (data not
shown).
Example 3: The SOD1 G93A Genotype Affects the Survival of Motor
Neurons in Culture
[0241] ALS is a late onset, progressive neurodegenerative disease,
and mice carrying the human SOD1G93A transgene develop symptoms as
a consequence of motor neuron loss after several weeks. Therefore,
it seemed possible that motor neurons derived from ES cells might
display neurodegenerative properties only after they have been
maintained in culture for a prolonged length of time. To determine
the period of time that ES cell derived motor neurons can survive
in culture, we dissociated day 7 Hb9GFP and SOD1G93A EBs and plated
the resulting mixture of GFP positive and negative cells at two
different densities in the presence of neurotrophic factors
(Wichterle et al., 2002) (FIG. 2). We observed that the number of
GFP positive cells decreased precipitously during the first two
weeks, and then continued to decrease over the following weeks.
However, GFP positive cells could still be detected in both HB9GFP
and SOD1G93A derived cultures 54 days after plating (data not
shown). Although GFP positive cells were present in both cultures
at later time points, the number of cells expressing GFP decreased
more rapidly in the SOD1G93A (FIG. 2B) cultures than in Hb9GFP
control cultures (FIG. 2A).
[0242] To confirm the affect of the SOD1G93A genotype on the number
of GFP positive cells, we differentiated both the SOD1G93A and
Hb9GFP ES cells into motor neurons, plated equal numbers of cells
at two different concentrations (8.times.10.sup.5 (n=3) and
4.times.10.sup.5 (n=3) EB cells per well) and counted the number of
GFP positive neurons in the cultures at 2 and 4 weeks (FIG. 2C-F).
Under both plating conditions, significantly fewer GFP positive
cells were observed in the SOD1G93A cultures at both 2 and 4 weeks
(FIG. 2C-F).
Example 4: Histopathological Hallmarks of ALS can be Observed in ES
Cell-Derived Motor Neurons
[0243] Hb9GFP and SOD1G93A ES cells differentiate into motor
neurons at a similar efficiency, but the cultures show differences
in the number of GFP positive cells over time. Thus, a pathological
process may underlie the preferential loss of GFP positive cells in
the SOD1G93A cultures. To investigate these processes and to
determine whether they mirror events that occur during the
progression of ALS, we examined motor neurons in culture for the
presence of histopathological hallmarks of the disease. Motor
neurons in ALS patients and transgenic mice carrying the SOD1G93A
allele accumulate protein inclusions that are recognized by
antibodies specific to the SOD1 protein (Boillee et al., 2006;
Bruijn et al., 19970. We therefore determined whether aggregation
of the mutant SOD1 protein accompanies the loss of GFP positive
motor neurons in SOD1G93A cultures by staining with antibodies
specific to the human SOD1 protein at 7, 14 and 21 days following
EB dissociation (data not shown). At 7 and 14 days following
dissociation both the wild-type SOD1 protein and the mutant
SOD1G93A protein were localized broadly and evenly in the cytoplasm
of GFP positive motor neurons (data not shown). However, at 14 days
punctate structures staining brightly with the SOD1 antibody could
be observed in a small proportion of motor neurons expressing the
SOD1 G93A protein, (data not shown).
[0244] When cultures were examined 21 days after dissociation, a
shift in protein localization was observed in the SOD1G93A motor
neurons (FIG. 3D). In 43/54 (79.75.+-.7.75%) of the motor neurons
selected at random for analysis by GFP expression, the SOD1G93A
protein localized to inclusions in the perinuclear space, in the
cell body and also in the neural processes (FIG. 3D). When control
motor neurons expressing wild-type SOD1 were examined 21 days after
differentiation (FIG. 3D), we observed inclusions in a smaller
proportion of cells 23/64 (35.93.+-.0.43%). The inclusions in motor
neurons expressing the SOD1G93A allele were also significantly
larger in area (FIG. 6A), significantly longer (FIG. 6B) and
displayed a higher optical density, suggesting that they contained
more SOD1 protein at a higher concentration (FIG. 6C).
[0245] The levels of ubquitinated proteins are significantly
elevated in motor neurons of ALS patients and SOD1G93A transgenic
animals during neural degeneration (Bruijn et al., 1997; Ince, P.
G., et al., Amyotrophic lateral sclerosis associated with genetic
abnormalities in the gene encoding Cu/Zn superoxide dismutase:
molecular pathology of five new cases, and comparison with previous
reports and 73 sporadic cases of ALS. J Neuropathol Exp Neurol 57,
895-904, 1998; Wang, J. et al. Copper-binding-site-null SOD1 causes
ALS in transgenic mice: aggregates of non-native SOD1 delineate a
common feature. Hum Mol Genet 12, 2753-64, 2003; Watanabe, M. et
al. Histological evidence of protein aggregation in mutant SOD1
transgenic mice and in amyotrophic lateral sclerosis neural
tissues. Neurobiol Dis 8, 933-41, 2001). Examination of the ES
cell-derived motor neurons revealed an increase in staining with
anti-ubiquitin antibodies, relative to other cells in the culture.
This staining often colocalized with the SOD1 protein inclusions
(data not shown).
[0246] The death of motor neurons in ALS patients and transgenic
mice carrying mutant SOD1 genes occurs through activation of
programmed cell death pathways. Apoptosis in these cells has been
proposed to be mediated through the release of cytochrome c and the
activation of caspase-3 (Pasinelli, P., Houseweart, M. K., Brown,
R. H., Jr. & Cleveland, D. W. Caspase-1 and -3 are sequentially
activated in motor neuron death in Cu, Zn superoxide
dismutase-mediated familial amyotrophic lateral sclerosis. Proc
Natl Acad Sci USA 97, 13901-6, 2000; Raoul, C. et al. Motoneuron
death triggered by a specific pathway downstream of Fas.
potentiation by ALS-linked SOD1 mutations. Neuron 35, 1067-83,
2002). Examination of the SOD1G93A motor neurons 14 days after
dissociation of EBs, revealed that some neurons expressed activated
caspase-3 and contained diffuse cytoplasmic staining with
cytochrome c specific antibodies (data not shown). Thus, the
SOD1G93A motor neurons appear to initiate cell death pathways in
vitro that are similar to those activated in vivo during the course
of disease (Pasinelli et al., 2000).
