U.S. patent application number 12/664387 was filed with the patent office on 2010-11-25 for targeted cell death.
Invention is credited to Robert H. Miller, Stephen M. Selkirk.
Application Number | 20100299770 12/664387 |
Document ID | / |
Family ID | 40130220 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100299770 |
Kind Code |
A1 |
Selkirk; Stephen M. ; et
al. |
November 25, 2010 |
TARGETED CELL DEATH
Abstract
The present invention provides compositions and methods for
studying neuropathy. The compositions and methods provided herein
are particularly useful for screening agents of therapeutic and/or
diagnostic potential.
Inventors: |
Selkirk; Stephen M.;
(Mayfield, OH) ; Miller; Robert H.; (Cleveland
Heights, OH) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
40130220 |
Appl. No.: |
12/664387 |
Filed: |
June 12, 2008 |
PCT Filed: |
June 12, 2008 |
PCT NO: |
PCT/US08/66774 |
371 Date: |
June 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60943448 |
Jun 12, 2007 |
|
|
|
Current U.S.
Class: |
800/17 ; 435/325;
435/6.16; 536/23.1; 800/13; 800/14 |
Current CPC
Class: |
C12N 9/6475 20130101;
A61P 25/00 20180101; C07K 2319/70 20130101; A01K 67/0275 20130101;
G01N 33/50 20130101; C12N 2830/008 20130101; A01K 2267/0356
20130101; C12N 2830/002 20130101; Y02A 90/26 20180101; Y02A 90/10
20180101; C12N 15/8509 20130101 |
Class at
Publication: |
800/17 ;
536/23.1; 435/325; 800/13; 800/14; 435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12N 5/079 20100101
C12N005/079; A01K 67/027 20060101 A01K067/027 |
Claims
1. A recombinant nucleic acid molecule comprising: a nucleic acid
sequence encoding a cell death mediator protein (CDMP), wherein
said nucleic acid sequence is operably linked to a neural
cell-specific regulatory element.
2-11. (canceled)
12. A host cell comprising: a recombinant nucleic acid molecule
comprising: a nucleic acid sequence encoding a cell death mediator
protein (CDMP), wherein said nucleic acid sequence is operably
linked to a neural cell- specific regulatory element.
13-29. (canceled)
30. A transgenic animal comprising: a nucleotide sequence encoding
a cell death mediator protein (CDMP) operably linked to a cell
type-specific expression regulatory element; wherein said animal
exhibits a greater degree of neuropathy relative to an animal
without said nucleotide sequence.
31. The animal of claim 30, wherein said animal is a mammal,
primate, or rodent.
32. The animal of claim 30, wherein said animal is a mouse, rat,
guinea pig, dog, cat, rabbit, pig, chimpanzee or monkey.
33. The animal of claim 30, wherein said neuropathy comprises
neuronal demyelination and/or defect in the blood brain
barrier.
34. The animal of claim 30, wherein said animal exhibits an
increase in apoptotic oligodendrocytes or pericytes relative to
that of a control animal.
35. The animal of claim 30, wherein said CDMP is caspase 2, caspase
5, caspase 8, caspase 9, caspase10 or caspase 11.
36. The animal of claim 30, wherein said CDMP is a chimeric protein
comprising a binding domain for a FK506-type ligand, a FKBP 12-type
ligand, cyclosporin A-type ligand, tetracycline or steroid
ligand.
37. The animal of claim 30, wherein expression of said CDMP is
inducible.
38. The animal of claim 30, wherein expression of said CDMP is
ectopically confined to the central nervous system.
39. The animal of claim 30, wherein the apoptosis promoting
activity of said CDMP is inducible.
40. The animal of claim 39, wherein said apoptosis promoting
activity is specifically in the neural cells of said animal.
41. The animal of claim 39, wherein said activity is induced by a
chemical inducer of dimerization (CID).
42. The animal of claim 39, wherein said CDMP is an inducible
caspase 9 (iCP9).
43. The animal of claim 41, wherein said CID is AP20187.
44. The animal of claim 30, wherein said cell type-specific
expression regulatory element is a neural or mural cell specific
regulatory element.
45. The animal of claim 44, wherein said neural cell specific
regulatory element is a glial cell specific regulatory element.
46. The animal of claim 45, wherein said glial cell is an
oligodendrocyte, astrocyte, microglial cell, or Schwann cell.
47. The animal of claim 45, wherein said glial cell specific
regulatory element is from a gene selected from CC1, myelin basic
protein (MBP), ceramide galactosyltransferase (CGT), proteolipid
protein (PLP). oligodendrocyte-myelin glycoprotein (OMG), cyclic
nucleotide phosphodiesterase (CNP), NOGO, myelin protein zero
(MPZ), peripheral myelin protein 22 (PMP22), protein 2 (P2), GFAP,
AQP4, PDGFa, RG5, pGlycoprotein, neurturin (NRTN), artemin (ARTN),
persephin (PSPN), PDGFR-.alpha., DGFR-.alpha., or sulfatide.
48. The animal of claim 30, wherein said nucleic acid sequence is
operably linked to a second nucleic acid sequence encoding a marker
protein.
49. A method of screening for a biologically active agent that
modulates a phenomenon associated with a demyelination or blood
brain barrier disorder comprising: a) contacting a candidate agent
with a cell comprising a nucleic acid encoding a cell death
mediator protein (CDMP), wherein said nucleic acid is operably
linked to a cell-type specific expression regulatory element; b)
detecting an effect on said phenomenon; and, c) selecting said
agent as effective to modulate said phenomenon if the level of
activity of said CDMP is modulated relative to a control cell.
50. (canceled)
51. (canceled)
52. A method of screening for a biologically active agent that
modulates a phenomenon associated with a demyelination or blood
brain barrier disorder comprising: a) administering a candidate
agent to a non-human transgenic animal, wherein said phenomenon
occurs in said animal upon expression of a nucleic acid sequence
encoding a cell death mediator protein (CDMP); wherein said nucleic
acid sequence expression is regulated by a cell-specific expression
regulatory element; b) activating said CDMP to effect apoptosis in
at least one cell in said animal, wherein said cell is associated
with a demyelination blood brain barrier disorder; and, c)
detecting an effect of said agent upon said phenomenon.
53-68. (canceled)
69. A method for compiling a profile data set for characterizing a
phenomenon associated with multiple sclerosis(MS) or MS-associated
condition related to multiple sclerosis comprising: a) providing a
transgenic animal or cell comprising a nucleic acid encoding a cell
death mediator protein (CDMP), wherein said nucleic acid is
operably linked to a neuronal- or glial-specific expression
regulatory element; b) activating said CDMP thereby inducing
apoptosis; c) obtaining at least one surviving neuronal or glial
cell following said activation; and d) profiling RNA transcripts
and/or encoded products in said surviving glial or neuronal cell;
thereby compiling a profile data set characterizing a phenomenon
associated with multiple sclerosis or MS-associated condition
related to multiple sclerosis.
70-72. (canceled)
Description
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/943,448, filed Jun. 12, 2007, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Neuronal demyelination is a deleterious condition
characterized by a reduction of myelin in the nervous system. Vital
to both of the central (CNS) and peripheral (PNS) nervous system,
myelin encases the axons of neurons and forms an insulating layer
known as the myelin sheath. The presence of the myelin sheath
enhances the speed and integrity of nerve signal in form of
electric potential propagating down the neural axon. The loss of
myelin sheath produces significant impairment in sensory, motor and
other types of functioning as nerve signals reach their targets
either too slowly, asynchronously (for example, when some axons in
a nerve conduct faster than others), intermittently (for example,
when conduction is impaired only at high frequencies), or not at
all.
[0003] Neuronal tissue generally comprises neurons and supporting
glial cells. Glial cells outnumber neurons by about ten to one in
the mammalian brain. Glial cells may be divided into four types:
astrocytes, oligodendrocytes, Schwann cells and microglial cells.
The myelin sheath is formed by the plasma membrane, or plasmalemma,
of glial cells--oligodendrocytes in the CNS, and Schwann cells in
the PNS. During the active phase of myelination, each
oligodendrocyte in the CNS typically produce as much as
approximately 5000 .mu.m.sup.2 of myelin surface area per day and
approximately 10.sup.5 myelin protein molecules per minute (Pfeffer
et al. (1993) Trends Cell Biol. 3: 191-197). Myelinating
oligodendrocytes have been identified at demyelinated lesions,
indicating that demyelinated axons may be repaired with the newly
synthesized myelin.
[0004] Neuronal demyelination is manifested in a large number of
hereditary and acquired disorders of the CNS and PNS. These
disorders include, for example, Multiple Sclerosis (MS),
Progressive Multifocal Leukoencephalopathy (PML),
Encephalomyelitis, Central Pontine Myelolysis (CPM), Anti-MAG
Disease, Leukodystrophies: Adrenoleukodystrophy (ALD), Alexander's
Disease, Canavan Disease, Krabbe Disease, Metachromatic
Leukodystrophy (MLD), Pelizaeus-Merzbacher Disease, Refsum Disease,
Cockayne Syndrome, Van der Knapp Syndrome, and Zellweger Syndrome,
Guillain-Barre Syndrome (GBS), chronic inflammatory demyelinating
polyneuropathy (CIDP), and multifocual motor neuropathy (MMN). For
the vast majority of these disorders, there are no cures and few
effective therapies.
[0005] During Parkinsons's disease (paralysis agitans or shaking
palsy) cells of the brain appear to deteriorate for unknown
reasons. However, a role for inflammatory reactions has been
postulated to play a role in the pathogenesis of Parkinson's.
Parkinson's is a disorder of the brain characterized by shaking and
difficulty with walking, movement, and coordination. The disease
affects approximately 2 out of 1,000 people, and most often
develops after age 50. It affects both men and women and is one of
the most common neurologic disorders of the elderly. Parkinson's
disease is caused by progressive deterioration of the nerve cells
of the part of the brain that controls muscle movement (the basal
ganglia and the extrapyramidal area).
[0006] In addition to the loss of muscle control, some people with
Parkinson's disease become severely depressed. Although early loss
of mental capacities is uncommon, with severe Parkinson's the
person may exhibit overall mental deterioration (including
dementia, hallucinations, and so on). Dementia can also be a side
effect of some of the medications used to treat the disorder.
[0007] Amyotrophic Lateral Sclerosis (ALS) is a rapidly
progressive, invariably fatal, disorder causing loss of nervous
control of voluntary muscles because of destruction of nerve cells
in the brain and spinal cord resulting in loss of the use and
control of muscles. The nerves controlling these muscles shrink and
disappear, which results in loss of muscle tissue due to the lack
of nervous stimulation. Muscle strength and coordination decreases,
beginning with the voluntary muscles (e.g., those under conscious
control). The extent of loss of muscle control continues to
progress, and more muscle groups become involved. There may be a
loss of nervous stimulation to semi-voluntary muscles, such as the
muscles that control breathing and swallowing. Eventually, all
muscles under voluntary control are affected, and patients lose
their strength and the ability to move their arms, legs, and
body.
[0008] Motor neurons located in the brain, brainstem, and spinal
cord serve as controlling units and vital communication links
between the nervous system and the voluntary muscles of the body.
Messages from motor neurons in the brain (upper motor neurons) are
transmitted to motor neurons in the spinal cord (lower motor
neurons) and from them to particular muscles. In ALS, both the
upper motor neurons and the lower motor neurons degenerate or die,
ceasing to send messages to muscles. Unable to function, the
muscles gradually weaken, waste away (atrophy), and twitch
(fasciculations). Eventually, the ability of the brain to start and
control voluntary movement is lost. The cause is unknown.
[0009] MS is the leading cause of nontraumatic CNS morbidity in
young adults. The young age of onset and progressive nature of the
disease imposes an enormous economic and social burden on society.
Acute exacerbations in typical relapsing-remitting MS are the
manifestation of acute and focal inflammation and demyelination in
the CNS and have long been considered the primary pathology of MS.
These events are the target of currently approved therapeutic
agents. However, correlations of T.sub.2 inflammatory signal on
magnetic resonance (MR) images and disease progression are weak as
are the clinical characteristics during the relapsing-remitting
(RR) phase and subsequent progression of disability. Furthermore,
once irreversible disability is reached, the progression to further
disability is not affected by relapses, including those occurring
before or after the onset of irreversible injury.
[0010] In addition to permanent neurological disability due to
axonal loss, inflammatory demyelination plays a role in MS
pathogenesis. In contrast to inflammation, axonal loss typically
correlates with T1 black holes, decreased N-acetyl aspartate (NAA)
on magnetic resonance spectroscopy (MRS) and the degree of spinal
cord atrophy, which can correlate with clinical disability in
patients. These changes have been noted in patients as early as six
months after diagnosis, but in most patients the chronic, and
perhaps global, axonal injury breaches a clinical threshold at the
onset of the secondary progressive phase of the disease.
[0011] During the acute inflammatory stage of the disease
(clinically defined as relapsing/remitting MS, or RRMS),
inflammatory mediators likely contribute to axonal injury.
Associations have been made between the number of CD8+ T cells and
the extent of axonal damage and animal models tend to support this
implicating a CD8-MHC class I pathway of axon destruction
(Rivera-Quinones et al., (1998) Nat Med. 4:187-193). Further
support comes from pathology studies in which activated CD8 T cells
containing cytotoxic granules polarized toward the demyelinated
axons suggests direct CD8+ T cell toxicity. Macrophages and
microglial cells have been found in close proximity to degenerating
axons. These cell types play a role in the homeostatic mechanism of
removing debris from the CNS, however they also release
inflammatory mediators including proteases, cytokines and free
radicals such as nitrous oxide (NO). Finally, antibodies and
complement may also play a role in axon damage during acute
inflammation. Levels of anti-ganglioside antibodies were found to
be significantly higher in primary progressive MS (PPMS) than in
secondary progressive or RRMS and axons exposed to complement after
demyelination may activate the complement cascade directly.
[0012] The relationship between inflammation and neuron loss in MS
has not been fully delineated. There is a need to establish a model
of oligodendrocyte loss and subsequent demyelination that does not
rely on the induction of inflammation to specific CNS antigens
systemically or necessitate the use of a potent system adjuvant.
Such a model that utilizes antigens and inflammation typically
fails to recapitulate demyelination and neuronal loss identified in
MS patients.
[0013] An accurate animal model of axonal transection and neuronal
loss should mimic the pathological features identified in MS brain
specimens. This includes the identification of transected axons,
transected dendrites and neuronal apoptosis in acute cortical
lesions. The acute event should also result in measurable impaired
neurotransmission that is restored, to varying degrees, by the
redistribution of Na.sup.+ channels as has been identified in MS
pathology specimens. Chronic lesions should demonstrate a variable
degree of remyelination and smoldering persistent axonal loss
should be evident. Finally, neuron loss in the animal model should
be identified in regions anatomically distinct and temporally
distinct from original demyelinating lesions mimicking the effect
of the disease on NAWM. These features would provide an important
and accurate depiction of the effects on neurons identified in
pathology specimens from MS patients.
[0014] The delineation of the precise molecular mechanism and
pathogenesis of neuropathy and in particular neuronal
demyelination, has been hampered by the continued lack of effective
animal models. Thus, there remains a pressing need for composition
and methods to effect a robust screen for therapeutics directed to
neuronal disorders.
SUMMARY OF THE INVENTION
[0015] The present invention provides compositions and methods for
understanding the process of neuropathy and to identify and develop
bioactive agents for the treatment of neuronal demyelination. The
transgenic animal(s)/cell(s) of the present invention provide a
model system that can be utilized to elucidate mechanisms for
remyelination and for screening candidate bioactive agents. By
utilizing remyelination-specific expression of markers and other
means, the present invention can be utilized to assay test agents
for effects on remyelination, whether the effects are positive
(potentially therapeutic), or the effects are negative (potentially
deleterious). Furthermore, determining the effect of the test agent
upon a phenomenon associated with a remyelination may involve any
suitable methods known in the art, including but not limited to
those utilizing cell-based assays or techniques.
[0016] The present invention provides a recombinant nucleic acid
molecule comprising a nucleic acid sequence encoding a cell death
mediator protein (CDMP), wherein the nucleic acid sequence is
operably linked to a neural cell-specific regulatory element. The
nucleic acid sequence can also be operably linked to a second
nucleic acid sequence encoding a marker protein, such as GFP or
other fluorescent markers. The present invention also provides a
host cell comprising a recombinant nucleic acid molecule comprising
a nucleic acid sequence encoding a cell death mediator protein
(CDMP), wherein the nucleic acid sequence is operably linked to a
neural cell-specific regulatory element. The host cell can be a
neural cell or mural cell, for example, it can be neuronal or glial
cells. For example, the glial cells can be oligodendrocytes,
astrocytes, microglial cells or Schwann cells. The neuronal cells
can be cervical ganglion neurons, cortical neurons, serotonin
nuerons, dorsal root ganglion, nodose ganglion neurons, spinal
motoneurons, midbrain dopaminergic neurons, central noradrenergic
neurons or enteric neurons. The mural cells can be endothelial
cells, perictyes or smooth muscle cells. In some embodiments, the
host cell can be immune cells such as B or T lympocytes.
[0017] Also provided is a transgenic animal comprising a nucleotide
sequence encoding a cell death mediator protein (CDMP) operably
linked to a cell type-specific expression regulatory element,
wherein the animal exhibits a greater degree of neuropathy relative
to an animal without said nucleotide sequence. The animal can be a
mammal, primate, or rodent, such as a mouse, rat, guinea pig, dog,
cat, rabbit, pig, chimpanzee or monkey. The neuropathy can comprise
neuronal demyelination, such as multiple sclerosis. The animal can
exhibits an increase in apoptotic oligodendrocytes relative to that
of a control animal. The CDMP can also be ectopically confined to
the central nervous system.
[0018] The nucleic acid sequence encoding the CDMP can be
integrated into the genome of the host cell or animal.
Alternatively, the nucleic acid sequence encoding the CDMP can be
episomal. Furthermore, the recombinant nucleic acid molecule can be
delivered to the host cell or animal, or to generate a transgenic
animal by a viral vector, such as a lentivirus vector.
[0019] In one aspect, the CDMP can be caspase 2, caspase 5, caspase
8, caspase 9, caspase 10, or caspase 11. The CDMP can be a chimeric
protein comprising a binding domain for a FK506-type ligand, a FKBP
12-type ligand, cyclosporin A-type ligand, tetracycline or steroid
ligand. The expression and/or the apoptosis promoting activity of
the CDMP can be inducible, for example, such as an inducible
caspase 9 (iCP9). For example, the expression regulatory element
can be inducible, constitutive and/or cell type or tissue-specific.
In some embodiments, the expression and/or activity is specific for
neural cells, such as those in an animal. The activity of the CDMP
can be induced by a chemical inducer of dimerization (CID), such as
AP20187.