Example 5: SOD1G93A Glial Cells Adversely Affect Survival of ES
Cell Derived Motor Neurons
[0247] Both autonomous defects in motor neurons and toxic non-cell
autonomous interactions with other cell types in the spinal cord
have been implicated in ALS pathology (Bruijn et al., 2004; Clement
et al., 2003; Boillee et al., 2006). Only a subset of cells in EBs
became motor neurons under our differentiation conditions. We
therefore considered the possibility that cells within the EBs
might also develop into other cell types that normally associate
with neurons in the spinal cord. These additional cells might, as
suggested by chimeric experiments, contribute non-cell autonomously
to the loss of neurons in SOD1G93A cultures. Glial cells are
closely associated with motor neurons, and both are derived from a
common progenitor in vivo (Zhou, Q. & Anderson, D. J. The bHLH
transcription factors OLIG2 and OLIG1 couple neuronal and glial
subtype specification. Cell 109, 61-73, 2002). We therefore
addressed the possibility that glial cells are present in our
cultures. To determine if these cells, were also produced by our
differentiation protocol, we stained SOD1G93A cultures with
antibodies specific to the glial fibrillary acidic protein (GFAP)
(Bignami, A. & Dahl, D. Astrocyte-specific protein and
neuroglial differentiation. An immunofluorescence study with
antibodies to the glial fibrillary acidic protein. J Comp Neurol
153, 27-38, 1974). Indeed, GFAP positive cells were found in close
association with GFP positive motor neurons (data not shown). We
found that approximately 30% of the cells at 28 days were GFAP
positive (Hb9GFP: 31%, SOD1: 32%, SOD1G93A: 36%). Thus, it seemed
possible that the SOD1G93A glial cells in these cultures might be
adversely affecting motor neuron survival.
[0248] To examine this possibility, we differentiated the three ES
cell lines (Hb9GFP, SOD1 and SOD1G93A) into motor neurons and
plated them on established monolayers of primary glia isolated from
the cortex of neonatal mice with differing SOD) genotypes
(wild-type SOD1, and the mutant SOD1G93A) (Banker, G. & Goslin,
K. Culturing nerve cells, xii, 666, 11 of plates (MIT Press,
Cambridge, Mass., 1998). During the first seven days after plating
on either glial monolayer, motor neurons of all genotypes increased
in size and took on a more mature morphology (data not shown).
However, by 14 days after plating there was a 50% decrease in the
number of wild-type SOD1 derived motor neurons in co-cultures with
SOD1G93A glia compared to the same preparation of neurons plated on
wild-type SOD1 glia. Similarly, we did not see a significant
reduction in the number of Hb9GFP motor neurons when plated on SOD1
glia, but did see a reduction of 30% if the same neurons were
co-cultured with SOD1G93A glia (FIG. 4). These data suggest that
wild-type SOD1 glia provide a permissive environment for motor
neuron growth and differentiation, comparable to other
well-established in vitro systems for motor neuron culture (Ullian,
E. M., Harris, B. T., Wu, A., Chan, J. R. & Barres, B. A.
Schwann cells and astrocytes induce synapse formation by spinal
motor neurons in culture. Mol Cell Neurosci 25, 241-51, 2004;
Allen, N. J. & Barres, B. A. Signaling between glia and
neurons: focus on synaptic plasticity. Curr Opin Neurobiol 15,
542-8, 2005; Ullian, E. M., Christopherson, K. S. & Barres, B.
A. Role for glia in synaptogenesis. Glia 47, 209-16, 2004). In
contrast, when we co-cultured wild type motor neurons (SOD1 or
Hb9GFP) with glia from SOD1G93A mice, we observed a marked
reduction in survival (FIG. 4).
[0249] We next investigated the affect of the presence of the
mutant SOD1 transgene in motor neurons in the co-culture system.
When mutant SOD1G93A motor neurons were plated on wild-type SOD1
glial cells, there was a 27% decrease in the number of neurons
between 7 and 14 days. The loss of motor neurons increased to 75%
when the mutant SOD1G93A motor neurons were cultured with mutant
SOD1G93A glia (FIG. 4).
[0250] Together, our results show that the SOD1G93A genotype in
glial cells has a negative effect on motor neuron survival
regardless of the motor neuron genotype. However, a greater
negative effect is observed with the SOD1G93A motor neurons. Thus,
glial cells have a non-cell autonomous effect on motor neuron
survival and mutant motor neurons are more sensitive to the
effect.
[0251] To determine whether the differing influences of the
wild-type SOD1 and the mutant SOD1G93A glial cell cultures could be
explained by the presence of different proportions of distinct
glial cell types, we characterized the two populations by
immunostaining with established glial markers. We did not observe a
significant difference in the wild-type SOD1 and the mutant
SOD1G93A glial populations over time (FIG. 7).
Example 6: Methods
[0252] Examples 1 through 5 were performed using the following
methods:
[0253] Derivation of mouse Embryonic Stem Cells. ES cell lines were
derived from crosses between mice transgenic for Hb9:GFP (Jackson
lab, Stock Number:005029) and mice transgenic for SOD1G.sup.93A
(Jackson lab, Stock Number:004435) or SOD1.sup.WT (Jackson lab,
Stock Number:002297). Transgenic Hb9:GFP females were injected IP
with 7.5 units of pregnant mares' serum (Calbiochem) followed 46-50
h later with 7.5 units of human chorionic gonadotropin (HCG)
(Calbiochem). After administration of HCG, females were mated with
either SOD1G93A or SOD1 transgenic males. Females were scarified
three days later and blastocysts were flushed from the uterine horn
with mES cell media (Knockout-DMEM (GIBCO), 15% Hyclone Fetal
Bovine Serum (Hyclone), 10.000 unit Penicillin and 1 mg/ml
Streptomicin (GIBCO), 2 mM Glutamine (GIBCO), 100 mM non-essential
aminoacid (GIBCO), 55 .quadrature.mM beta-mercapto-ethanol (GIBCO),
1,000 units/ml leukocyte inhibiting factor (Chemicon)). Blastocysts
were then plated individually into one 10 mm-well of a tissue
culture dish containing a feeder layer of mitotically inactivated
mouse embryonic fibroblast, in the presence of mES cell media
supplemented with the MEK kinase inhibitor PD98059 (cell signaling,
inc). 48 hours after plating embryos, one half volume of fresh
media was added to each culture well. Starting three days after
plating, the culture media was changed.