[0020] The nucleic acid molecule comprising a nucleic acid sequence
encoding a (CDMP) can be operably linked to a mural or neural
cell-specific regulatory element. For example, the regulatory
element can be a neuronal or glial cell specific regulatory
element. The expression or activity of the CDMP encoded by the
nucleic acid sequence can be specifically in neuronal or glial
cells, such as in oligodendrocytes, astrocytes, microglial cells,
or Schwann cells. The glial cell specific regulatory element can be
from a CC1, myelin basic protein (MBP), ceramide
galactosyltransferase (CGT), proteolipid protein (PLP).
oligodendrocyte-myelin glycoprotein (OMG), cyclic nucleotide
phosphodiesterase (CNP), NOGO, myelin protein zero (MPZ),
peripheral myelin protein 22 (PMP22), protein 2 (P2), GFAP, AQP4,
PDGFR-.alpha., PDGF-.alpha., RG5, pGlycoprotein, neurturin (NRTN),
artemin (ARTN), persephin (PSPN), PDGFR-.beta., or sulfatide
gene.
[0021] In another aspect of the present invention are methods for
screening biologically active, or bioactive, agents. The present
invention provides a method of screening for a biologically active
agent that modulates a phenomenon associated with a demyelination
disorder comprising contacting a candidate agent with a cell
comprising a nucleic acid encoding a cell death mediator protein
(CDMP), wherein said nucleic acid is operably linked to a cell-type
specific expression regulatory element; detecting an effect on the
phenomenon; and, selecting the agent as effective to modulate the
phenomenon if the level of activity of said CDMP is modulated
relative to a control cell. The cell can be neuronal, such as a
glial cell, or mural cell. The glial cell can be an
oligodendrocyte, astrocyte, microglial cell, or Schwann cell.
[0022] Also provided herein is a method of screening for a
biologically active agent that modulates a phenomenon associated
with a demyelination disorder comprising: administering a candidate
agent to a non-human transgenic animal, wherein demyelination
occurs in the animal upon expression of a nucleic acid sequence
encoding a cell death mediator protein (CDMP); wherein the nucleic
acid sequence expression is regulated by a cell-specific expression
regulatory element; activating the CDMP to effect apoptosis in at
least one cell in the animal, wherein the cell is associated with a
demyelination disorder; and, detecting an effect of the agent upon
said phenomenon associated with said demyelination disorder. In
some embodiments, the animal is allowed to recover from the
demyelination prior to administration of the candidate agent. The
demyelination disorder can be characterized by a loss of
oligodendrocytes, astrocytes or Schwann cells in the animal and the
phenomenon associated with the demyelination disorder can be
characterized by a decrease in myelinated axons. In some
embodiments, the demyelination disorder is multiple sclerosis. In
another aspect, determining the effect of the agent can involve
PCR, immunoassay, hybridization assay or a combination thereof. The
candidate agent can be an antisense oligonucleotide, a peptide, an
antibody, a liposome, a small interfering RNA, a small organic
compound, or an inorganic compound.
[0023] In yet another aspect of the present invention, a method for
compiling a profile data set for characterizing a phenomenon
associated with multiple sclerosis(MS) or MS-associated condition
related to multiple sclerosis comprising providing a transgenic
animal or cell comprising a nucleic acid encoding a cell death
mediator protein (CDMP), wherein the nucleic acid is operably
linked to a neuronal- or glial-specific expression regulatory
element; activating the CDMP thereby inducing apoptosis; obtaining
at least one surviving neuronal or glial cell following the
activation; and profiling RNA transcripts and/or encoded products
in said surviving glial or neuronal cell; thereby compiling a
profile data set characterizing a phenomenon associated with
multiple sclerosis or MS-associated condition related to multiple
sclerosis is provided.
INCORPORATION BY REFERENCE
[0024] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the invention will be obtained by
reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings.
[0026] FIG. 1 illustrates engineered SIN lentiviral vectors
(p.DELTA.cmvICP9/mEGFP (FIG. 1B) and p.DELTA.mbpICP9/mEGFP). FIG.
1A provides a p.DELTA.cmv/mEGFP vector construct. The second
generation, p.DELTA.mbpICP9/mEGFP (FIG. 1C), has a MBP specific
promoter to limit gene expression to infected oligodendrocytes, and
its cell specific expression causes oligodendrocyte specific cell
death. The dual promoter feature allows for co-expression of GFP
independent of the upstream gene so that infected cells can be
easily identified using fluorescent microscopy. LTR=long terminal
repeat; pCMV=cytomegalovirus promoter sequence; iCP9=engineered
inducible caspase-9 gene sequence; pMND=modified LTR promoter
sequence; EGFP=enhanced green fluorescent protein gene sequence;
pMBP=the myelin basic protein promoter sequence.
[0027] FIG. 2 illustrates lentiviral vectors created with various
different tissue specific promoters to induce apoptosis in a
cell-type selective manner. The viral vectors typically result in
EGFP expression in infected cells. LTR=long terminal repeat;
pMND=viral promoter sequence; GFP=green fluorescent protein;
pCMV=cytomegalovirus promoter sequence; iCP9=inducible caspase gene
sequence.
[0028] FIG. 3 provides a photograph demonstrating that concentrated
lentivirus can be applied directly to the CNS and the extent and
area of infection can be determined based on the number of GFP
positive cells detected after sacrifice and thin section of the
brain or spinal cord. This data establishes the transfer mechanism
for specific cell death in vitro and in vivo
[0029] FIG. 4 is a graphical representation of certain embodiments
of the present invention. The figure demonstrates a general outline
of one method of the invention and certain applications for the
method.
[0030] FIG. 5 provides a photograph demonstrating cells infected
with control virus (top) remained viable after CID was added to
culture as demonstrated by persistence of GFP+ cells. Cells
infected with virus expressing iCP9 underwent apoptosis after CID
exposure (bottom) as demonstrated by the absence of GFP+ cells. The
persistence of cells in bright field demonstrates the specificity
of apoptosis to infected cells only. BF=Bright Field.
[0031] FIG. 6 is a graph showing cells exposed to various dilutions
of virus encoding the iCP9 gene and then CID was added to the
culture media. 4 hours later, Apoptosis ELISA was performed.
Apoptosis levels decreased with dilution of virus as fewer cells
were infected and expressed iCP9. Thus apoptosis correlated with
iCP9 gene expression. X-axis is virus dilution. Y-axis is ELISA
absorption at 405 nm.
[0032] FIG. 7 shows mixed cortical cultures derived from rat pups
infected with pLpMBP(iCP9)MG and then exposed to CID. Four hours
later cells were stained to identify cell-types. Morphology of MBP+
cells after CID exposure (+CID) was suggestive of apoptosis. Other
cells types maintained morphology consistent with a viable cell.
This indicates the virus induces cell death in MBP expressing
cells.
[0033] FIG. 8 shows pan purified GFAP+ cells (astrocytes) infected
with pLpGFAP(iCP9)MG and exposed to CID (+CID). GFP+ cells
underwent apoptosis indicated by the absence of EGFP+ cells four
hours after exposure to CID (upper photomicrographs). Parallel
cultures not exposed to CID (-CID) maintained a high number of
EGFP+ cells (lower photomicrographs).
[0034] FIG. 9 shows mixed cortical cultures were infected with
pLpPDGFR(iCP9)MG virus and exposed to CID (+CID). Four hours later
A2B5+ cells were identified undergoing apoptosis based on the loss
of viable cell morphology.
[0035] FIG. 10 shows results from injection of pLpGFAP(iCP9)MG
virus into the corpus callosum of rats.
[0036] Twenty-four hours after exposure to CID apoptosis is
identified at the site of infection with a loss of GFAP+ cells
(Right panels). However, infection followed by exposure to vehicle
failed to generate apoptosis or loss GFAP+ cells (left Panels).
This suggests that the virus results in the specific loss of GFAP+
cells (astrocytes) via apoptosis.
[0037] FIG. 11 shows pLpMBP(iCP9)MG virus injected into the corpus
callosum of rats to specifically ablate oligodendrocytes.
Twenty-four hours after exposure to CID (CID+) apoptosis was
identified at the site of infection via TUNEL stain (top panels)
and the absence of MBP staining cells (second row). The area that
fails to stain with anti-MBP antibody, stains positive with
anti-GFAP antibody indicating that cells persist in that area. The
area indicated by black arrows stains negative for both and
represents a tissue defect created by the needle insertion.
V=ventricle. The bottom panel show the extent of infection as all
infected cells are GFP+.
DETAILED DESCRIPTION
[0038] In the present invention, transgenic animals or cells
provide a model system for studying neuropathy. The model system
can be employed for identifying, assessing and/or quantifying
neuronal health and/or demyelination/remyelination. Furthermore,
compositions andmethods of the invention are utilized in various
embodiments for the identification and development of biologically
active agents that modulate, promote or reduce neuronal or
myelination health or maintenance.
[0039] One aspect of the invention encompasses selective induction
of cell death in particular cell types to assess the effect on
neuronal demyelination and/or remyelination. As such a neuronal
response to demyelination in vivo and in vitro can be defined
without artificially induced inflammation.
[0040] The loss of neurons is an important factor in the
progression of disability in patients with multiple sclerosis.
Furthermore, the loss of neurons may be largely independent of
inflammation and may occur after the disruption of the co-dependent
relationship between the neuron and the myelin producing
oligodendrocyte cell.
[0041] Therefore in one embodiment, a system (e.g., an animal) is
provided to characterize the response of neurons to the loss of its
codependent oligodendrocyte cell. For example a modified retrovirus
is utilized to deliver an inducible suicide gene, or a gene that
encodes a cell death mediator protein, to oligodendrocytes in the
brain. When the inducible factor is administered to an animal, the
suicide gene induces the oligodendrocytes (myelin producing cells)
to undergo apoptosis, resulting in focal demyelination that is not
induced by inflammation. The response of neurons to the loss of
oligodendrocytes and myelin is then characterized. By understanding
the response of neurons to demyelination, interventions which
protect neurons after this event can be developed.
[0042] Cells such as neural cells can be targeted in a subject or
in cell culture for controlled cell death. In some embodiments,
such cells are isolated from the transgenic animals of the
invention for further study, for example, for assays which are
conducted in a cell-based or cell culture setting, including ex
vivo techniques.
[0043] Various aspects of the invention provide methods for
assessing pathological changes in the CNS, such as oligodendrocyte
or neuronal loss, which manifest clinically.
[0044] In some aspects of the invention, a non-human transgenic
animal are engineered using methods known in the art to provide
expression of a cell death mediator protein that is operably linked
to a cell-type specific or inducible expression regulatory element
(e.g., promoter/enhancer). In some embodiments, cells, human or
non-human can be engineered to express one or more cell death
mediator proteins that operably linked to a cell-type specific or
inducible expression regulatory element (e.g., promoter/enhancer).
Such regulatory elements useful in various aspects of the invention
are described more fully herein, as well as various cell types that
can be targeted with compositions/methods of the invention.
[0045] In one embodiment, expression of one or more cell death
mediator protein in a non-human transgenic animal ("Test Animal")
of the invention, results in a neuropathy or exacerbated neuropathy
in such an animal.
[0046] In yet another embodiment, expression of one or more cell
death mediator protein is in the Test Animal's central nervous
system or peripheral nervous system.
[0047] In one aspect of the invention, expression of one or more
cell death mediator proteins (CDMPS) results in degeneration of
neurons in a Test Animal. In some embodiments, such neurons include
cervical ganglion neurons, cortical neurons, serotonin neurons,
dorsal root ganglion, nodose ganglion neurons, spinal motoneurons,
midbrain dopaminergic neurons, central noradrenergic neurons or
enteric neurons.
[0048] In various embodiments, cells that comprise cell death
mediator nucleic acid constructs of the invention are, but not
limited to, neural cells or mural cells.
[0049] In some embodiments, such cells are glial cells, including,
but not limited to, oligodendrocytes, astrocytes, microglial cells
and/or Schwann cells. In other embodiments, such cells include, but
are not limited to, pericytes, endothelial cells and/or smooth
muscle cells.
[0050] Another aspect of the present invention is directed to
methods for determining the response of neurons and
oligodendrocytes in vivo after selective and limited cell death.
For example, the nucleic acid constructs of the present invention
(e.g. nucleic acid constructs encoding a cell death mediator
protein operably linked to a cell/tissue-specific expression
regulatory element) can be applied to adult rats which result in a
model system to assess the response of neurons to lesions in the
CNS that is not complicated by antigen/adjuvant stimulated
inflammation. Furthermore, the present invention provides
compositions and methods that allow for control of lesion size,
lesion location, lesion number and the temporal relationship
between lesions.
General Techniques
[0051] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:
A LABORATORY MANUAL, 2.sup.nd edition (1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series
METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY
MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
Definitions
[0052] As used in the specification and claims, the singular form
"a," "an," and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0053] The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid" and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA of any sequence, isolated RNA of any sequence, nucleic
acid probes, and primers. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
If present, modifications to the nucleotide structure may be
imparted before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labelmg component.
[0054] A "nucleotide probe" or "probe" refers to a polynucleotide
used for detecting or identifying its corresponding target
polynucleotide in a hybridization reaction.
[0055] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of a PCR, or the enzymatic cleavage of a polynucleotide
by a ribozyme.
[0056] The term "hybridized" as applied to a polynucleotide refers
to the ability of the polynucleotide to form a complex that is
stabilized via hydrogen bonding between the bases of the nucleotide
residues. The hydrogen bonding may occur by Watson-Crick base
pairing, Hoogstein binding, or in any other sequence-specific
manner. The complex may comprise two strands forming a duplex
structure, three or more strands forming a multi-stranded complex,
a single self-hybridizing strand, or any combination of these. The
hybridization reaction may constitute a step in a more extensive
process, such as the initiation of a PCR reaction, or the enzymatic
cleavage of a polynucleotide by a ribozyme.
[0057] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed into mRNA and/or the process by
which the transcribed mRNA (also referred to as "transcript") is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectedly referred to as "gene product." If the polynucleotide is
derived from genomic DNA, expression may include splicing of the
mRNA in a eukaryotic cell.
[0058] "Differentially expressed," as applied to nucleotide
sequence or polypeptide sequence in a subject, refers to
over-expression or under-expression of that sequence when compared
to that detected in a control. Underexpression also encompasses
absence of expression of a particular sequence as evidenced by the
absence of detectable expression in a test subject when compared to
a control.
[0059] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" refers to either natural and/or
unnatural or synthetic amino acids, including glycine and both the
D or L optical isomers, and amino acid analogs and
peptidomimetics.
[0060] As used herein, "myelinating cell" refers to those cells
capable of producing myelin which insulates axons in the nervous
system. Exemplary myelinating cells are oligodendrocytes
responsible for producing myelin in the central nervous system, and
Schwann cells responsible for producing myelin in the peripheral
nervous system.
[0061] The term "remyelinating" or "remyelination" refers to
regeneration of myelin, e.g., in response to a demyelination
insult.
[0062] A "subject," "individual" or "patient" is used
interchangeably herein, which refers to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, Humans, farm animals, sport animals,
and pets. Tissues, cells and their progeny or a bialogical entity
obtained in vivo or cultured in vitro are also encompassed.
[0063] The "biologically active agents" or "bioactive agents" that
are employed in the animal model or cell culture assays described
herein may be selected from the group consisting of a biological or
chemical compound such as a simple or complex organic or inorganic
molecule, peptide, peptide mimetic, protein (e.g. antibody),
liposome, small interfering RNA, or a polynucleotide (e.g.
anti-sense). Furthermore, such agents include complex organic or
inorganic molecules can include a heterogeneous mixture of
compounds, such as crude or purified plant extracts.
[0064] A "promoter element" is a regulatory sequence that promotes
transcription of a gene that is linked to such a sequence. The
regulatory sequence can include enhancer sequences or functional
portions thereof.
[0065] A "control" is an alternative subject, cell or sample used
in an experiment for comparison purpose. Cell Death Mediator
Proteins (CDMPs)
[0066] The present invention provides methods and compositions
comprising cell death mediator proteins (CDMPs) expressed in a cell
or tissue specific manner. For example, expression may be specific
to the CNS or PNS, or to specific neural or mural cells. The
expression can be in an animal, and such expression in an animal
can cause demyelination, for example, by inducing cell death
specifically in cells with roles in myelination or remyelination.
Expression can also be in vitro. The expression of the cell death
mediator proteins can be inducible, in vivo or in vitro.
[0067] The cell death mediator protein has a role in mediating cell
death, or apoptosis. The CDMPs may affect apoptosis directly or
indirectly, for example, by modulating the activity of proteins
that directly affect apoptosis. For example, the CDMP can be SMAC
s(second mitochondria-derived activator of caspases), IAPB
(inhibitor of apoptosis proteins), caspases, or modulators of them.
In other embodiments, the CDMP can be modulators of the TNF (tumor
necrosis factor) receptor and other death receptor signaling
pathways, such as Fas Receptor, and TRAIL receptor pathways. CDMPs
can also be activators of caspases, including Granzyme B, or
modulators of Granzyme B.
[0068] In various embodiments, the cell death mediator protein is
encoded by a nucleic acid construct. In some embodiments, the
nucleic acid construct can encode one or more CDMPs. The CDMPs can
be the same or different. For example, a single nucleic can encode
two of the proteins, such as two sequences encoding caspase 9 in
tandem, or can encode caspase 9 and caspase 3. The nucleic acid
construct of the present inventnion can encode caspase 2, 5, 8, 9,
10 or 11, their proenzyme forms, or derivatives thereof. In further
embodiments, the sequence encoding such caspase protein(s) is
modified to include a dimerization domain reactive with a
cross-linker compound. Thus in some embodiments, a wild type
caspase sequence is modified to produce a chimeric sequence
comprising the dimerization domain selectively reactive to a
cross-linker compound. Examples of such dimerization domains
include those disclosed in U.S. Patent Application No. 20050187177
and U.S. Pat. No. 6,984,635, the relevant portions of which are
incorporated herein by reference in their entirety.
Dimerization
[0069] In some embodiments, dimerization activates a biological
process (e.g, apoptosis via activation of caspase 9), and various
chimeric proteins can be utilized. The chimeric proteins are
recombinant in that the various domains are heterologous to one
another (derived from different sources, e.g. not found linked
together in nature). Recombinant DNA constructs which comprise
heterologous components, e.g, encoding a particular domain or
expression control sequence, which are not found directly linked to
one another in nature, are used genetically engineer target host
cells in vitro or in vivo. Cells thus engineered contain at least
one such chimeric protein or a first series of genetic constructs
encoding the chimeric protein(s). One such DNA construct encodes a
chimeric protein comprising (a) at least one receptor domain
(capable of binding to a selected ligand) fused to (b) a
heterologous additional ("action") protein domain. The ligand is
capable of binding to two (or more) receptor domains within the
chimeric proteins, preferably with a Kd value ranging from
approximately 10.sup.-6 to <10.sup.-9 and is preferably a
non-protein compound having a molecular weight less than
approximately 1 kDa, 5 kDa, 10kDa, 15 kDa or 20 kDa. The receptor
domains of the chimeric proteins so oligomerized may be the same or
different (i.e., homodimerization or heterodimerization). Upon
exposure to the ligand and receptor oligomerization, the chimeric
proteins can initiate a biological process (e.g., complement
cascade). The encoded chimeric protein may further comprise an
intracellular targeting domain capable of directing the chimeric
protein to a desired cellular compartment, e.g., a sequence
directing the protein to associate with the nucleus.