[0254] Four to five days after plating, ICM-derived outgrowth were
observed, dislodged from the rest of the cells with a Pasteur
pipette, washed once in a drop of PBS and then incubated for 10
minutes in a drop of 0.25% trypsin at 37 C. The ES cells clumps
were then gently dissociated with a Pasteur pipette filled with
mouse ES cell media and transferred onto a fresh layer of
fibroblasts in a 10 mm tissue culture well. For routine culture,
the mouse ES cells are generally split 1:6 with a solution of 0.25%
trypsin (GIBCO) every 2-3 days.
[0255] Generation of Chimeric embryos. Chimeric embryos were
generated as previously described (Hogan, B. in "Manipulating the
mouse embryo: a laboratory manual", xvii, 497 p. (Cold Spring
Harbor Laboratory Press, Plainview, N.Y., 1994). Blastocysts were
collected from the uterus of non-transgenic pregnant females mice
3.5 days postcoitum. The ES cells were injected (about 10 for each
blastocyst) using a microinjection pipette with a diameter of 12-15
.mu.m applying a brief pulse of the Piezo (Primetech, Ibaraki,
Japan) on one side of the blastocyst and pushing the needle through
the zona and trophectoderm layer into the blastocoel cavity. Ten
injected blastocysts were transferred to each uterine horn of 2.5
days postcoitum pseudopregnant Swiss females that had mated with
vasectomized males. Recipient mothers were sacrificed at 10.5 days
postcoitum, and embryos were quickly removed from the uterus and
placed in a dish with cold PBS for whole mount analysis of GFP
fluorescence.
[0256] Differentiation of mES cells into motor neurons. Mouse ES
cells were differentiated into motor neurons according to methods
previously described (Boillee et al., 2006). The ES cells were
grown at 70-80% confluence in a 10 cm plate (Falcon) in mouse ES
cells media. To form EB's, cells were washed once with a PBS
solution to eliminate the traces of media and then incubated with 1
ml of 0.25% trypsin (GIBCO) for 5-10 minutes at room temperature.
After this the cells were resuspended in 10 ml of DM1 media
(DMEM-F12 (GIBCO), 10% Knockout serum (GIBCO), pen strep, glutamine
(GIBCO) and 2-mercaptoethanol (GIBCO)), they were counted and
plated at a concentration of 200.000/ml in Petri dishes (Falcon).
After 2 days the embryoid bodies were split from 1 dish into 4 new
Petri dishes containing DM1 medium supplemented with RA (100 nM
.mu.M; stock: 1 mM in DMSO, Sigma) and sonic hedgehog (300 nM,
R&D Systems). The media is changed after 3-4 days. On day 7 the
embryoid bodies were dissociated in single cells suspension. The
EBs were collected in a 15 ml falcon tube, centrifuged at 1000 rpm
for 5 min, washed once with PBS and incubated in Earle's Balanced
Salt Solution with 20 units of papain and 1000 units of
Deoxyribonuclease I (Worthington Biochemical Corporation) for 30-60
minutes at 37.degree. C. The mixture was then triturated with a 10
ml pipette and centrifuged for 5 minutes at 1000 RPM. The resulting
cell pellet was washed once with PBS and finally resuspended in
supplemented F12 media (F12 medium (GIBCO) with 5% horse serum
(GIBCO), B-27 supplement (GIBCO), N2 supplement (GIBCO)) with
neurotrophic factors (GDNF, CNTF, and BDNF (10 ng/ml, R&D
Systems)). The cells were then counted and plated on
Poly-D-Lysine/Laminin CultureSlides (BD biosciences) or on a layer
of primary glia cells. For the motor neurons survival experiments,
GFP positive cells with visible axons and dendrites were counted at
different time points after plating (7, 14, 21, 28 days).
[0257] Polymerase chain reaction. All PCR reactions were performed
using an MJ Research Thermal Cycler, and TaKaRa Ex Taq HS (Takara)
enzymes. For the SOD1 genotyping of the newly derived ES cell lines
and transgenic mice the forward primer: CAT CAG CCC TAA TCC ATC TGA
(SEQ ID NO:1) and reverse primer: CGC GAC TAA CAA TCA AAG TGA (SEQ
ID NO:2) amplified a 236 bp fragment in the 4.sup.th exons of the
gene. As an internal control a set of primers that amplified a 324
bp fragment of the IL-2 gene (forward: CTA GGC CAC AGA ATT GAA AGA
TCT (SEQ ID NO:3), reverse: CAT CAG CCC TAA TCC ATC TGA (SEQ ID
NO:4)) were used. The annealing temperature for this reaction was
60.degree. C. for 35 cycles. For GFP a set of primers (forward: AAG
TTC ATC TGC AAC ACC (SEQ ID NO:5), reverse: TCC TTG AAG AAG ATG GTG
CG (SEQ ID NO:6)) that amplified a fragment of 173 bp of the gene
were used, with an annealing temperature of 60.degree. C. for 35
cycles.
[0258] FACS. For FACS analysis a BD biosciences LSRII flow
cytometer was used. The embryoid bodies were dissociated with
papain and resuspended in cold PBS with 2% FBS, Calcein blue
(INVITROGEN.TM.) was used to assay the cell viability. The cells
were then analyzed using a non transgenic mouse embryonic stem cell
lines as a negative control. The FACS Diva software package (BD
Biosciences) was used for data analysis
[0259] Glia Cultures. Glia monolayers were obtained from P2 mice
born from matings between transgenic SODG93A. Tissue was isolated
in Calcium and Magnesium Free-Hanks's BSS (HBSS). Under a
dissecting microscope the cortex was isolated and carefully striped
of the meninges. The tissue was split in small pieces then
transferred to a 50 ml centrifuge tube in a final volume of 12 ml
of HBSS. Tissue digestion was performed using trypsin--EDTA (GIBCO
BRLno.25200) and 1% DNAse (Sigma no.DN-25) at 37.degree. C. for 15
min, swirling the mixture and periodically. We collected the
dissociated tissue and triturated using a fire polish Pasteur glass
pipette and filtered the combined supernatant through a 72 .mu.m
nylon mesh (NITEX 100% polyamide Nylon Fiber TETKO Inc.) to remove
any undissociated tissue. The filtered material was centrifuged at
1000 rpm for 5 min to pellet the cells, resuspend in 2 ml of Glia
medium (Minimum Essential Medium with Earl's salts, GIBCO BRL no.
11095-080, 20% Glucose, Penicillin-streptomycin, GIBCO BRL
no.15145-014, and 10% Horse Serum GIBCO BRL no.26050-070) and cell
number was counted. The yield from one brain was generally enough
to plate one T75 flask (Falcon no.3084). Once monolayers were
confluent (generally in 10 to 14 days) cells were replated on 24 or
12 well multiwell dishes over poly-D-Lysine (0.5 mg/ml for 30 min
RT) coated glass cover slips.