[0070] Examples of the types of ligands to which the chimeric
proteins may bind include an FK506-type ligand, a cyclophilin type
ligand (e.g., cyclosporin A-type ligand), tetracycline or a steroid
ligand. Such binding causes oligomerization of homotypic (the same)
or heterotypic (different) chimeric protein molecules. Examples of
such ligands and/or receptor domains are disclosed in U.S. Pat.
Nos. 5,534,418, 5,002,753, 5,298,429, 6,235,872, 6,656,971,
7,196,182, 7,101,357, 7,109,317, 7,153,685 and 7,169,564, the
disclosures for each of which is incorporated by reference herein
in its entirety; see also, Straathof et al., Blood, (2005)
105:4247-4254; Belshaw et al., Proc Natl Acad Sci. (1996)
93:4604-4607.
[0071] Thus, utilizing methods described herein, target cells can
comprise a DNA construct encoding a chimeric protein comprising (i)
at least one receptor domain capable of binding to a selected
oligomerizing ligand and (ii) another protein domain, heterologous
to the receptor domain, which encodes caspase 9. Hence, following
exposure to the selected ligand, oligomerization of caspase 9
expressed in such target cells can induce the apoptosis program,
killing the cells comprising the DNA construct.
[0072] Caspase 9 is a protein that typically functions as a dimer
after cytochrome C and ATP dependent interaction with apoptotic
protease-activating factor 1 (Apaf-1). The dimerization of caspase
9 allows the polypeptide to activate downstream effectors molecules
ultimately resulting in apoptosis of the cell (Springer, J Biochem
Mol. Biol. (2002) 35:94-105).
[0073] Thus, in various embodiments, the cell death mediator
protein is a caspase 9 chimeric protein comprising binding domains
for a FK506-type ligand, cyclosporin A-type ligand, tetracycline or
steroid ligand. In one embodiment, the caspase 9 comprises a FKBP12
binding domain. In further embodiments, binding domains utilized in
chimeric constructs (e.g., caspase 9) may be optimized to bind a
ligand (e.g., chemical inducer of dimerization).
[0074] An inducible caspase 9 cDNA (iCP9) was engineered by linking
the caspase 9 cDNA sequence (GenBank AH002 818), after removal of
the caspase recruitment domain (CARD), to a FK506 binding protein
(FKBP) sequence (GenBank AH002818). The absence of the CARD
sequence prevents physiologic dimerization of the protein and
thereby prevents spontaneous initiation of the caspase cell death
cascade. The fusion to the FKBP sequence allows for chemically
induced aggregation after the administration of a chemical inducer
of dimerization (CID). The CID, AP20187 (ARIAD Pharmaceuticals,
Cambridge, Mass.) is a nontoxic synthetic FK506 analog that has
been altered to prevent interaction with endogenous FKBPs. The CID
system has been utilized in unrelated systems previously both in
vitro and in vivo (Straathof et al., Blood. (2005)
105:4247-4254).
[0075] In various embodiments of the invention, nucleic acid
constructs of the invention are delivered to a cell(s) (culture or
in vivo) via a viral vector, including but not limited to
adenovirus, adenovirus associated virus, murine leukemia virus,
lentivirus, foamy virus, rabies virus or other viral vectors known
in the art, such as those disclosed in U.S. Pat. No. 6,982,082.
Regulated Expression
[0076] In various aspects of the invention, cell- or
tissue-specific and/or inducible expression regulatory elements are
operably linked to cell death mediator proteins to effect selective
cell death upon expression. As described above, tissue specific and
cell specific regulatory sequences are available for expressing
transgenes in the central nervous systems. The regulatory sequences
allow ectopic expression of transgenes in the central nervous
system or peripheral nervous system in particular cell types. For
example, selective death can be achieved in cells such as, but not
limited to, oligodendrocytes, microglial cells, Schwann cells or
astrocytes.
[0077] Exemplary expression of regulatory sequences include
regulatory sequences selected from genes including but not limited
to CC1, myelin basic protein (MBP), ceramide galactosyltransferase
(CGT), myelin associated glycoprotein (MAG), myelin oligodendrocyte
glycoprotein (MOG), oligodendrocyte-myelin glycoprotein (OMG),
cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein
zero (MPZ), peripheral myelin protein 22 (PMP22), protein 2 (P2),
GFAP, AQP4, PDGFR-.alpha., PDGF-.alpha., RG5, pGlycoprotein,
neurturin (NRTN), artemin (ARTN), persephin (PSPN), sulfatide, 2
(VEGFR2), superoxide dismutase (SOD1), tyrosine hydroxylase, neuron
specific enolase, parkin gene (PARK2), parkin coregulated gene
(PACRG), neuron-specific T.alpha.l .alpha.-tubulin
(T.alpha.l),vesicular monoamine transporter (VMAT2), and
.alpha.-synuclein (SNCA), PDGFR-.beta., or proteolipid protein
(PLP).
[0078] Additional examples of neural cell-specific promoters are
known in the art, such as disclosed in U.S. Patent Application
Publication Nos. 2003/0110524; 2003/0199022; 2006/0052327,
2006/0193841, 2006/0040386, 2006/0034767, 2006/0030541; U.S. Pat.
Nos. 6,472,520, 6,245,330, 7,022,319 and 7,033,595, the relevant
disclosures of which is incorporated herein by reference; See also,
the website <chinook.uoregon.edu/promoters.html>; or
<tiprod.cbi.pku.edu.cn:8080/index> (listing promoters of
genes specific to certain cell/tissue); and Patterson et al., J.
Biol. Chem. (1995)270:23111-23118.
[0079] Thus one aspect of the invention is the utilization of
inducible/cell type specific expression regulatory elements for
temporal control of cell death, which in turn is utilized to assess
the effects of particular cells on neuronal health/maintenance or
assess effects of candidate molecules on neuronal
health/maintenance, as associated to neuropathies such as MS, ALS
and Parkinson's.
[0080] Expression of cell death mediator proteins can also be
temporally regulated by utilizing expression systems other than
those utilizing cell/tissue-specific promoters (e.g., where an
effector molecule is administered locally). Therefore, in some
embodiments, a gene encoding a cell death mediator protein can be
operably linked to a controllable promoter element, such as a
tet-responsive promoter. For example, where and when desired an
inducible agent (e.g., tetracycline or analog thereof) can be
administered to cells or a subject to induce expression of cell
death mediator protein in a cell/tissue specific manner (e.g., mere
tetracycline is delivered in a localized/limited manner). Such a
system can provide tight control of gene expression in eucaryotic
cells, by including the "off-switch" systems, in which the presence
of tetracyclin inhibits expression, or the "reversible" Tet system,
in which a mutant of the E. coli TetR is used, such that the
presence of tetracyclin induces expression. These systems are
disclosed, e.g., in Gossen and Bujard (Proc. Natl. Acad. Sci.
U.S.A. (1992) 89:5547) and in U.S. Pat. Nos. 5,464,758; 5,650,298;
and 5,589,362 by Bujard et al.
[0081] Additional examples of inducible promoters include but are
not limited to MMTV, heat shock 70 promoter, GAL1-GAL10 promoter,
metallothien inducible promoters (e.g., copper inducible ACE1;
other metal ions), hormone response elements (e.g., glucocorticoid,
estrogen, progestrogen), phorbol esters (TRE elements), calcium
ionophore responsive element, or uncoupling protein 3, a human
folate receptor, whey acidic protein, prostate specific promoter,
as well as those disclosed in U.S. Pat. No. 6,313,373; see also,
online at <biobase/de/pages/products/transpor.html>
(providing a database with over 15,000 different promoter sequences
classified by genes/activity); and Chen et al. Nuc. Acids. Res.
2006, 34: Database issue, D104-107.
[0082] Yet other inducible promoters include the growth hormone
promoter; promoters which would be inducible by the helper virus
such as adenovirus early gene promoter inducible by adenovirus E1A
protein, or the adenovirus major late promoter; herpesvirus
promoter inducible by herpesvirus proteins such as VP 16 or 1CP4;
promoters inducible by a vaccinia or pox virus RNA polymerases; or
bacteriophage promoters, such as T7, T3 and SP6, which are
inducible by T7, T3, or SP6 RNA polymerase, respectively.
[0083] In other embodiments, constitutive promoters may be
desirable. For example, there are many constitutive promoters
suitable for use in the present invention, including the adenovirus
major later promoter, the cytomegalovirus immediate early promoter,
the .beta. actin promoter, or the .beta. globin promoter. Many
others are known in the art and may be used in the present
invention. In yet further embodiments, a regulatory sequence can be
altered or modified to enhance expression (i.e., increase promoter
strength). For example, intronic sequences comprising enhancer
function can be utilized to increase promoter function. The myelin
proteolipid protein (PLP) gene comprises an intronic sequence that
functions as an enhancer element. This regulatory element/region
ASE (antisilencer/enhancer) is situated approximately 1 kb
downstream of exon 1 DNA and encompasses nearly 100 bp. See Meng et
al. J Neurosci Res. (2005) 82:346-356.
[0084] Furthermore, where expression of the transgene in a
particular subcellular location is desired, the transgene can be
operably linked to the corresponding subcellular localization
sequence by recombinant DNA techniques widely practiced in the art.
Exemplary subcellular localization sequences include, but are not
limited to, (a) a signal sequence that directs secretion of the
gene product outside of the cell; (b) a membrane anchorage domain
that allows attachment of the protein to the plasma membrane or
other membraneous compartment of the cell; (c) a nuclear
localization sequence that mediates the translocation of the
encoded protein to the nucleus; (d) an endoplasmic reticulum
retention sequence (e.g. KDEL sequence) that confines the encoded
protein primarily to the ER; (e) proteins can be designed to be
farnesylated so as to associate the protein with cell membranes; or
(f) any other sequences that play a role in differential
subcellular distribution of a encoded protein product.
[0085] In some embodiments, markers for distinguishing genetically
modified cells can be detected. Such markers include, but are not
limited to, CC1, myelin basic protein (MBP), ceramide
galactosyltransferase (CGT), myelin associated glycoprotein (MAG),
myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin
glycoprotein (OMG), cyclic nucleotide phosphodiesterase (CNP),
NOGO, myelin protein zero (MPZ), peripheral myelin protein 22
(PMP22), protein 2 (P2), galactocerebroside (GalC), sulfatide,
PDGFR-.beta., PDGFR-.alpha., PDGF-.alpha., and proteolipid protein
(PLP).
[0086] In various embodiments, animals in which selective cell
death, such as selective apoptosis of oligodendrocytes, is induced
can also be assayed for effects on demyleination/remyelination
status. For example, demyelination/remyelination phenomena can be
observed by immunohistochemical means or protein analysis as known
in the art. For example, sections of the test animal's brain can be
stained with antibodies that specifically recognize an
oligodendrocyte marker. In another aspect, the expression levels of
oligodendrocyte markers can be quantified by immunoblotting,
hybridization means, and amplification procedures, and any other
methods that are well-established in the art. e.g. Mukouyama et al.
Proc. Natl. Acad. Sci. (2006)103:1551-1556; Zhang et al. (2003),
supra; Girard et al. J. Neuroscience. (2005) 25: 7924-7933; and
U.S. Pat. Nos. 6,909,031; 6,891,081; 6,903,244; 6,905,823;
6,781,029; and 6,753,456, the disclosure of each of which is herein
incorporated by reference.
[0087] In yet other embodiments, animals in which selective cell
death occurs in cells important for maintenance of the blood brain
barrier, such as in pericytes. For example, expression a caspase,
such as caspase 9 or iCP9, under the control of the PDGFR-.beta.
promoter in animals can be used to assay for effects on the blood
brain barrier (BBB), such as its permeability, maintenance, or
integrity. For example, sections of the test animal's brain can be
stained with antibodies that specifically recognize BBB markers,
such as tight junction proteins. For example, markers that may be
detected include occludin and claudins, such as claudin 2, claudin
5, claudin 6, claudin 7, claudin 10, claudin 12, claudin 15, and/or
claudin 19. In another aspect, the expression levels of tight
junction protein marker can be quantified by immunoblotting,
hybridization means, and amplification procedures, and any other
methods that are well-established in the art.
[0088] BBB integrity or permeability may also be assayed by using
indicators such as any dye, marker, or tracer known in the art that
is utilized to determine, visualize, measure, identify or quantify
blood-brain barrier permeability. Non-limiting examples include,
Evans Blue and sodium fluorescein. Examples of such indicators will
be apparent to one of ordinary skill in the art, and include
essentially any compound that is unable to traverse an intact BBB,
but is capable of traversing a more permeable BBB, as well as
capable of being identified, measured or quantified.
[0089] Indicators can be enzymes, tracers or markers utilized to
determine BBB permeability changes, with non-limiting examples as
follows:
TABLE-US-00001 Enzyme Functions observed Dopa-decarboxylase Convert
L-Dopa to dopamine Monoamine oxidase-B Inactivates catecholamines
(5-HT) Pseudocholinesterase Deacetylates heroin to morphine
Cytochrome P450 O-Demethylates codeine to morphine UDP- Metabolizes
1-naphthol Glucuronosyltransferase Epoxide hydrolase Reacts with
epoxides (Benzo[a]pyre 4,5-oxide) Renin Angiotensinogen to
Angiotensin I Dipeptidyl dipeptidase Enkephalin metabolism ACE
Enkephalin, angiotensin I, neurotensin, and bradykinin metabolism
Aminopeptidase A Metabolism of angiotensin Aminopeptidase M (N)
Opioid degradation (N-terminal Tyr) Glutamyl aminopeptidase Convert
angiotensin II to angiotensin III Enkephalinase* Enkephalin,
Endothelin, and bradyknin (neutral Endopeptidase degradation 24.11)
Endopeptidase* Dynorphin, neurotensin, bradykinin, (Endopeptidase
24.15) angiotensin II, and LHRH degradation
.gamma.Glutamyltranspeptidase Convert leukotriene C4 to leukotriene
D4 Alkaline phosphatase purine and pyrimidine metabolism *Enzymes
in choroids plexus; ACE: angiotensin converting enzyme; LHRH:
luteinizing hormone releasing hormone
[0090] Additional examples of dyes, tracers or markers include
dextran, biotin, fibrinogen, albumin, blood globulin's using
Coons's reaction, Texas Red conjugated dextran (70,000 da MW),
Na(+)-fluorescein (MW 376) or fluorescein isothiocyanate (FITC)
labelled dextran (MW 62,000 or 145,000), or FITC-labeled dextran of
molecular mass 10,000 Da (FITC-dextran-10K).
Transgenic Animals
[0091] In one aspect of the invention, a transgenic animal is
generated having stably integrated into the genome a transgenic
nucleotide sequence encoding a neural cell-specific regulatory
element operably linked to a gene encoding a protein of interest.
The expression of the gene can be under the control of an inducible
promoter. In some embodiments, the cell-specificity is to neural
cells (e.g. glial cells, preferably astrocytes, oligodendrocytes
and/or Schwann cells; or neuronal cells, such as cervical ganglion
neurons, dorsal root ganglion cells, nodose ganglion neurons,
spinal motor neurons, midbrain dopaminergic neurons, central
noradrenergic neurons and enteric neurons).
[0092] In a preferred embodiment, the gene encoding a protein of
interest encodes a cell death mediator protein. Thus, a transgenic
animal of the present invention can comprise a nucleotide sequence
encoding a cell death mediator protein (CDMP) operably linked to a
cell type-specific expression regulatory element; wherein the
animal exhibits a greater degree of neuropathy relative to an
animal without the nucleotide sequence encoding the CDMP operably
linked to a cell type-specific expression regulatory element. The
neuropathy may be a neuronal demyelination, such as multiple
sclerosis. The transgenic animal may exhibit an increase in
apoptotic oligodendrocytes relative to that of a control animal,
and the expression of CDMP can be ectopically confined to the
central nervous system. In some embodiments the neuropathy is due
to a defect in the blood brain barrier, or due to defects in both
the BBB and demyelination. For example, the neuropathy may be
Amyotropic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Immune
Dysfunction Muscular Central Nervous System Breakdown, Muscular
Dystrophy (MD), Alzheimer's disease, Pakinson's disease,
Huntington's disease, Brain Ischemia, Cerebral Palsy, Corticobasal
Ganglionic Degeneration, Creutzfeldt-Jakob Syndrome, Dandy-Walker
Syndrome, Dementia, Vascular Encephalitis, Encephalomyelitis,
Epilepsy, Essential Tremor, Kuru-Landau-Kleffner Syndrome, Lewy
Body Disease, Machado-Joseph Disease, Meige syndrome, Migraine
Disorders, poliomyelitis, Multiple System Atrophy, Meningitis,
Drager Syndrome, Tourette Syndrome, Hallervorden-Spatz Syndrome,
Hydrocephalus, Oliovopontocerebellar atrophies, Supranucleal Palsy,
or Syringomyelia.
[0093] In one embodiment, the cell death mediator protein (CDMP) is
caspase 2, 5, 8, 9, 10 or 11. In another embodiment, one or more of
such cell death mediator proteins are expressed in a cell, such as
a neural cell or mural cell. For example, the CDMP can be targeted
to the neural cells of an animal, such as glial cells. The neural
cell can be, but not limited to, oligodendrocytes, astrocytes,
Schwann cells, or microglial cells. Targeting can also be to the
mural cells of the animal, for example mural cells such as, but not
limited to, pericytes, endothelial cells, or smooth muscle cells.
In some embodiments, a combination of one or more target cells is
selected (e.g., neural and mural, or different neural cells, or
different mural cells).
[0094] In some embodiments, expression of the CDMP is inducible.
For example, CDMP may be operably linked to a regulatory element
with an inducible promoter. The activity of the CDMP, such as
promoting apoptosis, may also be inducible. For example, a viral
vector encoding a CDMP that is induced by a CID can be directly
injected into the spinal cord or brain of a non-human adult animal
(e.g., rat) and a CID is administered. The CID can be administered
concurrent with, or subsequent to, administration of the viral
vector.
[0095] The extent of focal areas of oligodendrocyte loss, resulting
from focal infection, can be controlled by the amount of virus
administered. The response of surviving cells to the selective loss
of oligodendrocytes can be assessed using histological, molecular
and electrophysiologic assasys, which are apparent to one skilled
in the art. The extent of lesion size (for example, as measured by
oligodendrocyte death) can be correlated with axonal loss at the
lesion site. Similarly, by evaluating neurons whose axons pass
through the region of cell loss, it can be determined whether a
demyelinating threshold exists that once surpassed results in the
loss of distant neurons. Therefore, methods of the invention
provide insights into the molecular response following CNS
oligodendrocyte cell loss and allow identification of candidate
agents to effect temporal and environmental-based approaches to
neuronal protection in multiple sclerosis. In one embodiment, the
viral vector is a lentiviral vector. In a further embodiment, the
viral vector is an iCP9 lentivirus (further described below).
[0096] The animal models of the present invention encompass any
non-human vertebrates that are amenable to procedures yielding a
neuronal demyelination condition in the animal's nervous systems,
including the central and peripheral nervous system. Preferred
model organisms include, but are not limited to, mammals, primates,
and rodents. Non-limiting examples of preferred models are rats,
mice, guinea pigs, cats, dogs, rabbits, pigs, chimpanzees, and
monkeys. The test animals can be wildtype or transgenic. In one
embodiment, the animal is a rodent. In yet another embodiment, the
animal is a mouse. In another embodiment, the animal is from a
simian species. In yet another embodiment, the animal is a marmoset
monkey, which is commonly utilized in examining neurological
disease (e.g., Eslamboi, A. Brain Res Bull. (2005) 68:140-149;
Kirik et al. Proc. Natl. Acad. Sci. (2004) 100:2884-89).
[0097] Transgenic animals can be broadly categorized into two
types: "knockouts" and "knockins". A "knockout" has an alteration
in the target gene via the introduction of transgenic sequences
that result in a decrease of function of the target gene,
preferably such that target gene expression is insignificant or
undetectable, e.g. by replacing a portion of the target gene with
sequences unrelated to the target gene. A "knockin" is an animal
having an altered expression of a target gene, e.g., by operatively
inserting a regulatory sequence of the target gene; or is an animal
expressing modified copy of the target gene, e.g., by replacing the
target gene with a modified copy. Modifications can be deletion or
mutation of the target gene. The knock-in or knock-out animals can
be heterozygous or homozygous with respect to the target genes.
Both knockouts and knockins can be "bigenic," also known as double
knock-in or double knock-out. Bigenic animals have at least two
host cell genes being altered. In one embodiment, bigenic animal
carries a transgene encoding a cell-specific cell death mediator
protein and another transgenic sequence that encodes cell-specific
marker genes. The transgenic animals of the present invention can
broadly be classified as Knockins. In some embodiments, specific
cell types of the animals may be targeted. For example, the target
cells can be neural or mural. In various embodiments, neural cells
include oligodendrocyte, Schwann cells, microglial cells or
astrocytes, and mural cells include endothelial cells, pericytes or
smooth muscle cells.
[0098] Advances in technologies for embryo micromanipulation now
permit introduction of heterologous DNA into fertilized mammalian
ova as well. For instance, totipotent or pluripotent stem cells can
be transformed by microinjection, calcium phosphate mediated
precipitation, liposome fusion, retroviral infection or other
means. The transformed cells are then introduced into the embryo,
and the embryo develops into a transgenic animal. In a preferred
embodiment, developing embryos are infected with a viral vector
containing a desired transgene so that the transgenic animals
expressing the transgene can be produced from the infected embryo.
In another preferred embodiment, a desired transgene is coinjected
into the pronucleus or cytoplasm of the embryo, preferably at the
single cell stage, and the embryo is allowed to develop into a
mature transgenic animal. These and other variant methods for
generating transgenic animals are well established in the art and
hence are not detailed herein. See, for example, U.S. Pat. Nos.
5,175,385 and 5,175,384.
[0099] Accordingly, the present invention provides a method of
using animal models for detecting and quantifying remyelination in
a cell-specific manner. In such an embodiment, the method comprises
the steps of: (a) inducing cell death in a cell type-specific
manner by expression of cell death mediator protein in a cell; (b)
allowing time for cell death to occur; (c) determining modulation
of myelination/remyelination in the animal. The transgenic animals
may also be used for screening bioactive agents, determining the
modulation of myelination or remyelination of bioactive agents.
Animal Studies
[0100] Animal models are utilized with one or more methods of the
invention to assay selective or controlled cell death in target
cells related to neuropathy phenomenon (e.g. a demyelinating
disorder). The phenomenon can be associated with a demyelination
disorder characterized by a decrease in myelinated axon, a
reduction in the levels of oligodendrocyte markers, astrocyte
markers or Schwann cell markers. The demyelination disorder can be
genetic, or inflicted by a pathogen or virus.
[0101] The animal models may be used to screen for bioactive agents
that modulate a neuropathy. For example, the application of the
model system disclosed herein, when applied to the rat CNS,
provides a more accurate model that allows for the study of the
neuronal response to demyelination in vivo and the development of
treatments of demyelination disorders.
[0102] The present invention provides nucleic acid constructs
encoding a cell death mediator protein operably linked to a
cell/tissue-specific or inducible promoter that are administered to
an animal to achieve expression of the cell death mediator protein
and hence selective ablation of the target cells. Such expression
can be achieved via ectopically maintained transgene delivery
vehicles or such transgenes can be incorporated into the genome of
the animal using methods known in the art. For example, expression
could be achieved episomally or through stable integration of the
nucleic acids encoding the CDMP.
[0103] In various embodiments, a nucleic acid construct comprising
a gene encoding a cell death mediator protein ("suicide gene") is
operably linked to an expression regulatory element, which is
cell/tissue-specific or inducible. In various embodiments, the
target cell is a neural cell or mural cell. The neural cell can be,
but not limited to, oligodendrocytes, astrocytes, Schwann cells, or
microglial cells. Mural cells include, but are not limited to,
pericytes, endothelial cells, and smooth muscle cells. In some
embodiments, a combination of one or more target cells are selected
(e.g., neural and mural, or different neural cells, or different
mural cells).
[0104] In yet other embodiments, the target cell is an immune cell,
such as, but not limited to, B lymphocyte or T lymphocyte cell. In
yet a further embodiment, B lymphocytes and/or T lymphocytes are
not target cells.
[0105] In one aspect of the invention, the cell death mediator
protein is a caspase protein, including but not limited to, caspase
2, 5, 8, 9, 10 or 11. In one embodiment, nucleic acid constructs of
the invention comprise at least two different caspase proteins,
which can be expressed in target cells. The CDMP may be chimeric,
for example, CDMP with a binding domain for FK506-type ligand,
cyclosporin A-type ligand, tetracycline or steroid ligand. In one
embodiment, the caspase 9 comprises a FKBP12 binding domain. In
further embodiments, the apoptosis promoting activity of the CDMP
may be inducible, for example, binding domains utilized in chimeric
constructs (e.g., caspase 9) may be optimized to bind a chemical
inducer of dimerization, which promotes caspase 9 activity and
thus, apoptosis.
[0106] In another aspect of the invention, methods of testing a
biologically active agent for myelination/remyelination modulation
activity is provided.
[0107] In one embodiment, a method for testing a candidate agent
for modulation of neuropathy associated phenomenon comprises
inducing cell death in a Test Animal by expression of the cell
death mediator protein, allowing sufficient time for assessing
effects on myelination/remyelination, administering a test
bioactive agent and determining the effect on
myelination/remyelination as compared to without administration,
thus determining whether the test agent enhances/reduces
myelination/remyelination.
[0108] Thus, in some embodiments, the method comprises the steps
of: (a) inducing cell death in a cell type-specific manner; (b)
assessing demyelination insult in the transgenic animal of the
invention; (c) administering a test agent to the animal; (d)
optionally detecting and/or quantifying expression of cell-specific
marker gene(s) before and after step (c); (e) detecting if and how
much remyelination has occurred in step (d); (f) determining the
test agent to have remyelination modulation activity if
remyelination is enhanced or diminished (e.g., by histological,
histochemical, biochemical assays or by measuring expression of
remyelination-specific marker proteins which can be up- or
down-regulated in response to administration of the test agent). In
various embodiments, detection comprises histochemical or
biochemical assays known in the art. In some embodiments, detection
is made at various time points and administration of the test agent
can be repeated during the course of the assay, as well as using
different dosing regimens.
[0109] In another embodiment, a method of testing for a compound
that modulates a phenomenon associated with a neuropathy comprises
administering a candidate agent to a test animal as described
herein, enhancing demyelination in the test animal by inducing
expression of a cell death mediator protein, determining whether
administration of the candidate agent results in enhanced or
reduced remyelination, thus determining that the test agent
modulates a phenomenon associated with a neuropathy if
remyelination is enhanced or reduced. In one embodiment, practice
of the method determines whether the candidate agent modulates
cell-death mediated demyelination/remyelination.
[0110] For example, in one such embodiment, a method for testing a
biologically active agent that modulates a phenomenon associated
with a neuropathy comprises (a) administering a candidate agent to
an animal comprising a transgene encoding a death mediator protein
operably linked to an inducible/cell-specific expression regulatory
element; (b) inducing expression of the cell death mediator protein
thus effecting cell death; and (c) determining if the candidate
agent enhances myelination or remyelination. In some embodiments,
detection is made at various time points and administration of a
test agent can be repeated during the course of the assay, as well
as using different dosing regimens. In one embodiment the
neuropathy is a demyelinating disorder (e.g., MS). Levels of
myelination or remyelination can be compared to control animals,
and determined by methods including, but not limited to,
histological, histochemical, biochemical assays or by measuring
expression of remyelination-specific marker proteins which can be
up- or down-regulated in response to administration of the test
agent. For example, the effect of an agent upon a phenomenon
associated with a demyelination disorder can involve an
immunoassay, hybridization assay or PCR assay.
[0111] In various embodiments, detection comprises histochemical or
biochemical assays known in the art. In some embodiments, detection
is made at various time points and administration of the test agent
can be repeated during the course of the assay, as well as using
different dosing regimens.
[0112] The present invention also provides a method of testing a
candidate agent for effects on neuron maintenance, neuron death, or
neuron growth, or glial cell maintenance, glial cell death or glial
cell growth.
[0113] Immunocytochemistry and histological study can be used to
determine the effects on neuronal or glial maintenance, growth or
death. In some embodiments, the expression of the
remyelination-specific marker protein in the test animal can be
compared to a control or reference animal. In other embodiments,
the expression of the cell-specific marker protein in the test
animal is compared to measurements made at various time points in
the same animal, to determine onset or progress of neuron death or
growth.
[0114] The candidate agent of the present invention may be tested
by methods described herein for remyelination promoting activity,
or conversely, remyelination inhibiting or reducing activity. For
example, the method can comprise the steps of: (a) inducing
demyelination insult in the transgenic animal of the invention
through expression of a cell death mediator protein in one or more
particular cell types, wherein the one or more cell types affects
myelination or neuronal support; (b) allowing time sufficient to
effect myelin repair occur, as evidenced by expression of myelin
cell-specific marker gene(s); (c) administering a candidate agent
to tne animal, before, during and/or after steps (a) and/or (b);
(d) detecting the elect of the administered candidate on
remyelination, if any.
[0115] In some embodiments, a candidate agent is administered
before, during or after the inducing demyelinating insult step, for
example inducing the insult by inducing the expression and/or
activity of one or more CDMPs. In one embodiment, a candidate agent
is administered before inducing demyelinating insult. In another
embodiment, a candidate agent is administered during induction of
demyelinating insult. In yet another embodiment, a candidate agent
is administered after induction of demyelinating insult.
[0116] In some embodiments, a candidate agent is administered
before, during or after the allowance of time sufficient to effect
myelin repair. In one embodiment, a candidate agent is administered
immediately after insult. In another embodiment, a candidate agent
is administered during the time during which myelin repair can
occur. In yet a further embodiment, a candidate agent is
administered after myelin repair has occurred.
[0117] In some embodiments, a candidate agent is administered from
about 1 to about 24 hours after insult. In some embodiments, a
candidate agent is administered from about 1 to about 30 days after
insult. In various embodiments, a candidate agent is administered
from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23 or 24 hours after insult. In various
embodiments, a candidate agent is administered from about 1, 2, 3,
4, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In yet a
further embodiment, a candidate agent is administered from about 1
to about 12 months.
[0118] While the amount of time required for developing
remyelinated axons varies among different animals, it generally
requires at least about 1 week, more often requires at least about
2 to 10 weeks, and even more often requires about 4 to about 10
weeks.
[0119] In any of the methods directed to screening a candidate
agent, it should be understood that one or more candidate agents
can be screened simultaneously. In various embodiments, a candidate
agent is identified as enhancing remyelination, where remyelination
and/or expression of myelin specific marker proteins is enhanced or
increased.In some embodiments, the expression of the cell-specific
marker protein in the test animal can be compared to a control or
reference animal. In other embodiments, the expression of the
cell-specific marker protein in the test animal is compared to
measurements made at various time points in the same animal, where
an earlier time point can be used as a reference or control time
point. In yet other embodiments, the expression of
remyelination-specific marker proteins is measure in the test
animal and a control or reference animal, in determining whether a
candidate agent has remyelination inhibiting or reducing activity.
Such an agent can be categorized as a remyelination inhibitor or
remyelination toxin.
[0120] Remeylination can be ascertained by observing an increase in
the cell-specific expression of a marker gene/gene product (e.g.,
in the central or peripheral nervous system), such as by expression
of a marker protein (e.g. EGFP). In one or more methods herein,
where demyelination or myelination is sought to be identified,
various markers are available in the art. Exemplary markers for
identifying myelinating cells include, but are not limited to, CC1,
myelin basic protein (MBP), ceramide galactosyltransferase (CGT),
myelin associated glycoprotein (MAG), myelin oligodendrocyte
glycoprotein (MOG), oligodendrocyte-myelin glycoprotein (OMG),
cyclic nucleotide phosphodiesterase (CNP), NOGO, myelin protein
zero (MPZ), peripheral myelin protein 22 (PMP22), protein 2 (P2),
galactocerebroside (GalC), sulfatide, PDGFR-.beta., PDGFR-.alpha.,
PDGF-.alpha., and proteolipid protein (PLP).
[0121] Subsequent to insult, such as after induction of apoptosis
in myelinating cells, and after sufficient time for remyelination
to occur, fluorescence of the marker proteins may be detected using
in vitro or in vivo methods known in the art for detection of
fluorescence in small animals. In vivo fluorescence can be detected
and/or quantified utilizing devices available in the relevant art.
For example, pulsed laser diodes and a time-correlated single
photon counting detection system coupled to a visualization system
can be used to detect the level of fluorescence emission from
tissues. (Gallant et al., Annual Conference of the Optical Society
of America (2004).; Contag et al, Mol. Microbiol. 18:593-603 (1995;
Schindehutte et al., Stem Cells 23:10-15 (2005)). To avoid a large
signal from back-reflected photons at the tissue-air interface, the
detection point is typically located at 3 mm to the right of the
source point. Wavelength selection of both laser and filters is
dependent on the fluorescent marker of choice. Where biological
tissue absorption is low, fluorescent signals from larger tissue
depths (e.g., a few to several centimeters depending on laser
power) can be detected for in vivo imaging.
[0122] Mice to be imaged may be anesthetized with isoflurane/oxyten
and placed on the imaging stage. Ventral and dorsal images can be
collected for various time points using imaging systems available
in the relevant art (e.g., IVIS imaging system, Xenogen Corp.,
Alameda, Calif.). Fluorescence from various target tissue can be
imaged and quantified. For example, signal intensity can be
presented in text or figures as a means +/-standard error about the
mean. Fluorescence signals can be analyzed by analysis of variance
with post hoc t tests to evaluate the difference between
fluorescence signal for a given marker at time zero and each
subsequent time point.
[0123] Fluorescence visualization, imaging or detection can be made
using methods known in the art and described herein, supra.
Visualization, imaging or detection can be made through invasive,
minimally invasive or non-invasive techniques. Typically,
microscopy techniques are utilized to detect or image fluorescence
from cells/tissue obtained from the transgenic animals, from living
cells, or through in vivo imaging techniques. Supra, "General
Methodologies".
[0124] Luminescent, fluorescent or bioluminescent signals are
easily detected and quantified with any one of a variety of
automated and/or high-throughput instrumentation systems including
fluorescence multi-well plate readers, fluorescence activated cell
sorters (FACS) and automated cell-based imaging systems that
provide spatial resolution of the signal. A variety of
instrumentation systems have been developed to automate detection
including the automated fluorescence imaging and automated
microscopy systems developed by Cellomics, Amersham, TTP, Q3DM,
Evotec, Universal Imaging and Zeiss. Fluorescence recovery after
photobleaching (FRAP) and time lapse fluorescence microscopy have
also been used to study protein mobility in living cells.
[0125] Visualizing fluorescence (e.g., marker gene encoding a
fluorescent protein) can be conducted with microscopy techniques,
either through examining cell/tissue samples obtained from an
animal (e.g., through sectioning and imaging using a confocal
microscope), examining living cells or detection of fluorescence in
vivo. Visualization techniques include, but are not limited to,
utilization of confocal microscopy or photo-optical scanning
techniques known in the art. Generally, fluorescence labels with
emission wavelengths in the near-infrared are more amenable to
deep-tissue imaging because both scattering and autofluorescence,
which increase background noise, are reduced as wavelengths
increase. Examples of in vivo imaging are known in the art, such as
disclosed by Mansfield et al., J. Biomed. Opt. 10:41207 (2005);
Zhang et al., Drug Met. Disp. 31:1054-1064 (2003); Flusberg et al.,
Nat. Meth. 2:941-950 (2005); Mehta et al., Curr Opin Neurobiol.
14:617-628 (2004); Jung et al., J. Neurophysiol. 92:3121-3133
(2004); U.S. Pat. Nos. 6,977,733 and 6,839,586, each disclosure of
which is herein incorporated by reference.
[0126] One example of an in vivo imaging process comprises one week
before the in vivo imaging experiment, the dorsal hair in telogen
is depilated (about 2.5 cm.times.2.5 cm area) using a depilatory
agent (Nair, Carter-Wallace Inc.). On the day of the imaging
experiment, the mouse is anaesthetized and placed with its dorsal
skin on a microscope coverslip on the microscope stage. The
depilated area of the epidermis is illuminated by a 50 W mercury
lamp and scanned using an inverted laser scanning confocal
fluorescent microscope (Zeiss LSM 510) with a .times.10 objective
and an LP 520 emission filter (Zeiss). A laser, such as Argon laser
(488 nm) and a .times.10 objective can image fluorescence
emissions, progressively more effectively from deep tissue up to
the epidermal cells. By utilizing enhanced emitters or longer
wavelength emitters, the sensitivity for deeper tissue imaging can
be enhanced. Alternatively small animals, such as mice can easily
be scanned/imaged utilizing various different positions (e.g.,
dorsal, ventral, etc.). In vivo imaging has been effective even
with deep tissue regions, such as liver. (e.g., Zhang et al.,
supra).