[0260] Immunocytochemistry analysis. The cells were fixed with 4%
paraformaldehyde-PBS, blocked and permeabilized with BSA
(1%)-Triton X 100 (0.1%). After incubating overnight with the
following antibodies: mouse monoclonal anti-Tuj1 (Covance), Islet 1
Islet2, RC2 (Developmental Studies Hybridoma Bank University of
Iowa, Iowa, USA) anti-sod (SIGMA), S100 (Chemicon), CNPase (Abcam)
and rabbit anti-HB9 (Tom Jessell, Columbia University), GFAP
(Chemicon) anti-ubiquitin (DAKO); goat anti-Vimentin (Chemicon);
rat anti-CD 11b (Abcam) (see Table 1)
Table 1 describes primary antibodies used. Antiserum (host
species), working dilution and source.
TABLE-US-00002 Antiserum Dilution Source Tuj 1 (anti-Mouse) 1/1000
Covance Islet 1 (anti-Mouse) 1/1 Hybridoma Bank ChAT (anti-Goat)
1/100 Chemicon Hb9 (anti-Rabbit) 1/1000 Jessel Lab GFAP
(anti-Rabbit) 1/1000 Chemicon S100 (anti-Mouse) 1/100 Chemicon RC2
(anti-Mouse) 1/1 Hybridoma Bank Vimentin (anti-goat) 1/100 Chemicon
CD 11b (anti-Rat) 1/100 Abeam hSOD1 (anti-Mouse) 1/200 Sigma
Caspase 3 (anti-Rabbit) 1/2000 BD Pharmingen Ubiquitin
(anti-Rabbit) 1/200 DAKO Cytochrome C (anti-Mouse) 1/200 Abeam
[0261] The cells were then incubated with Donkey anti-rabbit
conjugated to Cy3 (1:100; 2 h) and Donkey anti mouse conjugated to
Cy5 second antibodies (1:100; 2 h; Jackson ImmunoResearch (West
Grove, Pa., USA.). After mounting the samples in Vectashield
(Vector Labs, Burlingame, Calif., USA), confocal or epi-fluorescent
microscopy was performed using Olympus FV 1000, 40.times. and
60.times. oil immersion objective 1.45NA or fluorescent microscope
Olympus IX70. Image acquisition was performed using FLUOVIEW
software 4.0 for relative fluorescence analysis, all settings such
as exposure time, magnification and gain were maintained constant
for all samples. Offline analysis of relative intensities for all
the samples was done using Metamorph 4.5 (Downingtown, Pa., USA).
Only cells having morphological features of neurons (i.e., phase
bright soma and several neurites) were considered for subsequent
analysis.
[0262] Neuronal Density. HB9-GFP positive motor neurons were
counted for condition studied (Zeiss microscope, 40.times. 1.3 NA,
oil immersion objective). The density of neurons, normalize as a
percent of initial number counted at 7 DIV or 14 DIV was
established for all conditions studied. These experiments were
carried out three times as independent experiments.
[0263] Data analysis. The data was obtained from control and SOD1
G93A motor neurons in parallel conditions (sister plates) to reduce
dispersion. Statistical analysis were performed using Student's
t-Test or ANOVA and are expressed as arithmetic mean.+-.S.E.M.;
t-test values of * P<0.05, ** P<0.01, *** P<0.005 were
considered statistically significant. Each set of data presented
was performed in sister cultures to reduce variability. Similar
significances were found expressing the data in cumulative
distributions plots. Therefore, we chose to present the data as
mean.+-.E.M. to simplify its presentation. The kinetic analysis was
done using MiniAnalysis 5.0 (Synaptosoft).
Example 7: Human ES Cell Lines are Sensitive to SOD1G93A Glia
[0264] To generate a large supply of motor neurons from human ES
cells for the study of ALS we adapted a recently reported method
for the production of these cells within embryoid bodies (EBs)
(Singh Roy, N. et al. Enhancer-specified GFP-based FACS
purification of human spinal motor neurons from embryonic stem
cells. Exp Neurol. December; 196(2):224-34, 2005) (FIG. 8A).
Undifferentiated, self-renewing HuES 3 ES cells (Cowan, C. A. et
al. Derivation of embryonic stem-cell lines from human blastocysts.
N Engl J Med. March 25; (13): 1353-6, 2004) were dissociated into
small clumps using collagenase treatment and then allowed to
spontaneously differentiate in suspension for 14 days (FIG. 8A).
Staining of the resulting EBs with the neuronal progenitor marker
PAX6 (data not shown) demonstrated that a substantial percentage
(29%+/-16%, FIG. 12a, b) contained cells differentiating down the
neuronal lineage. To direct these progenitors towards an anterior
and ventral motor neuron identity, we cultured the EBs another 14
days in the presence of retinoic acid (RA) and a small molecule
agonist of the sonic hedgehog (SHH) pathway. Under the influence of
these morphogens the population of PAX6 positive progenitors
expanded (45%+/-15%; FIG. 1b; FIG. 12a, b) and expression of the
ventral progenitor markers NKX6.1 and ISL1/2 was induced (FIGS. 12A
and 12B). To promote motor neuron differentiation and survival, we
then transferred these 28 day-old EBs to media containing
neurotrophic factors for a final 14 days. After 42 days of
differentiation, the number of progenitors expressing PAX6 and
NKX6.1 had begun to decline (FIGS. 12A and 12B), while the number
of cells expressing ISl1/2 continued to increase (FIGS. 12A and
12B). In addition, expression of the HB9 transcription factor,
which is expressed in maturing post-mitotic motor neurons, was
detected in 8% of all cells (FIGS. 12A and 12B). Furthermore, when
plated on laminin, these EBs elaborated impressive neuronal
processes (FIG. 8).
[0265] To further characterize the putative motor neurons contained
within these EBs, the 42 day-old EBs were dissociated with papain
and the resulting cells plated directly onto glial mono layers
prepared from the cortex of neonatal mice (data not shown). We
found, as had been previously reported with neurons derived from
mouse ES cells (Song, H. et al. Astroglia induce neurogenesis from
adult neural stem cells. Nature. May 2; 417(6884):39-44, 2002) that
culturing human ES cell derived neurons on a glial monolayer
promoted their survival (data not shown). Co-staining of cells with
antibodies specific to a neuronal form of tubulin (Tuj1) and the
transcription factors Hb9 and Isl1/2 (data not shown), as well as
co-staining for Hb9 and choline acetyl transferase (Chat) (data not
shown) confirmed that many neurons isolated from these EBs were
differentiating towards a motor neuron identity.