[0127] Cell/tissue sections mounted with Vectashield mounting
medium with DAPI (Vector Laboratories) can be visualized with a
Zeiss Axioplan fluorescence microscope. Images can be captured
using a Photometrics PXL CCD camera connected to an Apple Macintosh
computer using the Open Lab software suite. Fluorescence of
different wavelengths is detected and quantified by counting
positive cells within the median of the corpus callosum, confined
to an area of approximately 0.04 mm.sup.2. Additional methods for
detecting and measuring levels of fluorescence from tissue/cell in
vitro utilizing fluorescence or confocal microscopy are known in
the art and can be utilized in detecting or measuring fluorescence
from one or more marker proteins disclosed herein above.
[0128] In another example, neural cells can be imaged with an
Axiovert S100 TV inverted microscope fitted with Ludl filter wheels
(CarlZeiss, Thornwood, N.Y., USA) in the epifluorescence excitation
and emission paths, and a cooled charge-coupled device (CCD) camera
(Micro-MAXO; Roper Scientific, Trenton, N.J., USA) can be used to
collect the images. Specific excitation and emission filters and a
common dichroic element can be used to isolate the signals of the
two different fluorescent proteins (HQFITC and Texas Red excitation
and emission filters, and FITC/Texas Red V3 dichroic; Chroma
Technology, Brattleboro, Vt., USA). The filter wheels and camera
can be controlled by software (e.g., IPLabs software, Scanalytics,
Fairfax, Va., USA). Sets of the red and green fluorescent images
can be collected to analyze the relative percentage of cells that
have red or green fluorescence. The images may be analyzed and
prepared for publication with IPLabs and Adobe InDesign software.
Manipulations of the images may be confined to merging the
grayscale images of the red and green fluorescent proteins to
create RGB color files, adjusting the brightness/contrast of the
final printouts to match most closely what is observed through the
microscope and adding lettering and a scale bar. Fluorescence
microscopy apparatus are known in the art and commercially
available. See, e.g., website at
<confocal-microscopy.com/website/sc_llt.nsf>
[0129] In an alternative embodiment, fluorescence detection is
directly from the retina or cornea. The retinal site is a
non-invasive locus for study of systemic toxicity. The cornea is
particularly well suited to assessing toxicity of substances
applied directly to an organ containing glial cells without
invading the body. Therefore, fluorescence emitted from neural
cells differentially expressing a marker protein can be detected by
using confocal microscopy of the retina or cornea by training the
laser beam onto the desired region and detecting the level of
fluorescence emitted.
[0130] Moreover, demyelination/remyelination phenomena can be
observed by immunohistochemical means or protein analysis known in
the art. For example, sections of the test animal's brain can be
stained with antibodies that specifically recognize an
oligodendrocyte marker. In another aspect, the expression levels of
oligodendrocyte markers can be quantified by immunoblotting,
hybridization means, and amplification procedures, and any other
methods that are well-established in the art. e.g., Mukouyama et
al., Proc. Natl. Acad. Sci. 103:1551-1556 (2006); Zhang et al.,
supra; Girard et al., J. Neurosci. 25:7924-7933 (2005); a U.S. Pat.
Nos. 6,909,031; 6,891,081; 6,903,244; 6,905,823; 6,781,029; and
6,753,456, the disclosure of each of which is herein incorporated
by reference.
[0131] In another aspect, cell/tissue from the central or
peripheral nervous system can be excised and processed for the
protein, e.g., tissue is homogenized and protein is separated on an
SDS-10% polyacylamide gel and then transferred to nitrocellulose
membrane to detect marker proteins. Fluorescent protein levels can
be detected utilizing primary antibody/antisera (e.g., goat
polyclonal raised against a particular marker protein; BD Gentest,
Woburn, Mass.) and peroxidase-conjugated secondary antibody (e.g.
rabbit anti-goat IgG, Sigma-Aldrich). Chemiluminescence is detected
using standard reagents available in the art to detect and
determine levels of fluorescence marker proteins in tissue
samples.
[0132] Therefore, if a candidate therapeutic/drug or test bioactive
agent is being assayed in one or more methods of the invention,
then it can be determined if there is an overall difference in
response to the drug compared at different time points, as well as
compared to reference or controls.
Demyelination
[0133] In one aspect of the present invention, compositions and
methods of the invention are utilized to effect focal
demyelination, without a requirement for systemic antigen delivery,
or adjuvant priming to initiate an immune response. In various
embodiments of the invention, expression of cell death mediator
proteins in a cell-specific manner is utilized for inducing cell
death in target cells and/or neuronal loss. Expression and/or
activity of the cell death mediator protein can also be
inducible.
[0134] The CDMP can be SMACs (second mitochondria-derived activator
of caspases), IAPs (inhibitor of apoptosis proteins), caspases, or
modulators of them. In other embodiments, the CDMP can be
modulators of the TNF (tumor necrosis factor) receptor and other
death receptor signaling pathways, such as Fas Receptor, and TRAIL
receptor pathways. CDMPs can also be activators of caspases,
including Granzyme B, or modulators of Granzyme B.
[0135] In various embodiments, the cell death mediator protein is
encoded by nucleic acid constructs. In some embodiments, the
nucleic acid construct can encode one or more CDMPs. The CDMPs can
be the same or different. For example, a single nucleic can encode
two of the proteins, such as two sequences encoding caspase 9 in
tandem, or can encode caspase 9 and caspase 3. The nucleic acid
construct of the present inventnion can encode caspase 2, 5, 8, 9,
10 or 11, their proenzyme forms, or derivatives thereof In further
embodiments, the sequence encoding such caspase protein(s) is
modified to include a dimerization domain reactive with a
cross-linker compound. Thus in some embodiments, a wild type
caspase sequence is modified to produce a chimeric sequence
comprising the dimerization domain selectively reactive to a
cross-linker compound. Examples of such dimerization domains
include those disclosed in U.S. Patent Application No. 20050187177
and U.S. Pat. No. 6,984,635, the relevant portions of which are
incorporated herein by reference in their entirety.
[0136] Furthermore, selective cell death in vivo or in vitro can be
achieved by use of a dual promoter, self-inactivating (SIN)
lentiviral vector constructed to allow tissue specific expression
of the suicide gene or CDMP, and a marker gene (e.g., eGFP). In one
embodiment, cells such as oligodendrocytes are targeted using a
nucleic acid construct of the present invention, as shown in FIG.
1. The resulting vector allows for tissue specific expression of a
suicide gene and the marker gene eGFP. Therefore, the vector allows
for infection of post-mitotic cells such as oligodendrocytes.
Furthermore, the vector can be utilized to produce high viral
titers to facilitate efficient application in vivo. Thus in one
embodiment, the result of such cell specific expression is
oligodendrocyte specific cell death.
[0137] The dual promoter feature allows for co-expression of GFP
independent of the upstream gene so that infected cells can be
easily identified using fluorescent microscopy. Other fluorescent
markers known in the art can be used instead of GFP.
[0138] In one embodiment, a nucleic acid delivery vehicle is
utilized to deliver a sequence encoding a cell death mediator
protein (or suicide protein) to cells in vitro. Delivery can also
be to the CNS, in a focal pattern. There are several advantages to
this system.
[0139] First, the location of the lesion can be pre-determined,
allowing for accurate detection of associated neurons and cellular
responses. Second, the size of the lesion produced can be
controlled and altered in subsequent experiments. This is
significant as there may be a threshold of oligodendrocyte loss
that must be breached to propagate the death of associated neurons.
Thirdly, the model allows for the ability to designate the exact
timing of demyelination.
[0140] Therefore, the temporal description of neuronal response at
the cellular and molecular levels can be achieved. Furthermore, the
temporal relationship of one lesion to a second lesion produced at
a different location in the CNS can be examined to determine if the
time between demyelinating lesions is a significant factor in
determining the type of CNS response. In addition, the delivery
vector incorporates cell-specific expression regulatory sequent
resulting in cell type-specific (e.g., oligodendrocyte specific)
expression of the cell death mediator protein. This feature ensures
that the experimentally imposed death of cells is limited to the
desired cell type population with minimal bystander effect.
Finally, the model is amenable to modification and incremental
levels of complexity.
[0141] For example, in one embodiment, the iCP9 is cloned into a
lentiviral plasmid and a MBP promoter sequence is cloned upstream
of the iCP9 gene. The resultant lentiviral vector,
p.DELTA.mbpICP9/mEGFP, is used to create replication incompetent
virus (see Example 1). Thus, the vector can be utilized to define
neuronal response over a period of time or at specific time points
as a result of one or more particular cell types (e.g., neural
and/or mural cells, as desired) by selection of
cell/tissue-specific or inducible expression regulatory
sequences.
[0142] In various embodiments, utilizing methods of the present
invention, the response of neurons to oligodendrocyte loss can be
defined over time. This can be accomplished by observing changes in
known survival, trophic and apoptotic pathways as well as through
microarray analysis to identify novel responses to such events.
Furthermore, classification of the defined neuronal response to
environmental stress includes the expression of apoptosis inducing
factors, anti-apoptosis factors, neurotrophic factors and
neurotrophic related transcription factors.
[0143] Therefore, in various embodiments animals/cells of the
present invention can be utilized to screen various
factors/compounds to determine if such factors/compounds
enhance/diminish neuron maintenance or health. For example,
multiple transcription factors are expressed in neurons in response
to external stimuli. Two highly conserved and well defined
transcription factors, NF-.kappa.B and cAMP response element
binding protein (CREB), are expressed when neurons are stressed and
subsequently activates, in a partially defined manner, an extensive
number of downstream targets. NF-.kappa.B is modulated by
physiologicai and pathological conditions including stroke, cardiac
arrest and global ischemia, seizure and experimental exposure to
glutamate, glucose deprivation and .beta.- amyloid peptide. This
highly conserved response to variable insults suggests a basic
cellular response that is pertinent to neuron survival and control
of apoptosis. Similarly, CREB is activated in response to a vast
array of physiological stimuli. Initial in vitro studies and later
in vivo studies utilizing CREB null mice suggest that CREB is
necessary for survival of multiple neuronal subtypes. CREB is
activated in response to hypoxia, ischemia and oxidative stress in
multiple rodent models and inactivation of CREB during these
stressors typically exacerbates neuronal cell death.
[0144] Both CREB and NF-.kappa.B induced cellular response appear
to primarily act in a manner that ultimately supports neuron
survival. Similarly, neurotrophic factors support the growth and
survival of neurons. This group of molecules includes, but is not
limited to: glial derived neurotrophic factor (GDNF), ciliarly
neurotrophic factor (CNTF), brain derived neurotrophic factor
(BNDF), nerve growth factor (NGF), NT-3 and NT 4/5. These factors
are thought to be upregulated after acute CNS insult. Furthermore,
oligodendrocytes are thought to provide trophic support to neurons.
In vivo observations describing the loss of oligodendrocytes in
acute MS lesions, in transgenic mice lacking PLP and after,
irradiation of oligodendrocyte precusors at birth, have been
corroborated by in vitro studies. Specifically, the addition of
oligodendrocytes precursor cells or their conditioned media to the
substantia nigra significantly enhanced neuronal survival.
Similarly, optic nerve oligodendrocyte precursor cells or their
cultured media significantly enhanced retinal ganglionic cell
survival. NGF, BDNF, GDNF, Neuregulin and NT-3 have all been
identified in oligodendrocyte cultured media.
[0145] In contrast to neuronal survival, programmed cell death of
neurons may be an important response to acute oligodendrocyte loss.
Fas, a member of the death receptor family, induces apoptosis when
bound by its ligand FasL by activation of the caspase cascade. In
the brain, cortical neurons express Fas and in vitro, these cells
rapidly undergo apoptosis after Fas activation. In models of
stroke, Fas expression is upregulated in neurons, which
co-localizes with the expression of caspase 8. Furthermore, if FasL
is absent, stroke-induced brain damage is reduced. In vitro
cultures of motor neurons have demonstrated a similar reliance upon
Fas activation for initiation of programmed cell death as motor
neurons deprived of neurotrophic support in vitro were maintained
in culture only after the Fas/FasL interaction was abolished.
[0146] Finally, the expression of anti-apoptotic factors may be an
important aspect of the response to acute demyelination. The
apoptotic process of naturally occurring cell death is highly
conserved among species, and studies in the nematode C. elegans
initially led to the identification of a molecule essential to this
process and subsequently allowed for the identification of a
mammalian homolog, E4BP4. This molecule has been shown to be
expressed by motor neurons at the time of naturally occurring cell
death in developing brain. Moreover, in vivo overexpression
increases the number of neurons innervating targets suggesting that
the molecule prevents the naturally occurring apoptotic
process.
[0147] Therefore, in various embodiments, an anti-apoptotic
compound can be administered at various time points to cells or
animals of the present invention to determine anti-apoptotic
compound's effects on neuron maintenance. The anti-apoptotic
compound can have known activity against a cell death mediator
protein (e.g., anti-caspase 9) or it can be a candidate agent that
is screened to determine anti-apoptotic activity.
[0148] Thus, by defining the neuronal response over time to
oligodendrocyte loss, it can be determined what compounds affect
the cascade of events that ultimately leads to neuronal loss, or
the rescue of neurons from cell death. The information gained from
defining spatial and temporal expression patterns or molecules is
critical in designing appropriate functional studies and dissecting
the cellular interactions that underlie neural response to
injury
[0149] In one embodiment, methods of the present invention are
utilized to assay oligodendrocyte-neuron interaction that is free
from systemic complications in order to define the molecular
response of surviving neurons (e.g., Example 2). The transection of
axons and subsequent neuronal loss in acute and chronic MS lesions,
as well as in NAWM (normal appearing white matter), is a relatively
recently defined aspect of this disease. Evidence suggests that the
continual loss of neurons accounts for the accumulation of
disability in patients.
Cell-Based Screening Assays
[0150] In some aspects of the present invention cell culture is
utilized in one or more methods of the invention. Target cells can
be derived from a subject and transformed (e.g. genetically
modified), or the transgenic animals of the invention can be the
source for cell/tissue culture. In another aspect, the cells of the
present invention may be cells derived from cell lines.
[0151] In various embodiments, the practice of the present
invention may involve cell-based assays for providing a comparison
of the expression of a gene or gene product or the activity of the
gene product in a test cell (e.g., transgenic oligodendrocyte or
Schwann cell) relative to a control cell. The test cell used for
this invention can be isolated from central or peripheral nervous
systems, and includes cell culture derived therefrom and the
progeny thereof, and section or smear prepared from the source, or
any other samples of the brain that contain, for example,
oligodendrocytes or Schwann cells or their progenitors. Where
desired, one may choose to use enriched cell cultures that are
substantially free of other neuronal cell types such as, but not
limited to, neurons, glial cells, microglial cells, and astrocytes.
Various methods of isolating, generating or maintaining matured
oligodendrocytes and Schwann cells are known in the art and are
exemplified herein.
[0152] In one embodiment, a method is provided for compiling a
profile data set for characterizing a phenomenon associated with MS
or MS-associated condition comprising providing a non-human
transgenic animal or cell comprising a transgene encoding a cell
death mediator protein, wherein the transgene is operably linked to
a neuronal- or glial-specific expression regulatory element;
activating the cell death mediator protein, thereby inducing
apoptosis; obtaining at least one surviving neuronal or glial cell
following the activation; and profiling RNA transcripts and/or
encoded products in the surviving glial or neuronal cell; thereby
compiling a profile data set characterizing a phenomenon associated
with MS or MS-associated condition. In other embodiments, this
method provides for compiling a profile data set for characterizing
phenomenon associated with ALS or Parkinson's disease.
[0153] In some embodiments, the cell death mediator protein is
caspase-9 or caspase-11. In one embodiment, activating caspase-9
comprises inducing dimerization of caspase-9. In another
embodiment, activating caspase-11 comprises autoactivation.
[0154] In various embodiments, the cell type-specific expression
regulatory element is from a gene selected from a group including
one or more of CC1, myelin basic protein (MBP), ceramide
galactosyltransferase (CGT), myelin associated glycoprotein (MAG),
myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin
glycoprotein (OMGP), cyclic nucleotide phosphodiesterase (CNP),
NOGO, myelin protein zero (MPZ), peripheral myelin protein 22
(PMP22), protein 2 (P2), GFAP, AQP4, PDGF.alpha., RG5,
pGlycoprotein, neurturin (NRTN), artemin (ARTN), persephin
(PSPN),sulfatide, 2 (VEGFR2), superoxide dismutase (SOD1), tyrosine
hydroxylase, neuron specific enolase, parkin gene (PAKK2), parkin
coregulated gene (PACRG), neuron-specific T.alpha.l .alpha.-tubulin
(T.alpha.l),vesicular monoamine transporter (VMAT2),
.alpha.-synuclein (SNCA), PDGFR-.alpha., PDGFR-.beta., or
proteolipid protein (PLP).
[0155] In one embodiment, the present invention provides a method
of identifying a candidate biologically active agent that modulates
remyelination. The method involves the steps of obtaining or
isolating transgenic cells from transgenic animals of the present
invention that are capable of cell-differential expression of cell
death mediator protein, culturing such cells; contacting a
candidate agent with the cultured cells; detecting an altered
expression of a gene or gene product or an altered activity of the
gene product relative to a control cell, the gene or gene product
being correlated to modulation of cell death by the cell death
mediator protein; and selecting the agent as a candidate if the
level of expression of said gene or gene product is modulated
relative to said control cell.
[0156] In another embodiment, an agent is determined to be a
candidate agent if the number of target cells undergoing cell death
is modulated by addition of the candidate agent as compared to
control cells.
[0157] In another embodiment, the present invention provides a
method of identifying a biologically active agent that promotes
remyelination. The method comprises the steps of obtaining,
isolating and culturing target cells from a demyelinated lesion
present in a transgenic animal of the present invention; contacting
a candidate biologically active agent with the cultured cells; and
detecting an altered expression or an altered activity of a
transgene encoding a cell death mediator protein; and selecting the
agent as a candidate if the level of expression of the gene or gene
product, or the level of activity of the gene product is increased
relative to the control cell. For example, if a candidate agent
enhances cell death mediating activity of the cell death mediator
protein, then the candidate can promote demyelination, whereas if
said candidate agent reduces cell death mediating activity then the
agent can promote remyelination.
[0158] In certain embodiments, it may be preferable to employ
myelinating cells from young subjects whose nervous systems are
actively undergoing myelination. In other embodiments, it may be
preferable to use remyelinating cells derived from adult
oligodendrocyte precursors in demyelinated lesions, including but
not limited to, lesions inflicted by pathogens or physical
injuries, and lesions caused by toxic agents such as cuprizone.
[0159] In one embodiment, high density cortical cultures are
transfected with replication-deficient lentivirus expressing iCP9.
After addition of a CID (chemical inducer of dimerization) and
subsequent death of oligodendrocytes, microarray analysis of gene
transcription can be used to assess the expression of novel factors
in surviving cultured cells. The extent of oligodendrocyte cell
death in these cultures will be systemically varied to identify
molecular responses related to the severity of cell destruction.
Therefore, in various embodiments, one or more molecular targets
modulating cell death are identified.
[0160] Various genetic vehicles suitable for the present invention
are available in the art. They include both viral and non-viral
expression vectors. Non-limiting exemplary viral expression vectors
are vectors derived from RNA viruses such as retroviruses, and DNA
viruses such as adenoviruses, foamy virus, rabies virus and
adeno-associated viruses. Non-viral expression vectors include but
are not limited to plasmids, cosmids, and DNA/liposome complexes.
The genetic vehicles can be engineered to carry regulatory
sequences that direct tissue specific, cell specific, or even
organelle specific expression of the exogenous genes carried
therein.
[0161] In some embodiments, target cells can be co-transfected with
multiple genetic vemcies two vectors each of which comprises gene
constructs encoding a desired product and gene constructs encoding
one or more reporter genes).
[0162] Furthermore, if desired a wide variety of subcellular
localization sequences or signals have been characterized and are
applicable for directing organelle specific expression of
transgenes. For instance, subcellular localization sequence can be
any one of the following: (a) a signal sequence that directs
secretion of the gene product outside of the cell; (b) a membrane
anchorage domain that allows attachment of the protein to the
plasma membrane or other membraneous compartment of the cell; (c) a
nuclear localization sequence that mediates the translocation of
the encoded protein to the nucleus; (d) an endoplasmic reticulum
retention sequence (e.g. KDEL sequence) that confines the encoded
protein primarily to the ER; (e) a protein of interest can be
farnesylated, such that the protein will be membrane associated; or
(f) any other sequences that play a role in differential
subcellular distribution of a encoded protein product.
[0163] If desired, the genetic vehicles can be inserted into a host
cell (e.g., myelinating cells such as oligodendrocytes or Schwann
cells) by any methods known in the art. Suitable methods may
include transfection using calcium phosphate precipitation,
DEAE-dextran, electroporation, or microinjection.
[0164] The selection of an appropriate control cell or tissue is
dependent on the test cell or tissue initially selected and its
phenotypic or genotypic characteristic which is under
investigation. Whereas the test remyelinating cell is contacted
with a test compound, then a control cell or tissue may be a
non-treated counterpart. Whereas the test remyelinating cell is a
test cell detected post demyleination, the control cell may be a
non-treated counterpart. It is generally preferable to analyze the
test cell and the control in parallel.
[0165] As discussed in the sections above, these cells are useful
for conducting cell-based assays for elucidating the molecular
bases of neuronal remyelination conditions, and for assaying agents
effective for inhibiting neuronal demyelination or promoting
remyelination.
[0166] In some aspects of the invention, transgenic cells can be
obtained from the transgenic animals of the invention, cultured and
expanded, transduced with a gene encoding a target protein, and
implanted or reintroduced into the source animal or some other
animal. In such ex vivo methods, the transgenic cells can be
transfected with a gene encoding a biologically active agent (e.g.,
gene encoding a test product) that can be inducibly produced for
example, so as to assay the test gene/protein for modulation of
marker gene expression/production. Such modulation can be assayed
in cell-culture as described herein above. Alternatively,
transduced cells are reintroduced into the subject animal, where
marker gene expression can be assayed and compared to a control or
reference, where the cells transplanted are not transduced, do not
express a vector-borne product of interest, express a vector-borne
product of interest in a time controlled manner (e.g., inducible
expression) or express the product of interest constitutively
(e.g., CMV promoter).
[0167] For example, glial cells (e.g., oligodendrocytes or Schwann
cells) can be derived from nerve biopsies. Cells can be expanded in
culture (e.g., utilizing proliferating medium composed of DMEM
containing 10% heat-inactivated fetal bovine serum (FBS) and
supplemented with antibiotics, recombinant Neu differntiation
factor (NDF), insulin and forskolin (1 ug/ml). Furthermore, cells
can be sorted from non-transgenic nerve cells utilizing the
fluorescence labels provided by the transgene(s) (e.g., FACS). For
transduction, cells can be tranfected with various vector vehicles
known in the art that will deliver a product of interest.
Therefore, in various embodiments, the nucleic acid constructs of
the invention are also operably linked to one or more sequences
encoding a marker protein.
[0168] Vectors that can be utilized with one or more composition or
methods of the present invention include derivatives of SV-40,
foamy virus, rabies virus, adenovirus, lentivirus,
retrovirus-derived DNA sequences and shuttle vectors derived from
combinations of functional mammalian vectors and functional
plasmids and phage DNA. Eukaryotic expression vectors are well
known, e.g. such as those described by P J Southern and P Berg, J
Mol Appl Genet 1:327-341 (1982); Subramini et al., Mol Cell. Biol.
1:854-864 (1981), Kaufinann and Sharp, J Mol. Biol. 159:601-621
(1982); Scahill et al., PNAS USA 80:4654-4659 (1983) and Urlaub and
Chasin PNAS USA 77:4216-4220 (1980), which are hereby incorporated
by reference. The vector used in the methods of the present
invention may be a viral vector, such as a retroviral vector, such
as replication deficient adenoviruses. For example, a "single gene
vector" in which the structural genes of a retrovirus are replaced
by a single gene of interest, under the control of the viral
regulatory sequences contained in the long terminal repeat, may be
used, e.g., Moloney murine leukemia virus (MoMu1V), the Harvey
murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV)
and the murine myeloproliferative sarcoma virus (MuMPSV), and avian
retroviruses such as reticuloendotheliosis virus (Rev) and Rous
Sarcoma Virus (RSV), as described by Eglitis and Andersen,
BioTechniques 6(7): 608-614 (1988), which is hereby incorporated by
reference. Preferably, the vector of the present invention is a
lentivirus vector.
[0169] In one embodiment, an engineered inducible caspase 9 (iCP9)
cDNA sequence is cloned into a lentiviral vector under the control
of the myelin basic protein gene promoter sequence FIG. 1C.
Subsequently, replication incompetent lentivirus can be produced
from this plasmid and applied to cellular and animal systems.
Although the virus will result in infection of all cell types
exposed, expression of the iCP9 gene will be limited to
oligodendrocytes because of the cell specific promoter system. The
addition of a chemical inducer of dimerization (CID) will result in
the cellular death of only oligodendrocytes. This approach will be
highly effective at specifically killing oligodendrocytes both in
vitro and in vivo. One of skill in the art recognizes that other
CDMPs may be substituted for iCP9, such as other caspases or their
derivatives, that other promoter sequences may be substituted for
the MBP promoter.In another aspect of the present invention,
microarray or other expression profiling processes known in the art
are utilized to identify a gene or sets of genes that are
upregulated or downregulated in response to cell death. In various
embodiments, the expression data sets can be compiled for various
and particular time points, including before, during and after
induction of cell death (e.g., Example 5).
Bioactive Agent
[0170] A biologically active agent or bioactive agent effective to
modulate neuronal remyelination is intended to include, but not be
limited to, a biological or chemical compound such as a simple or
complex organic or inorganic molecule, peptide, peptide mimetic,
protein (e.g. antibody), liposome, small interfering RNA, or a
polynucleotide (e.g. anti-sense).
[0171] A vast array of compounds can be synthesized, for example
polymers, such as polypeptides and polynucleotides, and synthetic
organic compounds based on various core structures, and these are
also contemplated herein. In addition, various natural sources can
provide compounds for screening, such as plant or animal extracts,
and the like. It should be understood, although not always
explicitly stated, that the active agent can be used alone or in
combination with another modulator, having the same or different
biological activity as the agents identified by the subject
screening method.
[0172] When the biologically active agent is a composition other
than naked RNA, the agent may be directly added to the cell culture
or added to culture medium for addition. As is apparent to those
skilled in the art, an effective amount must be added which can be
empirically determined. When the agent is a polynucleotide, it may
be introduced directly into a cell by transfection or
electroporation. Alternatively, it may be inserted into the cell
using a gene delivery vehicle or other methods as described
above.
[0173] A wide variety of labels suitable for detecting protein
levels are known in the art. Non-limiting examples include
radioisotopes, enzymes, colloidal metals, fluorescent compounds,
bioluminescent compounds, and chemiluminescent compounds.
[0174] Candidate biologically active agents identified by the
subject methods can be broadly categorized into the following two
classes. The first class encompasses agents that when administered
into a cell or a subject, reduce the level of expression or
activity of a cell death mediator protein(s). The second class
includes agents that augment the level of expression or activity of
a cell death mediator protein(s).
Pharmaceutical Compositions
[0175] The methods of the present invention can be utilized to
select a biologically active agent that can subsequently be
implemented in treatment of demyelination disorders. The selected
biologically active agents effective to modulate remyelination may
be used for the preparation of medicaments for treating
demyelinating disorders. In one aspect, an identified/selected
biologically active agent of this invention can be administered to
treat neuronal demyelination inflicted by pathogens such as
bacteria and viruses. In another aspect, the selected agent can be
used to treat neuronal demyelination caused by toxic substances or
accumulation of toxic metabolites in the body as in, e.g., central
pontine myelinolysis and vitamin deficiencies. In yet another
aspect, the agent can be used to treat demyelination caused by
physical injury, such as spinal cord injury. In still yet another
aspect, the agent can be administered to treat demyelination
manifested in disorders having genetic attributes, genetic
disorders including but not limited to leukodystrophies,
adrenoleukodystrophy, degenerative multi-system atrophy, Binswanger
encephalopathy, tumors in the central nervous system, and multiple
sclerosis.
[0176] Various delivery systems are known and can be used to
administer a biologically active agent of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
expression by recombinant cells, receptor-mediated endocytosis
(see, e.g., Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432),
construction of a therapeutic nucleic acid as part of a retroviral
or other vector, etc. Methods of delivery include but are not
limited to intra-arterial, intra-muscular, intravenous, intranasal,
and oral routes. In a specific embodiment, it may be desirable to
administer the pharmaceutical compositions of the invention locally
to the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion during
surgery, by injection, or by means of a catheter. In certain
embodiment, the agents are delivered to a subject's nerve systems,
preferably the central nervous system. In another embodiment, the
agents are administered to neural tissues undergoing
remyelination.
[0177] Administration of the selected agent can be effected in one
dose, continuously or intermittently throughout the course of
treatment. Methods of determining the most effective means and
dosage of administration are well known to those of skill in the
art and will vary with the composition used for therapy, the
purpose of the therapy, the target cell being treated, and the
subject being treated. Single or multiple administrations can be
carried out with the dose level and pattern being selected by the
treating physician.
[0178] The preparation of pharmaceutical compositions of this
invention is conducted in accordance with generally accepted
procedures for the preparation of pharmaceutical preparations. See,
for example, Remington's Pharmaceutical Sciences 18th Edition
(1990), E. W. Martin ed., Mack Publishing Co., PA. Depending on the
intended use and mode of administration, it may be desirable to
process the active ingredient further in the preparation of
pharmaceutical compositions. Appropriate processing may include
mixing with appropriate non-toxic and non-interfering components,
sterilizing, dividing into dose units, and enclosing in a delivery
device.
[0179] Pharmaceutical compositions for oral, intranasal, or topical
administration can be supplied in solid, semi-solid or liquid
forms, including tablets, capsules, powders, liquids, and
suspensions. Compositions for injection can be supplied as liquid
solutions or suspensions, as emulsions, or as solid forms suitable
for dissolution or suspension in liquid prior to injection. For
administration via the respiratory tract, a preferred composition
is one that provides a solid, powder, or aerosol when used with an
appropriate aerosolizer device.
[0180] Liquid pharmaceutically acceptable compositions can, for
example, be prepared by dissolving or dispersing a polypeptide
embodied herein in a liquid excipient, such as water, saline,
aqueous dextrose, glycerol, or ethanol. The composition can also
contain other medicinal agents, pharmaceutical agents, adjuvants,
carriers, and auxiliary substances such as wetting or emulsifying
agents, and pH buffering agents.
Examples
Example 1
Production of iCP9 Virus
[0181] To Produce Replication Incompetent Virus.
[0182] FIGS. 1 and 2 depicts the general schematic for the transfer
vectors used herein. The first generation self inactivating
lentiviral vector pLpMG was modified by insertion of either the
cytomegalovirus promoter sequence (pCMV), a fragment of the rat
Myelin Basic Protein promoter (pMBP), a fragment of the rat glial
fibriallary acid protein promoter (pGFAP) or a fragment of the
platelet derived growth factor receptor (PDGFR)-alpha promoter
(pPDGFR). The resulting vectors (pLpCMVXMG, pLpMBPXMG, pLpGFAPXMG,
pLpPDGFRXMG) were further modified by ligation of the iCP9 cDNA
sequence, obtained via PCR using the original vector (kind gift of
Dr David Spencer, Baylor Medical Center) as template, was cloned
downstream of the new promoter sequence.
[0183] The three vectors, pLpCMV(iCP9)MG, pLpMBP(iCP9)MG,
pLpGFAP(iCP9)MG, pLpPDGFR(iCP9)MG, were then used to generate high
titer, replication incompetent lentivirus. Viral titer was
determined against 293T cells followed by FACS analysis for EGFP
expression as all vectors co-express EGFP. All titers used in vivo
were at least 10.sup.9 colony forming units per ml. This resulted
in a vehicle to transfer the expression of the inducible suicide
gene (iCP9) to cells both in vitro and in vivo while enabling
identification of infected cells through EGFP expression as it will
be driven off a viral promoter sequence within the same vector. It
also limited the gene expression to specific cell types based on
which promoter was used, which gives the system cell type specific
ablation in a time controlled manner.
[0184] 293T cells were transfected with the p.DELTA.mbpICP9/mEGFP
vector along with plasmids encoding the gag-pol and RD114 envelope
protein using lipofectamine (Invitrogen, Carlsbad, Calif.).
Forty-eight hours after transfection viral supernatant were
harvested and either directly applied to target cells or snap
frozen and stored at -80.degree. C.
[0185] To induce timely cell death. Collected virus was used to
transduce primary oligodendrocyte cultures that have been
established. Cells were cultured and evaluated using fluorescent
microscopy for the expression of GFP. The extent of GFP positive
cells correlated with the viral infection efficacy. Cell protein
isolation was conducted and protein isolates were subjected to
Western blot transfer to detect stable expression of the iCP9
protein. The resultant membrane was probed with anti-caspase 9
antibody (R&D Systems Inc. #AF8301). Once the stable expression
of iCP9 was confirmed at the protein level wan western mot
techniques, the CID was applied to these cells in culture at a 10
nM concentration. This induced the cellular death of infected
cells. Cell death was detected by viewing cultures under the
fluorescent microscope and by using Methylene Blue (Sigma)
viability staining of cells in culture after exposure to the CID.
All experiments were run in parallel using the astrocyte-derived
U87 cell line as a negative control.
[0186] To induce apoptosis in the presence of the CID and
demonstrate the cell type selectivity, lentivirus was applied to in
vitro cultures. Apoptosis was initially induced in 293T cells using
the pLpCMV(iCP9)MG virus which produces constitutive expression of
iCP9. EGFP is also produced from the same viral plasmid and
promoter. After the addition of CID, EGFP-positive cells
(corresponding to cells transfected with virus) rapidly underwent
apoptosis and were not identified in culture. However, cells not
transfected with virus (EGFP negative) persisted in culture. In
identical cultures transfected with control virus, pLpCMVXMG,
EGFP-positive cells persisted after exposure to CID (FIG. 5). A
Cell Death Detection ELISA Plus (Roche Inc) was also performed to
determine the level of apoptosis in culture after cells infected
with various dilutions of virus were exposed to CID. The ELISA
system detects histone-complexed DNA fragments that are produced by
cells undergoing apoptosis. The ELISA data shows a correlation
between infection, subsequent gene expression and cell death (FIG.
6).
[0187] Possible alternative cell death mediator systems include the
use of an E coli-derived cytosine deaminase gene, the HSV-tk system
and a transgenic CD20, which can be activated by a monoclonal
chimeric anti-CD20 antibody to induce apoptosis.
[0188] Results demonstrated efficient infection of target cells
(e.g., primary oligodendrocytes) with the p.DELTA.cmvICP9/mEGFP
derived lentivirus. The infection rate should approach
approximately 100% of target cells in culture based on preliminary
data. Furthermore, using an MBP promoter sequence to drive
oligodendrocyte specific expression of genes, GFP is detected in
the oligodendrocytes and not in the U87 control cell line (or other
control cell lines, e.g., NIH 3T3). Using iCP9 systems with CIDs,
the administration of the CID to cultures is expected to yield
rapid and efficient cell death in the oligodendrocytoma cell line,
but not in the U87 cell line.
[0189] Addition of CID to in vitro system. The CID was added to the
mixed cortical culture described above at a final concentration of
10 nm in Neurobasal medium with 50 ng/ml NGF. The initial time to
oligodendrocyte death was thus established as described above and
represents the first time point for the analysis of neuronal
response. The second time point represented the subacute response
at 24 and 72-hour analysis. Finally, a chronic response to
oligodendrocyte loss was analyzed at 1 week, 2 weeks and 2 months
post-CID administration.
Example 2
Oligodendrocyte Loss
[0190] The iCP9 cell death system was applied to oligodendrocyte
primary cell cultures and the efficacy of cell death, as well as
the time course, was defined. Second, the system was applied to
high-density cortical cultures to model the acute loss of
oligodendrocytes that occurs in MS.
[0191] Using this system the response of neurons to oligodendrocyte
loss was defined over time. This was accomplished by observing
changes in known survival, trophic and apoptotic pathways as well
as through microarray analysis to identify novel responses to such
events.
[0192] Establish primary oligodendrocyte cultures. Enriched
populations of oligodendrocytes were isolated from Fischer P2 rats.
Forebrain was dissected in Hank's buffered salt solution. Tissue
will be cut into approximately 1-mm pieces in a poly
L-lysine-coated 25 cm.sup.2 flask, and then incubated at 37.degree.