[0266] To ensure that the appearance of motor neurons within these
EBs was dependent on the influences of RA and SHH, we repeated our
differentiation scheme in the absence of one or both of these
morphogens and counted the number of HB9 positive cells (FIG. 8B).
When SHH or RA activity were removed individually, the frequency of
cells expressing HB9 fell to 0.7% (+/-0.2) and 1.1% (+/-0.5%)
respectively. If both signaling molecules were omitted, less than
0.2% of the dissociated cells expressed HB9 (0.17%+/-0.07%). We
further confirmed the robustness of our approach for generating
motor neurons by differentiating six independent human ES cells
lines and then quantifying the number of HB9 positive cells within
the resulting EBs (FIG. 8C). We found that HuES1, HuES3, HuES5 and
HuES9 ES cell lines all differentiated with a similar efficiency
(HuES1: 7.1%+/-1.8%; HuES 3: 8.5%+/-0.5%; HuES 5: 4.7%+/-0.8%; HuES
9: 7.7%+/-1.5%), while HuES 12 cells differentiated at a lower
efficiency (2.8%+/-1.3%) and HuES 13 cells at a higher efficiency
13.9% (+/-3.8%). These results are consistent with a recent report
that suggests independent human ES cell lines can have varying
abilities to differentiate into certain cell types (Osafune, K. et
al. Marked differences in differentiation propensity among human
embryonic stem cell lines. Nat Biotechnol. March; 26(3):313-5,
2008).
[0267] In order to identify living motor neurons in cultures of
differentiating human ES cells, we generated a stable transgenic
human ES cell line in which sequences coding for the green
fluorescent protein (GFP) were under the control of the murine Hb9
promoter (Wichterle et al., 2002) (FIG. 9). To validate that this
transgenic cell line accurately reported HB9 expression, we
differentiated the cells, plated them on glial monolayers and
co-stained with antibodies specific to GFP and HB9. HB9 expression
was observed in 95% of GFP positive cells (FIG. 13). We next
investigated whether these GFP positive cells expressed other
markers that would be consistent with a maturing motor neuron
identity (FIG. 13). We observed considerable overlap between GFP
and expression of NKX6.1 (data not shown) but no co-expression with
NKX2.2 (data not shown), confirming that GFP positive cells had
acquired the correct dorsal-ventral identity (Jessell.TM.. Neuronal
specification in the spinal cord: inductive signals and
transcriptional codes. Nat Rev Genet. October; 1(1):20-9, 2000).
Additionally these cells expressed ISL1/2 (data not shown) and ChAT
(data not shown) but no longer expressed the progenitor marker PAX6
(data not shown). Antibody co-staining experiments also
demonstrated that GFP positive cells did not co-express makers
found in other neuronal subtypes such as the interneuron markers
CHX10 (data not shown) and LHX2 (data not shown).
[0268] The results that we have described thus far confirm that it
is possible to reproducibly generate a large supply of human motor
neurons from embryonic stem cells. We next sought to use these
human neurons to ask whether they, like their mouse counterparts,
are sensitive to the non-cell autonomous effect of glial cells
overexpressing a mutant SOD1 gene product. To this end, we
dissociated 42 day-old EBs and plated the resulting cells on
primary glial monolayers derived from either SOD1G93A transgenic or
control mice (FIG. 10A). After 10 days a significant difference
(p<0.05) in the number of HB9 positive motor neurons was seen
between the two culture conditions (FIG. 10B) is already
appreciable. In cultures containing SOD1G93A glia less than half as
many motor neurons remained (131+/-53) as in cultures containing
non-transgenic control glia (269+/-44) (FIG. 10B). The deficit in
motor neuron survival in co-cultures with SOD1G93A glia became even
more pronounced after 20 days (FIG. 10C). We next sought to confirm
that the toxic effect of glia we observed in our initial
experiments was due to the action of the mutant SOD1 protein rather
than mere SOD1 protein over-expression. Motor neuron preparations
were generated from the Hb9::GFP human ES cell line and co-cultured
for 20 days with non-transgenic glia or glia which either
over-expressed the wild-type human SOD1 protein or the mutant
SOD1G93A protein (data not shown; FIG. 10D). There was no
discernable difference between the number of GFP positive motor
neurons present in culture with the non-transgenic Glia (304+/-60;
data not shown; FIG. 10D) or with glia over-expressing the wild
type SOD1 protein (328+/-30; data not shown; FIG. 10D). In
contrast, there was a highly significant reduction (p<0.01) in
the number GFP positive motor neurons (127+/-16; FIG. 10D) present
in culture with the SOD1G93A Glia, confirming that the non-cell
autonomous effect of glia was mediated through the mutant SOD1
protein.
[0269] In both patients and mice carrying mutant alleles of the
SOD1 gene, intracellular aggregation of the SOD1 protein is often
documented and has been associated with motor neuron death (Boillee
et al., 2006). We therefore wondered whether the toxic effect of
glial cells expressing the mutant SOD1 protein that we observed was
a downstream consequence of protein aggregation. To address this,
we separately cultured primary mouse glia and mouse ES cell derived
motor neurons carrying the same SOD1G93A transgene and stained the
cultures with antibodies specific for the human SOD1 protein. After
21 days in culture, the SOD1 protein in mouse motor neurons was
observed to aggregate into cytoplasmic and perinuclear inclusions
(data not shown) (Di Giorgio, F. P., Carrasco, M. A., Siao, M. C.,
Maniatis, T. & Eggan, K. Non-cell autonomous effect of glia on
motor neurons in an embryonic stem cell-based ALS model. Nat.
Neurosci. 10, 608-614, 2007). In contrast, even after more than 90
days in culture, the SOD1 protein was found to be broadly and
diffusely localized in the cytoplasm of all glial cells (data not
shown), suggesting that the mutant protein is mediating its effect
in these cells through a mechanism independent of protein
aggregation.
[0270] ALS leads to the specific degeneration of motor neurons.
Therefore, if the toxic effect of glial cells that we have observed
is relevant to ALS then we might expect that other spinal cord
neuronal types such as interneurons would not be sensitive to it.