C. for 15 minutes in a humidified 5% CO.sub.2 room air incubator in
0.01% trypsin and 10 .mu.g/ml DNase. Following this incubation
period, DMEM medium supplemented with 20% fetal calf serum (FCS)
was added to the tissue mixture and then left to proliferate for 10
days in a humidified 5% CO.sub.2 room air incubator. After ten days
oligodendrocyte precursor cells (A2B5.sup.+) was collected by
shaking the flask over-night at 200 rpm at 37.degree. C. The cells
that remain adherent to the flask were cultured in high-glucose
DMEM supplemented with fibroblast growth factor (FGF) and platelet
derived growth factor (PDGF) to a final concentration of 10 ng/ml
for one week. Afterwards, FGF and PDGF were removed from the
culture medium allowing for A2B5+ cells to differentiate into O4+
premyelinating oligodendrocytes over 3 to 7 days and then to O4+
/and MBP+ mature oligodendrocytes after 7-10 days. The status of
cellular differentiation was monitored by noting the change in
cellular morphology in culture. Differentiation into mature, MBP+
oligodendrocytes can be easily identified with microscopic
examination. To confirm the differentiation, the pattern was
observed with light microscopy as changes in cell morphology,
anti-A2B5, anti-O4 and anti-MBP antibodies were utilized in basic
immunohistochemical staining protocols using an aliquot of cultured
cells at various time points in the differentiation pathway.
[0193] Once this system and differentiation pattern was confirmed,
A2B5.sup.+ primary cell cultures were cultured in
p.DELTA.mbpICP9/mEGFP viral supernatant for 8 hours. After eight
hours supernatant was removed and replaced with DMEM/FGF PDGF
medium for 24 hours. Cells were then be visualized under
fluorescent microscopy to determine the efficacy of infection.
Infected cells expressed EGFP and were readily identified using the
proper fluorescent filter system.
[0194] After efficient infection with pimbpICP9/mEGFP is confirmed,
aliquots of cultured GFP.sup.+, A2B5.sup.+ cells are allowed to
differentiate in culture as described above. After mature,
MBP.sup.+ oligodendrocytes were identified in culture, the CID is
added to a final concentration of lOnM in DMEM/20% FCS. Maintaining
multiple cultures allowed for the analysis of apoptosis at various
time points after addition of the CID. Methylene Blue staining of
cultures was undertaken at 1, 4, 12 and 24 hours after the CID is
added to culture supernatant. Methylene blue positive cells are
thus identifiable and represent viable cells. This experiment was
run in duplicate using A2B5.sup.+ cells that have not been exposed
to p.DELTA.mbpICP9/mEGFP viral supernatant, and therefore, should
not undergo apoptosis. Therefore, time to death for 100% of cells
was established after addition of the CID.
Example 3
High Density Mixed Cortical Cultures
[0195] High density mixed cortical cultures were established after
isolation of cerebral cortices from 3 Fischer rats as previously
described. Cerebral cortices were removed and place in ice-cold
Hanks' balanced salt solution, centrifuged and digested with
trypsin at 37.degree. for 10 minutes. Tissue was centrifuged and
resuspended in minimal essential medium with Earle's salt
(Invitrogen) containing heat-inactivated fetal bovine serum and
horse serum. Cell suspension was passed through cell strainers and
plated. After three hours the medium is changed to Neurobasal
medium supplemented with B27 MinusAO (Invitrogen). Cultures were
maintained for seven days and then immunohistochemical staining was
performed to verify the composition of the cultures.
[0196] The mixed primary cortical cultures (consisting of all CNS
cell types) were infected with pLpMBP(iCP9)MG and exposed to CID
and then analyzed for cell death of oligodendrocytes (MBP+ cells).
The MBP promoter was designed to induce cell death only in MBP+
cells, the majority of which are mature oligodendroctyes. Prior to
CID expression cells appeared morphologically normal and cell
counts of control cultures and virally transfected cultures did not
demonstrate significant differences between cell types indicating
that viral transfection did not significantly alter the
constitution of the culture. After CID exposure MBP-positive,
EGFP-positive cells appeared disrupted relative to MBP-positive,
EGFP-positive cells not exposed to CID (FIG. 7). Furthermore, A2B5
(olidgodendrocytes precursor cells)/EGFP positive cells, GFAP
(astroyctes)/EGFP positive cells and O4 positive cells
(oligodendrocyte precursor cells)/EGFP positive cells appeared
morphologically normal before and after CID exposure.
[0197] To test the specificity of the GFAP promoter system, GFAP+
pan purified cultures (cultures of purified astroctyes) were
transfected with pLpGFAP(iCP9)MG and exposed to CID. Pan purified
GFAP cells infected with pLpGFAP(iCP9)MG were exposed to CID and 4
hours after a decrease in EGFP positive cells was demonstrated
while analogous cultures not exposed to CID maintained a high level
of EGFP positive cells (FIG. 8). These data confirmed that the GFAP
promoter drives iCP9 expression resulting in apoptosis of
astrocytes after CID exposure.
[0198] To confirm the cell specificity of the PDGFR-.alpha.
promoter system, rat-derived primary cortical cultures were
infected with pLpPDGFR(iCP9)MG virus. In mixed primary cortical
cultures (cultures containing all CNS cell types), A2B5-positive
cells (A2B5 expression overlaps with PDGFR expression and is used
in place of anti-PDDGFR antibody as it is less labor intensive)
were identified undergoing apoptosis with altered morphology, while
other cell types appear unaffected (FIG. 9). This provides data
supporting the oligoprecursor-specificity of the PDGFR-.alpha.
promoter system.
Example 4
Analysis of Neuronal Response to Acute Insult
[0199] Four major classes of responses: apoptosis inducing factors,
anti-apoptosis factors, neurotrophic factors and neurotrophic
related transcription factors, is analyzed. Analysis of
neurotrophic factors is carried out using commercial ELISA assays
for GDNF, CNTF, BNDF, NGF, NT-3 and NT 4/5 according to
manufacturer's protocol. Twenty-four hour supernatant from mixed
cortical cultures are collected after apoptosis at time points
described and from mixed cortical cultures that have not been
administered the CID, which serve as the control. All samples are
run in duplicate and final data represents the mean of the two
values.
[0200] Analysis of neurotrophic related transcription factors
NF-.kappa.B and CREB activation are carried out using the Trans-AM
P-CREB and NF-.kappa.B p65 kits (Active Motif Europe, Rixensart,
Belgium) according to manufacturer's instructions. The Trans-Am
assays measures the level of the active forms of phosphorylated
CREB and NF-.kappa.B contained in cell extracts able to bind
specifically to oligonucleotide containing the cyclic AMP-response
element and the NF-.kappa.B consensus site (5'-GGGACTTTCC-3')
coated to 96-well plates. A secondary horseradish
peroxidase-conjugated antibody provides a sensitive colorimetric
readout that will be quantified using the ELISA plate reader
(BIORAD) at 450 nm. All samples are run in duplicate. Cell extracts
are derived from mixed cortical cultures following induced
apoptosis at time points defined previously. Control cell extracts
consist of mixed cortical cultures not exposed to the CID.
[0201] The number of neurons undergoing apoptosis following the
loss of oligodendrocytes in mixed cortical cultures is quantified
using a TdT-mediated dUTP nick end labeling assay (TUNEL assay,
Roche, Indianapolis, Ind.) according to the manufacturer's
protocol. The number of positive cells are quantified using
microscopy and compared to the number of positive cells in cultures
not exposed to the CID. Immunohistochemical (IHC) staining is
carried out using anti-FAS and anti-NOS antibodies to quantify
expression in neurons from mixed cortical cultures after loss of
oligodendrocytes and compared to cultures that have not been
exposed to the CID. Staining is conducted using the VECTASTAIN ABC
kit (Vector Laboratories Inc., Burlingame, Calif.) per
protocol.
[0202] Antiapoptotic molecules may play a role in neuronal survival
or conversely a decrease in expression may contribute to neuronal
loss. A novel factor, E4BP4, appears to play an important
antiapoptotic function in neurons in the CNS. Therefore, the level
of expression of E4BP4 is examined via immunohistochemistry (IHC)
staining in neurons undergoing apoptosis following the loss of
oligodendrocytes in mixed cortical cultures and compared to
cultures that have not been exposed to the CID. IHC staining is
carried out as described above.
Example 5
Identification of Unique Molecules Regulated by Acute
Oligodendrocytes
[0203] The acute loss and chronic absence of oligodendrocytes
represents a unique pathological state in the central nervous
system, and therefore, novel genes are upregulated and identified
in neurons in response to these events. To detect these genes, a
microarray analysis is undertaken. Total RNA is isolated using the
RNeasy Mini Kit (Qiagen, Valencia, Calif.) from the remaining
culture after induced apoptosis of oligodendrocytes at various time
points after the insult. To produce the control array, total RNA is
isolated in a similar manner from a culture that has not be exposed
to oligodendrocytes death in vitro by withholding the CID. RNA
processing and analysis is carried out by the core facility of the
Case Western Medical School using standard protocols. Scanned
output files will be visually inspected for hybridization
artifacts. Arrays are scaled to an average intensity and then
analyzed using Affymetrix Microarray 5.0 software. Genes are
considered upregulated if the expression is changed >1.5-fold
relative to control RNA.
[0204] Microarray results are confirmed with immunohistochemical
staining of cultures to demonstrate increase expression level if
antibodies are available. Where antibodies are not available,
quantitative RT-PCR is performed based on the gene sequence to
confirm the upregulation of gene expression. Briefly, .beta.-actin
serves as baseline gene expression. Primers are purchased based on
cDNA sequence analysis. Real-time PCR is performed using a BioRad
iCycler, and the computer calculates the standard curve for the
threshold cycle. The mean threshold cycle is calculated from three
wells for each sample, and the mean TC and standard curve are used
to extrapolate the sample mRNA quantity. In each cell culture mRNA
is quantified as a proportion of .beta.-actin mRNA, and the mean
proportions from control cultures are compared.
[0205] Administration of p.DELTA.mbpICP9/mEGFP virus to
oligodendrocyte precursor cells result in nearly 100% GFP positive
cells. The expression of iCP does not occur until oligodendrocytes
mature and the administration of the CID results in cell death of
oligodendrocytes over 4 to 6 hours based on published data in other
cells types (Straathof et al., 2005). Initial analysis of
pre-defined pathways such as that to ischemia or hypoxia will be
evident in culture neurons in the acute phase after oligodendrocyte
loss, as there is most likely a common response to noxious events
in neurons. However, the more time that passes from oligodendrocyte
loss the response can deviate from known injury response.
Furthermore, the microarray analysis of neurons following the loss
of oligodendrocytes results in the upregulation and identification
of 2 to 4, 2 to 6, or 5 to 10 novel and relevant genes compared to
control neurons. Such a profile is identified 72 hours to one week
after oligodendrocyte loss, rather then in the acute phase.
Example 6
Animal Model
[0206] The virus described above, p.DELTA.mbpICP9/mEGFP, which
contains the inducible caspase 9 sequence was applied to the CNS of
adult rats to create an in vivo model of CNS demyelination.
Administration of the viral vector allowed effective transfer of
genetic information in a variable area of parenchyma based on the
rate of administration (convective distribution) and viral titer.
The data demonstrated that areas of the CNS are effectively
infected with lentivirus. First, adult Fischer rats were
anaesthetized by intraperitoneal injection o f ketamine
hydrocholoride (80 mg/kg) and xylazine (4 mg/ml) and placed in a
sereotactic frame. A midline scalp incision is made. Access to
brain parenchyma was accomplished by plaement of a right-sided burr
hole through the skull, at various predetermined coordinates
depending on the desired location of the lesion. Lentivirus was
administered in serum free media using a sterile 10 .mu.l Hamilton
syringe with a No. 32S- gauge needle at a rate dependent upon the
target area size (convective distribution of virus is most
effective at rates of 0.1 to 0.5 .mu.l/minute). The volume of viral
vector (e.g., lentivirus) delivered depends upon the infection
efficacy as determined herein and the target area size. After
injection, the needle is left in place for five minutes and then
slowly withdrawn over the next four minutes. The skin was closed
with sutures.
[0207] For virus administration to the spinal cord an incision was
made in the thoracic spine followed by a lamenectomy to expose the
spinal cord. A No. 32S-gauge needle was passed into the posterior
column at predetermined coordinates. At various times after
injection of virus the CID was administered via intraperitoneal
injection resulting in apoptosis of the infected region of the
CNS.
[0208] Verification of reversible physiologic dysfunction after
acute demyelination. Based on the data a viral vector was utilized
to successfully infect areas of the CNS and effectively express
transgenes. The physiologic manifestation of acute oligodendrocyte
death was initially observed in rats with brain lesions over time.
For lesions in the spinal cord a more precise recording mechanism
was employed. The functional integrity of axons in the dorsal
columns is examined in vivo using somatosensory evoked potential
recordings (SSEP).
[0209] At various time points after the administration of the CID
rats were anesthtized as described previously for SSEP recording.
SSEPs were recorded from a screw electrode over the right
somatosensory cortex referenced to an Ag/AgCl disk electrode placed
under the hard plate while the contralateral sciatic nerve is
stimulated at 1 Hz (0.2 ms pulse duration and 40 mA constant
current intensity for an average of 200 sweeps). A ground electrode
was placed on the scalp transdermally. SSEP amplitude is measured
from the first negative peak to the positive peak. Response latency
was measured as the time between the onset of stimulus and the
first peak. The amplitude and latency values was recorded as the
mean of three independent measures. These measures were repeated at
1, 2, 7 and 14 days after the administration of the CID to
establish a measurable pattern of CNS damage and repair.
Immunohistochemical analysis of acute lesions. In order to evaluate
the cellular changes associated with iCP9 oligodendrocyte cell
death, animals were sacrificed at various time points following the
administration of the CID.
[0210] Rat brains or spinal cords were snap frozen in isopentane
for 20 seconds and then stored at -80.degree. C. until sectioning.
Coronal thin sections of the brain and axial sections of the spinal
cord at 10-.mu.m thickness were generated using a cryostat
(-20.degree. C.). Initially this was conducted at 1-day post death
to determine the success of CID induced cell death. Afterwards, 1,
2, 7 and 14-day post CID animals were sacrificed and examined using
fluorescent microscopy, luxol fast blue staining and hematoxylin
and eosin (H&E) staining. Thin section of brain and cord were
examined via immunohistochemical staining using various antibodies
to determine the extent of inflammation (anti-LCA antibody,
anti-ED1) and gliosis (anti-GFAP antibody) that has occurred.
[0211] The photograph in FIG. 2 demonstrates that concentrated
lentivirus can be applied directly to the CNS and the extent and
area of infection can be determined based on the number of GFP
positive cells detected after sacrifice in thin sections of the
brain or spinal cord. This data establishes the transfer mechanism
for specific cell death in vitro and in vivo that is the basis of
the model proposed herein.
Example 7
Identification of Axonal Transaction and Neuronal Apoptosis in
Acute Lesions
[0212] Thin sections are used to detect focal neuron damage at the
site of the lesion in the form of transected axons or apoptotic
neurons. Immunohistochemical staining is conducted on thin sections
encompassing demyelinating lesions using anti-amyloid precursor
protein (APP) antibody. This identifies disturbances of axonal
transport and transection as APP accumulates at the ends of such
axons. Similarly, Bielschowskys silver impregnation staining is
utilized to detect neurons and transected axons. Fixed sections are
stained in prewarmed (40.degree. C.) 10% silver nitrate for ten
minutes and then washed in PBS. Ammonium hydroxide is added to the
silver nitrate solution and slides incubated for 30 minutes at
40.degree. C. after which time slides will be placed directly in
developer working solution (40% formaldehyde, citric acid, nitric
acid solution) for one minute.
[0213] The reaction is halted in 1% ammonium hydroxide, washed in
PBS and then incubated in 5% sodium thiosulfate for 5 minutes.
Finally, slides are dehydrated and mounted. Using this staining
technique, transected axons can be identified and the number of
axons are counted and compared to control animals that have been
infected with virus but have not been administered the CID and
therefore do not have focal areas of demyelination. Neurons are
counted in and surrounding acute lesions at various time points
after cell death and compared to thin sections derived from animals
that have received virus but not CID and therefore do not have
focal areas of demyelination. A 0.01 mm.sup.2 field, defined by an
ocular morphometric grid, taken throughout the middle of each
lesion area or a distant, normal area are selected for examination.
In this field APP positive fibers or Bielschowskys silver
impregnated fibers are counted under a 100.times. objective.
Example 8
Identification of Distant Neuronal Loss after Acute Demyelinating
Lesions
[0214] In order to determine whether demyelination in the CNS can
result in the death of distant neurons with axons transversing the
lesion, neurons are examined in the contralateral red nucleus
following the production of demyelinating lesions in the lateral
fasiculus of the spinal cord at various time points. The number of
neurons in the red nucleus of rats after the induction of
oligodendrocyte death in the spinal cord are compared to the number
in the red nucleus of rats infected with virus that have not
received the CID. Also, a TdT-mediated dUTP nick end-labeling assay
(TUNEL assay, Roche, Indianapolis, Ind.) is utilized to detect
apoptotic activity. This is conducted on thin sections according to
the manufacturer's protocol. Subcortical Lesion Burden and Spinal
Cord Atrophy. Extensive spinal cord atrophy is a well defined
feature of MS. Demyelinating events in distant areas of the CNS
induce spinal cord atrophy and that this induction is related to
the extent of demyelinating burden is detemrined.
[0215] Therefore, as described previously a viral vector (e.g.,
iCP9), is delivered to subcortical regions of the CNS and delivery
of the CID results in areas of demyelination. Eight months after
the original insult rats are sacrificed, spinal cord removed, snap
frozen, thin sections prepared and H&E stained. Spinal cord
diameter is measured and compared to control animals that are
infected in the same manner with the iCP9 virus, but will not have
received the CID and therefore, will not have acute demyelinating
lesions. The number of lesions, location, as well as the time
interval between lesions can all be varied to produce greater
degrees of demyelinating area and frequency to maximize the
possibility of detecting atrophy in the spinal cord.
[0216] Finally, results identify changes in survival or apoptotic
factors or unique genes are verified using the in vivo model. This
is carried out using immunohistochemical staining. Results can also
be assayed in the in vivo model, which provides another physiologic
substrate to study neuronal response to demyelination. The acute
loss of oligodendrocytes in the spinal cord can result in an acute
physiological dysfunction and measurable changes in SSEP
recordings. Specifically, an attenuation of amplitude and increase
in latency are identified. This corrects to baseline over a time
course that are determined as the rat recovers from the initial
insult. Using Luxol Fast Blue staining after cell death, an area of
acute demyelination is defined. The extent and type of inflammatory
infiltrate is determined using immunohistochemical staining.
[0217] Thus, a mild nonspecific inflammatory infiltrate can ensue
and dissipate over several weeks. As has been demonstrated in
pathological specimens from MS patients, transected axons are
localized within the acute lesion. Finally, a decrease in the
number of viable neurons in the red nucleus after the induction of
multiple, distant demyelinating lesions are observed. The time
course and extent of loss can occur slowly over time, e.g., may be
identified from about 6 to 12 months after demyelination.