During our characterization of human ES cell derived motor neurons
we noted that additional neurons expressing the transcription
factors CHX10 and LHX2, indicative of V2 and D1 interneuron
differentiation, were also produced (data not shown). To test
whether these neuronal types were affected by co-culture with
mutant glia, we dissociated 42 day-old EBs, plated equal numbers of
cells on either SOD1G93A glia or non-transgenic glia (FIG. 11A-D)
and after 20 days of culture stained for Tuj1 and either LHX2 (FIG.
3) or CHX10 (FIG. 11D). We found that neurons expressing either of
these interneuron markers were unaffected by culture with mutant
glia (FIG. 11B-C), in striking contrast to the sensitivity of motor
neurons to this culture environment.
[0271] To determine if the toxic effect of mutant glial cells was
the consequence of a specific activity within this cell type rather
then a general property of any cell over expressing the SOD1G93A
mutation, we plated motor neuron preparations on mouse embryonic
fibroblasts (MEFs) prepared from SOD1G93A and non-transgenic
sibling embryos (data not shown). After 20 days of co-culture we
did not observe a significant difference between the number of
HhB9, Ttuj1 double positive motor neurons present on SOD1G93A MEFs
(204+/-28) or non-transgenic MEFs (197+/-23) (data not shown),
consistent with the hypothesis that astrocytes are specifically
responsible for the non-cell autonomous effect we observed (Di
Giorgio et al., 2007; Nagai et al., 2007).
Example 8: Methods
[0272] Example 7 was performed using the following methods:
[0273] Growth of human Embryonic Stem Cells.
[0274] The HuES cell lines were obtained from Doug Melton and
cultured as described by Cowan et al. (Cowan et al., 2004). The
hESCs were maintained on a feeder layer of inactivated mouse
embryonic fibroblasts (GlobalStem) in human ES cell media (KO-DMEM
(Gibco), 10% KO Serum Replacement, 10,000 units Penicillin and 1
mg/ml Streptomicin (GIBCO), 2 mM Glutamine (GIBCO), 100 mM
non-essential amino acids (GIBCO), 55 .quadrature.mM
beta-mercapto-ethanol (GIBCO), 10% Plasmanate (Bayer), 10 ng/mL
bFGF2 (GIBCO)). The cells were cultured at 37.degree. C. and 5%
CO.sub.2. Media was replaced daily for the duration of hESC
expansion and the cells in these conditions were passaged every 5-7
days using a solution with 0.05% trypsin (GIBCO).
[0275] Differentiation of human Embryonic Stem Cells into motor
neurons. For differentiation into motor neurons, the cells were
allowed to reach 80-90% confluency, washed once with PBS, and then
incubated for 15 minutes at 37.degree. C. in a solution of 1 g/L
Collagenase IV (GIBCO) in DMEM-F12 (GIBCO).
[0276] Using a cell scraper, the ES cell colonies were scraped and
washed off the plate, centrifuged for 5 minutes at 1000 RPM and
resuspended in human ES cell media without bFGF2 or plasmanate in
low attachment 6-well plates.
[0277] After 24 hours, the cells had aggregated to form embryoid
bodies (EBs), and the media was changed to remove debris by
centrifuging the EBs and resuspending in fresh in human ES cell
media without bFGF2 or plasmanate in low attachment 6-well plates.
EBs were cultured as such for 13 more days, with half of the media
changed every two days, and a complete media change every week.
After 14 days, the EBs were induced toward a caudal and ventral
identity using retinoic acid (1. M, Sigma) and an agonist of the
Shh signaling pathway (1 .mu.M) in N2 media: 1:1
DMEM:F-12+Glutamate (Gibco), 10,000 units Penicillin and 1 mg/mL
Streptomicin (Gibco), 1% N2 Supplement (Gibco), 0.2 mM ascorbic
acid (Sigma-Aldrich), 0.16% D-(+)-Glucose (Sigma-Aldrich), BDNF (10
ng/ml, R&D Systems), for another 14 days. The EBs were then
matured for a final 14 days in N2 media with GNDF (10 ng/mL,
R&D Systems). After 42 days of differentiation, the EBs were
dissociated. To dissociate the EBs, they were centrifuged at 1000
rpm for 5 min in a 15 ml falcon tube, and then washed once with PBS
to eliminate residual media. The EBs were then incubated for 60
minutes at 37.degree. C. in Earle's Balanced Salt Solution with 20
units of papain and 1000 units of Deoxyribonuclease I (Worthington
Biochemical Corporation). EBs were triturated using a 2 mL
serological pipette every 15-20 minutes during this incubation.
When an almost single cell suspension was achieved, the cells were
centrifuged for 5 minutes at 1000 RPM. The resulting cell pellet
was washed once with PBS and then resuspended in N2 media with
neurotrophic factors (GDNF, and BDNF (10 ng/ml, R&D Systems)).
These cells were then counted and plated on Poly-D-Lysine/Laminin
CultureSlides (BD biosciences) or on a layer of primary glial
cells. Depending on the experiment, motor neurons or interneurons
were counted 10 or 20 days after plating.
[0278] Generation of the HuES 3 Hb9::GFP cell line. To generate the
Hb9::GFP HuES 3 cell line, HuES 3 cells were electroporated with a
plasmid containing a neomycin resistance cassette and the coding
sequence of Green Fluorescent Protein under transcriptional control
of a 9 kb murine Hb9 promoter restriction fragment. The plasmid was
a kind gift of Hynek Witcherle (Columbia University) and was a
modification of the construct described in Witcherle et al. (2002).
The elctroporation was performed as described in Zwaka P T. et al.
(Zwaka T P, Thomson J A. Homologous recombination in human
embryonic stem cells. Nat Biotechnol. March; 21 (3):319-21,
2003).
[0279] Undifferentiated HuES 3 cells were grown as described below.
Once the cells reached 80-90% confluency, they were dissociated in
trypsin and counted. Approximately 1.0.times.10.sup.7 were
resuspended in 0.7 mL of human ES cell media and mixed with 0.1 mL
of the same media containing 30 mg of linearized vector. This mix
of cells and DNA was then transferred to a 0.4 cm cuvette and
exposed to a pulse of 320 V, 200 mF at room temperature. After 10
minutes at room temperature the cells were plated on a 10 cm dish
of MEF, and 48 hours after electroporation the cells were switched
to media containing G418 (50 mg/mL, GIBCO). Selection media was
changed daily for 14 days, after which we picked and expanded 24
resistant human ES cell colonies. In order to assay GFP expression,
we differentiated six of these resistant clones into motor neurons
and immunostained for GFP and HB9 co-expression. Two of these
clones gave rise GFP positive cells that elongate green axons,
however only one clone was validated by immunoreactivity to the Hb9
antibody and used in subsequent experiments.