Example 9
Transgenic Animal Expressing Caspase-9
[0218] A transgenic mouse expressing caspase-9 under the control of
myelin basic promoter is created by first generating a transgenic
targeting vector construct. Various nucleic acid elements are
incorporated to ensure the expression of caspase-9 in mouse. A
synthetic intronic element is placed in front of caspase-9 cDNA for
proper processing of pre-mRNA originated from the vector. A poly
adenylation signal is incoroporated at the end of caspase-9 cDNA
for proper processing of mRNA. To allow inducible expression of
caspase-9, a stop codon floxed by two LoxP sites (Cre-recombinase
recognition sites) are incorporated between the myelin basic
promoter and the synthetic intron. The vector is linearized for
efficient integration of the vector into the genome and injected
into a number of pronuclei by microinjection. The injected
pronuclei are implanted into pseudopregnant FVB/N strain mice. The
pups are screened for the integration of the injected vector(s)
into the host genome. Pups positively identified in the screen are
weaned and mated with a transgenic mice containing ER-Cre
transgene. The pups from the mating are screened for animals
harboring both the caspase-9 and ER-Cre transgenes. Tamoxifen is
peritoneally injected to induce the expression of Cre protein from
ER-Cre transgene. The excision of LoxP site and the resulting
expression of caspase-9 in myelin sheath are confirmed in an
immunofluorescence staining using anti-caspase-9 antibody.
Example 10
Cell Specific Induction of Apoptosis in vivo
[0219] The system was tested in vivo to ensure that cell specific
apoptosis could be induced and the timing of cell ablation
controlled as it was in vitro. To this end, virus was injected into
the corpus callosum of adult Fischer rats and then three weeks
later CID was administered into the ipsilateral ventricle. Rats
were sacrificed 24-hours thereafter, brains removed in whole, fixed
and cyrosectioned for analysis. Rats injected with pLpGFAP(iCP9)MG
(GFAP promoter limits expression to astrocytes) and then CID were
sacrificed 24-hours later and demonstrated TUNEL positive staining
at the site of virus infection (demarcated by EGFP positive cells
as the vector results in constitutive expression of EGFP). The
TUNEL (TdT-mediated dUTP-X nick end-labeling) system adds a tag on
to DNA that has been fragmented during the apoptosis cascade
allowing for labeling using standard immunohistochemistry. Thin
sections derived from control rats receiving glycerol rather than
CID were TUNEL negative. Consecutive thin sections were then
stained with anti-GFAP antibody to identify astrocytes. GFAP+ cells
were not detected at the area of viral infection in thin sections
derived from rats administered CID, in contrast to sections derived
from control rats receiving glycerol (FIG. 10). This confirmed that
in vivo the iCP9 system induces apoptosis only after CID
administration and effectively ablates GFAP+ astrocytes.
[0220] In a similar manner, rats injected with pLpMBP(iCP9)MG
(designed to ablate oligodendrocytes) and exposed to CID failed to
stain with anti-MBP antibody at the site of infection, while thin
sections from control rats infected with pLpMBP(iCP9)MG and
administered glycerol appeared normal after immunohistochemical
staining using anti-MBP antibody. TUNEL positive cells were
identified at the site of infection after exposure to CID, but not
in controls exposed only to glycerol (FIG. 11).
[0221] These data confirmed that the iCP9 system is able to induce
apoptosis in vivo in a time controlled fashion and appears specific
to cell-type based on the promoter sequence utilized.
Example 11
Cellular Response to Acute Oligodendrocyte Apoptosis in the CNS
[0222] The demyelinating and repair process in rats both in the
spinal cord and the brain is defmed by using rat pups, 3 days old
(P3), that are anesthetized by intraperitoneal injection of
ketamine hydrocholoride (80 mg/kg), acepromazine (2.1 mg/kg), and
xylazine (4 mg,/ml) and placed in a stereotactic frame (Stoelting
Co). A 10 .mu.l Hamilton syringe with a No. 26S- gauge needle is
passed through the soft skull 0.5 mm anterior and 0.5 mm lateral of
bregma at a depth of 0.2 mm Five microliters of pLpMBP(iCP9)MG
virus (selectively ablates oligodendrocytes) in serum free media is
injected using a 10 .mu.l Hamilton syringe with a No. 26S-gauge
needle at a rate of 2 .mu.l/min. For rats receiving injections into
the thoracic spinal cord, a 10 .mu.l Hamilton syringe with a No.
26S- gauge needle is manually passed through the spinal cord. Virus
is then injected at the same rate as in brain by way of a
microinjection system (Harvard Co). After injection, the needle is
be left in place for five minutes and then slowly withdrawn over
the next four minutes.
[0223] Three weeks after injection of the virus, 5 .mu.l of CID (10
nm) is injected into the ipsilateral ventricle (-0.8 mm anterior
and 1.4 mm lateral to bregma at a depth of 3.6 mm) which results in
the apoptosis of infected oligodendrocytes. Every other day,
beginning on the day of CID injection, animals receive an
intraperitoneal injection of 100 mg/kg BrdU labeling mix (Sigma)
which incorporates into proliferating cells and allows for their
identification using immunohistochemistry. Rats are sacrificed at
days 1, 7, 14, 21 and 28 post-CID.
[0224] Control rats are injected with virus in the same manner as
the experimental animals but receive an injection of glycerol (CID
is diluted in glycerol) rather than CID. Rats are deeply
anesthetized and perfused transcardially first with 150 ml of 0.9%
NaCl saline solution followed by equal volume of ice-cold 4%
paraformaldehyde. Whole brain and/or spinal cord is then removed
and post-fixed in paraformaldehyde for at least 4 hours followed by
cryoprotection in 30% sucrose until tissue sinks to the bottom of
the container. Samples are then frozen in OCT and cryosectioned at
10 to 20 .mu.m on superfrost plus slides. This experiment is
repeated in the spinal cord in the same manner.
[0225] Immunohistochemisry. Tissue derived from 1, 7, 14, 21 and 28
day post-CID animals is examined using fluorescent microscopy to
identify the area of infection. In order to define the area of
acute demyelination and subsequent remyelination, black gold
staining of myelin is conducted on thin sections after the
induction of acute oligodendrocyte death at each time point. The
time to demyelination in this experiment is defined as the time
point at which the area of infection does not stain with black
gold, and the time to remyelination in this experiment is defmed as
the time point after demyelination that black gold stains the area
of infection with equal intensity as the neighboring unaffected
myelin, is noted for each animal and a mean and standard deviation
calculated. These data is compared to the time to demyelination and
the time to remyelination for control animals. Statistical analysis
is not conducted as the control is not expected to
demyelinate/remyelinate. However, these data points serve as
controls for future experiments.
[0226] Thin sections of brain and/or spinal cord is examined to
determine the extent of inflammation using anti-CD45 antibody (T
and B cells), anti-ED1 antibody (microglial cells), anti-CD68
antibody (macrophages) and ghosis using anti-GFAP antibody
(astrocytes). This is done at each time point and qualitively
compared to labeled thin sections derived from equivalent control
animals.
[0227] Sections are also stained with anti-nestin antibody
(pluripotent stem cell), anti-NG2+ antibody (glial committed stem
cell), anti-PDGFR-alpha (total pool of OPCs), anti-04+ antibody
(early OPC lineage when O1 negative), anti-O1 antibody (late OPC
lineage) and anti-MBP antibody (mature oligodendrocyte) to identify
stem cell and oligodendrocyte precursor cell mobilization and
incorporation into the dynamic lesion site. These antibodies
identify different cells along the pathway from CNS stem cell to
mature oligodendrocyte. Thin sections are then examined using
fluorescent microscopy and labeled cells counted in a 0.01 mm.sup.2
field defined by an ocular morphometric grid under a 100.times.
objective. Four distinct locations within the lesion are randomly
selected and counted and an average cell number and standard
deviation for each section at each time point will be calculated.
This is compared to the corresponding site in the contralateral
hemisphere, which does not have a demyelinating lesion and to
control animals which were infected with virus but did not receive
CID and therefore should not have a demyelinating lesion. The area
for counting in the control animal is the site of viral injection
identified by the presence of EGFP+ cells. The average cell count
for each area is compared using a paired t-test. This is repeated
for each antibody used at each time point to determine which
oligodendrocyte subtype migrates to the lesion and the temporal
distribution of this migration.
[0228] Rats are injected with BrdU labeling mix after CID exposure.
BrdU incorporates into the DNA of cells during division and allows
for the identification of cells that divided by the application of
anti-BrdU antibody and standard immunohistochemistry. After
completion of staining for cell types, thin sections are labeled
with anti-BrdU antibody with a secondary antibody distinct from
that used in the initial labeling procedure. The overlap of signals
identify the proliferating cell types. The double stained sections
are compared to control sections at analogous time points. If the
same proliferating cell type is identified in the control sections
then double stained cells are counted in the entire ipsilateral
hemisphere of four sections and averaged and compared to average
counts derived from control sections which can then be
statistically compared using a standard t-test. If double labeled
cells are absent in controls the quantitative measure is presence
or absence of the double labeled cells.
[0229] The above experiments are also carried out in the spinal
cord with comparable controls that receive viral injection into the
spinal cord but vehicle (glycerol) rather than CID. The preparation
of animals for spinal cord demyelination differs in that the CID is
injected into the cisterna magna rather than the ipsilateral
ventricle. This is accomplished by palpation along the spinal cord
to the base of the skull followed by passage of the Hamilton
syringe into this space.
Example 12
The Response of Neurons to the Acute Loss of Oligodendrocyte in
vivo
[0230] Identification of axonal transection in acute lesions.
Demyelinating lesions are produced as describe in Example 11.
Animals are sacrificed at time points determined by data from
Example 11. Control rats are injected with virus in the same manner
as the experimental animals but receive an injection of glycerol
(CID is diluted in glycerol) rather than CID. Rats are sacrificed
and prepared as described. In order to characterize the response of
axons within the lesion, thin sections are labelled with
anti-neurofilament (NF) antibody and anti-amyloid precursor protein
(APP). The former identifies axons in the lesion while the later
identifies disturbances of axonal transport and transection. NF+
axons and APP positive axons are counted using a 0.01 mm.sup.2
field defined by an ocular morphometric grid are counted under a
100.times. objective. Four distinct areas within the demyelinated
lesions are counted and averaged.
[0231] A percentage or transected axons (APP+) is calculated by
dividing the average APP+ cell count by the mean number of axons in
the lesion (NF+). These data is compared to control animal counts
conducted at the site of infection in a comparable manner. Counting
of neurons is conducted at days 1, 7, 14, 21 and 28 post-CID. A
simple t-test is used to determine statistical significance between
control and experimental counts at any given time point.
[0232] Identification of distant neuronal loss after acute
demyelinating lesions. In order to determine whether demyelination
in the CNS can result in the loss of neurons in spatially distinct
regions of the CNS, neurons are examined in the substantia nigra
following the induction of a demyelinating lesion in the lower
thoracic spinal cord. Rats are prepared as described but the site
of the lesion is the thoracic spinal cord and CID is injected into
the cisterna magna as described previously. Controls rats are
injected with virus but receive a glycerol injection into the
cisterna magna rather than CID. Rats are sacrificed at the same
time points (1, 7, 14, 21 and 28 days) and whole brain and spinal
cord removed and prepared as described. The spinal cord is
sectioned and stained with black gold to confirm the presence of a
demyelinating lesion. Brain sections incorporating the substantia
nigra, identified by its anatomical location and appearance, is
labeled with anti-NF1 antibody and neurons counted as described
above. The average number of neurons in the substantia nigra of
rats after the induction of oligodendrocyte death in the spinal
cord is compared to the average number in the substantia nigra of
control rats which do not have a demyelinating lesion. Similarly, a
TUNEL stain is performed on thin sections of the brain that
encompass the substantia nigra derived from rats with demyelinating
lesions in the spinal cord to detect active apoptosis of neurons.
The TUNEL (TdT-mediated dUTP-X nick end-labeling) system adds a tag
onto DNA that has been fragmented during the apoptosis cascade
allowing for labeling using standard immunohistochemitry. If TUNEL
positive cells are identified within the substantia nigra they are
counted. As before the TUNEL stain results are compared to staining
and counts in the substantia nigra of control rats and average
counts compared with a t-test. These experiments determine if
distant loss of oligodendrocytes effects the survival of unrelated
neurons in the CNS.
[0233] Determination of the relationship between demyelinating
burden and neuron loss. The loss of distant axons in normal
appearing brain may be dependant on the extent of demyelination in
the CNS. To test this, the size of demyelinating lesions and the
number of demyelinating lesions is altered. P3 rats are injected
with virus in the thoracic spinal cord and into the bilateral
corpus callosum. Rats are allowed to mature and at P20 rats are
injected with CID as described. Rats are sacrificed at day 28 post
CID injection and tissue from the brain and spinal cord prepared as
described above. The time point for analysis may vary, but is
completed after the lesions remyelinate. Control rats receive the
identical viral injections but receive glycerol rather than CID at
P20. As described above, after lesion induction, which is
multifocal in this example, the integrity of neurons in the
substantia nigra is examined via TUNEL assay and neuron counting.
The number of viable neurons and apoptotic cells per 0.01 mm.sup.2
field defined by an ocular morphometric grid under a 100.times.
objective is averaged from 4 separate fields within the substanta
nigra in the same section and compared to control animals counted
in the same manner. Cell counts are compared using a standard
t-test.
[0234] Verification of reversible physiologic dysfunction after
acute demyelination. The functional integrity of axons in the
dorsal columns is examined in vivo using somatosensory evoked
potential recordings (SSEP). At various time points after the
administration of the CID rats are anesthetized as described
previously. SSEPs are recorded from an electrode inserted into the
spinal cord above the lesion referenced to an Ag/AgCl disk
electrode placed under the hard plate while the contralateral
sciatic nerve is stimulated at 1 Hz (0.2 ms pulse duration and 40
mA constant current intensity for an average of 200 sweeps). A
ground electrode is placed on the scalp transdermally. SSEP
amplitude is measured from the first negative peak to the positive
peak. Response latency is measured as the time between the onset of
stimulus and the first peak. The amplitude and latency values are
recorded as the mean of three independent measures. These data is
collected at time points, but also includes a baseline prior to
demyelination, a data point after demyelination and one after
repair has occurred histological. The control data is obtained by
recording from the controlateral dorsal columns in the same rat.
The mean amplitude and latency value from the lesion side
(experimental side) is compared to the contralateral dorsal column
(control side) using a standard t-test.
Example 13
The role of Oligodendrocyte Precursor cells in the Remyelination
Process in the Adult Rat
[0235] To selectively ablate OPCs, a novel viral vector was created
using vector strategies described. Based on cell morphology
changes, that A2B5 labeled cells undergo apoptosis after infection
with the pLpPDGFR(iCP9)MG and CID exposure as described above. Pan
purified A2B5+ cells (culture enriched with A2B5+ by capturing
cells with antibodies) and P3 derived mixed cortical cultures
(methods to describe cultures described earlier) cultures grown in
parallel are infected but not exposed to CID and serve as a control
for labeling and ELISA studies. Both sets of cultures are stained
with anti-A2B5 antibody and TUNEL stain to determine the
specificity of cell ablation. A2B5 expression overlaps with
PDGFR-.alpha. expression and is easier to label in vitro and
therefore serves as a surrogate marker of PDGFR-.alpha.+cells.
Mixed cortical cultures are also stained with anti-GFAP (astrocyte
label) and anti-MBP (oligodendrocytes label) to ensure that other
cells types are not affected by infection with this viral
construct. The pan-purified A2B5+ cell cultures are exposed to
serial dilutions of pLpPDGFR(iCP9)MG virus and exposed to CID.
Cultures are then subjected to the Cell Death Detection Elisa Plus
(Roche Inc) system according to manufacturer protocols (described
in preliminary work) in order to quantify apoptosis levels. This
confirms that PDGFR+ cells are the only cells ablated after
infection and CID exposure. All cultures are then run in triplicate
to ensure reproducibility.
[0236] Upon confirmation of efficacy and specificity, the
pLpPDGFR(iCP9)MG virus will be injected into P3 rat brains or
thoracic spinal cord. Animals will be allowed to mature and at age
P30 lysolethicin will be injected into the ipsilateral corpus
collosum, at the site of original virus infection. Lysolethicin
(LPC) is a detergent that will result in rapid cell membrane
destruction and acute loss of myelin with axon preservation.
Twenty-four hours after LPC is injected into the rat brain, animals
will be sacrificed to confirm that the experimental procedure has
resulted in the expected in vivo paradigm. The presence of a LPC
induced demyelinating lesion within the area of original
pLpPDGFR(iCP9)MG infection will be confirmed by identifying the
area of viral infection on thin sections using fluorescent
microscopy. Subsequently, sections with EGFP+ cells will be stained
with black gold to confirm the presence of a superimposed
demyelinating lesion. I propose the use of 4 animals to confirm
that I am able to reproducibly create a LPC lesion within the area
of original viral injection. There is no control and no statistical
analysis for this experiment.
[0237] Once the position of infection and demyelination are
confirmed, the experiment is repeated with the addition of CID to
ablate PDGFR+OPCs 24 hours after the injection of LPC. Animals are
sacrificed at days 1, 7, 14, 21 and 28 to determine the effect of
OPC ablation on myelin repair. The controls for these studies are
animals infected with pLpPDGFR(iCP9)MG virus at P3 with a
superimposed LPC lesion at P30 but receive glycerol rather then CID
thereafter. Therefore they should have a demyelinating LPC induced
lesion but no OPC ablation and should repair in a pre-defined
manner.
[0238] Animals with superimposed LPC demyelinating lesions and
subsequent OPC ablation (experimental group) are compared to
animals with superimposed LPC lesions and no OPC ablation (control
group) using the time to remyelinate data point identified with
black gold staining (see Example 11). Furthermore, the time
required for OPC migration to the site of LPC lesion is also
recorded and compared between groups. Both the time to remyelinate
and the time to OPC migration is recorded for each animal and
statistical means of each group compared using standard t-test
analysis. As described in Example 11, sections are stained for the
presence of oligodendrocyte cell types, astrocytes and inflammatory
cells. Cells are counted on labeled sections per the protocol
described in Example 11 and means calculated and compared to the
average cell counts in control groups using a standard t-test.
[0239] Finally, injecting all animals with BrdU every other day
after CID allows for the identification of proliferating cells as
described. After completion of staining for cell types, thin
sections are then labeled with anti-BrdU antibody with a secondary
antibody distinct from that used in the initial labeling procedure.
The overlap of signals identifies the proliferating cell types. The
double stained sections are compared to control sections at
analogous time points. If the same proliferating cell type is
identified in the control sections then double stained cells are
counted in the entire ipsilateral hemisphere of four sections and
averaged and compared to average counts derived from control
sections which can then be statistically compared using a standard
t-test. If double labeled cells are different than those identified
in control animals there is no quantification.
[0240] This experiment can be repeated in the spinal cord.
[0241] The present invention is not limited to the embodiments
described above, but is capable of modification within the scope of
the appended claims. Those skilled in the art will recognize, or be
able to ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein.
* * * * *