[0280] Immunocytochemistry Analysis.
[0281] Cells were fixed with 4% para-formaldehyde for 30 minutes at
room temperature. After fixation, the cells were washed 3 times
with PBS for 10 minutes and then treated for 1 hour in a blocking
solution (PBS (Cellgro), donkey serum (10%, Jackson
Immunoresearch)) plus Triton X (0.1%, Sigma) for permeabilization.
After blocking, the cells were incubated overnight at 4.degree. C.
with primary antibodies: mouse anti-beta tubulin III (Covance);
rabbit anti-beta tubulin III (SIGMA); Pax6, Nkx6.1, Nkx2.2, Isl1,
Hb9 (DSHB); Chx10, Lhx2 (Santa Cruz Technologies); ChAT (Chemicon);
rabbit anti-GFP conjugated Alexa fluor 488 (Molecular Probes); in
the blocking solution. After the overnight incubation the cells
were washed 3 times in PBS for 10 minutes. Localization of antigens
was visualized by incubating for 1 hour at room temperature using
the respective secondary antibodies (Alexa fluor 594 or 488;
Molecular Probes). Finally, the samples were washed again in PBS 3
times and mounted using a solution with or without DAPI. Images
were taken using a fluorescent Olympus IX70 microscope.
[0282] Primary Glial Cultures.
[0283] P1-P3 mouse pups transgenic for SOD1G93A, SOD1WT or
non-transgenic pups were sacrificed by using an approved method of
euthanasia. Under a dissection microscope, the parenquima were
isolated and the meninges were carefully stripped away with fine
forceps. The tissue was then dissected into small pieces and
transferred to a solution containing 12 ml of HBSS, 1.5 ml of
trypsin (GIBCO) and 1% DNAse (Sigma) and incubated at 37.degree. C.
for 15 min, swirling the mixture periodically. The supernatant
containing the dissociated cells was then collected and 3 ml of
serum was added to inhibit trypsin activity.
[0284] The cells were then centrifuged at 1000 rpm for 5 min,
resuspended in Glia medium: (Minimum Essential Medium with Earl's
salts (GIBCO), D-(+)-Glucose 20% (Sigma), Penicillin-streptomycin
(GIBCO), 10% Horse Serum (GIBCO)) and plated at the concentration
of 80,000 cells per mL in T75 flasks (Falcon). After the glia
reached confluency, they were replated onto Poly-D-Lysine/Laminin
CultureSlides (BD biosciences).
[0285] Data analysis. Statistical analysis was performed using
Student's t-Test and are expressed as arithmetic mean.+-.S.D.;
t-test values of * P<0.05, ** P<0.01, were considered
statistically significant.
Example 9. Identification of Candidate Genes Involved in SOD1G93A
Glial Toxicity
[0286] To better understand how the expression of a mutant gene
that causes ALS can perturb the normal phenotype of astrocytes, and
to identify genes that may have a role in their toxic effect on
motor neurons, we used oligonucleotide arrays to compare the global
gene expression profiles of glia overexpressing the mutant SOD1G93A
protein with two different sets of controls: non-transgenic glia
and glia overexpressing the wild type form of the human SOD1
protein.
[0287] We identified 135 genes whose expression was significantly
(P<0.001) increased more than 2 fold in SOD1G93A glia when
compared to non-transgenic glia. Of these 135 genes, 53 genes were
exclusively up-regulated in the mutant glia, and not in glia
over-expressing the wt SOD1 protein (FIG. 14A). We found that 13 of
these 53 genes (24%) have previously been identified to have a role
either in inflammatory or immune processes. Genes overexpressed
more than 2 fold (P<0.001) in SOD1G93A glia with respect to WT
glia and SOD1WT glia are listed in Table 2. We narrowed our
analysis to a subset of these genes deemed to be of particular
interest because of their known role as pro-inflammatory factors
and their substantially increased expression in mutant glia (FIG.
14B). The prostaglandin D2 (PGD2) receptor was up-regulated more
than 14 fold in SOD1G93A glia compared to the control sample. Three
different cytokines were also shown to be over expressed in mutant
glia: Mcp2, Cxcl7, and Rantes. Also found to be highly (>13
fold) up-regulated in these microarrays, was the gene encoding
glial maturation factor beta (GMFb), which has been shown to induce
a pro-inflammatory state in astrocytes (Zaheer et al., J.
Neurochem. 101:364-376, 2007). Finally, we found that the
expression of SHH and the SHH responsive genes NKX2.2 and DBX2 was
modestly increased in the mutant glia, suggesting that this
signaling pathway might be activated in response to the actions of
the mutant SOD1 protein.
[0288] Microarray Analysis.
[0289] Glia were derived from P1-P3 mouse pups as described above.
Once the cells reached confluence, total RNA was isolated using
Trizol (Invitrogen) from three different biological replicates for
each type of glia. RNA was amplified by one round of T7
transcription using the Illumina TotalPrep RNA Amplification Kit.
Illumina Bead Array Reader. Analysis was done using the Illumina
Bead Studio Program.
[0290] Data Analysis.
[0291] Statistical analysis was performed using Student's t-Test
and are expressed as arithmetic mean.+-.S.D.; t-test values of *
P<0.05, ** P<0.01, were considered statistically
significant.
TABLE-US-00003 TABLE 2 Genes overexpressed in SOD1G93A mutant glia
SYMBOL SEARCH_KEY Fold diff. G93A vs N.T Gene function Serpina1b
NM_009244.2 17.64285714 protease Ptpn7 scl0320139.8_83 15.5
signaling/immuno response Zc3hdc1 NM_172893.1 14.61111111 unknown
Ptgdr NM_008962.2 14.125 inflammation Gmfb NM_022023.1 13.07692308
inflammation Abca5 NM_147219.1 11.45454545 signaling Dbx2
scl0223843.1_155 11.35714286 transcription factor Rab6b
scl0270192.9_201 6.363157895 signaling Cutl1 scl013047.4_12 5.4
transcription factor Ada NM_007398.2 4.962962963 metabolic Ncoa6ip
NM_054089.2 4.654545455 unknown Ifi35 scl40880.4.1_8 4.369047619
inflammation Rabl2a NM_026817.1 4.081081081 unknown AI481214
scl0004149.1_262 3.84 unknown Cygb NM_030206.1 3.833333333
transport Dfy NM_010045.1 3.305084746 inflammation Chodl
scl48930.7.1_272 3.186440678 structural Nrxn1 scl0001711.1_8
3.178723404 signaling Defb11 NM_139221.1 3.164556962 immuno
response Rusc2 scl25518.12_3 3.086787565 unknown Nrxn1
scl0001711.1_8 3.023584906 signaling Matn4 NM_013592.2 3.006147541
structural Xlr3a NM_011726.1 2.837696335 unknown Ccl8 NM_021443.1
2.776623377 inflammation Timd4 scl41638.9.1_29 2.760869565 immuno
response Osr2 scl47995.5.83_129 2.738461538 transcription factor
9130213B05Rik scl27589.4_81 2.6 unknown Reck scl053614.23_117
2.594262295 signaling Olfr116 NM_146632.1 2.574712644 unknown
A230098A12Rik scl36723.20_445 2.505050505 unknown Shh
scl28000.7.1_29 2.475247525 signaling Fpr1 scl50268.2.1_13
2.447058824 inflammation Cxcl7 NM_023785.1 2.43324937 inflammation
Dnajb3 scl16502.1.45_71 2.429906542 structural Defb10 NM_139225.1
2.348387097 immuno response Apoa2 NM_013474.1 2.335526316 metabolic
Col1a2 scl012843.30_10 2.326599327 structural 1700030B17Rik
scl16712.12_256 2.319796954 unknown Atp9a scl18294.24.1_12
2.317307692 metabolic Ccl5 NM_013653.1 2.299539171 inflammation
Slc39a14 scl00213053.1_19 2.288590604 transporter Saa3
scl31343.5.1_35 2.25437788 inflammation 3632451O06Rik
scl45626.8_445 2.203812317 unknown Atrnl1 scl0226255.12_147
2.14556962 unknown Alms1 scl29818.18.1_0 2.137614679 unknown Nkx2-2
scl18553.4.1_4 2.112612613 transcription factor Prss19 NM_008940.1
2.076923077 signaling Hist1h4k NM_178211.1 2.038550501 unknown
Ephb2 scl0013844.2_257 2.017094017 receptor Syt12 NM_134164.2
2.016666667 signaling Foxq1 NM_008239.3 2.016666667 transcription
factor Sfrs16 scl31678.22.1_32 2.01518785 unknown Lancl1
NM_021295.1 2.010822511 immuno response Mlp scl0017357.2_67
2.010471204 signaling
Example 10. Human ES Cell Derived Motor Neurons can be Used to
Identify Neurotoxic Factors
[0292] In order to investigate the possible involvement of
candidate factors and signaling pathways in the glial mediated
neurotoxicity we have observed, we tested the effect of these
candidate gene products, or molecules that activate them, on motor
neuron survival in co-cultures with wild type glial cells.
Non-transgenic glia were individually pretreated for 1 day with
either one of the three cytokines MCP2, Cxcl7, or Rantes; with
GMFb; an agonist of SHH pathway; or with PGD2. Glia were pretreated
for 1 day with either MCP2 (100 ng/ml; Peprotech), Cxcl7 (100
ng/ml; Peprotech), Rantes (100 ng/ml; Peprotech), GMFb (250 ng/ml;
Peprotech), an agonist of Shh pathway (1 mM), PGD2 (10 mM; Chayman
Chemical) or MK 0524 (10 mM; Chayman Chemical). After the
pretreatment for 24 hours, a cellular preparation containing
Hb9::GFP human ES cell-derived motor neurons, dissociated from EBs,
was added to the glia at the concentration of 30,000 cells/well.
Replicate cultures were individually maintained for 20 days in the
presence of each of the 6 factors, fixed, and the numbers of GFP
positive motor neurons quantified.
[0293] We found that treatment with GMFb did not significantly
affect the number of human ES cell derived motor neurons compared
to the control condition (95%+/-9%). Likewise, the presence of any
one of the three cytokines (Rantes, Cxcl7 and Mcp2), or the SHH
agonist, did not seem to negatively affect the number of GFP
positive motor neurons (respectively 108%+/-20%; 102%+/-12%;
103%+/-8%; 97%+/-12%) (FIG. 20C). However, when the cells were
treated for 20 days with PGD2, we found a dramatic decrease in the
number of motor neurons compared to the control condition
(19%+/-2%; p<0.01) (FIG. 14C), suggesting that prostaglandin D2
signaling contributes to motor neuron toxicity in this system.
Example 11. Inhibition of the Prostaglandin D2 Receptor Rescues
Motor Neuron Loss
[0294] To determine if there was a direct relationship between the
toxic effect of prostaglandin signaling on motor neurons and the
SOD1G93A glial mediated neurotoxicity, we tested whether a specific
antagonist of the prostaglandin D2 receptor, MK 0524 (Sturino et
al., J. Med. Chem., 50:794-806, 2007), could counteract or
ameliorate the toxic effect of mutant glia on motor neurons.
SOD1G93A glia and wt glia were pretreated for 1 day with the
prostaglandin D2 receptor inhibitor, human motor neurons were
added, and cultures were maintained for 20 days both in the
presence and absence of the drug. We found that the presence of MK
0524 did not affect motor neuron numbers when they were co-cultured
with wildtype glia. (100%+/8%, FIG. 14D). However, when human motor
neurons plated on SOD1G93A glia were treated with the inhibitor,
there was a statistically significant (p<0.05) increase in the
number of GFP positive neurons (32%, relative to untreated neurons
plated on the same glia) (FIG. 14D). In a similar experiment using
another prostaglandin D2 receptor DP1 specific antagonist BWA868C,
BWA868C significantly reduced cell death of motor neurons that were
cultured on SOD1G93A glia (FIG. 14F). This protection property was
specific for DP1 receptor and not for the DP2 receptor as a DP2
receptor specific antagonist, BAY-u3405, had no effect on promoting
survival of motor neurons cultured on SOD1G93A glia (FIG. 14E).
[0295] These experiments suggest that inhibitors of PGD2 signaling
do not generally act to promote motor neuron survival and instead
act to specifically counteract the toxic effects of glial cells
carrying the ALS mutation.
[0296] The foregoing description is to be understood as being
representative only and is not intended to be limiting. Alternative
methods and materials for implementing the invention and also
additional applications will be apparent to one of skill in the
art, and are intended to be included within the accompanying
claims.
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